CARBON(14) TRACER STUDIES ON THE MECHANISM OF BIOSYNTHESIS OF PENICILLIN

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The Penneylvanla State College The Graduate School Department of Chemistry .CARBON FOURTEEN TRACER STUDIES ON THE MECHANISM OF BIOSYNTHESIS OF PENICILLIN

A Dissertation by John Moore Tome

Submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

June 1951

ACKNOWLEDGMENT Four pharmaceutical companies, Abbott Laboratories, Eli Lilly and Company, Parke, Davis and Company, and The Upjohn Company, have sponsored the Antibiotics Project as a Joint program between the Departments of Bacteriology and Chemistry. I wish to thank Doctors R. B, Wagner and H. D. Zook, directors of the chemical portion of the project, for their constant advice and encouragement throughout the course of this research. I wish to thank members of the Department of Bacteriology, Doctor R. W. Stone, as director of the program, and Doctor Esther L. Martin, Mr. C. W. Godzeskl, and Mr. J. J. Berky, as co-workers, without whose efforts this research would not have been possible. I also wish to thank the contributing companies for the fellowship under which I worked for three years.

0

:r'"40

TABLE OF CONTENTS Introduction

.............. . . ,

1

Historical A. General

. . ........ .

3

B. Metabolism . . . . . . . . . . .

k

C . Isolation

6

......... ..

Discussion

8

A. General Aspects ..........

10

................

17

B. Procedures • • • • • C. Results

...

Experimental

A. Fermentation . . . . . . . . . .......... ..

35 38

B.

Isolation

C.

De gr ad ati on..................

52

D.

Special Techniques and Reagents

57

Sutanary and Conclusions .

........

Bibliography . . . . . . . . . . . .

6k

66

I. UttBOUJCTION

Heady availability of Isotopes In the past five years has lead to Investigation of heretofore unsolvably complex problems.

Encouraging results have been obtained In many fields;

however, the most outstanding advances have been made In the biological sciences, where life Itself Is the problem.

Study and

understanding of the simpler forms of living matter are yielding building blocks from which more difficult problems can be reached. Within this simpler life group, molds play an Important role as Is evidenced In the production of antibiotics.

As the

first major antibiotic, penicillin Initiated a new era In medical practice.

Although the structure of penicillin has been established,

no commercial chemical synthesis has been accomplished, and thus mold fermentation remains the single method of production.

The

metabolic process by which the mold forms penicillins Is suf­ ficiently complex so as to eliminate most methods now used for resolution of such problems. Study of the mechanism of the biosynthesis of penicillin is Important for several reasons:

an insight Into mold metabolism

may lead to a better understanding of life processes; and a known mechanism may give rise to production of new types of penicillin or higher production of present types. Hiere, then, Is a pertinent complex problem which may yield to solution by means of the Isotope tracer technique.

This dissertation has two objectives: first, to devise methods and procedures to apply the Isotope tracer technique to the study of penicillin biosynthesis, and second, to demonstrate their feasibility by obtaining fundamental data on the mechanism of biosynthesis of penicillin. complished.

Both objectives have been ac­

3

H.

HISTORICAL

A.

General The structure of penicillin was determined through the

combined efforts of many scientists during the years 19^3

(l).

Using as a basis, degradation, X-ray crystallographic, and physical measurement studies, the ^3-lactam structure below was proposed as the most promising.

The final evidence needed, unequivocal

chemical synthesis, could not be accomplished.

S— 0(CH,)a B-CO-BB-OB— OH O^C — N— CH-COOH

Recent work by Sheehan (2) in preparing a phenyl substituted penicillin by established reactions provides the final proof of the

-Inctam-thiazolidine ring structure which had been the major

point of controversy in previous work.

Chemical synthesis of the

commercial penicillin in use today still has not been accomplished. Many types of penicillin have been Identified (3A>5>6). The only -variation In the penicillin molecule that can be made without loss of biological activity is In the R group shown In the structure.

Identification of these different types of penicillin

Is done by degradation or by variation In microbiological assay (7 ) known as differential assay. In most fermentations are:

The common types of penicillin found

k

R group

Differential Assay (M,pyogenes/B •subt it is )

Penicillin Designation

1.0

Benzyl

G

2-perrtenjl

F

.65

n-heptyl

K

.35

Penicillin G is the type used pharmaceutically. A large number of Pane1111a cultures produce penicillin (8 ,9 ); however, Penlcllllum chryaogenum, strain Q-176, has been found to give the highest yield of penicillin and is, therefore, used commercially. Corn steep solids is the basis of the normal fermentation medium but it has not been chemically defined and thus would not be satisfactory for metabolism studies.

