THE RACEMIZATION OF AMINO ACIDS IN AQUEOUS SOLUTION BY ACETIC ANHYDRIDE

BY VINCENT DU VIGNEAUD AND CURTIS E. MEYER

(From the Laborator?/ of Physiological Chemistry, University of Illinois, Urbana)

(Received for publication, July 21, 1932)

In a previous investigation du Vigneaud and Sealock (1) have reported that the sodium salt of acetyl-l-tryptophane in aqueous solution at 35-40” is completely racemized by acetic anhydride within a few hours. The ease of racemization and particularly the mild conditions under which the reaction occurred seemed to us to offer an excellent method for racemizing amino acids if the reac- tion should prove to be a general one. The present investigation was therefore undertaken to extend the study to other representa- tive amino acids and further to study in greater detail the condi- tions under which the reaction will take place.

The amino acids selected for the work were methionine and cys- tine, the sulfur-containing amino acids, glutamic acid represent,ing the dibasic acids, arginine as an example of the basic amino acids, tyrosine and phenylalanine as representatipes of the monoamino acids, and finally proline which contains a secondary amino or imino group.

The sodium salts of the acetyl derivatives of all these amino acids with the exception of proline were racemized in aqueous solu- tion by acetic anhydride. In the case of proline no racemization occurred. With cystine some decomposition resulted with the separation of free sulfur, although the reaction was carried out at only 36”. When free acetyl-l-cystine in aqueous solution without any NaOH was treat.ed with acetic anhydride, no racemization occurred, nor was there any decomposition. Furthermore, the acetyl-l-cystine was not racemized or decomposed by the alkali alone under the above conditions.

The lack of racemization of the proline and the splitting out of sulfur from cystine during its racemization we regard as being very

295

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

296 Racemization of Amino Acids

significant from the standpoint of the mechanism of the reaction. This question of the mechanism will be dealt with more fully in a later publication.

In order to obtain some idea of the rate of racemization, a large amount of acetylgutamic acid was subjected to the racemization procedure, and, at stated intervals, samples were removed and the decrease in optical activity determined. Since the rotation of acetyl-d-glutamic acid is rather small and therefore not suitable for showing small differences in rotation, the samples were hydrolyzed and the glutamic acid separated as the hydrochloride. The free glutamic acid was then isolated and dissolved in 1 equivalent of hydrochloric acid for the determination of the rotation. The change of rotation with respect to time is shown in Chart I. Within 15 minutes the material was 50 per cent racemized, showing that the racemization proceeded very rapidly at first. After that the rate of the reaction gradually decreased. At 8 hours the ma- t,erial isolated was completely racemized. It is possible, of course, that a small amount of active material might have still remained at the 8 hour point and in the isolation some fractionation might have occurred. The yields obtained in the isolations, however, precluded the possibility of this affecting significantly the results. Furthermore, an experiment was run to test the possibility of frac- tionation by taking a mixture of 0.10 gm. of d-glutamic acid ([ar]i5 = +30.52”) and 0.90 gm. of dl-glutamic acid. The hydro- chloride was isolated and the free glutamic acid then prepared as in the original experiment. The glutamic acid isolated had a rotation of [LY]:’ = +3.10”, which shows that practically no frac- tionation occurred.

The ease of racemization differed somewhat with the various amino acids. For example, during the same time interval that led to a complete racemization of tryptophane, only partial racemi- zation of acetylglutamic acid resulted.

It was of some importance from a preparative as well as a theo- retical standpoint to determine whether or not the racemization was confined to the acetyl derivatives of the amino acids. The formyl derivative of I-phenylalanine was therefore tried. Racemi- zation occurred, although not as readily as in the case of the acetyl derivative. For instance the acetyl compound was completely racemized within 24 hours, and probably much sooner, whereas the

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

V. du Vigneaud and C. E. Meyer 297

formyl compound which originally had a specific rotation of +75” still showed a rotation of +17” at 24 hours. It was necessary to treat the material for 48 hours longer to racemize it completely. This work had been carried out at 36”. At a higher temperature racemization occurs of course much more rapidly. At 76” the racemization was complete within 12 hours.

The fact that racemization can be obtained with the formyl derivative makes possible its direct racemization, where the inac- tive formyl compound is desired for resolution purposes.

