I. Oxidation of Gluconic Acid with Oxides of Nitrogen. II. A Procedure for the Synthesis of DL-Valine Containing a Labeled Carbon Atom

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P U R D U E U N IV E R S IT Y

T H IS IS TO C ER T IF Y T H A T T H E T H E S IS P R E P A R E D U N D E R M Y S U P E R V IS IO N

Harold A. Price

BY

E N T IT L E D

I. OXIDATION OF GLUCONIC ACID WITH OXIDES OF NITROGEN.

II. A PROCEDURE FOR THE SYNTHESIS OF DL-VALINS COTTTAÏNINO A LABELED CARBON ATOM C O M P L IE S W IT H T H E U N IV E R S IT Y R E G U L A T IO N S O N G R A D U A T IO N T H E S E S

A N D I S A P P R O V E D B Y M E A S F U L F IL L IN G T H IS P A R T O F T H E R E Q U IR E M E N T S

FO R THE D EG REE O F

Doctor of Pli 11 -oaopbyi

P r o f e s s o r in C h a r g e o f T h e s is

H ear of S chool or D epartm ent

TO T H E L IB R A R IA N

-tsTTHS T H E S IS IS N O T TO B E R E G A R D E D A S C O N FID E ]

»& DT OHABGB

GRAD. SCHOOL FORM 9-*3-49— OM

OXIDATION OF GLUOONIC ACID WITH OXIDES OF NITROGEN

A PROCEDURE FOR THE SYNTHESIS OF DL-VALINE CONTAINING A LABELED CARBON ATOM

A Thesis Submitted to the Faculty of Purdue University

by Harold A. Price

In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

June, 1950

ProQuest N um ber: 27714154

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uest ProQuest 27714154 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKNOWLEDGMENT The author wishes to express his sincere appreciation to Dr. Ed. F. Degerlng for his contribution to this work which also included the selection of the thesis subjects.

The author

wishes to express his gratitude to The Sugar Research Foundation and the Indiana Elks As­ sociation for their financial support of these projects through the Purdue Research Foundation. Thanks are due also to the many persons who aided in the completion of this work.

TABLE OF CONTENTS Page ABSTRACT

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

1

Part 1, ,Oxidation of Gluconic Acid with. Oxides of Nitrogen*..................... Part 2.

*.

A Procedure for the Synthesis of QL-

Vallne Containing a Labeled Carbon Atom I.

OXIDATION OF GLUCONIC ACID WITH OXIDES OF NITROGEN. . ......

Introduction. Reagents Used.

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

Discussion of Results. Conclusion. Bibliography.

xiv 1 1

Experimental.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II.

i

.......

7 3 17

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

26

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

28

A PROCEDURE FOR THE SYNTHESIS OF DL-VALINE CONTAINING- A LABELED CARBON ATOM. . ....... Introduction........... Reagents Used.

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

Experimental.......... Butyl But oxy a ce tat e ................ Ethyl Phenoxyacetate...... Phenoxyacetone........ 1-Phenoxy-2-methyl-2-propano l................. Butyl Ch 1 oromethyl Ether....................... sec-Butyl Chloromethyl Ether......... 2-Ethylbutyl Chloromethyl Ether............... Chloromethyl 2-Octyl Ether. .......... Chloromethyl Isopropyl Ether............. Chloromethyl Octyl Ether...................... Amyl Chloromethyl Ether........................ Chloromethyl 2-Methylbutyl Ether. .......... Butoxyacetonitrlle............................. sec-Butoxyacetonitrile........

30 30 33 35 35 36 37 37 39 39 40 40 41 41 4l 42 42 42

TABLE OF CONTENTS (Cont.) Page (2-Ethylbutoxy )acetonitrile................... laopropoxyaoetonitrile......... Octyloxyacetonitrile............ Amy 1 oxy a cetonitrl le ........... (2-Methylbutoxy )acetonit ri le.................. ............... (2-0ctyloxy )aeetonitrile. Butoxyaoetone . ......... I sop r o poxy a ee t one ............... (2-Methylbutoxy )acetone........................ (2-0ctyloxy) acetone............................ (2-Ethylbutoxy )acetone................... 1-But oxy-2-me thy 1-2-propanol.................. 1- (2-Methylbutoxy )-2-methyl-2-propanol..... Isobutyraldéhyde. ...... 5-Isopropylhydantoin........ DL-Valine. ........................... Ethyl laonitroaomalonate.................. Ethyl Acetamidomalonate....... 1.15 M laopropylmagnesium Iodide. ...... Isobut y rie Acid .......

43 43 43 44 44 44 44 45 46 46 47 48 49 50 51 52 54 55 55 55

Discussion..........

56

Conclusion......

67

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

69

VITA............. .........................................

LIST OF TABLES Table 1.

Page Time Study of the Oxidation of 0.05 Mole of ....

Gluconic Acid with 35^ Nitric Acid. 2.

9

Oxidation of 0.05 Mole of Gluconic Acid with 0.2 Mole of Nitric Acid at Various Concentrat ions

3.

......

10

Oxidation of 0.1 Mole of Gluconic Acid with Various Amounts of Nitric Acid........

11

4.

Effect of Adding Nitrogen Tetroxide at Intervals.•.

5«

Oxidation of deIta-Gluc onolac tone with NgOA at Various Mole Ratios...........

6.

20

Effect of KNOp on the Oxidation of delta-Gluconolactone with N 2O4 ..........

7*

19

24

Effect of Added Moisture on the Oxidation of delta-Gluconolactone.

.......

24

8.

Preparation of Ohioromethyl Ethers , ROGHpCl........

59

9.

Preparation of Alkoxyacetonitrlles, ROCHpGN........

59

10.

Preparation of Alkoxyacetones, ROGHpGO.GH^.........

61

11.

Preparation of Isobutyraldéhyde.

63

12.

Preparation of 5-Isopropylhydantoln.

13*

Preparation of Isobutyrlc Acid by Carbonation

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

of 1.15 M Isopropylmagnesium Iodine..............

64

67

LIST OF FIGURES Figures

Page

1.

Apparatus for Carrying Out NpOA Oxidation.........

2.

Oxidation of delta-Gluconolactone with NpOA at 350G. In 0014......................................

15

21

Department of Chemistry and Purdue Research Foundation Purdue University, Lafayette, Indiana

OXIDATION OF GLUCONIC ACID WITH OXIDES OF NITROGEN* ♦This is an abstract of a portion of a thesis submitted by Harold A* Price to the Faculty of Purdue University in par­ tial fulfillment of the requirements for the Degree of Doctor of Philosophy, June, 1950. This project was sponsored by the Sugar Research Foundation. By Harold A. Price with Ed. F. Degering

ABSTRACT Although the literature contains numerous references to the oxidation of carbohydrates, such as glucose, starch and galactose, relatively little has been reported on the conversion of gluconic acid to saccharic acid.

The prepara­

tion of the latter from gluconic acid and delta-gluconolactone was suggested by the apparent specific oxidation of the primary hydroxyl groups in certain glycosides with nitric acid (1 , 2 ) and nitrogen tetroxide (3*4,5 ,6 ), and by (1)

Dietz, Ph.D. Thesis, Purdue University, 1941.

(2)

Mench, Ph. D. Thesis, Purdue University, 1944.

(3)

Mench and Degering, Proc. Indiana Acad. Bel. . 5 5 . 69 (1946).

(4)

Unruh and Kenyon, THIS JOURNAL, 64, 127 (1942).

(5)

Xackel and Kenyon, THIS JOURNAL, 64, 121 (1942).

(6 )

Maurer and Drefahl, Ber. . 7 5 B . 1489 (1942); 80* 94 (1947).

il

the importance of using saccharic acid (7) in pharmaceutical (7)

Mehltretter, U.S. Patent 2,436,659 (Feb. 24, 1948).

compounds and preparations, In resins and plastics, and as a food acid. (8 )

Nitrogen tetroxide, gluconic acid (8,9) and

Forgea* Glark and Gaetrock, Ind. Eng. Ghem., 32, 107 (1940)*

(9)

Wells, Moyer, Stubbs, Herrick and May, ibid. , 2£, 653 (1937).

the corresponding lactone are readily available materials. The principal objective of this work, therefore* was the attainment of a high yield o f saccharic acid through the action of nitrogen tetroxide on gluconic acid and deltagluconolactone under various experimental conditions. The use of dilute nitric acid as an oxidant for the preparation of saccharic a d d from gluconic acid was reported by Kiliani (10), vfeo found 5-ket oglu conic acid among the (10) Klllanl, Ber. . SSB. 75, 2817 (1922); S M ,

2016 (1923).

products ii&en he allowed the reaction to proceed at room temperature*

After liberating g lue on! e a d d from Its calcium

salt * M e n d (2 ) obtained a 23% yield of saccharic acid upon treatment with nitric acid for one and one-half hours.

In

this investigation* a 50% yield of this compound was accom­ plished by the addition of potassium nitrite to the reaction mixture.

ill

UiKlBXt QBEttSiin aoscllA Iona* tiiG oxldaLlon ot de It»g -r gluconolaotone wltii nltrogen tetroxide was effected In a manner suggested by tbe work of Maurer and Drefah.1 (6), who oxidized galaotoae» methyl glucoslde and methyl galactoside to muelo sold and the corresponding substituted uronlc acids, respectively* In the presence or absence of a diluent and similar to that employed in the work of Mench and Deger­ ing (2,3)# who converted glucose and starch to saccharic acid and alphaslinked polyanhydroglucuronlc acid*.respective­ ly.

Greater than 50^ yields of saccharic acid from gluconic

acid and the cor responding, lactone have neither been, reported nor obtained during the course of this investigation. EXPERIMENTAL Materials.--delta—Grlueonolactone (Ohas. Pfizer and Go.) was dried in a vacuum desiccator over phosphorus pentoxlde at room temperature and 10 mm* pressure.

Nitrogen, tetroxide

(Solvay Process Company) was dried over phosphorus pentoxlde prior to its use. Oxidation of Gluconic Acid with Nitric Acid in the Presence of Potassium Nitrite. — When gluconic acid was oxidized with 35% nitric acid in the presence of potassium nitrite* the amount of original carbon returned as potassium acid saccharate was 4-9» 96%»

A mixture of 40 g. (0.1 mole )

of technical 50^ gluconic acid, 6 g. of distilled water,

25*7

(0.4 mole), of concentrated nitric acid and 10 g. of

potassium nitrite was placed in a 200-ml. round-bottomed

iv

flask fitted, with a Friedrich condenser-

The reaction mix­

ture was cooled In Ice-water for three hours before It was heated In a boiling water-bath during a period of two hours. Then the mixture was cooled in a n lee-bath ^ treated with solid potassium hydroxide to about pH 11 and filtered, to remove inorganic salts.

After standing four hours» the

alkaline solution was acidified with glacial acetic acid to pH 3-4-

The precipitate was collected in a Buchner funnel,

washed» with 50% ethanol and dried.

The yield of potassium

acid saccharate was 12.40 g. Oxidation of delta-Slueonolactone in Carbon Tetrachloride. — The apparatus consisted of a 500-ml. three -necked flask, to which a glass stirrer fitted with a lubricated ball joint, a dry-lee-cooled finger-type condenser and a water-Jacketed burette were attached* delta-Glue o no lactone (17-81 g - , 0.10 mole) and 250 ml. of dry carbon tetrachloride were placed in the flask, and the required quantity of liquid nitrogen tetroxide, the weight being calculated from density and'temperature data, was delivered through the water- jacketed burette cooled with ice-water*

After the reaction mixture was stirred constantly

for a selected length of time at 35 t 0.1°G-, it was cooled in ice to retard further reaction.

The nitrogen tetroxide

was swept out of the solvent by a stream of air, and finally the carbon tetrachloride was decanted-

A solution of the

residue in 40 ml* of water was washed with two 30-ml. portions of ether, made alkaline with solid potassium hydrox-

V

Ide and allowed to stand overnight.

Finally, the dark

alkaline solution was acidified with glacial acetic acid to pH 3-4.

After the mixture was sufficiently cooled* potassium

acid saccharate was collected in a Buchner funnel, washed with $0% ethanol and dried. Oxidation of deIta-Gluconolactone with Nitrogen Tetr­ oxide in the Absence of a Diluent.— delta-Oluconolactone (17♦ 81 g. , 0.10 mole) and 80 ml. of liquid nitrogen tetroxide were placed in a 200^ml. three-necked flask, which was fitted with a glass stirrer and a dry-ice-cooled condenser.

The

reaction mixture was stirred for two and one-half hours at room temperature.

At the end of this time, the excess oxi­

dant was evaporated with a current of air.

After the residue

was processed in the manner described previously, a 39.5^ yield (9*8 g . ) of potassium acid saccharate was obtained. RESULTS AND DISCUSSION When gluconic acid (G* 1 mole, 50^ solution) was oxidized with 35-40^ nitric acid, the reaction mixture was allowed to reflux in a hot water-bath for one and one-half hours and then chilled immediately to retard further reaction. After the solution was treated in the usual.manner, 35-46^ yield of potassium acid saccharate was obtained as shown in Table 1, b u t n o distinct trend is evident. Potassium nitrite was tried as a "Catalyst” because it was reported as such in the nitric acid, oxidation of glucose and galactose.

In one case, 10 g. of potassium

Vi

Table 1.

Oxidatian of 0*1 Mole ©f Gluconic Acid with Various Amounts of Nitric Acid,

Nitric Acid moles %

Yield of KH Saccharate % (Crude) 6*

0^4

35

10,35

41,7

0,5

35

11.55

46. 5

0,5

35

10.30

41.5

0,5

35

9*80

39*4

0,5

35

9.0

36.3

0.6

35

10.30

41.5

0,7

35

8.80

35.4

0.4

40

11.35

45.7

0.5

40

10.10

40.7

0.6

40

11.44

46.1

0.7

40

9*10

36.6

0.81

40

10.35

41.7

nitrite was added to a chilled mixture of 0,1 mole of glu­ conic acid and 0,4 mole of 35^ nitric acid.

The reaction

mixture was allowed to reflux in a hot water-bath for two hours and treated in a manner described previously to give 12,40 g* of potassium acid saccharate or 49»96/£ of theory. According to the observations of Unruh » Yackel and Kenyon (4*5) and Mench and Degering (3) * nitrogen tetroxide appears specific in its oxidizing action»— attacking only the primary hydroxyl grouping and leaving the secondary hydroxyls

vil

Intac t .

This suggested that this oxldl zing agent would se rve

admirably for the conversion of delta-gluconolaotone to saccharic acid In greater than 50% yields. One approach toward finding the optimum experimental conditions concerned the study of the effect of adding the oxidant in portions over an Interval of several hoursAccordingly, 0.1 mole of delta-# lue o no lac t one was treated with 0.2 mole of nitrogen tetroxide dissolved in 250 ml. of dry carbon tetrachloride at 35°C.

The reaction was found to

be rather slow when the mole ratio of the oxidant to the lactone is less than 2:1»

Thus, it was evident that this

procedure does not have any apparent advantage over the single addition of nitrogen tetroxide at the outset. The results listed i n Table 2 combine the study of two more variables in the experimental conditions of this reaction, namely, variation of the mole ratio of oxidant to the lactone and time of reaction.

According to Figure 1,

the effect of increasing the mole ratio is twofold:. the rate of reaction increases and comparable yields of saccharic acid are obtained in less time.

The maximum yield, although

less than 50%, was achieved in approximately ten hours when the mole ratio was 2:1; whereas, the optimum yield of sac­ charic acid was obtained in half the time when the concentra­ tion of the oxidant was quadrupled.

Thus, the time of

reaction is a function of the mole ratio of the oxidizing agent to the lactone. Another significant fact Illustrated in Figure 1 is that

vlli

Table 2.

Oxldatl.on of delta~01uc onolac tone with

at

Various Mole Ratios. Time hr.

Mole Ratio: N204/0 6h 10°6

Yield of KH Saccharate g. % (Orude)

5 6 7 8 9

2 2 2 2 2

3*08 6.57 8.93 10.50 10.80

12.41 26.47 35.98 42.31 43-50

9 10 11.5 3*5 5

2 2 2 3 3

11.52 10.83 10.50 3*01 9*65

46.41 43.65 42.31 12.13 38.88

6 7 9 3 3*5

3 3 3 4 4

10.97 11*57 11.40 1.66 5*88

44.20 46. 62 45.93 6.69 23.69

5 5 5 6 8

4 4 4 4 4

lie 01 11.52 11.20 11*55 12.06

44.36 46.41 45.13 46.54 48.59

2 2.5 3 4 6

8 8 8 8 8

1.40 5*03 8.40 11.76 11.10

5.51 20.27 33.84 47.38 44.72

the Induction period Is diminished with a 0onoomitant In­ crease In the ratio of nitrogen tetroxide to delta—aluoonolactone.

The work of Kenyon, et. al.

