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Best practices for supporting and expanding undergraduate research in chemistry
 9780841232846, 0841232849, 9780841232839

Table of contents :
Content: PrefaceEarly Career Experiences1. The FUTURE Program: Engaging Underserved Populations through Early Research Experiences2. Four-Year Research Engagement (FYRE) Program at the University of Oklahoma: Integrating Research in Undergraduate Curriculum 3. Another Round of Whiskey for the House: Community College Students Continue Research on Experimental New Flavors of Whiskey 4. Transforming Second Semester Organic Chemistry Laboratory into a Semester Long Undergraduate Research Experience 5. Embedded Research in a Lower-Division Organic Chemistry Lab Course Upper Divison Opportunities6. Developing an Integrated Research-Teaching Model 7. Theory and Experiment Laboratory: Modeling the Research Experience in an Upper-Level Curricular Laboratory 8. Integrating Research into the Curriculum: A Low-Cost Strategy for Promoting Undergraduate Research 9. Peptidomimetics from the Classroom to the Lab: Successful Research Outcomes from an "Upper-Level" Class at a Primarily Undergraduate Institution 10. Translation of Chemical Biology Research into the Biochemistry Laboratory: Chemical Modification of Proteins by Diethylpyrocarbonate 11. Leveraging Student Interest in Environmental Topics for UndergraduateResearch in an Interdisciplinary Environmental Research Cluster Programs and Curriculum Reform12. Overview of a Flexible Curriculum and the Impact on Undergraduate Research 13. Transformative Impact of a Comprehensive Undergraduate ResearchProgram on the Department of Chemistry at the University of North Carolina Asheville 14. Leveraging NSF-CREST Center Funding To Support Undergraduate Research at Multiple Hispanic Serving/Minority Institutions 15. Institutionalizing Undergraduate Research and Scaffolding Undergraduate Research Experiences in the STEM Curriculum Mentoring and Assessment16. Engaging Early-Career Students in Research Using a Tiered Mentoring Model 17. Best Practices in Mentoring Undergraduate Researchers for Placement in an International Setting 18. Assessing Undergraduate Research in Chemistry 19. Senior Undergraduate Research and Assessment at Florida Southern College 20. Implementing Best Practices to Advance Undergraduate Research in Chemistry Editors' Biographies Indexes

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Best Practices for Supporting and Expanding Undergraduate Research in Chemistry

ACS SYMPOSIUM SERIES 1275

Best Practices for Supporting and Expanding Undergraduate Research in Chemistry Bridget L. Gourley, Editor DePauw University Greencastle, Indiana

Rebecca M. Jones, Editor George Mason University Fairfax, Virginia

Sponsored by the ACS Division of Chemical Education

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Library of Congress Cataloging-in-Publication Data Names: Gourley, Bridget L., editor. | Jones, Rebecca M. (Chemistry professor), editor. | American Chemical Society. Division of Chemical Education. Title: Best practices for supporting and expanding undergraduate research in chemistry / Bridget L. Gourley, editor (DePauw University, Greencastle, Indiana), Rebecca M. Jones, editor (George Mason University, Fairfax, Virginia) ; sponsored by the ACS Division of Chemical Education. Description: Washington, DC : American Chemical Society, [2018] | Series: ACS symposium series ; 1275 | Includes bibliographical references and index. Identifiers: LCCN 2018015181 (print) | LCCN 2018018384 (ebook) | ISBN 9780841232839 (ebook) | ISBN 9780841232846 (alk. paper) Subjects: LCSH: Chemistry--Study and teaching (Higher) | Chemistry--Research. Classification: LCC QD453.3 (ebook) | LCC QD453.3 .B4745 2018 (print) | DDC 540.71/1--dc23 LC record available at https://lccn.loc.gov/2018015181

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2018 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

Contents Preface .............................................................................................................................. xi

Early Career Experiences 1.

The FUTURE Program: Engaging Underserved Populations through Early Research Experiences .............................................................................................. 3 Amanda J. Reig, Kathryn A. Goddard, Rebecca E. Kohn, Leslie Jaworski, and David Lopatto

2.

Four-Year Research Engagement (FYRE) Program at the University of Oklahoma: Integrating Research in Undergraduate Curriculum .................... 23 Naga Rama Kothapalli

3.

Another Round of Whiskey for the House: Community College Students Continue Research on Experimental New Flavors of Whiskey ......................... 33 Regan Silvestri

4.

Transforming Second Semester Organic Chemistry Laboratory into a Semester Long Undergraduate Research Experience ........................................ 47 Andrew J. Carr, Ryan J. Felix, and Stephanie L. Gould

5.

Embedded Research in a Lower-Division Organic Chemistry Lab Course ..... 65 Lee J. Silverberg, John Tierney, and Kevin C. Cannon

Upper Divison Opportunities 6.

Developing an Integrated Research-Teaching Model ......................................... 83 Robert E. Bachman

7.

Theory and Experiment Laboratory: Modeling the Research Experience in an Upper-Level Curricular Laboratory ............................................................ 101 Bridget L. Gourley

8.

Integrating Research into the Curriculum: A Low-Cost Strategy for Promoting Undergraduate Research .................................................................. 119 Sanchita Hati and Sudeep Bhattacharyya

vii

9.

Peptidomimetics from the Classroom to the Lab: Successful Research Outcomes from an “Upper-Level” Class at a Primarily Undergraduate Institution .............................................................................................................. 143 Danielle A. Guarracino

10. Translation of Chemical Biology Research into the Biochemistry Laboratory: Chemical Modification of Proteins by Diethylpyrocarbonate .... 165 Laura M. Hunsicker-Wang and Mary E. Konkle 11. Leveraging Student Interest in Environmental Topics for Undergraduate Research in an Interdisciplinary Environmental Research Cluster ............... 181 Neelam Khan, Sang H. Park, David P. Pursell, and Kathryn Zimmermann

Programs and Curriculum Reform 12. Overview of a Flexible Curriculum and the Impact on Undergraduate Research ................................................................................................................ 211 Bridget L. Gourley 13. Transformative Impact of a Comprehensive Undergraduate Research Program on the Department of Chemistry at the University of North Carolina Asheville ................................................................................................ 227 Bert E. Holmes, Amanda L. Wolfe, Sally A. Wasileski, and George L. Heard 14. Leveraging NSF-CREST Center Funding To Support Undergraduate Research at Multiple Hispanic Serving/Minority Institutions ......................... 243 Kimberley R. Cousins, Timothy Usher, Douglas C. Smith, Renwu John Zhang, Paul K. Dixon, and Sara Callori 15. Institutionalizing Undergraduate Research and Scaffolding Undergraduate Research Experiences in the STEM Curriculum .............................................. 259 Mitch Malachowski, Jeffrey M. Osborn, Kerry K. Karukstis, Jillian Kinzie, and Elizabeth L. Ambos

Mentoring and Assessment 16. Engaging Early-Career Students in Research Using a Tiered Mentoring Model ..................................................................................................................... 273 Sarah M. Hayes 17. Best Practices in Mentoring Undergraduate Researchers for Placement in an International Setting ....................................................................................... 291 J. C. Goeltz and R. S. Duran 18. Assessing Undergraduate Research in Chemistry ............................................ 301 Rebecca M. Jones

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19. Senior Undergraduate Research and Assessment at Florida Southern College ................................................................................................................... 311 Deborah Bromfield Lee and An-Phong Le 20. Implementing Best Practices to Advance Undergraduate Research in Chemistry .............................................................................................................. 335 Rebecca M. Jones Editors’ Biographies .................................................................................................... 345

Indexes Author Index ................................................................................................................ 349 Subject Index ................................................................................................................ 351

ix

Preface In 1922, William A. Noyes, the founder of Chemical Abstracts and Priestley Medalist, stated “proper methods of conducting undergraduate research should train the student in the use of chemical literature and be taught personal initiative in attacking a problem (1).” Noyes’s words are emblematic of the appreciation that the American Chemical Society (ACS) and chemists, in general, have for the significance of undergraduate research. Beginning in 1983, the ACS Committee on Professional Training (CPT) Guidelines included this statement; “the Committee strongly endorses carefully designed programs of undergraduate research.” Additionally, CPT has increased the number of hours of laboratory instruction that can be met by participation in research from about 15% to 45% of the required hours. Undergraduate research symposia at national ACS meetings have become standard programming in the Division of Chemical Education (CHED) with over 1000 students presenting each year. Without question, undergraduate research is significant to chemists. Undergraduate research is also significant within the general context of higher education. In the first decade of the 21st century, the American Association of Colleges and Universities (AAC&U) published multiple works examining high impact practices, but none focus exclusively on undergraduate research (2, 3). In 2009, the manuscript Broadening Participation in Undergraduate Research: Fostering Excellence and Enhancing Impact, edited by Mary K. Boyd, and ACS’s own Jodi L. Wesemann, became one of the first to chronicle the best practices of colleges and universities supporting this high impact practice (4). While the previous publications were general with respect to discipline, Lopatto’s Science in Solution: The Impact of Undergraduate Research on Student Learning (5) and Laursen, et al.’s Undergraduate Research in the Sciences: Engaging Students in Real Science (6) effectively established the value of undergraduate research across a range of scientific disciplines. Though valuable and impactful in their own right, none of these well-respected publications provide concrete examples from the individual or departmental level or offer readers specific ideas they might adapt at their own institutions. This symposium volume helps fill the gap between generalized or holistic assessments and individual classroom/laboratory innovations, which can serve as models for adoption. The aim of this volume is to share a collection of best practices currently employed by faculty and administrators to support and expand undergraduate research in chemistry at their colleges or universities. As members of ACS, as well as serving chemistry councilors for the Council on Undergraduate Research, we were motivated to bring individuals together to discuss best practices for supporting and expanding undergraduate research by organizing symposia at xi

recent spring national ACS meetings. At the 249th ACS National Meeting in Denver, CO (2015), the focus was on expanding opportunities and broadening participation. In 2016, at the 251st ACS National Meeting in San Diego, CA, the focus was on supporting and expanding undergraduate research opportunities in chemistry. The symposium at 253rd ACS National Meeting in San Francisco, CA (2017) had a general theme of advancing undergraduate research. This volume has chapters from selected speakers who contributed during each of these three meetings. We have organized this volume into four parts: Early Career Experiences, Upper Division Opportunities, Program and Curricular Reform, and Mentoring and Assessment. Chapters in the first section describe initiatives that serve students during what would typically be the first two years of a typical collegiate curriculum. These chapters focus both on programmatic initiatives that facilitate students early research participation and curricular innovations that embed research into the teaching laboratory. The second section provides a variety of approaches related to courses typically taken during students’ junior and senior years. In some cases, laboratories associated with particular courses have been revised into a full semester research experience, while in other cases the laboratory is structured to take an interdisciplinary approach or to expose students to the various elements of the research process. The third section, Programs and Curriculum Reform, provides chapters focused on shifting the overall curriculum within one or more institutions for transformative change. Chapters include approaches to intentional scaffolding of curricula and leveraging external funds. No volume would be complete without addressing the key issues of Mentoring and Assessment, our final section. Mentoring chapters discuss tiered approaches and issues related to international research experiences. The final two chapters provide models of assessing undergraduate research. Overall, this volume provides a snapshot of curricular and programmatic best practices in engaging a broad spectrum of students in undergraduate research. Readers interested in additional innovative ideas are invited to also review abstracts from our symposia, “Advancing Undergraduate Research: Best Practices and New Innovations” at the 255th ACS National Meeting in New Orleans, LA (2018) as well as the 2015-17 symposia archives for those speakers who were unable to contribute to this volume. As editors of this collection, we are grateful to the chapter authors for their contributions and the numerous reviewers who carefully read and thoughtfully commented on each submission. We also would like to acknowledge the help we received from the ACS Books and Technica Editorial staff, with notable gratitude to Courtney Coppage, for her efficient communication and support. We hope readers of this volume appreciate the breadth of ways we can engage chemistry students in undergraduate research. Additionally, we hope faculty will realize the synergistic opportunities to enhance their own scholarly agendas while serving students. In chemistry, teaching and research have never been mutually exclusive; undergraduate research effectively engages both.

xii

References 1. 2. 3.

4.

5. 6.

Kinkead, J. What’s in a Name? A Brief History of Undergraduate Research. CUR Q. 2012, 33 (1), 20–29. Elgren, T. E.; Hensel, N. Undergraduate Research Experiences: Synergies between Scholarship and Teaching. Peer Rev. 2006, 8 (1), 4–7. Kuh, G. High-Impact Educational Practices: What They Are, Who Has Access to Them, and Why They Matter; American Association of American Colleges & Universities: Washington, DC, 2008. Broadening Participation in Undergraduate Research; Boyd, M. K., Wesemann, J. L., Eds.; Council on Undergraduate Research: Washington, DC, 2009. Lopatto, D. Science in Solution; Council on Undergraduate Research: Washington, DC, 2010. Laursen, S. Undergraduate Research in the Sciences: Engaging Students in Real Science, 1st ed.; Jossey-Bass: San Francisco, 2010.

Bridget L. Gourley, Ph.D. Percy Lavon Julian Professor of Chemistry and Biochemistry Department of Chemistry and Biochemistry DePauw University 602 South College Avenue Greencastle, IN 46135-1900 (765) 658-4607 (telephone) (765) 658-6084 (fax) [email protected] (e-mail)

Rebecca M. Jones, Ph.D. Associate Professor, Department of Chemistry and Biochemistry STEM Accelerator, College of Science George Mason University 4400 University Drive, MSN 3E2 Fairfax, VA 22030 (703) 993-1084 (telephone) (703) 993-1055 (fax) [email protected] (e-mail)

xiii

Early Career Experiences

Chapter 1

The FUTURE Program: Engaging Underserved Populations through Early Research Experiences Amanda J. Reig,*,1 Kathryn A. Goddard,2 Rebecca E. Kohn,2,4 Leslie Jaworski,3 and David Lopatto3 1Department of Chemistry, Ursinus College, Collegeville, Pennsylvania 19460, United States 2Department of Biology, Ursinus College, Collegeville, Pennsylvania 19460, United States 3Department of Psychology, Grinnell College, Grinnell, Iowa 50112, United States 4Present Address: College of Arts & Sciences, Arcadia University, Glenside, Pennsylvania 19038, United States *E-mail: [email protected]

The FUTURE (Fellowships in the Ursinus Transition to the Undergraduate Research Experience) program at Ursinus College provides early research opportunities to members of underserved populations in STEM (science, technology, engineering, and mathematics). As part of the Parlee Center for Science and the Common Good at Ursinus College, the goal is to encourage and equip these students to become the scientific and civic leaders of the future. The program, established through a grant from the Howard Hughes Medical Institute, targets underserved students (underrepresented minorities, students with disabilities, women, and first-generation college students) prior to, or immediately following, their first year at Ursinus. These students, designated as FUTURE mentees, complete a four-week summer research experience mentored by an upper-class student (FUTURE mentor) and a faculty member. The FUTURE mentees also engage in a 1-credit course entitled “Science and Mathematics in Society” and take part in social programs designed to build community. Following the program,

© 2018 American Chemical Society

the FUTURE participants are expected to present their research findings at a regional or national conference and are strongly encouraged to continue research in subsequent semesters. From 2013 to 2017, 82 students (41 mentees and 41 mentors) and 22 faculty from a variety of STEM disciplines participated in the program. FUTURE mentees show greater persistence as STEM majors than their underserved peers (89.7% compared to 64.5% by junior year and 91.7% compared to 65.1% at graduation) and nearly all (90.2%) continue their involvement in undergraduate research. In retrospective surveys completed immediately after the program, both mentees and mentors reported gains in all categories assessed, including laboratory skills, understanding of their discipline, and confidence in explaining their projects. Learning gains were also assessed using the SURE Follow-Up survey, which showed the FUTURE participants had gains above national means in nearly all categories measured. The success of the FUTURE program demonstrates how early access to a short four-week, research intensive program can improve outcomes for underrepresented students and provides a simple model by which this can be achieved.

Introduction In 2012, Ursinus College established the Parlee Center for Science and the Common Good with support from a Howard Hughes Medical Institute (HHMI) Undergraduate Science Education Program grant. The objectives of the Parlee Center are to (1) provide opportunities for students to think, speak, and write about the impact of science on the common good, and (2) cultivate the next generation of scientific leaders with a focus on students from underserved populations (USPs). Underrepresented minorities (URMs), women in chemistry, computer science, mathematics, or physics, first-generation college students, and students with disabilities were included as USPs. The FUTURE (Fellowships in the Ursinus Transition to the Undergraduate Research Experience) program was created specifically to address the second objective above, and was designed based on studies which show that early involvement in undergraduate science research is an effective way to improve retention and outcomes for students from underserved populations (1, 2). For example, an extensive quantitative study by Jones et al. found that “participating in science research during the first two years or for more than three terms is associated with about a 240% increase in a student’s odds of graduation in biology with a [competitive] GPA (1).” In addition, frequent, positive contact with faculty and advanced peers in a research setting was shown to increase the academic and social integration of students from USPs and, as seen above (1), improved their retention in science (2). These results echo numerous studies which have shown that social integration is vital in the persistence and retention of minority students (3–5). By providing research 4

experiences to students from USPs very early in their undergraduate careers, the FUTURE program builds on the robust culture of undergraduate research at Ursinus (nearly 70% of STEM majors collaborate with faculty on original research) and leverages the close-knit scientific community that exists on our small, residential campus to encourage and equip them to become the scientific leaders of the future.

Program Overview The FUTURE program is a four-week, residential, paid opportunity for students from USPs during the summer prior to or immediately following their first year at Ursinus. FUTURE mentees receive a stipend of $1250 and free campus housing for the duration of the program. From 2012 to 2017, eight students were selected to be FUTURE mentees each summer, distributed evenly across students who had just completed their first year and those who would matriculate in the fall. The FUTURE program specifically targets USP students at the beginning of their undergraduate careers with the goals of improving their persistence as STEM majors, increasing their participation in undergraduate research, and expanding the number who enter STEM careers. Many summer undergraduate research programs, including Ursinus’ own Summer Fellows program, require students to have completed their sophomore year of college. Unfortunately, many USP students have already left STEM before reaching that point. USP students with interests in the following STEM disciplines and majors at Ursinus were invited to apply: biochemistry and molecular biology (BCMB), biology, chemistry, computer science, health and exercise physiology, mathematics, neuroscience, physics, and psychology. The FUTURE program is comprised of three main components: (1) a facultyand student-mentored research experience, (2) enrollment in the 1-credit course “Science and Mathematics in Society”, and (3) social programming to connect students with the campus community. The FUTURE program is overseen by a small committee that includes STEM faculty and the Assistant Director of Residence Life who oversees first-year students. The committee is chaired by a faculty program coordinator who receives a stipend or a course release.

Participant Selection Each year, applications for FUTURE faculty/mentor teams are solicited at the end of the fall semester. Faculty are asked to certify that that will be available for the duration of the program and will participate in the program activities (teach in the course, attend discussions, and contribute to assessment), identify a current research student whom they believe would make a good FUTURE mentor, and provide a brief research description that would be understandable to a student completing high school. FUTURE mentors are asked to certify their availability and participation in program activities, describe their prior research experience, reflect on their desire and ability to serve as a mentor, and discuss their career 5

goals. Selections are made based on academic performance, experience with the research project, and potential for excellence in mentoring. Students from USPs are strongly encouraged to apply to be FUTURE mentors, and special consideration is given to individuals who participated as FUTURE mentees previously. The FUTURE mentees are selected in two rounds. Current first-year students are invited to apply early in the spring semester after nomination by a faculty member. These students are asked to certify their availability for the duration of the program and their willingness to participate in the program activities, describe any prior research experiences (clearly noting that no prior experience is required), discuss their strengths and weaknesses when working on group projects, explain why they want to participate in the program and how their participation would benefit their career goals, and have a letter of recommendation submitted by a faculty member on their behalf. Incoming students who express an interest in one or more of the program fields and qualify as a member of an USP are identified with assistance from the Admissions Office and sent invitations to apply late in the spring semester. These students complete an application similar to that for the first-year students, and top candidates are given a phone interview prior to final selections. We have typically accepted students with moderate to strong backgrounds in STEM who have not yet participated in an independent research experience, reasoning that students with the potential to succeed but who lack experience would benefit the most from early integration into our research laboratories. As part of the application process, students are asked to rank the available projects based on the descriptions provided by the participating faculty mentors. The FUTURE mentees are assigned to a project based on their preference, intended major, and career goals. Mentored Research Experience Each FUTURE mentee is matched with a research team consisting of a faculty member and a FUTURE mentor. The FUTURE mentor is a rising junior or senior with at least one semester of prior research experience with the faculty member. The FUTURE mentors are full participants in our paid, residential Summer Fellows research program, in which they collaborate with faculty on research projects. The FUTURE program is timed to overlap with weeks 4–7 of the eight week Summer Fellows program. This allows the FUTURE mentor three weeks to work on the project before the FUTURE mentee arrives, giving them time to prepare to integrate their mentee into the research experience. The FUTURE mentors then have one final week to focus on preparing a required research paper and oral presentation without mentoring obligations. The FUTURE mentor is responsible for working with the FUTURE mentee daily, assisting them in understanding the project, conducting experiments, and analyzing data. Faculty mentors have responsibility for designing and/or overseeing the research project, interacting with the FUTURE mentor and mentee on a frequent basis, providing guidance and feedback on research progress, creating and maintaining a collegial and supportive environment for the students, and assisting them with the production of their final presentations and papers. 6

During the program, the FUTURE mentees spend approximately 35 hours per week in the research lab and participate fully in all aspects of the research project, including reading journal articles, designing and carrying out experiments, and analyzing and interpreting data. On the last day of the program, a mini symposium is held in which each FUTURE mentee gives a 10-minute oral presentation summarizing the goals and results of their project to an audience of students, faculty, friends, and family. The FUTURE mentees also prepare a written report, typically 5-8 pages in length, which must be submitted by the end of the fall semester. These two aspects of the program provide the mentees with early practice in oral and written scientific communication, building skills that they will use frequently in their future coursework and research experiences.

Science and Mathematics in Society Course In parallel with conducting research, FUTURE mentees take a one-credit pass/fail course called “Science and Mathematics in Society.” The course is designed to introduce them to the academic experience of Ursinus and equip them with tools they will need to be successful researchers. The course meets for approximately one hour four times per week (M, Tu, Th, and F). No class is held on the final Thursday of the program to give students additional time to prepare for their presentations, which take place on the last Friday morning. The course covers a wide range of topics, including laboratory safety, how to read scientific journal articles, how to analyze and interpret data, and how to give a scientific presentation, ethical dilemmas in research, diversity and bias in STEM, and communicating science to the public. One of the four meetings each week is set aside for large group discussions (including all FUTURE mentees, mentors, and faculty) over lunch. These large group discussions are centered on a book or excerpts from a book that everyone reads in preparation for the meeting, and model for the FUTURE mentees the type of academic experience they can expect in our first-year seminar program known as the Common Intellectual Experience (CIE). Texts used for this component of the course have included Thinking, Fast and Slow by Daniel Kahneman (6), The Demon Haunted World by Carl Sagan (7), How I Killed Pluto and Why It Had It Coming by Mike Brown (8), and Genome: The Autobiography of a Species in 23 Chapters by Matt Ridley (9). Books are provided for all FUTURE participants, including faculty, and are distributed several weeks before the program begins. As part of the course, the mentees have the opportunity to spend half a day visiting a nearby academic or industrial research facility. Trips have included tours of laboratories at the Perelman School of Medicine at the University of Pennsylvania in Philadelphia, PA, the Lewis Katz School of Medicine at Temple University in Philadelphia, PA, and GlaxoSmithKline in Collegeville, PA. The course is co-taught by all faculty participants in the FUTURE program, with each faculty mentor covering 1-2 class days. Each faculty mentor is asked to assign the mentees either a pre- or post-class assignment (or both), which encourages the students to continue their academic engagement in the evenings and on the weekends. Due to the diversity of disciplines represented in the 7

program, faculty are requested to use sources and examples from a range of fields in their lessons. Faculty volunteer for or are assigned to specific topics based on their interests and availability, and are encouraged to include their FUTURE mentor in the class activities.

Social Programming and Residential Life Mentees are required to live on campus for the entire four weeks of the FUTURE program. To ensure that they are integrated into the campus community and are well-supported in their residential experience, the program has a dedicated, paid FUTURE Coordinator of Student Programs. The FUTURE Coordinator is a senior student majoring in one of the fields covered by the program who also has previous experience as a Resident Assistant on campus. The role of the FUTURE Coordinator is to provide assistance and emotional support to students, enforce campus and program policies, facilitate conflict resolution as needed, and coordinate the social programming for the FUTURE mentees. Evening and weekend activities are designed to build community amongst the FUTURE mentees and with other campus residents, familiarize students with the campus and surrounding area, and have fun. The FUTURE Coordinator has a $500 budget with which to fund programming, including transportation costs. Activities include scavenger hunts, game nights, movie nights, a beach trip, a day trip to Philadelphia, and other activities aligned with student interests. FUTURE mentors are invited and encouraged to join the social activities. The FUTURE students have consistently reported that a highlight of the program are weekly dinners for the FUTURE mentees, mentors, and faculty and their families hosted at a faculty mentor’s home.

Mentor Training Based on feedback following the first year of the program, a structured training program was instituted for the FUTURE mentors. The mentors participate in four 1-hour discussions on mentoring that occur weekly beginning one week prior to the arrival of the FUTURE mentees. Topics include what it means to be a mentor, strategies for working with students from diverse backgrounds, overcoming challenges in mentoring their FUTURE mentees, and reflecting on their role as a mentor. Resources used in these discussions include “Adviser, Teacher, Role Model, Friend: On Being a Mentor to Students in Science and Engineering” from the National Academies Press (10), “Mentoring across Cultures” by Betty Neal Crutcher (11), and Nature’s “Guide for mentors (12).” Each discussion is facilitated by one of the faculty participants. Through these meetings, the FUTURE mentors gain confidence in their mentoring skills and are encouraged to be a role model of excellence and balance in their academic and social lives. 8

Post-Summer Activities and Opportunities Although FUTURE is a nominally a four-week summer research program, support and mentoring for the FUTURE mentees do not end when the summer is over. All FUTURE mentees are encouraged to continue with undergraduate research, whether in partnership with the same faculty mentor or on a new project. In addition, there is an expectation that the FUTURE mentee and mentor will jointly present the results of their work at a regional or national conference within 1-2 years. Funds are made available to offset student travel costs to facilitate this goal. The FUTURE mentees are also urged to assume leadership roles as FUTURE mentors or in other capacities on campus in subsequent years.

Program Sustainability In its initial format, the FUTURE program was fully-funded by HHMI at an operating budget of approximately $115,000 per year for 8 teams (faculty, mentor, and mentee). This included all stipends ($2500 per student mentor, $2500 per faculty member, and $1250 per mentee), housing costs for each mentor ($1080 for eight weeks) and mentee ($540 for four weeks), research supply costs per mentor/mentee team ($1500 over the summer and an additional $2000 if the mentee continued research the following academic year), travel funds to present work at an external conference ($2400), a stipend and housing costs for the undergraduate FUTURE coordinator ($4600), and funds for social programming and books for the 1-credit course ($1000). Through endowed funds raised to support the Parlee Center and its associated programs in perpetuity, the FUTURE program at Ursinus will be sustained at a level of approximately $10,000 per team ($6250 in stipends for faculty, mentor, and mentee; $1600 in housing costs; $1500 in supply and travel funds; and $800 towards the FUTURE coordinator and social programming costs). For institutions that already have paid summer research programming options in place, the costs to provide a FUTURE-like program can be relatively minimal. The additional required expenses would be mentee stipends (half a typical student summer stipend; $1250 at Ursinus) and housing ($540 for four weeks at Ursinus), FUTURE coordinator stipend ($3000) and housing (5 weeks for $675), and social programming costs ($500-$1000). A minimum of six mentees is recommended to ensure a critical mass for community building. To incentivize faculty participation, faculty stipends, if not already provided through current summer research funding, and supply and/or travel funds are recommended. However, the specific amounts can be easily adjusted to accommodate available resources. We also found faculty were self-motivated to participate for the opportunity to train research students early for long-term collaboration and to contribute to current diversity initiatives on campus.

9

Participant Data Table 1 provides a summary of demographic data of the FUTURE participants from 2013 to 2017. A total of 82 Ursinus students took part in the program, 41 as FUTURE mentees and 41 as FUTURE mentors. This corresponds to eight pairs of students per year except for 2016 when nine pairs of students participated. Note that several participants qualified under more than one USP category. The majority of the FUTURE mentees were URM or first-generation college students (or both). The population of FUTURE mentors, selected from junior and senior STEM majors, is less diverse but is consistent with overall campus diversity, which averaged 13.1% URM and 25.9% first-generation students for 2015-2017. It is also important to note that the percentage of USPs serving as FUTURE mentors has gone up significantly over the five years that the program has run (0%, 12.5%, 12.5%, 50%, 37.5%), due in large part to five former FUTURE mentees having now served as FUTURE mentors between 2015 and 2017. The high percentage of female FUTURE mentees is reflective of the FUTURE applicant pool, which was 79% female.

Table 1. Demographics of FUTURE Participants from 2013 to 2017 FUTURE Mentees (n = 41)

Demographic

9

22%

13

32%

Female

32

78%

28

68%

Underrepresented Minority

22

54%

5

12%

First-Generation College Student

23

56%

8

20%

Student with Disabilities

4

10%

1

2%

Female in Chemistry, Computer Science, Math, or Physics

4

10%

2

5%

41

100%

14

34%

Male

Total Underserved Populationa a

FUTURE Mentors (n = 41)

Participants who meet more than one demographic criterion are included in each count.

Program Assessment A number of strategies were used to assess the FUTURE program in order to improve the participant experience, measure learning gains for the FUTURE mentees and mentors, and track outcomes for all participants. Short-term perceptions were evaluated through focus groups and anonymous pre- and post-program surveys administered within the first three and last three days of the research experience, respectively, for FUTURE mentees and mentors. Written 10

and oral responses on the strengths and weakness of the program were discussed with the FUTURE faculty and reviewed by an external assessment consultant on an annual basis. These discussions informed changes made to the program and led to several improvements each year. Approximately one year after completing the FUTURE program, all FUTURE participants were asked to complete the SURE (Survey of Undergraduate Research Experiences) Follow-Up survey. This retrospective survey uses approximately 47 questions to measure the student’s growth as a scientist, their understanding of how science is done, and whether the experience has affected their career choice or the way that they approach their academic coursework (13, 14). Finally, the achievements, research coursework, and graduation and employment outcomes for all participants in the FUTURE program were tracked and compiled on an annual basis. All surveys were vetted by the external assessment consultant, approved for use by the Ursinus College Institutional Review Board (IRB) and administered using Qualtrics (via Ursinus or Grinnell College) according to the requirements established by the US Department of Health & Human Services.

Pre- and Post-Program Survey Results Pre- and post-program surveys were created based on the public SALG (Student Assessment of their Learning Gains; https://www.salgsite.org/) instrument “HHMI Undergraduate Research, Summer 2011” created by Gudrun Willett. In a pre-program survey, the FUTURE mentees were asked to comment on their general expectations for their summer research experience, describe what they expected to learn through the program, and to use a Lichert scale to rank their abilities on over 20 different categories related to scientific research. Questions included “I can figure out the next step in a research project”, “I understand how my upcoming research will contribute to the accumulated knowledge in my field”, “I am confident in my ability to contribute to science”, “I am confident in my ability to do well in future science courses”, “I am confident that I know what everyday research is like”, “I know how to read a primary research article”, and “I am able to work independently in a research lab.” The post-program survey then asked the mentees to self-reflect on their abilities both before and after the FUTURE program on the same 27 categories. Mentees were also asked to comment on their mentoring support, aspects of the 1-credit course, and components of the program to keep or remove. The survey results were overwhelmingly positive, with mentees reporting significant post-program gains in most categories assessed. A few areas of dissatisfaction for the mentees were consistently noted each year, including too much or too little social programming, a lack of opportunities to interact with the rest of the student research community, and restrictions placed on the mentees (e.g., a dorm curfew and limited off-campus travel). Our efforts to optimize the level of programming continue, but satisfaction with programming may reflect differences in participant preferences from year to year. While unpopular, the modest residential restrictions on the mentees were deemed necessary to ensure the well-being of the pre-matriculation students. 11

The mentees uniformly gave high praise for the trips to local research facilities, commenting that “the field trip to UPenn was awesome; we were able to learn about a life as a graduate student who conducts research and able to observe their working environment and what they actual do on daily bases” and “the field trips to GSK and Temple were very interesting and helpful to get to network with other people in this field and outside Ursinus.” In addition, the mentoring relationships fostered by the program proved beneficial both in and out of the research lab. One mentee noted “even though I learned so much in research, the most important thing to me the people I’ve met through this experience. Many upperclassmen offered to help me with the classes I’ve been taking, or offered their opinions as to what classes I could take to get where I want to go in life”, while another commented that “I gained relationships with professors as well as lifelong friendships with future scientist[s] in this program. I also gained a more confident outlook on my future success in science and the many options I have at Ursinus College.” The program also changed mentees’ minds about their careers in STEM. One wrote that the summer program “completely opened my eyes to what science entails and completely changed the direction of my career path. I fell in love with changing the world through scientific discoveries and critically thinking of future experiments.” Other mentees commented that “[i]t made me realize that I want to do research as a career” and “I am much more likely to enroll in a graduate program.” In another comment, a mentee showed how the connections s/he made through this experience may not have happened without the FUTURE program, writing “I am so glad I got the chance to research so early in my college career; I absolutely loved being in the lab, and I think I would have been too afraid or hesitant to ask a professor if I could join his/her lab freshman year.” The FUTURE mentors completed similar pre- and post-program surveys and also showed gains (although smaller in magnitude) in all categories assessed. The smaller gains are to be expected based on the previous academic and research experiences of the mentors. When asked what skills they gained by participating in the program, the mentors consistently reported that they gained patience, organization, clearer communication skills, leadership, and a better understanding of their own research project. One mentor illustrated the benefits of mentoring a new student this way: “I loved having the opportunity to teach someone else how and why we were doing experiments. It forced me to really understand what and why I was doing it to be able to effectively communicate it.” Other mentors remarked on how their mentoring experience would benefit them in the future, saying: “the biggest skill I gained from the experience was being able to help others (my mentee) understand research and the logic behind what we were doing. Next year I have to TA as part of my PhD program and this has been a positive influence that will help me”, and “[b]eing given the opportunity to mentor definitely allowed me to grow as a scientist. I am much more confident in my abilities to explain lab procedures and rationales, and also more confident in my ability to foster a positive lab environment. I would like to run a lab in the future, so these qualities were definitely important ones to gain.” As previously mentioned, responses from the mentor surveys in the program’s first year (2013) indicated that mentorship training was desired. The mentor 12

training described in the program overview was established the following summer and has been routinely praised by the FUTURE mentors since that time. The workshops have allowed the mentors to see that “all the other mentors were going through similar things as I was, even though we had different majors” and “being able to talk about these skills made me reflect upon what I was doing well, and what things I could incorporate in to the act of mentoring.” The greatest challenge in the program, as reflected by mentee, mentor, and faculty responses, has been balancing the research, academic, and social components of the program. Over the five summers that the program has run, we have adjusted the course meeting times to maximize the hours mentees and mentors can spend on their research by scheduling them first thing in the morning or over the lunch hour. We have also limited the classwork in the final week of the program to ensure the FUTURE mentees have ample time to complete their project and prepare for their oral presentations on the last morning of the program. Each summer also presents a unique challenge when it comes to the social programming organized by the FUTURE Coordinator. Each group of FUTURE mentees has different interests and it is important to find a balance of enough activities to keep them engaged and build community, but not have them be overscheduled.

SURE Follow-Up Survey Results SURE Follow-Up survey results are available for 30 of the 33 FUTURE mentees and 26 of the 33 FUTURE mentors who participated in the program between 2013 and 2016 (91% and 79% response rates, respectively). Data for the 2017 cohort will not be available until Fall 2018 as the survey is administered approximately one year after completion of the FUTURE program. The survey is administered at this time point because it has been shown that with time student opinions about their experiences sharpen, their perceptions are influenced by dissemination activities related to the research project, and they report behavior changes in the direction of greater independence and motivation (15). In the first part of the survey, students are asked to self-report on their postgraduate plans. Participants overwhelming responded that they “had a plan for postgraduate education before - plan has not changed” (41% of mentees, 46% of mentors) or that they “considered postgraduate education - research experience confirmed decision” (34% of mentees, 31% of mentors). Four mentees and one mentor reported that the research experience changed their mind to either pursue or consider pursuing postgraduate education. When asked to evaluate their summer learning experience, 77% of the FUTURE mentees and 58% of the FUTURE mentors reported that “Summer lab was fantastic—I learned a lot.” An additional 17% of FUTURE mentees and 33% of FUTURE mentors indicated that “Summer lab is the way to learn about science.” Nearly all of the mentees and mentors reported that they would take future courses in the same department as the research experience (93% and 96%, respectively, compared to 63% nationally) and that the research experience was likely to influence their behavior in those courses (96% and 88%, respectively, compared to 82% nationally). 13

The second part of the survey asks students to report their learning gains across 21 categories, where 1 is no or very small gain and 5 is a very large gain. The results for the FUTURE program are shown in Figure 1, with means and 95% confidence intervals reported for the FUTURE mentees (n ≤ 30) and mentors (n ≤ 26) compared to the national data set (n ≤ 1300). The FUTURE mentees report substantial learning gains in all categories assessed by the SURE Follow-Up survey. The greatest reported gains by the FUTURE mentees were in tolerance for obstacles faced in research, ability to analyze data and other information, learning laboratory techniques, and giving effective oral presentations. Even given the relatively large confidence intervals due to the small sample size, the FUTURE mentees registered learning gains higher than the national results in ability to analyze data, understand primary literature, learning ethical conduct, skills in oral presentations and scientific writing, and self-confidence. These results suggest the lessons taught in the 1-credit “Science and Mathematics in Society” course, which include scientific ethics, reading primary literature, science writing, and oral presentations, along with the oral and written requirements for the program, are effective at increasing student learning in these areas. The FUTURE mentors reported mean gains at or above the national Follow-Up survey results in most categories. Like the FUTURE mentees, the mentors report the greatest absolute gains in tolerance for obstacles faced in research and skill in giving effective oral presentations. They also report high gains in readiness for more demanding research, understanding how scientists work on real world problems, skill in science writing, and learning to work independently. The largest gains for the FUTURE mentors compared to the national means were in their potential for science teaching and skills in science writing and oral presentation. We find the large gain in teaching potential particularly exciting as it certifies the value in their experience mentoring their FUTURE mentee. The FUTURE mentors did not score themselves as high in understanding how knowledge is constructed, self-confidence, and understanding how scientists think. One explanation may be that as rising juniors and seniors, the FUTURE mentors often have significant previous academic and research experience and thus may feel strong in these areas prior to their participation in the FUTURE program.

14

Figure 1. SURE III Follow-Up survey results for FUTURE mentees (closed circles, n ≤ 30) and FUTURE mentors (closed diamonds, n ≤ 26) from 2013 to 2016. These data are compared to the national means for the Follow-Up survey (open squares, n ≤ 1300) collected from 2009 to 2013. Error bars represent the 95% confidence intervals. FUTURE mentees show mean gains greater than FUTURE mentors in nearly all categories, and both populations report mean gains equal to or greater than the national means in nearly all categories.

15

Outcomes Participant outcomes, including persistence as STEM majors, post-program research experiences, grade point averages, and post-graduation destinations have been tracked for all FUTURE mentees and are summarized in Tables 2 and 3. In all cases, comparison data is provided for STEM majors (defined as students majoring in a field supported by the FUTURE program, excluding FUTURE mentees) and USP STEM majors at Ursinus. The results are very positive, with the FUTURE mentees recording better outcomes than the STEM majors, and specifically the USP STEM majors, at Ursinus on all metrics.