We have repeatedly tried to racemize the free acetylamino acid instead of the sodium salt in aqueous solution with acetic anhy- dride. Only slight racemization, if any, occurred. We found no

I? 4 6 8

CHART I. The racemixation of amino acids in aqueous solution by acetic anhydride.

racemization with acetylglutamic acid, and in the case of acetyl- phenylalanine only a relatively slight decrease in rotation resulted from 120 hours action, whereas the sodium salt was completely racemized within 24 hours. It is also very interesting that Behr and Clarke (2) obtained very little racemization when they acety- lated tyrosine by adding acetic anhydride to a suspension of tyro- sine in water at 90-95.” This lack of racemization of the free acetylamino acid and the fact that racemization of the sodium salt takes place in the presence of water are quite in contrast to the catalytic racemization of Bergmann and Zervas (3) in which the free acetylamino acid dissolved in glacial acetic acid is racemized by acetic anhydride in the complete absence of water.

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Racemization of Amino Acids

Although the sodium salts of the acetylamino acids in water have an alkaline reaction towards litmus, the reaction mixture becomes acid to litmus immediately upon the addition of acetic anhydride. Since we have shown that the racemization is a time reaction, it is apparent that the racemization must take place in acid solution. It should follow from this that the racemization should take place about as readily for a mixture of the free acetylamino acid and an equivalent of sodium acetate as for the sodium salt of the acetyl- amino acid. This was shown by experiment to be the case, acetyl- I-phenylalanine being used.

The greater ease of the racemization of the sodium salt in con- trast to the free acid might be due either to a specific effect of the sodium ion or to too great an acidity which might prevent or hin- der the racemization. To test the former possibility, racemiza- tion of the free acid was attempt’ed in the presence of 1 mol of NaCl in place of the sodium acetate. Very little racemization resulted, which showed that the reaction was not due to some catalytic effect of the sodium ion. To test whether the reaction was dependent on the hydrogen ion concentration the sodium acetate experiment was repeated but 1 mol of sulfuric acid was added. There was only a small decrease in rotation. Finally, to rule out entirely the question of the sodium ion and to test whether a buffer other than sodium acetate might work, racemization was carried out with 1 mol of pyridine in place of the sodium hydroxide. Complete race- mization resulted within 24 hours. Furthermore, since mono- acetyl-d-arginine is not as acidic in reaction as the acetyl deriva- tives of the monoamino acids or the dibasic acids, it occurred to us that it should be capable of racemization without any other base present. As was expected, complete racemization occurred.

N-Acetyl-l-tyrosine was prepared in excellent yield by acetylat- ing an alkaline solution of tyrosine with acetic anhydride. The compound crystallized in rod-like crystals from water. The Mil- Ion reaction was positive and the analytical data indicated a mono- acetyl derivative. The melting point of 152-154’ (corrected) is somewhat higher than that reported by Takenaka (4) which was 146-148” for monoacetyl-Z-tyrosine prepared from the ethyl ester of acetyl-Z-tyrosine. The specific rotation of our product [cr]i6 = +47.50” for a 2 per cent aqueous solution agrees well with that found by Behr and Clarke (2), which was 46.2’ for a 4.4 per cent

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

V. du Vigneaud and C. E. Meyer 299

solution in water for their acetyl-Z-tyrosine prepared by the addi- tion of acetic anhydride to an aqueous suspension of tyrosine. By a procedure very similar to the one we have employed Berg- mann and Zervas (3) obtained diacetyl-Z-tyrosine, which crystal- lized in needles and melted at 170’.

When tyrosine was acetylated under the conditions necessary to bring about racemization we obtained the diacetylated inac- tive tyrosine. The difference between this and the above proce- dure was that in the racemization reaction we employed much more acetic anhydride and less sodium hydroxide. The diacetyl- dl-tyrosine crystallized in tufts of small needles, melted at 170° (corrected), and gave a negative Millon reaction.