(11), on the mechanism

(11)- McGee, Fowler, Taylor, Unruh and Kenyon, THIS JOURNAL, 6£, 355 (1947).

Ix Time in Hours 10

40

30

20

8:1 4:1 3:1 2:1

Fig. 1.

Oxidation of delta-G-luconolactone with N^O^ at 35° In CC14

X

inaralvdLng the oxidation of cellulose by this oxidizing agent may offer an explanation for this induction period*

They

found that the formation of the nitrate ester is the primary step, which is followed by rapid denitration-carboxylation when traces of nitric acid are present.

Accordingly, the

following reactions may occur: -0H20H + N 204 --- » 3 HN02 ___ ^ H 20 * 2 N204

-GH20N02 * HN02

HN05 ♦ 2 NO * H 20 --- >

-0H2ONO2 * N2C>4

2 HN03 4. N205

(HNQ^) ^

* HN02 * N203

Potassium nitrite was tried as a "catalyst11 for the oxidation of delta-gluconolactone.

The results in Table 3

Indicate that the presence of this Inorganic salt tends to accelerate the formation of saccharic acid.

In these experi­

ments, 0*2 mole of nitrogen tetroxide was allowed to react with 0.1 mole of the lactone in the presence of 4 g. of potassium nitrite at 35°G*

Although less time was required

to achieve comparable results, the yield of saccharic acid was not significantly improved with the utilization of this 11catalyst11. The significance of the results in Table 4 is that added moisture causes the carboxylation step to take place sooner * but the yield of saccharic acid Is still less than 50%.

According to the work of Kenyon, et al*

(11), the

xi

Table 3*

Effect, of KNOg on the Oxidation of delta-Oluconolactone with $ Yield of KH Saccharate (With KNOg) (Without KNOg)

Time hr. 4

15-96

-----

5

28.77

12.41

6

37.11

26.47

7

34.25

35.98

Table 4.

Effect of Added Moisture on the Oxidation of delta-Gluconolactone.

Time hr.

Mole Ratio ^2^4/^6^10°6

% Yield of KH Saccharate ^Wlth Added HgO)

(Without HgO)

3~5

4

43.96

23.69

5

4

46.94

46.41

5

4

44.60

4 4 .36

6

4

45.77

46.54

3

8

41.62

33.84

4

8

48-07

47.38

5

8

44.44

increase in the concentration of nitric acid in the oxldl zing mixture promotes faster conversion of nitrate ester to the carboxyl groups When 0.1 mole of delta-gluconol&ctone was treated with

xll

a n ,excess of nitrogen tetroxide In the absence of a diluent for two and one-half hours* the yield of potassium acid saccharate was 39.5$.

This is almost twice the amount of

product obtained when 0.1 mole of the lactone was allowed to react during the same length of time with 0.8 mole of the oxidant dissolved in carbon tetrachloride.

The advantage

of employing nitrogen tetroxide without a diluent is that the starting material and the oxidation products remain dispersed throughout the course of the reaction. The results of this investigation on the oxidation of delta-gluconolactone to saccharic acid indicate that nitrogen tetroxide may not be a specific oxidizing agent since the yields were not greater than 50^.

It is probable that the

suitable experimental conditions are yet to be discovered. Among the other possible products of this reaction* oxalic acid was found in small amounts. SUMMARY Saccharic acid has been obtained by the oxidation o f gluconic acid with 35-40/& nitric acid.

The best yield

(49.96^) was accomplished In the presence of potassium, nitrite. The oxidation of delta-gluconolactone by nitrogen tetroxide to saccharic acid has been tried under a variety of conditions.

Good yields (40-50^) of product were achieved

in correspondingly less reaction time when the mole ratio of the oxidant

ROOH20(GH3 )2OMgI + HgO

--->

ROGH2G(GH3 )2OH * H* --- > (OH3 )2GHOHO * HON

R00H20(0H5 )20I%I

>>

(GH3 )2OHGHO > ROH (CH3 )20HGH(OH)GN

(CH,)oGHGH(0H)GN + (NHA )oG0^

^

ROGH2G (OH3 )20H + % I . O H

--->

/GONH (0H,)o0HGH ) > NH, * 2 HpO

^ ^

(c h ,)2o h c h

'COBH... B a (O H )g I (

moo

'X'Mran

HgO

^NHGO

J

» *0H_aH0H ( m o )00-H . BaCO, > KH

In this case, the carrier for Isotoplc carbon Is methyl Iodide, which is readily available or can be prepared in 80-90% yield from labeled barium carbonate, using high vacuum technique (4). (4)

Tolbert, THIS JOURNAL, 62, 1529 (194?). In order to study the feasibility of this synthesis,

several alkoxyacetones, namely, iaopropoxyacetone, butoxyacetone, (2-methylbutoxy )acetone, (2-ethylbutoxy)acetone

XV1

and (2-octyloxy)acetone, were prepared according to the following equations (5 »6,7)$ (5)

Barnes and Budde, THIS JOURNAL, 68., 2339 (1946).

(6 )

Gauthier, Comnt* rend. . 143. 831 (1906); Ann, chlm. oh v s .

(7)

[8j , 16, 289 (1909).

Henze, Duff, Mathews, Melton and Forman, THIS JOURNAL, 64, 1222 (1942).

ROH + HOI + HGHO

ROGHgOl ♦ HgO

ROGHgGl ♦ GuGN ROGHgGN * GH^Mgl

ROGHgGN ---^

R0GH2G ( : N % I ) G H 3 + 2 HgO

GuGl

ROGHgC (:NMgl )GH^ -Hf*.

ROGHgGO.GH^ ♦ NH3 * % I . O H

The first step was reported to occur in yields of 50-70% when equivalent quantities of certain alcohols and para­ formaldehyde were treated with an excess of dry hydrogen chloride.

Substitution of trioxane, a more soluble com­

pound , for paraformaldehyde improved the yields which were as much as 91%, provided sufficient time elapsed to allow maximum conversion of the corresponding acetals of formalde­ hyde to the desired products.

In one case, bis(2-ethyl-

butoxy)methane was isolated and later converted to 2-ethylbutyl chloromethyl ether after further treatment. The alkoxyacetonitriles were prepared according to a modified procedure of Gauthier (6 ) in yields of 58-95%. Excellent recovery of the desired nitrile from the reaction

xvli

mixture was possible by distillation with the aid of a highboiling diluent. Table 1.

Preparation of Ohloromethyl Ethers, ROGH^Gl*

R-

% Conversiôn

B.p.°G.

n20D

laopropyl

61

98-101

—

Butyl

39-3

122-128

—————

seewButyl

60

119

—————

Amyl

79+5

65/33 mm.

1.4251

2-MethyIbuty1

80.8

61/35 mm.

1.4240

2-Ethylbutyl

35-89

62-63/19 mnu

1.4320

Octyl

65.2

77-79/6 mm.

I.4363

2-Octyl

91

68-69/6 mm.

1.4345

Table 2.

— — —

Preparation of Alkoxyacetonltriles, ROGHgGN.

R0-

% Yield

B.p.0C.

n2°D

laopropoxy

71

70/55 mm.

1.3968

Butoxy

62.8

72-73/28 mm.

1.4079

sec-Butoxy

58.5

63.5/17 mm.

1.4066

Amyloxy

89.5

77/15 mm.

1.4149

2-Methylbutoxy

95-2

60/7 mm.

1.4138

2-Ethylbut oxy

84

62.5/3 mm.

1.4220

Octyloxy

80.9

105-106/7 mm.

1.4283

2-Octyloxy

90.7

102-103/11 mm. 1.4266

xvîli

Th.d preparation of a Ikoxy acetone s was achieved by treatment of correaponcUng alkoxyacetonitrile s with méthyl­ magnésium, iodide.

This reaction has been investigated, by

Henze and coworkers (7,8), who reported yields of 44-48^. (8)

Henze and Rigler, THIS JOURNAL, £6, 1350 (1934).

Barnes and Budde (5) claim that they were able to improve this reaction in some preparations by addition of the nit rile to the Grignard reagent at ^50°0.

The latter technique was

applied In the preparation of (2-methylbutoxy)acetone, (2-ethylbutoxy )acetone and (â-octyloxy )acetone with conversions of 31-48^.

A 60» 6% yield of the latter was obtained

when the reaction mixture was allowed to reflux slightly during the addition of me thy Imagne slum iodide.

The lower

yields may be attributed to a yside reaction and the oxidiz­ ability of these ketones in air. Table 3»

Preparation of Alkoxyacetones, ROCH^GO.GH^.

R0-

% Yield

B.p.°G.

n20D

laopropoxy

42.3

56-57/40 mm.

1.4007

Butoxy

40.2

69-70/20 mm.

1.4100

2 -Me thy Ibut oxy

23-31

67-68/15 mm.

1.4158

2 -Ethy Ibut oxy

38-55

72-73/9 mm.

1.4228

2-0ctyloxy

44-60.6

94-95/7 mm.

1.4262

xix

The first step in this synthesis of valine involves the addition of méthylmagnésium iodide to an alkoxyacetone to give mono substituted Isobutylene glycol.

According to

the work of Barnes and Budde (5), yields of 50-60^ of the desired products are possible with this reaction.

Hurd

and Ferletz (9) reported an 88^ yield of 1-phenoxy-2-methyl(9)

Hurd and Ferletz, THIS JOURNAL, 68, 38 (1946).

2-propanol; Sommelet (10) was able to obtain 68% of 1-ethoxy(10) Sommelet, Gomet, rend. . 143. 827 (1906); Ann, chlm. ohvs. [83

, £, 484 (1906).

2 -methyl - 2-propanol.

However^ during the course of this

investigation, 1-butoxy-2-methyl-2-propanol and 1 - (2-methyl­ butoxy) -2-methy1-2-propanol were prepared from the correspond­ ing ketones in 49% and 42% yields, respectively.

The former

was also obtained.In 70-83% yields from butyl butoxyacetate and méthylmagnésium Iodide*

Better results may be possible

if the alkoxy radical Is small. Heating of 1-alkoxy-2-methyl-2-propanol in the presence of acids, such as dilute mineral acids, formic acid and oxalic acid, gives rise to the formation of isobutyraldé­ hyde through a mechanism much like that for the plnaoolpinaeolone rearrangement (11).

This method Is similar to

(11) Wheland, “Advanced Organic Chemistry,11 2nd. ed., John Wiley and Sons, Inc., New York, N. Y. , 1949, pp. 451-535.

XX

that originally employed by Somme let (10 ) and more recently by Barnes and Budde (5),

This rearrangement of 1-butoxy-

2-methyl-2-propanol, 1-isopropoxy-2-methyl-2-prop ano1, 1phenoxy-2-methy1^2-propanol, 1-(2-methylbutoxy)-2-methyl2-propanol and 1^(2-ethylbutoxy)-2-methy1-2-propanol in the presence of formic, oxalic, dilute hydrochloric and dilute sulfuric acids gave 33-78^ of isobutyraldéhyde ascertained by gravimetric determination as the bisulfite addition .com­ pound or the p-nitrophenylhydrazone»

Since the formation

of the ^-iiltrophenyIhydrazone is not quantitative, higher yields of the aldehyde are possible. The conversion of isobutyraldéhyde.to 5-isoprapylhydantoin is similar to the method described by Gaudry (12), (12) Wo

Gaudry, Can. J. Research. 24B, 301 (1946). report ed an 84$ yield W e n the intermediate cy anohydri n

was made via the sodium bisulfite ,product*

This method was

modified slightly by the formation of cyanohydrin directly from isobutyraldéhyde and hydrogen cyanide, thereby eliminat­ ing the long process of continuousether extraction.

By

either procedure, however, the maximum yield was found to be approximately 85$»

Greater than 1C$ excess of hydrogen

cyanide appeared to be undesirable because a lower yield of the desired product and excessive coloration were observed. The final step of this synthesis is the hydrolysis of 5-isopropylhydantoin to valine, usually with barium hydroxide. The hydantoin and barium hydroxide octahydrate were dissolved

xxi

In boiling water and transferred into a Gariue tube, which was sealed and heated to 15 0^-160° G » in a Gariue furnace for one-half to three hours*

After barium was separated as the

carbonate, valine was obtained as pure white crystals from a concentrated .solution*

As muoh as 85$ of this amino acid

was produced by this reaction under the experimental con­ ditions described herein. EXPERIMENTAL Ghloromethvl Ethers.— Isopropyl, butyl, sec-butyl, amyl, 2-methyIbutyl, 2-ethylbutyl, and octyl ohloromethyl ethers were prepared according to the procedure described for 2-octyl ohloromethyl ether* 2-Octyl Ghloromethv 1,Ether*-— In a 1-1. three-necked flask fitted with a precislon-bore glass stirrer and a gas delivery tube, hydrogen chloride was passed into a mixture of 390.7 g* (3 moles) of 2-oetanol, 90.1 g. (1 mole) of trioxane, 50 ml. of benzene and 50 ml. of concentrated hydrochloric acid during an interval of six hours*

At the

end of this ,time, the light-colored solution was dried over anhydrous calcium, chloride.

After the solvent was removed,

the desired.product was distilled from a 1-1. Claisen flask at 68-69°G.

(6 mm. ).

The yield was 487 g* (91% conversion).

(2-Meth.v Ibutoxv )acet oni t rile.--Ohloromethyl 2-methylbutyl ether (330 g. , 2.42 moles ) was added.drop wise with stirring to 233 g*

(2.6 moles) of powdered cuprous cyanide,

which was heated simultaneously until the reaction began.

xxii

After the addition was complete, the reaction mixture was heated to 120°C. for another hour.

The mixture was diluted

with 400 ml. of dibutyl phthalate and distilled at 6l-64°C. (8 m m . ). at 60°a.

After redistillation, the desired nitrile boiled

Anal. 11.01.

The yield was 292*7 &* (95*2%).

(7 mm. )-

Galcd. for G ^ H ^ O N :

Found;

G, 66.10; H, 10.30; N,

C, 66.05; H, 10.15; N, 11.12.

se c-Butoxyacetonitrile. ■— This compound was prepared in a similar manner from 175*3 6* (1.43 moles) of secbutyl chloromethyl ether and 183 6* (2.04 moles) of powdered cuprous cyanide. Anal. Found;

The yield was 94.7 g.

Galcd. for CgH ^ O N :

(58. 5% )•

C, 63*68; H, 9*80; N, 12.38.

C, 63*45; H, 9*52; N, 12.38.

(2-Ethylbut oxy )acet onit rile.■— This nit rile was .prepared from 197.8 g. (1.31 moles) of 2-ethylbutyl chloromethyl ether and 144 g. (1.61

moles ) ofcuprous cyanide.

was 155*4 g. or 84% of

theory.

Anal. Found:

Galcd. for

Gq H ^ O N ;

G , 68.04; H,

The yield

10.71;

N, 9*92.

G , 68.15; H, 10.42; N, 9*86.

(2-Methvlbutoxv)acetone.--This ketone was prepared according to the procedure of Barnes and Budde (5) in 22.830*9% yield. Anal.

Galcd. for

OgH-^Og: 0, 66.62; H,

11.18.

Found;

G, 66,80; H, 11.35* (2-Ethylbutoxy lacet one.— This ketone was prepared ac­ cording to the procedure of Barnes and Budde (5) in 38-55% yields.

xxiii

Anal,

Galcd. for G g H ^ O g i

G, 68.31; H, 11.4?.

Found:

G, 68.50; H, 11.58. The semicarbazone, prepared In the usual manner (13 )> (13)

Shriner and Fuson, HIdentification of Organic Compounds, John Wiley and Sons, Inc., New York, N. Y . , 1948, p. 167

melted at.91»5-92.5°G. Anal.

(cor.).

Galcd. for G^QH^OgN^:

N, 19*52.

Found;

N,

19»52* (2-Octyloxv)acetone.--This compound was prepared accord­ ing to the procedure of B a m e s and Budde. (5) in 48.6% yield. Anal.

Galcd, for

G, 70.92; H, 11.91*

Found;

G, 71.05; H, 12.05. The semicarbazone melted at 67»5-68.5°G *

(cor.).

Anal.

Found;

Galcd. for Gi2^25°2^3s

17*27*

N,

17.40. l-Butoxy-2-methyl-2-pronanol,•— Procedure A.

Butyl

butoxyacetate (94.1 g ♦ , 0.5 mole ) dissolved in 200 ml. of dry ether was added dropwise to a stirred solution of méthyl­ magnésium iodide, which, was prepared in the usual manner from 29 g* (1.2 gram-atom) of magnesium, 170 g. (1.2 moles) of methyl iodide and 300 ml. of dry ether.

During the addition

the reaction mixture was cooled in an ice-bath.

After the

addition was complete, the mixture was allowed to attain room temperature and stirred constantly for another hour.

The

reaction mixture was carefully hydrolyzed with 50 ml. of. water and 300 ml. of 12% hydrochloric acid in this order.

xxiv

The ethereal layer was separated and later combined with a 200-ml. ethereal extract of the aqueous layer.