Table 2. Outcomes for FUTURE Mentees Compared to Ursinus STEM Majors and USP STEM Majors Outcome

FUTURE Mentees

Ursinus STEM Majors All

USP

Persisted as STEM majora

89.7% (26/29)

65.5% (658/1004)

64.5% (222/344)

Four-year graduation rate in STEMb

91.7% (11/12)

60.9% (312/499)

65.1% (105/175)

Engaged in one or more semesters of research

90.2%c (37/41)

67.9%d (248/365)

71.4%d (90/126)

3.43 ± 0.30 (n = 10)

3.30 ± 0.40 (n = 365)

3.25 ± 0.39 (n = 126)

Mean GPA at graduatione

Percentage of 2012–2015 cohorts who intended to major in STEM upon matriculation and had a declared STEM major by fall of their junior year b Percentage of 2012–2013 cohorts who intended to major in STEM upon matriculation and graduated with a STEM degree in four years c Percentage of FUTURE mentees (2012–2017 cohorts) who enrolled in at least one semester of research following the FUTURE program d Percentage of STEM majors (2012–2013 cohorts) who enrolled in at least one semester of research by the fall term of their fourth year e Includes students from 2012 and 2013 cohorts; reported values are means ± standard deviations a

Persistence in STEM is a significant concern for students from USPs and a major goal of the FUTURE program. We measured persistence as a STEM major by comparing the number of students who intended to major in a STEM field at matriculation to the number of those students who had declared a STEM major by fall of their junior year. For the 2012–2015 cohorts, these values averaged 65.5% for all STEM majors, and 64.5% for STEM majors from USPs. The results are particularly poor for URM STEM majors, at 48.8%. In contrast, FUTURE mentees from these same cohorts had a persistence rate of 89.7% (26/29). Of the three students who did not continue in STEM, two withdrew from the college and one is now pursuing a non-STEM major. The four-year graduation rate in STEM was determined by comparing the number of students who intended to major in a STEM field at matriculation to the number of those students who graduated 16

with a degree in STEM by the end of their fourth year. Twelve students who participated in FUTURE in either 2013 (the first year of the program) or 2014 have reached their graduation year. Of these students, 11 graduated with a STEM degree (91.7%) and one student withdrew from the college. This four-year STEM graduation rate is very favorable compared to the campus numbers for STEM and USP STEM students in the same cohorts (Table 2). The high persistence and four-year STEM graduation rates for the FUTURE mentees support the assertion that early engagement in an authentic research experience can lead to better academic and social integration in college for students from underserved populations. Early intervention is key since by junior year approximately 35% of students who matriculated intending to major in STEM have either changed to other disciplines (16%) or are no longer enrolled at Ursinus (19%). The FUTURE program is also very successful at encouraging students to continue to engage in undergraduate research during subsequent semesters. While the rates of undergraduate research are high at Ursinus (averaging 67.9% for all STEM majors, and 71.4% for USP STEM majors for the 2012 and 2013 cohorts), connecting the FUTURE mentees with the research culture at Ursinus very early leads nearly all of them (37/41, or 90.2%) to enroll in subsequent research courses. As part of this continued research experience, 64% of the FUTURE mentees from 2013 to 2016 have had the opportunity to present their work at regional or national conferences in their field at least once. Five FUTURE mentees were later selected to serve as FUTURE mentors, demonstrating their growth as researchers and as leaders in STEM. As noted previously, this has also increased the percentage of USPs serving as FUTURE mentors. The mentored research experience appears to provide a supportive learning community that has a positive effect on the academic performance of the FUTURE mentees. The mean grade point average (GPA) at graduation for FUTURE mentees in the 2012 and 2013 cohorts was 3.43 ± 0.30 (n = 10). This compares favorably to all STEM majors (3.30 ± 0.40, n = 365) and USP STEM majors (3.25 ± 0.39, n = 126) for these same cohorts. The close student-faculty connections built during the program and the knowledge gained from the 1-credit course likely also contribute to the better academic performance by the FUTURE students. Graduates who participated in the FUTURE program as either a mentee or mentor have gone on to a wide range of career paths (Table 3). A majority of the FUTURE mentees are either employed in their field (36.4%) or are currently pursuing post-graduate studies in STEM or medicine (45.5%). The overwhelming majority of FUTURE mentors are also either employed in their field (32.3%) or are pursuing post-graduate studies in STEM or medicine (51.7%). The rates of post-graduate study for FUTURE participants is noteworthy as only 27.8% of all Ursinus STEM graduates from 2014 to 2016 were pursuing post-graduate studies in STEM or medicine. While data from additional cohorts will be needed to confirm these results, they suggest that the early exposure to research experiences may be influencing the career paths of the FUTURE participants.

17

Table 3. Post-Graduation Destinations for FUTURE Mentees and Mentors FUTURE Menteesa (n = 11)

FUTURE Mentorsb (n = 31)

Ursinus STEM Majorsc (n = 610)

Employed in STEM

36.4%

32.3%

40.5%

Post-Graduate Studiesd

45.5%

51.7%

27.8%

(Graduate School)

(27.3%)

(32.3%)

(19.0%)

(Medical School)

(9.1%)

(12.9%)

(7.6%)

(Post-Baccalaureate Program)

(9.1%)

(6.5%)

(1.2%)

9.1%

0

2.6%

0

9.7%

11.8%

9.1%

3.2%

4.7%

Other

0

0

6.4%

Unknown

0

3.2%

6.2%

Post-Graduate Destination

Service/Volunteer Employed not in STEM Seeking employment

Includes FUTURE mentees who graduated in 2015 or 2016 Includes FUTURE mentors who graduated in 2014, 2015, or 2016 c Includes Ursinus STEM majors who graduated in 2014, 2015, or 2016 d Percentages are the sum of graduate school, medical school, and post-baccalaureate program values a

b

Impact of the FUTURE Program on Faculty Participants From 2013 to 2017, 22 individual faculty participated in the FUTURE program. Of those 22 faculty, 11 have served as a faculty mentor more than one time. Faculty were incentivized to participate in the program through a stipend ($2500) and funds to cover research supplies during the summer ($1500) and over the academic year ($2000) should the FUTURE mentee and mentor continue their work. Survey responses from faculty participants indicated that the program had positive impacts on their research programs and mentoring skills. Several faculty participants noted that mentees who were brought in to the research environment in their first year and continued to work on the project in subsequent years were highly productive and made significant contributions to their scholarship. One faculty participant said “having a student start training on clinical skills so young, allowed me the opportunity to train him extensively and help him with his research projects. He was able to complete several smaller pilot projects over the years which all built up to his final honors project. Each of his pilot projects has provided me valuable pilot data that I will use when on my Pre-Tenure leave this fall writing my grant proposal.” For other faculty, the program changed their perception about working with at-risk students: “FUTURE showed me how immensely valuable early immersion in meaningful research experiences can be for underrepresented students. Because of this program, I feel very confident inviting at-risk students into my research lab as early as the summer prior to their sophomore year. I plan to continue this practice for the remainder of my career.” 18

The mentoring discussions also proved beneficial for faculty in addition to the students: “In the two summers that I participated I mentored the mentors—that helped my own growth as a mentor. Doing the readings and participating in the conversations with the students about what makes a good mentor was useful.”

Table 4. Scholarly Presentations and Peer-Reviewed Publications Including FUTURE Mentees and Mentors Scholarly Work

Number

Regional/national presentation including FUTURE mentor coauthor

35

Regional/national presentation including FUTURE mentee coauthor

27

Peer-reviewed publication including FUTURE mentor coauthor

6

Peer-reviewed publication including FUTURE mentee coauthor

3

It is important to note that participation in the FUTURE program did not hinder research productivity by the faculty participants, and in several cases enhanced faculty scholarship. The timing of the four-week program allows the FUTURE mentor and faculty to make significant progress on their project while bringing in a new student during highly productive research weeks. Table 4 summarizes the number of scholarly presentations and peer-reviewed publications to date that include FUTURE mentee and/or mentor coauthors. Publications appeared in Letters in Biomathematics, NeuroReport, Perception, Proceedings of the International Symposium on Biomathematics and Ecology, International Journal of Vascular Medicine, and Journal of Occupational and Environmental Medicine. Conference presentations have included the National Meeting of the American Chemical Society, the Society for Neuroscience Annual Meeting, the American Society for Cell Biology Annual Meeting, the Annual Meeting of the Society for Experimental Biology, the Annual Joint Mathematics Meetings, the Entomological Society of America Annual Meeting, and the Annual Meeting of the Biophysical Society, among others. These results highlight the productivity that can be gained by creating long-lasting student-faculty partnerships in research like those fostered by the FUTURE program.

Conclusions The FUTURE program is a successful example of how undergraduate research experiences can be used to effectively engage, retain, and support students in STEM from underserved populations. By connecting these students with an authentic research experience very early in their undergraduate careers, the mentees gain knowledge, confidence, and practical skills that help them to navigate the challenges of college. A dedicated mentoring team and a deliberately-balanced combination of academic, research, and social activities to create a well-rounded experience appear to be vital to the program’s success. 19

While a four-week program is shorter than many undergraduate research experiences, the reported outcomes suggest that this is a sufficient amount of time to build community, fully engage with a research project, and gain academic skills that help the mentees to be successful in college. Importantly, all the FUTURE participants, including the faculty and student mentors, benefited from their involvement. As many colleges and universities currently run summer research programs, the addition of a FUTURE-like module would be a relatively easy and inexpensive way to recruit and improve outcomes for STEM students from USPs.

Acknowledgments The authors would like to thank past and present members of the FUTURE program committee (Rebecca Lyczak, Reese McKnight, Simara Price, Jordan Toy, and Charlene Wysocki) for the time and effort they devoted to creating, improving, and sustaining the FUTURE program. We also extend our gratitude to Robert Dawley and Akshaye Dhawan for their work as members of the Parlee Center steering committee, Kathy Pusecker (Director of the University of Delaware Center for Teaching and Learning Assessment) for her guidance and assistance with program assessment, and Annemarie Bartlett and Whitney Hawkins for providing institutional data. Finally, we greatly appreciate the Howard Hughes Medical Institute and Donald and Joan Parlee for their past and future financial support of the Parlee Center and the FUTURE program at Ursinus College.

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Jones, M. T.; Barlow, A. E. L.; Villarejo, M. Importance of undergraduate research for minority persistence and achievement in biology. J. Higher Educ. 2010, 81, 82–115. Gregerman, S. R.; Lerner, J. S.; von Hippel, W.; Jonides, J.; Nagda, B. A. Undergraduate student-faculty research partnerships affect student retention. Rev. Higher Educ. 1998, 22, 55–72. Gummadam, P.; Pittman, L. D.; Ioffe, M. School belonging, ethnic identity, and psychological adjustment among ethnic minority college students. J. Exp. Educ. 2016, 84, 289–306. Hausmann, L. R. M.; Schofield, J. W.; Woods, R. L. Sense of belonging as a predictor of intentions to persist among African American and white firstyear college students. Res. Higher Educ. 2007, 48, 803–839. Pittman, L. D.; Richmond, A. University belonging, friendship quality, and psychological adjustment during the transition to college. J. Exp. Educ. 2008, 76, 343–362. Kahneman, D. Thinking, fast and slow; Farrar, Straus and Giroux: New York, 2013. Sagan, C.; Druyan, A. The demon-haunted world: Science as a candle in the dark; Ballantine Books: New York, 1997. 20

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Brown, M. How I killed Pluto and why it had it coming; Spiegel & Grau: New York, 2012. Ridley, M. Genome: The autobiography of a species in 23 chapters; Harper Perennial: New York, 2006. Institute of Medicine, National Academy of Sciences, and National Academy of Engineering. Adviser, Teacher, Role Model, Friend: On Being a Mentor to Students in Science and Engineering; The National Academies Press: Washington, DC, 1997. Crutcher, B. N. Mentoring across cultures. Academe 2007, 93, 44–48. Lee, A.; Dennis, C.; Campbell, P. Nature’s guide for mentors. Nature 2007, 447, 791–797. Lopatto, D. Survey of undergraduate research experiences (SURE): First findings. Cell Biol. Educ. 2004, 3, 270–277. Lopatto, D. Undergraduate research experiences support science career decisions and active learning. CBE-Life Sci. Educ. 2007, 6, 297–306. Lopatto, D. Exploring the benefits of undergraduate research: The SURE survey. In Creating effective undergraduate research programs in science; Taraban, R., Blanton, R. L., Eds.; Teacher’s College Press: New York; pp 112–132.

21

Chapter 2

Four-Year Research Engagement (FYRE) Program at the University of Oklahoma: Integrating Research in Undergraduate Curriculum Naga Rama Kothapalli* Department of Chemistry and Biochemistry, The University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019, United States *E-mail: [email protected]

The Four-Year Research Engagement (FYRE) program has been developed to promote STEM undergraduate research at the University of Oklahoma (OU). The FYRE program is a multi-year program for students interested in a STEM career path and is composed of FYRE1 – FYRE4. This program parallels and significantly adds to the course curriculum of OU STEM students with an emphasis on developing research skills. In this program, we expect a diverse population of undergraduates including first-year, under-represented and transfer students to conduct semester-long, mentored research internships. The FYRE program is built on the foundation of a very successful campus-wide first-year research experience, existing since 2012. The first-year research experience (FYRE1 program) allowed selected OU first-year students to conduct a mentored, semester-long experimental project in an OU research laboratory. In the second year, we focus on the STEM engagement of students through a FYRE2 seminar course where discussing the role of science and technology in transforming society and the world. During the third and fourth years (FYRE3 and FYRE4), the content of the seminar courses emphasize tangibly important skills and content for a future in STEM. This project is a scalable program to engage students in meaningful chemical research that can readily incorporate independent inquiry. © 2018 American Chemical Society

Introduction The preparation and training of a diverse technical workforce will allow the United States to remain a world leader in innovation and technology. This requires a significant change in the education of undergradutaes interested in the STEM fields to include interdisciplinary research exposure that combines STEM methods and theories with societal context to motivate student learning. There is an emerging consensus that one path forward towards implementing these systemic changes in undergraduate education is discipline-based research (1). With the rapidly evolving technological scene, it is imperative that students understand the need to assess and promote the transfer of knowledge and skills within and across disciplines as they major in different STEM fields (2). One of the primary requirements for interdisciplinary conversation among undergraduates is an avenue for them to meet and discuss their research with their peer group on a regular basis. At the University of Oklahoma, a four-year research engagement program is one such avenue, which brings together students from different disciplines with research interest to discuss research and scientific developments.

The Four-Year Research Engagement (FYRE) Program The Four-Year Research Engagement (FYRE) program was designed as a gateway program for undergraduates at the University of Oklahoma (OU) interested in conducting research in science, technology, engineering and mathematics (STEM) fields. As a gateway program, FYRE allows incoming first-years (freshmen and transfer students) to obtain research experience at the start of their academic program at OU. This program was officially launched in 2016 with multi-level institutional support (i.e. faculty, departmental, college and provost level support). The FYRE program, detailed below, is a multi-year program designed to mentor and train all OU students interested in a STEM career path. The FYRE program parallels but is independent of the curriculum in different STEM majors. The current FYRE program is built on the foundation of a very successful campus-wide first year research experience, established in 2012. The evolving FYRE program is designed to help OU students, in their first year, participate in research related activities and continue until graduation. The FYRE1 program, instituted at OU from 2013-2016, on a small scale was successful in accelerating students’ interest in STEM research and in building a critical mass of interdisciplinary undergraduate researchers. We believed the impact of the program could be significantly increased by: 1) extending the benefits of the FYRE1 program into a multi-year experience and; 2) broadening the population of participating students from only Honors students to all undergraduates interested in STEM professions. The new FYRE program is a multi-year program that offers students different support based on their year in the program. The program is divided into FYRE1 (first-year experience), FYRE2 (second-year engagement) and FYRE3 (third-year engagement), and FYRE4 (fourth-year engagement). A schematic of the current program is shown in Figure 1. 24

Figure 1. Schematic representation of FYRE Program. FYRE1 engaged selected OU first-year students to conduct a mentored, semester-long experimental project in an OU research laboratory for which students earn three credit hours with a grade. The FYRE1 program is a campus-wide initiative with students in many different STEM majors participating as well as non-majors. The primary goals of the FYRE1 program are: 1) to provide a safe, constructive, instructive, relevant and immersive experience in experimental research; 2) provide a gateway to understanding applied scientific research and its relationship to theoretical learning; and 3) to create a critical mass of undergraduate on a STEM professional trajectory at the University of Oklahoma. FYRE1 students, matched based on their research interests, conduct a semester of basic experimentation in research groups across 14 different OU STEM departments: Chemistry and Biochemistry, Biology, Microbiology and Plant Biology, Mathematics, Physics, Psychology, Health and Exercise Science, Electrical Engineering, Biomedical Engineering, Aerospace and Mechanical Engineering, Civil Engineering and Environmental Sustainability, Geology and Geophysics, Anthropology, and Meteorology. Examples of the research conducted and presented by the students at the end of semester poster session can be seen on our website http://fyre.oucreate.com. In 2016, the FYRE1 program expanded to target STEM students that had recently transferred to OU. Since feedback from the small number of students gaining research experience was very positive, we have introduced this as a regular component of the program. For students who are unsure of their interests or research capabilities, they meet with the instructor to discuss a variety of publications to determine their interests prior to joining a group. Additionally, we have secured funding to provide assistantships to a few students during summer to promote additional participation. The FYRE2 program will be for all students who have either completed the FYRE1 program, or for any student that has completed a full semester of undergraduate research. We have observed that approximately half of the FYRE1 students remain active as undergraduate researchers in their second year. To help 25

with students’ development as research professionals, we introduced a 1-credit hour seminar course to keep them engaged in the process of research. As STEM interested students in their second year are still likely in a formative stage of STEM-development, the FYRE2 seminar course focuses on the role of science and technology to transform society and the world. The FYRE3 and FYRE4 years of the program are designed to emphasize skills that are important and tangible for a future STEM professional. The FYRE3 and FYRE4 courses focus on developing professional leadership skills, such as teamwork and communication (FYRE3) and problem solving in innovative research (FYRE4). These skills have been described as key skills for future STEM career success (3). The FYRE3 course focuses on leadership and communication in STEM fields, while FYRE4 course focuses on the nuts-and-bolts of how innovation and research are conducted in STEM fields. Some of the topics covered in these seminar courses are grant writing, venture capital funding, intellectual property, and building research groups. These skill development courses are designed to offer assistance in writing summer fellowship applications, applying to graduate programs, and interviewing for STEM jobs. Additionally, individual STEM advisement provided by the FYRE program continues throughout FYRE3 and FYRE4. FYRE is a “True Center-of-Mass” of STEM Development and Undergraduate Research at the University of Oklahoma. The FYRE program currently enhances all other STEM programs at the University of Oklahoma by serving as the source for STEM-research focused undergraduates on campus. The FYRE program acts as an ideal informational conduit to make students aware of the other STEM opportunities on campus, such as the TRiO programs, the Ronald McNair Scholars program, the Chemistry Learning Community, OK-LSAMP, OU Undergraduate Research Day, the Oklahoma Journal of Undergraduate Research, and many other initiatives. The individual STEM advising provided by the FYRE program ensures that the students are aware of other opportunities to advance in their STEM development, both a OU and at other institutions.

Student Assessments The FYRE program currently allows us access to a relatively large, diverse population of undergraduates in a multitude of STEM disciplines to help us understand the factors that influence a student’s decision to pursue, persevere or abandon STEM disciplines. Our program design allows for conducting such a study, where the participating STEM student population is tracked and studied from their first-year at OU through their graduation and one year beyond. Recently our proposal to explore the correlations between possessing a growth-learning mindset and successfully launching a STEM research professional track has been funded through NSF. Because our program garners participation of students from various disciplines in STEM fields, we aim to gain a comprehensive understanding of students’ attitudes and mindsets, and the influence of having engaged in research, may have on their undergraduate education, as well as, their careers beyond. The assessments designed will allow us to conduct both qualitative and quantitative analysis using surveys and student interviews. 26

Chemistry Research for Non-Chemistry Majors While research is essential or almost a necessity for students who desire to be successful in STEM careers, it is equally important for non-STEM majors to be exposed to research practices. The advantages have been described in detail in many publications and have been summarized (4–7). One of the common approaches to make research more approachable to non-STEM majors is to make research more relatable and associated with everyday life. To promote participation in chemical research by undergraduates who are not declared chemistry majors and/or non-STEM majors, we created a project called “The Molecular Gastronomy of Coffee” in which students evaluate the flavor components of coffee: an integral part of many people’s lives. We have been able to provide research experience exploring student-driven questions about chemical nature and composition of coffee.

Figure 2. A typical GC-MS chromatogram had over 100 detectable compounds (M = 106).

Coffee is one of the most widely consumed beverages in the world, with an estimate of 100 million people drinking coffee daily in United States. The existence of convenient home-brew methods has not decreased the popularity of coffee-shop chains and growth of artisanal coffee roasters and growers. Every 27

person who partakes in coffee has their own ideas and preferences about best coffee brewing method and how to enjoy it. Several factors affect the complex flavor of any cup of coffee: plant species, country of origin, processing, roasting, grinding, storage, and brew method (8, 9). Additionally, how coffee affects an individual is a combination of chemical, physiological, and physical factors that is unique to an individual consuming coffee. Using chemical techniques both organic and analytical, students can identify chemical flavor components of coffee. The results obtained will provide insight about the variables that affect coffee and help to design brewing methods for specific taste profiles. During this project, students established a methodology to isolate and quantify concentrations of volatile flavor components of coffee samples. The extracts were subsequently processed using gas chromatography mass spectrometry (GCMS). A typical chromatogram obtained after a GCMS run is shown in Figure 2. Using various parameters, students could identify the flavor components of coffee and how they differed depending on the processing of the coffee from a bean stage till it is brewed. Initially, we helped students to learn the theoretical and experimental aspects of organic extraction (both liquid-liquid [LLE] and solidphase [SPME]) as well as GCMS (10, 11). Using previously published methods like liquid-liquid extraction; some compounds could be quantified in an absolute manner. Our students also tested other methods like solid-phase micro-extraction method to determine if more compounds could be isolated to pinpoint the flavor profile of coffee under various conditions. A representative trace of SPME run overlaid with LLE run is shown is Figure 3.

Figure 3. A typical SPME chromatogram had over 80 detectable compounds, while typical liquid-liquid extraction chromatogram had over 140 detectable compounds. 28

A comparative analysis of LLE and SPME showed that each method was optimum for eluting specific kinds of compounds. LLE was optimal for eluting less volatile compounds towards the end of GCMS run, while SPME was ideal for highly volatile compounds early in the GCMS run. While the number of compounds identified was different, the overlap of compounds allowed us to identify 8 compounds shown in Figure 4 to determine exact concentrations of the other compounds detected (12). They were also able to correlate the flavor of coffee to each of components. Independently, students were asked to pose a question and determine the changes in the levels of these 8 components. The examples of the questions asked are shown in Table 1.

Figure 4. A flavor profile subset of 8 compounds is presented here with their structures, flavor characteristics and flavor threshold.

Overall, this study has revealed several novel aspects in coffee brewing including the alterations in the flavor quality of coffee depending on the ground size and the roasting methods. Additional variables considered were purchase conditions, storage and brewing temperature. The results revealed how the concentration of flavor components differ based on these conditions. As a part of the research project, we also had the students discuss their findings with local coffee roasters and were delighted to have their scientific posters displayed in the coffee shops for more that a week. The students were also able to establish collaborations with the coffee shops to gain access to various kinds of coffee grounds and roasting instruments. As a result, the FYRE program continues working collaboratively with a couple of local roasters to help them improve products. These results have been presented at two national meetings hosted by American Society of Mass Spectrometry (ASMS) (13, 14) and two of our students have written their senior thesis based on their findings in this project. 29

Table 1. Examples of Independent Inquiry Questions of Students and Results Obtained Question asked and variable tested

Results obtained

Effect of brewing water temperature on coffee flavor Variable: Temperature

Observed a proportional increase number and amount of flavor components with water temperature, specifically hot coffee brewing. Cold brew extracted more components that hot brew.

Difference in flavors between whole bean vs. pre-ground Variable: Ground size

Compared to pre-ground, whole bean coffees have flavor components in greater relative amounts, and also contained some novel compounds. More components were detected in dark roasted; flavors masked by 2-furan methanol

Key differences in instant coffee vs. brewed coffee Variable: Time of preparation

The concentration of flavor components in the instant coffee were not significantly lower than that of the Brazilian coffee except for 2-(hydroxymethyl)furan.

Differences between caffeinated & decaffeinated flavors in green and roasted coffee beans

Coffee beans are decaffeinated when green using water, dichloromethane or ethyl acetate. Both green and roasted decaffeinated coffee contained more variety and abundance of flavor components compared to caffeinated. 2-methoxy-4-vinylphenol was the only compound identified in green and roasted coffee.

Comparing flavor profiles of commercially available types of Folgers

Folgers packages 7 different roasts and this study compared flavor profiles of 4 different roasts. Compounds that did show variation were more concentrated in the milder roast and contained “sweet” flavor notes.

Comparing single-serve coffee cups to normal coffee brew.

All coffee had similar amounts of phenol with the light roast having more 2-furan methanol and significantly less butyrolactone compared to the medium roast.

Utilizing everyday ingredients like coffee to develop research projects allow us to broaden participation of students in chemistry research. Non-STEM majors participating in this program gain valuable skills in critical and creative thinking. This approach can be adapted to use various commonly consumed items as test topics with students asking questions regarding their composition as well as decomposition. One of the advantages of this approach is that it can be introduced to first-year students and provide a continuing experience.

Acknowledgments As the program director, I would like to acknowledge the support of the University of Oklahoma Honors College, College of Arts and Sciences and all the participating faculty. The funding to continue this program and conduct student assessments are provided by NSF IUSE grant (NSF#1726889). Special thanks to 30

Dr. Halterman and Dr. Foster for designing the coffee project to help broaden the scope of this program. Some of the data presented here has been contributed by Ms. Emily Erdman and Ms. Stephanie Allred.

References 1.

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Beach, A. L.; Henderson, C.; Finkelstein, N. Facilitating change in undergraduate STEM education. Chang. Mag. High. Learn. 2012, 1383, 52–59. Bradforth, S. E.; Miller, E. R.; Dichtel, W. R.; Leibovich, A. K.; Feig, A. L.; Martin, J. D.; Bjorkman, K. S.; Schultz, Z. D.; Smith, T. L. University learning: Improve undergraduate science education. Nature 2015, 523, 282–284. Ramsey, K.; Baethe, B. The Keys to Future STEM Careers: Basic Skills, Critical Thinking, and Ethics. Delta Kappa Gamma Bull. 2013, 80, 26–33. Sandeen, C. High-Impact Educational Practices: What We Can Learn from the Traditional Undergraduate Setting. Continuing Higher Educ. Rev. 2012, 76, 81–89. Lopatto, D. Undergraduate Research Experiences Support Science. CBE Life Sci Educ. 2007, 6, 297–306. Linn, M. C.; Palmer, E.; Baranger, A.; Gerard, E.; Stone, E. Undergraduate research experiences: Impacts and opportunities. Science 2015, 347, 1261757–1261757. Harackiewicz, J. M.; Barron, K. E.; Tauer, J. M.; Elliot, A. J. Predicting Success in College: A longitudinal Study of Achievement Goals and Ability Measures as Predictors of Interest and Performance from Freshman Year Through Graduation. J. Educ. Psychol. 2002, 92, 247–255. Shibamoto T. An overview of coffee aroma and flavor chemistry. In 14th International Scientific Colloquium on Coffee; San Francisco, 1991; Association Scientifique Internationale du Cafe: Paris, France, 1992; pp 107–116. Flament I. Coffee Flavour Chemistry, 1st ed.; John Wiley and Sons: West Sussex, England, 2002; pp 81 Barrionuevo, W. R.; Lanças, F. M. Comparison of Liquid-Liquid Extraction (LLE), Solid-Phase Extraction (SPE), and Solid-Phase Microextraction (SPME) for Pyrethroid Pesticides Analysis from Enriched River Water. Bull. Environ. Contam. Toxicol. 2002, 69, 123–128. Ebeler, S. E.; Terrien, M. B.; Butzke, C. E. Analysis of Brandy Aroma by SolidPhase Microextraction and Liquid-Liquid Extraction. J. Sci. Food Agric. 2000, 80, 625–630. Rodrigues, N. P.; Bragagnolo, N. Identification and Quantification of Bioactive Compounds in Coffee Brews by HPLC–DAD–MSn. J. Food Compos. Anal. 2013, 32, 105–115. 63RD ASMS Conference on Mass Spectrometry and Allied Topics. J. Am. Soc. Mass Spectrom. 2015, 26, 1–236. 64TH ASMS Conference on Mass Spectrometry and Allied Topics. J. Am. Soc. Mass Spectrom. 2016, 27, 1–266.

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Chapter 3

Another Round of Whiskey for the House: Community College Students Continue Research on Experimental New Flavors of Whiskey Regan Silvestri* Division of Science and Mathematics, Lorain County Community College, 1005 North Abbe Road, Elyria, Ohio 44035, United States *E-mail: [email protected]

Students at Lorain County Community College in Elyria, Ohio have partnered with a local company, Cleveland Whiskey, to conduct research studies on experimental new flavors of whiskey. Industry partner Cleveland Whiskey continues to create experimental flavors of bourbon via their innovative technology of rapid pressure aging. Members of the undergraduate research group have used gas chromatography-mass spectroscopy (GC-MS) to profile the volatile flavor compounds in the new experimental flavors of whiskey provided by the industry partner. First, the flavor compounds resulting from rapid pressure aging with traditional American oak were mapped. Second, flavor compounds including black cherry, apple, hickory, sugar maple and honey locust were identified and profiled; these products have since been commercialized. Finally, Chinese baijiu liquors have been aged as whiskeys to impart wooden barrel flavors, in an effort to modify these spirits into something more akin to the western palate. Students working on the project are excited about the topic and pleasantly surprised with the straightforward nature of the work owing to its practical and applied characteristics. Through it all, the student body of the entire college has been captivated with the story of the students working on the “science of the flavor of whiskey” project. This unique example of innovative community-partnered research at a community college offers a model for other schools to follow.

© 2018 American Chemical Society

Introduction This is the story of how a successful undergraduate student research program can be synthetized at a community college: from inception, to involvement, to funding and sustainability. Beginning with a chance encounter between a chemistry professor and the founder of a start-up whiskey company, an independent student research program in analytical chemistry was created and has continued to fluorish. A vibrant independent student research program for undergraduates has since been created and is blossoming. Students who would not normally be considered “ready for research” are now enthusiastic to become involved in the “flavor of whiskey” project, which has quite the buzz on campus, and students have made significant contributions over the past years.

Round 1: Beginnings Incidental Connection and Pseudo-Accidental Program Inception The entrepreneurial start-up company Cleveland Whiskey was founded in 2009 based on the revolutionary production technology of rapid “pressure aging.” Although the specific details of the patented “pressure aging” process are proprietary, the procedure essentially involves placing new spirit in a stainless steel vessel with pieces of charred wood of controlled surface area. The stainless steel vessel is then sealed, and the head space above the liquid is subjected to a precisely defined cycling in pressure that forces the alcohol into the wood, extracting compounds from the wood which naturally flavor the whiskey (1). This revolutionary technology accelerates the aging/maturing process of whiskey from a few years to a few days. As the company was founded on the basis of an innovative new technology for manufacturing, Cleveland Whiskey is essentially a technology company. It’s therefore no surprise that a chance encounter between the Founder and CEO of a technology company and a chemistry professor would quickly result in the establishment of a cooperative research project using gas chromatography-mass spectroscopy (GC-MS) to study the flavor compounds in bourbons produced using this new technology. While the encounter happened by chance, the ensuing inception of the program organically proceeded and was therefore only pseudo-accidental. Initial Work: Comparisons with Traditionally Aged Bourbon Whiskeys, and likewise bourbons, are chemically complicated mixtures of flavor compounds. Arguably, a substantial portion of the flavor is imparted during maturation when the spirit is aged, traditionally in oak barrels, for up to 10 years or more. During this time, the clear distillate becomes flavored and colored with compounds that leach into the spirit from the charred oak barrel (2). The result is a chemically complex mixture of a large number of flavor compounds (up to hundreds), all present in very low quantities (circa ppm). The flavor and fragrance industry therefore recognizes GC-MS as a routine technique 34

for application to study such complex mixtures (3, 4). Techniques for improving chromatographic resolution (5), optimizing separation (6), and quantifying results (7) are commonplace for GC-MS studies of flavor compounds in spirits. The cooperative project described herein between the community college and the industry partner was initiated by using GC-MS to identify and quantify the volatile flavor compounds present in varieties of rapid pressure-aged bourbons alongside traditionally aged bourbons. The objective was to compare the flavor profiles of bourbons produced by rapid pressure aging to bourbons which had been traditionally aged. In doing so, it was observed that bourbons produced by rapid pressure aging were nearly identical to bourbons which had been traditionally aged, the only observable difference being that bourbons produced by rapid pressure aging included the addition of steric acid, a compound with little to no associated flavor (8).

Round 2: Unprecedented Bourbon Flavors The Uncommon Barrel Traditionally, oak barrels have been used for aging whiskey because oak is a hard, durable wood that enables the barrel to maintain its integrity over the long aging period, thus minimizing evaporation of the volatile product. Owing to the pressure-aging technology that Cleveland Whiskey has developed, barrels have become antiquated and Cleveland Whiskey is therefore no longer confined to aging solely with oak wood. Subsequently, Cleveland Whiskey has proceeded to mature distillate with other varieties of wood generating new experimental flavors of whiskey that are completely original, unprecedented, and only made possible via the innovative technology of accelerated pressure-aging. Some of these unprecedented bourbon whiskey flavors include black cherry, apple, hickory, sugar maple, and honey locust, to name a few. These unprecedented bourbons are naturally flavored with compounds extracted from the various unique woods used for maturing via pressure aging. The technology of pressure-ageing essentially allows the capture of previously untapped natural flavors. Chemistry students at the college are always thrilled to receive samples of experimental whiskey flavors that are not yet commercially available!

Tasty Results for Tasty Bourbons Undergraduate students at the community college have used routine straight injection GC-MS to identify and profile the distinct flavor compounds that are leached from the various woods into these uniquely flavored bourbon whiskies (9). In general, it can be said that the various woods impart largely the same flavor compounds to the bourbons, however in relatively different quantities (8). Table 1 lists the peak assignments for the GC-MS chromatogram traces along with the corresponding flavors of the compounds thereby identified. 35

Table 1. GC-MS Peak Assignments for Unprecedented Flavors of Bourbon Peak assignments for GC-MS chromatogram traces of bourbon aged with unprecedented woods, and corresponding flavors of the compounds. Retention Time (minutes)

Compound

Flavor

2.05

furanone

sweet, fruity, caramellic, brown maple note

2.10

branched butanol/ pentanol

sweet, fruity, fusel oil, apricot, banana, apple, wine

2.20

branched butanol/ pentanol

sweet, fruity, fusel oil, apricot, banana, apple, wine

2.30

glycolate

fruity, green, pineapple, fusel

2.40

phenyl acetaldehyde

honey, sweet, floral, chocolate and cocoa, with a spicy nuance

2.75

furfural

woody, sweet, bready, nutty, caramellic with a burnt astringent nuance

2.90

isoamyl acetate

sweet, banana, fruity with a ripe estery nuance

3.10

glycolic acid

very mild buttery

3.30

benzoic acid

faint balsamic

3.40

5-methylfurfural

sweet, brown, caramellic, grain, maple-like

3.50

ethyl hexanoate

sweet, pineapple, fruity, waxy, banana

4.25

phenethyl alcohol

floral, sweet, rosey and bready

4.55

ethyl octanoate

sweet, waxy, fruity, pineapple, creamy, fatty, mushroom, cognac notes

4.70

decanoic acid

fatty citrus, waxy fruit

4.80

stearic acid

none

4.85

decanol

Waxy, fatty, tart, perfumistic, floral orange, sweet clean watery

4.90

phenethyl acetate

sweet, honey, floral, rosy, slight green nectar, fruity body

5.05

ethyl cyclohexane propionate

Fruity, sweet, pineapple, peach, pear, honey, caramallic, maple and cocoa Continued on next page.

36

Table 1. (Continued). GC-MS Peak Assignments for Unprecedented Flavors of Bourbon Peak assignments for GC-MS chromatogram traces of bourbon aged with unprecedented woods, and corresponding flavors of the compounds. Retention Time (minutes)

Compound

Flavor

5.10

benzoic acid

faint balsamic

5.25

furanone

sweet, fruity, caramellic, brown maple note

As such, the flavor profile for each of these uniquely flavored bourbons has been determined, allowing comparisons between the different flavors of wood. For example, Figure 1 shows an overlay of the chromatogram trace for a bourbon finished with black cherry wood as compared to one finished with American oak. Accordingly, it can be seen that cherry bourbon has more ethyl octanoate, a compound known to impart a sweet fruity flavor, than does traditional oak flavored bourbon. Further, it can be seen that cherry bourbon has less phenethyl alcohol, a compound known to impart a floral and bready flavor, than does traditional oak bourbon (10).

Figure 1. Overlay of GC-MS traces of cherry and oak aged bourbons. 37

The comparison of cherry to oak aged bourbon is demonstrated herein as only one example of the types of data that can be produced. Comparisons between the relative quantities of the various flavor compounds have been carried out in detail for all of the bourbons aged with these various unprecedented woods. Essentially, the routine technique of straight-injection GC-MS has been applied to yield straightforward and valuable results: analytical descriptions of the flavor profiles of new flavors of bourbons.

Round 3: Chinese Baijiu Spirit Flavored as American Whiskey Motivation China and Japan are seen in general by American industries as enormous market opportunities. The American bourbon industry is certainly no exception, which is currently looking toward Asia with much anticipation for continued exponential growth (11). Baijiu is a popular Chinese spirit distilled from fermented sorghum. While this clear liquor is considered strong-in-exotic-flavors by the western palate, its flavor is considered as something of a standard by the eastern palate. In an effort to modify the flavors of baijiu only slightly, samples of various baijiu spirits have been matured with wood. The motivation is to create a new product for the eastern market: a recognizable flavor (baijiu) with an American twist (wood). Hence, experimental samples have been produced of various Chinese baijiu spirits flavored to taste more similar to American bourbon by aging with various woods. This has been accomplished by subjecting Chinese baijiu liquors to the novel accelerated aging process of pressure-aging, to mature the spirit in 24-48 hours and impart wooden barrel flavors. By processing Chinese baijiu liquor via this innovative technology of rapid pressure aging, the clear spirit becomes colored and flavored with the wood in the short time of a few days.

Terra Incognita: An American Styled Chinese Spirit? The student research group, now well versed in applying GC-MS to determine flavor profiles of new flavors of spirits, turned their attention to these newly created liquors: American-styled Chinese spirits. Multiple different imported samples of commercially available baijiu liquors were used as starting material, and each was matured by finishing with charred American oak, black cherry, and honey locust wood. Table 2 lists the complete peak assignment for the GC-MS chromatogram traces of these wood aged baijiu spirits, along with the corresponding flavors of the compounds thereby identified. While a wood matured Chinese baijiu may be complete terra incognita, some published results do exist for GC-MS peak assignments of flavor compounds in traditional neat baijiu (12–14).

38

Table 2. GC-MS Peak Assignments for Oak Aged Chinese Baijiu Spirit Peak assignments for GC-MS chromatogram traces of Chinese baijiu spirits aged with charred American oak and corresponding flavors of the compounds. Retention Time (minutes)

Compound

Flavor

2.10

branched butanol/ pentanol

sweet, fruity, fusel oil, apricot, banana, apple, wine

2.15

2-pentanol

musty, fusel, white wine, banana, apple, melon rind, tropical nuances

2.20

five carbon alcohol

fusel, fermented, fruity, bready, cereal, cognac

2.40

ethyl butanoate

fruity, sweet, apple, fresh and lifting

2.50

methyl butanoate

fusel, impacting, fruity, cultured dairy, acidic

2.55

propanoic acid

acidic, dairy with a pronounced fruity lift

2.70

furfural

woody, sweet, bready, nutty, caramellic with a burnt astringent nuance

2.85

1-hexanol

green, fruity, apple-skin

2.95

ethyl pentanoate

sweet, strawberry, pineapple, tropical fruit

3.20

2,4-dimethyl-3pentanone

acetone-like

3.40

hexanoic acid

cheesy, fruity, fatty, goaty

3.50

ethyl hexanoate

sweet, pineapple, fruity, waxy, banana

3.80

benzyl alcohol

chemical, fruity with balsamic nuances

3.85

unbranched 6-8 carbon alcohol

green, earthy, fruity, oily

4.00

ethyl heptanoate

pineapple, banana and strawberry with a spicy, oily nuance

4.45

phenyl ethanone

sweet fruit, almond, cherry, honeysuckle, jasmine, strawberry, orange

4.50

ethyl octanoate

sweet, waxy, fruity, pineapple, creamy, fatty, mushroom, , cognac notes

Table 2 shows clearly that wood aged Chinese baijiu flavor is dominated by a series of unbranched aliphatic ethyl ester compounds. A complete and perfectly sequential series of unbranched ethyl esters is observed: from ethyl butanoate, through ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, to ethyl octanoate (15). 39

Differing Results for Differing Starting Materials While the identities of the various imported commercially available baijiu spirit starting materials was kept confidential in this blind study, a striking difference was seen between the responses of the various starting materials to rapid pressure-aging. Figure 2 shows an overlay of the GC-MS chromatogram traces for an unaged baijiu as compared to the same baijiu which has been oak aged. For this baijiu starting material, known only as ID number 6882, no significant differences were measured by GC-MS upon aging. While the aged baijiu is certainly colored (visually) and flavored (aroma) with wood as compared to the unaged clear/transparent liquor, the observation of similar GC-MS traces leads to the conclusion that the differences between the two samples are largely in non-volatile compounds and would better be measured by high pressure-liquid chromatography (HP-LC).

Figure 2. Overlay of GC-MS traces of unaged and oak aged baijiu sample ID number 6882. Figure 3 shows an analogous overlay (unaged versus oak aged) of the GC-MS chromatogram traces for a different baijiu starting material, known in this blind study only as starting material ID number 60701. For this baijiu starting material, a slight increase in the concentration of ethyl hexanoate was observed upon aging. Accordingly, it can be inferred that the increased content of ethyl hexanoate in charred oak aged baijiu imparts a sweet and fruity nuance to the spirit, relative to that of the traditional unaged baijiu. 40

Figure 3. Overlay of GC-MS traces of unaged and oak aged baijiu sample ID number 60701.

Figure 4. Overlay of GC-MS traces of unaged and oak aged baijiu sample ID number 654B. 41

Figure 4 shows yet another analogous overlay (unaged versus oak aged) for yet a third baijiu starting material, known in this blind study only as starting material ID number 654B. For this baijiu starting material, an even more substantial increase was observed in the concentration of ethyl hexanoate upon aging. Further, substantial increases upon aging were also observed in the concentrations of ethyl butanoate and a five carbon alcohol. Clearly, sample 654B was more altered in flavor by the pressure-aging process than the other starting materials (15). Knowledge of the identity of these blind samples would likely lead to inferences as to why different baijiu starting materials respond to the pressure-aging process differently.