An observation made in our racemization work on phenyl- alanine which might be significant from a metabolic standpoint is worthy of attention. The acetyl derivative of our d-phenylalanine had a negative rotation of [a], = -51” while Knoop and Blanc0 (5) reported pract,ically the same negative rotation for the acetyl derivative of naturally occurring Z-phenylalanine. After the feeding of inactive acetylphenylalanine to a dog, the latter investigators isolated from the urine an acetylphenylalanine having a levorota- tion. They came to the conclusion that the body could oxidize the acetyl derivative of the unnatural form more readily than that of the natural enantiomorph. These investigators did not report the isolation of the free phenylalanine from-the acetyl derivative, although they did prepare the acetyl derivative of an active phenyl- alanine which they received from H. Fischer and Schoeller. This acetyl derivative they reported as having a rotation of [Q.], = -51X’, but likewise they do not state the rotation of this free phenylalanine. It must follow that the acetyl derivative isolated from the urine was predominantly that of d-phenylalanine and that the body oxidizes more readily the acetyl derivative of the natur- ally occurring form! in contrast to the conclusion of Knoop and Blanco.

We hope to study this question further by the feeding of the acetyl derivatives of both the I- and d-phenylalanines.

It might also be pointed out that the acetyl derivative of the racemized naturally occurring methionine was identical in physical properties with the acetyl derivative of the racemic methionine synthesized by the method of Windus and Marvel (6).

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

300 Racemization of Amino Acids

EXPERIMENTAL

Preparation of Acetyl-SMethionine

The dl-methionine was prepared by the method of Windus and Marvel (6). To an ice-cold solution of 5 gm. of dl-methionine in 10 cc. of water and 16.75 cc. of 2 N NaOH, 10 cc. of 2 N NaOH and 1 cc. of acetic anhydride were added. The solution was kept cold in an ice bath and, during the addition of the acetic anhydride, it was shaken vigorously. Eight such additions were made at 2 minute intervals. After the solution had stood at room tempera- ture for 20 minutes an amount of 6 N H&SO4 corresponding to the NaOH used was added.

After removal of the water and acetic acid by vacuum distilla- tion, the acetylmethionine was extracted with hot absolute ethyl acetate. After evaporation of the ethyl acetate, the compound was recrystallized from the minimum amount of ethyl acetate, and 5.2 gm. of beautiful prismatic needle-like crystals were obtained. The product melted at 114-115’ (corrected). The acetylmethio- nine can also be crystallized from water.

Analysis 3.578 mg. substance: 0.226 cc. N at 24.5’ and 751 mm.

C,HI,08NS. Calculated, N 7.32; found, N 7.16

Preparation of Acetyl-l-Methionine

The I-methionine obtained from a tryptic digest of casein as described by du Vigneaud and Meyer (7) was acetylated as de- scribed above, but ethyl alcohol was used in the extraction of the acetylmethionine. After recrystallization from water the prod- uct melted at 111-111.5° (corrected) and had a specific rotation of [a]? = -16.1”.

Analysis 3.126 mg. substance: 0.212 cc. N at 28.5’ and 742 mm.

C1H1303NS. Calculated, N 7.32; found, N 7.49

Racemixation of I-Methionine

1 gm. of I-methionine was dissolved in 3.71 cc. of 2 N NaOH and diluted to 7 cc. with water. To this solution 2.2 cc. of acetic anhy- dride were then added in three equal portions with vigorous shak- ing. After the solution had stood at. 37” for 5 hours, an amount of

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

V. du Vigneaud and C. E, Meyer 301

6 N H,SOI equivalent to the NaOH used was added. After the solution was evaporated to dryness in vacua the residue was ex- tracted with hot ethyl acetate. 1.20 gm. of acetyl-dl-methionine were obtained. The product was identical with the acetyl deriva- tive prepared from the synthetic dl-methionine described above.

Analysis 3.330 mg. substance: 0.219 cc. N at 27.5” and 747 mm.

C,H,,O,NS. Calculated, N 7.32; found, N 7.34

Rate of Racemization of Acetyl-d-Glutamic Acid

Preliminary experiments indicated that glutamic acid was more resistant to racemization than either tryptophane or methionine. Furthermore, since we wished to determine the rate of racemiza- tion, it was felt advisable to prepare first the active acetyl-d- glutamic acid according to the method of Bergmann and Zervas (3) and study the racemization of the acetyl derivative rather than to start with the free amino acid as we did in the case of methionine. Because of the low specific rotation of acetyl-d-glutamic acid, it was necessary in studying the degree of racemization to hydrolyze the acetyl compound and to use the free glutamic acid for the ro- tation determination. For the latter the glutamic acid was dis- solved in water containing 1 equivalent of hydrochloric acid.