After the

ethereal solution was dried over Drierlte., the solvent was removed by distillation, and the residual liquid was rectified through a 24*in. helix-packed column.

The desired product

boiled at 170°C. (750 mm.); n20D 1.4147* Procedure B.

In a 500-ml. three—necked flask fitted

with a dropping funnel, a reflux condenser and a Hershberg stirrer, 14 g.

(0.107 mole) of butoxyacetone dissolved in

100 ml. of dry ether was added dropwise to a stirred solution of méthylmagnésium iodide, vdiich was prepared in the usual manner from 3*65 g. (0.15 gram-atom) of magnesium, 14,2 g. (0.1 mole) of methyl iodide and 100 ml. of dry ether.

The

solution was cooled in an ice-bath during the addition. After the addition was complete, the reaction mixture was allowed to attain room temperature and stirred for another hour.

The mixture was again cooled in an ice-bath and de­

composed by addition of 50 ml. of 18% hydrochloric acid.

The

ethereal layer was separated, and the aqueous layer was ex­ tracted with three 50-ml. portions of ether.

The combined

extracts were dried over anhydrous magnesium sulfate. , After the ether was removed by distillation, the desired product boiled at 51-52°0. 7.21 g.

(12 mnu); n20D 1.4145.

The yield was

(49.3%).

Anal.

Galcd. for

O' 65*71; H, 12.41.

Found;

G, 65*87; H, 12.45*

1-(2-Methvlbutoxv)-2-methyl-2-propanol.— A solution

XXV

of méthylmagnésium iodide prepared in the usual manner from 12.2 g.

(0.5 gram-atom) of magnesium, 44 g.

(0.31 mole) of

methyl iodide and. 200 ml. of dry ether was added dropwise to a well-stirred solution of 43.25 g* (0.3 mole) of (2methyIbutoxy )acetone in 150 ml. of dry ether.

During the

addition the mixture was cooled in an ice— salt bath.

After

the addition was complete, the reaction mixture was allowed to attain room temperature overnight.

The mixture was again

cooled in an ice-bath and decomposed by addition of 32 g. of ammonium chloride dissolved, in 100 ml. of water.

The

ethereal layer was separated, and the aqueous layer was extracted with five 75-ml. portions of ether.

The combined

extracts were washed with water and dried over Drierlte. After the solvent was evaporated under reduced pressure, the desired product boiled at 55^56°G• (5 mm, ); n2% The yield was 20.4 g. Anal.

1.4178.

(42.4%).

Galcd. for

G, 67*45; H, 12.58.

Found:

0, 67»50; H, 12.53* Isobutvraldehvde.--The following is a typical procedure for the synthesis of isobutyraldéhyde by rearrangement of a monosubstituted isobutylene glycol.

A mixture of 9*81 g.

(0.067 mole) of 1-butoxy-2-methy1-2-propano1 and 15 g • (0.12 m o l e ) of oxalic acid dihydrete was heated gently to approximately 115°G . for five hours.

Simultaneously, the

distillate was collected up to 65°G. In a 100-ml. volumetric flask cooled in ice.

After the receiver was filled to the

mark with dilute ethanol solution, one-tenth aliquots were

XXVI

treated with 1.85 g. of p-nitrophenylhydrazine dissolved in 100 ml. of 3 N hydrochloric acid.

On the basis of this

gravimetric determination, the yield was 75%* 5—XsonronvIhvdantoin (12).— Under a good h o o d , 10 ml. (0.26 mole) of hydrogen cyanide dissolved in 10 ml. of cold ether was added to a cooled solution of 18.03 g . (0.25 mole) of freshly distilled isobutyraldéhyde, a few drops of triethyl amine and 10 ml. of ether.

The resulting cyanohydrin

was transferred to a 200-ml. Morton-type flask containing a mixture of 58 g. (0.6 mole) of ammonium carbonate in 120 ml. of 50% aqueous methanol solution.

The mixture was heated

at 50-55°G. and stirred constantly for six hours.

At the

end of this time, the temperature was raised to 80°0. for another hour to decompose excess ammonium carbonate.

After

the solution was allowed to cool, the white crystals of 5isopropyIhydantoin were collected in a Buchner funnel and washed with a small amount of ethanol.

Additional amount

of the product was obtained by evaporation of the filtrate. The yield of 5-isopropyIhydantoin was 18.5-30.1 g. (52-84.5% of theory); m.p. 143-145°G. DL-Valine (12).— A mixture of 14.22 g.

(0.1 mole) of

recrystallized 5-isopropylhydantoln and 47-5- g* (0.15 mole) of barium hydroxide octahydrate was dissolved in 100 ml. of boiling water and transferred to a Oarlus tube (25 x 19 x 600 mm.).

The tube was sealed and heated at 150-l60°0. for

one-half to three hours in a Oarius furnace.

After room

xxvii

temperature was attained, the tube was opened, and the con­ tents were filtered and washed with hot water.

The filtrate

was treated with 10 g. of ammonium carbonate to precipitate the barium remaining in solution.

The mixture was then heated

rapidly to boiling point to remove excess ammonium,carbonate and filtered hot-

After this filtrate was evaporated to

dryness in vacuo over a steam cone, and residue was dissolved in a minimum amount of hot water, and an equal volume of ethanol was added.

The solution was kept in a refrigerator

for twelve hours to crystallize DL-valine.

The yield of the

desired amino acid was 6.5-10.0 g. or 56-85% of the theoreti­ cal amount. SUMMARY A procedure for the synthesis of labeled DL-valine is described wherein an isotopic carbon atom can be incorporated in the fourth position. The following new compounds were prepared; aeetonitrlle, (2-methylbutoxy)acetonitrile,

sec-butoxy-

(2-ethylbutoxy)-

acetonitrile, (2-methylbutoxy)acetone, (2-ethylbutoxy)acetone, (2-octyloxy)acetone, l-butoxy-2-methyl-2-propanol, and 1(2-methylbutoxy)-2-methyl-2-propanol.

I.

OXIDATION OF GLUCONIC ACID WITH OXIDES OF NITROGEN

INTRODUCTION The oxidation of gluooae and related compounds with nitric acid and oxides of nitrogen has been known for many years to produce acidic derivatives such as saccharic, tar­ taric, 5-ketogluconic and oxalic acids, but the yields of these reactions have been too low to be commercially feasible. The Importance of saccharic acid (18) for use in pharmaceuti­ cal compounds and preparations, in resins and plastics and as a food acid, and also the apparent specific oxidation of the primary hydroxyl groups in certain glycosides with nitric acid (6,19) and nitrogen dioxide (17,29,31) suggested the preparation of saccharic acid from gluconic acid and deltagluconolactone by treatment with these oxidizing agents. This review and study is concerned mainly with the oxidation of gluconic acid and the corresponding lactone when nitric acid and nitrogen tetroxide are used as the oxidizing agents.

Although a survey of the literature re­

veals numerous references to the oxidation of carbohydrates, such as glucose and starch, relatively little work has been published on the conversion of gluconic acid to saccharic acid.

Good summaries of the literature in reference to the

oxidation of glucose and related compounds with nitric acid and nitrogen tetroxide have been compiled by Dietz (6), Mench (19), and B e m t s e n (3)*

2

Gluconic acid is readily available as a 50^ solution or In the form of its derivatives,, calcium gluconate and delta-gluconolactone*

Although gluconic acid may be prepared

from glucose by means of oxidation with bromine (2) or with electrolytlcally generated bromine (2*7*12,13), it is pro­ duced industrially by fermentation of glucose with Aspergillus nlaer (24*30)*

Under the best conditions of air flow, agita­

tion and air pressure found by Wells, et al.

(30), as much

as 97$ yields of gluconic acid, based on the glucose con­ sumed, can be obtained in eighteen hours from 15$ glucose solutions» The use of dilute nitric acid as an oxidant for the preparation of saccharic acid from gluconic acid was reported by Klllani (15) in 1922»

When the reaction was allowed to

proceed at room temperature * he found 5-hetoglueonlc acid as one of the products.

Mench (19) performed an experiment in

which he converted calcium gluconate to the corresponding free sold by removing the calcium ion with sulfuric acid and finally allowed the filtrate to reflux with a known amount of nitric acid for one and one-half hours.

Under these

experimental conditions, 23*1$ yield of saccharic acid was obtained as the potassium acid salt. The most common method of preparing saccharic acid has been the oxidation of glucose with nitric acid in the absence of catalysts such as the vanadic and molybdlc salts. Probably the most extensive studies on the nitric acid oxidation of glucose can be accredited to Klllani (15)* who

3

was able to obtain approximately an 18^ y i e l d .of potassium acid saccharate (16).

Dietz (6) and Mench (19) found experi­

mental conditions,that enabled them to produce potassium acid saccharate in much, better yields than those of Klllani. In a recent patent, Mehltretier (18) described a process for producing saccharic acid with a maximum yield of 45*7/6 of the theoretical amount under the best experimental conditions found.

A solution of glucose is added gradually to a n excess

of 50-70$ nitric acid solution and allowed.to react during an, interval of one hour.

Subsequently, saccharic a d d is

isolated as the potassium acid salt in a manner described later in this manuscript»

The formation o f s m a l l .amounts of

oxalic and 5--ketoglueenie acids has been established in most of these investigations; however, under optimum; conditions saccharic acid is the predominant product when, nitric acid is utilized in non^oataly11c oxidations of glucose. , The presence of catalysts, especially vanadium com­ pounds, tends to accelerate the oxidation so that tartaric acid and increased yields of oxalic acid •can be obtained in addition to saccharic acid.

Mench (20) studied the catalytic

effects of various metals, their salts and oxides, and found vanadium pentoxide to have extremely great activity in the oxidation o f .glucose to tartaric and oxalic acids.

A number

of patents (5,8,10,23,26,27,28) describe processes wherein glucose or glucose^containing materials can be oxidized to saccharic, tartaric and oxalic acids with nitric acid in the presence of catalysts, such as vanadic and molybdlc acids.

4

In moat instances, the yields that are reported have little likelihood of commercial Interest,

Hachlhama and Fujlta

(24) reported that the heating of sucrose with 30% nitric acid at 85°G. for fifteen hours yields 28^ of saccharic acid and 8,1$ of oxalic acid; whereas, the treatment of starch with 35$ nitric acid under similar conditions produces 49«1$ and 10,1$ of these acids, respectively.

With the addition

of small amounts of vanadium salts as catalysts, they claimed that approximately the same results are attained in five hours at 70°G. The apparent specificity which nitrogen tetroxide exhibits in the oxidation of certain glycosides and the fact that in recent years it has become readily available at low cost suggested Its use for the preparation of saccharic acid from gluconic acid and delta*-gluconolaatone.

In order to

determine which of the possible oxidation products of glucose would undergo further oxidation, Bernt sen (3) treated deltagluconolactone and gluconic acid with nitrogen tetroxide and obtained saccharic acid.

Other references pertaining to the

oxidation of gluconic acid or delta-gluoonolactone with this oxidant have not been found.

In fact, Riebsomer (25) , In

his review of the reactions of nitrogen tetroxide with or­ ganic compounds, reported relatively few examples of the oxidation of carbohydrates. As early as 1897» Cohen and Calvert (4) found benzaldehyde after treatment of benzyl alcohol with a mixture of nitrogen trloxide and nitrogen tetroxide, but upon re-

5

investigation of this work Base and Johnson (l) converted benzyl alcohol entirely to benzoic acid when an excess of the oxidizing agent was utilized-

They considered the formation

of benzaldehyde as the first step of the reaction-

Later,

Kenyon and Beyl (14) patented the production of acids from alcohols by the use of this reagent, e.g-, adipic acid from cyclohexanolMenoh (19) reported the reaction of glucose with nitro­ gen tetroxide at various times of oxidation and with different inert solvents, and he was able to attain yields of saccharic acid up to 40%.

B e m t s e n (3) conducted similar studies under

a variety of conditions— namely, time of reaction, amount of nitrogen tetroxide, temperature of reaction, choice of inert media, use of nitrogen tetroxide alone, use of inorganic salts and catalysts.

Under suitable conditions, up to 50$

of the original carbon in glucose was returned as saccharic acid. Mench and Degering (21) treated starch with nitrogen tetroxide in carbon tetrachloride to produce a n e w type of oxidized starch containing, predominantly, uronic acid resi­ dues.

They employed a procedure similar to that of Maurer

and Drefahl (17), who claimed to have obtained a 75$ yield of.mucie acid from the oxidation of galactose with nitrogen tetroxide in an inert diluent, such as carbon tetrachloride or chloroform.

The advantage of this method is that the

oxidation is readily controlled to yield products of any desired carboxyl content.

The factors involved are the time

6

reaction a n d :the molar ratio of oxidant t o .starch. The oxidation of cellulose, with nitrogen tetroxide to a polyanhydroglucuronic acid, as observed by Unruh, Yackel, and Kenyon (29,31)# can serve as another example that this reagent preferentially attacks the primary hydroxyl groups in certain glycosides*

The carboxyl content, which can be

as high as 25%, was found to depend upon the ratio of the oxidant to cellulose and the contact time. Kenyon* et al. (22), after making a fairly exhaustive study of the reaction, suggested a mechanism of the oxidation of cellulose in the presence of carbon tetrachloride.

It is

believed that oxidation with nitrogen tetroxide involves at least two steps, the first of which is nitration.

As it is

virtually impossible to prepare anhydrous cellulose, some nitric acid will be present in any nitrogen tetroxide scheme for oxidizing this substance.

Thus the following mechanism

is suggested* 2 B204 + H 20

»

(C6H 1005 )x + x HNO

2 HN03 + M 203 --- *

(C6H 90 5N02 )x + x HgO

According to their observations, the most significant result is that the nitrogen value reached maxima after a relatively short reaction time and then more slowly decreased to very low amounts.

It appeared evident to Kenyon and his associates

that the presence of nitric acid together with nitrogen tetr-

7

oxide favors rapid dénitration^carboxylation*

Additional

facts supported the conclusion that the presence of nitric acid in the oxidation mixture catalyzes the conversion of nitrate ester to the carboxyl group. REAGENTS USED J. T. Baker Chemical C o . Acetic A c i d , O.P. Nitric Acid# G.P. Potassium Qarbonat e a h h y d . # C.P . Potassium Hydroxide, C.P. Union Carbide & Carbon Corp. Carbon Tetrachloride Ethanol Eastman Kodak Co. Gluconic Acid, 50% tech. Mai1inekrodt Chemical Works Ethyl Ether Phosphorus Pentoxide Potasslum.Nitrite Chas. Pfizer & Co.. Inc. deIt a-Gluconolactone The Solvav Process Co. Nitrogen Tetroxide

8

EXPERIMENTAL A*

Nitric Acid Oxidation of Gluconic Acid

Varying the Time of Reaction.— Table 1 gives the results of experiments wherein a technical grade of 50$ gluconic acid was oxidized with 35$ nitric acid.

The actual concentra­

tion of the gluconic acid solution was found to be 50»7$ when weighed samples were titrated with standard sodium hydroxide solution* A mixture of 20 g. (0.05 mole) of 50$ gluconic acid, 12.8 ml.

(0* 2 mole ) of concentrated nitric acid (d ^

1.42 )

and 8.2 ml. of water was placed In an 8 -in. test tube and heated during a designated interval in a water-bath main­ tained at 85°0*

The reaction mixture was then cooled in

ice water, made slightly alkaline with solid potassium hydroxide and diluted to a total volume of 35 ml.

After

standing approximately one hour, the alkaline solution was acidified with glacial acetic acid.

At the end of sixteen

hou r s , crystallized potassium acid saccharate was Isolated by filtration, washed with a small quantity of cold water and dried in air. The results of the work shown in Table 1 do not show any distinct trend*

One may note# however, that the optimum

yields of saccharic acid can be accomplished in one to two hours under suitable experimental conditions. Varying the Concentration of Nitric Acid.— In the attempt to make an approximate determination of optimum

9

Table 1*

Time Study of the Oxidation of 0.05 Mole of G-luconlo AoId with 35^ Nitric Acid. N.E.

Run

Reaction time (hours)

1

0.5

2.86

23.1

250.4

2

0.5

3.96

31.9

252.8

3

1.0

4.25

34.2

251.4

4

1.0

4.37

35.2

255.6

5

1.5

4.14

33-4

253.6

6

1.5

4.25

34.2

256.6

7

2.0

4.41

35.5

252.2

8

2.0

4.08

32.9

256.7

9

2.0

4.82

38.8

254.1

10

2.5

4.25

34.2

253.8

11

2.5

3.98

32.1

254.8

Yield of KH saccharate (grams) (%)

experimental conditions In the nitric acid oxidation of gluconic acid, variation of the initial concentration of nitric acid was suggested.

In a typical experiment, 20 g.