New Flavors Imparted in Wood Aged Baijiu Spirit The higher concentration of ethyl hexanoate observed in charred oak aged baijiu can be inferred as to impart a sweet and fruity nuance to the spirit relative to the unaged spirit. If however this is desirable may be debated by whiskey enthusiast endlessly in a taste test over a glass neat, a glass on ice, etc. Further, it is clear that wood aged Chinese baijiu flavor is dominated by a series of unbranched ethyl esters, as a complete series from ethyl butanoate, through ethyl pentanoate, ethyl hexanoate, and ethyl heptanoate, to ethyl octanoate is observed (15). These ethyl esters impart various fruit flavor nuances to the oak aged baijiu. One might easily be led to speculate about the creation of these compounds via the formation of ester linkages between ethyl alcohol and various carboxylic acids; it is presumed that these linkages are being prompted by the increased pressure experienced during pressure aging.

Looking Back with Hindsight Vision Impact on Students Certainly we can presume it likely that none of the students will become flavor scientists. What value does participation in the program bring these students? The overwhelming majority of the students who have thus far participated in the program are health-science students. Of the students involved in the research, only two are studying to be chemists and one a science teacher. This experience has given them a genuine understanding of the way that analytical data is generated and processed. I envision a scenario in their future careers where they say: “Let’s send a sample to the lab. If we talk to the people in the lab they can advise on what appropriate test to run.” This experience has left the students with a predisposition that analytical data can be interpreted to provide useful information. Further, every student who has been through the program has presented their work publicly at a local or national conference. The true value for the students of participation in the program has been the opportunity to speak publicly about their research. They learn how to succinctly convey highly technical information to a non-expert audience. Arguably, this is an invaluable life skill. 42

Lessons Learned Having an industrial partner has been essential for developing a research program at a community college which is both practical and applied. The initial chance encounter with Cleveland Whiskey led to the initiation of a collaboration to establish this program. Students find the work concrete and easily relatable, as they are generating practical and useful information. Since students find the work concrete and easily relatable, we are able to recruit students into the program in their first semester on campus. Specifically, students in their first semester of General Chemistry need not understand the theoretical basis of GC-MS to understand how the instrument can be applied to provide useful information on a practical topic. The lesson learned is that undergraduate research need not and in-fact should not be reserved for upperclassmen students simply because they have advanced to upper level coursework. As students begin to conduct independent research, faculty efforts can shift to identifying opportunities for students to present publicly and coaching students on public presentation skills. Indeed, the experience of presenting such highly technical information to a non-expert audience, and conveying the information succinctly, has been the most valuable experience gained by the students who have participated in the program. Finally, we believe that the large amount of success which this program has enjoyed is due predominately to one word, “whiskey.” The choice of a topic is paramount for securing student engagement, especially in an “optional” undergraduate research program. Owing to the “whiskey” connection, students are enthusiastic to become involved and they enjoy the experience.

Summary Students find the work on this project conceptually straightforward. The work is practical and applied, and therefore easily relatable; this is owing to collaboration with an industrial partner. Whereas academic research is often seen as conceptually abstract, students find this work concrete, and are thereby encouraged when they are able to quickly contribute to the work. The analytical technique is well established, it can be applied to unique samples, and as such yields practical and useful information. The innovative production technology of rapid pressure-aging allows endless possibilities to innovate new flavors of whiskey. Essentially, the technology opens possibilities for endless untapped flavors from unprecedented woods, where creativity is now the only limit to innovation. This work can continue into the future in unlimited different directions bound only by creativity. A vibrant independent student research program for undergraduates has been created and is blossoming, with students working independently on various aspects of the project as part of a collaborative research group (16). Essentially, a research group based on the graduate school model has been established at a two-year community college, with a well-equipped and well-funded laboratory, and students performing substantial project based independent research on an 43

ongoing and self-perpetuating basis. It’s challenging enough for four-year colleges to offer substantial research experiences to undergraduate students, usually in their upperclassman years and traditionally in the form of independent studies projects. Yet here, at a two-year college where the challenge is even more substantial because students are only on campus for their freshman and sophomore years, a flourishing program has been established where students are performing not just a single isolated independent studies project, but ongoing research within the structure of a research group working on cooperative topics. A graduate style “research group” (Figure 5) has been established at a community college, and is thriving. Students are enthusiastic to become involved in the “science of the flavor of whiskey” project which has quite the buzz on campus. While it is real science, it’s also real fun.

Figure 5. Lorain County Community College “Whiskey Students” Research Group 2016-2017. (L-R): Selena Vazquez, Christopher Wright, Clayton Mastorovich, Professor Regan Silvestri, Daniel McKeighen, Katie Nowlin, Valerie Gardner, Heather Ketchum. (Photograph by Ronald Jantz).

Acknowledgments Grateful thanks for financial support from the American Chemical Society Collaborative Opportunities Grants program, the American Chemical Society Two-Year College Faculty/Student Travel Grant Awards program, the Lorain County Community College Foundation Campus Grants program, the Lorain County Community College Center for Teaching Excellence, the NASA Ohio Space Grant Consortium, the Ohio Academy of Sciences, Ohio Means Internships and Co-Ops, Choose Ohio First, and to Cleveland Whiskey, for their corporate sponsorship and collaboration. 44

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Patent: Lix, T. S. Whiskey making method; U.S. Patent 0149423A1, June 13, 2013. Gill, V. A whisky tour. Chemistry World 2008December, 40–44. Scott, D. Aroma study of potable spirits. PerkinElmer Application Note; 2013; pp 1−4. Analyzing alcoholic beverages by gas chromatography. Restek Technical Guide; 2002; Lit. Cat. No. 59462, pp 1−16. Improved resolution of alcoholic beverage components by packed column GC. Supelco Bulletin 790C; 1996; pp 1−4. Wittrig, R. E.; Macnamara, K.;, Mosesman, N. Optimizing the capillary GC separation of acids, esters, and other flavor components in distilled liquor products. Restek Technical Guide; 2002; pp 1−8. Qualitative comparison of whisky samples using fast GC/TOFMS. LECO Corporation Form No. 203-821-200; 2008; pp 1−4. Nowlin, K.; Gardner, V.; Mastorovich, C.; Wright, C.; Silvestri, R. GC-MS analysis of unprecedented whiskey flavors created by a novel aging process. Proceedings of the Society for Applied Spectroscopy Sixtieth Annual May Conference, May 18, 2016; p 63. Silvestri, R. Chemistry of whiskey flavor. American Chemical Society Undergraduate Blog Reactions; April 6, 2016; https:// www.acs.org/content/acs/en/funding-and-awards/grants/collaborative -opportunities-grants/independent-student-research--flavor-of-whiskey.html ?_ga=2.257423691.1516371979.1521126994-1467036413.1516377539 (accessed March 15, 2018). Widener, A. Imparting whiskey wisdom. Chemical and Engineering News 2016May2, 40. Horn, J. Why Japan is the best country for whisky right now. Playboy 2016June15. Zheng, Y.; Sun, B.; Zhao, M.; Zheng, F.; Huang, M.; Sun, J.; Sun, X.; Li, H. Characterization of the key odorants in Chinese zhima aroma-type baijiu by gas chromatography−olfactometry, quantitative measurements, aroma recombination, and omission studies. J. Agric. Food Chem. 2016, 64, 5367–5374. Xu, H.; Xu, X.; Tao, Y.; Yuan, F.; Gao, Y. Optimization by response surfacemethodology of supercritical carbon dioxide extraction of flavour compounds from Chinese liquor vinasse. Flavour Fragrance J. 2015, 30, 275–281. Wu, J.; Zheng, Y.; Sun, B.; Sun, X.; Sun, J.; Zheng, F.; Huang, M. The occurrence of propyl lactate in Chinese baijius (Chinese liquors) detected by direct injection coupled with gas chromatography-mass spectrometry. Molecules 2015, 20, 19002–19013. Ketchum, H.; Silvestri, R. GC-MS analysis of Chinese baijiu liquor flavored as American bourbon whiskey. Ohio J. Sci. 2017, 117, 6. Silvestri, R. Distilling lessons from a whiskey research program. Chemistry 2016November, 12–13. 45

Chapter 4

Transforming Second Semester Organic Chemistry Laboratory into a Semester Long Undergraduate Research Experience Andrew J. Carr, Ryan J. Felix, and Stephanie L. Gould* Department of Chemistry, Austin College, Sherman, Texas 75090, United States *E-mail: [email protected]

At Austin College, organic chemistry is a multi-section, large enrollment class that serves as a gateway to the chemistry, biochemistry, and biology majors. In an effort to provide students an early authentic research experience in this sophomore-level course, a semester long research project was developed for the second semester course. This project provides students with practice searching and reading primary literature, organizing and conducting a multi-step synthesis, collecting, sharing, and analyzing data, and writing a formal lab report in the style of the Journal of Organic Chemistry. Scaffolded instruction on writing and teamwork skills enabled students to learn and practice these skills within the new formative environment of the lab.

Introduction In 2011, the local environment in the science division at Austin College was teeming with enthusiasm for rethinking curriculum. The chemistry department faculty were active participants in an extensive series of professional development events centered on integrating High-Impact Practices (HIPs) into courses (1, 2). There are ten HIPs that are key types of actions that Universities and Colleges engage in that have demonstrated impacts on achievement and retention (3, 4). One HIP is providing research opportunities for all students. It has also © 2018 American Chemical Society

been shown that early research is particularly impactful for science students (5). Diverse groups of students benefit from the HIP approaches, such as undergraduate research, collaborative projects, and taking multiple courses as a cohort, without hurting traditional students (6–8). Traditional research models are expensive; therefore, the course-embedded research project approach was explored as a way to increase access to research opportunities (9, 10) and provide training for traditional research experiences (11). The Council on Undergraduate Research published a book in 2007 titled: Developing & Sustaining a Research-Supportive Curriculum: A compendium of Successful Practices (12), which provided examples of how other institutions used their curriculum to support traditional research in their departments. The examples outlined in the book provided inspiration to think creatively about how laboratory relates to the accompanying lecture course. Organic chemistry I and II are ideal courses at Austin College to explore an early research opportunity because these classes serve as gateway courses for several majors (chemistry, biochemistry, and cell and molecular biology), the chemistry minor, and for pre-medical/pre-allied health students. Organic chemistry II is a multi-section course enrolling 60-70 students annually, and is taught only in the spring semester. Each lecture section has roughly 25 students and the laboratory sections are capped at 20 students each. Three lecture sections are offered in the spring semester, each taught by one of this paper’s authors. Four or five lab sections are available. Students are comingled, meaning students from any lecture may enroll in any lab. A faculty member or a full-time permanent lab instructor teaches each laboratory section, assisted by one or two teaching assistants. Teaching assistants are junior and senior-level students who help answer questions and troubleshoot issues that arise during lab. The faculty member is responsible for all pre-lab instructions, in-lab supervision, and grading. Teaching assignments are made to align with the needs of the department, any of the authors and the full-time lab instructor may teach any of the sections and it varies year-to-year which instructor teaches which section. All students earning a chemistry or biochemistry major are required to participate in a traditional research experience to complete the major. Students fulfill this requirement through on-campus academic-year and summer research opportunities or in off-campus National Science Foundation supported research experiences for undergraduates (REU) and REU-like opportunities. Organic chemistry II provided a unique opportunity in the curriculum to prepare students for the rigors of undergraduate research At the same time, the STEM Teaching and Research (STAR) Leadership program (13) at Austin College was expanding. This program teaches professional and leadership skills from within science courses by focusing on the skills of communicating ideas, problem solving, collaborative work, foresight and planning, and acting responsibly. A realistic research experience as a part of the organic chemistry II laboratory was an ideal setting to integrate teamwork, communication skills, and foresight and planning. Lab assignments and STAR activities are standardized across all lab sections and agreed upon before the beginning of the semester. The organic chemistry team (faculty and full-time instructor) meets weekly to coordinate and discuss upcoming 48

assignments. All members of the team received the same training for STAR and collaboratively design the activities for organic chemistry II. Planning for the semester long research project began in 2011 by extending the short 2-week synthesis project already in the schedule. The current 13-week scaffolded research experience seeks to develop important research, teamwork, and communication skills. Student teams write throughout the semester to complete drafts of paper components, with the final goal of creating a research quality presentation and paper similar to one that would be submitted to The Journal of Organic Chemistry.

Turning Ideas into Practice Choosing a Research Project Careful consideration was made when choosing an appropriate research project to use for this transformation. Ideally, a project will have similar chemical reactions to those being learned in class, have relatively simple synthetic procedures, and have products that can be recrystallized. Within the department, Dr. Andrew Carr’s independent research synthesizing and studying organogelator compounds (14) was an ideal starting point. Organogelator compounds form gels when heated with organic solvents. The solvent is contained within the supramolecular structure of the gel. The quality of an organogelator compound is measured by how much solvent can be gelled by a known quantity of the compound, known as the critical concentration. The first organogelator (compound 1) synthesized in Dr. Carr’s laboratory, shown in Figure 1, is a symmetric compound with two urea groups and two aromatic rings to guide stacking of the molecules. The long alkyl tails (R) form entanglements, creating strong gels when heated with organic solvents (15).

Figure 1. Chemical Structure of Original Carr Organogelator. 49

The target compounds (compound 6 with varying tail lengths) for the research project were a new class of organogelators with a similar core and only one alkyl tail (R) on each side of the compound, shown in Figure 2. The synthetic pathway for compound 1 was modified for the target compound. A readily available starting material (Vanillin, compound 2) was used. The synthetic pathway includes an alkylation to give compound 3, oxime formation (compound 4) and reduction to an amine to give compound 5. In the last step compound 6 is synthesized by forming the urea compound. The overall This research was modified for the teaching laboratory so that each step could be completed within a scheduled 3-hour laboratory. The chemistry employed is familiar to second semester sophomores with an initial SN2 reaction, followed by an oxime formation, then a reduction, and ending with the urea formation. Each reaction is either taught explicitly in organic chemistry I and II or is very similar to chemistry that is taught. Intermediates can be purified by recrystallization, preventing the need for column chromatography which is expensive due to solvents, silica, and time. The generalized synthetic pathway can be used for several semesters by changing the tail (R), alternating the starting material between vanillin (shown) and isovanillin, or starting with a different aldehyde starting material.

Figure 2. Synthesis of Spring 2016 Compounds.

50

Transforming the Lab Structure The course started as a traditional lab with “cookbook” style experiments and was transformed into a 13-week course-embedded research experience, as illustrated in Table 1, over five years. The traditional lab contained standard experiments such as nitration of a methyl benzoate, a Grignard reaction, and a condensation reaction. Two weeks of the semester were spent synthesizing known starting materials for Dr. Andrew Carr’s summer research students. In 2012 and 2013, the synthesis experience was extended to three weeks and students began synthesizing new starting materials instead of previously studied starting materials. In 2014 the lab was expanded to a five-week, 4-step synthesis project synthesizing and testing new organogelator molecules. The timeline in 2014 was optimistic and ultimately unrealistic. In spring 2015, the experience was expanded to nine weeks of the semester and the synthesis project was transformed into the synthesis, characterization, study, and reporting of new organogelators. Between 2016 and 2017, the chemical information lab was adapted to be part of the research experience and a techniques lab was introduced. In the current format, teams of four students synthesize two target molecules during the semester long project. Modeled after the way a new research student joins a synthetic research lab, the teams are each assigned two compounds and provided with the synthetic procedures needed to conduct the experiments. Each year the procedures are modified to accommodate new starting materials and/or procedural optimizations done in the previous year. Within each section, five target molecules are synthesized. There is overlapping data collected throughout the section by having two teams assigned to a target. For example team A will have the 10- and 12-carbon tail target molecules, while team B will have the 12- and 14carbon tail target molecules. Teams share their data within the laboratory section via Google Sheets for final analysis. Each section has the same target molecules, so there are 4-5 replicates done annually. The semester schedule was designed to move students through the project while providing scaffolding of the research process along the way, shown in Table 1. The first two weeks of the semester guide the students through chemical searches in SciFinder, reading and understanding primary literature, and presenting information they found. The first wet lab, week 3 of the semester, is a technique lab to familiarize with the techniques and protocols they will use all semester. This lab includes learning to use a rotoevaporator and separatory funnel, how to run a reaction in an inert atmosphere, and a reminder of how to run TLC plates. During week 4 to week 9, students synthesize the assigned compounds. The final week of the synthetic stage is designed as a flex day. Mimicking a traditional research project, the flex day is built into the schedule in case mistakes are made and/or synthesis steps need to be repeated. Students have two weeks to test the ability of their compounds to gel organic solvents, for example, hexanes, toluene, and octane, and two weeks to prepare their presentations and final reports. After a few iterations (2015 and 2016) the week 12 data analysis day was added to help students make deep connections between their data, the class data, and their introductions. Providing lab time and some guidance enabled students 51

to produce a much better final report. During the final week of the semester, each team presents their data, the class data, and their interpretations.

Table 1. Weekly Schedule of Organic Chemistry II Laboratory Week

2012

2014

2015

2017

1

Chem Info (SciFinder)

Chem Info (SciFinder)

Chem Info (SciFinder)

Chem Info (SciFinder)

2

Presentations

Presentations

Presentations

Presentations

3

Aromatic Nitration (EAS)

Iodination of Vanillin (EAS)

Iodination of Vanillin (EAS)

Techniques Lab

4

Aromatic Nitration (EAS)

Iodination of Vanillin (EAS)

Iodination of Vanillin (EAS)

Synthesis

5

Rates of Bromination

Rates of Bromination

Synthesis

Synthesis

6

Grignard Reaction

Grignard Reaction

Synthesis

Synthesis

7

Grignard Reaction

Grignard Reaction

Synthesis

Synthesis

8

Qualitative Analysis

Synthesis Project

Synthesis

Synthesis

9

Qualitative Analysis (acid/base extractions)

Synthesis Project

Synthesis

Flex Day

10

Synthesis Project

Synthesis Project

Flex Day

Gelation Tests

11

Synthesis Project

Synthesis Project

Gelation Tests

Gelation Tests

12

Condensation Reaction

Synthesis Project

Gelation Tests

Data Analysis and Q&A Day

13

ACS Exam and Check-out

Gelation Tests

Presentations

Presentations

Integrating Writing Prior to 2014, no formal writing was required for the entire semester. Invigorating the lab by converting to a research experience provided an opportunity to develop writing as a part of the laboratory. The approach was developmental. Students needed scaffolded guidance through each step of writing the report. The final product is a full journal article in the style of the Journal of Organic Chemistry. Table 2 shows the overall schedule for writing. 52

Table 2. Schedule of Writing and Professional Skills Development Week

Writing Emphasis

STAR Emphasis

1

Finding Journal Articles

2

Analyzing Journal Articles

Presentations

3

How to write introductions Journal Analysis Due

Problem solving in teams

4

Outline of introduction due

Self-reflection

5

Preparing synthetic schemes; Introduction drafts due

6

Experimental discussion; Draft of schemes due

7

Conflict and feedback in teams Peer feedback

8

Draft of Experimental due

9

Self-reflection

10 11 12

Writing Results and Discussion sections

Peer feedback

13

Final paper due

Self-reflection

During the first week of the semester students are taught to find journal articles using SciFinder. During the second week, they are asked to prepare a journal article analysis by answering the following questions about a single article:

• • • • •

What was the main goal of this paper? What methods were used to collect data to analyze this goal? What conclusions did the author(s) make in this paper? What data was used to support the conclusions? Can this journal article be used to support my introduction? How?

Each individual in the team must use a different article for their analysis, providing the team with four analyses in preparation for writing an introduction. During the third week, specific instructions about how to write an introduction are provided to the students. Students are encouraged to consult LabWrite, a National Science Foundation supported website designed by professors at North Carolina State University to help students write better reports (16). Outlines of their introductions are due the following week and a full draft of the introduction is due a week later. The authors developed a set of questions for evaluating the content of an introduction: 53

• • • • • • • • •

• • • •

Is context established for the overall lab? Is the scientific concept being studied mentioned in the first few sentences? Is current literature regarding the scientific concept summarized? Is the literature citied connected to the scientific concept adequately? Does the introduction include a figure of the original gelator synthesized in Dr. Carr’s laboratory? Are the main objectives of the study discussed? Is the method to study those main objectives discussed? Do the authors adequately connect the main objectives to the overall scientific concept? Does the introduction include a figure showing the structure of the new compounds being synthesized? (may be included in the figure with the original gelator) Are the figures labeled properly? Are the citations in endnote format? Are the citations in ACS J.O.C. format? Is the entire introduction written in 3rd person?

Teaching assistants peer-evaluate each draft introduction submitted with the questions above. Students have the questions as a part of the instructions for the assignment. In subsequent weeks, the art of drawing schemes and preparing experimentals for each reaction is taught. Students have access to ChemDoodle through a site-license paid for by the college. During the second to last week of the semester, students are taught how to do data analysis and write a results and discussion section with a whole lab session dedicated to doing the analysis as a class. Systematic and scaffolded instruction of writing components has dramatically improved the quality of the final reports.

Teaching Teamwork The STAR Leadership Program was integrated into the organic chemistry sequence starting in 2013. During the fall and spring semesters, the skills of teamwork, providing feedback, and self-reflection are emphasized, illustrated by the schedule outlined in Table 2. During the first semester, students learn about the importance of effective teamwork and how to bring a team back to task. The second semester is focused on practical issues surrounding teamwork and communication. Beginning with the first week of synthesis, teams are expected to meet ahead of the lab and answer pre-lab planning questions. The questions are designed to make the team think through how they will divide the labor each week and support one another. Asking teams to meet before the lab to think through these critical aspects reduced the chaos and confusion during the lab sessions. Team members are expected to help each other throughout that day’s lab. The whole team is responsible for all of the work. Slowly, the students learn that they are capable 54

of much more work, and of a higher quality, working as a team than the sum of their individual work. Students are asked to take the Kirton Adaptor-Innovator instrument (17, 18) to think through their own problem solving predispositions and how working with someone who approaches problems differently will impact teamwork. The instrument arranges students on a spectrum to help students understand their preferred approach to solving problems. At the ends of the spectrum, adaptor and innovator, people tend to solve problems in dramatically different ways, while people in the middle of the spectrum move more easily between the two approaches. Students share their placement on the spectrum with their team and talk about how their unique strengths can help their team. A second goal for this activity is to develop empathy towards differences. The most effective teams are made up of a variety of problem solving techniques (19). The following week students are asked to write a reflection about how their team completed the first two tasks (SciFinder, presentations, and the techniques lab). They must comment on the division of labor, communication within the team, focus, trust, and decision-making; as well as describe how the different types of problem solvers communicate in their team. Reflections are graded based on the quality of the reflection, not the specfic reflection they make. High quality reflections are specific, use examples, and provide ideas for future improvements. Finally, students engage in activities to learn about the cycle of conflict and how to reduce conflict before it becomes a crisis. Effective feedback to teammates is demonstrated by the professors. Students are asked to consider the cycle of conflict from a misunderstanding, to a simmering resentment, to a crisis (20). Students have the opportunity to think through strategies to recognize the early stages of conflict and find solutions before a crisis begins. Following this instruction students are given two opportunities to give positive feedback and an area of improvement feedback to their teammates. Students write self-reflections after each feedback. Reflections need to comment on the feedback they received and their thoughts on the improvements that need to be made. Students are also graded on their feedback; feedback must be specific and actionable. On occasion, a team will have a serious crisis of communication or a conflict that they are unable to handle by themselves. To help mediate these situations the faculty member meets with the team and establishes the lines of communication. Grievences are aired and solutions are developed. The team then must prepare a list of ground rules by which everyone will abide.

Outcomes Outcomes for the transition to an early course-embedded research experience were measured using two instruments. First, the widely used Classroom Undergraduate Research Experience (CURE) instrument, developed by Dr. David Lopatto at Grinnell College (21, 22), was administered to the second semester organic chemistry students in 2015 and 2017. Significant changes to the course design (techniques lab added, data analysis day, more STAR and writing scaffolding, etc.) were introduced in 2016 as a result of the 2015 survey. 55

The authors used 2016 to make these changes. Not all of the changes were implemented successfully; therefore, CURE data was collected for a second time in 2017 after significant reworking of the changes. Changes ranged from simplifying the techniques lab from 5 techniques to 4 in order to finish in the time allotted to reworking the writing assignments for clarity. The second instrument was designed by the authors to understand student impressions of laboratory experience in comparison to traditional labs students have taken at Austin College. CURE Results The CURE survey asks students to provide basic demographic information (Table 3), rank their perceived gains as a result of this course on course elements (Table 4) and learning elements (Table 5), as well as overall impressions of the course (Table 6). Student responses of their perceived gains from the laboratory experience are on a 5-point scale from 1, indicating no or very small gain, to 5, indicating a very large gain. Reported values shown in Table 4-6 include all students, nationwide, who took the CURE survey in spring 2017 and Austin College students who took the survey in spring 2015 and spring 2017. Typically CURE survey items have a standard deviation of approximately ±1. Unfortunately, the survey results provided by the CURE team did not provide specific standard deviation for the items in Table 4 or Table 6. The standard deviation for items in Table 5 vary from 1.00 to 1.23.

Table 3. Gender and Ethnicity Self-Reported Data from CURE Survey Percent of Organic Chemistry II Students who self-report as...

2015 Austin College Students (n≤53)

2017 Austin College Students (n≤64)

Male

55.7%

28.1%

Female

44.3%

71.9%

Asian American

35.9%

36.1%

Black or African-American

5.7%

4.9%

Hispanic/Latino

5.7%

6.5%

White

39.7%

37.7%

Two or more races

9.4%

8.2%

Other

1.8%

6.6%

Foreign National

1.8%

0.0%

Self-reported gender and demographic data is shown in Table 3. This information is provided to give a snapshot of the population that participated in organic chemistry II during the transformation toward a course-embedded research project. It is important to recognize the significant change in gender balance from 2015 to 2017. Looking at the gender balance of organic chemistry 56

II over the past five years, it can be concluded that 2017 is an anomaly. Austin College has maintained a 50-55% female to 50-45% male total gender balance over the past five years. Usually organic chemistry II has between 45-55% female students and 45-55% male.

Table 4. Self-Reported Gains on Course Elements in Organic Chemistry II Laboratory 2017 Students Taking CURE (n≤9718)

2015 Austin College Students (n≤53)

2017 Austin College Students (n≤64)

Lab or project where no one knows the outcome

2.51

3.68

3.79

A project where students have input into process or topic

3.94

3.67

4.23

A project entirely of student design

3.63

2.95

3.33

Work individually

3.42

3.14

3.24

Work as a whole class

3.28

3.19

3.52

Work in small groups

3.98

4.11

4.31

Become responsible for a part of the project

4.03

4.04

4.42

Read primary scientific literature

3.71

3.81

4.40

Write a research proposal

3.60

3.28

3.88

Collect data

4.05

4.10

4.25

Analyze data

4.12

4.27

4.38

Present results orally

3.73

4.08

4.27

Present results in written papers or reports

3.86

4.25

4.37

Critique work of other students

3.38

3.49

3.61

Course Element

The data in Table 4 demonstrates that students in the course believed they gained experience in areas that were part of the design of the course. The research opportunity is introduced as an example of how scientists make discoveries. Students appear to understand this goal as demonstrated by their responses to the “Lab or project where no one knows the outcome” prompt. Austin College students show fewer gains than the national average on the question “works independently.” This is consistent with the goals of the course to teach teamwork and collaboration as a normal part of science. Comparing Austin College data from 2015 to 2017 provides interesting differences that likely result from the modifications made to the curriculum during the 2016 spring semester. For example, in 2015 students were asked to read and prepare an introduction based on primary literature. The following year 57

the literature analysis assignment described above was introduced. Scaffolding the primary literature assignment was necessary because the students had little to no prior experience reading chemistry primary literature. In the data, students in 2017 indicated 0.60 more gain on the “reading primary literature” question.

Table 5. Self-Reported Gains on Learning Elements of this Organic Chemistry II Laboratory 2017 Students Taking CURE (n≤9603)

2015 Austin College Students (n≤53)

2017 Austin College Students (n≤64)

Skill in interpretation of results

3.72

4.14

3.75

Tolerance for obstacles faced in the research process

3.70

4.13

3.92

Readiness for more demanding research

3.63

3.92

3.77

Understanding how knowledge is constructed

3.64

4.02

3.62

Understanding the research process

3.69

3.95

3.81

Ability to integrate theory and practice

3.64

3.81

3.81

Understanding how scientists work on real problems

3.77

4.02

3.91

Understanding that scientific assertions require supporting evidence

3.80

4.05

4.04

Ability to analyze data and other information

3.88

4.31

3.96

Learning laboratory techniques

3.89

4.27

4.28

Ability to read and understand primary literature

3.60

4.16

3.94

Skill in how to give an effective oral presentation

3.41

4.13

3.69

Skill in science writing

3.58

4.13

3.85

Self-confidence

3.48

3.89

3.41

Learning Element

Another example of increased gains was in student abilities in analyzing data. In 2015, students were expected to analyze their data and the class data on their own, bringing questions to their lab instructor as needed. After 2015, a laboratory session was dedicated to teaching students how to analyze their data. During the session, students work in their lab teams and as a class to find trends in their data. This change is apparent in the data in two questions. First “Work as a whole class” has a 0.33 gain from 2015 to 2017 and also “Analyze data” has a 0.12 gain. Finally, a gain of 0.22 was observed from 2015 to 2017 in the question “Critique work of other students.” This gain is likely from changes made to help students better understand how to provide feedback in an effective manner to their 58

peers. Additionally, changes to the reflection prompt require students to write about the feedback and what they will do differently. Table 5 shows perceived gains in ability as a result of this organic chemistry II lab. Similar to Table 4, gains are reported on a 5-point scale from 1, indicating no or very small gain, to 5, indicating a very large gain. Data is presented from all 2017 students who took the CURE survey nationwide and Austin College students who took the survey in 2015 and 2017.

Table 6. Post Course Overall Assessment 2017 Students Taking CURE

Element

2015 Austin College Students

2017 Austin College Students

This course was a good way of learning about the subject

4.12

4.36

4.24

This course was a good way of learning about the process of scientific research

4.20

4.54

4.43

This course had a positive effect on my interest in science

3.97

4.25

4.02

I was able to ask questions in class and get helpful responses

4.17

4.24

4.13

Austin College students universally report lower gains in 2017 than in 2015. There are many possible reasons for this, including but not limited to, students in 2017 are more comfortable with learning, the teaching of these learning elements was less explicit, or dramatic changes to the curriculum in pre-requisite courses over the past two years. The most likely cause is the third one: curriculum changes in pre-requisite courses. Over the course of three years more faculty became engaged in integrating HIPs practices into the classroom and more faculty joined the STAR Leadership Program. Integration of STAR into general chemistry and general biology, both first year courses, occurred in the 2015-2016 academic year. Students enrolled in organic chemistry II during spring 2015 did not have the enhanced curriculum in courses taken the year before. The more that students are exposed to this type of learning the less any one individual course will change a student’s perception of that learning aspect. The data is still at or above the average reported value for all students taking the CURE instrument nationwide. The last part of the CURE instrument asks students to rate four statements in regard to their own satisfaction with the research experience being embedded into a course based on a 5-point scale. Results are shown in Table 6. Both in the 2015 and 2017 data sets, Austin College students report an average rating of 4 or above; however, the students in 2017 report slightly less satisfaction (approximately 0.1). 59

Student Impressions The authors administered a self-designed student impressions instrument to students who finished the spring 2015 and spring 2017 organic chemistry II course in order to evaluate the new laboratory format. Students took the survey three to four months after the conclusion of the course in September of the same year. Fifty-seven students were invited to participate in the survey in 2017 yielding 27 responses (47.4%). In 2017 an additional 53 students were invited to participate in the survey resulting in 27 responses (50.9%). Data is organized by impressions of the overall course (Table 7) and impressions of the skills taught within the course (Table 8). Students could respond to each statement as strongly agree, agree, disagree, and strongly disagree. Strongly agree and agree answers are aggregated as positive responses in each table.

Table 7. Student Impressions of Overall Course Positive Response 2015 (n=27)

Positive Response 2017 (n=27)

I enjoyed participating in the Chem 222 research experience.

25 (92.6%)

23 (85.2%)

I would like the professors to continue to offer a research experience in Chem 222 to future classes.

25 (92.6%)

23 (85.3%)

As a result of the Chem 222 research experience, I am more likely to enroll in a course that I know will have a research experience laboratory than a course with a traditional laboratory component.

18 (66.7%)

21 (77.8%)

As a result of the Chem 222 research experience, I am more likely to pursue additional research opportunities (could be in other disciplines).

21 (77.8%)

21 (77.8%)

I was more engaged with problem solving during the Chem 222 research experience than I was in a more traditional laboratory (Chem 111, 112, 221, etc.).

23 (85.2%)

22 (81.5%)

Statement

Student impressions about the course are important to understand because a course embedded research experience is resource intensive and the cost changes based on the starting materials needed for the particular target molecule. The first question “I enjoyed participating in the Chem 222 research experience” received overwhelmingly positive responses, 92.6% in 2015 and 85.2% in 2017. This next 60

question “I would like the professors to continue to offer a research experience in Chem 222 to future classes” received positive responses as well, 92.6% in 2015 and 85.2% in 2017. These affirmative response rates give evidence that spending the time and resources on this type of lab experience is impactful for Austin College students. Approximately two-thirds of respondents in 2015 and three-fourths of respondents in 2017 are more likely to pursue additional courses with embedded research experiences and research experiences in general. Finally, greater than 80% of students indicated that they felt more engaged in problem solving during the research experience rather than a traditional lab for each year. The second set of data from the student impressions instrument, Table 8, centers around skills that students receive explicit direction and instruction for during the laboratory sessions. Skills development beyond the execution of scientific technique is consistent with the developmental model that has been implemented in this course. The data shows respondents believe this course makes them more comfortable working in teams, resolving conflict, reading primary literature, drawing conclusions from scientific data and presenting data orally and in written form. It is not surprising that reading and understanding primary literature is the lowest of the skills because steps have been taken between 2015 and 2017 to improve instruction in this area. More development on assignments is necessary to help students understand and use primary literature.

Table 8. Student Impressions of Skills Developed Positive Response 2015 (n=27)

Positive Response 2017 (n=27)

Working collaboratively in a laboratory setting

23 (85.2%)

24 (88.9%)

Resolving conflicts within a team in a laboratory setting

23 (85.2%)

22 (81.5%)

Reading and using primary literature

21 (77.8%)

17 (63.0%)

Drawing conclusions from experimental scientific data

22 (81.5%)

20 (74.1%)

Presenting scientific data orally to my classmates

19 (70.4%)

21 (77.8%)

Presenting scientific data in written form.

24 (88.9%)

21 (77.8%)

As a result of the Chem 222 Research Experience I am more comfortable…

The student impressions instrument gives tremendous information about the value of a course-embedded research experience to Austin College students in Organic Chemistry II. With this level of response, the authors believe transforming the laboratory into the embedded research opportunity has been successful. 61

Transferability The model described herein is successful at a small, private liberal arts college, in a large enrollment gateway course. Because of the multi-section lab arrangement, there is an opportunity for this model to be transferred to larger universities and community colleges. Organic chemistry I and II are only offered in fall and spring, respectively. Students only have one choice of type of class to take. The class is taught at the level that chemistry majors need to move on with a chemistry degree. All majors, minors, and pre-med/pre-allied health students take the same course. Success at Austin College was made possible by appropriately designing the synthetic pathway, using a developmental model similar to when a student joins a research lab, and well-trained laboratory supervision. Recognizing the inexperience of sophomore-level students means providing intentional instruction on technique. Research projects must be chosen to fit into the laboratory time frame. Providing synthetic conditions results in all students performing the same types of reactions, minimizing chaos in the laboratory. Additionally, only procedures that fit within the resources of the lab (physical and financial) can be selected. Finally, training and supervision are important. At Austin College, one faculty member and 1-2 undergraduate TAs supervise each laboratory. The TAs participated in the experience as a student within the last two years. In a community college setting it is likely that a professor will supervise the labs. In a large university the TAs are more likely to be graduate students. While this format is more freeform and less prescribed, TA training prior to the semester starting can enable proper supervision.

Conclusions The sophomore-level organic chemistry II laboratory at Austin College was successfully transformed from a traditional “cook-book” style lab to an embedded research opportunity. A result of this transformation is enabling more students to have a research opportunity early in their scientific careers. Students are learning important technique and communication skills in this format. Outcomes were measured through the CURE survey from Dr. Lopatto at Grinnell University. Austin College students indicated they perceived a gain in course elements and learning elements as a result of this course. While the data was universally positive, some variations from 2015 to 2017 data is likely a result of faculty teaching pre-requisite courses in similar formats after 2015. An instrument designed at Austin College measured overall student impressions. Similar to the CURE results, the student impression results were all positive in the areas that were emphasized. Teaching in this manner will continue at Austin College because of the positive responses from students.

62

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Chapter 5

Embedded Research in a Lower-Division Organic Chemistry Lab Course Lee J. Silverberg,*,1 John Tierney,2 and Kevin C. Cannon3 1Pennsylvania

State University, Schuylkill Campus, 200 University Drive, Schuylkill Haven, Pennsylvania 17972, United States 2Pennsylvania State University, Brandywine Campus, 25 Yearsley Mill Road, Media, Pennsylvania 19063, United States 3Pennsylvania State University, Abington Campus, 1600 Woodland Road, Abington, Pennsylvania 19001, United States *E-mail: [email protected]

This chapter describes how research has been embedded into the second-year organic chemistry laboratory course at three satellite campuses of the Pennsylvania State University. The course, CHEM 213, is a two-credit course in the Spring semester which meets for six hours per week. Approximately the last third of the course is an original research project in synthetic organic chemistry. The projects are small pieces of larger projects, and are novel and lead to publications, particularly benefiting the undergraduates concerned. Examples of the research projects and their implementation are described here.

Introduction The value of Science, Technology, Engineering and Math (STEM) research with undergraduates has been well-documented in recent years (1–12). For example, students are more satisfied and learn more (1), they integrate knowledge learned in courses (2), minority and female students are guided towards a scientific career (1), institutions attract more students (1), and students are better prepared for (2) and more likely to enroll in graduate school (3). One way of getting students involved in research is through “Course-embedded Undergraduate Research Experiences” or “CUREs” for short (13–24). This approach has seen rapid expansion in recent years. The authors of this chapter have been collectively © 2018 American Chemical Society

using this approach in the sophomore organic chemistry laboratory course at smaller campuses of Pennsylvania State University (PSU) for several decades. Herein, our experiences are detailed.

Course Structure Unlike most institutions, PSU’s Organic Chemistry Lab (CHEM 213) is not offered with the first semester of Organic Chemistry lecture (CHEM 210), but is introduced as a two credit course (six lab contact hours) with the second semester of lecture (CHEM 212). This results in students with a better theoretical background when they enter the lab. In the courses run by the present authors, approximately the first third of the course is devoted to learning the essential skills in an organic chemistry lab: safety, melting points, distillations, liquid-liquid extractions, recrystallization, chromatography, and spectroscopy (when available). The second third of the course consists of standard organic reactions. These are typical “cookbook” lab procedures, which give them experience in running reactions, reinforce the techniques previously learned, and allow them to see reactions that have been discussed in the lecture course. The last third of the class, four to five weeks, is a research project. At 6 hours per week, this gives them 24-30 hours of time to do a research project, enough time to get something significant accomplished. The students’ lab notebooks now become permanent documentation of research, which emphasizes the importance of taking good notes. The unknown outcome means that a) sometimes difficulties are encountered which much be overcome through careful thought and perseverance; and b) a successful result is exciting to the students. Communication of scientific results is one of the important aspects of the course. Students write lab reports, in the format of chemistry journals, for the various experiments during the semester. At the conclusion of the semester, the students do a poster or oral presentation of their research. Thus the students get training and feedback on both written and oral communication.