To 20 gm. of acetyl-d-glutamic acid dissolved in 105.8 cc. of 2 N NaOH (2 mols), 140 cc. of acetic anhydride and enough water to make a total volume of 350 cc. were added.. The flask was kept at 38” during the course of the experiment.

At various intervals 35 cc. portions were removed and each worked up in the following manner. In order to decompose the acetic anhydride and hence fix the length of time for the racemiza- tion reaction, 45 cc. of 6 N NaOH were added. 48.5 cc. of 6 N HzSO* were next added and the solution evaporated to dryness in vacua. The acetylglutamic acid was extracted from the Na2S04 with hot absolute ethyl alcohol. After distillation of the alcohol, the compound was hydrolyzed by remixing with 10 volumes of 2.5 N HCI for 1 hour. The glutamic acid hydrochloride was isolated by saturating the ice-cold solution with HCl gas. After crystal- lization seemed complete, an equal volume of absolute alcohol was added, the solution filtered, and the precipitate washed with a few cc. of alcohol and ether. A 90 per cent yield of the glutamic acid

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Racemization of Amino Acids

hydrochloride was obtained. For the determination of the rota- tion the free glutamic acid was isolated and then dissolved in 1 equivalent of HCl. This procedure gave more consistent results than were obtained by determining the rotationof the hydrochloride as isolated. A control run with acetyl-d-glutamic acid in which no acetic anhydride was used showed no decrease in rotation whatso- ever by this procedure. The results are shown in Chart I.

Preparation of Formyl-dl-Phenylalanine

Synthetic dl-phenylalanine was formylated by the method worked out by H. T. Clarke’ for cystine. 28.3 gm. of dl-phenyl- alanine were dissolved in 300 cc. of 85 to 90 per cent formic acid and the solution warmed to 45’. 100 cc. of acetic anhydride were added gradually, the temperature rising to 70”. The solution was allowed to stand at this temperature for about 15 minutes and was then cooled in an ice bath and 200 cc. of water added. Crystal- lization of the formyl derivative began immediately. 20 gm. of formyl-dl-phenylalanine were obtained, which upon recrystalliza- tion gave 18 gm. of the pure compound melting at 168-169” (cor- rected). Fischer and Schoeller (8) report a melting point of 168% 169.8’. By evaporation of the original mother liquor to a small volume and recrystallization of the material, 12.8 gm. more of the pure compound were obtained. The total amount of purified formyl-dl-phenylalanine was 91 per cent of the theoretical yield which is 10 per cent higher than that obtained by the formylation method of Fischer and Schoeller (8).

Resolution of Formyl-dl-Phenylalanine

The formyl-dl-phenylalanine was resolved by means of the bru- tine salt according to the directions given by Fischer and Schoeller (8). Both the formyl-d-phenylalanine and formyl-l-phenylalanine were obtained in excellent yields. Fischer reports that if the mother liquor from the brucine salt of the formyl-d-phenylalanine is kept free from moisture and placed in an ice box for a few days, the brucine salt of the levo compound will begin to crystallize out. We have obtained better yields by evaporating the mother liquor in 2racuo to dryness and recrystallizing the residue from a minimum

1 Personal communication.

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

V. du Vigneaud and C. E. Meyer 303

amount of water. The brucine salt of the formyl-d-phenylalanine melted at 149-150” (corrected) and had a specific rotation of [LY]~’

-35.3”, while the brucine salt of the levo isomer melted at l-93’ and had a specific rotation of [LY]~ = -7.3”. Fischer did not report the rotation of the levo isomer.

The free formyl derivatives were freed from the brucine salts in the usual fashion and obtained in a 93 per cent yield. The formyl- d-phenylalanine melted at 167’ and had a rotation of [a]“,” = -75.9” in 95 per cent ethyl alcohol, which agree with the values given by Fischer and Schoeller [S).

Preparation of d-Phenylalanine

The free d-phenylalanine was prepared by hydrolysis of the formyl derivative by refluxing for 70 minutes with 12 volumes of 1 N HBr. The hydrobromide which was obtained by evapora- tion in vacua to dryness was dissolved in absolute alcohol and NH*OH added until the solution was neutral towards litmus. The phenylalanine crystallized out immediately. The crystalliza- tion was completed by cooling in an ice bath. A yield of 80 per cent of the theoretical was obtained. The d-phenylalanine had a rotation of [ali = +33.5” which is slightly lower than the maxi- mum of +35.1’ reported by Fischer and Schoeller (8).