(0.05 mole) of technical 50% gluconic acid and 12.8 ml. (0.2 mole) of concentrated nitric acid were placed In an 8-in. test tube, and sufficient distilled water was added to adjust the oxidant to the desired strength.

The mixture was

heated during an interval of two hours in a water-bath main­ tained at 85°0.

At the end of this time, the reaction mix­

ture was cooled in an ice-bath, made alkaline with solid

10

potassium hydroxide, diluted to a total volume of 40 ml. and later acidified with glacial acetic acid.

After sixteen

hours, crystallized potassium acid saccharate was isolated by filtration, washed quickly with a small quantity of cold water and dried in air.

The results in Table 2 are somewhat

erratic and show no distinct trend.

One explanation may be

the incomplete recovery of saccharic acid as the potassium acid salt from the reaction mixture. Table 2.

Oxidation of 0. 05 Mole of Gluconic Acid with 0.2 Mole of Nitric Acid at Various Concentrations.

Run

Nitric acid (% concn.)

Yield of KH saccharate (grams) (#)

N.E.

1

25

3.85

31.0

255-6

2

30

3-79

30.5

247-6

3

35

4.82

38.8

254.1

4

35

3.40

27-4

248.2

5

40

3.79

30.5

256.4

6

40

3.71

29-9

246.4

7

45

4.46

36.0

256.6

8

45

2.80

22.6

247-3

Varying the Mole Ratio:

Nitric Acid/Glue onic Acid.—

In a 200-ml. flask fitted with a reflux condenser, 0.10 mole of gluconic acid and a quantity of concentrated nitric acid designated in Table 3 were placed, and sufficient water was added to adjust the acid concentration to the desired per-

11

Table 3«

Oxidation of 0.1 Mole of Gluconic Acid with Various Amounts of Nitric Acid.

Nitric acid (moles) W

Yield of KH saccharate (grams) (%)

N.E.

0.4

35

10.35

41.7

249.6

0.5

35

o cvj • CM H

—— — —

262.5

11.55

46.5

248.2

0.5

35

10.30

41.5

251.5

0.5

35

9.80

39-4

249.5

0.5

35

9-0

36.3

254.1

0.6

35

10.30

41.5

246.4

0.7

35

8.80

35-4

252.1

0.4

40

11.35

45.7

249.2

0.5

40

10.10

40.7

249.0

0.6

40

12.80

— ———

277-8

11.44

46.1

248.2

0.7

40

9.10

36.6

250.6

0.81

40

11.85

----

284.8

centage.

Water in 50^ gluconic acid was also taken into

account.

The flask was immersed in a boiling water-bath

during an interval of one and one-half hours.

The mixture

was then cooled in an ice-bath and made alkaline with solid potassium hydroxide.

If any characteristic crystals of

potassium nitrate formed at this stage, the alkaline solution was filtered.

After standing approximately sixteen hours

12

(overnight for convenience) to saponify the lactone ring completely, the dark basic solution was acidified to pH 3-4 with glacial acetic acid*

When sufficient- time elapsed

to permit optimum crystallization of the product , potassium acid saccharate was removed by forced filtration, washed with 50^ ethanol and dried in air* The results listed in Table 3 do not exhibit enough sign!finance to indicate the trend In the yield of potassium acid saccharate when the mole ratio of nitric acid to glu­ conic acid is changed from 4 to 8:1 under the experimental conditions described herein*

Mench (19) and Dietz (6)

found that optimum yields of saccharic acid from glucose can be obtained when twice the theoretical amount of nitric acid is employed*

Similarly, 4 moles of 35-40$ nitric acid

should be sufficient to provide optimum yields of saccharic acid from one mole of gluconic acid as 2 moles of nitric acid are theoretically required according to the following equations

HOOa(OHOH)4OH2OH + 2 HNCy

___„

HOOC (CHOHl^COOH + 2 HNOg + HgO.

Experiments in which the mole ratio of the oxidant to glu­ conic acid is leas than four were not performed.

The results

in Table 3, however, do indicate that it is possible to achieve 46$ yields of potassium acid saccharate when twice the required amount of nitric acid Is used. To improve the recovery of potassium acid saccharate, the addition of an equal volume of ethyl alcohol to the

13

crystallizing medium was suggested (11).

This not only

accompli shed an apparent increase in the yield through more complete precipitation but also tended to lower the purity of the product , as shown by the h i g h neutral equivalent values in three instances.

On the basis of 248.2, the

neutral equivalent of pure potassium acid saccharate, the yields were calculated. Oxidation of Gluconic Acid in the Bresence of Potassium Nitrite.— When gluconic acid was oxidized with 35% nitric acid in the presence of potassium nitrite, the amount of original carbon returned as potassium acid saccharate was 49.96%.

A mixture of 40 g. (0.1 mole) of technical 50%

gluconic acid, 6 g. of distilled water, 25.7 ml.

(0.4 mole)

of concentrated nitric acid and 10 g. of potassium nitrite (assay 87%) was placed in a 200-ml. round-bottomed flask, which was fitted with a Friedrich condenser.

The flask was

cooled in ice-water for three hours, and finally the reaction mixture was heated in a boiling water-bath during a period of two hours.

At the end of this interval, the mixture

was cooled in an ice-bath, treated.with solid potassium hydroxide to about pH 11 and filtered to remove crystallized potassium nitrate.

After standing four hours, the alkaline

solution was acidified with glacial acetic acid to pH 3-4. The thick precipitate was collected in a Buchner funnel, washed with 50% ethanol and finally dried under reduced pressure.

The yield of potassium a d d saccharate was 12.40

g. , and the empirical neutral equivalent value was 254.2.

14

B.

Oxidation of dalta-Grlucono lac tone and D1 methylene -Dglueonlc Acid with Nitrogen Tetroxide. Oxidation of deIta-Glueonolactone In the Presence

of Carbon Tetrachloride.— The apparatus employed for the oxidation of delta-gluconolactone in the presence of carbon tetrachloride as the diluent Is shown in Figure 1.

delta-

G-luconolactone was dried under reduced pressure at room temperature prior to its use.

Nitrogen tetroxide was dried

over phosphorus pentoxide during storage in Pyrex bromine bottles. de It a -G-lucono lactone (17-31 g. , 0.10 mole) and 250 ml. of dry carbon tetrachloride were placed into the 500-ml. three-necked flask, and the required quantity of precooled liquid nitrogen tetroxide (d^ 1.49) was delivered through the water-jacketed burette cooled with ice-water.

After the

reaction mixture was stirred constantly for a selected length of time at 35 ± 0.1°0., It was cooled In ice in order to retard further reaction.

The unused nitrogen tetroxide was

swept out of the solvent by a stream of air, and finally the carbon tetrachloride was decanted. in 40 ml. of water.

The residue was dissolved

The resulting solution was washed with

two 30-ml. portions of ether, made alkaline with solid potas­ sium hydroxide and allowed to stand overnight to effect complete saponification of the lactone ring.

Finally, the

dark alkaline solution was acidified with glacial acetic acid to pH 3-4.

After the mixture was sufficiently cooled.

15

14/35

18/9

$ 24/40

Fig. 1.

24/40

Apparatus for Carrying Out NgO^ Oxidation

16

potassium acid saccharate was collected in a Buchner funnel, washed with 50^ ethanol and dried under reduced pressure. Oxidation of delta-Crluconolactone in the Absence of a Diluent .--delta-Gluconolactone (17^81 g. , 0.10 mole ) and 80 ml. of precooled liquid, nitrogen tetroxide were placed in a 200-ml. three»-necked flask which was fitted with a motor-driven glass stirrer and a dry-ice-cooled condenser shown in Figure 1.

The reaction mixture was stirred during

an Interval of two and one^half hours at room, temperature. At the end of this time, the excess, oxidant was evaporated with a. current of air.

After the residue was processed in

the manner described previously, 39« 5% yield (9*8 g. ) of potassium acid saccharate was obtained* Preparation of 2,4; 3,5- Dime thy lene - D-g lue onic Acid* — This compound was prepared by a method similar to that of Zlef and Scattergood (32).

delta-Gluconolactone (178.1 g . ,

1 mole) and 90 g. (1 mole) of trioxane were dissolved in 250 ml. of concentrated hydrochloric acid in a 1-1. glassstoppered bottle.

The solution was placed on a shaking

machine for twenty-four hours.

After the reaction mixture

was diluted to a total volume of 500 ml. , it was shaken for another sixty-five hours.

The product was separated, and

recrystallized from water in the form of colorless needles which melted at 2l6-217°G.

(uncor.).

The yield of the desired

product was 145* 8-164*5 6* or 65-74.7$ of the theoretical amount.

Attempted Oxidation of 2,4; 3,S-Dlmethylene-D-gluconlc

17

Acid with Nitrogen Tetroxide»•— The apparatus for this experi­ ment is shown in Figure 1.

2,4;3,5-Dimeth3rlene-D-gluconic

aoid (22 g . , 0.1 mole) and 250 ml. of dry carbon tetrachloride were placed in the 500-ml. three ^-necked flask, and 50 ml. (0.8 mole, d^^ 1.47) of p r e c e d e d liquid nitrogen tetroxide was added through a water-Jacketed burette.

The reaction

mixture was stirred.constantly during an interval of five hours while the temperature was maintained at 35 ± 0.1°0. The excess oxidant was removed from the solvent by a current of air, and the solid material was collected on a Buchner funnel and dried.

The solid substance weighed 21.75 g.

The

empirical neutral equivalent of 216.7 indicates that the desired reaction hardly occurred under these experimental conditions. Other attempts to obtain an oxidized product were also unsuccessful. DISCUSSION OF RESULTS This discussion pertains only to the results obtained from the oxidation of delta-gluconolaotone by nitrogen tetr­ oxide with or without carbon tetrachloride as the diluent. The following conditions were considered:

addition of oxi­

dant at intervals, variation of the mole ratio of nitrogen tetroxide to delta-gluconolactone, time of reaction, effect of inorganic salts, influence of added water, and the pre­ sence or absence of a diluent.

In all cases when carbon

tetrachloride was employed as the diluent, the temperature

18

of the reaction was maintained at 35 ± O#l°0* by means of an oil bath controlled by a mercury thermostat. One approach toward the optimum experimental conditions consisted of the determination of the effect of adding nitrogen tetroxide in portions over an interval of several hours* as shown in Table 4.

The results indicate that the

reaction tends to be rather slow when the mole ratio of nitrogen tetroxide to delta-gluconolactone is less than 2:1. Thu s , It can readily be seen that adding the oxidant at intervals does not have any apparent advantage over the single addition of nitrogen tetroxide at the outset. The results listed in Table 5 illustrate another variable in the experimental conditions of this reaction and are presented graphically in Figure 2.

According to the graph

of yield versus time, the effect of increasing the mole ratio is twofold:

the rate of reaction increases and compar­

able yields of saccharic acid are obtained in less time. Although an increase in the ratio of nitrogen tetroxide to delta-gluconolactone is concomitant with a more rapid forma­ tion of saccharic acid at 35°0. in carbon tetrachloride, yields greater than 50% of the desired product have not been realized.

On the other hand, the maximum yield is reached

in approximately ten hours when the mole ratio is 2:1; whereas, the optimum yield of saccharic acid can be obtained in half the time when the concentration of the oxidant Is quadrupled*

Thus the time of reaction bears a direct relation­

ship with the mole ratio of the oxidant to the lactone.

19

Table 4*

Effect of Adding nitrogen Tetroxide at Intervals.

Total Addition of time (moles) (hrs. (hours) elapsed) 6

Yield of KH saccharate N.E. (grams ] w

0.10 0.10

0 3

1.15

4.65

6

0.20

0

6.57

26.47

6.5

0.10 0.10

0 3

1.67

6.73

0.15 0.15 0.15

0 2 4

21.89

44.10

257.7

8

0.20

0

10.50

42.31

254.3

9

0.10 0.10

0 6

10.50

42.31

253.1

0.10 0.10

0 6

9.0

36.30

255.0

9

0.20

0

10.80

43.50

256.0

9

0.10 0.10

0 6

11.80

0.10 0.10

0 5

0.10 0.10

11.5 24

8

9

9 12

Remarks

259.0

0.20 mole of gluconolactone.

47.54

255.7

5 g. KgOO^ added.

11.30

45.53

254.8

13.8 g. ^ 2 ^ 3 sd&Gd.

0 6

11.30

45.53

256.1

0.20

0

10.50

42.31

256.1

0.20

0

8.20

33.00

254.7

2 ml. AcpO added.

-

Another significant fact illustrated in Figure 2 is that the induction period is diminished with a concomitant increase in the ratio of nitrogen tetroxide to delta-glucono­ lactone.

The investigation by Kenyon, et al.

(22), of the

20

Table 5*

Oxidation of delta-G-luoonolactone with N^O^ at Various Mole Ratios. Yield of KH Saccharate i N.B. Grams

Time hr.

Mole ratio: NgO^/CgH-LQOg

5 6 7 8 9

2 2 2 2 2

3.08 6.57 8.93 10.50 10.80

12.41 26.47 35.98 42.31 43.50

255-7 259.4 261.1 254.3 256.1

9 10 11.5 5-5 5

2 2 2 3 3

11.52 10.83 10.50 3.01 9.65

46.41 43.65 42. 31 12.13 38.88

257.3 253.6 256.0 254.2 255-6

6 7 9 3 3.5

3 3 3 4 4

10.97 11.57 11.40 1.66 5.88

44.20 46. 62 45.93 6 .69 23.69

253.7 256.9 254.0 252.9 254.9

5 5 5 6 8

4 4 4 4 4

11.01 11.52 11.20 11.55 12.06

44.36 46.41 45.13 46.54 48.59

257.1 255-4 250.8 250.7 256.6

2 2.5 3 4 6

8 8 8 8 8

1.40 5.03 8.40 11.76 11.10

5.51 20.27 33.84 47.38 44.72

251.5 256.4 257.7 257.4 255.9

mechanism involving the oxidation of cellulose by this oxidizing agent may offer an explanation for this induction period.

Thus the initial period during which little or no

oxidation is observed may be analogous to the formation of nitrate ester in cellulose as the primary step.

The presence

of nitric acid, which is one of the compounds formed during

Time in Hours i

10

pi H 4

»

---^

HN03

H20 ♦ 2 N 204

►

-ch2ono2 The formation

+ N204

- c h 2o n o 2 +

hno2

+ 2 NO + H20 2 HN05 * N205

(HNO-z) —- 2

^

—002H * hno2 4»N20^•

of nitric oxide (b.p. -151*8°C.)is inferred

from the observation of reddish-brown fumes leaving the dryice-cooled condenser, since the gas was colorless prior to its exposure to the air.

The presence of nitrogen trioxide

(b.p. 3*5°0.) was established by the characteristic blue color of the condensate. In most of the reactions between delta-gluconolactone and nitrogen tetroxide at 35°C. in dry ca,rbon tetrachloride, the attainment of a state as nearly anhydrous as possible in the reaction mixture was believed to be essential for the obtainment of excellent yields of saccharic acid.

Because

both the product and the starting material are practically insoluble in carbon tetrachloride, the presence of water formed in situ was thought to hasten the agglomeration of these substances.

Consequently, the reduction of surface

23

area of ttie solid, reactant meant less efficient use of the oxidant*

This condition could presumably be counteracted

by very rapid stirring.

In an attempt to render the react­

ing mixture free of water as it is formed, anhydrous.potassium carbonate was utilized.

If the relative insolubility of the

latter In most organic solvents and the uniform dispersion of nitrogen tetroxide in carbon tetrachloride are considered, water under these experimental conditions is more apt to react with the oxide of nitrogen to form nitric acid and nitrogen trioxide.

According to the results in Table 4,

however, the presence of potassium carbonate seems to increase the yield of saccharic acid.

Berntsen (3) also found this

to be the case when glucose was treated with nitrogen tetr­ oxide and potassium carbonate in the absence of a solvent. Potassium nitrite was tried as a wcatalyst" because It was reported as such in the nitric acid oxidation of glucose. The results listed in Table 6 Indicate that the presence of this inorganic salt tends to initiate the oxidation of delta-gluconolactone sooner.

In these experiments,, 0.2 mole

of nitrogen tetroxide was allowed to react with 0.1 mole of the lactone in the presence of 4 g. of potassium nitrite at 35°G«

Although less time was required to achieve comparable

results, the yield of saccharic acid was not significantly improved with the utilization of this "catalyst". Table 7 illustrates by comparison the influence of added moisture upon the formation of saccharic acid.

In the course

of these experiments, 0.1 mole of delta-gluconolactone was

24

Table 6.

Effect of KN02 on the Oxidation of delta-Gluconolactone with #2^4"

% Xield of KH Saccharate (With KNOg) (Without KNOg)

Time hr.

Table 7.

4

15.96

5

28.77

12.41

6

37.11

26.47

7

34.25

35.98

Effect of Added, Moisture on the Oxidation of delta-G-luconolactone.

Time hr.