Embedded Research Finding Appropriate Projects The CHEM 213 research project has to draw on the laboratory skills and techniques already learned early on in the semester. In any research synthesis, additional techniques not already learned can be introduced to the more advanced and adept students in the group. Introducing synthetic research into the organic classroom and laboratory produces excitement among the students, particularly when they realize the compounds they are preparing are novel, publishable, and possibly bioactive. Each student in a group can be given a slight variation on a synthesis with regards to a substituent in the molecule, leading to a group of related compounds in a series. The resulting compounds can then undergo further study using spectroscopic techniques or biological testing which encompasses further involvement on the part of the student with potential interdisciplinary collaborations. The project becomes shared between the students, and the students 66

start to experience the aspect of working in the environment of a scientific team rather than as individuals. Planning and approach are very important. The students, no matter how academically talented, are not going to have the level of expertise and experience to do projects that are overly complicated and advanced. Further, they should not be entrusted with projects where the potential hazards are high. So in planning the research to be carried out, it is important to keep in mind what they are going to be able to do, and able to do safely. The research must be relatively simple to perform, and in “bite-size” chunks, giving them a chance of accomplishing something in the limited time frame during which they conduct research. The synthetic path chosen should have a reasonable degree of success regardless of a student’s lab skills in order for the student not to become frustrated by failure. Ideally, each piece becomes publishable, if not by itself, then as part of a larger effort. Collaboration The three authors of this chapter collaborate with each other and with scientists at other institutions as well. Research for us is a lesson in long-distance team building. With three synthetic organic chemistry groups, it becomes easier to effectively complete more experiments since we can combine our data for a publication. We have additional collaborators to obtain instrumental data, including NMR spectroscopy, infrared spectroscopy (IR), mass spectrometry (MS), and X-Ray crystallography. We collaborate with computational chemists on mechanistic and structural investigations. We work with biologists for biological testing (vide infra). Simply put, we reach out in many different directions to put our ideas into practice. However, it must be understood that this need for collaboration means work will not get done quickly. Patience is important. In order to advance our work at a reasonable pace, we sometimes assume that an experiment has worked based on observation, thin layer chromatography (TLC), and melting points until we eventually obtain more indicative data such as an NMR spectrum. Sometimes those assumptions are wrong and we get a surprise (vide infra). Examples During a kinetic study (25) of the formation of substituted 2,3-diaryl-1,3thiazolidin-4-ones from the reaction of imines with thioglycolic acid, a reaction originally demonstrated by Surrey (Figure 1) (26), it became apparent that there was a wide array of substituted 1,3-thiazolidin-4-ones that had not been prepared, but could be prepared by second-year organic chemistry students relatively easily. In addition, physical organic chemistry studies utilizing IR, NMR and MS data could be also be achieved – further incorporating information that the students had learned in the lecture portion of the class. The move to a synthetic project proved to be much more doable within the confines of the equipment available at the campus, and fruitful for incorporation into the organic chemistry lab course. 67

Figure 1. Synthetic scheme for the formation of substituted 2,3-diaryl-1,3-thiazolidin-4-ones.

The spectroscopic data that was used initially tracked the chemical shift variation of the methine proton (at C2), methylene protons (at C5) and the 13C NMR chemical shift data at C2, C4 and C5 in the 1,3-thiazolidin-4-one ring (Figure 2) based on substituents in the phenyl rings. It was in this phase of the project, in the early 1990s, that undergraduate students were incorporated into the project as part of the CHEM 213 undergraduate lab.

Figure 2. Nomenclature and atom positions in 1,3-thiazolidin-4-one ring. 68

The starting materials for these syntheses are relatively cheap and readily available. The one downside is that the thioglycolic acid is somewhat malodorous and has to be handled carefully. However, it is pointed out to the students that thioglycolic acid is used in permanent waving solutions in women’s hairdressing salons, and some students recognize the odor. The first publication where undergraduate students were intimately involved in the work appeared in Magnetic Resonance in Chemistry in 1996 (27). Soon after, a steady stream of published data appeared on the different series of compounds shown in Figure 3 (28–33). At the same time additional features, such as computational work (34), have further been incorporated into the research, enhancing the students’ research experience. An array of substituted 1,3-thiazolidin-4-ones were tested (R. Farrell, Penn State York; E. Dudkin, Penn State Brandywine) for biological activity against HT 1080 mice tumor cells. The data is, as yet, unpublished but enough positive activity resulted that a PSU Invention Disclosure was filed (35).

Figure 3. Series of 1,3-thiazolidin-4-ones that have been synthesized by undergraduates. This project extended to the Penn State Abington campus in 2009 when one of the coauthors (KC) began to incorporate research into CHEM 213. Students enrolled in organic chemistry lab at Abington synthesized N-cyclohexyl 1,3-thiazolidin-4-ones (36) and 2-(o-substituted phenyl)-1,3-thiazolidin-4-ones (37) (Series 8, 9, and 10, Figure 3) as part of their practical examination for the course. Presently, six of the compounds from Series 9 are being tested at the group of Eric Ingersoll at Abington for anticancer activity against a HeLa cell line. The syntheses of novel 1,3-thiazolidin-4-ones are no longer being pursued, but the syntheses of 1,3-thiazolidin-4-ones from Series 1, 2, 8, 9, and 10 in Figure 3 are still part of the course-work since these compounds are being used in other undergraduate-based research (vide infra). On two occasions, the reaction sequence shown in Figure 1 did not go according to plan, leading to very interesting new areas of investigation. In the first instance, when, in the presence of trichloroacetaldehyde, the amine used was switched from an arylamine (Series 7, Figure 3) to cyclohexylamine, the resulting product produced was an N-formyl compound instead of the corresponding 1,3-thiazolidin-4-one (Figure 4) (38). However, use of an appropriate Lewis acid allowed for the formation of the 1,3-thiazolidin-4-one. The second reaction that gave an interesting, but yet to be characterized polymeric product, was the reaction of p-hydroxybenzaldehyde with aniline. On addition of the thioglycolic 69

acid a thick glassy orange mass resulted. Initial work indicates the possible coupling of imine units with the loss of the hydroxyl group as water (Figure 5) (39).

Figure 4. Reaction of chloral, cyclohexyl amine, and thioglycolic acid without a Lewis acid.

Figure 5. Possible polymerization product from the C-p-hydroxy imine. The synthesis of novel 1,3-thiazolidin-4-ones in organic chemistry lab provided a library of compounds which serve as a basis for an ongoing undergraduate research projects involving Oxone® oxidation to produce the corresponding sulfoxide or sulfone compounds (Figure 6) (37, 40, 41). This library enables a thorough investigation of substituent effects on the oxidation of these compounds.

Figure 6. Oxidation of 1,3-thiazolidin-4-ones. The 2-(m- and p-substituted phenyl)-3-cyclohexyl-1,3-thiazolidin-4-one series produced in organic lab has also been reacted with Ph3SnCl to produce 1:1 1,3-thiazolidin-4-one tin complexes (42); similar organotin complexes have demonstrated antifungal activity against Ceratocytis ulmi, the cause of Dutch Elm disease (43, 44). The 1,3-thiazolidin-4-one ligand is coordinated to the Sn metal center via the oxygen atom of the C4 carbonyl (Figure 7). Currently, studies 70

utilizing IR and NMR data are being conducted by undergraduate researchers to track the chemical shift variation of the methine proton (at C2), methylene protons (at C5) and the 13C NMR chemical shift data at C2, C4 and C5 in the 1,3-thiazolidin-4-one ring.

Figure 7. Preparation of organotin complexes of 1,3-thiazolidin-4-ones.

Upon joining PSU in 2009, one of the authors (LJS) opened two lines of synthetic research. The first was a progression from some research done while working as a process chemist in the pharmaceutical industry. In that work, the very reactive epoxides of (+)-2-carene had been studied (45). The goals of the new research were to study the aziridines and halonium ions of (+)-2-carene and of α- and β-pinene (Figure 8).

Figure 8. Cyclopropyl and cyclobutyl aziridines and halonium ions. X = NTs, Br+, Cl+. 71

CHEM 213 students in Spring 2011 did experiments involving the halonium ions. Some very interesting results were obtained with the halonium ions, which were eventually included in a paper published in 2015 (46). The second track involved the synthesis and reactivity of 2,3-diaryl-1,3thiaza-4-one heterocycles in collaboration with a coauthor (JT). The initial work (Spring 2010) was on preparation of a series of 2,3-diaryl-1,3-thiazolidin-4-ones (Figure 3, Series 3). This resulted in a publication (33). Then studies of S-oxidation of 2,3-diaryl-1,3-thiazolidin-4-ones (Figure 3, Series 3) and 3-benzyl-2-phenyl-1,3-thiazolidinones (Figure 3, Series 5) were done (Figure 9) (Spring 2012). In this project, the lack of easy access to an NMR at that time caused some surprises. Using Oxone® and judging by TLC, it was believed that by controlling the temperature (0 °C or room temp.) and the amount of Oxone® (1.5 or 3.0 equivalents) either the sulfoxide or sulfone could be produced. However, when the NMRs were eventually obtained, it turned out that all of the reactions had been highly selective towards the sulfoxide. The full series has not been published, but a paper detailing the crystal structures of one sulfide and its corresponding sulfoxide has been published (47). As discussed earlier, the work has also been extended at Penn State Abington, where it has been shown that production of the sulfone requires heating and a large excess of Oxone® (37, 40, 41).

Figure 9. S-Oxidation of 1,3-thiazolidin-4-ones.

A downturn in the number of students led to only one student doing research in Spring 2013 and the course not being offered in Spring 2014. The student in 2013 was involved in initial attempts to prepare 2,3-diaryl-2,3-dihydro-4H-1,3benzothiazin-4-ones from an imine and a thioacid. This was not successful, and it was known in the literature that the six-membered rings were more challenging to make by this method than the five-membered rings and that the N-aryl rings were more difficult to prepare than the N-alkyl rings (48, 49). During further studies in the summer of 2013, it was found that 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P) (50–53) worked well (Figure 10). This procedure has proven to be very general for six- and seven-membered 2,3-diaryl-1,3-thiaza-4-ones (54, 55). Students in Spring 2015 and Spring 2016 prepared 2,3-diaryl-2,3-dihydro-4H-1,3-benzothiazin-1,3-ones (Figure 10). One student also began attempts at reducing the carbonyl in these types of compounds. 72

Figure 10. Synthesis of 2,3-diaryl-2,3-dihydro-4H-1,3-benzothiazin-4-ones.

The Spring 2017 class worked on several aspects. Some worked on synthesis of 2,3-diaryl-2,3-dihydro-4H-1,3-benzothiazin-1,3-ones, while others worked on the synthesis of 2,3-diaryl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-ones (Figure 11) and on the S-oxidations of previously prepared 2,3-diaryl-1,3-thiaza-4-ones, using Oxone® to prepare sulfoxides, and potassium permanganate to synthesize the sulfones.

Figure 11. Synthesis of 2,3-diaryl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-ones.

Most of the specific work done in CHEM 213 from 2013-2017 at Schuylkill has not yet been published, but it will be. It has been included in a variety of presentations (56–60) and posters (61–64). Other pieces of these projects have been completed by students not enrolled in CHEM 213. This research has been well received by the undergraduates. The chemistry is readily done using standard organic techniques. Since the interest is in the compounds themselves, yields are not critical. The compounds are novel and the data produced in these studies has continually proven to be of interest to 73

the scientific community, as seen by the publication record. Initial screening (H. Sobhi, Coppin State U.) of six 2,3-diphenyl-1,3-thiaza-4-ones (Figure 12) (54) has shown some to have significant activity against fungi Scedosporium (Lomenstospora) prolificans and Cryptococcus neoformans (58, 60). Knowing that their compound(s) may be of medicinal use is exciting to the students.

Figure 12. 2,3-Diphenyl-1,3-thiaza-4-ones (54) tested for antifungal activity (58, 60).

Conclusion Our collective experience with CUREs in sophomore organic chemistry laboratory has been very successful both in learning outcomes for the students and in publications, which benefit both the students and the faculty. This approach assures that every student who comes through the Organic Chemistry sequence will get a genuine research experience. Most students are grateful for this opportunity. It also happens relatively early in their college education, and may lead them to seek out other research opportunities.

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Basu, A.; Lee, J.; Chapdelaine, A. The advantages and disadvantages of embedding undergraduate research into the curriculum. CUR Q. 2011, 32, 3. Wenzel, T. J.; Larive, C. K.; Frederick, K. A. Role of undergraduate research in an excellent and rigorous undergraduate chemistry curriculum. J. Chem. Educ. 2012, 89, 7–9. Brownell, J. E.; Swaner, L. E. Five High-Impact Practices: Research on Learning Outcomes, Completion, and Quality; Association of American Colleges and Universities: Washington, DC, 2010. Kuh, G. D. High-Impact Educational Practices: What They Are, Who Has Access to Them, and Why They Matter; Association of American Colleges and Universities, Washington, DC, 2008. Laursen, S.; Seymour, E.; Hunter, A. Learning teaching and scholarship: fundamental tensions of undergraduate research. Change: The Magazine of Higher Learning 2012, 44, 30–37. Laursen, S.; Hunter, A.; Seymour, E.; Thiry, H.; Melton, G. Undergraduate Research in the Sciences: Engaging Students in Real Science; Jossey-Bass: San Francisco, CA, 2010. Randall, D. C.; Wilbur, F. H.; Burkholder, T. Two models for an effective undergraduate research experience in physiology and other natural sciences. J. Adv. Physiol. Educ. 2004, 28, 68–72. Sadler, T. D.; Burgin, S.; McKinney, L.; Ponjuan, L. Learning science through research apprenticeships: a critical review of the literature. J. Res. Sci. Teach. 2010, 47, 235–256. Powell, N. L.; Harmon, B. B. Course-Embedded Undergraduate Research Experiences: The Power of Strategic Course Design. In The Power and Promise of Early Research; Murray, D. H., Obare, S. O., Hageman, J. H., Eds.; ACS Symposium Series 1231; American Chemical Society: Washington, DC, 2016; pp 119−136. Bakshi, A.; Patrick, L. E.; Wischusen, E. W. A framework for implementing course-based undergraduate research experiences (CUREs) in freshman biology labs. Am. Biol. Teach. 2016, 78, 448–455. Clark, T. M.; Ricciardo, R.; Weaver, T. Transitioning from expository laboratory experiments to course-based undergraduate research in general chemistry. J. Chem. Educ. 2016, 93, 56–63. Corwin, L. A.; Graham, M. J.; Dolan, E. J. Modeling course-based undergraduate research experience: an agenda for future research and evaluation. CBE Life Sci. Educ. 2015, 14, 1–13es1. Olimpo, J. T.; Fisher, G. R.; Dechenne-Peters, S. E. Development and Evaluation of the Tigriopus Course-based undergraduate research experience: impacts on students’ content knowledge, attitudes, and motivation in a majors introductory biology course. CBE Life Sci. Educ. 2016, 15, 1–15ar72. Rodenbusch, S. E.; Hernandez, P. R.; Simmons, S. L.; Dolan, E. L. Early engagement in course-based research increases graduation rates and completion of science, engineering, and mathematics degrees. CBE Life Sci. Educ. 2016, 15, 1–10ar20. 75

19. Wang, J. T. H. Course-based undergraduate research experiences in molecular biosciences – patterns, trends, and faculty support. FEMS Microbiol. Lett. 2017, 364, 1–9. 20. Shortlidge, E. E.; Bangera, G.; Brownell, S. E. Each to their own CURE: faculty who teach course-based undergraduate research experiences reports why you should teach a CURE. J. Microbiol. Biol. Educ. 2017, 18, 399–408. 21. Large, D. N.; Albasrawi, H. K.; Timmons, S. C. Incorporation of course-based undergraduate research experiences in the organic chemistry and biochemistry laboratory. American Chemical Society Central Regional Meeting, Dearborn, MI, 2017; Abstract 200. 22. Antos, J.; Reed, S. A. Design, synthesis and evaluation of small molecule sortase inhibitors: A CURE for the undergraduate organic chemistry lab. American Chemical Society National Meeting, San Francisco, CA, 2017; Abstract CHED-2034. 23. Brown, L. C. Course-based undergraduate research experience (CURE) in organic chemistry. American Chemical Society National Meeting, San Francisco, CA, 2017; Abstract CHED-1937. 24. Weaver, G. Course-based undergraduate research experiences as a “CURE” for what ails science education for the 21st century. American Chemical Society National Meeting, San Diego, CA, 2016; Abstract CHED-209. 25. Tierney, J. The formation of 2,3-disubstituted thiazolidin-4-ones from S-α′-aminomercaptoacetic acid derivatives. J. Heterocycl. Chem. 1989, 26, 997–1001. 26. Surrey, A. R. The preparation of 4-thiazolidones by the reaction of thioglycolic acid with Schiff bases. J. Am. Chem. Soc. 1947, 69, 2911–2912. 27. Tierney, J.; Houghton, G.; Sanford, K.; Mascavage, L.; McCoy, M.; Findeisen, A.; Kilburn, J. Effects and conformational analysis of some substituted 2,3-diphenyl-1,3-thiazolidin-4-ones. Magn. Reson. Chem. 1996, 34, 573–576. 28. Issac, R.; Tierney, J.; Mascavage, L. M.; Findeisen, A. Synthesis, spectroscopic and structural properties of novel substituted 2-trichloromethyl-3-phenyl-1,3-thiazolidin-4-ones. Heterocycl. Commun. 1996, 2, 227–232. 29. Tierney, J.; Sheridan, D.; Kovalseky, C.; Mascavage, L. M.; Sheely, C.; Schneider, K. Substituent effects for some substituted 3-benzyl-2-phenyl1,3-thiazolidin-4-ones using 1H and 13C NMR. Heterocycl. Commun. 2000, 6, 105–111. 30. Tierney, J.; Mascavage, L. M.; Sheridan, D.; Gorbecheva, D.; Ripp, M.; Son, S. A preliminary study on predicting the 13C chemical shifts for a series of disubstituted 2,3-diphenyl-1,3-thiazolidin-4-ones. Heterocycl. Commun. 2005, 11, 215–222. 31. Tierney, J.; Colasante, N.; Eagles, J.; Kelly, S.; Lehmicke, G.; Lucas, O.; Mannikarottu, C.; Mehmood, O.; Nguyen, M. D.; Rai, V.; Tsai, J.; Mascavage, L. M.; Lagalante, A. F. A comparison of substituent effects using 13C chemical shifts for a series of substituted 2-phenyl-3-pyridyl-1,3thiazolidin-4-ones compared to similarly substituted 1,3-thiazolidin-4-ones. Heterocycl. Commun. 2007, 13, 335–342. 76

32. Tierney, J.; Koyfmann, V.; Cannon, K.; Mascavage, L. M.; Lagalante, A. F. A second study - predicting the 13C chemical shifts for a series of substituted-3(4-methoxyphenyl)-2-phenyl-1,3-thiazolidin-4-ones. Heterocycl Commun. 2008, 14, 453–460. 33. Silverberg, L. J.; Bear, E. R.; Foose, K. N.; Kirkland, K. A.; McElvaney, R. R.; Cannon, K.; Tierney, J.; Lascio, S.; Mesfin, K.; Mitchell, D.; Sharkey, S.; So, L.; Treichel, J.; Waxman, M.; Lagalante, A. Verifying the predictability of 13C chemical shifts for a series of substituted-2-(4-chlorophenyl)-3-phenyl1,3-thiazolidin-4-ones. Int. J. Chem. (Toronto, ON, Can.) 2013, 5, 121–127. 34. McGarity, D.; Tierney, J.; Lagalante, A. An anomalous Hammett correlation for a series of substituted 3-benzyl-2-phenyl-1,3-thiazolidin-4-ones. Int. J. Chem. (Toronto, ON, Can.) 2014, 6, 12–18. 35. Tierney, J.; Farrell, R.; Dudkin, E. PSU Invention Disclosure No. 2007-3295. 36. Cannon, K.; Mascavage, L. M.; Kistler, K.; Tierney, J.; Yennawar, H.; Lagalante, A. F. An experimental and theoretical conformational study of a series of substituted 3-cyclohexyl-2-phenyl-1,3-thiazolidin-4-ones. Int. J. Chem. (Toronto, ON, Can.) 2013, 5, 46–56. 37. Cannon, K. C.; Gandla, D.; Lauro, S.; Silverberg, L. J.; Tierney, J.; Lagalante, A. Selective synthesis of ortho-substituted 3-cyclohexyl-2phenyl-1,3-thiazolidin-4-one sulfoxides and sulfones by S-oxidation with Oxone®. Int. J. Chem. (Toronto, ON, Can.) 2015, 7, 73–84. 38. Mascavage, L. M.; Tierney, J.; Sonnet, P. E.; Dalton, D. R. The HoffmannSchiff dichotomy: on the reaction between chloral and amines. Arkivoc 2010 (viii), 278–284. 39. Messick, M. A. Production and analysis of 2-(p-hydroxyphenyl)-3-phenyl1,3-thiazolidin-4-one and 3-(p-hydroxyphenyl)-2-phenyl-1,3- thiazolidin-4one. Schreyer Honors Thesis, Pennsylvania State University, 1999. 40. Cannon, K. C.; Costa, M.; Pepper, M.; Toovy, J.; Selinsky, R. S.; Lagalante, A. F. Selective synthesis of substituted 3-aryl-2-phenyl-1,3thiazolidin-4-one sulfoxides and sulfones by S-oxidation with Oxone®. Int. J. Chem. (Toronto, ON, Can.) 2017, 9, 1–11. 41. Cannon, K. C.; Alkurdi, A.; Himel, H.; Kurochka, I.; Liu, S.; Costa, M.; Sundberg, B.; Lagalante, A. F. Selective Synthesis of ortho-Substituted 2aryl-3-phenyl-1,3-thiazolidin-4-one Sulfoxides and Sulfones by S-Oxidation with Oxone®. Int. J. Chem. (Toronto, ON, Can.) 2017, 9, 87–97. 42. Cannon, K.; Costa, M.; Ongari, A.; Tierney, J. Synthesis and characterization of 3-cyclohexyl-2-aryl-1,3-thiazolidin-4-one triphenyltinchloride complexes. American Chemical Society Middle Atlantic Regional Meeting, Hershey, PA, 2017; Abstract 364. 43. Smith, F. E.; Hynes, R. C.; Tierney, J.; Zhang, Y. Z.; Eng, G. The Synthesis, Molecular and crystal structure of the 1:1 adduct of triphenyltin chloride with 2,3-diphenylthiazolidin-4-one. Can. J. Chem. 1995, 73, 95–99. 44. Eng, G.; Whalen, D.; Musingarimi, P.; Tierney, J.; DeRosa, M. Fungicidal and spectral studies of some triphenyltin compounds. Appl. Organomet. Chem. 1998, 12, 25–30. 45. Silverberg, L. J.; Resnick, T. M.; Casner, M. L. U.S. Patent 6,867,335, 2005. 77

46. Silverberg, L. J.; Kistler, K. A.; Brobst, K. A.; Yennawar, H.; Lagalante, A.; He, G.; Ali, K.; Blatt, A.; Foster, S. B.; Grossman, D. B.; Hegel, S.; Minehan, M. J.; Valinsky, D. M.; Yeasted, J. G. Reactions of the halonium ions of carenes and pinenes: an experimental and theoretical study. Eur. J. Chem. 2015, 6, 430–443. 47. Yennawar, H. P.; Hullihen, P. D.; Tierney, J.; Silverberg, L. J. Crystal structures of 2,3-bis(p-chlorophenyl)-1,3-thiazolidin-4-one and trans-2,3bis(p-chlorophenyl)-1,3-thiazolidin-4-one-1-oxide. Acta Crystallogr. 2015, E71, 264–267. 48. Surrey, A. R.; Webb, W. G.; Gesler, R. M. Central nervous system depressants. The preparation of some 2-aryl-4-metathiazanones. J. Am. Chem. Soc. 1958, 80, 3469–3471. 49. Loev, B. 2,3-Dihydro-4H-1,3-benzothiazin-4-ones. J. Org. Chem. 1963, 28, 2160. 50. Wissmann, H.; Kleiner, H. J. New peptide synthesis. Angew. Chem. Int. Ed. 1980, 19, 133–134. 51. Dunetz, J. R.; Xiang, Y.; Baldwin, A.; Ringling, J. General and scalable amide bond formation with epimerization-prone substrates using T3P and pyridine. Org. Lett. 2011, 13, 5048–5051. 52. Unsworth, W. P.; Kitsiou, C.; Taylor, R. J. K. Direct imine acylation: rapid access to diverse heterocyclic scaffolds. Org. Lett. 2013, 15, 258–261. 53. Kitsiou, C.; Unsworth, W. P.; Coulthard, G.; Taylor, R. J. K. Substrate scope in the direct imine acylation of ortho-substituted benzoic acid derivatives: the total synthesis of (±)-cavidine. Tetrahedron 2014, 70, 7172–7180. 54. Silverberg, L. J.; Pacheco, C. N.; Lagalante, A.; Cannon, K. C.; Bachert, J. T.; Xie, Y.; Baker, L.; Bayliff, J. A. Synthesis and spectroscopic properties of 2,3-diphenyl-1,3-thiaza-4-one heterocycles. Int. J. Chem. (Toronto, ON, Can.) 2015, 7, 150–162. 55. Silverberg, L. J.; Tierney, J.; Pacheco, C.; Lagalante, A.; Bachert, J. T.; Bayliff, J. A.; Bendinsky, R. V.; Cali, A. S.; Chen, L.; Cooper, A. D.; Minehan, M. J.; Mroz, C. R.; Noble, D. J.; Weisbeck, A. K.; Xie, Y.; Yang, Z. Synthesis and spectroscopic properties of a series of novel 2-aryl-3-phenyl-2,3-dihydro-4H-1,3-benzothiazin-4-ones. Arkivoc 2016 (vi), 122–143. 56. Silverberg, L. J.; Yennawar, H. P.; Tierney, J.; Pacheco, C. N.; Cannon, K. C.; Lagalante, A.; Bachert, J. T.; Baker, L.; Bayliff, J. A.; Bendinsky, R. V.; Cali, A. S.; Chen, L.; Cooper, A. D.; Coyle, D. J.; Dahl, J. R.; Minehan, M. J.; Mroz, C. R.; Singh, H.; Xie, Y. Studies on cyclic six- and seven-membered 2,3-diaryl-1,3-thiaza-4-ones. National Meeting of the American Chemical Society, Denver, CO, 2015; Abstract ORGN 456. 57. Silverberg, L. J. Studies on 1,3-thiaza-4-one heterocycles. 3rd Science Symposium: Innovation of Science, Nanotechnology, Human Health and Environment for a Global Society. Coppin State University, Baltimore, MD, 2015. 58. Silverberg, L. J.; Yennawar, H.; Pacheco, C.; Lagalante, A.; Sobhi, H.; Alemany, K.; Bachert, J.; Baker, L.; Bandholz, K.; Bayliff, A.; Bendinsky, R.; Bradley, H.; Buchwalter, M.; Cali, A.; Cardenas, O.; Chen, L.; 78

59.

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Colburn, B.; Cooper, A.; Coyle, D.; Dahl, J.; Felty, M.; Fox, R.; Islam, J.; Kimmel, E.; Koperna, S.; Lawler, M.; Moyer, Q.; Mroz, C.; Noble, D.; Perhonitch, K.; Reppert, H.; Singh, H.; Verhagen, C.; Vidal, R.; Weisbeck, A.; Xie, Y.; Yang, Z. More studies on cyclic six- and seven-membered 2,3-diaryl-1,3-thiaza-4-ones. American Chemical Society Middle Atlantic Regional Meeting, Hershey, PA, 2017; Abstract 290. Silverberg, L. J. Providing early research experiences for undergraduates. National Meeting of the American Chemical Society, San Francisco, CA, 2017; Abstract CHED 1996. Silverberg, L. J.; Yennawar, H. P.; Pacheco, C. N.; Lagalante, A.; Sobhi, H.; Alemany, K. B.; Bachert, J. T.; Baker, L. M.; Bandholz, K. E.; Bayliff, J. A.; Bendinsky, R. V.; Bradley, H. G.; Buchwalter, M. J.; Cali, A.; Cardenas, O. A.; Chen, L.; Colburn, B. K.; Cooper, A. D.; Coyle, D. J.; Dahl, J. R.; Felty, M.; Fox, R.; Islam, J. M.; Kimmel, E. G.; Koperna, S. E.; Lawler, M. K.; Moyer, Q. J.; Mroz, C. R.; Noble, D. J.; Perhonitch, K. C.; Reppert, H. E.; Singh, H.; Verhagen, C. R.; Vidal, R. M.; Weisbeck, A. K.; Xie, Y.; Yang, Z. More studies on cyclic six- and seven-membered 2,3-diaryl-1,3-thiaza-4ones. National Meeting of the American Chemical Society, San Francisco, CA, 2017; Abstract ORGN 399. Silverberg, L. J.; Yennawar, H. P.; Cannon, K.; Pacheco, C.; Singh, H. Studies on cyclic six- and seven-membered 2,3-diaryl-1,3-thiaza-4-ones. Gordon Research Conference on Heterocyclic Compounds, Newport, RI, 2014. Silverberg, L. J.; Yennawar, H. P.; Tierney, J.; Pacheco, C.; Bendinsky, R. V.; Cali, A. S.; Coyle, D. J.; Minehan, M. J.; Singh, H.; Cooper, A. D.; Chen, L. Studies on 2,3-diaryl-2,3-dihydro-4H-1,3-thiazin-4-ones with a fused arene. Gordon Research Conference on Heterocyclic Compounds, Newport, RI, 2014. Yennawar, H.; Silverberg, L.; Tierney, J.; Cannon, K.; Bendinsky, R.; Cali, A.; Coyle, D.; Minehan, M. crystal structures of 1,3-thiaza-4-one heterocycles. XIII International Union of Crystallographers Congress, Montreal, Canada, 2014. Abstract. Acta Crystallogr. 2014, A70 (Supplement), C1777. Noble, D. J.; Yang, Z.; Silverberg, L. J. Reduction and oxidation of 2,3-diaryl-1,3-thiaza-4-ones. American Chemical Society Middle Atlantic Regional Meeting, Hershey, PA, 2017; Abstract 109.

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Upper Divison Opportunities

Chapter 6

Developing an Integrated Research-Teaching Model Robert E. Bachman* Department of Chemistry, The University of the South, 735 University Avenue, Sewanee, Tennessee 37383, United States *E-mail: [email protected]

The goal of this education project is to convert a traditional skill-building laboratory sequence within an inorganic chemistry course into an authentic research experience. This approach has developed a natural teaching-research nexus that provides students with valuable intellectual growth and faculty with recognition for both their teaching and scholarship. Given that research experiences involve an integrated intellectual exploration, students in the course learn an array of valuable skills, such as literature research, literature reading, teamwork, and scientific communication, and both synthetic and analytical techniques. The faculty member teaching the course has gained new leads for their overall research program and initial results that have even led to public presentation.

Introduction Over the last four decades, faculty across higher education have come to recognize that Undergraduate Research (UR) experiences are an advantageous educational touchstone for undergraduate students. One of the earliest recognitions of this learning paradigm was the launching of the Council on Undergraduate Research (CUR) in 1978 by a small community of chemistry leaders. CUR developed one of the earliest definitions of undergraduate research: “An inquiry or investigation conducted by an undergraduate student that makes an original intellectual or creative contribution to the discipline.” Interestingly, the American Chemical Society Committee on Professional Training describes a similar definition but also notes the importance of faculty mentorship/teaching © 2018 American Chemical Society

providing intellectual growth of students that are engaged in research (1). The educational value of undergraduate research has been examined extensively, especially by Lopatto (2, 3). As part of the overall expansion of UR activity, many faculty at Primarily Undergraduate Institutions (PUIs) moved from focusing their work almost entirely on teaching toward a more even balance of teaching and research efforts. This transition was worthwhile because it produced a significant amount of valuable scientific knowledge and provided many young potential scientists the opportunity to gain research experience earlier in their educational career. Increased national recognition of UR as a “high impact practice” (4) has led faculty and administrators at large research-intensive doctoral institutions to develop UR opportunities for students, in many cases these schools have launched a campus-wide UR office and program. While the overall recognition of UR has provided many students an important opportunity, the growth of this practice has reached a capacity problem in many settings. At the larger schools, it is frequently obvious that all students in a chemistry program cannot find a home in a faculty member’s research group. Even at smaller schools, this capacity problem can occur as the number of majors grows. At one point, I attempted to manage eight students with effectively different small projects in a given semester. Not surprisingly, this approach was challenging for the students and myself. Scheduling the individual learning time needed for each student was the most significant challenge. The students often had to attempt a procedure or technique for the first time with only a short introductory explanation. Individually teaching and training so many students created a natural stress in terms of balancing teaching and scholarship goals, especially since the mentorship/supervision of research students is not part of the teaching load at Sewanee. At a few institutions, the acknowledgement of work balance related to UR mentorship has provided some level of load compensation; however, faculty at many institutions routinely support UR without official compensation because their research program benefits from having research assistants and because they find internal contentment as a mentor to future scientists. Given the obvious capacity problems and the related work stress, I began to explore how the traditional segmented landscape of courses and research could be bridged in the inorganic chemistry course (Figure 1), which contains both three lecture blocks and a weekly afternoon laboratory. I hoped that formally joining these two realms would decrease the perceived divide between class-related knowledge and “real” science felt by many students and faculty, increase student UR capacity, and decrease faculty workload stress. As an added benefit, this integrated approach would provide a way to naturally link the “High Impact Practice” (HIP) (4–6) of UR to other HIPs such as collaborative projects, capstone experiences, and writing-intensive (WI) communication. The inorganic course was already designated as a required major-level writing-intensive course so it was expected that integrated a research experience into the writing process would provide a variety of communication opportunities. For example, students would be able to present initial research as a poster at our campus research symposium and develop a journal style manuscript reporting their progress on a research question. 84

Figure 1. A visual approach of how research and teaching can be integrated.

In the research milieu, a variety of oral communication learning experiences are available, such as in-lab idea sharing, collaborative group-meeting research conversations, and discussing their research progress via a poster in a public venue.

Course Development The inorganic chemistry laboratory began originally with traditional “cookbook” laboratories based on well-tested and repetitive experiments. This time-honored approach began with classic experiments in areas such as coordination complex syntheses (synthesis of cis/trans isomers of [Co(en)2Cl2], reactivity studies (nitro to nitrito isomerization), and solid-state synthesis (1-2-3 Superconductor) (7–10). An early step toward the research approach began with labeling the course as a major-related WI course; students at Sewanee are required to complete at least one WI course in the major. This first step led to asking students to write all “lab reports” as parts of a typical ACS journal article and utilizing review and revision techniques. The details of this approach are described below. Additionally, I decided to replace one or two traditional experiments with an open-ended inquiry experiment over multiple weeks. This approach refocused the student work away from cookbook thinking. As you can see in Table 1, this small move toward the complete research-focused course began by inserting a two-week research experience in weeks 12 and 13 in the 2010 course. That initial project was connected to my ongoing primary research focus at that point, with the students making simple derivatives of platinum-centered liquid crystalline compounds (11). When this two week period of inquiry was introduced, it was immediately visible that the students found tremendous excitement about the idea of possibly making a novel material that had never existed before. Even though every one of the students’ trials technically failed, they all indicated that the work gave them a significant insight into the world of research—delving into the literature to find a new idea or approach, the value of trial and error, and the willingness to push through a challenge. In their own way, these research failures were a success because an initial idea was explored within the safety-net of the teaching realm, a place where success is often measured by knowing that students understand and value a learning experience.

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Table 1. Weekly Schedules for the Inorganic Laboratory and Writing Example from 2010

2015 to 2017

1

The Three R’s: Reading wRiting and Research

Discussion of Physical Techniques

2

Acylation of Ferrocene

The Three R’s: Reading wRiting and Research

Develop an Abstract for a Literature Paper

3

Separation of Acylated Ferrocene Products

Computational Chemistry and Choice of Project Framework

Results and Discussion of Computational Experment

4

Synthesis of Superconductive Oxide

Synthesis and Characterization of Metal Complexes

Initial Experimental Section (ACAC)

5

Characterization and Analysis of Superconductive Oxide

Synthesis and Characterization of Metal Complexes

Introduction (ACAC)

6

Computational Chemistry

Synthesis and Characterization of Metal Complexes and Research Proposal

Proposal for Research

7

Synthesis of Geometric Isomers

Data Analysis and Writing Workshop

Full Experimental Section as well as Results and Discussion (ACAC)

8

Reactivity and Characterization of Geometric Isomers

Research Project

9

Synthesis of Linkage Isomers

Research Project

10

Reactivity and Characterization of Linkage Isomers

Research Project

11

Synthesis of ACAC Complexes

Research Project

12

Synthesis of a Metallomesogen

Research Project

86

Week

2015-2017 Writing

Initial Experimental Section

Initial Results and Discussion

Week

Example from 2010

2015 to 2017

2015-2017 Writing

13

Characterization of a Metallomesogen

Research Project and Poster Preparation

Introduction and Conclusions

14

Presentations

Research Project and Poster Preparation

Poster Development

15

Presentations

Poster Presentation at Scholarship Sewanee

Final Manuscript

87

To build on this partial success, I endeavored to convert the laboratory portion of the course into a full semester “internship” organized much like the developmental steps many mentors use with students when they join their research group, both during the summer and in the academic year. The initial portion of the 14-week laboratory period (Table 1) was converted into a series of group meetings to expand, explore and discuss several useful skills: (a) the wide array of characterization methodologies available and how they are used, (b) how to search and read the scientific literature, (c) how to write scientific communication, and (d) how computational chemistry can aid and expand experimental science. The next steps for the students are to develop their synthetic and characterization skills, and to imagine and design a small part of a larger research effort. After the initial orientation week of the analytical tools that will be available, an initial orientation to literature searching and the planned research frameworks are initially presented in the second lab meeting (“Reading, wRiting and Research”). Two example frameworks derived from my laboratory, and a third framework that the co-instructor of the course in 2017 are presented in Scheme 1. One reason multiple frameworks are developed is so that the students can discover a project in an area of potential intrinsic interest; the other is that a variety of any instructors’ novel research ideas can be explored and tested.

Scheme 1. Three Potential Project Frameworks Derived from the Instructors’ Laboratories: (a) Selective bromination of a [M(acac)3] complex and Suzuki coupling to a naphthalenedimide dye. (b) Potential synthesis routes for an array of potential drug derivative compounds related to KP1019—[Ru(ind)2Cl4]–. (c) Synthesis of new catalysts for amide preparation. 88

Generalized representations such as those shown in Scheme 1 are used in the initially framework presentations so that the students see the overarching ideas and goals and engage in an open discussion about both the possible underlying questions and potential “bite-sized” projects that can be explored for the research portion of the course. Short project synopses of these discussions and initial literature leads are also posted to the course website, allowing the students time choose a topic of interest. Here are a few examples of how the frameworks in Scheme 1 connect lead toward the instructors’ research goals. The framework in Scheme 1a is to develop a new class of inorganic-organic conjugate dyes capable of harvesting solar energy. The first goals of this work have been (a) to develop a more useful monobromination of metal acetylacetonate complexes (12) and (b) to conjugate the resulting complex to a naphthalenediimide photodye via a Suzuki coupling (13). The hope is that this project appeals to students interested in materials and environmental questions. While the bromination approach has proved difficult, a series of Suzuki conditions using other useful model compounds (for example, 5-bromophenanthroline) have led to preliminary results being moved into my research laboratory. The framework in Scheme 1b focuses on the development of derivatives associated with a ruthenium-centered anti-cancer drug, usually referred to as KP1019, that reached early clinical trials but did not move beyond that point (14). At this point the project is focused on synthesis; however, it could be easily expanded in terms of reactivity studies or even biological assays if two faculty begin to collaborate in the research realm and/or the project might be integrated into a potentially related course; for example, biochemistry or molecular biology. This project helps students see the relationships between chemical synthesis, pharmacology, and medical care. The framework in Scheme 1c is aimed at developing new catalytic processes capable of preparing amides in gentle and environmentally useful ways (15). In the first year of utilizing this framework students have improved the proposed synthetic methodologies aimed to create potential catalysts, and even attempted to test catalytic behavior for the new compounds. Once this initial couple of orientation weeks are carried out, the students begin their experiential learning with an in-house written expanded version of the traditional syntheses of metal acetylacetonate complexes. To make this experiment more inquiry-driven, the instructor(s) specifically do not fully state what the complexes’ structures are, and pose questions such as that shown in Scheme 2, “How does the acetylacetonate ligand bond to the metal?” Each pair of students begins by choosing two metals from the listed options (typically, Cr, Mn, Fe, Co, and Al) and then carries out the appropriate syntheses provided (16–18). Each complex has a slightly different historical methodology, and as a result the students gain extra hands-on experience by following multiple protocols simultaneously. Each complex is usually prepared by at least two student teams so that the students can compare the outcomes. Once the samples are made, each team begins by checking purity (i.e., by melting point determination) and developing a purification method (i.e. recrystallization), if needed. Next comes a full battery of analytical characterization—IR, UV-Vis, NMR (diamagnetic and paramagnetic Evans methodology), and solid-state magnetometry, using a Gouy 89

balance. The development of a computational model using Spartan is suggested as an optional exercise to examine the bonding question raised earlier (Scheme 2). This allows the students to return to the computational skills they began to gain in the initial computational session a few weeks earlier.

Scheme 2. Hypothetical Metal-Ligand Bonding Options Once the initial analytical work and group comparisons are complete, the students and the instructor(s) work together to consolidate data for all the metal acetylacetonate complexes in an analysis and writing workshop. This discussion provides an opportunity to practice data sharing and replicate checking. The consolidated spectroscopic information also allows the students to understand structure-property relationships and spectral analysis trends more completely. The group discussion provides teaching moments about how to tabulate data in a style often used in the synthetic chemistry literature for experimental reporting and how to develop a narrative of the results and their meaning, both spoken and written. The gathered data is also brought into the lecture portion of the course to explore the intellectual understanding of how transition metal complexes, and matter of all kinds, interacts with electromagnetic radiation in ways that provide information about structure and properties.