Several synthetic media of known composition

have been developed (10,11) which satisfy the nutrient requirements for penicillin formation and these have been used in metabolism studies. B.

Metabolism General factors affecting the fermentation process, such as

P^ requirements and control, have been studied by Jarvis and Johnson (12).

Addition of surface active agents necessary for good fermen­

tation provides an increase in penicillin yield.

This effect has

been discussed by Goldschmidt and Koffler (13). Phonylacetic acid added to the fermentation medium substan­ tially increases the yield of penicillin G.

N-substituted phenyl-

acetamide additives increases the yield even further.

To prove that

these precursor compounds actually are Incorporated in the penicillin molecule deuterophenylacetyl-N^-dl-valine, has been tested (lU). The resulting penicillin contains the deuterium Isotope while the

5

content is very low.

This evidence Indicates that the phenyl-

acetyl precursor Is utilized while the attached nitrogen Is not. Recently Craig, et al (15) added phenylacetaalde-l-C^ to produce the correspondingly labeled penicillin which was then used to in­ vestigate commercial penicillin production by the Isotope dilution method.

The addition of substituted acetic acids using this pre­

cursor effect has been exploited to vary the R group and produce many new penicillins Mineral requirements for fermentation are reflected In the composition of the synthetic media.

Mich work has been done In the

study of the chemical elements needed and the effect of changes in their concentration (10,16,17 ,18). Inorganic sulfate is the most readily utilized source of sulfur for incorporation into penicillin.

This Is supported by

the fact that when sulfate Is the only source of sulfur, penicillin is still formed in high yield.

.Also, two groups of workers (19,20)

have used radioactive sulfur, S35, as sulfate and have obtained radioactive penicillin.

If Inorganic sulfide is added with the

sulfate, the sulfate is preferentially used (3 ,2 1 ).

Dons of the

organic sulfur compounds that have been tested (3 ,2 1 ) have equaled Inorganic sulfate for penicillin production.

Cysteine and methio­

nine do approach, however, the results obtained with sulfate.

In­

organic sulfur is best utilized from the oxidized state; organic sulfur, from the reduced state. Inorganic nitrogen salts supply the entire nitrogen require­ ments of the fermentation.

It is Interesting to note that the nitrogen

needs are Just the reverse of those for sulfur In that the reduced,

ammonium, nitrogen is more available than the oxidized, nitrate, nitrogen for metabolism (ll).

Most of the organic nitrogen com­

pounds tried hare been amino acids.

Their effect on stimulation

of penicillin yield has either been inconclusive or negative (21). Various degradation products from penicillin itself have been added to the fermentation medium vith the hope of increasing the yield; however, the results have been negative (3 )• C•

Isolation The tvo major processes, the solvent process and the carbon

process, developed for the isolation of penicillin from the fer­ mentation broth have been concisely described by 'Whitmore and co-workers (22).

Modifications of these tvo methods are used

commercially today. The solvent process utilizes the acidic character of peni­ cillin far numerous transfers between aqueous solution and organic solvents.

By using optimum conditions for penicillin transfer, the

undesirable pigments and mold metabolites are removed, thus allowing the pure penicillin to be isolated. The carbon process involves the removal of the penicillin from the fermentation broth by absorption on activated carbon. Recovery from the carbon is accomplished with aqueous acetone followed by flash evaporation to reduce the volume.

After this

initial purification, several steps of the solvent process are used to give the pure penicillin.

7

A mixture of several types of penicillin is Isolated "by these methods.

Any work which requires a single penicillin must also

include provisions for their separation.

Commerdaily, penicillin

G is the type required and this is separated from other penicillins by means of salt formation (l).

Methods have heen developed whereby

several types of penicillin can be separated and recovered.

These

include absorption chromatography, partition chromatography, and counter-current -distribution. Absorption chromatography on alumina (23) for penicillin separation has been used extensively but because of inactivation of certain types of penicillin, namely, penicillin F, the procedure was abandoned In favor of the milder conditions of partition chromatography. Two types of partition chromatography have been used suc­ cessfully.