Mole Ratio N204/°6H1006

% Yield of KH Saccharate (With Added HgO) (Without HgO)

3-5

4

43. 96

23.69

5

4

46.94

46.41

5

4

44.60

44.36

6

4

45.77

46.54

3

8

41.62

33.84

4

8

48.07

47-39

5

8

44.44

— ——— —

allowed ^to react In the presence of two drops of water with either 0*4 or 0«8 mole of nitrogen tetroxide dissolved In carbon tetrachloride *

It can readily be seen that the yields

of the desired product comparable to those obtained under

25

apparently anhydrous conditions were achieved In correspond­ ingly less time at the early stage of the reaction*

Accord­

ing to the work of Kenyon, et al* (22), the increase in the concentration of nitric,acid in the oxidizing mixture pro­ motes faster conversion of nitrate ester to the carboxyl group. As oxidation of certain carbohydrates with nitrogen tetroxide in the absence of a diluent has been mentioned in the chemical literature, oxidation of delta-gluconolactone under similar conditions was thought desirable.

After, treat­

ment of 0,1 mole of the lactone with an excess of nitrogen tetroxide at room temperature for two and one-half hours, the yield of potassium acid saccharate was 9.8 g. or 39.5^ of theory.

This represents almost twice the amount of pro­

duct obtained when 0,1 mole of delta-gluconolactone was allowed to react with 0,8 mole of the oxidant dissolved in carbon tetrachloride during the same length of time.

The advantage

of employing nitrogen tetroxide without a diluent is that the starting material and the products of oxidation remain dis­ persed throughout the course of the reaction. The original hope that nitrogen tetroxide might be s p e d f l c for the oxidation of delta-gluconolactone to saccharic acid has not been fulfilled*

Other products formed may in­

clude compounds such as guluronic, 5-ketogluconic, tartaric and oxalic acids.

Attempts to Isolate any of these possible

products other than oxalic acid were not successful.

At

least 3% of the original carbon was found to be oxalic acid.

26

which was obtained by treatment of the filtrate from the isolation of potassium acid saccharate with calcium ace­ tate.

The crude calcium oxalate was reprecipitated before

it was analyzed with standard permanganate solution. The product isolated from the oxidation of deltagluconolactone .according to the procedures mentioned has been established as potassium acid saccharate*

The empirical

neutral equivalents are, for the most part, slightly above the theoretical value of 248.2, which indicates that little or no potassium acid tartrate was present*

The observations

of Berntsen (3)» who ascertained the identity of the salt by several methods of analysis, substantiate that this product is potassium acid saccharate. Phases of this problem which warrant further study Include the determination of the presence of nitrate ester, if any, in the initial stages of the gluconolactone-nitrogen oxide mixture, the oxidation of dime thy lene-D-gluconlc acid under comparable conditions, and the isolation and Identifi­ cation of other products of the reaction. CONCLUSION Saccharic acid has been obtained by the oxidation of gluconic acid with 35$ nitric acid.

The best yield (49.96$)

of potassium, acid saccharate was effected when potassium nitrite was employed as a "catalyst". The oxidation of delta-gluconolactone by nitrogen tetroxide to saccharic acid has been tried under a variety

27

of conditions.

Good yields (40-50%) of product were achieved

in correspondingly less reaction time when the mole ratio of the oxidant to delta-gluconolactone was Increased from 2 to 8:1.

Similar results were obtained in the presence of added

moisture* potassium nitrite and nitrogen tetroxide without a diluent»

Experimental conditions whereby greater than 30%

yields of saccharic acid are possible have not been found. Oxalic acid was found in small but definite amounts to be present in the oxidation mixture. 2,4; 3»5’-Dimethylene-D-gluconic acid was prepared, and the oxidation, of this compound with nitrogen tetroxide was at­ tempted.

28

BIBLIOGRAPHY 1*

Basa and Johnson, J. Am* Qhem* Soc. , 46, 456 (1924).

2.

Bernhauer and Iglauer, Blochem. Z. , 249, 227 (1932).

3-

Berntsen, Ph. D. Thesis, Purdue University, 1949.

4.

Cohen and Calvert, J. Chem* Soc., %1, 1050 (1897)*

5*

Diamalt Akt.^Ges., Brit. Patent 108,494 (Aug. 4,

1917);

C. A., 11, 3278 (1917). 6 * Dietz, Ph. D. Thesis, Purdue University, 1941. 7*

Pink and Summers, Trans. Electro chem. Soc. , %4, 625 (1938).

8 . Graf and Jacoby, Can. Patent 233,734 (Aug. 21, 1923) ; Ç. A., II, 3190 (1923). 9*

Hachihama and Fu jit a, J. Soc. Chem. Ind. , Japan, 3 8 , Suool. binding 744 (1933); 0* A., 3 0 , 4033 (1936).

10.

Hales, U.S. Patent 2,419,019 (April 15, 1947); C. A., 41, 4803 (1947).

11.

Isbell and Frush, Bur. Standards J. He search, 6 , 1145 (1931).

12.

Isbell and Hudson, Bur. Standards J. Research. %, 327 (1932).

13.

R. 0. Hockett, private communication.

14.

Kenyon and Heyl, U. S. Patent 2,298,387

(Oct. 13, 1942);

0. A., 51, 1449 (1943). 15.

Klliani, Ber.. B B B . 75, 2817 (1922).

16.

Klllanl, Ber. , 5 6 B . 2016 (1923).

17.

Maurer and Drefahl, Ber., 7 6 B . 1489 (1942).

29

18.

Mehltratter, U.S. Patent 2,436,659 (Feb. 24, 1948).

19.

Mench, Ph. D. Thesis, Purdue University, 1944.

20.

Mench, M. S. Thesis, Purdue University, 1942.

21.

Mench and Degering, Proc. Indiana Acad. Scl.,

5 5 , 69

(1946). 22.

McGee, Fowler, Taylor, Unruh and Kenyon, J. A m . Ohem. §oc., 6£, 355 (1947).

23.

Odell, U.S. Patent 1,425,605 (Aug. 15, 1922).

24.

Forges, Glark, and Gastrock, Ind. Eng,. Ghem* .

3 2 . 107

(1940). 25.

Riebsomer, Ghem. Rev. . 36., 157 (1945).

26.

Sanders, U^S. Patent 2,419,038 (April 15, 1947); 0. A., 41, 4804 (1947).

27.

Soltzberg, U.S. Patent 2,380,196 (July 10, 1945).

28.

Stokes, U.S. Patent 2,257,284 (Sept. 30, 1941).

29.

Unruh and Kenyon, J. A m . Ghem. Soc. , 6 4 . 127 (1942).

30.

Welts, Moyer, Stubbs, Herrick and May, Ind. Eng. Ghem. , 22, 653 (1937)•

31.

Yackel and Kenyon, J. A m . Ghem. Soc. . 64, 121 (1942).

32.

Zlef and Scattergood, J. Am. Ghem. Soc. . 6 9 . 2132 (1947).

30

II*

A PROCEDURE FOR THE SYNTHESIS OF DL-VALINE CONTAININGA LABELED CARBON ATOM

INTRODUCTION One of the many phases of cancer research is a study of the metabolic fate of amino acids, particularly the essential amino acids or those required preformed in the normal diet, in both cancerous and n o c a n c e r o u s tissues* For this work the investigator is provided with a very use­ ful tool, the isotopes of carbon which can be Incorporated within the amino acid molecule*

Thus, it is possible to

follow these labeled compounds in the course of their metab­ olism by the use of sensitive instruments, such as the mass spectrograph for carbon-13 and the Geiger-Mueller counter for carbon-14 (7)* This investigation is confined to the improvement of known methods and the development of a new synthesis of valine wherein it is desirable to Introduce the tagged carbon in a position other than the carboxyl or acidic group. Although many methods are available now, relatively few can be employed conveniently with economical use of isotopic carbon on a very small scale*

This problem is also limited

to the use of readily available materials, such as labeled carbon dioxide which is the least expensive in the form of barium carbonate.

Thus, it is advisable to devise a synthetic

route that will afford an over-all yield, based on the isotopic starting materials, as high as possible.

31

Prior to th.© past decade, very little was reported on the synthesis of valine, although the preparation of many other amino acids had received much attention.

Marvel (29)

treated Isovaleric acid with bromine in the presence of phosphorus trichloride and obtained 87.5-88.6# of alnhabromolsovaleric acid, which was converted to DL-vallne with aqueous ammonia in 47-«-48# yield. by Cheronls, et al.

The latter step was studied

(9>4-0), who were able to contribute some

improvements by utilizing ammonium carbonate in place of aqueous ammonia. Another method that may be of interest involves the reaction between a benzenediazonlum salt and isopropylacetoaeetlc ester (12,13).

The resulting phenylhydrazone,

alpha-keto1sovaler1c acid phenylhydrazone, after saponifica­ tion with alcoholic sodium hydroxide was found to be reduced with zinc dust in alcoholic solution to DL-vallne.

The

over-all yield was calculated to be approximately 50#. In 1946, G-audry (16) reported a comparative study of the synthesis of valine by the St re eke r method under different experimental conditions.

He observed that the

optimum yields were possible when equimolar amounts of isobutyraldéhyde and potassium cyanide were treated with an excess of ammonium chloride»

The resulting alpha-ami no -

nit rile was hydrolyzed to 53*5-62.5# of valine.

He was able

to achieve a 65# yield of this amino acid through a modified Bucherer synthesis (6,20,35) which involves treatment of isobutyraldéhyde cyanohydrin with a solution of ammonium

32

carbonate to give 5-isopropylhydantoin*

The latter can be

hydrolyzed to valine with barium hydroxide as described later in this manuscript. Many amino aolds have been prepared by alkylation of a cetamidomalonlc ester (15 >28,43) with the appropriate alkyl halide with varying degrees of success*

According to

the work of Snyder, Shekleton and Lewis (42), however, the preparation of derivatives of isoleucine and valine by alkylation of ethyl acetamidomalonate with secondary butyl bromide and isopropyl bromide, respectively, results in very poor yields.

This difficulty may be attributed to

the steric effects of secondary halides and the bulky aeetamidomalonic ester.

On the other hand, Albertson and Tullar

(2,3,46) obtained 6>S% of ethyl oc -acetamido- ac -cyano-y? methylbutyrate from the alkylation of ethyl acetamidocyanoacetate with isopropyl bromide.

An over-all yield of 53%

of valine was achieved after acid hydrolysis of the inter­ mediate*

Apparently, the cyano group offers less hindrance

to the entering isopropyl group.

Similarly, Ehrhart (10)

reported a synthesis of valine from methyl phenylacetamldocyanoacetate and isopropyl iodide. The synthesis of valine may also be accomplished by application of the Hofmann degradation procedure (24) and the Gurtius reaction (14), starting with cyanoacetic ester and an Isopropyl halide• Race ml c valine can be resolved easily by an enzyme, Isolated from the hog kidney, which acts rapidly and asym-

33

metrically upon tfcie N-acylated derivative (34-).

The aoyl

radical Is completely hydrolyzed from the L-form, leaving the N-acylated D-form Intacta

The free L-valine can be

separated by addition of alcohol» leaving the soluble Nacylated D-valln^ In the mother liquor to be hydrolyzed by a mineral acid. REAGENTS USED Allied Chemical & Dye Corporation Benzene American Cvanamld Company Hydrogen Cyanide J. T . Baker Chemical Company Acetic Acid, O.P., glacial Ammonium Carbonate Ammonium Chloride, C.P. Barium Hydroxide Octahydrate Cuprous.Cyanide Formic Acid, C.P., 90% Potassium Carbonat e , C.P. Potas slum Hydroxide, C.P. Silver Sulfate Sodium Carbonate, C.P. Sodium Chloride, C.P. Sodium Bisulfite, C.P. Sodium Hydroxide, C.P. Sulfuric Acid, C.P.

34

Carbide & Carbon Cheoilcals Corporation Acetone Acetic Anhydride Carbon Tetrachloride Dlbutyl Ehthalate Ethanol » absolute 2-Ethylbutanol Isopropyl Alcohol Methanol Columbia Organic Chemicals Co., Inc. Amyl Alcohol Iaopropy1 Iodide Methyl Iodide Central Scientific Company Cuprous Cyanide Dow Chemical Company Magnesium* turnings E. I. du Pont de Hemours and Company, Inc. Trioxane Eastman Kodak Company 2-Butanol Ghloroacetic Acid Ohio ro a cet one 2-Methylbutano1 Octanol 2-0ctanol p-NitrophenyIhydra zine

35

Paraformaldehyde Semiearbazide Hydrochloride B-Toluene sulfonic Acid Trioxane General Chemical Division Hydrochloric Acid, 20° Be' Malllnckrodt Ghemlcal Works Galclum Ghlorlde, anhydrous Guprous Cyanide Ether, anhydrous Magnesium Sulfate, anhydrous Phenol Platinum Chloride Sodium Sodium Nitrite Toluene The Ma theson Company, Inc. Butanol Isobutyraldehyde

EXPERIMENTAL Butyl Butoxyacetate.— Glean sodium (92 g . , 4 gramatoms) was added in small portions to 1300 ml. of dry butanol in a 3-1* round-bottomed flask fitted with a reflux condenser. Then 189 g* (2 moles) of chloroacetic acid dissolved in 200 ml. of dry butanol was added slowly through the condenser

36

at such a rate as to maintain the boiling point of the mix­ ture*

After the mixture was allowed to cool to room temper­

ature, it was acidified with hydrochloric acid and filtered from the precipitated sodium chloride* . The filtrate and 500 ml. of carbon tetrachloride were placed in a 3-1. round^-bottomed flask, which was fitted with a continuous water-extractor, and allowed to reflux for eighteen hours.

After carbon tetrachloride and butanol were

removed by distillation at atmospheric pressure., the residual liquid, was transferred to a 1-1. flask and recti fled through a 24-in. helix-packed column* 77°a*

(3 mm. ); n20D 1.4200.

The-desired ester boiled at The yield was 244.8 g. (65% of

theory). Ethyl Phenoxyaoetate.— To a mixture of 142 g. (1.5 moles) of chloroacetic acid partially neutralized with 42 g. (0.3 mole) of potassium carbonate, 151 g. (1.6 moles) of phenol and 200 ml. of water, a solution of 164 g. of potassium hydroxide (assay 85-8%) in 250 ml. of water was added. The reaction mixture was heated in a 1-1. flask at 80°0. for two hours.

After the mixture cooled, the solid material was

collected In a Buchner funnel, dissolved in 300 ml. of water and treated with 150 ml. of concentrated hydrochloric acid. The yield of phenoxyacetic acid was 201.2 g. (88%); m.p. 97.5-98.5°0. A solution of 201.2 g. (1.32 moles) of phenoxyacetlc acid, 290 ml. of ethanol, 250 ml. of carbon tetrachloride and 1 g. of p-toluenesulfonic acid was boiled for ten hours in a

37

1-1* flask fitted with a continuous water-extractor#

At

the end of this period# the solvent was removed by distilla­ tion#

The residual oil was rectified through a 24-in# helix-

packed column#

Ethyl phenoxyacetaie boiled at 98-99°G .

{2 mm.); n20D 1.5038.

The yield was 169-3 g. (78.25? based

on phenoxyacetlc acid u s e d )#

A fraction boiling at 117°C#

(2 m m # ) contained 18#5 g* of unreacted phenoxyacetlc acid* Fhenoxvacetone.— A mixture of 55 6* (0*58 mole) of phenol, 46*3 g* (0# 5 mole) of chloroaoetone, 70 g. of potas­ sium carbonate and 350 ml* of acetone was allowed to reflux for two hours in a 1-1. flask fitted with a reflux condenser. After the mixture was allowed to cool and filtered, the sol­ vent was removed by distillation at atmospheric pressure. The rosi dual oil was rectified through a 24-in# helix-packed column.

Phenol (17«7 g# ) was recovered at 51°G.

and the fraction boiling at 51-8l°C.

(2 mm. ) ,

(2 mm# ) was dissolved

in 100 ml. of ether, washed with 50 ml* of \0% sodium hydrox­ ide solution and treated with a concentrated solution of sodium bisulfite (35 g # )•

The addition product of phenoxy-

acetone weighed 69*8 g* (55%)* 1-Phenoxy-2-methyl-2-propanol*— Procedure A.

In a

1-1* three-neeked flask fitted with a precision-bore glass stirrer, a reflux condenser and a 250-ml. dropping funnel, 0*27 mole of phenoxyacetone dissolved in 200 ml. of dry ether was added dropwise to a stirred solution of méthyl­ magnésium iodide, which was prepared in the usual manner from 42*5 g# (0.3 mole) of methyl iodide, 7*7 g*

(0.32 gram-

38

atom) of magnesium and 250 ml. of dry ether.

During the

addition the mixture was g o o led in an iee-bath.

When the

reaction was complete* the reaction mixture was decomposed by addition of 40 g. of ammonium chloride dissolved in 200 ml. of water.