Writing Aspects of the Course The course has been designated as a writing-intensive (WI) requirement within the chemistry major, and as such the students are offered the opportunity to resubmit edited versions of their writing after an initial review by the instructor. In the initial years of developing this approach, the use of peer review was attempted, but the students repeatedly indicated that peer review was overly challenging and time-consuming. Upon moving back to instructor review and requested revision, 90

the use of both a writing contract and a writing rubric have been implemented. In all approaches, the main goal was to guide the students to the realization that rewriting and revision are important skills in scientific discourse. Throughout the first seven weeks of the course, the students begin their development of scientific writing skills by creating individual sections of a “full paper” manuscript in a somewhat unusual order (see Table 1)—abstract, experimental, results and discussion, and introduction. This order begins the writing process by formulating a summary that explains what was done, what was discovered, and what is “big picture” of a research effort. Students repeatedly indicate that the “big picture” writing needed for a well-framed introduction is the most difficult part of scientific writing, which is why that is left to the end. It is also hoped that this order helps the students gain more insight into the research framework they have chosen, and the small research goals they have developed. The first writing assignment (see Table 1) begins with reading and discussing a short literature paper, typically a communication, with its abstract excised so that each student writes their own abstract for the paper without referring the one found on the publication website or SciFinder. The goal of this initial exercise is having students note key features of a shorter literature work and develop a clear summary of the outcomes. Once they have completed the initial assignment, the next step is to formulate their understanding of how experimental findings can be communicated. First, they draft a “results and discussion” section for the initial computational experiment they have explored, which is focused on questions related to the formation of molecular orbitals and molecular structure. This writing exercise allows the students to evaluate their computed results by connecting the ideas to theoretical topics under discussion in the lecture portion of the course and by comparing their own results to literature sources. Once the students begin the metal acetylacetonate complex synthesis, the next writing goal is drafting an experimental section in the traditional third-person and past passive voice used in many journals. As the analytical and spectroscopy data on the complexes is collected, the students explicitly revise the experimental section and add the accumulated spectral information in the format used in many synthetic manuscripts (e.g. “1H NMR (CDCl3, ppm): 4.55 (1H), 2.15 (6H)….”). In the last two weeks of the initial pre-research period, the students are asked to draft two assignments. The first is an introduction to present the metal acetylacetonate complex synthesis. The second assignment is developing a research proposal that maps out the procedures they will attempt in the research weeks and the goals of this work as they seem them. Both of these exercises help the student to consider questions such as “What is the work focused on?” and “Why does the reader care?” As the research project part of the course begins, the students are asked to assemble the sections developed for their metal acetylacetonate complexes as a complete manuscript, and to complete as many revision suggestions as possible. They know that they will need to develop a public poster presentation and write a full manuscript about their research work at the end of the course. As such, they value the relatively easy opportunity to assemble this manuscript while primarily focusing on their research. 91

The last week of the course caps the research process by preparing a poster for our campus symposium, “Scholarship Sewanee.” Being ready to present the poster and discuss their work strongly motivates the students to compile all of their research results, accepting the good, bad, and ugly data as part of the process. To reexamine and analyze their research data, they convert the poster information into a manuscript. While the poster is authored by a pair, or in a few cases two pairs working on a related question, the final manuscripts are solitary writing tasks. Interestingly, the instructor(s) have noticed that each student of a team usually chooses to focus their manuscript on different points of discussion related to the overall project outcomes. For example, one team member focuses on how the spectral data defines the product made and the other focuses on unexpected results, positive or negative.

Outcomes Two survey-related tools were utilized to examine the potential impacts of this integrated teaching-research model. The more straightforward instrument was the on-campus teaching evaluation, which offers open questions related to topics like “was the course intellectually stimulating” and allows optional questions such as “was the laboratory experience valuable.” The Classroom Undergraduate Research Experience (CURE) survey developed by Lopatto (19, 20), which uses a traditional 5-point scale, was utilized to hopefully discover more detailed insights about what intellectual and experiential growth the students have gained in this course. The CURE survey asks the students about their perceptions in three areas—course elements, intellectual growth benefits, and attitudes about science. The course elements portion uses a pre vs. post self-reflection approach to assess both prior knowledge and learning gains in the course. While the questions’ wordings are identical in both sections, the answer scales provided for the students are different. The “preflection” examines prior experience by using a scale ranging from 1 = “no experience/inexperience” to 5 = “significant previous experience.” The post-survey focuses on the learning gains by using a scale ranging from 1 = “no to little gain” and 5 = “large gain.” In contrast to the pre vs. post approach of the course element portion, the intellectual growth benefits portion only measures post-experience expectations, ranging from career exploration to problem solving and analysis. Tables 2 and 3 examine the averaged data of the three years (2015 to 2017) of CURE data accumulated since the course redesign. A rationale for using an average is to more accurately compare the small data sets at Sewanee (4 to 12 submitted surveys) with the nation-wide data. A weighted averaging of the oncampus data was utilized to minimize any potential skewing of the average by the smallest class’s averages. That said, examination of an individual course year’s data indicates that there are no significant outliers from year to year. Moreover, the averaging of both the local data (24 participants) and the national data (>27K participants) generate relatively small standard deviations, for the national data, less than ±0.1; for the local data, less than ±0.5. As such, comparison of the local 92

and national data should provide insight with regard to what impacts the course might provide. Because of the pre vs. post process of the CURE course elements portion, I was intrigued with the possibility that calculating a “differential value” between the students’ “learning gains” and their “prior experience” (differential = learning gain – prior experience) would highlight additional insights into the potential educational impacts of this integrated research-teaching model. Both the local (Sewanee) and national data for these differential values, as well as the established CURE data, are compared in Table 2.

Table 2. Self-Reported Experiences and Learning Gains Element

Prior Experience

Learning Gains

Post-Pre Differential

Sewanee / National

Sewanee / National

Sewanee / National

4.13

3.45

2.36

3.30

-1.77

-0.16

A

Scripted lab or project where students know outcome

B

Lab or project where only instructor knows outcome

3.75

3.32

3.08

3.42

-0.68

0.09

C

Lab or project where no one knows the outcome

3.04

2.52

4.06

3.41

1.01

0.89

2.46

3.01

4.17

3.87

1.71

0.86

D

A project where students have input into process or topic

E

A project entirely of student design

2.00

2.55

3.56

3.57

1.56

1.03

F

Work individually

3.17

3.61

2.93

3.40

-0.23

-0.20

G

Work as a whole class

2.42

3.14

2.89

3.24

0.48

0.10

H

Work in small groups

4.00

3.85

4.17

3.91

0.17

0.06

I

Become responsible for a part of the project

3.75

3.81

4.13

3.94

0.38

0.13

J

Read primary scientific literature

3.04

3.11

3.92

3.62

0.87

0.51

K

Write a research proposal

1.54

2.52

4.29

3.50

2.75

0.98

L

Collect data

3.29

3.67

4.17

3.94

0.88

0.27

M

Analyze data

3.29

3.60

4.25

4.05

0.96

0.45

N

Present results orally

2.46

3.15

3.76

3.64

1.31

0.48

Continued on next page.

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Table 2. (Continued). Self-Reported Experiences and Learning Gains Element

Prior Experience

Learning Gains

Post-Pre Differential

Sewanee / National

Sewanee / National

Sewanee / National

O

Present results in written papers or reports

3.21

3.42

4.04

3.76

0.83

0.33

P

Present posters

2.34

2.93

4.08

3.34

1.75

0.41

S

Critique work of other students

2.54

2.95

3.16

3.31

0.62

0.36

T

Computer modeling

2.37

2.32

3.04

3.12

0.67

0.80

I hypothesized that the differential values for these course elements would correlate to both the students’ understanding of how the course is structured and their own educational growth. Figure 2 provides easy visualization of the relative differential values between all the local and national data (right column in Table 2). As can be seen in Figure 2, the differential values for most of the survey items are slightly, or significantly, higher for the local data relatively to the national. Of specific note, the differential values for “student input” and “student design” are approximately twice as high as their national differentials. Additionally, all the items related to communication show differential values between two to four times larger than the national data. Consequently, it appears that this course design leads to significant learning gains in important research traits—exploration of the unknown and communication of discovery.

Figure 2. Differential Between Self-reported Prior Experience and Learning Gains. 94

Interestingly, this differential value approach led to the intriguing discovery that only two items in the national data, and three in the Sewanee data, (others that focus on the lecture portion of the course have been omitted) have a negative differential value—scripted labs, working individually, and project where the instructor knows the outcome. This set of correlations suggests that shifting to a research-based experience has made an impact on student perspectives relating to the purpose of a laboratory experience. These negative differentials suggest that students perceive little to no gains in these course elements, which are to some degree inverse to the research experience goals. For example, the differential value for the “scripted lab” is significantly different between the local and national data (-1.77 vs. -0.16), which suggests that students do not generally sense the partially scripted experiences in the course even with some partial cookbook procedures present early in the course. In a similar vein, the differential for “a lab or project where only the instructor knows the outcome” also is negative for the local data, while the national is slightly positive (-0.68 vs. 0.09). This difference highlights that the students in the course understand they are working on research that has no prior instructor insight and see the work as not containing traditional “cookbook” learning. The intellectual growth benefits portion of the survey unfortunately does not provide a pre vs post comparison; it is only contains a post-experience survey. As can be seen in Table 3, again comparing the averaged data for three years, the local and national data are relatively similar in most cases. The national data for each year is reported with a standard deviation of approximately ±1, which suggests that students across all participating institutions have a wide array of different views. Interestingly, the three-year averages for both the local and national data have significantly smaller deviations (approximately 0.1), which suggests views from year to year are relatively stable overall. The Sewanee data are slightly higher than the national information in many categories, but it is unlikely most of the differences between the two datasets is statistically significant given the relatively small deviations for the three-year averaging. The one item with a notable distinction is “skill in science writing.” The local data is 0.65 higher than the national data, which makes sense given the integration of the inorganic course into our campus-wide WI program. Other smaller differences (approximately 0.2 higher) can be seen for “Understanding how scientists work on real problems,” “Self-confidence,” “Understanding how scientists think,” and “Learning to work independently.” These general trends hint that the course is providing creative and experiential learning in the research realm. The CURE survey also includes some overall assessment from the student view, Table 4. As with the “Intellectual Growth” the report from Lopatto’s team reports an annual standard deviation of approximately ±1, suggesting the student beliefs about an overall class are somewhat varied but that the overall view of the classes is favorable.

95

Table 3. The Self-Reported Intellectual Growth Benefits Learning Element

Sewanee

National

Skill in interpretation of results

3.65

3.62

Tolerance for obstacles faced in the research process

3.62

3.59

Readiness for more demanding research

3.62

3.51

Understanding how knowledge is constructed

3.60

3.53

Understanding the research process

3.65

3.57

Ability to integrate theory and practice

3.57

3.55

Understanding how scientists work on real problems

3.86

3.67

Understanding that scientific assertions require supporting evidence

3.76

3.71

Ability to analyze data and other information

3.65

3.79

Understanding science

3.76

3.69

Learning ethical conduct

3.23

3.30

Learning laboratory techniques

3.81

3.79

Ability to read and understand primary literature

3.62

3.48

Skill in how to give an effective oral presentation

3.05

3.29

Skill in science writing

4.10

3.45

Self-confidence

3.52

3.35

Understanding how scientists think

3.67

3.51

Learning to work independently

3.65

3.43

Becoming part of a learning community

3.67

3.55

Clarification of a career path

2.94

3.08

Confidence in my potential as a teacher

3.11

3.05

As previously stated, three years of this data was averaged to attempt a clearer comparison. Given the typical annual standard deviation for the national data, it is not surprising that the local data is not significantly different than the national. The slightly lower value related to the course being a “good way of learning about the subject” probably occurs because the student career demographics vary widely in the small sample (24 students) that have taken the course. However, the small increases in “learning about the process of scientific research” and being “able to ask questions…get helpful responses” might hint that students have valued the experiential and collaborative learning environment that the course is focused on. 96

Our open-ended course evaluation questions provide a qualitative comparison to the quantified student perception measures in the CURE survey (Table 4). Table 5 collates testimonials from students in all three years of the new lab format.

Table 4. Self-Reported Views About Value of the Course Sewanee

National

This course was a good way of learning about the subject

3.7

4.1

This course was a good way of learning about the process of scientific research

4.4

4.1

This course had a positive effect on my interest in science

4.0

3.9

I was able to ask questions in this class and get helpful responses

4.4

4.1

Table 5. Student Testimonials Provided in Course Evaluations “I really liked doing the research project in the lab. It made me think about what I was doing and why as opposed to just blindly following instructions given to me.” “This a perfect example of how to teach a student to be independent.” “I really liked the research based class since it gives a more applicable way of teaching students how to actually use the knowledge learned in class in a lab setting.” “I really liked doing the KP1019 lab and how it was a lot like a research lab. However, I think it would be beneficial to have more time to work on it” “I learned a lot in this course, one of my favorite things was the way in which the lab was run, giving us what felt like an actual taste of what research is like in real life.”

These quotes indicate that several students deeply valued the opportunity to experience the research world within the course. Interestingly, while many students did not provide this type of positive feedback, none really provided strong negative comments. Rather, they made small requests such as the one above that asks for more research time. Toward that end, the instructor(s) have intentionally pared down the scripted exercises in the courses. For example, the first year of the course included a second two-week “practice” laboratory, the synthesis of KP1019. Removing that exercise provided both an additional week of research time and the data analysis workshop in the current form of the course. 97

Annual course modifications like this, as well as updating the research frameworks, have benefited not only the students but also the faculty members involved. Expanding the research period has helped provide fuller partial answers for the instructors’ original research questions. These partial answers both refine the presented frameworks and the instructor’s own research goals, helping to forge a link between teaching and research ambitions.

Conclusions This inorganic chemistry course has become a link between teaching and research, both for the students and the faculty member. It helps address the capacity issue associated with undergraduate research by providing all chemistry majors on campus with a “mini-REU” research experience. The CURE data itself, the differential value approach being explored here, and the individual course evaluation testimonials strongly indicate that the students perceive the laboratory to provide educationally beneficial and intellectual growth. Examining the primary student outputs—lab writing, poster presentations, verbal communication in the laboratory—it is also clear that the students are learning a range of laboratory techniques, spectroscopic analysis, and important communication skills through this educational laboratory experience. From a faculty perspective, this approach has provided an integrated link between both the work of teaching and the work of scholarship. Several new research ideas initiated in the course have grown and flourished in my research lab over the last three years. Moreover, my co-instructor for the last year gained new initial data for their research program. Additionally, this link has lead to some of the valuable educational impacts for students that Astin (6) noted as essential years ago: (a) quality and quantity of student interactions with faculty outside the classroom and (b) level of student involvement. This course also reconnects to Boyer’s long-term goal of reconsidering scholarship (21) and hopefully balancing two of the priorities in faculty work, teaching and research.

Acknowledgments Dr. Evan Joslin has worked on this project as a co-instructor and by providing a research framework for several students in the most recent year of the course. Dr. Bridget Gourley and Dr. Rebecca Jones are also thanked for providing valuable discussion focused on the integration of research and teaching at several American Chemical Society national meetings.

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Undergraduate Professional Education in Chemistry; American Chemical Society: Washington, DC, 2015; p 16. Lopatto, D. Undergraduate Research as High-Impact Student Experience. Peer Rev. 2010, 12, 27–30. Lopatto, D. Science in Solution: The Impact of Undergraduate Research on Student Learning; Research Corporation: Tuscon, AZ, 2009. Kuh, G. D. High-impact educational practices: What they are, who has access to them and why they matter; Association of American Colleges and Universities: Washington, DC, 2008. Kuh, K. D.; O’Donnell, K. Ensuring Quality & Taking High-Impact Practices to Scale; Association of American Colleges and Universities: Washington, DC, 2008. Astin, A. W. What Matters in College: Four Critical Years Revisited. JosseyBass: San Francisco, CA, 1993. Girolami, G. S.; Rauchfuss, T. B.; Angelici, R. J. Synthesis and Technique in Inorganic Chemistry: A Laboratory Manual, 3rd ed.; Univeristy Science Books: Sausalito, CA, 1999; pp 17−26. Work, J. B. Inorg. Synth. 1946, 2, 221. Broomhead, J. A.; Dwyer, F. P.; Hogarth, J. W. Inorg. Synth. 1957, 6, 183. Richen, D. T.; Glidewell, C. Linkage Isomerism: An Infrared Study in Inorganic Experiments: Second, Completely Revised and Enlarged Edition; Woolins, J. D., Ed.; Wiley VCH Verlag: Weinheim, Germany, 2003; pp 31−33. Cocker, T. M.; Bachman, R. E. Mol. Cryst. Liq. Cryst. 2004, 408, 1–19. Collman, J. P.; Moss, R. A.; Maltz, H.; Heindel, C. C. J. Am. Chem. Soc. 1961, 83, 531–534. Pirtsch, M.; Paria, S.; Matsuno, T.; Isobe, H.; Resier, O. Chem. Eur. J. 2012, 18, 7336–7340. Cebrian-Losantos, B.; Reisner, E.; Kowol, C. R.; Roller, A.; Shova, S.; Arion, V. B.; Keppler, B. K. Inorg. Chem. 2008, 47, 6513–6523. Zhang, Y.; Chen, C.; Ghosh, S. C.; Li, Y.; Hong, S. H. Organometallics 2010, 29, 1374–1378. Glidewell, C. Metal Acetylacetonate Complexes: Preparation and Characterization in Inorganic Experiments; Woollins, J. D., Ed.; Wiley VCH: Weinheim, Germany, 2003; pp 149−159 Szafran, Z.; Pike, R. M.; Singh, M. M. Microscale Inorganic Chemistry, A Comprehensive Laboratory Experience; John Wiley and Sons, Inc.: New York, 1991; pp 224−227. Shalhoub, G. M. J. Chem. Ed. 1980, 57, 525–526. Denofrio, L. A.; Russell, B.; Lopatto, D.; Lu, Y. Linking student interests to science curricula. Science 2007, 318, 1872–1873. Lopatto, D.; Alvarez, C.; Barnard, D.; Chandrasekaran, C.; Chung, H.-M.; Du, C.; Eckdahl, T.; Goodman, A. L.; Hauser, C.; Jones, C. J.; Kopp, O. R.; Kuleck, G. A.; Mcneil, G.; Morris, R.; Myka, J. L.; Nagengast, A.; Overvoorde, P. J.; Poet, J. L.; Reed, K.; Regisford, G.; Revie, D.; 99

Rosenwald, A.; Saville, K.; Shaw, M.; Skuse, G. R.; Smith, C.; Smith, M.; Spratt, M.; Stamm, J.; Thompson, J. S.; Wilson, B. A.; Witkowski, C.; Youngblom, J.; Leung, W.; Shaffer, C. D.; Buhler, J.; Mardis, E.; Elgin, S. C. Undergraduate research. Genomics Education Partnership. Science 2008, 322, 684–685. 21. Boyer, E. L. Scholarship Reconsidered: Priorities of the Professoriate The Carnegie Foundation for the Advancement of Teaching; John Wiley: New York, 1990.

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Chapter 7

Theory and Experiment Laboratory: Modeling the Research Experience in an Upper-Level Curricular Laboratory Bridget L. Gourley* Department of Chemistry and Biochemistry, 602 South College Avenue, DePauw University, Greencastle, Indiana 46135, United States *E-mail: [email protected] Phone: 765-658-4607.

While independent faculty mentored research projects are considered the gold standard for the undergraduate research experience (Laursen, S.; Hunter, A.B.; Seymour, E.; Thiry, H.; Melton, G. Undergraduate research in the sciences: Engaging students in real science; Jossey-Bass: San Francisco, CA, 2010), having all departmental majors gain experience with how new knowledge is built on existing literature and previous data has an important place in advancing undergraduate research and the education of students. This paper describes a round robin approach to a series of multi-week projects in a required upper-level laboratory for majors. As teams advance from one experiment to the next they build on the work of the previous team. The pedagogical vision is shared, how teams learn about the previous groups work is explained, sample projects are noted and insights about student gains based on students oral and written work are distributed.

© 2018 American Chemical Society

Introduction Institutional and Departmental Context The course being described in this paper is a capstone course for the chemistry major at DePauw University, a four-year residential private liberal arts institution located in Greencastle, Indiana. The institution, founded in 1837 by the Methodist Church has a 4-1-4-1 academic calendar with three-week terms in both January and May. The student body of 2200 students is 53% women, 20% declared minority students and 9% international. The first-year to second-year retention rate hovers near 90% and the four-year graduation is approximately 80% (1). The department has approximately 35 majors graduate per year of which five are chemistry majors and the other 30 are biochemistry majors. As with most departments of chemistry and biochemistry we serve the institution in a variety of ways, including approximately 200 unique students per year taking 100-level laboratory courses in the department each year, serving not only our own majors but biology and geoscience majors along with those pursuing a variety of pre-health interests. Additionally, the department regularly contributes to the first-year seminar program. Certified by the American Chemical Society (ACS), the department has 8 and 1/3 full-time equivalents (FTE) assigned. In 2002, the department launched a completely new curriculum shifting from the traditional linear path (Figure 1) through the curriculum to a curriculum that allows for multiple entry points and many trajectories (Figure 2). The old curriculum had the typical hierarchical year of general chemistry, year of organic chemistry, sophomore level courses in inorganic and analytical all required before taking a year of physical chemistry, instrumental methods of analysis and an advanced inorganic course.

Figure 1. Schematic illustrating pre-2002 curriculum illustrating linear path through the chemistry major. 102

Figure 2. Flowchart of pathways through the current introductory core.

DePauw uses a course credit system in which one course earns one credit and 31 credits are required for graduation. Non-laboratory courses typically meet for three hours per week and full credit laboratory courses in the department are scheduled for three class hours and three laboratory hours each week. In the catalog a DePauw credit is described as equivalent to a four-hour course at institutions with a credit hour distinction. The curriculum now begins with an introductory core of 4.25 credits taken by both the chemistry and biochemistry majors. A summary of requirements is given in Table 1. The core includes three separate full credit courses with laboratory that introduce students to the way inorganic, organic and biochemists view the discipline. Structure and Properties of Inorganic compounds (Chem 130) and Structure and Properties of Organic Molecules (Chem 120) can be taken in either order. Structure and Function of Biomolecules (Chem 240) requires Chem 120. The fourth course in the core, Thermodynamics, Equilibrium and Kinetics (Chem 260) has Chemical Stoichiometry (Chem 170), the 0.25 credit portion of the core and either Chem 120 or Chem 130 as pre-requisites. After the core chemistry majors select 1.5 course credits from three categories of advanced courses, Chemical Reactivity, Chemical Analysis, and Theoretical and Computational Chemistry. Course selections must include a laboratory course in each category. Students may also take elective courses in the Biochemistry category for the additional 0.5 credits of electives required for the major. Chemical Reactivity houses both inorganic and organic synthesis and reaction mechanisms. Chemical Analysis includes topics typically found 103

in analytical and instrumental methods courses. Theoretical and Computational Chemistry incorporates the components of Physical Chemistry and is where the Theory and Experiment upper-level 0.5 credit laboratory course described below is positioned. Other major requirements include the senior comprehensive exam based on the literature and mandatory seminar attendance.

Table 1. Requirements for the Chemistry Major Introductory Core (4.25 credits) • Structure and Properties of Organic Compounds (Chem 120) (1 credit w/lab) • Structure and Properties of Inorganic Molecules (Chem 130) (1 credit w/lab) • Stoichiometric Calculations (Chem 170) (0.25 credit; self-paced) • Structure and Function of Biomolecules (Chem 240) (1 credit w/lab) • Thermodynamics, Equilibrium and Kinetics (Chem 260) (1 credit w/lab) taken by both Chemistry and Biochemistry majors Categories for Advanced Courses • Chemical Reactivity (Mechanism and Synthesis) • Chemical Analysis (Analytical Chemistry) • Theoretical and Computational Chemistry (Physical Chemistry) • Biochemistry Chemistry Majors • 4.25 core credits • 4.0 cognate courses credits • 1.5 credits in each (including a lab in each category) o Chemical Reactivity o Chemical Analysis o Theoretical and Computational Chemistry • 0.5 additional elective credit • Senior Comprehensive Exam based on the chemical literature and seminar attendance

Course Overview Theory and Experiment (Chem 460) has three laboratory hours and one hour of recitation (or classroom time) per week. It is the only course in the Theoretical and Computational Chemistry category with a laboratory and so is effectively required of all chemistry majors, involving those who complete a senior research project and those who do not. A 300-level Chemical Kinetics, Chemical Thermodynamics, or Quantum Chemistry course is a pre- or co-requisite, helping to ground the experimental work. Students typically take this course as juniors or seniors. In early years of the new curriculum, the course was offered annually; over the last four years, the offering has been reduced to only even numbered years. The largest enrollment was nine students, while five or six is typical, and the course has been offered with as few as three students, in which they worked alone rather than in teams. 104

The catalogue description helps define for students the focus and approach of the course: “This project based laboratory will develop skills in asking fundamental questions about chemical behavior, deciding which theories can be used to explain that behavior, and then designing and implementing experiments to answer these questions (2).” In designing the course, I was motivated by a desire to provide all chemistry majors with some experience with how scientific progress is made, particularly since research is not required for the minimal B.A. The course should also give students practice building their own experiment based on the chemical literature and model experimental problem solving; in other words helping students learn how scientists establish confidence that data is meaningful as they learn a new experimental methodology. Additionally, I wanted the revised course to help students recognize core instrumentation that can be used to address multiple types of scientific questions. And finally, I aimed to create a paradigm that would keep the course dynamic and avoid getting stale. Considering how the rotation of experiments is envisioned, it is helpful to have a sense of some overarching structural issues. In a fourteen-week semester the first week of laboratory is an introductory week where we overview the course, check-in, and discuss laboratory safety. Some years, there are three projects each four weeks in length, while other years four projects are conducted each three weeks in length. The last laboratory session is used for clean-up and check out. Students work in groups of two or three and complete a group formal laboratory report for each experiment. Additionally, each group gives two oral presentations, a project update and a project finding during the recitation meetings over the course of the semester. Each student also writes an individual “memo to the boss” for each experiment performed. Table 2 provides the schedule for an example semester in which three four-week experiments were completed.

Round Robin Project Rotation During the first week, discussions about group dynamics are conducted reminding students of a previous course in the curriculum, Chem 260, where the laboratory work was completed in teams that stayed together throughout the semester and a series of ethical case studies about effective group work were discussed. Once teams for the semester are formed, the rotation begins with each team reviewing the project choices, handouts or procedural reference available on Moodle (our course management software) and submitting their ranked preferences. The instructor assigns each team a first experiment to avoid having more than one team on any experiment. In addition to avoiding duplicate need for the same equipment, assignment guarantees there are enough different experiments in the rotation to avoid a team rotating back to a previously performed experiment. For the second, project teams rotate to a project that was completed by another team during the first round (Figure 3).

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Table 2. Theory and Experiments (Chem 460) Semester Schedule Semester Week

Main Recitation Activity

Laboratory Activity

1

Course intro, laboratory safety, oral & written report expectations

Written laboratory report workshop, Error propagation discussion

2

Project 1 (P1) week 1

Mathematica exercise

3

P1 week 2

Update presentations P1

4

P1 week 3

Software tips for laboratory reports

5

P1 week 4

Findings presentations P1

6

Project 2 (P2) week 1

Grant proposal workshop

7

P2 week 2

Update presentations P2

8

Spring Break

No class

9

P2 week 3

Grant proposal review panel activity

10

P2 week 4

Findings presentations P2

11

Project 3 (P3) week 1

Introduction to convolution

12

P3 week 2

Update presentations P3

13

P3 week 3

Convolution exercises

14

P3 week 4

Findings presentations P3

15

Clean-up, check out

Back-up presentations day

Figure 3. Illustration of laboratory round robin with sample experimental activity and procedural reference (3–7). 106

As a result of the project update and project findings presentations and class conversation teams and the instructor mutually agree on a rotation that guarantees that each group rotates to a new project. A key element of the approach is that teams share lessons learned during their oral presentations and provide recommendations for next steps. When a second group rotates onto the new experiment, they must first repeat an aspect of the first team’s work to demonstrate mastery of the technique and then decide on a new direction or extension of the work generating data that moves the project forward in some way; two examples follow. In one experiment, students measure the diffusion coefficients via laser diffraction; the first group might complete the experiment as described in J. Chem. Ed. (7) and the next team might choose to either study a different chemical system or perhaps refine the experimental design to improve the quality of the data. In the bomb calorimetry experiment, the first team might perform a relatively traditional estimate the resonance energy of a series of polycyclic aromatic hydrocarbons and a second group might decide to expand the series or shift to a completely different question that might logically be addressed via another calorimetric method, such as bomb, semi-micro, or solution calorimetry. For the third project, teams may rotate to a third experiment in the rotation or bring in a new experiment they would like to design. Groups may choose from an additional list of potential new experiments or submit an experiment from J. Chem. Ed. or other primary literature for approval. The additional list of potential experiments includes a few J. Chem. Ed. references that aim to inspire students to think about finding references for their own ideas. Criteria for experiment approval include that the institution owns the necessary instrumentation and, in the instructor’s best estimation, it is plausible to set up the experiment. Students must also demonstrate proof of concept and have sufficient data to write a formal laboratory report in the number of weeks allotted. Years when four experiments are included in the rotation, students have two potential opportunities to move to an experiment not previously done by other class teams. In the past, a new experiment introduced in the third rotation has been subsequently chosen in the fourth rotation by another team. As this course sits in the Theory and Computational Chemistry category for the major, it makes sense to choose experiments with an emphasis on physical chemistry. In any given year, there are a variety of approaches to kinetics, thermodynamics and spectroscopy, which sometimes is dependent upon changes in departmental instrumentation. Approximately 20 different experiments have been attempted over the ten separate offerings of the course to date. Kinetics experiments have included a variety of spectroscopic measurements of concentration as a function of time, method of initial rates, quenching and stopped-flow kinetics experiments. A Langmuir isotherm, calorimetry and three component phase diagrams are most often explored thermodynamic themes. Traditional quantum spectroscopy rounds out the other common experiments. Considering the successes and failures, there are seven experiments that are considered “tried and true” favorites and the initial first round offerings are typically selected from this list.

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Vision for Project Work-flow Project work-flow is designed to model the research process and give students experiences similar to reporting out in research group meetings typically experience in graduate programs (Figure 4). Presentations during the recitation meeting time and regularly encouraging peer feedback further develop a collaborative group meeting atmosphere.

Figure 4. Progression of project activities illustrating how the various steps model the research process.

During the first two weeks of the project, teams set up their apparatus, make necessary solutions and gather enough data to demonstrate they have a working experiment. At the end of the second week, teams give an oral project update (described in more detail in the Oral report section below). One required component of the reporting is a description of planned next steps and/or challenges they are currently facing. Immediately following the report is a brainstorming session to address any challenges the team is facing or to ask probing questions that help teams refine their planned next steps. This interaction is similar to what might be experienced in a research group meeting at the graduate level when particular group members are updating others. The final two weeks of the project are spent collecting additional data to refine results. Groups are encouraged to begin writing the introductory and procedural sections of their formal laboratory reports. At the end of the last week of the project, teams prepare a second oral report, referred to as ‘project findings’ where they summarize results but are not yet required to have completed their error analysis. This is an opportunity to present their conclusions to the class who then function as a research group by asking questions about the data and conclusions, helping the team to think through what they need to discuss in order to produce a robust analysis in the formal report. 108

Formal reports are due early in the week following the experiment and, by that time, an error analysis and propagation is expected. Additionally, individual written ‘memo to the boss’ reports follow by the end of the week following. These memos are designed to ask students to use metacognitive thinking to address what they have learned over the course of the experiment, gain insight into the individual member contributions to the team’s work, and assure that all group members understand the project. Also, both oral reports serve to scaffold the formal report writing, with the project updates report helping organize the groups thinking with regard to their introduction and procedural sections and the project findings helping to frame the data analysis and conclusions.

Oral Report Foci and Purposes Project Updates Each team is expected to prepare a presentation of no more than ten minutes where they discuss what their experiment is measuring and how they are approaching the measurement. They are expected to explain some of the background theory, describe problems they have experienced to date, and suggest plans to address those challenges. These presentations are expected to take advantage of DyKnowTM, a tabletbased software used throughout the semester in this and related courses, that allows for mark-up and lecture capture of prepared slides for more informal interactions. Teams may have a couple of slides prepared that they annotate and we capture for their reference as well as all members of the class. Students can insert a blank slide to sketch ideas or hand write key equations or calculations. This contributes to a more informal conversation while allowing everyone to reference the content generated later as they reflect on next steps. As the semester progresses and the class hears a second and third group present background on the same project, teams begin to share additional aspects of the theoretical background so as to not just repeat what was heard in the previous round of project update presentations. As a result, by the time the rotations are completed, students gain an appreciation for a number of experiments, in many cases more than just those they performed during the semester.

Project Findings These presentations are more polished and likely have a collection of prepared slides. Teams are expected to share results requiring that their calculations are mostly complete. Teams typically show a sample calculation and then provide appropriate graphical or tabulated data summaries. It is a great opportunity for teams to organize the data and get feedback from their peers about whether the tables and/or figures they have prepared are effectively communicating what the laboratory group is hoping to illustrate. There is an expectation teams will provide 109

preliminary conclusions and float data interpretations to get feedback from their class peers who are the audience for the presentations. When the error analysis and propagation are still in progress, teams are expected to discuss sources of error. Hopefully this leads to discussion about whether or not they have identified the major sources of error to help focus their propagation calculations. Also, it often helps students determine whether the random nature of different trials or inherent measurement limitations define the outcome of their project. Finally, teams are expected to offer suggested future directions for the project. Since, in the next rotation, another group continues from where the presenting group ended the presenting group is encouraged to respond to the prompt, “if you had another three or four weeks to work on the project, what would you be most interested in exploring next and why.” The next group is not obligated to take the suggested direction, but the response gives a place to start. The groups are encouraged to consider and discuss the suggested next steps given the following week they need to move the project forward rather than just reproduce the same experiment. Because the course is designated to meet the DePauw requirements for a speaking and listening intensive course, students are also asked to complete a peer feedback survey for the project finding results. Comments from the peer feedback surveys are compiled and shared with the presenting team along with instructor feedback and scoring. Class members are asked to provide brief feedback to the following prompts:

• • • • •

Briefly summarize the presenters’ main results. What were the presenters’ main sources of error? Based on the presentation, what issues do you expect the presenters to raise in the discussion portion of their formal laboratory report? What was particularly helpful to you about the way the presenters shared their information? What else could the presenter have done to further develop your understanding of the experiment?

This survey is shared with the class when we discuss oral reporting and serves to further help frame the expectations for project findings. There is also a written course handout provided to students that further elaborates on oral report expectations and grading .

Written Report Contents Written handouts with expectations for both types of reporting are provided to students at the beginning of the semester.9 Additionally, a few sample anonymized formal reports, with an A, B and C level proficiency, from past semesters are provided. In a workshop fashion, students assess/score the reports based on 110

the rubric and we discuss their decisions between their first project update and project findings presentations. This usually leads to a better understanding of the complexity involved in both writing quality reports and evaluating reports.

Formal Written Reports These reports are traditional formal laboratory reports where students need to provide a title, abstract, introduction, experimental section, analysis of results, conclusion section, appendices and references. This is at least the second time in students experience in our curriculum where as a team they have had to compose a formal group report that included some of their own experimental design. Such an approach gives them further experience with an important skill used by most scientists today in academic, industrial, and national laboratory settings.

Informal “Memo to the Boss” These informal individual writings ask students to conversationally explain a project and how it fits into a bigger picture; provide a summary of the team results and describe their significance; address problems they encountered and suggestion resolutions; overview logical next steps and discuss what is the “personal value added.” The informal style of this writing piece is designed to help them practice the skill of casually discussing scientific work and find the balance of being able to conversationally use technical terminology. This particular piece is most clearly demonstrated when students explain how the project fits into a bigger picture. Summarizing results and describing significance is designed to assess whether or not they individually have taken ownership of the project and corresponding data analysis. Discussing the problems encountered, possible solutions, and logical next steps gives them the opportunity to agree with or deviate from the team consensus. Perhaps the most useful part is the value added section, which asks them to do metacognitive thinking about their own learning and how they have developed as a result of their work on this particular project. In some semesters, I have requested a student self-evaluation of their contribution and a description and evaluation of other members’ contributions. I value having students acknowledge their individual inputs. Also, at the beginning of the semester, those students who are worried about having to work in a group and whether or not everyone will “pull their weight” appear to appreciate knowing they have an official opportunity to provide feedback. However, I have found that this type of reporting often leads to some awkward group dynamics as the semester progresses. Consequently, in more recent iterations of the course, I have opted to omit those sections. I am considering adding back the self-reflection component of their own contribution to the group without the description and evaluation of others. 111

Project Grading Each project is worth 200 points divided up as follows: • • • • • •

30 points for actual laboratory work, e.g. time spent, quality and quantity of data 20 points for submitting laboratory notebook pages each week 25 points for the oral group project updates 35 points for the project findings presentation 60 points for the written formal team report 30 points for he written informal individual “memo to the boss”

The oral group project update, project finding presentation and written team report each receive one score received by every member of the team. Usually every team member also earns the same score with regard to the 30 points for laboratory work, although if I see a team member putting in noticeably more effort than other team members there may be a differential. Laboratory notebook pages and the “memo to the boss” are each graded based on the quality of the individual student’s submission. Use of Recitation (or Class) Time In addition to providing time for the project update and project findings presentations and discussion, alternate weeks are used to review error propagation, discuss effective presentation and graphical representation of data; workshop evaluating sample written reports; discuss software tips and tricks (including Microsoft Excel, Mathematica and other relevant software packages); examine grant proposals and conduct a mock grant proposal panel to expose students to another form of scientific peer review; and to learn about deconvolution of signals in data. The depth of coverage of these topics depends upon the skills students bring to the course, the post-baccalaureate plans of the students, and general student interest. For example, in some years, software tips and tricks consumed multiple recitation meetings while deconvolution of data was never really discussed. In other years, the grant proposal workshop and mock panel review spanned more than two class hours and software tips and tricks received little time.

Outcomes Some of the best evidence for the value of this approach comes from the “personal value added” section of the individual informal reports. In the quotes that follow all emphasis is added. The purpose of the italics and bold within the quotes is to highlight the portion of comments that specifically demonstrate achieving goals that motivated this course design. In a few cases, there are isolated quotes that speak to one motivation; more often the quotes speak collectively to a couple of the motivations. 112

I lead with a quote from several years ago that highlights the valued added to students when they pick-up the work of a previous group and move the project forward; it shifts student perspective from thinking of laboratory experiments as just something they need to get through as to having value. Additionally, it speaks to student’s freedom to put his or her own mark on the experiment. Finally I believe that being allowed to take what the previous group had completed compliments what we are able to put into the experiment. It was especially exciting when the previous groups data was so close to ours and still varied so much from the theoretical to find the “mistake” or misunderstanding of the manipulations of the data. – Student Fall 2010 As a physical chemist, I am always disappointed when students only see some of our major instrumentation such as NMR, IR, and GC-MS as tools solely for organic structure determination since that is the way in which they first encountered their utility in our curriculum. Evidence, such as the next quote, that demonstrate students gain new appreciation for the types of questions that can be answered a particular piece instrumentation is particularly gratifying as an instructor. My only experience with the NMR was to determine compounds based on ppm shifts. Other than figuring out the shifts of each component, it was interesting in a sense to find a new way to calculate the mole ratios just using peak integration. … It allowed me to think outside the box and to assume possibilities I wouldn’t have thought of otherwise.” – Student Spring 2016 In a variety of ways, the next few quotes demonstrate students becoming metacognitive about their learning and engaging in the overarching reflection that we want all students to do in all laboratory settings. By doing this experiment, I learned the importance of planning before the lab. My productivity of the first two lab time was not very high, but after I made plan before I went to the lab, the following two lab time has a really high productivity. [sic] – Student Fall 2012 From the Langmuir isotherm I plotted, I realized that I probably need not have done so many trials near the concentration where I assumed the surface sites were saturated because there wasn’t much variation in the Raman intensities for very similar concentration. – Student Fall 2012 I believe this lab deeply increased my own personal value: asking myself if the data made sense was the most contributing factor to this value increase. – Student from Fall 2010

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Sometimes there were gains not anticipated at the outset. For example, while I always strive to help students understand the value of error analysis and propagation, most students see the task as drudgery and seldom really think about what the results of error propagation are telling them about their experiment. These next quotes illustrate student intellectual growth as a result of error analyses. This experiment allowed me to increase my knowledge of error analysis and how to apply it to new theories. The hand out given for this lab did not contain error statistics for their data. This allowed our group to formulate our error entirely based on where we personally thought error could have occurred. – Student from Fall 2010 The other aspect of this lab that I found to be useful was in understanding error propagation. Our goal of factoring in identified indeterminate sources of error by eliminating the thousandths and ten-thousandths place in our mass data caused huge errors to occur for measurements that were less than one gram. This simply suggests that a sample with mass of less than one gram have fewer “parts” that can be counted as significant. Thus, apparently small deviations lead to huge errors for measurements significantly less than one gram. – Student Fall 2010 My biggest take away from this experiment came from the data analysis and calculations. X and Y’s presentation emphasized the amount of work that went into their calculations in order to have accurate dissociation constants, so I was expecting a grueling calculation process. Then I reread the sample report on Moodle and noticed for their calculations they assumed a 5% protonation rate, and ignored the dissociation calculation for individual samples. Initially I was wary of this assumption and its impact on the final results, so I ran the calculation using X and Y’s data in order to determine how much the values actually varied as a result of the different calculation methods. The mass percentages had a percent difference of less than 1% for every single piece of data. Since the difference between the two methods was less than 1% I felt comfortable using the 5% protonation assumption for all of my calculations. The lesson here was that assumptions can provide results that are equally as valid (to the same significant figures), and they can make the calculations significantly less time consuming. – Student Spring 2016 This last quote demonstrates how many quotes focus on more than one of the core goals. While this student’s remarks are address error propagation calculations, they also provide a rich example of deep thinking about calculations and that perhaps it is unwise to just launch in to brute force a set of calculations. This quote also demonstrates a student who is invested in their learning, even 114

to the point of redoing the calculations of a previous group in the rotation; this would be very unlikely without this course’s pedagogical focus. Finally, two last quotes demonstrate students delving deeper into the analysis of their data and recognizing skills that will allow them to be more independent researchers. These quotes also illustrate students making multiple gains within a single experiment and being conscious of these gains. This was not an easy lab at all, because our data often differed significantly from the predicted outcome. Therefore, we constant had to redo trials to get better results. However, this lab challenged us to think about our data and to reason out solutions [to] problems more than the XXX lab did. – Student Spring 2015 We had to try things out four times and get to the right ways. I think we have practice one of the most important skills in this experiment. When we had problems we suggested the ways to solve them. Even if it did not work for the first few times we still kept trying until we came up with the right solutions. This is a very important quality that we developed and will maintain in the future. Also touching on new things and being able to learn them are also impressive in this lab. As I mentioned earlier we did not have experience in YYY. Then we started reading literatures and asked questions and finally understood the things going on in this lab. In the future we would acquire new knowledge faster because we had experience with that. [sic] – Student Spring 2016 (Non-native English speaker) Many of the quotes above (and others not included in this manuscript) allude to the individual gains in experimental problem solving and figuring out whether or not the data being collected is providing meaningful results. There are no student comments that directly demonstrate that this round robin approach keeps the course from becoming stale. However, after the first rotation, I am never quite sure what students might choose to address as they build on what the previous team accomplished in the first round. This is perhaps the best evidence that this approach has kept the course from becoming boring. Personally, the course has kept me, as the instructor, intellectually on my toes and fully engaged in the wonder of discovery with the students. A couple of the quotes indirectly demonstrate that students are making links between the approach of this course and the progress of science more generally. A few comments hint at the value of utilizing the chemical literature to advance the experiment. Usually about 50% of the teams in any particular semester will choose to test an experiment their cohort has not done as the last experiment in the rotation. Most of the time they choose an experiment (or at least a twist on one) that has not been previously performed at DePauw. As there are two presentations with each project, all students gain from the inclusion of a new experiment by others.