Racemization of Sodium Salt of Formyl-LPhenylalanine

Preliminary experiments demonstrated that the formyl deriva- tive was fairly resistant to racemization, and therefore a longer period of time for the reaction to take place was indicated. 1 gm. of the formyl-l-phenylalanine was dissolved in 2.6 cc. of 2 N NaOH. 3 cc. of water and 5 cc. of acetic anhydride were added and the solution kept at 37” for 24 hours. The formyl-Z-phenylalanine re- covered had a specific rotation of +17”, showing that although considerable racemization had occurred, the reaction was much slower with the formyl derivative t.han with the acetyl derivative, the racemization of which will be shown later. 0.5 gm. of the above partially racemized product was treated again with the cor- responding amounts of reagents and kept at 37” for 48 hours. The formylphenylalanine obtained after this treatment was com- pletely inactive. By carrying out the reaction at 76” the racemiza- tion was complete within 12 hours.

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

304 Racemization of Amino Acids

Attempts to racemize the formyl-Z-phenylalanine without using any sodium hydroxide were entirely negative. The formyl- phenylalanine recovered after standing with the acetic anhydride and water for 84 hours possessed practically the original rotation.

Preparation of Acetyl-d-Phenylalanine

4.5 gm. of d-phenylalanine were dissolvedin 10.9 cc. of 2~ NaOH. The solution was cooled in an ice bath and eight additions of 9 cc. of 2 N NaOH and 0.9 cc. of acetic anhydride were made at intervals of 2 minutes, the mixture being continually shaken. After the solution had been allowed to stand at room temperature for some time, 27.64 cc. of 6 N H2S04 were added and the solution evap- orated in vacua until the acetyl derivative began to crystallize out. 5.27 gm. of small rectangular plate-like crystals were obtained, the yield being 92 per cent of the theoretical. The product, after recrystallization from water, melted at 172” (corrected) and pos- sessed a specific rotation of [cr]i6 = -51” in absolute alcohol. Knoop and Blanc0 (5) reported a melting point of 170” and a rota- tion of -51.8’ for l-acetylphenylalanine in absolute alcohol. They no doubt had the acetyl derivative of cl-phenylalanine.

Racemization of Acetyl-d-Phenylalanine

The racemization of the acetyl-cl-phenylalanine was carried out as in the case of the formyl derivative, corresponding amounts of sodium hydroxide and acetic anhydride being used. After the mixture had stood for 24 hours at 37”, an amount of H&O4 equiva- lent to the sodium hydroxide used was added, the mixture evapor- ated to dryness, and the acetylphenylalanine extracted with alcohol. The recovered product was completely inactive. Evi- dently the acetyl derivative is more readily racemized than the formyl. The inactive acetylphenylalanine melted at 150-151’ and crystallized in needles. Rnoop and Blanc0 (5) reported a melting point of 151”.

A run was made as above without the sodium hydroxide. The mixture was allowed to stand at 37” for 24 hours. At the end of that period the acetic anhydride was decomposed with excess NaOH, and H&SO4 was added equivalent to the NaOH. The acetyl derivative which was then recovered as described above showed practically no change in rotation. Even after allowing

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

V. du Vigneaud and C. E. Meyer 305

the acetic anhydride to act for 120 hours, the aeetylphenylalanine showed a specific rotation of -48” whereas that of the original material was -51”.

Similar experiments were carried out with, in place of the sodium hydroxide, the equivalent amounts of the various substances to be tested. Experiments were carried out with sodium chloride, sodium acetate, sodium acetate plus sulfuric acid, and pyridine. The sodium chloride and sodium acetate plus sulfuric acid experi- ments led to only little racemization, whereas the sodium acetate and pyridine experiments gave complete racemization.

Racemization of Monoacetyl-d-Arginine

Monoacetyl-d-arginine was prepared according to the directions of Bergmann and Zervas (3). The crystalline product melted at 269-270’ (corrected) and had a specific rotation of [CX]~~ = f7.72” in water.