The ethereal layer was, separated* and. the

aqueous layer was extracted with 200 ml. of ether.

After

the combined extracts were dried over Drierite, the solvent was evaporated under reduced pressure.

The crude yield of

l-phenoxy-2-methyl-2-propanol was 33*5 S* (74.7%)* Procedure B.

In a 1-1. three-necked flash equipped

with a dropping funnel, a reflux condenser and a precisionbore glass stirrer, 90.1 g. (0.5 mole) of ethyl phenoxyacetate dissolved in 200 ml. of dry ether was added dropwise to a stirred soItu 1 on of méthylmagnésium 1o d i d e w h i c h .was pre­ pared in the usual :manner from 29 g.

(1.2 gram-atoms ) of

magnesium, 170 g * (1.2 moles ) of methyl iodide and 300 ml. of anhydrous ether.

During the addition the reaction mix­

ture was cooled in an ice-b&th.

After the, addition was com­

plete , the mixture was allowed to attain room temperature and stirred constantly for another hour.

The reaction

mixture was carefully hydrolyzed with 50 ml. of water and 300 ml. of 12^ hydrochloric acid in this order.

The ethereal

layer was separated and later combined with a 200-ml. ethereal extract of the aqueous layer.

After the ethereal solution

was dried over Drierite, the solvent was removed by distil­ lation, and the residual liquid was rectified through a 24in. hel^x-p&cked column.

The desired product boiled at

39

87-88°G.

(3 mm. ).

The yield was 61.2 g. or 73*1% based on

ethyl phenoxyacetate,. Butyl Chloromethvl Ether (11).— Gaseous hydrogen chlor­ ide was passed into a mixture of 224*3 8- (3 moles) of nbutanol and 120 g. of paraformaldehyde in a 1-1. three­ necked flask equipped with a precision-bore glass stirrer and a gas delivery tube*

The reaction mixture was cooled

in an ice-bath during the addition.

After approximately

three hours, the mixture became saturated with hydrogen chloride and separated into two layers.

The upper layer was

filtered through Super-Gel to remove excess paraformaldehyde and dried over anhydrous calcium chloride.

The crude pro­

duct was distilled from a 500-ml. Glalsen flask and boiled at 122-128o0 .

Some difficulty was encountered because para­

formaldehyde solidified in the condenser.

The yield was

144.5 g. (39•3% conversion). se c-But rl Chloromethyl Ether (11).— In a 1-1. threenecked flask fitted with a precision-bore glass stirrer and a gas delivery tube, hydrogen chloride was passed into a solution of 224.5 g# (3 moles) of 2-butanol and 115 8* moles) of trioxane which was cooled in an ice-bath.

(1*26

After

approximately three hours, the solution became saturated with hydrogen chloride.

It was filtered from the unreaeted

trioxane and dried over anhydrous calcium chloride.

The

crude product was rectified to give 220.6 g. {60% conversion) of sec-butyl chloromethyl ether boiling at 119°0.

(740 mm. )

and 28 g. of material, presumably the corresponding acetal.

40

vftilch boiled at 158°C»

(740 mm,)*

S-Ethylbutvl Ohioromethyl Ether,— In a 1-1. three­ necked flask fitted with a precision-bore glass stirrer and a gas delivery tube, hydrogen chloride was passed into a cooled mixture of 408.8 g.

(4 moles) of 2-ethyIbutanol and

126.7 g. (1.39 moles) of trioxane during a period of three hours.

The light brown solution was dried over anhydrous

calcium chloride.

The crude product was rectified through

a 24-in. helix-packed column to give 214.3 g. (35*6^ conver­ sion) of 2-ethylbutyl chloromethyl ether boiling at 48°0. (10 m m , ) and 191 g. of the corresponding acetal which boiled at 100°0.

(8 mm.).

The acetal was converted to the chloromethyl ether upon further treatment.

In a 500-ml. three-necked flask

fitted, as described previously, hydrogen chloride was passed Into a mixture of 184 g. (0.85 mole) of bis(2-ethyIbutoxy)methane, 25*5 g* (0.28 mole) of trioxane and 50 ml. of con­ centrated hydrochloric acid for a period of three hours. After the resulting solution was dried over anhydrous calcium chloride, 228.7 g. (89*5^ conversion from the acetal) of 2ethylbutyl chloromethyl ether was distilled from a 500-ml. Claisen flask at 62-63°0.

(19 mm.); n20D 1.4320.

Ghloromethvl 2-0ctyl Ether.— In a 1-1. three-necked flask fitted with a precision-bore glass stirrer and a gas delivery tube, hydrogen chloride was passed Into a mixture of 390.7 g.

(3 moles) of 2-octanol, 90.1 g. (1 mole) of tri­

oxane, 50 ml. of benzene and 50 ml. of concentrated hydro­

41

chloric acid without cooling during an interval of six hours.

At the end of this time, the light-colored solution

was dried over anhydrous calcium chloride.

After the sol­

vent was removed, ohloromethy 1 2-i-octyl ether was distilled from a l-l. Glaisen flask at 68-69°C.

(6 mm.); n ^ D 1.4345•

The yield was 487 S* (91% conversion). Chloromethyl Isonronvl Ether (23)«— This compound was prepared, according to the procedure for chloromethyl 2octyl ether, from 90.1 g. of paraformaldehyde, 180.3 g. (3 moles) of isopropyl alcohol and excess hydrogen chloride. The crude product (215 g . X was distilled from a 500-ml. Claisen flask at 97-104oC.

After redistillation, 199 6»

(61% conversion) of chloromethyl isopropyl ether was obtained at 98-101°C.

(750 mm.).

Chloromethyl Octvl Ether.— ?Thla compound was prepared by treatment of a mixture of 390.7 g* (3 moles) of n-octanol, 55*1 S* of trioxane, 35 6* of paraformaldehyde, 100 ml. of toluene and 50 ml. of concentrated hydrochloric acid with excess hydrogen chloride at 20°C. for six hours.

The desired

product boiled at 77-79°C.

The yield

was 348.8 g.

(6 ram. ); n ^ D 1.4363*

(65-2% conversion).

The corresponding acetal

(131*5 g. ) was obtained at 145-150°C.

(6 mm.).

Amyl Chloromethyl Ether.— This compound was prepared by treatment of a mixture of 247 g * (2.8 moles) of amyl alcohol, 84 g. of paraformaldehyde, 50 ml. of toluene and 30 ml. of concentrated hydrochloric acid with excess hydrogen chloride at 25°0. for six hours.

The desired product boiled

42

at 65°G. (33 mm.); n20D 1.4251.

The yield was 302.7 g. (79*3^

conversion). Ghloromethvl 2-Methvlbutvl Ether.— This compound was prepared by treatment of a mixture of 265 g. 2 -methyIbutano 1 , 90.1 g.

(3 moles) of

(1 mole) of trioxane, 50 ml. of

benzene and 50 ml. of conoentrated hydrochloric acid with excess gaseous hydrogen chloride at 20°0. for six hours. desired product boiled at

6l0G. (35 m m . ); n20D 1.4240.

yield was 330.8 g. (80.8^

conversion).

The The

Butoxvacetonltrlle (2 5 ).--In a 500-ml. three-neeked flask fitted with a reflux condenser and a Hershberg stirrer, a mixture of 107*8 g. (0.88 mole) of butyl chloromethyl ether and 100 g. (1.11 mole) of powdered cuprous cyanide was heated until the reaction started.

Then the source of

heat was removed, and the reaction controlled by means of intermittent cooling with an ice^bath.

When the reaction

subsided, the mixture was heated again to approximately 120°0. for another hour.

Ether (200 ml. ) was added to

the

cooled mixture, and the product was separated by filtration with t h e .aid of Super-Gel.

The desired nitrile was distilled

from a 200-ml. Claisen flask at 72-73°C. 1.4079*

(28 mm.);

The yield was 62.5 g* or 62.8^ of theory.

sec-Butoxyacetonitrile.— According to the procedure described previously, this compound was prepared from 175* 3 g. (1.43 moles) of seç-buty 1 chloromethyl ether and 183 g* (2.04 moles) of powdered cuprous cyanide. nit rile boiled at 63* 5°G.

The desired

(17 mm.); n ^ D 1.4066.

The yield

43

was 94.7 g. or 58 .5/6 of theory. Ami. Found:

Galed. for 06H llONî

°* 63*68; H, 9.80; N, 12.38.

G, 63.45; H, 9.52; N, 12.38.

(2-Ethvlbutoxy)acefronltrile.^^Aoaordlnp; to the proce­ dure described previously, this compound was prepared from 197-8 g. (1.31 moles) of 2-ethylbutyl chloromethyl ether and 144 g.

(1.61 moles) of powdered cuprous cyanide.

nit rile boiled at 62.5o0. (3 mnu); n ^ D 1.4220.

The desired The yield was

I55.4 g. or 84% of theory. Anal. 9.92.

Galcd. for CqH150N:

Found;

0, 68.04; H, 10.71; N,

0* 68.15; H> 10.42; N, 9 .8 6 .

Isopropoxyacetonltrlle.--Ghloromethvl isopropyl ether (185 g . , 1.7 moles) was added dropwise to 170 g.

(1.9 moles)

of powdered cuprous cyanide heated to about 120°G. reaction was very exothermic.

The

After the reaction mixture

was processed in the manner, described previously, 119.5 g. (71% yield) of isopropoxyacetonltrlle was obtained at 70°G. (55 am.-); n20D 1 .3968. Octvloxyacetonitrile.-— In a 1-1 . three-necked flask fitted with a reflux condenser, a Hershberg stirrer and a 500-ml. dropping funnel, 347.1 g. (1.94 moles) of chloro­ methyl octyl ether was added dropwise to 197 g • (2.2 moles ) of powdered cuprous cyanide.

The mixture was heated simul­

taneously to about 120°G. until the reaction began and for another hour after the addition was complete.

After the

mixture was cooled and decanted, the remaining crude product was obtained by distillation in vacuo.

The desired nitrile

44

was distilled from a 500-ml* Olaisen flask at 105-106°C* (7 mm.); n20B 1.4283.

The yield was 301 g. or 80.9# of

theory. Annrloxyaoetonitrlle.--Aooordinp; to the procedure for octyloxyaeetonltrile, this compound was prepared from 300 g.

(2.2 moles) of amyl chloromethyl ether and 220 g.

(2.45 moles) of powdered, cuprous cyanide. nitrile boiled at 77°0.

The desired

(15 mm.); n20D 1.4149.

The yield

was 250.3 g. or 89.5# of the theoretical amount. (2 -Me thy Ibut o xv )ace tonlt rile. — Ohio ro methyl 2-methylbutyl ether (330 g . , 2.42 moles) was added dropwise with stirring to 233 g. (2.6 moles) of powdered cuprous cyanide, which was heated simultaneously until the reaction began. After the addition was complete, the reaction mixture was heated to 120°C. for another hour.

The mixture was diluted

with 400 ml. o f dibutyl phthalate and distilled at 6l-64°0. (8 mm.). at 60°0.

After red!stillation, the desired nitrile boiled (7 m m . ); n20D 1.4138.

Anal. Found;

Galed. for GyH^^ON:

The yield was 292.7 g. (95*2%). G, 66.10; H, 10.30; N, 11.01.

0, 66.05; H, 10.15; H, 11.12.

(2-Ootyloxy)acetonitrile (5)•— According to the pro­ cedure for (2-methylbutoxy)acetonitrile, this compound was prepared from 485*9 S* (2.72 moles) of chloromethyl 2-octyl ether and 260 g. (2.9 moles) of powdered cuprous cyanide. The desired nitrile boiled at 102-103°C. 1.4266.

(11 mm.); n2^D

The yield was 417.6 g. or 90.7 % of theory.

Butoxyacetone.— In a 1-1. three-necked flask fitted

45

with a reflux. coB&enser, a 500-ml, dropping funnel and a Hershberg stirrer, 61.3 g.

(0.54 m ole) of butoxyacetonitrile

dissolved in 200 ml. of dry ethyl ether was added dropwise to a stirred solution of méthylmagnésium iodide, which was prepared in the usual manner from 14.6 g. of magnesium, 81 g. ml. of dry ether.

(0.6 gram-atom)

(0.57 mole) of methyl iodide and 300 During the addition the reaction mixture

was cooled in an ice-salt bath.

After the addition was

complete, the mixture was allowed to stand at room tempera­ ture for sixteen hours.

Then the mixture was cooled in an

ice-bath and decomposed by addition of 250 ml. of 18$ hydro­ chloric acid.

The ethereal layer was separated, and the

aqueous layer was extracted with two 100-ml. portions of ether.

The combined extracts were washed with 100 ml. of

water, dried over anhydrous magnesium sulfate and treated with powdered zinc to remove iodine.

After the solvent was

removed by distillation, 20.3 g. (40.2$) of butoxyacetone was distilled from a 200-ml. Claisen flask at 69-70°C. (20 mm.); n20D 1.4100. Isopronoxvacetone.--Accordln# to the procedure described previously, this compound was synthesized from 115*1 g. (1.16 m o les) of isopropoxyacetonitrile dissolved in 400 ml. of dry ether and méthylmagnésium iodide, which was prepared in the usual manner from 31* 6 g. (1.3 g ram-atoms ) of magnesium, 180 g. ether.

(1.27 moles ) of methyl iodide and 400 ml. of dry The desired ketone boiled at 56-57°C.

na 0 D 1.4007.

The yield was 57 g. (42.3#)-

(40 mm.);

46

X2»Met.hylbutoxy )acetone*— In a 5-1 * three-necked flask fitted with a reflux condenser, a Hershberg stirrer and a dropping funnel, 276.4 g.

(2.17 moles) of (2-methylbutoxy)-

acetonit rile dis solved In 200 ml. of dry ether was added dropwise to a well-stirred solution of méthylmagnésium Iodide, which was prepared from 58.3 6 * (2.4 gram-atoms) of magnesium, 389 g.

(2.6 moles) of methyl iodide and 1500 ml. of dry ether.

The solution was allowed to reflux slightly during the addi­ tion.

After the addition was complete, the mixture was

allowed to stand fifteen hours.

The mixture was cooled in

an ice-bath and decomposed by addition of 1 1 . of 1@$ hydro­ chloric acid.

The ethereal layer was separated, and the

aqueous layer was extracted with five 100-ml. portions of ether.

The combined extracts were washed with 10% sodium

bicarbonate and finally dried over anhydrous potassium car­ bonate and Drier!t e . g.

After the solvent was removed, 71.3

(22*8%) of the desired ketone was obtained at 67"68°Cî.

(15 m m . ); n

po

D 1.4158.

Khen méthylmagnésium iodide was added to the nitrile at -50 t 10°C., 30.9% yield of (2-methylbutoxy)acetone was obtained. Anal.

Calcd. for OgH^^Og:0, 66.62; H, 11.18.

Pound:

G, 66.80; H, 11.33(2-0 ctyloxy)acetone.--In a 1-1 . three-necked flask fitted with a reflux condenser, a Hershberg stirrer and a 500-ml. dropping funnel, 84.6 g. (0*5 mole) of (2-octyloxy)acetonltrlle dissolved in 250 ml. of dry ether was added

47

dropwise to a stirred solution of méthylmagnésium iodide » vfaich was prepared in, the usual, manner from 15 6 * (0*62 gram-atom) of magnesium, 75 g* (0*53 m o l e ) of methyl iodide and 3Q0 ml. of dry ether.

The solution was maintained at

-50 * 10°G. in a bath of dry ice and acetone during the addition.

After the addition was complété, the reaction

mixture was allowed to attain room temperature and to stand four hours longer.

Then the mixture was oooled to -10 r 5°G.

and decomposed by addition of 240 ml. of 18% hydro oh lor 1 c acid.

The ethereal layer was separated, and the aqueous

layer was extracted with two 100-ml. portions of ether.

The

combined extracts were washed with 10% sodium bicarbonate and finally dried over anhydrous magnesium sulfate. solvent, was removed by distillation,

After the

(2-octyloxy)acetone was

obtained a t .94-95°a. (7 mm.); n20D 1.4262.

The yield was

45*3 g. or 48.6% of the theoretical amount. In other experiments wherein the reaction mixture was allowed to reflux slightly during the addition of the nitrile, the yield of the desired ketone was 44.1-60.6%.

Better re­

sults were observed also with smaller runs. A nal.

Galed. for C11H22P 2 *

G, 70.92; H, 11.91.

Found:

0 , 71 »0 5 ; H, 1 2 .05. The semicarbazone, prepared in the usual manner (39)» melted at 67.5-68.5°0 . (cor.). A n al.

Galed. for GtgHg^OgN^:

N, 17.27.

Found:

N,

17.40. (2-Eth vlbutoxv )acetone.— (2-Ethylbutoxy) acetonltrlle

48

(70.6 g., 0.5 mol©) dissolved In 200 ml. of dry ether was added dropwise to a well-stirred solution of méthylmagnésium iodide, which was prepared in the usual manner from 14.115.0 g. of magnesium, 81 g. (0.57 mole) of methyl iodide and 300 ml. of dry ether.