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Conclusions This approach to an upper-level laboratory engages all departmental majors at DePauw in a opportunity to experience the scholarly process and to learn how new knowledge is built on existing literature and previous data. This course develops students’ ability to resolve issues with data collection. Students come to appreciate the value of digging into the literature to see what others have done related to their project or might suggest new directions. Students also learn the process of building on the work of others. Students gain confidence they have built a collection of skills, intuition, and knowledge that will facilitate their ability to design and conduct experiments moving forward. Students become natural speakers about their work in front of a group. Faculty members can learn a great deal about where students are by asking them to reflect and share what they have learned. Summarizing the peer feedback, as well as providing instructor comments within a day of the project finding presentations, helps teams strengthen the quality of their formal reports. Asking students to self-evaluate their personal overall contribution to the group’s work provides additional useful metacognitive development. Asking students to provide reflection about other group member’s contributions, while potentially providing the instructor useful information and group member accountability, can also set up awkward group dynamics among team members as the semester progresses. Finally, this approach to an upper-level laboratory creates a dynamic and interesting class, enjoyed by both the students and the instructor.

Acknowledgments I gratefully acknowledge the DePauw University Theory and Experiment (Chem 460) students over the past 15 years. These students have been instrumental in helping me to refine handouts, rubrics, and experiments. In addition, I thank my departmental colleagues, tenured, tenure-track and term, who have helped develop this curriculum and prepared students for this upper-level laboratory course.

References 1. 2.

3. 4.

DePauw University quick book 2017. https://www.depauw.edu/about/quickfacts/ (accessed March 12, 2018) DePauw University catalog. https://www.depauw.edu/academics/ departments-programs/chemistry-and-biochemistry/courses/ (accessed March 12, 2018) Garland, C. W.; Nibler, J. W.; Shoemaker, D. P., Experiments in physical chemistry, 8th ed.; McGraw Hill: New York, 2009; pp 436-446. Annis, D. A.; Collard, D. M.; Bottomley, L. A. Bromination of disubstituted arenes: Kinetics and mechanism: GC-MS experiments for the insturumental analysis and organic chemistry labs. J. Chem. Ed. 1995, 72, 460–462. 116

5.

6. 7.

8. 9.

Karukstis, K. K.; Avrantinis, S. K.; Boegeman, S. L.; Conner, J. N.; Hackman, B. M.; Lindsay, J. M.; Mandel, A. L.; Miller, E. J. Spectroscopic determination of ternary phase diagrams. J. Chem. Ed. 2000, 77, 701. Claasen, R.; Wolcott, R.; Reinbold, P. E. Study by NMR of a three-component system. A physical-organic experiment. J. Chem. Ed. 1978, 55, 542–544. Rinehart, F. P.; Sattar, S. Diffusion of CsCL in aqueous glycerol measured by laser refraction: A physical chemistry laboratory. J. Chem. Ed. 1998, 75, 1136–1138. Handout for students describing oral presentation requirements available from the author upon request. Handout for students describing written report requirements available from the author upon request.

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Chapter 8

Integrating Research into the Curriculum: A Low-Cost Strategy for Promoting Undergraduate Research Sanchita Hati and Sudeep Bhattacharyya* Department of Chemistry, University of Wisconsin-Eau Claire, 101 Roosevelt Avenue, Eau Claire, Wisconsin 54702, United States *E-mail: (S.B.): [email protected] E-mail: (S.H.) [email protected]

Undergraduate research provides students with hands-on training where they can apply classroom learning to solve original research problems and develop new skills. It not only enhances the problem solving and analytical skills of the students, but also promotes collaboration and teamwork among them. Additionally, it fosters an open learning environment that encourages students to respect diversity and inclusivity. Considering the invaluable benefits of engaging undergraduates in collaborative research, we have integrated authentic discovery-guided classroom projects in our chemistry curriculum. A project-based biophysical chemistry laboratory course, which is offered to the biochemistry and molecular biology majors in their senior year, is described in this chapter. For this biophysical chemistry course, the theoretical study of the relationship between structure, dynamics, and function of proteins is integrated with the discovery-based labs utilizing computer modeling and simulations. Modern computational tools are introduced and computer-based laboratory protocols including novel research projects are developed to help the students gain an in-depth understanding of the role of proteins’ dynamics in their function. The students analyze their own findings in the term papers, aiming to go beyond the standard article summary or literature review. Finally, results of these research projects are communicated in peer-reviewed journals.

© 2018 American Chemical Society

Introduction One of the main goals of a liberal college education is to expand one’s intellectual horizons by understanding the diverse and complex nature of the physical world. Research opportunities in colleges offer evidence-based learning − an invaluable tool to achieve that goal. In particular, involvement in collaborative research provides students with excellent opportunities to step into the real-world situations: they get to use the classroom learning to explore active areas of research. It is evident that engagement in collaborative research generates several important educational outcomes (1). For example, it has been observed that the retention and degree completion rates are higher for the students who are engaged in collaborative research (2). Such involvement also impacts their career plans, as reflected in their improved preparedness for taking on challenges, competitiveness for future employment, and readiness for the pursuit of advanced degrees. In addition to higher grade point average (GPA) and graduate record examination (GRE) scores, undergraduate research experience is now considered a prerequisite for admission to many graduate and professional schools. Students in STEM (Science, Technology, Engineering and Math) fields often obtain research experiences through summer internships in a research lab. However, there are two main limitations of the collaborative undergraduate research conducted in traditional research lab setting (Scheme 1): i) only a few selected students get the opportunity to conduct collaborative research with a faculty mentor, as it is difficult to scale up due to limited recourses, such as limited research lab space or funding for research supplies, and ii) there is an increase in workload for faculty mentors conducting research with the undergraduate students. Most universities do not have enabling policies such as course release, reduced service obligations, or teaching credit for faculty who are involved in mentoring undergraduate students. On the other hand, incorporation of research into the curriculum provides research opportunities to many more students, including students from under-represented backgrounds or those students who do not thrive in traditional coursework. It is important to note that these groups of students are relatively hesitant to actively seek research opportunities by networking with their professors. Consequently, as has been observed on many occasions, they remain unaware of their own potential. In this scenario, faculty-driven undergraduate research opportunities could be provided to a larger group of students by expanding undergraduate research from the traditional lab setting to the classroom setting (Scheme 1). This also helps the research mentors as they can avoid overloading their work schedule. They can still pursue their research interests and provide high-impact learning opportunities to a significantly larger number of students at the same time. In order to provide research experiences to many students at a time, we decided to incorporate research into our chemistry curriculum. We started with our biophysical chemistry course (CHEM 406, 4 credits), a capstone course taken by biochemistry/molecular biology majors. It consists of one two-hour lab period per week with the class size of no more than 24 students. Earlier, the lab was only focused on protein structure and function, where the students used some common software like Rasmol (3) and Pymol (4) for molecular visualization, high-quality 120

rendering, and graphic generation. Also, software like PoseView (5) was used to examine the active-site pocket of enzymes and identify the catalytically important residues. In order to introduce authentic research problems, we have redesigned the lab to focus on proteins’ structure-dynamics-function relationship as our own research is focused on protein dynamics. Several new but free online software and computational packages (vide infra) are introduced to provide students with an in-depth understanding of role of protein dynamics in their function - the missing link between structure and function.

Scheme 1. Expanding Undergraduate Research beyond Traditional Lab Setting

Learning Goals and Objectives The overarching goal is to provide the students with conceptual understanding of the course materials with the help of discovery-guided and inquiry-based research projects (Table 1). Through these projects, they are expected to develop a thorough understanding of structure-dynamics-function relationship in proteins. They also learn how to use software/computational programs for protein structure visualization, protein modeling and structure assessment, and for studying protein dynamics. In addition, students get opportunities to develop intellectual and practical skills (Table 1). The new lab experiments are designed in such a manner that the students could study structure, dynamics and functions of known protein systems through guided questions, and subsequently follow-up the investigation with an original research problem. This allowed us to provide students with hands-on and experiential learning that can’t easily be done through textbooks or in traditional lecture classes, as it requires an authentic setting where the students can work on a real scientific problem and not just solve preset exercises to obtain the known results. 121

Table 1. Learning Goals and Objectives Goal

Objectives

Learning biophysical chemistry through solving authentic research problem

■ Gain the conceptual understanding of the course materials ■ Visualize and classify protein structure ■ Understand the role of non-covalent interactions in protein folding ■ Study protein dynamics and their role in functions ■ Learn the use of software/computational programs for exploring protein structure and dynamics

Understanding structure-dynamicsfunction relationship in protein

■ Develop homology models and assess structural qualities of 3D models ■ Understand the types of non-covalent interactions that govern protein functions ■ Characterize backbone flexibilities and studying their impact on substrate selectivity and catalysis

Developing intellectual and practical skills

■ Develop critical and creative thinking ■ Improve written and oral communication ■ Foster inquiry and analysis-based learning ■ Develop teamwork and problem solving ■ Respect and value diversity

Plan of Activities for Fifteen Weeks Semester The 15-week lab is divided into four modules (Table 2). In the first three modules (modules I to III), the students are introduced to modern computational tools through newly designed computer-based laboratory protocols. These protocols allow them to visualize the secondary, super-secondary, and tertiary structures of proteins, analyze non-covalent interactions in protein-ligand complexes, and develop three-dimensional (3D) structural models (homology model) for new protein sequences. Additionally, the students learn to evaluate the structural qualities of 3D structures, and study the proteins’ intrinsic dynamics to understand their role in substrate binding and catalysis. In the fourth module, the students are assigned with an authentic research problem, where they apply their classroom learning and laboratory skills (acquired through modules I − III) to answer conceptual biophysical questions. All computations are carried out using dual-core personal computers using Intel® Core™ i5 processors. Preparation of Manuscript As mentioned earlier, a total of three term papers are assigned to the students, including the one for the final project in a 15-week-long semester. The students write the term papers following the format of the Journal of Biophysical Chemistry. They are encouraged to have the following sections in their term papers: abstract, introduction, methods, results (tables, figures and figure legends), discussion, conclusions, and bibliography. The instructor/faculty mentor provides written feedback to all the students to improve their writing 122

and analytical skills. After successful completion of the semester, the student who scores the highest grade in the end-of-semester project (Term Paper III) is provided with an opportunity to write the manuscript. This student can enroll in an Independent Study course in the following semester, with the responsibility to compile the manuscript using the results of all other students. During the writing process, the student works closely with the instructor, discussing ideas and clarifying queries. All students who had participated in the research project are included as coauthors of the manuscript.

Table 2. The Plan for Fifteen Weeks Semester Module

Week

Activities

I

1-4

Visualization of three-dimensional structure of proteins using Visual Molecular Dynamics (VMD) package; TERM PAPER I

II

5-7

Homology modeling and structural assessment using Swiss-Model webserver; TERM PAPER II

III

8 - 10

Analysis of protein motions using Normal Mode Analysis ([email protected])

IV

11 - 14

Research project and oral presentation; TERM PAPER III

15

Term paper III due

Module I. Structure Analysis The main objective of this module is to visualize the three-dimensional structure of proteins and understand the relationship between the protein structure and its function. Visual Molecular Dynamics (VMD) package is used in this course, which is a powerful molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3D graphics and built-in scripting (6). Moreover, VMD is available at no cost for use in academic settings. The first day of the lab is dedicated to get familiar with the VMD program using the VMD tutorial. The VMD tutorial helps students learn graphical representations to display the tertiary structures as well as secondary structural elements and side chains of proteins. It also enables students to portray the bound substrate in the active site, and to analyze non-covalent interactions between ligand and the protein. In the following weeks, students are instructed to use the VMD program to perform in-depth study of special structural features of protein. For example, zinc-binding protein is used to study protein-DNA interactions (Figure 1), hemoglobin to examine the interaction between porphyrin ring and helix bundles, and the existence of hydrophobic pore and water channel in aquaporin to visualize how the side-chains are oriented to allow the flow of nonpolar/polar molecules through the channel. In addition, they use VMD to 123

analyze geometric features (e.g. phi/psi angles, hydrogen bonding, salt-bridges, and distance between two functional sites of a protein) and local and global fluctuations using simulated trajectory conformations (e.g. conformational changes, movement of loops and domains, etc.). Students use different "Drawing Methods" for graphical representation of the secondary, super-secondary and tertiary structures of protein and the bound small molecules. For example, in the case of zinc finger protein (PDB code: 1A1F), students display the protein and double stranded DNA and analyze the structural differences between these two biomolecules. Specifically, they visualize the location of side chains in protein alpha helix versus DNA double helix. They also look at the surface and charge compatibilities between these two biomolecules to analyze the non-covalent forces that favor DNA-protein interactions (Figure 1). Similarly, in the case of hemoglobin, students visualize the four subunits of hemoglobin, assessing the orientation of helix bundles and the site of each porphyrin ring. They also analyze residues surrounding the porphyrin ring by creating a small subset by selecting only those residues, which are within 5 - 10 Å of porphyrin ring. Through this exercise, students get familiarity with the 3D structure of hemoglobin and attributes of amino acids surrounding the porphyrin ring. In module I, students also analyze additional protein systems to have better understanding of the interplay of protein structure and function. This also includes conformational changes upon substrate binding [example: GTP- and GDP-bound Ras protein (PDB code: 1PLL and 121P, respectively); Ras protein acts as a molecular switch to control cellular process and undergoes a conformational change when bound GTP is hydrolyzed to GDP] and structural classification of proteins [visualizing different families of proteins (Helical, Sheet, and Mixed Helical/Sheet Proteins)]. Each year, the students are also challenged with newly reported protein structures.

Figure 1. DNA-Protein interactions in zinc-finger protein (PDB code: 1A1F). Reproduced with permission from reference (14). Copyright © 2016 John Wiley & Sons, Inc. (see color insert) At the end of module I, a new protein is assigned to each student for their TERM PAPER I, where the student describes the structure-function relationship through proper display of the assigned protein’s structure and identification of 124

structural features that governs its function. Taken together, the first module is meant for the students to develop familiarity with the VMD program and have an in-depth understanding of proteins’ structure and how they regulate their functions.

Module II. Homology Modeling As knowledge of a protein structure is key to understand its function, the focus of the first module is on protein structure visualization to engender deep insight into the relationship between protein structure and function. However, for many proteins the experimental structures have remained unknown. Therefore, the second module is designed to teach the students how to develop 3D models of proteins for which the experimental structures are unavailable. In this module, students use homology modeling to develop 3D structural model for a new protein sequence using template protein(s), which is/are homologous protein(s) with known 3D structure(s). The homology modeling lab starts with an introductory lecture on the importance of homology modeling, the criteria for safe homology modeling, and the steps involved in generating the 3D model structures. In this module, students are introduced to many online servers: Clustal Omega (7) and Basic Local Alignment Search Tool (BLAST) (8) for sequence alignment of proteins, Swiss-Model (9) for generating 3D model of new protein sequence, DALI (10) for proteins structure alignment, PROCHECK and ANOLEA for structure assessment (bond angel, bond distance, dihedral angles) and bad contacts, respectively (9). As the first step of homology modeling is to identify the template protein(s), the students first conduct BLAST search (8) to identify a suitable template protein(s) for the assigned target protein. PSI-BLAST (Position-Specific Iterative Basic Local Alignment Search Tool) is used to search for template proteins with known experimental structure by restricting the search only to the PDB database. Pair-wise sequence alignments between target and template proteins are conducted to obtain the sequence identity and sequence similarity. Potentially suitable templates are chosen based on BLAST score (< 0.001) and reasonable sequence identity (> 30%) (11, 12). To understand why 30 % sequence identity is reasonable for a homologous protein to consider as a good template, students are assigned a task, in which they compare structure and sequence identity for different pairs of proteins. Generally, the overall folding of protein pairs that exhibit variable degrees of sequence identity is compared. Through this exercise students learn about proteins’ structural modularity and robustness. They get the idea that a high degree of structural homology could exists between proteins (of length 200 amino acids or greater) even when the sequence identity is low (30 %) (11, 13). This exercise helps students to understand the criteria of safe homology and that 3D structural models could be generated for a target protein even if the sequence identity between template and target proteins is not significantly high. The homology modeling is performed using the fully-automated homology modeling server known as SWISS-MODEL (14, 15). The input files for homology modeling are the sequence alignment file of target and template proteins in FASTA format, which can be generated using the Clustal Omega (7), and the PDB codes 125

for the template proteins. The internal routines like ANOLEA and PROCHECK of the SWISS-MODEL server are used for structural assessment of the 3D models (14). ANOLEA (Atomic Non-Local Environment Assessment) performs energy calculations to evaluate the "Non-Local Environment" of each heavy atom in the protein molecule. The input for this analysis is a PDB file containing one or more protein chains. The output is an energy profile, which gives an energy value for each amino acid of the protein; High Energy Zones (HEZs) in the profile represents errors in the protein structure. PROCHECK evaluates the stereochemical quality of a protein structure and identifies disfavored phi/psi angles, unusual bond lengths and bond angles, steric clashes, very large RMSD among templates, etc. Once they understand the criterion of template selection, students perform the homology modeling to generate the 3D model structure of a protein of unknown structure. For example, the crystal structure of Escherichia coli (Ec) prolyl-tRNA synthetase (ProRS) is not available yet, so the homology model of this protein was developed in one year. Ec ProRS catalyzes the covalent attachment of proline to the 3´-end of the tRNAPro, an essential reaction in protein biosynthesis. Ec ProRS is a multi-domain enzyme. Therefore, the students got the opportunity to study not only the secondary and super-secondary structural elements but they also visualized the different domains and their folding. The structures of other bacterial ProRSs are known. As a first step, the amino acid sequence of the Ec ProRS was gathered from NCBI (National Center for Biotechnology Information) and then PSI-BLAST was performed to identify template protein(s). The PSI-BLAST search was restricted to PDB database while searching the template proteins. Students then developed the 3D model structure of the Ec ProRS (target protein) using SWISS-MODEL. Through this process, they found Enterococcus faecalis (Ef) ProRS as a potential target with which Ec ProRS bear significantly high sequence identity (65%). The 3D model structure of Ec ProRS obtained from SWISS-MODEL was found to possess identical folding with very similar secondary and supersecondary structural elements compared to the template protein (Figure 2).

Figure 2. The template, Ef ProRS (left) and the target protein, Ec ProRS (right). Adapted with permission from reference (14). Copyright © 2016 John Wiley & Sons, Inc. (see color insert) 126

The quality of the model structure of Ec ProRS was assessed through ANOLEA and PROCHECK. The ANOLEA plots identified only a few high energy regions for the 3D model structure of Ec ProRS suggesting existence of local unfavorable interactions, which were absent in the template protein, Ef ProRS (13). Analysis of Ramachandran plots revealed ~86 % of residues fell into the most-favored regions, ~10% in the additionally allowed regions and 0.6% fell into generously allowed regions (13). Only a few residues in the model structures fell into the restricted regions. Students also noted that few residues possessed distorted main-chain bond lengths and angles. These small deviations could be fixed through further energy minimization. Overall, the 3D model structures were satisfactory, and students concluded that Ef ProRS was a good template for generating the homology model structure of Ec ProRS. At the end of the second module, students are assigned the second term paper on homology modeling (Term Paper II). For this term paper, each student is assigned a protein sequence with unknown structure. They apply the knowledge gained in this module to develop the 3D model structure of the assigned protein, assess their stereochemical qualities, and finally write the term paper.

Module III. Normal Mode Analysis As a sequel to the 3D structural analysis and homology modeling covered in modules I and II, respectively, module III is introduced to provide an understanding of protein dynamics and their significance in protein functions. Proteins are intrinsically dynamic in nature and undergo transition between different conformational substates (16). Protein motions, on various timescales, are believed to play important roles in substrate recognition and catalysis. Protein dynamics help enzymes to achieve enormous rate enhancement (17–21); the intrinsic flexibility of a protein is either responsible for presenting an active site conformation conducive to catalysis or directly influencing the bond breaking and bond forming processes during catalysis. To provide in-depth insight into the relationship between protein dynamics and function, all-atom and coarse-grained simulations are widely used. As all-atom molecular dynamics simulations are computationally more expensive, we decided to use coarse-grained normal mode analysis for our biophysical labs. Normal mode analysis characterizes all possible vibrations that a protein can undergo around its equilibrium conformational state. The low-frequency vibrations typically correspond to collective motions, while the higher frequency modes represent local deformations (22, 23). The coarse-grained NMA is a powerful tool to identify biologically relevant conformational dynamics from protein structure with no limit in time-scale or system size. Prior to the first NMA lab, students receive an introductory lecture on the theory and applications of NMA. A user-friendly web-based tool, [email protected], is used to study protein dynamics (24). The [email protected] provides three main pieces of information regarding backbone Cα atoms dynamics: i) the backbone flexibility; ii) the direction and magnitude of protein displacement, and iii) the correlated and anticorrelated motion between residue pairs and various protein segments. 127

Students use [email protected] server to analyze the low-frequency modes (vibrational motions) of an allosteric enzyme, adenylate kinase (PDB code: 4AKE). For this lab, the default settings of [email protected] are used to perform the following analyses – "Atomic Displacement Analysis" to identify the flexible region of the protein, "Correlation Matrix Analysis" to identify the protein segments engaged in correlated/anticorrelated motions, and finally, "Mode Visualization" and "Vector Field Analysis" to obtain the direction of collective motions of the protein. The normalized squared atomic fluctuation for each Cα atom in a protein is obtained from the "Atomic Displacement Analysis". Peaks in an atomic fluctuation profile correspond to flexible regions of proteins. The correlated or anticorrelated motions between residue pairs of distant structural elements are determined from the dynamic cross-correlation matrix (DCCM). In a DCCM, the correlation coefficient values range from −1 to +1; a negative correlation value indicates anticorrelated motion, whereas a positive value identifies correlated patterns of dynamics between two Cα atoms. As the low-frequency motions are biologically more relevant, directions of protein motions in the first 3 lowest-frequency modes are obtained from the NMA study and displayed and analyzed using VMD following the instruction provided in the [email protected] site. Once students are familiar with the single NMA to analyze the low-frequency vibrational modes, which requires only the PDB file of the protein, they perform comparative NMA. Comparative NMA is useful for comparing the dynamics of two or more different proteins or the same protein from different species to understand the conservation of intrinsic dynamics across species. The comparative analysis is performed using an aligned FASTA sequence file and corresponding PDB coordinate files of proteins to be compared. For comparative analyses of the dynamic features, the Mustang program (25), which employs the structure-based sequence alignment algorithm to generate multiple sequence alignments of proteins is used. From the comparative analysis, normalized squared atomic fluctuation profiles and the dynamic similarity (measured as root mean squared inner product (RMSIP) and Bhattacharyya coefficient (BC) (26) are obtained. RMSIP measures the similarity of atomic fluctuations between proteins by comparing their normal modes, whereas BC compares the covariance matrices obtained from the normal modes (27–29). Values of BC and RMSIP vary between 0 to 1 and represent the amount of overlap between the collective dynamics of two or more proteins; a value of 1 represents maximum overlap. As mentioned earlier, a simple protein system, Ec adenylate kinase, is used first for NMA. This enzyme catalyzes the Mg-dependent nucleotide phosphoryl exchange reaction ATP + AMP ⇋ 2ADP. It comprises of three domains (Figure 3) − the LID domain, residues 118–160; the NMP (nucleoside monophosphate) domain, residues 30–67; and the central CORE domain, residues 1–29, 68–117, and 161–214. Adenylate kinase undergoes a large conformational change upon substrate binding which favors its catalytic function. The LID and the NMP domains are intrinsically very dynamic in nature and undergo an "open" to "closed" conformational change upon substrate binding (Figure 3A); in the "closed" conformation, substrates are situated in a catalytically favorable environment. For this exercise, students first visualize the crystal structure of the enzyme to identify the secondary and super-secondary structural elements 128

present in this protein. Students then study the intrinsic dynamics of this protein by performing NMA with the open conformation of this protein (4AKE.pdb). They first analyze the normalized atomic fluctuation profile (Figure 3B); peaks in an atomic fluctuation profile correspond to the relatively more flexible regions of a protein. Figure 3 revealed two distinct flexible regions; residues 125-155 of the LID domain are the most flexible part of the protein. Similarly, residues 30-60 (NMP domain) are also observed to be very flexible, whereas the CORE domain is quite rigid. The flexibilities of these two domains are experimentally observed and found to be essential for substrate binding and catalysis by this enzyme.

Figure 3. A) The "open" (PDB code: 4AKE) and "closed" (PDB code: 1AKE) conformations of the adenylate kinase. The substrate induced conformational changes from "open" to "closed" are apparent from the "blue" (substrate-unbound) and "green" (substrate-bound) color representation of the LID domain and the NMP domain. B) Atomic fluctuation of each Cα atom in adenylate kinase. The x-axis represents amino acid numbers, while the y-axis represents the normalized displacement corresponding to each amino acid. Peaks in an atomic fluctuation profile correspond to flexible regions of proteins. Adapted with permission from reference (14). Copyright © 2016 John Wiley & Sons, Inc. (see color insert) The [email protected] analysis also enabled students to visualize the direction of the domain motions and have some understanding of the amplitude of their displacement from the equilibrium position (Figure 4). As it is evident from Figure 4A and B, the movements of the LID and NMP domains are in opposite direction. The extent of movement of the LID domain from its original position is greater than the NMP domain as is apparent from the magnitude of the vectors representing the direction of protein motions. The anticorrelated motion between these two domains is also observed by analyzing the DCCM. The blue region shown by rectangle box in Figure 4C indicates the anticorrelated motion between the two domains. Through this exercise, students for the first time get an opportunity to visualize the dynamical nature of proteins. They also understand how movements of different structural elements of proteins are important for their functions such as substrate binding and positioning reactants into proper orientation for effective catalysis. 129

Figure 4. A) The three domains of the adenylate kinase (PDB code: 4AKE), B) the direction of movements of the three domains in mode 9, and C) DCCM plot showing correlated/anticorrelated motions between Cα atoms of adenylate kinase. Both axes represent the amino acid residue index. Each cell in the DCCM shows the correlation between the motions of two amino acid residues (Cα atoms) on a range from -1 (anticorrelated, blue) to 1 (correlated, red). The rectangle box indicates the anticorrelated motion between residues 120-150 and residues 1-80. Adapted with permission from reference (14). Copyright © 2016 John Wiley & Sons, Inc. (see color insert)

Module IV. End-of-Semester Research Projects Once students gain experience in protein visualization, homology modeling, and studying protein dynamics through the exercises in modules I-III, they are assigned an original research problem for their third term paper. In this section, we are describing some of the end-of-semester projects that were conducted by biophysical chemistry students in three successive years, 2012-2014. The results of these projects were published in peer-reviewed journals (30–32).

Project 1. Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases This project was conducted by a class of 20 students over five weeks. AARSs are an important family of enzymes that play a key role in protein biosynthesis. They catalyze the ligation of an amino acid to its cognate tRNA molecule (33). They are modular enzymes and most of them have two main domains - the catalytic domain, where the adenylate formation and aminoacylation of tRNA take place and the anticodon binding (ACB) domain recognizes and binds the correct tRNA. Some AARSs also have evolved to have additional domains (insertion and/or extension). These domains assist AARSs in substrate recognition, catalysis, and/or editing (hydrolyzing) misacylated tRNAs. There are 20 common AARSs, which are divided into two broad classes of 10 each - class I and class II. Traditional classification of AARSs is based on protein sequence and structure similarities (34). 130

The big question for the research project was - are these two classes of AARSs display distinct patterns of motions? As protein dynamics is an intrinsic property, it was hypothesized that AARSs could be classified based on their intrinsic dynamics. Students investigated if AARSs of a given class exhibit similar patterns of motions, which are distinctively different from the dynamic patterns of enzymes of another class. A thorough comparison of the intrinsic dynamics of class I and II enzymes was conducted by 20 students. They characterized the functional relevance of the collective motions in these enzymes. The tasks given to the students were as follows: i) learn about AARSs family, ii) collect sequence and structure of the assigned AARS from three different species, iii) develop structural model for the assigned AARS if the structure is not known for all the three species, iv) perform structural characterization and assessment, v) analyze the intrinsic dynamics of the assigned AARS, vi) compile and share their results (oral presentation), vii) participate in classroom discussions to explore if AARSs could be classified based on their intrinsic dynamics, and viii) write the term paper. Students first analyzed the structural difference between the two distinct classes of AARSs by visualizing the class I Thermus thermophilus leucyl-tRNA synthetase (Tt LeuRS) and Class II Ef ProRS structures using VMD (13). They observed that there is a distinct difference in the folding of the catalytic core of class I and II enzymes. The catalytic core of class I synthetases has the Rossmann fold, consisting of a central five-stranded parallel-β sheet connected by α helices. On the other hand, the catalytic domain of class II synthetases is composed of six-stranded antiparallel β-sheet flanked by loops and α helices. As there were 20 students working on this project, with 20 different AARSs, each student performed the NMA of an AARS. For this study the Ec enzymes were considered. Homology models were first generated for the three AARSs (ProRS, SerRS and TrpRS) for which the crystal structure of Ec were not available. The intrinsic dynamic patterns were compared by analyzing the DCCM for each AARS. A clear distinction between class I and II enzymes was made based on the collective dynamics of the ACB domain with respect to the catalytic domain. For class II enzymes, the ACB domain motion is predominantly anticorrelated with respect to the catalytic domain. On the other hand, the ACB domain of class I enzymes exhibits a mix of correlation and anticorrelation motions with respect to the catalytic domain. These differences in dynamic patterns between class I and II enzymes are expected as the mode of interactions of class I enzymes with tRNA differ significantly from that of class II enzymes. Also, it was observed that the insertion domain of both class I and II enzymes predominantly exhibits anticorrelated motion with the catalytic domain. Students explained that the very existence of the anticorrelated motion between insertion (editing) and catalytic domains allows the 3′-end of tRNA molecules to access the catalytic domain for aminoacylation. Additionally, anticorrelated movement between these two domains favors the translocation of the misacylated-tRNA from the central catalytic domain to the insertion (editing) domain during the editing process (30). Taken together, the students used their knowledge of homology modeling and protein dynamics to study a real scientific problem. Their work demonstrated that AARSs can be grouped into two distinct classes based on their intrinsic dynamics. 131

It was observed that the intrinsic dynamics based classification is similar to the traditional classification based on sequence-structure homology (30). Although traditional classification of AARSs based on sequence and structure has been useful, the study of AARSs dynamics have provided better insight into the catalytic processes (tRNA binding and release) of AARSs. This work resulted into a peer-reviewed publication (30).

Project 2. Comparison of Intrinsic Dynamics of Cytochrome P450 Proteins To Understand if the Difference in Atomic Fluctuations Is Related to Substrate Specificity Cytochrome P450 (CYP) enzymes catalyze the monooxygenation reaction:

These heme containing-enzymes receive electrons from NADH/NADPH via electron transfer proteins. CYP proteins are classified into six broad classes based on their electron transfer partner − bacterial, CYB5R/cyb5, FMN/Fd, microsomal, mitochondrial, and P450 only (35). These proteins possess very similar structure but catalyze a wide-range of structurally diverse substrates of endogenous and exogenous origin. So, the research problem we studied was − Is there any correlation between atomic fluctuations and substrate recognition? As protein dynamics play an important role in molecular recognition and catalytic activity, students hypothesized that there should be some correlation between differences in intrinsic dynamics of CYP proteins and the substrate specificity. They performed NMA of five classes of CYP proteins to characterize their intrinsic dynamics; one CYP family of enzymes, FMN/Fd class, was not included because of the lack of X-ray crystal structures. This project was conducted by a class of 17 students. Therefore, each group of 3 or 4 students was assigned one class of CYP proteins to study for their term paper III. Three proteins, each from three different species, were studied for each CYP class. The CYP enzymes share a common tertiary fold with < 25% sequence identity. Students first performed the qualitative analysis of structural and dynamic similarities. The conservation of the overall tertiary structure of CYP proteins was observed on visualizing proteins from different CYP classes using VMD (31). Pair-wise protein structure comparison is performed using the Dali Server (10). Students then performed the NMA analysis and their study revealed some important dynamic features of these proteins. The DCCM obtained from the NMA analysis of individual proteins revealed strikingly similar patterns of correlated/anticorrelated motions among all of the CYP proteins studied. Patterns of the correlated motions between residues surrounding the heme cofactor are very similar across CYP family. However, a scrutiny of DCCM of five families of CYP proteins revealed some noticeable differences in correlated motions between residues in the heme and substrate binding pocket. Also, the atomic displacement fluctuation profiles of different class of CYP proteins were compared and a perceptible difference in the flexibility of Cα atoms of structural elements surrounding the heme cofactor and substrate binding pocket was noted 132

(Figure 5). These differences in residue fluctuations are believed to be crucial for substrate selectivity in these enzymes. Students concluded that the local dynamical differences between different classes of CYP proteins allow these enzymes to catalyze reaction involving structurally diverse substrates. In addition to the qualitative analyses, students also conducted the quantitative analysis of sequence, structure and dynamic similarities in CYP proteins. Structural similarity was obtained from the pairwise structural comparison of CYP proteins (10). Significant structural similarities were observed between CYP proteins; the root-mean-square-deviation (RMSD) varies from 2.8 – 3.6 Å. Dynamic similarities between different classes of CYP proteins were obtained by computing the RMSIP and BC, the quantitative measures of dynamic similarity between proteins. The comparative study revealed that CYP enzymes share a strong dynamic similarity (RMSIP > 55% and BC > 80%) despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the CYP superfamily. The CYP enzymes are mainly monooxygenases, and the presence of high dynamical similarities among these enzymes suggests their similar catalytic function, which depends upon the heme cofactor and the electron transfer protein partner. However, the local dynamical differences between different classes of CYP proteins explain how these enzymes can catalyze reactions involving wide-varieties of substrates with different size and chemical properties. In this project, undergraduate students dealt with a fundamental question regarding the role of protein dynamics in substrate recognition. While teaching the thermodynamics and kinetics of ligand-protein interactions, we usually emphasized on the non-covalent interactions and structural requirements for substrate binding. Through this study, students obtained first-hand exposure to the role of protein dynamics in substrate binding. They learned that differences in local fluctuations modulate substrate specificity in this very important superfamily of these enzymes. This work also resulted into a peer-reviewed publication (31).

Project 3. Comparisons of the Intrinsic Dynamics of Enzymes Involved in Metabolic Pathways and Develop a Dynamic-Based Tool for the Functional Identification of Proteins This project was assigned to a class of 14 students. For their project, they studied the intrinsic dynamics of enzymes involved in primary metabolic pathways. The main objective of this project was to investigate if the dynamic information could be used for the functional characterization of unknown proteins. The coarse-grained NMA was performed to examine the intrinsic dynamic patterns of 24 different metabolic enzymes. Enzymes from six species were chosen for this NMA study. If six X-ray crystal structures were not available for any enzymes, homology models were generated and used for dynamic analysis. By comparing DCCMs it was observed that each metabolic enzyme exhibits unique patterns of motions, which are conserved across multiple species and functionally relevant. Analysis of DCCMs revealed that they are visibly identical for a given enzyme family but significantly different from DCCMs of other protein families (32). Students were also successful in functional identification 133

of six unknown proteins by matching the DCCM of six unknown proteins to the DCCM of a set of known proteins (32). Their work demonstrated that the DCCMs of proteins could be used for functional classification of proteins as well as correct identification of unknown proteins based on their intrinsic mobility patterns. This work resulted into presentations at national meetings and a peer-reviewed publication (32). Similar projects are being designed to provide hand-on experiences with modern computational tools while investigating authentic research problems. These enriched, experiential, and collaborative learning experiences are extremely valuable for students going to graduate school or entering the workforce.

Figure 5. The fluctuations of Cα atoms of different CYP proteins. The various helices are designated by letter A to L. Reproduced with permission from reference (32). Copyright © 2015 The Protein Society. 134

Discussion The Boyer Report (36) on “Reinventing Undergraduate Education”, published by the Carnegie Foundation in the late 1990s, had stressed the importance of a “research-based education.” It was pointed out that a “learning-as-inquiry” model of education has better learning outcomes than the traditional lecture, which encourages students to be passive learners and suppresses student-centered enquiry. To develop transferable skills of critical thinking, reasoning, problem-solving, communication, initiative, and teamwork, students should be provided with active learning opportunities. One of the best ways to provide active learning opportunities is to engage students in original and meaningful research projects. This chapter describes our efforts to provide authentic research experiences to many students by integrating research into the chemistry curriculum. The primary objective of the biophysical chemistry course is to explore protein functions at the molecular level while studying the principles of thermodynamics and kinetics. Therefore, the interplay of protein structure, dynamics, and function has been the focus of the lab component of this course. We redesigned the lab course in such a way that the students get to learn the course materials by solving original research problems. New computational experiments and protocols were developed as a part of the biophysical lab by integrating structural and dynamic features of proteins to understand their functions. Each year, a new project was introduced where students performed computational experiments to probe a hypothesis. In particular, the students were guided to explore hypothesis-driven new biophysical concepts such as dynamics as a tool for classification of proteins, functional identification of new proteins based on dynamics, and the role of dynamics in substrate selection by enzymes. Recently, new projects on drug design have been introduced. Students are pursuing docking and molecular simulations to explore if inhibitors based on unique protein dynamics could be used to target the desired enzymes. The four modules that are described in this chapter were designed to provide an in-depth understanding of the role of protein dynamics in its function and to impart hands-on experience in modern computational tools. Module I was dedicated to protein structures, where the focus was to characterize the four different levels of protein structure and understand the role of non-covalent interactions in protein-ligand complexes. The VMD program was introduced for visualization and analysis of protein structures. Through guided questions, the students received a thorough understanding of the role of protein structure and types of non-covalent interactions that govern protein functions. In module II, homology modeling was introduced, where the students learnt how to develop 3D model structure of a protein of unknown structure from its sequence. In addition, they also learnt how to assess the quality of the homology model structure using different software, available online. Finally, in module III, the use of NMA enabled them to characterize the backbone flexibilities as well as understand how these fluctuations impact substrate selectivity and catalysis. They visualized and analyzed different low-frequency motions and their role in substrate selectivity and catalysis. After completion of the three modules, students were challenged 135

with an original research problem, where they used the lab skills acquired through modules I-III to solve the assigned problem. An important result of our endeavor was that we were able to provide an authentic research experience to many more students, who otherwise would not have had such an opportunity through traditional research lab setting. During the period 2012 to 2014, only 22 out of 51 students enrolled in CHEM. 406, had the opportunity to conduct research in traditional lab setting. The rest, i.e. 29 more students (57% of the total enrollment), could obtain research experience exclusively through this course (in the classroom setting). Apart from making the collaborative research experience accessible to a much larger group of students, some definitive learning outcomes of the redesigned course were also noted: a) through these lab experiments and assignments, students were able to gain molecular-level understanding of interplay of protein structure, dynamics, and function; b) the discovery-guided end-of-semester research project enabled students to apply the classroom learning and lab skills to investigate new scientific problems; c) incorporation of three term papers (Table 2) helped students to improve their communication skills, specifically writing skills. Students’ performances were assessed based on their ability to i) relate their project topic with the contemporary advances in protein dynamics as supported by literature, ii) analyze their data and relate that to the core questions of the assigned project, iii) provide a rational explanation of their findings, and finally iv) prepare a complete report in a professional manner. The assessment results from 2012–2014 demonstrated a very satisfactory performance by our students. The relative number of students, who obtained higher overall course grades (≥ 85%) were consistent; thirteen out of twenty students in 2012, thirteen out of seventeen students in 2013, and nine out of fourteen students in 2014. The high grades indicated a good understanding of the core biophysical concepts among a significantly high number of the students. Similarly, assessment of students’ performance in their final term paper was also quite satisfactory; fifteen out of twenty students in 2012, seventeen out of seventeen students in 2013, and twelve out of fourteen students in 2014 scored ≥ 85% in the final term paper. Furthermore, their ability to communicate scientific ideas was also evaluated through final oral presentation and a significantly high number of students scored ≥ 90%: sixteen out of twenty students in 2012, ten out of seventeen students in 2013, and eleven out of fourteen students in 2014. These numbers reflect that a significant majority of the students developed strong intellectual and practical skills besides conceptual understanding of the course materials. Finally, the three peer-reviewed publications (Table 3), which emerged out of this process, also provide a clear evidence of the students’ in-depth understanding of structure-function-dynamics relationship in proteins. Overall, we were successful in providing a research environment in the classroom setting, where students participated and collaborated in challenging research problems, shared their data, prepared reports in professional manner, and finally, got credited as a co-author in a research paper (Table 3). Through this collaborative research work, students also got opportunities to interact with diverse group of students, which is expected to promote their ability to respect and value diversity. This model of fostering high-impact learning experience is 136

easily implementable as one can provide research experience to many students without overloading the work schedule. Although a thorough study to evaluate the long-term impact of this model on students’ performance is required, the present assessment of three years is noteworthy and could serve as inspiration to the “research in classroom” movement.