One utilises the regular chromatographic column with

buffered silica gel as the solid phase (1^,23 ,2k); the other Involves paper partition chromatography (25 ) but has the disadvantage of accommodating only minute quantities. A technique of counter-current distribution has been de­ veloped by Craig (26,27) for separation of small quantities of organic material.

This technique, known as the Craig technique,

has been applied to the separation and purification of penicillin (28,29).

8

HI. A.

DISCUSSION General Aspects Certain basic aspects of the biosynthesis of penicillin

have been investigated.

The most available sources of sulfur and

nitrogen have been shown to be inorganic sulfate and salts.

ammonium

The only variable part of the molecule, the precursor

moity, can originate from the incorporation of vhole organic molecules without their alteration. The major problems remaining in the penicillin biosynthesis are; (1 ), the source of the carbon found in the ^ -lactmm-thiasolidine fused ring system, and (2 ) the mechanism by which this carbon is incorporated. By use of a chemically defined medium- (10), the source for this carbon can be limited to lactose, glucose, acetic acid and antifoam agent.

Addition to this medium of one and tvo carbon

molecules which are building blocks in other biosynthetic processes, and which are deemed necessary to the penicillin fermentation should prove promising for study. When labeled with isotopic carbon and added to the fer­ mentation, these molecules if part of the biosynthetic scheme should produce an lsotoplc penicillin.

Degradation of this lsotoplc

penibillin is necessary to show the final location of the isotope. By consecutively labeling various molecules in specific positions and noting the location of the labeled atom in the penicillin one can establish certain principles upon which a mechanism for biosyn­ thesis may be postulated.

9 Four labeled compounds were selected for study:

carbon

dioxide as sodium carbonate, sodium formate, and both carboxyland methyl—

labeled sodium acetate*

The role of carbon dioxide is of special importance* is evolved from the fermentations.

It

Since it is thought that all

organisms can utilise some CO2> even if they do not possess photosynthetic abilities, the degree of utilisation in the biosynthesis of penicillin is fundamental.

If carbon dioxide is utilised, then

the tracing of the radioactive carbon from a particular additive to a particular position in the penicillin molecule would be obcure, especially if that additive were degraded to carbon dioxide* Interest in the labeled sodium acetate is apparent from the need and use of acetic acid in the synthetic medium (10)* The importance of formic acid in mold metabolism has become in­ creasingly apparent (30,31).

For this reason its utilisation as

sodium formate by Panic 1Ilium has been investigated. Carbon-lij- has been selected as the Isotope best suited for these Initial experiments.

The short half-life of Carbon-11

eliminated its employment.

Economic factors ruled out the use of

Carbon-13, a non-radioactlve isotope, in this initial study. Fortunately, the

- labeled compounds desired for study

or materials for their preparation are commercially available. From the high cost of these, however, it becomes apparent that small scale fermentations would have to be used.

10

The problem can be defined as (l) to adapt the precautions necessary In handling Carbon-lit to the existing small-scale, penic1111 n-fermeixtat Ion procedurej(2 ) to modify methods of penicillin isolation to a semi-micro scale, (3 ) bo devise a scheme for degra­ dation of penicillin in vhich all parts of the molecule can be in­ vestigated from the small quantities available, and (It) with the aid study of the of these techniques to make an initial/mechanism of the biosynthesis of penicillin. B . Procedures 1.

Fermentation Modifications in the normal small-scale fermentation

far conversion to radio-tracer experiments are minor.

The sterlized,

lsotoplcally labeled compounds are added under aseptic conditions to the synthetic medium before and during the fermentation. During the eight days of Incubation, carbon dioxide Is evolved by the fermentation.

A hood system is employed vhereby a

continuous flow of air could be passed over the fermentation flasks and the effluent gases are bubbled through three consecutive con­ centrated potassium hydroxide absorbers.

Since the last absorber

contains less than l£ of the trapped radioactive carbon dioxide, almost complete recovery of the radioactive carbon is assured.

In

this way the health hazards Involved by having high concentrations of

in the air are minimized.

The total amount of radioactivity

recovered as carbon dioxide has been determined for each additive for possible correlation.

11

2.