The ,reaction mixture was processed

according to the procedure described for (2-octyloxy)acetone. The desired ketone boiled at 72-73°0-. (9 mnu); n ^ D 1.4228. The yield was 30 .1-43.7 g* or 38-55/6 of theory. Anal. Found:

Calcd. for

C, 68.31» H, 11.47.

C, 68.50 ; H, 11.58.

The semic arba z o n e p r e p a r e d in the usual manner (39), melted at 91.5-92.5°C. (cor.). Anal.

Calcd. for

;

N, 19*52.

Found:

N, 19* 52* l^Butoxv-2-methvl-2-propanol.— Procedure A.

Butyl

butoxyacetate (94.1 g., 0.5 mole) dissolved in 200 ml. of dry ether was added dropwise to a stirred solution of méthyl­ magnésium iodide, which was prepared in the usual manner from 29 g* (1.2 gram-*atoms) of magnesium, 170 g. methyl iodide and 300 ml. of dry ether-

(1.2 moles) of

The reaction mix­

ture was processed In the manner described for 1-phenoxy2-methy 1-2-propanol, Procedure B..

The crude product was

rectified through a 24-in. helix-packed column to give 24-34 g. of butanol (b.p. ll4°C./750 mnu; n20D 1.^991) and 51.261.Z g. (70.2-83.6/6) of l-butoxy-2-methyl-2-propanol. desired product boiled at 170°C. Procedure B.

The

(750 mm.); n20D 1.4147•

In a 500-ml. three-necked flask fitted

49

with a dropping funnel, a reflux condenser and a Hershberg stirrer, 14 g . (0*107 mole) of butoxyacetone dissolved in 100 ml* of dry ether was added dropwise to a stirred solution of méthylmagnésium iodide, which wasprepared in the usual manner from 3*65 g. (0*15 gram-atom) of magnesium, 14*2 g. (0.1 mole) of methyl iodide and 100 ml. of dry ether.

The

solution was cooled in an ice—bath during the addition* After the addition was complete, the reaction mixture was allowed to attain room temperature and stirred for another hour.

The mixture was again cooled in an ice-bath and de­

composed by addition of 50 ml. of 18$ hydrochloric acid.

The

ethereal layer was separated, and the aqueous layer was ex­ tracted with three 50-ml* portions of ether.

The combined

extracts were dried over anhydrous magnesium sulfate.

After

the ether was removed by distillation, the desired product boiled at 51“52°G. 7*21 g.

The yield was

(49*3$)*

Anal. Found;

(12 mm.); n ^ D 1.4145*

Calcd. for

C , 65*71; H, 12.41.

G, 65*87; H, 12.45*

1-(2-Methvlbutoxv)-2-methyl-2-propanol*— In a 500-ml. three-necked flask fitted with a reflux condenser* a Hersh­ berg stirrer and a dropping funnel, a solution of méthyl­ magnésium iodide prepared in the usual manner from 12.2 g* (0*5 gram-atom) of magnesium, 44 g* (0* 31 mole ) of methyl iodide and 200 ml. of dry ether was added dropwise to a well-stirred solution of 43*25 g* (0.3 mole) of (2-methylbutoxy)acetone in 150 ml. of dry ether.

During the addi-

50

tion the mixture was cooled In art ice-salt bath.

After the

addition was complete, the reaction mixture was allowed to attain room, temperature overnight.

The mixture was again

cooled in an ice-bath and decomposed by addition of 32 g • of ammonium chloride dissolved in 100 ml, of water.

The

ethereal layer was separated, and the aqueous layer was extracted with five 75-nil- portions of ether.

The combined

extracts?, were washed with water and dried over Drier it e . After the solvent was evaporated, under reduced pressure, the desired product boiled at 55-56°G (5 mm,); n ^ D 1,4178. The yield, was 20,4 g. or 42,4^ of theoretical amount. Anal,

Calcd, for OgHgoOg:

C, 67,45; H, 12.58,

Found;

0, 67,50; H, 12.53. XsobutvraIdehvde.— Frocedure A.

The fo 11 owing is a

typical procedure for the synthesis of isobutyraldéhyde by re a rrange ment of a monosubstituted laobutylene glycol.

A

mixture of 9.81 g. (0.067 mole) of l-butoxy-2-inethyl-2propanol and 15 6 . (0.12 mole) of oxalic acid dihydrate was heated gently to approximately 115°0 for.five hours.

Simul­

taneously , the di stillate was collected up to 65°0 , in a 100-ml. volumetric flask cooled in ice.

The yield of iso­

butyraldéhyde was determined g ravimet rlcally as the p-nitrophenyIhydrazone.

After the receiver was filled to the mark

with dilute.ethanol solution* one-tenth ..aliquots were t reated with 1.85 S» of p-nitrophenyIhydrazine dissolved in 100 ml. of 3 N hydrochloric acid. was 13%*

On this basis the apparent yield

51

Procedure B*

The following is a typical procedure for

the preparation of Isobutyraldéhyde from an alkoxyacetone. (2-Ethylbutoxy)acetone (15*82 g . , 0.1 mole) dissolved in 30 ml. of dry ether was added dropwise to a well-stirred solution of méthylmagnésium, iodide , which was prepared in the usual maimer from 3*4 g.

(0.14 gram-atom) of magnesium,

21.3 g. (0.15 mole) of methyl iodide and 70 ml. of dry ether. During the addit ion the mixture was oooled in an ice--salt bath.

After the addition was complete, the reaction mixture

was allowed to attain room temperature and stirred for another hour.

The mixture was again cooled in ice a n d ,decomposed

by addition of 15 g* of ammonium chloride dissolved in 50 ml. of water.

The ethereal solution was separated, and the

aqueous layer was extracted with four 15-ml. portions of ether. The ether was evaporated from the combined ethereal extracts under reduced, pressure.

The residual liquid was

mixed with 50 ml. of 10% hydrochloric acid I n a 200-ml. flask, which was iattached to 10-in helix^packed column, and heated to approximately 115°G.

Simultaneously, the distill­

ate was collected in the manner described previously.

On

the basis of p-nltrophenylhydrazone, the yield of isobutyr­ aldéhyde was 42.8-51*7%* 5-1 sopropvlhydantoin (16). — Under a good hood, 10 ml. (0.26 mole) of hydrogen cyanide dissolved in 10 ml. of cold ether was added to a cooled solution of 18.03 g* (0.25 mole) of freshly distilled isobutyraldéhyde, a few drops of trl-

52

ethy lamine and 10 ml» of ether*

The resulting cyanohydrln

was transferred to a 200-ml* Morton-type flask containing a mixture of 58 g* (0*6 mole) of ammonium carbonate in 120 ml. of 50/6 aqueous methanol solution.

The mixture was heated

at 50-55°o. and stirred constantly for six hours.

At the end

of this time, the temperature was raised to 8 0°0. for another hour to decompose excess ammonium carbonate*

After the solu­

tion was allowed to cool, the white crystals of 5-laopropylhydantoin were collected in a Buchner funnel and washed with a small amount of ethanol*

Additional amount of the product

was obtained by evaporation of the filtrate* 5-lsopropylhydantoin was 18.5-50.1 g.

The yield of

(52-84.5$ of theory);

m.p. 143-145°0* DL-Valine (16).— Procedure A. bottomed flask, 14.2 g» 80 g.

Into a 500-ml. round-

(0.1 mole) of 5-i sopropyIhydant o in ,

(0.25 mole) of barium hydroxide octahydrate and 100

ml. of water were placed.

A reflux condenser with a soda-

lime drying tube was attached to the flask.

After the mix­

ture was boiled under reflux for twenty-four hours, it was transferred to a 4C0~ml. beaker and treated with carbon dioxide to precipitate the barium in solution.

The precipi­

tate was removed by filtration and washed with hot water. After the flit rate was retreated with more carbon dioxide and refiltered, the clear solution was evaporated.to dryness in vacuo.

The crude product was dissolved in 65 ml. of hot

water, and 75 ml. of ethanol was added.

The solution was

chilled for twenty-four hours to induce crystallization of

53

DL-valine, «blcti was oolleoted on a Buchner funnel and washed with ethanol.

The mother liquor was reworked to

give more of the.product.

The yield of DL-valine was 8.3 g«

(71% of theory). Procedure B.

A mixture of 14.22 g.

(0.1 mole) of re-

crystallized 5-isopropylhydantoin and 47*5 S*

(0.15 mole)

of barium hydroxide octahydrate was dissolved in 100 ml. of boiling water and transferred to a Oarlus tube (25 x 19 x 600 ram.).

The tube was sealed and heated at 150— 160°0.

for one-half to three hours in a Car lus furnace.

After room

temperature was attained, the tube was opened, and the con­ tents were filtered and washed with hot water.

The filtrate

was treated with 10 g. of ammonium carbonate to precipitate the barium remaining in solution.

The mixture was then

heated rapidly to boiling point to remove excess ammonium carbonate and filtered hot.

After this filtrate was evap­

orated to dryness in vacuo over a steam cone, the residue was dissolved in a minimum amount of hot water, and an equal volume of ethanol was added.

The solution was kept in a

refrigerator for twelve hours to crystallize DL-valine.

The

yield of the desired amino acid was 6 .5-10.0 g. or 56-85% of the theoretical amount. Procedure 0.

Under the atmosphere of dry nitrogen,

100 ml. of absolute ethanol was distilled over calcium hydride directly into a 200-ml. three-necked flask, which was fitted with a precision-bore glass stirrer and a reflux condenser.

To this 1.27 g.

(0.055 gram-atom) of clean sodium.

54

11*95 g. (0.055 mole) of ethyl aoetamldonialonate, and 8 .57 S* (0.05 mole) of dry isopropyl iodide were added in the order given.

This mixture was heated at reflux temperature

with stirring for fourteen hours.

At the end of this tim e ,

the solvent and unreaoted isopropyl iodide were removed by evaporation under reduced pressure.

The residual oil was

treated with 75 ml. of concentrated hydrochloric acid and allowed to reflux for three hours.

The brown solution was

reduced in volume in vacuo and adjusted to pH 6 with ammonium hydroxide.

After an equal volume of ethanol was added, the

solution was allowed to stand in the refrigerator to induce crystallization.

Colorless crystals of DL-valine were sepa­

rated by filtration, washed with ethanol and dried.

The

yield was 12.5-16.2% of the theoretical amount, Ethyl I sonitrosomalonate»— In a 3-1 . three-necked flask fitted with a thermo mete r and a motor-driven Hershberg stirrer, 400 g. (2,5 mo lea) of ethyl malonate, 450 g. (7*5 moles) of glacial acetic acid and 25*5 g. of acetic anhydride were placed.

(0.25 mole)

This mixture was main­

tained below 20°0 . while a solution df 518 g. (7*5 moles) of sodium nitrite in 710 ml. of water was added during an interval of one hour.

After stirring was continued for

five hours longer, the solution was extracted with five 500-ml. portions of ether.

These extracts were washed with

five 150-ml. portions of 10% sodium carbonate solution followed by two 250-ml. portions of water and dried over anhydrous calcium chloride.

The amount of crude product

55

obtained after removal of the solvent in vacuo was 442 .5 6 » Ethvl Aoetamldonialonate.— Grade ethyl isonitrosomalonate (I8.9 g . ) was dissolved in a solution of 50 ml. of glacial acetic acid and 20 ml. anhydride.

(0.88 mole)

(0.22 mole) of acetic

Platinum oxide. (0.6 g . ) was added, and the mix­

ture was subjected to hydrogenation at 50 lbs. pressure. After two hours the theoretical amount of hydrogen was ab­ sorbed.

The mixture was decanted from the catalyst and the

solvent removed by evaporation.

The yield of white, crystal­

line ethyl a cet ami domalonat e was 12.5-18.0 g. m.p. 95.0-96.5°0.

(54-77/0 »

The yield was based on ethyl malonate.

1.15 M 1 sopropyImagneslum Iodide.— This Or 1guard reagent was prepared in the conventional manner from 30 ml.

(0.3

mole) of isopropyl iodide, 9*6 g. (0.4 gram-atom) of magnesium and 175 ml. of absolute ether.

The molarity was determined

by titration with standard hydrochloric acid (1 8 ). Isobutvrlc Acid.-— The apparatus consisted of a large test tube (300 mm. x 38 mm.) to which was attached a 125ml. separatory funnel and a. 50-ml. carbon dioxide generator connected by a small drying tube.

After the apparatus was

evacuated ,to approximately 2 mm. pressure, carbon dioxide, which, was generated by dropping concentrated sulfuric acid upon anhydrous barium or sodium carbonate of known weight, was passed through anhydrous magnesium perchlorate into the reaction tube.

The latter was cooled to -750C ., and a known

quantity of 1.15 M i sopropy Imagne slum iodide was introduced. After a certain period of cooling, the reaction mixture was

56

agitated.

At approximately 0°C. the mixture was carefully

hydrolyzed with 10 ml. of water and enough 6 N sulfuric acid to dissolve all the magnesium, hydroxide.

The mixture was

extracted with six 25-ml. portions of ether.

After the

ether was removed, the residue was steam distilled.

Iso-

butyric acid in the distillate was determined by titration with standard sodium hydroxide solution, using phenophthalein as the indicator. DISCUSSION Several syntheses of valine were considered, but most of them seemed to have at least one undesirable reaction along the line.

The synthetic route. Illustrated by the

following series of equations, was selected because it appears to have the features required in the preparation of labeled compounds. ROCHgOO.CH,

*

3?51 >

H2°

ROOH2G(OH3 )2OH * H*

(CH3 )2OHOHO * HON

RO0H20(CH3 )2OMs I ROOHgO(CHj )gOH * Mgl.OH

--->

---»

(OH3 )2OHCHO . ROH

(OH3 )2aHOH(OH)CN

(OH,)-OHOH(OH)ON * (NHaKOO, -> * ^ ^

/OO.NH — > (0H,)20H0H | ^ ^ x NH.OO * NH3 * 2 H 20

57

/OO.HH (0H3 )20H0H I * B a (O H )o * HgO X NH.OO

--->

(0H3 )gOHOH(NHg)COgH 4* BaCO^ ♦

In this case, methyl iodide is designated as the labeling agent.

Labeled methyl iodide is available or can be pre­

pared in a special apparatus with yields of 80-90% from barium carbonate (44).

Thus, a synthesis of DL-valine,

containing an isotopic carbon in the fourth position, is provided as shown by the following formula:

c*h5chch (m 2 )aogH . ch3 In order to study the feasibility of this synthesis, several alkoxyacetones, namely, isopropoxyacetone, butoxy­ acetone,

(2-methylbutoxy)acetone, (2-ethylbutoxy)acetone and

(2-octyloxy)acetone, were prepared according to the follow­ ing reactions (4,17,22): ROH + HOI 4. HOHO

^

ROGHgOl + GuCN ROOHgGN + CH^Mgl

ROCHgGl * HgO ROGHgON * GuCl

--->

ROGH2G(:NMgI)GH3 + 2 HgO

ROGHgG ( :NMgI )0H3 -V

ROGHgGO.CH3 ♦ NH3 4- Mgl.OH

The first step was reported to occur in yields of 50-70% when equivalent quantities of certain alcohols and para­ formaldehyde were treated with an excess of dry hydrogen

58

chloride (11,22).

The results listed In Table 8 were found

to be more favorable when trioxane was substituted In place of paraformaldehyde.

This fact may be attributed to the

higher solubility of the former In water and the alcohols. Acetals of formaldehyde are by-products of this reaction and can be isolated if the reaction time is not sufficiently long to allow complete conversion to corresponding chloro­ methyl ethers.

In one case, bis(2-ethyIbutoxy)methane was

isolated and converted to 2-ethylbutyl chloromethyl ether after further treatment. The alkoxyacetonitriles were prepared according to a modified procedure of G-authier (1 7 )* who treated a number of chloromethyl ethers with cuprous cyanide.

This reaction

is very exothermic and, in some instances, occurs without the application of heat.

If the chloromethyl ether is added

slowly to h o t , stirred cuprous cyanide, excellent yields of the desired product are possible, as shown in Table 9*

Loss

of product during separation from the reaction mixture can be avoided,usually by distillation with the aid of a highboiling diluent such as dibutyl phthalate.

As well as the

author is aware, sec-butoxyacetonitrile, (2-methylbutoxy)acetonltrlle and (2-ethy Ibutoxy) acetonit rile are compounds which have not been reported in the literature. The preparation of alkoxyacetones, which are designated as starting materials In this synthesis of valine, was accomplished by treatment of corresponding alkoxyaeeto-

59

Table 8.

Preparation of Chloromethyl Ethers # ROCH2 CI.