Table 3. End-of-Semester Projects and Their Outcomes Year

Biophysical concepts/hypothesis

Number of students

Contributions to Biophysical Community

2012

Intrinsic dynamics as a tool for AARS classification

20

The Protein Journal (2014)

2013

Intrinsic dynamics to probe substrate selectivity in Cytochrome P450

17

Protein Science (2015)

2014

Intrinsic dynamics to identify unknown protein families

14

Cogent Biology (2017)

Students’ Feedback Analysis of students’ responses, collected from the course evaluation, showed that they enjoyed the research-based biophysical chemistry lab. These responses were collected from an anonymous survey conducted at the end of each semester. In general, students found computational lab experiments highly exciting and extremely useful. They were fascinated by the scope of VMD and [email protected] in viewing protein structures and depicting their collective motions, respectively. As it happens with any research, students experienced both joy and frustrations, while carrying out their research projects. Some students felt overwhelmed in the beginning of the computational lab. In particular, a small number of students felt uncomfortable with the VMD program in the beginning, but slowly, through collaboration with other students, they managed to learn the VMD commands and enjoy the overall process of investigating a real scientific problem. Some typical students’ comments about the lab include: “VMD and [email protected] are cool. It was neat to see the structure and movements of protein molecules, which helped me to have better understanding of how proteins work.”; “The lab is honestly very well done. I feel like I have a better understanding of databases and software that are utilized in research and industry.”; “All the interactive labs on VMD, SWISS-MODELING, and NMA are very beneficial. I feel like I have a 137

good command of those programs now.” Regarding term papers, the students had mixed feelings. Although most of the students liked the end-of-semester project and took ownership of the assigned research project, some students expressed frustrations regarding the time it took them to prepare the term papers. Some of the remarks related to term paper include: “The partitioning of topics in the lab is a good directive. Using papers to assess student’s understanding not only are a good indicator of the student’s knowledge but also develops scientific writing skills.” “The term papers are extremely helpful in understanding the direct applications of biophysical chemistry, which may go missed by some students. It’s just not the best use of time to always meet, especially when the papers do take time to write.” Overall, the students’ feedback was positive. The group of students who matriculated to doctoral programs or joined the work force especially appreciated the experience and reported that their research-based computational lab experiences had well-prepared them for their current job.

Conclusions Our work shows that the high-impact leaning experiences could be provided to many students by integrating research into the curriculum. Through these discovery-guided research projects, the students received intensive but satisfying lab experiences. Moreover, they gained an in-depth understanding of protein structure-dynamics-function relationships through these innovative lab experiments. They also got opportunities to be involved and contribute significantly in innovative research projects, which provided them with unique and valuable learning experiences. The process of solving research problems enabled students to learn to think critically. They also got opportunities to disseminate their research findings in the form of oral presentations as well as research papers, subsequently shared to the academic community. They got credited as a co-author on these published research papers. This is critically important as graduate and professional schools often expect the students to conduct research during their undergraduate careers. Our work also demonstrates that important findings can be made in the classroom through collaboration with undergraduates. Moreover, with the decline in funding and increase in faculty workload, the integration of research into the curriculum is a cost-effective strategy to provide high impact learning experiences to our students. We also observed that the hours spent by two students during an academic year [30 weeks (30 weeks x 2 students x 6 hours per week) + 3 weeks of Winterim (3 weeks x 2 students x 40 hours per week) = 600 hours] to complete a project like those described over here is comparable to the hours spent by fifteen students during one semester of biophysical class [15 weeks x 20 students x 2 hours per week = 450 hours). Overall, we are satisfied with the results of our efforts to integrate research into the biophysical chemistry lab curriculum, which not only provided high-impact learning opportunity to many more students but also resulted into important new findings.

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Acknowledgments We gratefully acknowledge the computational support from Learning and Technology Services of the University of Wisconsin-Eau Claire. The authors would like to thank the department of chemistry and the Office of Research and Sponsored Programs, University of Wisconsin-Eau Claire, for providing the financial support. The authors would also like to thank Ms. Clorice R. Reinhardt and Mr. Ajay Rai for careful reading of the manuscript and providing helpful comments and suggestions. Finally, the authors would like to thank the reviewer for the insightful and constructive comments.

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Chapter 9

Peptidomimetics from the Classroom to the Lab: Successful Research Outcomes from an “Upper-Level” Class at a Primarily Undergraduate Institution Danielle A. Guarracino* Chemistry Department, The College of New Jersey, 2000 Pennington Road, Ewing, New Jersey 08628, United States *E-mail: [email protected]

One of the challenges at a primarily undergraduate institution (PUI) is to afford meaningful research experiences to all students in the chemistry curriculum. With an ever-changing student population and those wishing to “try on” research, our department has creatively increased inclusion of all students in research through several possible upper-level classes. I will describe the incorporation of research into my Advanced Topics Chemical Biology course. Early on, students randomly choose a peptide from a list of new designs provided by the instructor. Throughout the semester, in the classroom and lab, they learn how peptides are made, synthesized and characterized, gaining experience at the bench. Near the conclusion of the course, they learn spectroscopic techniques to examine the secondary structure of their peptides. By this point they know the sequential and structural elements contributing to protein folding, what stabilizes natural and unnatural peptides, and can predict how their chosen peptide should behave. After testing the compounds, they describe the results in relation to their prediction, and compare across teams to determine the links between sequence and folding. Students write a communication-style final paper that encompasses their work. The lecture portion of the course has students applying the skills of chemical biology to novel problems. They prepare a final © 2018 American Chemical Society

presentation on a unique area within epigenetics, teaching the class and writing a question for their final. In both iterations of the class that I have taught we were able to publish our results in peer-reviewed journals with the students as authors. Overall, students enjoy the course and materials and I plan to include some new areas of inquiry into future versions of the course, continuing this positive trajectory.

Development of the Course Advanced Topics in Biochemistry: Chemical Biology The College of New Jersey is a public liberal arts college located in central NJ across 289 acres. It is ranked as the number one public institution in the northern region of the country by U.S. News & World Report and boasts approximately 7,000 full-time students. The Chemistry Department is ranked in the top 4% of chemistry programs nationally in graduating American Chemical Society (ACS) certified bachelor degrees. Our chemistry department is “undergraduate only” and has about 110-160 chemistry majors, 12 full time faculty and approximately $1 million of research grade instrumentation. As the numbers of students interested in research is steadily increasing, we as a faculty, in conjunction with the standards of ACS certification, have developed a series of advanced topics classes for interested junior and senior students. All chemistry majors need to take 2-3 advanced topics classes for the ACS-certified degree and we offer a range of interesting options across the analytical, biochemical, inorganic, organic, and physical subdisciplines. In addition, we have had exciting forays into materials science, food science, forensics, and instrumentation, as well. I have twice taught the Chemical Biology course I designed, once in 2013 with 13 students and again in 2015 with 12 students. The course meets for two 80 minute lectures a week and one 3 hour lab and has the co- or pre-requirement of a biochemistry course (with a laboratory). I have built in several research components into the laboratory and lecture portions of the course across the general topics covered. Chemical Biology is a broad discipline that is defined as the use of chemical techniques and tools applied to understanding and controlling a biological system including fixing or altering a biological problem. In designing my course, I took inspiration from the topics covered in the Chemical Biology courses I took for my graduate work and prepared my own materials, updating and including interesting modern ventures in the field. The first course objective we go through in lecture is synthetic biology and biological synthesis, covering an array of techniques available. To connect the lecture and laboratory components, students perform solid phase peptide synthesis of short novel peptides by hand in the lab, and also learn how to synthesize non-natural β3-amino acids through several synthetic steps. This hands-on work ties into their second course objective, which is the study of peptidomimetics and disruption of protein-protein interactions. The third course objective covers common techniques and assays used in Chemical Biology, and the lecture covers several that, when brought to the lab, make use of 144

our instrumentation. The students use high performance liquid chromatography (HPLC) to assess the purity of their synthesis of their peptide and β3-amino acid, the liquid chromatography mass spectrometer (LCMS) to determine the identity of their compounds based on mass, and then proton nuclear magnetic resonance (1H-NMR) spectroscopy on the β3-amino acid for further identification. As part of this area, we discuss the bridge between biophysical characterization of peptidomimetic folding and methods that assess the disruption of protein-protein interactions. In the laboratory we focus efforts on predicting and assessing peptide folding by examination of their compounds by circular dichroism spectroscopy. And finally, the fourth course objective is the study of genomics and proteomics, modern methods and information. While we have not covered this “hands on” in the lab, students perform a final literature research project on epigenetics. By assimilating all of these topics into cohesive “research” projects, the entire class becomes a team working towards the same goals, each contributing a portion of new and interesting materials.

Begininng the Process: Laboratory Research Goals When the class premiered in 2013, I asked a very specific lab question, answerable by the work the students would perform: do idealized α-helical and 14-helical sequences translate to opposite molecules? There are specific sequences in proteins that have been found to confer α-helicity, the most common form of secondary structure, to the peptides that are comprised of these particular amino acids (1, 2). With that concept in mind, I designed one hexa-, one septaand two octa-peptides with helix-promoting sequences, which would segregate oppositely charged, polar groups from hydrophobic groups upon peptide folding (Figure 1, peptide sequences). The use of leucine as a hydrophobic group might also lead to a possible leucine zipper, across two helices, indicating higher level structure among the folded groups (Figure 1, PT3) (1). PT4 replaced those leucines with alanines to see what the effects would be to overall helicity (Figure 1, PT4). PT2 incorporated two non-natural aminoisobutyric acid (Aib) residues that have been known to positively influence helical structure (Figure 1, PT2) (3). The field of β-peptides emerged in the past two decades as non-natural folding oligomers. β-peptides, which are comprised of β-amino acids with an extra carbon along the amino acid backbone, can fold into what is known as the 14-helix when substitution is on the third carbon (Figure 1) (4, 5). The 14-helix resembles the α-helix however its notable differences include left-handedness, a reversed macrodipole placing a net partial negative charge on the N-terminus, and the approximate three residues per turn. It has been shown that arranging the three faces of the helix such that one has hydrophobic groups (usually valines that can interdigitate), one has salt-bridging (in line with the reversed macrodipole), and the final one is kept free for substitution, led to the development of an ideal peptidomimetic scaffold (7–9). Therefore, in the interest of creating the smallest possible motifs for comparison, BP1 represents the smallest scaffold of an idealized β-peptide, the minimal 145

epitope for binding (Figure 1). BP2-4 represent the beta equivalents of the alpha PT2-4 peptides, to test whether folding of sequences can translate between α- and β-peptides. PT1, tests this in the opposite direction; does an idealized 14-helix still have any relevance when the sequence is placed in an α-peptide? Students who worked on these peptides examined not just whether their peptide would be helical, but also the relationship of similar sequences in different types of peptides, whether it led to helicity. There was much comparing across groups.

Figure 1. Top: The four α-peptides, depicted in helical-wheel format with hydrophobic and charged residues highlighted. Middle: structures of the aminoisobutyric acid amino acid and a representative β3-amino acid. Bottom: The four β-peptides, depicted in helical net diagrams with hydrophobic and charged residues highlighted. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis.

In 2015, on the heels of our inquiries from 2013, I instead branched the projects into three different directions. The first was taking the most folded α-peptide from the 2013 lab, PT3, and changing the potential salt bridging groups, examining different pairs. The original used lysine and glutamate arrangements, but now PT3R replaced lysine with arginine, whereas PT3D replaced glutamate with aspartate (Figure 2). Students who worked on these peptides would make comparisons to the original peptide as well as to each others’, and would research what might make one salt bridge more effective than another in helping a peptide fold (9–12). 146

Figure 2. Left: The α-peptides with potential salt-bridging residues shown. Right top: The new β-peptide scaffolds. Right bottom: β-turn/β-sheet scaffolds. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis. Additionally, on the heels of BP1’s success as a highly 14-helical, short βpeptide, I had other teams of students look into the idea of pi-pi stacking as a possibility for helping the 14-helix fold, in place of the valines, in peptide BP1Y (Figure 2). Additionally, on the helical face left free for substitution, I had them change the residues to leucines in an effort to target a particular protein-protein interaction that contributes to angiogenesis in certain cancers. The Hif1α helix has key leucines on one face of its structure, which mediates its binding to protein 147

CH1 (Figure 2, BPYHif) (14). Here, students would test if changing the residues of the scaffold, and attempting a new form of helical control, retained helicity, and if so, the helix could be further used in the future as a potential inhibitor. Finally, the last set of peptides were an α- and a β-peptide with same sequence, designed as a βturn/β-sheet model utilizing D-proline and glycine as turn initiatiors, threonine and tyrosine as potential intra-sheet points of interaction (HPA and HPB, respectively, Figure 2) (15–17). Students examining these would learn about spectroscopy signatures that indicate turn and sheet formations, as well as whether these minimal motifs can actually fold, followed by the comparison, again, between α- and βpeptides. The process begins with a “blind” sign-up, the first day of lab class. In the blind sign-up, each lab partnership chooses a β3-amino acid to synthesize, an αpeptide to synthesize and an α- or β-peptide to study with CD. The synthesized peptide and the peptide studied by CD may not be the same one for each group, which simply makes them study a larger variety of interesting compounds. I premake the peptides in question and purify about half the stock prior to the start of the semester, to ensure that there is enough peptide for them to perform the CD studies; their syntheses during the course are by hand and with the limited time may not yield much. Also, this ensures that there is compound available should they lose some as they work. I do not have them synthesize β-peptides in class as the building blocks are so precious. In the end, students choose their compounds of study in a random fashion, which works well as they have no preconceived notions as to whether their peptide will be “good” or “bad” at folding. This also makes for a fair distribution throughout the class; no one is sure which teams will have the “best” compounds. At this point, they cannot predict much since the choices are made at the beginning of the semester. Throughout the semester they will learn the contributing factors to folding, helicity, and sheet formation and will study peptidomimetics in the literature that were successful. From this, they can make predictions. Overall, their choices for β-amino acid and peptide synthesis will replenish our stocks. These processes will teach them how unnatural amino acids and peptides are made and can lead to further study.

Details of the Lab Modules Module 1: Synthetic Chemistry In the first module, which lasts about four lab periods of three hours each, students perform three synthetic steps to take a purchased α-amino acid, with necessary protecting groups on the N-terminus and side chain to a protected β3-amino acid (Figure 3). The steps can be broken into two reactions, the Arndt-Eistert homologation, which features the use of a specially-made glassware apparatus and the generation of the potentially explosive diazomethane, followed by the Wolff rearrangement, which, mechanistically, occurs via an interesting carbene intermediate (18). Between each step, students perform column chromatography to purify their product for the next reaction. As each team is working with a different β-amino acid, they likely experience unique polarities 148

and slightly differing results, therefore, while performing the same steps, their outcomes can differ and they need to use their insight to determine how to best purify their compounds. Additionally, in this module, students work on the solid phase peptide synthesis of their chosen peptide. Here, more than one group may have signed up for the same peptide, since they only work synthetically with the α-peptide designs. The general process has taken place with iterative steps by hand in a fritted syringe reaction vessel and is accomplished across three lab periods (Figure 3). Students complete the repetitive steps of deprotection and coupling protected amino acids, and end with capping the peptides with an alkyl group and cleaving them from the resin beads, followed by ether precipitation. The final products of both the β-amino acid and the α-peptide are freeze dried to a powder or oil prior to the next steps.

Figure 3. Top: Synthetic scheme to make an Fmoc-protected β3-amino acid. Bottom: Synthetic steps for solid phase peptide synthesis. 149

Module 2: Instrumental Analysis For the following steps, students analyze the results from their earlier work, identifying and purifying their β3-amino acid and α-peptide. In one laboratory period, students will prepare small samples of their protected β-amino acid in inert solvents such as deuterated chloroform and will perform 1H-NMR spectroscopy. A full analysis of chemical shifts, splitting patterns and comparisons to expected outcomes are performed by each team in regard to their unique amino acid. In another lab period, each team prepares a small sample of β-amino acid and, separately, their α-peptides for HPLC and LCMS analysis by dissolving them in a mixture of acetonitrile and water. The HPLC used is from the Agilent Technologies 1260 Infinity series and a semi-preparative column (Grace Vydac, 218TP C18, 250 mm× 10 mm, 10–15 μm) was used. Analysis uses a gradient of approximately 5% acetonitrile in water to 95% acetonitrile in water over thirty minutes. Analytical HPLC is used to estimate the overall percent purity for their reactions in the preparation of both compounds. Additionally, the LCMS provides the students with a look into the accuracy of their synthesis of both compounds, helping them identify the peptide and β-amino acid by mass and what other impurities might be present (e.g. a peptide chain missing an amino acid, or a side product). For mass spectrometry, they use the Agilent Technologies 1260 Infinity with 6130 Quadrupole Liquid Chromatography Mass Spectrometer. Each sample is passed through the analytical column (Agilent poroshell 120, EC-C18, 2.7 μm, 4.6 mm × 50 mm). In a third lab period of this module, students receive the purified peptide I had prepared that they signed up for the first week. Here, they prepare buffer, stocks and dilutions for the upcoming spectroscopy experiments. Prior to lab, students use the online “protein calculator” from the Scripps Research Institute and input their sequences to get a molar absorptivity, ε, in M-1cm-1 for their peptide at 280 nm. In class, they prepare the provided samples and use UV/Visible spectroscopy to obtain an absorbance for their stock concentration, also at 280 nm. Therefore, using Beer’s Law (A = εcl) they can equate the absorbance they achieve (A) with the molar absorptivity they determined prior to class (ε), and the pathlength of the cuvette they use (l, usually 1 cm) to determine the concentration of their peptide in molarity (c). From here they perform a range of serial dilutions of their peptide from higher to lower concentration, in duplicate, so they will be able to examine the effects of concentration on their CD signal, over a range of values. Once prepared, all of their samples are refrigerated, with their crude samples frozen to replenish the stocks, and their purified (provided) samples in diluted form ready for spectroscopy. Module 3: Biophysical Characterization of Peptide Folding Using CD Spectroscopy In the third module, students focus on using CD spectroscopy to assess the degree of secondary structural pattern for their chosen peptide. Students learn about circular dichroism, how circularly polarized light travels through a chiral medium, such as a peptide in buffer, tracing out an ellipse versus a circle, due 150

to the different abosorbance of left- and right-hand components of the light. Alpha helices have a well described observable pattern with a double minmum in ellipticity found around 208 nm and 222 nm, and a calculatable maximum expected ellipticity based on the number of amino acids (19). 14-helices are generally described by a minimum ellipticity around 214 nm with a maximum in the 195 nm region. There are assumptions made about percent 14-helicity, such as fingerprints for short versus long sequences, however a definitive calculation about maxima is not established as for α-helices (8). The small folded turns, which resemble β-sheets, have a matachable signature as well; minimum ellipticity broadly around 216-218 nm and a maximum around 200 nm (20). Students begin this module with a solid background of the factors in peptide amino acid sequences that affect helicity and secondary structure. Therefore, their first activity is to revisit the peptide they chose the first week of lab and, based on its design, hypothesize if they think it will be helical and why, or why not. Additionally, they will discuss whether they think there are quaternary structures forming, therefore, whether they will see a concentration dependence in their CD trials. Also, as the work will contain a thermal denaturation scan as well, they will hypothesize whether their peptide should exhibit a cooperative melting transition or if it simply is not folded well enough to view this. This information becomes a part of their pre-laboratory assignment that is graded individually per student, out of ten points towards their final laboratory grade. Students accumulate data on their samples directly using our Jasco J810 spectropolarimeter. The raw data simply lists the CD readout for each wavelength assessed and students process it for each of the samples they measure. Each team typically has six samples, three different concentrations of peptide in buffer in duplicate for statistical purposes. Students calculate the mean residue ellipticity (MRE) by normalizing their data with a buffer blank, dividing by the number of amino acid residues, the concentration of their peptide in μM and the pathlength of the CD cuvette (8). In their teams, students work to best plot their data and draw conclusions. Their final reports are graded in their teams, with approximately half of the twenty points devoted to their write up of the results and the methods. The other half of their reports were dedicated to a full background description of what they were using CD to accomplish, what the data obtained explains and how it relates to their earlier hypotheses. I impressed upon the students that whether they saw helicity (or some form of β-sheet) was not as important as if they could describe why they were or were not seeing it. With the different iterations of the course we obtained different outcomes, as to be expected. Each has led to exciting revelations that have opened the doors to new inquiry.

Outcomes Results from 2013 The initial work from class this first time it was taught had notable issues. The addition of trifluoroethanol (TFE) to buffer to aid helicity in studying small peptides is fairly common, but we had instead just tried a simple 1X phosphate 151

buffered saline (PBS) solution. Additionally, our peptides all had a free amine and carboxylic acid at the termini, however it is noted in the literature that capping the termini with an acetyl (at the N-terminus) and an amide (at the C-terminus) can aid in stability and folding (21, 22). Student results, therefore, had large error bars across the two trials, and also showed some unprecedented concentration dependence in the β-peptides (Figure 4). Therefore, to continue the work, even after the class had ended, I worked with three research students from my lab, one of whom had been in the class, and we resynthesized the peptides with the notable termini changes and brought them up in buffer containing TFE. Our results were later published in the Journal of Biomolecular Structure and Dynamics, and each student in the class was acknowledged for their early work on the peptides (6).

Figure 4. Uncapped PT3 (left) and BP1 (right) in 1X PBS buffer with no TFE, CD spectroscopy results across three concentrations.

Our results were important for shaping the course for the next time it was taught and also brought new insight into the field regarding primary sequence and secondary structure. Figure 5 shows how the most α-helical peptide indeed was PT3, designed to be most helical, and it did not show concentration dependence, therefore most likely was not forming a higher order structure such as the leucine zipper. PT2, with the Aib residues, and PT4, with the alanine replacement of leucine, were marginally helical whereas PT1, which transplanted the sequence most likely to form a 14-helix in a β-peptide into an α-peptide, showed no discernable helicity. Figure 6 shows the results from the β-peptides examined. Clearly, BP1 is most helical, as its general scaffold was designed as such, but BP2’s small unprecendented 14-helicity showed a bit of “cross-talk” between the α- and β-peptides. BP3 and BP4, both with the sequences most α-helical, showed no discernable helicity when placed in β-peptides. Therefore, general conclusions 152

indicated that there is little overlap between the sequences that form the best α-helices and those that form the best 14-helices. Interestingly, demonstrated in Figure 7, when each of the peptides with some evidence of secondary structure was heated in a CD melt, the α-peptides did not show much evidence of unfolding, most likely due to their low helicity to begin with, wheras BP1 showed the typical linear decrease seen for well-folded β-peptides. BP2, however, showed an odd gain in structure at increasing temperatures, until the final decrease (Figure 7). This is a pattern we would see again for one of the α-peptides studied in the future, and we hypothesized, at the time, that the peptide was not very folded and had gained the kinetic energy to sample the proper folding formation before being completely denatured.

Figure 5. PT1-4 (capped) CD spectroscopy results across three wavelengths in 1X PBS with 10% TFE. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis.

With new, short sequences of peptides developed to initiate folding into αor 14-helices, I was armed with information for the next iteration of the course, where we built upon these designs to test some new and unique peptides. 153

Figure 6. BP1-4 CD spectroscopy results across three wavelengths in 1X PBS with 10% TFE. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis.

Figure 7. Left: Summary of the thermal denaturation of BP1, BP2, and PT2-4. Right: Wavelength temperature scans for BP2. Reprinted with permission from ref. (6). Copyright 2015 Taylor & Francis. 154

Results from 2015

Figure 8. Top left and right are the CD spectroscopy results for PT3R and PT3D, respectively. Bottom is the CD spectroscopy result for HPA. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis.

The outcomes from the second iteration of the class were more immediately informative, with some new insights into secondary structural folding. Both trials of each concentration from the α-peptide groups, working on PT3R, PT3D and HPA, demonstrated evidence of secondary structure (Figure 8). The arginine substitution into PT3 to make PT3R improved helicity, and is hypothesized to do so due to the ability of arginine to make complex salt bridges (23, 24). The aspartate substitution to yield PT3D did not appreciably change helicity. Thermal denaturation of these peptides was analyzed in class, giving a slightly cooperative unfolding to PT3R, which indicates its higher original helicity (Figure 9). Once again, however, an odd increase in helicity upon heating was seen for a peptide, this time PT3D. I, personally, performed a temperature/wavelength scan on this peptide (Figure 9) and saw the minimum at approximately 220 nm “grow in” from 10 °C to about 40 °C, before the denaturation occurred. Here, we hypothesized, 155

based on the shape seen, that this could indicate an intermediate between a 310 helix on its way to the α-helix, as the unique shape of the curve, with large negative MRE at approximately 205 nm indicates 310-helicity (25, 26). HPA, our first short α-peptide sequence designed to fold into a β-turn/β-sheet showed some proper CD signature to indicate this was occurring. This was an exciting possibility from which to build upon for a new scaffold in the future (Figure 8).

Figure 9. Left: Thermal denaturation of PT3R and PT3D, at ~220 nm and 100 μM concentration. Right: PT3D temperature wavelength scans across 70 degrees. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis.

We prepared these results for publication, with the six students who worked on these particular peptides and summarized their results as part of class as contributing authors. Upon review of the submission of the follow-up paper for the Journal of Biomolecular Structure and Dynamics, it was suggested I examine the contributions from TFE across several percentages (Figure 10) and I saw that only 50% TFE truly exacerbates the α-helicity of PT3R, whereas 10% and 50% added helicity to PT3D, however not in as high a quantity. TFE is thought to either directly hydrogen bond with the helix to help stabilize it, or to weaken the interaction of the peptide with water, therefore allowing the intramolecular hydrogen bonds to form more readily (27). Additionally, it was suggested that I take a look at the effects of guanidinium hydrochloride (GuHCl), which breaks up salt bridges, on the helicity of the peptides without TFE (28). Any inference of structure was quickly removed with the presence of GuHCl, which was expected due to the importance of the salt bridging in the helical formations (Figure 10). These results, accumulated by myself outside of class, were added to the final paper and it was published in early 2017. 156

Figure 10. Top left: PT3R CD spectroscopy trials with varying TFE. Top right: PT3R CD spectroscopy trials with varying GuHCl. Bottom left and right: same for PT3D. Reprinted with permission from ref. (13). Copyright 2017 Taylor & Francis. However, as this was a research-based project for a class, not all data from every team was fruitful. The attempts to control β-peptide 14-helicity using tyrosine aromatic stacking led to a complete loss of 14-helicity (Figure 11). We hypothesized that too many changes to the scaffold were made and that, in the future, we can work off of these results and the initial ones for BP1 and try to build a different scaffold. Additionally, the HPB peptide did not show any evidence of β-sheet formation, more than likely not folding (Figure 11). As the α-peptides, however, gave repeatable and unique data, we did publish the results as a follow-up paper in the same journal as prior, with six student authors all from the class with their data and some analysis included.

Assessment of the Research Project Whether students would be published or not, properly predicted the results they achieved, were surprised by the results, or even received negative results, they each wrote a final paper at the end of the course using the Journal of the American Chemical Society (JACS) communication style. They were tasked with working in their lab teams to cover the entire semester’s materials, from synthesis to instrumentation to the biophysical analysis. This required students to find 157

common themes throughout their work, even if their β-amino acid and α-peptide did not coincide with the peptide they examined using CD. Also, it required students to discuss results across teams to make some generalizations about the sequence and structure connection. The final communication-styled paper was also an exercise in brevity as students used the proper JACS template and were only allowed as many figures, references and pages as a true communication. Therefore, they had to work on their quick communication skills and could not write everything from the full semester course but had to limit their reporting to what was imperative and important to include.

Figure 11. Top Left: CD spectroscopy results for BP1Y. Top Right: CD spectroscopy results for BPYHif. Bottom: CD spectroscopy results for HPB.

As mentioned earlier, we did successfully publish two research papers based on the work from each iteration of the course. While the first paper, published online in 2014, with full publication in 2015, reflected work begun in the 2013 class it was completed by myself and my research students so of the authors on the paper only one is a student from the class. However, our most recent publication, online as of January 2017, comes from results from the 2015 version of the course and features six students from the class, which amounted to half of the class! As 158

publication occurred long after the courses were completed, I kept that separate from any part of grading the students. Some of the students with top grades in the course were not actually authors on the paper, as the inclusion of students as authors had more to do with their successful work on the compounds they picked randomly, as well as their analyses, rather than their academic achievement.

“Research” Incorporated into the Classroom While much of the focus has been to discuss the laboratory outcomes of the course, in the interest of incorporating research aspects to the class across the board, the lecture portion of the course had a number of research aspects, as well. I taught from the primary literature, pulling on papers from the past couple of decades, including current work, to immerse the students in the field of Chemical Biology.

In-Class Assignments Throughout the semester, students had four in-class assignments where they would work in groups of 2-4 students and have about 20-25 minutes to answer open-ended questions based on the course materials. The best three out of four of these assignments would count towards their final grade, and they were generally out of 10 points with multiple, possible well-thought-out answers accepted. For example, in the 2015 edition of the course, students began with an assignment that revolved around a protein they had not heard about before, and were told to use a process they learned about, in the biological synthesis and synthetic biology section, to make up the protein, including the details of how they would use the process and all the steps. Then, they had to discuss the benefits of the process they chose. In a later assignment, students were provided a β3-peptide general scaffold with X written at all of the amino acid locations. They had just learned about a protein-protien interaction involved in the Severe Acute Respiratory Syndrome (SARS) virus fusion mechanism with a host cell and were tasked with designing two β-peptides that would inhibit this event, keeping in mind controlling both the helicity of the scaffold and recognition of the protein target. They needed to describe their reasons, as well, for placing the different residues where they put them on the scaffold. As this was actually unpublished work from my own graduate thesis, I was curious to see if they would come up with the very compounds I had made and studied, or if they would introduce new ones, which led to lively discussion! A third assignment involved students evolving an enzyme that could cleave a link between mRNA and puromycin in mRNA display, a technique covered in the common techniques and assays section of the class. With hints, I wanted them to focus on how to separate the mRNA that codes for the enzyme versus other, nonfunctional mRNAs in a library. Their final assignment involved yeast two hybrid screening, another assay and technique they had learned about. They were asked to describe what type of screen they would 159

choose, how it would react and how they would use it to generate an inhibitor to a particular well-studied protein-protein interaction involved in cancer. There were several options, leaving the answers open for students to combine assay and peptidomimetics work. These types of assignments were useful for students in that they applied knowledge from the course materials, posed novel “research” problems for students to work at solving, and also afforded them the opportunity to work in different groups. Often, I would hear student debating what they thought was a good answer, and why, and, as there were frequently more than one way to answer the questions, it was interesting to moderate such debates. Through this, students really “owned” the materials they were learning.

Literature Research and the Final Exam As a final project, students were given the task to work in their lab partnerships outside of scheduled class time and research the literature on a subtopic within the area of epigenetics. They signed up for a subtopic (i.e. environmental epigenetics, epigenetic therapeutics, epigenetics and inheritance, cancer epigenetics, epigenetics and stem cells and gene regulation and chromosome biology) on our course website, so each team had a unique area, and were tasked with giving a presentation on their topic the final week of class. Students were given 20-25 minutes to “teach” the class about their area, building off of the previous teams if necessary, and introducing a question that they would answer throughout their presentation. This question needed to be test-type in nature and would be included on their final, therefore each team was responsible for putting effort into their communication of the information as well as how the information would be assessed. Overall, their presentations needed to go through the general background of their subtopic, definitions, how the topic is approached technically, and the broader impacts of the topic, with several refernces from the primary literature. The test questions the students came up with were large in scope, ranging from “describe an advantage or disadvantage for a specific technique,” to “choose a therapeutic strategy and describe what it does,” and “which modification is best” for a specific aim, as well as “describe a mode and mechanism for epigenetic inheritance.” In addition, I created some multiple-choice and fill-in-the-blank type questions based on their work, and overall the epigenetics portion of their final was approximately 15% of the total covered materials. They so enjoyed taking the reins, I would like to increase this in the future! In both iterations of the course students showed an immersion into their literature research and delighted in hypothesizing where the field would go next. A couple of students, who continued on to graduate work after graduating from TCNJ, actually mentioned how the Chemical Biology course, and specifically their review of epigenetics, gave them direction in their graduate research in the field!

160

Grading the Course Overall, grading for the course was divided into several subsections. The research-inspired in-class assignments were 15% of the total grade (with the best 3 of 4 counting towards that), the two class exams, that pulled information from lecture from the primary literature, were 25% of the grade in total, the class presentation based on the literature research in epigenetics was 10%, the laboratory component, with its unique research-based structure, was 25% and the final exam, which the students participate in writing, was 25%. For the lab component, I did one notebook check during each module, looking for proper data collection, hypotheses and predictions, safety concerns described, and at the close of each module students wrote up a lab report. In the end, recall, students also submitted a final JACS communication-styled paper that summarized their entire semester’s lab work. Research, whether the act of studying something new through hands-on experimentation or by reading the literature and making conclusions based on deep and directed thought on the topics, is a part of each of these components in the lab and classroom. Therefore, through this course, and others in similar style at TCNJ, a valuable research experience can be brought to student participants.

Summary and Future Directions Using research-based laboratory projects asking novel questions with unique outcomes is a hallmark of the Chemical Biology course’s laboratory work. In the classroom, open-ended in-class group work, and the literature research of the final topic that students present and teach the class also brings research out of the lab. Together, from these research-aspects incorporated into the course, students contribute to their final by not only assimilating knowledge on a new topic but developing a way in which they and their peers can be assessed. Additionally, as a result of both iterations of the course, experimental materials were published in a peer-reviewed journal, adding to the breadth of the field. Taken as a whole, these facets have led to a successful Chemical Biology course! In the future, I will extend the topics in the classroom, with new in-class assignments and perhaps more possibilities for such group work. I will have the student presentations and “final questions” count more to their final grade as their work on these has proven exemplary. For the lab, building off of the helical scaffolds, we will continue to search for new ways to control small 14-helical β-peptides, and revisit our protein-protein target involved in cancer. As for the α-helices, as well as β-turn motifs, we will develop these short sequences further, examining well-tolerated substitutions and move into assay screens that could easily accommodate a classroom of students, such as antimicrobial vetting. Students will continue to train on the newest departmental instrumentation and, at the close of the future course I would have students themselves come up with the directions for future class iterations. What do they think is important to peptidomimetics and how would they design new compounds and pose new questions to incoming students? 161

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Chapter 10

Translation of Chemical Biology Research into the Biochemistry Laboratory: Chemical Modification of Proteins by Diethylpyrocarbonate Laura M. Hunsicker-Wang1 and Mary E. Konkle*,2 1Department

of Chemistry, Trinity University, San Antonio, Texas 78212, United States 2Department of Chemistry, Ball State University, Muncie, Indiana 47306, United States *E-mail: [email protected]

Chemical modification of proteins is an ideal system to use to integrate research and teaching. A common chemical modifier, diethyl pyrocarbonate (DEPC), was used to probe the reactivity of the ligating histidine residues in the Rieske protein toward small molecules in a research laboratory. The DEPC work in the research lab inspired a new teaching lab using DEPC to monitor modification of lactate dehydrogenase to teach students to use spectroscopic measurements, structural analysis, and modeling to predict where modifications would be found on the protein. In the original studies in the research lab, saturating amounts of DEPC were added to the samples, and so collection of new data by Biochemistry lab students using less than saturating amounts was used to inform the research lab. The results from the teaching lab led to a several new projects in the research lab that resulted in a publication. These examples demonstrate the usefulness of chemical modification in the research lab, the teaching lab, and the integration of of research and teaching.

© 2018 American Chemical Society

Introduction Today’s professor at a primarily undergraduate institution (PUI) has to maintain the precarious balance between research and teaching for both their own personal career development as well as for the benefit of their students. One way to alleviate this tension is to bring research projects into the teaching laboratory. Subsequently, one can use the results from the teaching lab to inform the next steps in the research laboratory. We present a case study of how chemical modification in the research laboratory presented numerous opportunities, both in pedagogical content and additional novel research discoveries (Figure 1).

Figure 1. Overall workflow.

Background One way to determine the functional impact of a particular amino acid in a protein is by methodically changing the amino acid identity at the genetic level through site-directed mutagenesis. However, the expense of the materials and equipment needed for this technique often make it prohibitive for the typical undergraduate biochemistry laboratory course. An alternative and complementary technique to introduce the effect(s) of changing the structure on protein function in the Biochemistry Laboratory is chemical modification. Chemical modification has the benefit of expanding the available organic functional groups past those encoded by DNA. Additionally, it provides a welcome pedagogical link between material taught in organic chemistry courses and its applications to the biochemistry laboratory. In contrast to the reactions carried out in an organic chemistry laboratory, the introduction of a macromolecule such as a protein introduces heterogeneity of reactivity that is both vexing in characterizing the products and rich in teaching opportunities. The reactivity of amino acid side chains in a protein can be influenced by a number of biophysical characteristics such as solvent accessibility, hydrogen bonding, and local pKa values. 166

Diethyl pyrocarbonate (DEPC) is a small molecule (Figure 2) that readily reacts with free amines in a pH-dependent manner and readily degrades in water to ethanol and carbon dioxide, two molecules that are relatively innocuous to biomolecules. Because many buffers used in the Biochemistry Laboratory contain a free amine, like tris(hydroxymethyl)aminomethane (Tris), care needs to be taken to find an inorganic molecule or non-nitrogen containing organic molecule which can stabilize at the desired pH but will not interfere with the experiment. This allows for an excellent teachable moment about characteristics of a buffer and the importance of knowing its chemical structure in addition to its common name and pKa.

Figure 2. DEPC molecule and the histidine adduct in the Rieske protein.

DEPC is most reactive towards the amino acids lysine and histidine (in the deprotonated state), but also reacts with cysteine and tyrosine residues. The lysine-DEPC adduct is irreversible in contrast to the carboethoxylated histidine-DEPC adduct which is reversible under high pH conditions or upon exposure to hydroxylamine (1). The histidine-DEPC adduct has the advantage of being a chromophore that absorbs at λ = 240-250 nm (ε = 3200 M-1cm-1). Since DEPC-reactive residues are often key in the mechanism of enzymatic activity, DEPC is used as both a probe for active sites that have no structural information available and/or as a deactivator (e.g. DEPC treatment of water is used to deactivate RNAse). The sites of modification can be identified by proteomic analyses using tryptic digestion and LC-MS/MS analysis in an analogous fashion to identification of post-translational modification (2–9). 167

Research Genesis Complex III in the respiratory electron transport chain accomplishes the oxidation of quinol and transfer of electrons to cytochrome c (10). One subunit of Complex III is the Rieske protein (RP), which contains a [2Fe-2S] cluster that is ligated by two cysteine and two histidine residues (11, 12). RPs couple electron transfer with proton movement across the membrane and thus have pH-dependent reduction potentials. These potentials can be as high as ~ +475 mV at low pH. Thermodynamic characterization of the Thermus thermophilus Rieske protein (TtRp) indicates a low-pH reduction potential of +161 mV with pKox1, pKox2, and pKred values of 7.85, 9.65, and 12.5, respectively (13). When RP is reduced, the electron is localized to the Fe atom bound to the histidine (10). At the time of the original experimental design, the molecular determinant(s) of the pH-dependence was unclear. The Hunsicker-Wang Laboratory, with Dr. Konkle as a postdoctoral researcher, decided to use DEPC as a probe for deprotonated histidine residues that also ligated the [2Fe-2S] cluster (Figure 2).

Original Research Outcomes A truncated form of RP from Thermus thermophilus (truncTtRP) and a mutant that removes the histidines that are not coordinated to the cluster, were modified at a variety of pH values (6.0, 7.0, 8.0) and the modification was monitored with both UV-visible and circular dichroism spectrophotometry over time. The accumulation of the His-DEPC chromophore was observed at λ= 250 nm. Additionally, the impact of modification on the environment local to the [2Fe-2S] cluster can be monitored by ligand to metal charge transfer (LMCT) bands (Figure 3). The DEPC-His chromophore signal (Figure 3b) does not discriminate between the ligating and non-ligating histidine residue. However, the LMCT bands report solely on modification of ligating residue(s) (Figure 3a) (5, 6).