1 gm. of the free acetyl-d-arginine was dissolved in water and 5 cc. of acetic anhydride were added. The mixture was kept at approximately 37” for 24 hours. At the end of 24 hours more water was added and the solution evaporated in vacua to a small volume. This procedure was repeated three times and finally the material was taken up in 5 cc. of water from which the acetyl- arginine crystallized out in irregular broken platelets upon the addition of acetone as described by Bergmann and Zervas (3). The material was optically inactive and melted at 108-110” (corrected). The compound contains 2 molecules of water of crystallization.

Analysis 3.290 mg. substance: 0.661 cc. N at 29” and 748 mm.

C~HI~OSN~.~HZO. Calculated, N 22.22; found, N 22.34

Preparation of N-Acetyl-l-Tyrosine

15 gm. of I-tyrosine were dissolved in 42.5 cc. of 2 N NaOH and 25 cc. of water. After cooling the solution in ice, 200 cc. of 2 N

NaOH and 20 cc. of redistilled acetic anhydride were added in eight equal portions with vigorous shaking and cooling between additions. After the mixture had stood at room temperature 40 minutes, 83.9 cc. of 6 N HzS04 were added. The mixture was allowed t.o remain in the refrigerator overnight, whereupon a sma.11

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

306 Racemization of Amino Acids

amount of unreacted tyrosine precipitated out. The filtrate was evaporated to dryness in vacua. The residue was then extracted with watery acetone and the extract evaporated to a thick syrup. Addition.of wat.er to the syrup caused it to crystallize in rod-like crystals. Crystallization from water gave 13.6 gm. of a product melting at 152-154” (correct,ed) which had a specific rotation for a 2 per cent aqueous solut.ion of [ol]“d; = 47.5“. The compound gave a positive Millon reaction.

Analysis 3.296 mg. substance: 0.184 cc. Ii at 26” and 746 mm.

CIIH,304N. Calculated, N 6.27; found, N 6.25

Preparation of Diacetyl-dl-Tyrosine

15 gm. of I-tyrosine were dissolved in 166.6 cc. of 1 N NaOH and to this solution were added 60 cc. of redistilled acetic anhydride in 5 cc. portions. After the mixture was allowed to stand at 40” for 4 hours, 27.81 cc. of 6 N H&SO4 were added, and the solution evap- orated and extracted with acetone as above. The residue left after the evaporation of the acetone was crystallized from water in tufts of small needles. 7.8 gm. were obtained. The recrystallized material melted at 168-170” (corrected) and was optically inactive. In contrast to the monoacetyl derivative this compound gave only a slight suggestion of color with the Millon reagent after 10 minutes at room temperature and only a weak test aft’er boiling.

Analysis 3.508 mg. substance: 0.170 cc. N at 30” and 746 mm.

C13H1bOjN. Calculated, N 5.28; found, N 5.36

Racemization of Diacetyl-l-Cystine

1 gm. of diacetyl-l-cystine prepared according to the directions of Hollander and du Vigneaud (9) was dissolved in 6.6 cc. of 1 N

NaOH and 3 cc. of redistilled acetic anhydride were added and the mixture kept at 3540” for 3 hours. The solution diluted to a con- centration of 0.2 per cent acetylcystine gave a specific rot,ation of [a]:’ = -10” in contrast t.o t.hat of the original diacetyl-l-cyst,ine which was [cr]:’ = -107.5”. After standing a day, the racemized solution of acetylcystine began to deposit a precipitate which was identified as free sulfur, indicat,ing that, some decomposition had

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

V. du Vigneaud and C. E. Meyer 307

taken place. We expect to investigate more closely this racemiza- tion and decomposition of cystine.

The rotation -16” no doubt does not represent accurately the rotation of the remaining acetylcystine in the above experiment. However, attempts to isolate the acetylcystine and determine its rotation were unsatisfactory because of the amount of decomposi- tion which had occurred. To show that racemization of acetyl- cystine really had occurred as well as decomposition, the racemiza- tion was carried out under even milder conditions, less acetic anhydride being used. The acetylcystine isolated from this reac- tion mixture was approximately 50 per cent racemized.