R

% Conversion

b.p.°C.

n20D

Isopropyl

61

98-101

————

Butyl

39.3

122-128

----

sec-Butvl

60

119

—---

Amyl

79.3

65/33 mm.

1.4251

2-Methylbutyl

80.8

61/35 mm.

1.4240

2-Ethylbutyl

35-89

62-1-63/19 mm.

1.4320

Octyl

65*2

77-79/6 mm.

1.4363

2-Octyl

91

68-69/6 mm.

1.4345

Table 9»

Preparation of Alkoxyaoetonitriles, ROGHgCN.

R0-

% Yield

b.p.°c.

n20D

Isopropoxy

71

70/55 mm.

1.3968

Butoxy

62.8

72-73/28 mm.

1.4079

sec-Butoxy

58.5

63*5/17 mm.

1.4066

Amyloxy

89.5

77/15 mm.

1.4149

2 -Me thy Ibut oxy

95.2

60/7 mm.

1.4133

2-Ethylbutoxy

84

62.5/3 mm.

1.4220

Octyloxy

80.9

105-106/7 mm.

1.4283

2-0ctyloxy

90.7

102-103/11 mm.

1.4266

nitriles with me thyImagnes lum iodide.

Under normal experi­

mental conditions, nitriles react with Grignard compounds to form imino compounds, which on hydrolysis give ketones

60

(30): RON + R'Mgl --- > RR* 0: NMgl RR10:NMgI + 2 HgO

--->

RR'CO 4. I % O H + NH^

The reaction of alpha-alkoxy nit r 1 le a with G-rignard reagents has been investigated by Henze and coworkers (22,23)* who have reported yields of 44-48%*

Barnes and Budde (4) claim

that they were able to improve this reaction in some prep­ arations by addition of the nitrile to the G-rignard reagent at -50oG*

The latter technique was applied in the prep­

aration of (2-methylbutoxy)acetone, (2-ethyIbutoxy)acetone and (2-octyloxy)acetone with conversions of 31-48%.

The

60.6% yield of the latter, as shown in Table 1 0 , was observed when the reaction mixture was allowed to reflux slightly during the addition of méthylmagnésium iodide.

In each

case, a high-boiling dark red oil remained in the flask after the desired product was distilled under reduced pressure. This material may be the result of a side reaction which is the condensation of the nitrile with itself under the in­ fluence of the Grignard reagent, which acts as a strong base.

This by-product appeared to lose its water of con­

stitution at the boiling point even under reduced pressure. Another possibility which should not be overlooked is the oxidizability of these ketones in air, thereby lowering the yield.

Thus, this reaction warrants further study,

although this is not a critical step in the synthesis of

61

Table 10.

Preparation of Alkoxyacetones, ROCOCO. CH-y

R0-

% Yield

n20D

b.p.°G.

Isopropoxy

42.3

56— 57/40 mm.

1.4007

Butoxy

40.2

69-70/20 mm.

1.4100

2- Me thyIbut oxy

22.8-30.9

67-68/15 mm.

1.4158

2-Ethylbutoxy

38-55

72-73/9 mm.

1.4228

2-Octyloxy

44-60.6

94-95/7 mm.

1.4262

labeled valine.

(2-Me thyIbutoxy)acetone, (2-ethyIbutoxy)-

acetone and (2-octyloxy)acetone have not been found in the literature. The first step in the present synthesis of valine in­ volves the addition of méthylmagnésium iodide to an alkoxya cetone to give monosubstituted isobutylene glycol.

Ac­

cording to the work of Barnes and Budde (4), the reaction of G-rignard reagents with some alkoxyketones results in yields of 50-60^ of the desired products.

Hurd and Perletz

(2 6 ) reported an 88% yield of l-phenoxy^2-methyl-2-propanol from phenoxyacetone and méthylmagnésium iodide.

Sommelet

(4l) ,was able to obtain. 68% of 1-ethoxy-2-methyl-2-propanol. However,.during the course of this investigation, 1-butoxy2-methyl-2-propanol and l-( 2-methylbutoxy)-2-methyl-2-propanol, which are believed to be new compounds, were prepared from the corresponding ketones in 49% and 42% yields, respective­ ly.

The former was also obtained in 70-83% yields from

butyl butoxyacetate and méthylmagnésium iodide.

This

62

reaction warranta further study as it is possible to achieve commendable results, especially if the alkoxy radical is small, e.g., methoxy and ethoxy.

Large groups tend to hinder

the addition of the Grignard reagent to the carbonyl group. Heating of l-^alkoxy-2-methyl^2-propanol in the presence of acids, such as dilute mineral acids, formic acid, and oxalic acid, gives rise to the formation of isobutyraldéhyde through a mechanism much like that for pinacol-pinacolone rearrangement (47).

This method is similar to that original­

ly employed by Sommelet (4l) and more recently by Barnes and Budde (4).

During the course of this investigation, this

rearrangement of the mono substituted isobutylene glycol was observed to take place in 33-78^ yields, which were ascer­ tained by gravimetric determination as the bisulfite addition compound or the

nit rophenylhydrazone.

The latter method

of determining the yield was found to be more reliable, since the presence of acid in the product is not detrimental to the, formation of p^nitrophenylhydrazone.

The average yield

of p-nltrophenylhydrazone from nine determinations with known quantities of pure isobutyraldéhyde was 93.13%»

Thus,

if t h i s .correction factor is taken into account , as much as 84$ of isobutyraldéhyde can be realized from the treatment of l-butoxy-2

(CH^ )2OHOH (OH )S O ^ a

(CH3 )2CHaH(O H )S03Na * NaGN

___^

(GH3 )2GHGH(0H)GN + NagSO^

64 /CO.NH

---»

(aH,)2GHCHfOH)CN + (KH4 )pOO, ^ ^

^

(CH^)pOHCH ^ ^

+ my

|

NH. CO

+ 2 HgO

This procedure requires the separation of the cyanohydrln by continuous ether extraction over one or two days prior to treatment with ammonium carbonate for best results.

This

synthesis was modified slightly by the formation of the cyanohydrln directly from isobutyraldéhyde and hydrogen cyanide in much less time.

In either case, the maximum

yield was found to be approximately 85^*

Greater than 10^

excess of hydrogen cyanide, however, appeared to be un­ desirable because a lower yield of the desired product and excessive coloration were observed. Table 12.

Preparation of 5-Isopropylhydantoln. HON (mole)

(

)gCO-^ (mole)

Yield (2)

0.25

0.3

0.6

52.2

0.25

0.37

0.6

20.9

0.25

0.26

0. 6

84.5

0.25

0.25

0. 6

80.0

H 6

0.11

0.24

83.8

O H

(CH5 )2CHCH0 (mole)

0.12

CVJ * o

75.7

0.1

0.11

0.24

54-87

65

The, final atep of this synthesis is the hydrolysis of 5-isopropylhydantoin to valine, usually with barium hydroxide. The hydantoin and .barium hydroxide octahydrate were dissolved in boiling water and transferred into a CSarius tube, which was sealed and heated to 150-l60o0. in a Carius furnace for a desired length of time.

After barium was separated as the

carbonate, valine was obtained as pure white crystals from a concentrated solution.

As much as 85^ of this amino acid

was produced by this reaction under the experimental condi­ tions described herein. Another synthesis of valine, which differs from the first in that isobutyraldéhyde is derived by another method, was Investigated and is illustrated as follows: RX

— > RCOOH

--->

RC0.01

>.

RGHO.

R = (CH3 )2CHThis synthetic route was selected on the basis of the relatively low cost of labeled barium carbonate, a source of carbon dioxide, and favorable carbonation of Grignard reagents.

The techniques involved in the carbonation of

isopropylmagnesium halides have been developed to the point whereby yields of 80-95% of the corresponding acid are possible (7,8,19#21,31,32,36,38,45).

Isobutyryl chloride

can be prepared from isobutyric acid and thionyl chloride in yields of 85-90% according to the procedures of Kent and McElvain (27).

On the other hand, that part of thia synthesis

which pertains to the transformation, RC0.01

-- ^

RGHO,

66

leaves much to be desired.

The conversion of acid chlorides

to corresponding aldehydes with hydrogen in the presence of palladium on barium sulfate, with or without a catalyst poison, is known as the Rosenmund reduction (l).

Although

good results have been recorded for the preparation of aromatic aldehydes, those of the aliphatic series appear to be unsatisfactory.

This fact was observed also by other

members of this Laboratory. At temperatures below -40°0., reactions of Grignard reagents have been known to give excellent yields of com­ pounds which ordinarily react further (33)» and at -20°G. absorption of carbon dioxide in the Grignard reagent occurs rapidly.

When the experimental conditions, namely, the

quantity of Isopropylmagnesium iodide, source of carbon dioxide and time of agitation, were varied, 79-86^ of isobutyric acid was obtained as shown in Table 13-

The barium

carbonate used in these experiments was prepared from solu­ tions of barium hydroxide and sodium carbonate of good grade.

Carbon dioxide was released from a sample of known

weight by treatment with concentrated sulfuric acid under reduced pressure. Another synthetic route for labeled valine that was attempted Involved the alkylation of acetamidomalonic ester, which was prepared by catalytic reduction of ethyl isonitroso malonate in the presence of acetic anhydride.

The most

difficult part of this synthesis is the alkylation of ethyl acetamldomalonate with a secondary halide such as isopropyl

67

Table 13 .

Preparation o£ Isobutyric Acid by Garbonation of 1.15 M Isopropylmagnesium Iodide.

Grignard Reagent ml.

Carbon Dioxide Mmoles source

15

9.14

15

9.63

BaCO^ «

13

10.0

10

10.0

10

10.0

it

20

10.0

11

20

10.0

i«

15

10.0

iodide.

Na^G03 11

NaHCO^

Agitation mln.

Yield

%

30

79.3

45

86.1

30

84.4

30

82.2

30

83.2

15

84.7

10

81.2

13

81.7

According to Snyder, Shekleton and Lewis (42), the

preparation of derivatives of isoleucine and valine by alkylation of ethyl acetamidomalonate with secondary butyl bromide and isopropyl bromide, respectively, affords poor results.

Attempts to produce valine by this method lead to

12.5-16. 2% yields. CONCLUSION

A procedure for the synthesis of labeled DL-valine is described.

More than k-0% over-all yield of this amino acid,

based on the alkoxyacetone, is possible. The following new compounds were prepared: acetonitrile,

sec-butoxy-

(2-methylbutoxy)acetonitrile, (2-ethylbutoxy)-

68

acetonitrile, (2-methylbutoxy}acetone, (2-ethylbutoxy)acetone,

(2-octyloxy)acetone, l-butoxy-2-methyl-2-propanol,

and. 1- (2-methylbutoxy )-2-methyl-2-propanol.

69

BIBLIOGRAPHY 1.

Adams, "Organic Reactions," John Wiley and Sons, Inc., New York, N. Y. , 1949, Vol. 4, p. 362.

2.

Albertson and Archer, U.S. Patent 2,479,662 (Aug. 23* 1949); 0. A., 4 4 , 1133 (1950).

3»

Albertson and Tullar, J. Am. Ghem. Soc., 6%, 502 (1945).

4.

Barnes and Budde, ibid., 68,

5*

Bruson and Me Cleary, U.S. Patent 2,169,578 (Aug. 15,

2339 (1946).

1939); G. A., 31, 9326 (1939). 6.

Bucherer and Lieb, J.

prakt. Ghem. . 141, 5 (1940).

7.

Galvin, Heidelberger, Reid, Tolbert and Xankwlch, "Isotopic Carbon," John Wiley and Sons, Inc., New York, N. Y . , 1949.

8.

Galvin and Lemmon, J. Am. Ghem. Soc. . 6 9 , 1232 (1947).

9.

Oheronls and Spitzmueller, J. Org. Ghem., 6, 349 (1941).

10.

Ehrhart, Ghem. Ber. . 82, 60 (1949).

11.

Farr en, Fife, Clark and Garland, J. A m . Ghem. Soc. , 4%, 2419 (1925).

12.

Feofllaktov and Zaitsev, J. Gen. Ghem. (U.S.S.R. ), 10, 1391 (1940); G. A. , 35., 3606 (1941).

13.

Ibid. , 1 3 , 358 (1943); G. A., 3 8 , 1211 (1944).

14.

Gagnon, Gaudry and King, J. Ghem. Soc. , 1944, 13.

15. Galat, J. Am. Ghem. Soc. , 6 9 . 965 (1947). 16.

Gaudry, Gan. J. Research, 243. 301 (1946).

17.

Gauthier, Gompt. rend. , 143. 831 (1906); Ann, chim. phys. ,

[8]

, 16, 289 (1909).

70

18.

Oilman, et al., J. Am. Ghem. Soc. , 4 5 , 150 (1923)•

19.

Oilman and Kirby, “Organic Syntheses,11 Coll. I, John Wiley and Sons, Inc., New York, N. Y . , 1941,

20.

Goldsmith and Tishler, U.S. Patent 2,480,644

p. 361. (Aug. 30,

1949); 0. A., 44, 2017 (1950). 21.

Heidelberger, Brewer and Dauben, J. A m . Chem. Soc, , 62, 1389 (1947).

22.

Henze, Duff, Ifethews, Melton and Forman, Ibid. . 64, 1222 (1942).

23.

Henze and Rig 1er, Ibid. . $6, 1350 (1934).

24.

Huang, Lin and LI, J. Chinese Chem. Soc. , 15 , 38 (1947); C. A., 42, 523 (1948).

25*

Hurd and Fowler, J. A m .

26.

Hurd and Perletz, Ibid. . 68 , 38 (1946) .

27.

Chem. Soc. , 6 1 , 249(1939).

Kent and McElvain, Org. Synthèses, 2 5 , 58 (1945).

28.

Locquln and Cerchez, Comot, rend. , 186, I360

(1928).

29*

Marvel, OrR. Syntheses, 20, 106 (1940).

30.

Mlgrdlehlan,.11Hie Chemistry of Organic Cyanogen Com­ pounds ,,l A. C. S. Monograph Series No. 105 # Reinhold Publishing Corp. , New York, N. Y. , 1947 , P* 251.

31.

Nahinsky, Rice, Ruben and Kamen, J. A m . Chem. Soc. , 6 4 , 2299 (1942).

32.

Nahinsky and Ruben, Ibid. , 6 3 . 2275 (1941).

33*

Newman and Smith, J. Org. Chem. , 1 3 , 592 (1948).

34.

Price, Gilbert and Greenstein, J. Biol. Chem., 1 7 9 . 1169 (1949).

35*

Rogers, Emmick, Tyran, Phillips, Levine and Scott, J.

71

Am* Ghem. Soc*, 7 1 > 1837 (1949)• 36.

Ruben, Allen and Nahinsky, Ibid., 64, 3050 (1942).

37.

Sabetay, Bull * soc * chim., [4]

38.

Sakami, Evans and Gurin, J. Am. Ghem. Soc.*, 6£, 1110

, 4 5 , 534 (1929)•

(1947). 39-

Shrlner and Puson, "Identification of Organic Compounds," John Sfiley and Sons, Inc., New York, N* Y. , 1948, p. 167.

40.

Sisler and Gheronis, J. Org.

41.

Sommelet, Gompt* rend. . 143. 827 (1906); Ann, chim. Phys. , L8]

42.

Ghem. , 6, 467 (1941).

, £, 484 (1906).

Snyder, Shekleton and Lewis, J. A m . Ghem. Soc. . 6 7 , 310 (1945).

43.

Snyder and Smith, ibid. , 66, 350 (1944).

44.

Tolbert, ibid,, 69, 1529 (1947).

4 5 . Weinhouse, Medes and Floyd, J. Biol. Ghem.,

1 5 5 . 143

(1944). 46.

Wlnthrop Chemical Go. , Inc,, Brit . Patent 621,477 (April 11, 1949); G. A . , 42, 6653 (1949).

47.

Wheland, "Advanced Organic Chemistry," 2nd.

ed. , John

Wiley and Sons, Inc., New York, N. Y . , 1949, pp. 451535.

VITA

Harold A. Price was born on May 27, 1919, in Glenwood, Minnesota.

He attended Thornton Township High School and

Junior College, Harvey, Illinois, where he graduated in 1937 and 1939, respectively.

From. 1941 to 1944, he attended

evening classes at the Illinois Institute of Technology, Chicago, Illinois.

In 1944 he transferred to the University

of Illinois, Urbana, Illinois, and was graduated with a B. S. degree in June of 1945*

After attending Indiana Uni­

versity for one year, he received his A. M. degree in October of 1946.

In September of 1946 he began his work at Purdue

University and was graduated with a Ph. D. degree in June of 1950.

He is a member of Alpha Chi Sigma, Phi Lambda Upsilon

and Sigma XI.

A portion of the following publication was

taken from his thesis for the B. S. degree;

"The Preparation

of Cyclopentenones from Lactones,11 Frank, Armstrong, Kwiatek and Price, J. Am. Chem. Soc. . JO, 1379 (1948).