Isolation The fermentation troth at the time of harvest contains

at least three types of penicillin, pigments, and proteinaceous material. The method used for isolation of the total penicillin content is a combination of the carbon process and the solvent process. In the initial step the penicillins are adsorbed on activated carbon and then eluted by 80$ aqueous acetone.

A four-

fifths-volume reduction is obtained by flash evaporation of the eluate.

Several transfers between aqueous buffer solutions and

chloroform complete the purification and concentration of the com­ bined penicillins.

The concentration is essential in preparing the

sample for the chromatographic separation. Partition chromatography is used to separate the three types of penicillin; namely, penicillins K,F, and G, found in the fermentation. penicillin.

This technique also removes the pigments from the

The solid phase for the chromatograph is silica gel

which buffered with an aqueous solution of potassium phosphate. The penicillin sample is added in chloroform; developed with the same solvent.

the chromatogram is

Twenty-five to thirty fractions

are obtained from the development and extrusion of the column*

As

shown in Graph 1 , the major portion of the color is removed first, then penicillin K and penicillin F.

The penicillin G usually remains

on the silica gel due to the limited amount of development. recovery of total penicillin through this stage is about 50$.

The It

had been hoped that a correlation could be found between the peniclllin-aesay values and the radioactivity-assay values on fractions

GRAPH 1

Chromatographic Separation of Panicillina CHjC1*OQNa Additive (Run IV) 4000 Relative Visual

3000

Color

(units)

6 5 4 3

1000 /—

\

2 1 0 1

3

5

7

9

11

13

15

17

19

Chromatographic Column Elution Fraction Number

21

23

25

Intensity

Penicillin

2000

of the chromatogram.

Unfortunately, the Intense radioactivity

in the pigments completely masked that vhich might he due to the penicillin.

This necessitated a further purification step.

Counter-current distribution has been shown to be readily applicable for the purification of penicillin (28).

This second

sensitive method of purification is applied to the almost pure penicillins from the chromatographic column.

The fractions used

for distribution are those containing a single penicillin, as determined by differential assay, with a minimum amount of pigment (See Graph l).

The resulting distribution over a range of twenty-

five samples is found to give good correlation between the distri­ bution curves for the penicillin-assay value and the radioactlvityassay value.

Since the pigment had obsoured the results from the

chromatograms, a determination of color in each of the countercurrent distribution samples is made.

The intensity of color as

determined in a Fisher Elefcrophometer was negligible.

Detailed

information is given in the experimental section pertaining to the development of the isolation process. 3.

Degradation The degradation and Isolation of fragments of radio­

active penicillin had to be done on small quantities in one single continuous experiment.

The procedure as developed is a combination

of many experiments originally used in the proof of structure of penicillin.

Certain modifications also had to be made to adjust for

the small scale of this work. The scheme for degradation is shown for penicillin G in Diagram 1.

Benzylpenicillin is hydrolyzed with sodium hydroxide to

14

DIAGRAM

1

DEGRADATION SCHEME ! II ^ S — C(CH,)a

O

*

III

-CO-NH-fCH— C H ; 0-C--- j-4J— CH-COOH i IV

Benzylpenicillin

/—


and ^ show the distributions for penicillins K,F, and G, respectively, for the sodium carbonate fermentation. The amount of radioactivity is very small and falls within the range of experimental error.

This establishes the fact that carbon

dioxide is not a carbon source for penicillin. The incorporation of carboxyl-labeled sodium acetate is indicated by the good correlation between the distribution curves of penicillin and radioactivity for penicillins K, F, and G as shown in Graphs 5, 6 , and 7f respectively. In the same manner incorporation of methyl-labeled sodium acetate is Indicated by the distributions shown for penlcilllnsE, F, and G in Graphs 8 , 9> and 10, respectively. Graphs 11, 12, and 13 show the same distribution correlation for penicillimK, F, and G, respectively, when labeled sodium formate was the fermentation additive. These data prove that the carboxyl and methyl carbons of acetic acid and the carbon in formic acid are metabolised by the penicillin mold and incorporated in all three types of penicillin.

20

GRAPH 2

NaaC1403 Additive (Run III)

300

200

o

Penicillin (units)

120 100

100

80 60 AD 20 0

3

6

9

12

18

21

Distribution Tube lfunbar

2

Radioactivity (counts par adnata) -

Q

Fmicillin (unit*) —

8

t

% $

O

& t

M

d.a.«>.60

Vj J

22

GRAPH

Na2C1