Preparation of Acetyl-l-Proline

10 gm. of I-proline isolated by Town’s copper salt method (10) from gelatin was dissolved in 43.5 cc. of 2 N NaOH. To this solu- tion kept cold in an ice bath, 98 cc. of 2 N NaOH and 9.8 cc. of ace- tic anhydride were added in seven equal portions. After the solu- tion had stood for about an hour at room temperature, 47.2 cc. of 6 N H&SO., were added and the solution evaporated in vacua. Water was added a number of times, each time followed by distil- lation to remove the acetic acid. It was finally reduced to dryness and the residue extracted with warm absolute alcohol. The alco- hol was evaporated and the residue recrystallized from 15 cc. of water. After filtration, the crystalline product was dried for 2 days over PzOs in a vacuum desiccator.. The weight of this prod- uct was 9.2 gm. It melted at 116-117’ (corrected).

Upon recrystallization from water diamond-shaped prisms were obtained melting at 81-82” (corrected) after being air-dried. It was shown that this compound contained 1 molecule of water of crystallization and that upon drying to constant weight in vacua at 58” the product melted at 116-117” (corrected). By recrystal- lizing the compound from absolute alcohol and ether, elongated triangular prisms melting at 115-117” (corrected) were obtained. The dry material had a specific rotation of [a]:’ = - 106.7”

Analysis 3.217 mg. substance: 0.257 cc. N at 27.5’ and 746mm.

CTH~~OZN. Calculated, N 8.91; found, N 8.90

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Racemization of Amino Acids

Attempted Racemization of Acetyl-l-Proline

To 4.71 gm. of acetylproline 15 cc. of 2 N NaOH and 19 cc. of acetic anhydride were added. The solution was allowed to stand at 38” for 30 hours. At the end of this period, 5 cc. of 6 N HzS04 were added and the solution evaporated in vacua to a small volume. 59 cc. of water were added and again distilled off. This was re- peated twice more before bringing the material to dryness. The residue then was extracted with warm alcohol and the acetyl- proline isolated as before. The material had the same crystalline form and melting point as given for the acetyl-Z-proline described above and was optically active. The specific rotation of the material was [a]:’ = - 105” which agrees with the specific rotation of the acet.yl-Z-proline already given.

SUMMARY

The racemization reaction found by du Vigneaud and Sealock with the sodium salt of acetyltryptophane in aqueous solution with acetic anhydride has been extended to other amino acids.

It has been found that certain representative amino acids such as glutamic acid, phenylalanine, methionine, tyrosine, and arginine can be completely racemized by this reaction. It has furthermore been shown that the formyl derivative can also be racemized.

The conditions of the reaction have been studied and the rate of reaction of acetylglutamic acid has been determined.

It has also been found that during racemization of acetylcystine by this method decomposition occurs.

No racemization was obtained with proline. The raeemized acetyl derivative of naturally occurring methio-

nine was shown to be identical with the acetyl derivative of the synthetic compound.

BIBLIOGRAPHY

1. du Vigneaud, V., and Sealock, R. R., J. Biol. Chem., 96,511 (1932). 2. Behr, L. D., and Clarke, H. T., J. Am. Chem. Sot., 64,163O (1932). 3. Bergmann, M., and Zervas, L., Biochem. Z., 203,280 (1928). 4. Takenaka, Y., Acta schol. med. univ. imp. Kioto, 4,367 (1922). 5. Knoop, F., and Blanco, J. G., 2. physiol. Chem., 146,267 (1925). 6. Windus, W., andMarvel, C. S., J. Am. Chem. Sot., 62,2575 (1930). 7. du Vigneaud, V., and Meyer, C. E., J. Biol. Chem., 94,641 (1931-32). 8. Fischer, E., and Schoeller, W., Ann. Chem., 367, 1 (1907). 9. Hollander, L., and du Vigneaud, V., J. Biol. Chem., 94,243 (1931-32).

10. Town, B. W., Biochem. J., 22,1083 (1928).

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Vincent du Vigneaud and Curtis E. MeyerACETIC ANHYDRIDE

ACIDS IN AQUEOUS SOLUTION BY THE RACEMIZATION OF AMINO

1932, 98:295-308.J. Biol. Chem. 

  http://www.jbc.org/content/98/1/295.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  ml#ref-list-1

http://www.jbc.org/content/98/1/295.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

by guest on May 15, 2018

http://ww

w.jbc.org/

Dow

nloaded from