Implementation into the Teaching Lab There were several factors that made translating this experiment into the Biochemistry Teaching Lab feasible. Spectroscopy is a satisfying module in the biochemistry teaching laboratory because results are evident quickly, and numerous companies supply accessible modules for UV-visible spectroscopy in the teaching laboratory. DEPC is an inexpensive and readily available reagent from several commercial sources. The side-reactions (mostly with water) result in the generation of carbon dioxide gas and ethanol, both of which are non-toxic. From an enzymology perspective, many enzymes contain an active site histidine or lysine residue. Therefore, DEPC can be used to illustrate the impact of chemical modification on enzymatic activity and to reinforce the concept of enzyme mechanism taught in a lecture setting. 168

Figure 3. a) Difference spectra of truncTtRP modified by DEPC and monitored by UV-visible spectroscopy. The arrows denote the DEPC-His chromophore and LMCT band. The raw data is seen in the inset. b) The rate of modification at pH 6.0 (triangles), 7.0 (circles), 8.0 (squares). Error bars shown are the S.E.M. of n=3 and the dashed lines are shown for illustration only. Adapted with permission from ref. (6). Copyright 2010 ACS.

Implementation Implementation 1: Modification of Lactate Dehydrogenase by DEPC Lactate dehydrogenase (LDH) is an enzyme of recent interest as a serum biomarker in cancer and HIV as well as a target for treating diabetes (14–17). There are numerous structures of LDH isozymes from a variety of tissues. The robust and well-documented spectrophotometric assay for LDH activity 169

of monitoring NADH production at λ = 340 nm makes it an ideal model system by which to examine the impact of DEPC modification on enzyme structure/function. The experiment consisted of two sections; pH dependence of chemical modification and enzymatic activity assay. The chemical modification (Week 1) and enzyme activity assays (Week 2) was done in groups of two-three students (Figure 4).

Figure 4. Workflow for determining the impact of DEPC modification on LDH enzymatic activity.

LDH Chemical Modification Each group made a buffer of either pH 6.0, 7.0, 8.0, and 9.0 to dilute a control sample of an aliquot of LDH or a matched sample to be modified by DEPC (Sigma Aldrich). The students were responsible for the calculations and pH stabilization of their assigned buffers. In our case, the LDH was purified from a beef heart as part of previous lab exercises, but it is also commercially available. The reaction was begun by the addition of 5 μL (neat) of DEPC to a microcuvette (Starna) containing LDH diluted into the appropriate buffer (375 μL total volume) and was monitored using an Agilent 8453 Spectrophotometer. The spectrophotometer was used to measure the absorbance at 280 nm (to monitor intrinsic protein absorbance of tryptophan, phenylalanine, tyrosine residues) and 250 nm (to monitor the accumulation of the histidine-DEPC adduct chromophore) every 15 s in Kinetics mode after the addition of DEPC. The appropriate buffer was used as 170

a blank. Each sample (both treated and untreated) was simultaneously dialyzed using separate labeled dialysis cassettes (Thermo Fisher Scientific) (eight total samples with control and modified samples at four different pH values) into one 2 x 4 L volume of 100 mM phosphate buffer at pH 8.0 at 4° C over 2 x 2 hour periods to remove ethanol from the samples. Due to the time constraints of the laboratory period, the instructor may need to change the dialysis buffer at the appropriate time. The samples were stored at -80°C for one week. For the data analysis for Week 1, the students must plot spectra for individual time points and contribute data to a shared plot of ΔAbs250 vs. time (Figure 5a).

Figure 5. Representative data from the DEPC modification of LDH in the teaching laboratory a) Kinetics of His-DEPC adduct accumulation at pH 6 (triangles), pH 7 (squares), pH 8 (triangles) b) Impact of DEPC modification at pH 6 (light gray), pH 7 (dark gray), pH 8 (open) on enzymatic rate of LDH. Errors shown are S.E.M. 171

LDH Activity Assay Briefly, the students were asked to titrate the amount of control LDH sample needed to achieve a rate between 0.2 – 0.4 ΔAU/min at λ = 340 nm (to monitor NADH degradation) upon the addition of pyruvate and NADH. All kinetic assays were done at 37° C in microcuvettes (375 μL total volume used) on an Agilent 8453 Spectrophotometer in Kinetics mode. Students assayed the modified samples using the same volume of LDH enzyme for comparison. The data analysis for Week 2 was that students must contribute data to a shared plot of activity vs. modification pH (Figure 5b).

Implementation 2: Molecular Modeling of Modification The sites of modification in truncTtRp by DEPC were analyzed using mass spectrometry (6). The ligating residue His154 was identified as being modified. Additionally, the sites of modification not accessible by spectroscopy (mostly lysine residues in this case) were also identified (Figure 6). One lysine residue in truncTtRP, Lys95, was not observed as modified. Lys95 was observed to be in either hydrogen-bonding or ion-pairing interaction with a glutamate residue in crystal structures of TtRP. Since crystal structures of LDH are available, students can be asked to predict the reactivity of histidine and lysine residues based on solvent accessibility and existing molecular interactions using free molecular modeling software (e.g. UCSF Chimera Package). The concept of solvent accessibility was difficult for students to infer from the crystal structures as only 20% of students in a representative lab section had a completely correct answer, 60% had a partially correct answer and 20% received no credit. This study presents an opportunity to improve upon pedagogy and consider additional tools beyond static crystal structures. The students were also asked to identify catalytic residues that would be vulnerable to DEPC modification. In contrast to the solvent accessibility question, 80% of the students answered this question correctly. In summary, the molecular modeling module was completed outside of class and familiarizes students with modeling software, requires critical analysis of lab results, and reinforces the structure/function paradigm.

Implementation 3: Refining Original Research Experimental Conditions The Rieske protein was a wonderful target for DEPC. There are strong spectroscopic changes that can be observed upon modification and therefore could be monitored using techniques that are already taught in a Biochemistry laboratory. All of the original studies were performed using exceedingly high amounts of DEPC (10 µL of neat DEPC into a 300 µL sample) and thus a laboratory experiment was designed for the Biochemistry lab to test what would happen if less DEPC was used. Students were divided into groups and assigned a set of conditions to test the DEPC reaction. They were given one of 2 different pH values and either 2.5 or 5 µL of DEPC added. Students then collected both 172

UV-Visible and CD spectra, and the groups shared their data to develop a profile of the effect of pH. Ultimately, what the students and the instructor learned was that even decreasing the DEPC amounts by ½ to ¼, the resultant spectra are identical. Therefore, in order to fully probe the effect of different amounts of DEPC, a method to achieve lower concentrations was needed. Fresh dilutions of the near DEPC into 200 proof ethanol proved to be the way to attain the lower number of equivalents. This method was then applied to the wild type protein and the H120Q/H162Q mutant in the research lab. Thus, the full study was supported by work carried out in the Biochemistry teaching lab (7).

Figure 6. Modification of truncTtRP identified using proteomic analysis. a) Crystal structure of TtRP illustrating the residues modified by DEPC b) Table describing the modified residues of TtRP in various forms. Adapted with permission from ref. (6). Copyright 2010 ACS.

Analyzing Modified Protein Using Isoelectric Focusing Gels, UV-Visible and CD Spectroscopies Chemical modification of proteins alters many properties of the proteins, and thus several analytical methods are available to characterize the modified protein. It is powerful to use these techniques in the context of a complex reaction rather than in a “canned lab”. The techniques that were utilized were UV-Visible and CD spectroscopies and Isoelectric Focusing gels. UV-Visible spectroscopy was used to monitor the formation of the DEPC adduct since there is a chromophore that forms upon reaction with the histidine (1). CD spectroscopy was used to monitor the changes at the [2Fe-2S] cluster, including the reduction that occurs following modification by DEPC (7). The students each monitored the two different reactions using both techniques and 173

could compare the effect of either pH or the different amounts of DEPC. For many students, CD is not a technique that they have typically used and if they have, it is usually monitoring the low wavelength regions for changes in secondary structure. Thus, the laboratory gives them an opportunity to use CD in a different way and to explore its capabilities in the context of chemical modification. It is also powerful for the students to compare the UV-Visible and CD results since the two techniques report on different aspects of the modification. The UV reports on the histidine-DEPC adduct formation, whereas the majority of the spectral change in the CD derive from the reduction of the cluster. Examining how these correlate leads to a deeper appreciation of the complexity of the reactions. Isoelectric focusing (IEF) gels were also utilized to characterize the products of the chemical modification. DEPC reacts with the lysines and the N-terminus which will lower the isoelectric point (pI) of the protein, resulting in a change that can be visualized using IEF gels. The students saved their CD samples after modification (stored at -80 C), and then loaded 5 µL of the reaction mixture onto the IEF gel. They then compared those samples to unmodified protein and to a ladder to determine the pI of the unmodified protein and how the pI changed. They could also determine if all of the protein sample was modified by noting if any protein with the pI of the unmodified protein was present on the gel. This exercise was especially helpful for students to appreciate the amount of modification that was taking place, as nearly all of the sample changed pI. As a multi-week lab, students were assigned one of two weeks to come to the lab and conduct the UV and CD experiments. The data from all of the students was shared so that the students could compare their data to others in the lab. Then in the final week, all the students were in the lab together running the IEF gels. Once all the experiments were completed, the students wrote up the report using their CD and UV data, the shared CD and UV data and their IEF gels.

Implementation 4: Extending to Other Proteins in Research and Teaching The CuA protein has also served as a great target for DEPC modification. The CuA protein is subunit II of cytochrome oxidase (Complex IV) of the ETC. It contains a dinuclear copper cluster where the two copper ions are bridged by two cysteines, each copper is ligated by one histidine, and a methionine and a glutamine carbonyl from the backbone complete the ligation environment (Figure 7) (18). Given its structural similarity to the Rieske protein, it is a good candidate for reactivity with DEPC. The protein was therefore subjected to reaction with 400 eq of DEPC at pH values 5-9. The reaction was again monitored using UV-visible and CD spectroscopies. The reaction is more extensive under higher pH reaction conditions which is consistent with a reactive histidine residue. A mutant, which removes the non-ligating histidine residues, H40A/H117A, was also tested. There was still modification when there were the only ligating histidine residues. Thus, all the data points to a ligating histidine being modified (19). In the research lab, all of these studies used 400 equivalents of DEPC, which is at the solubility limit of DEPC. 174

Figure 7. a) Structure of the ba3 cytochrome c oxidase from Thermus thermophilus (1XME). b) The CuA metal site with ligands and the important Asp 111 shown.

In order to take this project into the teaching lab, a laboratory protocol was developed in order to test what would happen when the protein was subjected to 1-200 equivalents of DEPC. Different groups were assigned lower values of DEPC. The teaching lab students discovered that the spectroscopic changes that occur in the CD spectrum happen with as few as 3 equivalents of DEPC! Thus, a very small amount of DEPC is needed to observe changes at the copper cluster. This result was completely unexpected, considering that for the Rieske protein, very little change in the spectrum was observed with 1-6 equivalents (7). Like the Rieske lab, students also analyzed their data using IEF gels and noted that the pI decreased with modification but with the lower amount of DEPC added, some unmodified protein remained. These results have led to further studies in the research lab which corroborate the results of the teaching lab and are being incorporated into an upcoming publication (19). In the biochemistry lab, each experiment is graded by students turning in a report that consists of results and discussion sections that are modeled off of manuscripts. Students present the data that they collect in the results section and then are asked to put their data in context in the discussion section. The discussion section is facilitated by the students answering a series of discussion questions. The answers to the questions are all written in paragraph form. These types of questions necessitated students looking beyond their own data and also forced them to use multiple types of data to understand what is happening in the reaction. 175

Assessment Discussion Questions from the CuA Chemical Modification (Similar Questions Were Asked about Rieske) A file with all the groups’ sets of UV and CD data was compiled and a plot of 240 vs. the number of equivalents was produced and included in the PowerPoint file. The file was put on a learning management system and the entire section had access to it. Answer the following questions looking at the data from across the section

1.

2.

3.

Looking at the A240 nm data from the UV allows us to determine the rates of modification (from A240 nm) since the 240 nm absorption is due to the histidine-DEPC adduct. Looking at the increase in equivalents across the section, what trends do you see in the rates of modification? Looking at the 440 nm data from the CD allows us to look at the changes localized at the cluster due to the modification. This statement is true because all of the changes in ellipticity in the visible region of the CD originate from the Cu2S2 cluster. Looking at the increase in equivalents across the section, what trends do you see in the changes at the cluster? Do the changes in the UV and CD correlate?

One desired student learning outcome for the lab would be a deep understanding of the techniques that are taught in the lab. To this end, the chemical modification should help students to better grasp the techniques that are used to analyze the products of the chemical modification. CD, UV-Vis and IEF gels are used for the analysis and the students that perform the chemical modification should perform better on an extension question on a final exam that asks them to explain a phenomenon that they had observed, but that we had not discussed directly. In two different semesters of the biochemistry teaching lab, students were asked the same question (with a slight change to wording to be appropriate for the given lab) about IEF gels on the final exam. One semester we used the IEF gel to help analyze the results of the chemical modification. In the other semester, the IEF gel was used to identify an unknown protein from a set of possibilities, using pI as the determinate factor. The question was worth 5 points and is given below. In the year when chemical modification was used, the average score was 3.6 out of 5, where the average answer from the year chemical modification was not used was 3.2. Thus, there is preliminary evidence that the use of chemical modification may help students better understand IEF gels. It will be interesting to compare more years of exams to see if this is a reproducible trend.

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Final exam questions: Semester without chemical modification (n= 18) You all ran pI gels of several proteins. If you looked carefully at the isoelectric focusing gel while it was running, you could actually see the brown bands of the heme-containing Myoglobin progressing through the gel. If you ran an SDS-PAGE gel of the same sample at the same concentrations, you would not be able to see those same brown bands progressing through the gel. Explain why using 1-3 sentences. Semester with chemical modification of the Cu A protein (n=10) If you looked carefully at the isoelectric focusing gel while it was running, you could actually see the purple bands progressing through the gel. If you ran an SDS-PAGE gel of the same sample at the same concentrations, you would not be able to see those same purple bands progressing through the gel. Explain why using 1-3 sentences. A second way to assess student engagement is to look at course evaluations. The following is an example quotation from the course evaluation of laboratory course when the chemical modification of Rieske was used. “I really liked the rieske lab. I think labs are far to guided at all levels with results able to be calculated before the lab is even done. I think more labs should be open ended and working without a net because that’s how it happens in "the real world". A second assessment of the student reception of the material is through a question on the final exam. The last exam question is always “What was your favorite lab and what was your least favorite lab this semester and why? (There is no wrong answer but you must answer which one AND why).” In the year when we did the chemical modification of CuA, 50% of the class answered that the chemical modification was their favorite lab or tied for their favorite lab. For perspective, the following year when chemical modification was not in the lab, the “favorite lab” was chosen by 28% of the class. Thus, a higher percentage of students chose the chemical modification lab as their “favorite” one.

Ideas for Extensions and Limitations There are several ways to implement or extend the given examples directly. One could envision modifying other commercially available proteins that would have critical histidine residues, such as carbonic anhydrase, RNAse, DNAse, etc. The limitations here are that the proteins need to have solvent accessible histidine residues to detect a spectroscopic change or a relatively open active site to detect changes in enzymatic activity after modification. For example, cytochrome c has an active site histidine, but it is not accessible and DEPC has been shown to only react with the surface histidine residues (4, 20). Perhaps more significantly, the authors believe that this can serve as an example of how to integrate the research and teaching purposes for both faculty and students. 177

References 1. 2.

3.

4.

5.

6.

7.

8.

9.

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11.

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13.

Miles, E. W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977, 47, 431–442. Borotto, N. B.; Zhou, Y.; Hollingsworth, S. R.; Hale, J. E.; Graban, E. M.; Vaughan, R. C.; Vachet, R. W. Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry. Anal. Chem. 2015, 87, 10627–34. Zhou, Y.; Vachet, R. W. Diethylpyrocarbonate labeling for the structural analysis of proteins: label scrambling in solution and how to avoid it. J. Am. Soc. Mass Spectrom. 2012, 23, 899–907. Zhou, Y.; Vachet, R. W. Increased protein structural resolution from diethylpyrocarbonate-based covalent labeling and mass spectrometric detection. J. Am. Soc. Mass Spectrom. 2012, 23, 708–17. Karagas, N. E.; Jones, C. N.; Osborn, D. J.; Dzierlenga, A. L.; Oyala, P.; Konkle, M. E.; Whitney, E. M.; David Britt, R.; Hunsicker-Wang, L. M. The reduction rates of DEPC-modified mutant Thermus thermophilus Rieske proteins differ when there is a negative charge proximal to the cluster. J. Biol. Inorg. Chem. 2014, 19, 1121–35. Konkle, M. E.; Elsenheimer, K. N.; Hakala, K.; Robicheaux, J. C.; Weintraub, S. T.; Hunsicker-Wang, L. M. Chemical modification of the Rieske protein from Thermus thermophilus using diethyl pyrocarbonate modifies ligating histidine 154 and reduces the [2FE-2S] cluster. Biochemistry 2010, 49, 7272–81. Li, S. Y.; Oyala, P. H.; Britt, R. D.; Weintraub, S. T.; Hunsicker-Wang, L. M. Reactive sites and course of reduction in the Rieske protein. J. Biol. Inorg. Chem. 2017, 22, 545–557. Jin, X. R.; Abe, Y.; Li, C. Y.; Hamasaki, N. Histidine-834 of human erythrocyte band 3 has an essential role in the conformational changes that occur during the band 3-mediated anion exchange. Biochemistry 2003, 42, 12927–32. Hondal, R. J.; Ma, S.; Caprioli, R. M.; Hill, K. E.; Burk, R. F. Heparinbinding histidine and lysine residues of rat selenoprotein P. J. Biol. Chem. 2001, 276, 15823–31. Trumpower, B. L.; Gennis, R. B. Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: the enzymology of coupling electron transfer reactions to transmembrane proton translocation. Annu. Rev. Biochem. 1994, 63, 675–716. Baum, H.; Silman, H. I.; Rieske, H. S.; Lipton, S. H. On the composition and structural organization of complex 3 of the mitochondrial electron transfer chain. J. Biol. Chem. 1967, 242, 4876–87. Rieske, J. S.; Hansen, R. E.; Zaugg, W. S. Studies on the electron transfer system. 58. Properties of a new oxidation-reduction component of the respiratory chain as studied by electron paramagnetic resononace spectroscopy. J. Biol. Chem. 1964, 239, 3017–22. Konkle, M. E.; Muellner, S. K.; Schwander, A. L.; Dicus, M. M.; Pokhrel, R.; Britt, R. D.; Taylor, A. B.; Hunsicker-Wang, L. M. Effects of pH on the 178

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Rieske protein from Thermus thermophilus: a spectroscopic and structural analysis. Biochemistry 2009, 48, 9848–57. Adeva-Andany, M.; Lopez-Ojen, M.; Funcasta-Calderon, R.; AmeneirosRodriguez, E.; Donapetry-Garcia, C.; Vila-Altesor, M.; RodriguezSeijas, J. Comprehensive review on lactate metabolism in human health. Mitochondrion 2014, 17, 76–100. Gallo, M.; Sapio, L.; Spina, A.; Naviglio, D.; Calogero, A.; Naviglio, S. Lactic dehydrogenase and cancer: an overview. Front Biosci. 2015, 20, 1234–49. Mahapatra, D. K.; Bharti, S. K.; Asati, V. Chalcone scaffolds as anti-infective agents: structural and molecular target perspectives. Eur. J. Med. Chem. 2015, 101, 496–524. Miao, P.; Sheng, S.; Sun, X.; Liu, J.; Huang, G. Lactate dehydrogenase A in cancer: a promising target for diagnosis and therapy. IUBMB Life 2013, 65, 904–10. Williams, P. A.; Blackburn, N. J.; Sanders, D.; Bellamy, H.; Stura, E. A.; Fee, J. A.; McRee, D. E. The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 A resolution. Nat. Struct. Biol. 1999, 6, 509–16. Devlin, T. H., C.; Hunsicker-Wang, L. M. TX DEPC Modification of CuA Protein; Trinity University, San Antonio, 2017. Konopka, K.; Waskell, L. Chemical modification of cytochrome b5, cytochrome c and myoglobin with diethylpyrocarbonate. Biochim. Biophys. Acta 1988, 954, 189–200.

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Chapter 11

Leveraging Student Interest in Environmental Topics for Undergraduate Research in an Interdisciplinary Environmental Research Cluster Neelam Khan, Sang H. Park, David P. Pursell,* and Kathryn Zimmermann School of Science and Technology, Georgia Gwinnett College, 1000 University Center Lane, Lawrenceville, Georgia 30043, United States *E-mail: [email protected]

Undergraduates are interested in applied research focusing on environmental issues. To capitalize on this interest, four faculty members at Georgia Gwinnett College formed an interdisciplinary environmental research cluster so as to encourage undergraduate participation in research on these issues. The four cluster faculty include a physicist, chemist, environmental engineer, and environmental toxicologist. The cluster faculty support undergraduate environmental research by sharing resources and students while working on varied aspects of a suite of projects. Over the past five years, more than 50 students have participated in environmental cluster research projects. Student researchers have been 62% women and 48% under-represented groups, as defined by the National Science Foundation. This chapter briefly highlights three recent projects: river water analysis through popular recreational canoe and kayak routes in Georgia; toxic substances air sampling and environmental justice; and production of biodiesel from waste cooking oil and grease from campus dining operations. Biodiesel project details are then presented to illustrate the depth of student research experiences.

© 2018 American Chemical Society

1. Introduction Georgia Gwinnett College (GGC) is a 4-year, public college in the University System of Georgia. The college was founded in 2006 and has rapidly grown to more than 12,000 students in just over 10 years. Admissions are non-competitive and most students are commuters who live and work in the greater metropolitain Atlanta area of north Georgia. The college is comprised of five schools, including the School of Science and Technology (SST), which offers academic programs in biology, chemistry, environmental science, exercise science, information technology, and math. As part of their academic program, all SST students complete a one semester internship with local industry/agency or independent research under the direction of faculty. As a new college, research facilities, instrumentation, and funding support are limited. In addition, 4-year college faculty have heavy teaching and service loads which inhibit, in a practical sense, faculty effort on research activities. While many studies highlight the significant and positive contributions of undergraduate research to increasing STEM retention (1–5), engaging our students in meaningful independent research projects presents logistical and financial challenges for faculty and the administration. To help overcome these challenges, four faculty (condensed matter physicist, environmental engineer, chemical physicist, environmental toxicologist) formed an environmental research cluster. Cluster faculty re-tooled their research programs to correspond to widespread student interest in environmental topics. The cluster faculty share facilities, instrumentation, resources, and grant proposal preparation. Each student has one official mentor, but to capitalize on faculty research expertise, cluster facuty work with all students on the varied aspects of their projects. This approach allows faculty to pool resources (both financial and temporal) to incorporate many students working on a project each semester. The cluster approach has proved successful in recruiting student researchers from GGC and the Gwinnett School of Math, Science, and Technology (GSMST), the local public, science magnet high school. From 2012 to 2017, 56 students of have conducted environmental research as shown in Table 1.

Table 1. Research Student Demographics Students

M

F

Wt

Bk

As

Hi

GGC

35

9

26

10

10

10

5

GSMST

21

9

12

2

8

10

1

Total

56

18

38

12

18

20

6

NSF Demographic Categories: Male (M), Female (F), White/non-Hispanic (Wt), Black/African-American (Bk), Asian (As), White/Hispanic (Hi). Other catagories not represented among our students

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Students have driven the research agenda by selecting topics and projects of particular interest to them. Cluster faculty then provide support (safety, techniques, and instrument training; supplies, chemicals, and solvents; project objectives, timelines, and reporting) to make the projects scientifically and experimentally meaningful student research experiences. Faculty meet regularly with the students throughout the semester to provide students hands-on assistance with particularly challenging techniques, work through issues as they arise, help maintain student focus on project objectives to ensure progress, and provide encouragement as students develop their research skills. A sampling of project titles listed below illustrates the breadth of student environmental interest.

• • • • •

• •

• • • •

Georgia Adopt-A-Stream Water Quality Monitoring Project Community Innovations Project-Air Quality and Environmental Justice Analysis of Waste Oil and Grease from the Campus Chick-fil-A for use as Biofuel Synthesis of Biodiesel from GGC’s Used Oil Using Size Averaging to Evaluate Heavy Metal Accumulation in Shells of Asiatic clams (Corbicula fluminea) as Biomarkers for Environmental Toxicity Designing Methods for Phytoremediation of Lead in the Georgia Gwinnett College Community Garden Elemental Analysis of the Organ systems of the Fetal Pig via Flame Atomic Absorption Spectroscopy and Inductively Coupled Plasma Mass Spectrometry Chemical Analysis via Atomic Absorption Spectroscopy of a Campus Ecosystem during Intense Construction Activity Chemical Analysis of Kudzu Roots via Atomic Absorption Spectroscopy Oil and Grease (O&G) Determination in Water on GGC Campus Ecosystem via FTIR Optimization of CO2 Adsorbents for Carbon Capture and Sequestration

2. Overview of Three Recent Projects The environmental research cluster has enabled undergraduates to participate in numerous community-based, public interest, environmental projects aligned with their interests. This chapter includes a brief overview of two ongoing projects, as well as an in-depth report of a third (and more complete project) demonstrating the breadth of student interests and strategies to engage them in meaningful, applied research.

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2.1. Analysis of Heavy Metal Content in the Conasauga, Oostanaula, and Coosa Rivers during Paddle Georgia 2016 This unique project combined components and collaborators from industry (PerkinElmer), academia (GGC), state entities (Georgia Adopt-A-Stream and Georgia Environmental Protection Division), and citizen scientists. Figure 1 illustrates the collaboration overlaid on the site plan for collection and analysis of 84 different water samples along the Coosa River Basin.

Figure 1. The Georgia river basins project forged a lasting collaboration between industry, government, and academia by integrating collection and anlysis of water samples for heavy metals at 84 sites along the Coosa River Basin. 184

Water samples from the Conasauga, Oostanaula, and Coosa Rivers were collected by volunteer citizen scientists during the Paddle Georgia Trip 2016. These citizens were trained and offered technical support by the Georgia Adopt-A-Stream (AAS) program, which is supported by the Georgia Environmental Protection Division (EPD) (6). During the 1970’s, the Coosa River Basin was contaminated by numerous sources, including the industrial carpet industry. During the period of 1960-1990, the river was viewed as unfit for recreational purposes (7). Participants in Paddle Georgia gathered samples for analysis of trace metals at 84 sites along the Coosa River Basin. Other data collected included air and water temperature, pH, dissolved O2 (ppm), conductivity (µS/cm), total hardness (ppm), total alkalinity, and turbidity (NTU). The samples collected by citizen scientists, aided and trained by Georgia AAS staff, were analyzed by two GGC undergraduates interning at the PerkinElmer Technical Center in Johns Creek, GA. PerkinElmer provided students initial ICP-MS training and access to the instrument. Faculty worked in concert with PerkinElmer personnel to mentor the students through their internship. Students prepared the unfiltered samples for hot block digestion in dilute HCl and HNO3. Quality assurance measures such as blank and spike recovery samples were included. Samples were analyzed for trace heavy metals and mineral composition on a PerkinElmer NexION 350D, using Kinetic Energy Discrimination (KED) with helium to remove polyatomic interferences. The undergraduate students presented this work as a poster at ACS regional and national conferences, as well as the Georgia AAS Confluence conference. The students brought their expertise back to the GGC campus in subsequent work supporting the environmental cluster using GGC’s recently acquired PerkinElmer Elan ICP-MS instrument. Although preliminary results of this project are outside of the scope of this manuscript, it is expected that this project will support a future publication involving undergraduate authors.

2.2. Measurement of Hazardous Air Pollutants and Policy Analysis in Metro Atlanta: Environmental Justice This project used the theme of environmental justice to create an interdisciplinary and collaborative research team that included students and faculty from GGC’s SST and School of Liberal Arts (SLA). Figure 2 graphically portrays the project’s collaborations. Environmental justice is broadly defined by the U.S. Environmental Protection agency as “the fair treatment and meaningful involvement of all people regardless of race, color, national origin, or income with respect to the development, implementation, and enforcement of environmental laws, regulations, and policies (8).” This project, internally funded by GGC’s Community Innovations Project (CIP) program, partnered two faculty and seven undergraduate students with an external advisor at the Atlanta-based environmental law firm, GreenLaw. 185

Figure 2. The environmental justice air pollution project fostered faculty collaborations between groups interested in social justice. This community-based inquiry sought to analyze gas-phase hazardous air pollutants, specifically polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), at sites selected by students based on community demographic parameters such as income, percent minority population, and linguistic isolation. The measurements were made using passive air samplers (PAS), which are a low cost alternative to traditional active sampling methods of gas-phase organic pollutants. Because PAS are low-cost and require no power, samplers of this type can be used to increase the spatial resolution of gas-phase PAHs studies, allowing for comparison concentrations in communities with differing socio-economic characteristics, or for continuous monitoring purposes (9). The concentrations of PAHs and PCBs in the gas-phase were compared to demographic data to investigate the issue of environmental justice in the Atlanta metropolitan region. The project served two primary objectives for the research students. In the first objective, GGC students experienced both field and laboratory work through deployment of PAS to sample PAHs and PCBs. The collaborative student team (two biochemistry, one environmental science, and four legal studies students) worked together in the laboratory to quantify the analytes in each sample, as well as conduct quality control experiments (laboratory and field blanks, spike 186

recovery experiments, response factors of deuterated internal standards). The students also worked collaboratively on the acquisition of US Census data for the specified sampling locations, including parameters such as percent minority populations, percent below poverty populations, and percent living in linguistic isolation. The combination of both lab and policy work for all students ensured experiences outside of students’ area of expertise and communication between social and physical scientists. In the second objective, the collaboration of students with an outside partner, as well as the communities in which they placed samplers, gave students exposure to professional development opportunities, networking, and practice in communicating the goals and methods of their projects to an audience outside of academia. Results relating to both the concentrations of hazardous air pollutants, demographic analysis, and student response to interdisciplinary research are beyond the scope of this chapter, but are expected to be published in a different venue.

2.3. Synthesis and Analysis of Waste Oil and Grease (O&G) from the Campus Chick-fil-A for Use as Biofuel The third highlighted project, and the focus of the remainder of this chapter, is the work of environmental cluster faculty, GGC students, GSMST students, GGC dining operations, and multiple external collaborators in synthesizing and characterizing biofuel from waste O&G. Figure 3 graphically portrays the biodiesel project.

Figure 3. The biodiesel project developed on and off campus collaborations in coverting waste O&G into useful fuel. 187

The interdisciplinary nature of this project engages students majoring in chemistry, biology, and environmental science, as well as young investigators from GSMST with interest in sustainability and renewable energy resources. Current challenges regarding traditional petrochemical sources of energy facilitate the need for alternative fuel sources, such as biodiesel. Biodiesel is an alternative fuel that has potential for reducing both greenhouse gas and hazardous emissions. Though many different types of feedstocks are available for the generation of biodiesel, it can be created from waste O&G. The project also addresses a possible avenue for a sustainable disposal method of campus waste O&G, since current disposal methods are both costly and may contaminate environmental matrices. The biodiesel project uses waste O&G from the Chick-fil-A restaurant on campus. Current objectives include research into the sustainability of a campus-wide biodiesel production effort, bench-scale biodiesel production using well developed transesterification reactions, and physical and chemical characterization using equipment and instrumentation available at GGC, much of which students have used directly in their other course work. The concept of biofuels is reasonably well developed and several undergraduate laboratory excercises or classes have focused on creating and/or characterizing biodiesel (10–13). However, the unique aspect of this project is to refine the bench scale production and characterization of biofuel from locally sourced (on-campus) waste oil and grease while simultaneously conducting a detailed cost-benefit analysis of scale-up opportunities as an environmentally sustainable process to fuel on-campus four-wheeled vehicles. The biodiesel project begain in Fall 2016 and results presented in this chapter cover work through Spring 2017. The project is ongoing and anticipated to continue for several additional semesters. Each semester faculty conducted safety, techniques, and instrument training for GGC undergraduate students and the high school students from GSMST. We have recently recalled students from previous semesters, who had become proficient with the various processes, to conduct training for the new research students. Faculty outlined the project goals and timelines for the semester, provided background references, and students self-organized and began work. Students synthesized all biodiesel, generated all data using equipment and instrumentation at GGC, and conducted analysis of the data. Faculty met regularly with students during each semester to discuss progress, suggest approaches to work through problems, replenish supplies, and suggest next steps. At the end of the each semester, the cluster faculty and all students met to consolidate results and analysis and provide guidance for the student presentation at the college wide research and creative activities symposium. Students then prepared and presented their work to faculty and students at the symposium.

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3. Details of the Biodiesel Project 3.1. Background The increase in industrialization and population has led to the increased need for energy. The primary sources of this energy are coal, petroleum, natural gas, hydro and nuclear (14). Projected world energy consumption indicates that liquid fuels, mostly petroleum-based, coal, and natural gas will be the dominant energy sources in future as shown in Figure 4 (15). The high price of petroleum products, decrease in fossil fuel reserves and atmospheric pollution created by use of petroleum-based fuels are just some of the motivation for alternate energy sources (14, 16–18). The use of vegetable oil as fuel originates with Rudolph Diesel, the inventor of the diesel engine. He fueled his engine with peanut oil in the Paris exhibition of 1900. Since then a number of studies have shown the promise of vegetable oil use as an alternate fuel for diesel engines. However, the high viscosity, low volatility, and poor cold flow properties of vegetable oils result in severe engine deposits. Polymerization of vegetable oils under high pressure and temperature, injector coking and piston ring sticking have also prevented direct use of vegetable oil as a fuel in diesel engines (17, 19–21).

Figure 4. Projected world energy consumption. Reproduced with permission from ref. (15). U.S. Energy Information Administration (May 2016).

Many researchers have worked to develop vegetable oil based derivatives that approximate the properties and performance of petroleum based diesel fuel. The most common method for production of biodiesel is transesterfication, shown in Figure 5, in which the vegetable oil or animal fat (triglyceride) reacts with a monohydride alcohol in the presence of a catalyst to create the corresponding mono alkyl esters (20, 22, 23). However, the high manufacturing cost of vegetable oil is a major barrier in the commercialization of biodiesel production. Waste cooking oil, which is virtually expense free compared to pure vegetable oil is 189

a promising alternative for the production of biodiesel (24, 25). Moreover, the production of biodiesel from waste cooking oil will reduce the challenge of its disposal and possible contamination of land and water resources. The ability to prepare diesel fuel from waste cooking oil would partly decrease the dependency on petroleum-based fuel, focusing on a more carbon neutral approach to energy (16, 22, 26). Biodiesel production using waste cooking oil and its characterization have been conducted by various researchers. Previous studies have compared the use of different types of catalysts, alcohol/oil mole ratio, temperature, reaction time, and pre-treatment methods for the production of biodiesel (27–30). In addition, different characterization methods have been employed to determine various chemical and physical properties of the generated biodiesel (16, 30–34).

Figure 5. General transesterification reaction.

3.2. Biodiesel Synthesis The biodiesel production via transesterification combines waste O&G (triglycerides), methanol, and sodium hydroxide to produce glycerol and three fatty acid methyl esters (FAMEs) (20, 27, 35–37). Students tried several stoichiometric proportions of reactants and obtained best results using 1 mol triglyceride to 6 moles methanol. Students combined filtered waste O&G (420 g) from Chick-fil-A, CH3OH (300 g), and dried NaOH (4.2 g, 1% mass of O&G) in the reaction apparatus shown in Figure 6a. The reaction proceeded at 65 °C for 2.5 hours under reflux with stirring. The reaction was paused and the bottom layer of glycerol was removed. The reaction then continued for an additional 45 minutes. Afterwards, the reaction solution was transferred to a separtory funnel, and it remained there overnight to enable further separation of glycerol as shown in Figure 6b. After the removal of glycerol, the remaining biodiesel solution was washed with 2 M HCl followed by DI water until the wash water achieved pH 7. The washed biodiesel was oven dried for 1 hour at 100 °C. Figure 6c shows the filtered waste O&G contrasted with the finished biodiesel product in Figure 6d. 190

Figure 6. Experimental set-up for biodiesel reaction: a) reaction vessel; b) post-reaction separation of methyl esters from glycerol and other waste; c) crude waste oil and grease from Chick-fil-A; d) finished biodiesel product.

3.3. Biodiesel Physical Characterization and Analysis After synthesizing biodiesel, students then conducted physical and chemical characterization of their product using many techniques they learned in their previous course work.

3.3.1. Biodiesel Density

Methods Injection systems, pumps, and injectors must supply an amount of fuel precisely adjusted to provide proper combustion, thus density is considered an important property of fuel (38). Density of biodiesel was determined by measuring the mass with increasing volume of 5 ml aliquots at three different temperatures.

Results Figure 7 presents student density plots and correspondingdensity values at 22.4 °C, 36.3 °C and 65 °C. The experimental value of density at 22.4 °C was compared to a calculated density determined by a linear combination of biosiesel composition fraction and literature density values. Composition fractions were determined via GC-MS analysis (see Section 3.4.1). Based on components of methyl oleate [cis-9] (69.5%), methyl linoleate [cis-9,12] (20.3%), methyl 191

palmitate (10.2%), methyl stearate (0.1%) the calculated density is 0.874 g/ml. This is compared to an experimental density of 0.85 g/ml, giving a 2.8% error. The density versus temperature trends in Figure 7 correspond with the expected trend of decrease in density as temperature increased, although the measured difference is small.

Figure 7. Mass versus volume plots and curve fits at 22.5, 36.3, and 65.0 °C, and biodiesel density determined from the plots.

3.3.2. Biodiesel Kinematic Viscocity

Methods. Fuel with high viscosity leads to poorer atomization upon injection into the combustion chamber, cold weather injection issues, and poor lubrication for the precision fit of fuel injection pumps, resulting in poor performance (39, 40). The American biodiesel standard (ASTM D6751) optimal kinematic viscosity for biodiesel at 40 °C ranges from 1.9 – 6.0 mm2/s (41). Students determined kinematic viscosity of their biodiesel following the ASTM D445 method at 40 °C using the Cannon-Ubbelohde viscometer (Figure 8a) and a PolyScience viscosity bath (Figure 8b). The viscometer and bath were operated at 40 °C, with 20 minutes provided for temperature equilibration. Efflux time (time for biodiesel to flow a certain distance in the viscometer under gravity) was then measured. Efflux time for biodiesel was recorded as it flowed from mark E through F in Figure 8a. 192

Figure 8. Viscosity apparatus: a) Ubbelohde viscometer with demarcations for positions used for measuring efflux time; b) temperature-controlled viscosity bath with viscometer set into the bath.

Results Using the Ubbelohde viscometer constant (0.04810 mm2/s2 for viscometer used for waste O&G, 0.01087 mm2/s2 for viscometer used for the biodiesel), students determined the kinematic viscosity of their biodiesel at 40 °C as 4.6 mm2/s using Equation 1. 193

The kinematic viscosity of students’ biodiesel lies well in the range for biodiesel viscosity according to ASTM D6751. Biodiesel kinematic viscosity of biodiesel was compared to that of waste used oil using the ASTM D445 method. Kinematic viscosity calculated for waste used oil was 42 mm2/s at 40 °C which is approximately 9 times higher than the viscosity of biodiesel. Table 2 summarizes kinematic viscosity results for waste O&G, student biodiesel, and ASTM D6751 biodiesel standards.

Table 2. Biodiesel Kinematic Viscosity at 40 °C Sample

Ave Efflux Time (s)

Kinematic Viscosity (mm2/s)

Filtered waste O&G

870

870*0.04810=42

Biodiesel

422

422*0.01087=4.6

Biodiesel ASTM D6751

----

1.9 – 6.0

3.4. Biodiesel Chemical Characterization and Analysis 3.4.1. Biodiesel Components via GC-MS

Methods Students analyzed biodiesel samples using the Shimadzu QP2010S Gas Chromatograph Mass Spectrometer (GC/MS) with HP 88 column (60 m x 0.25 mm, i.d., 0.20 µm film thickness) in scan mode to determine fatty acid methyl ester (FAME) composition. Analyte retention times and confirmations were compared to a standard mixture of 37 fatty acid methyl esters (FAMEs, in methylene chloride, Restek, Bellefonte, PA). 20 mg of student generated biodiesel was dissolved in 10 mL of methylene chloride (Fisher Scientific, HPLC grade) and injected as 2 µL in split mode (50:1) at a constant column flow of 2.0 mL/min. The GC oven temperatures were held at 175 °C for 10 min, ramped at 3 °C/min to 220 °C with a final hold for 5 min. The GC/MS interface was maintained at 250 °C and the MS ion source temperature was 230 °C.

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Results The FAME profile of the biodiesel generated from GGC waste cooking oils was determined by GC-MS analysis method described above. The retention times of individual peaks of the gas chromatogram were verified against a FAME standard mixture and individual FAMEs were identified using the MS database (NIST library data). A representative chromatogram, mass spectrum of major components, and biodiesel mixture composition are shown in Figure 9.

Figure 9. Representative gas chromatogram and mass spectrum.

Relative percentages of FAMEs were calculated from the total ion chromatogram by integration and results are presented in the Table 3. The student biodiesel from waste O&G consists of 10.2 wt.% of methyl palmitate (C16:0), 69.5 wt.% of methyl oleate (C18:1), 20.3 wt.% of methyl linoleate (C18:2), and