A GENERAL REACTION OF AMINO ACIDS.

BY H. D. DAKIN AND RANDOLPH WEST.

(From Xcarborough-on-Hudson and the Medical Clinic, Presbyterian. Hospital, Columbia University, New York.)

(Received for publication, April 5, 1928.)

In the course of some experiments relative to the great variation shown by the lability of sulfur in cystine and its derivatives, it was desirable to prepare diacetylcystine, a substance which has not hitherto been described. Since the usual reagents gave indifferent results it was decided to t.ry a mixture of acetic anhydride and pyridine. Upon warming the mixture on a steam bath, the cys- tine went into solution in the course of 2 or 3 hours and on re- moving the excess of reagents it was expected that the residue, which did not crystallize, would be mainly diacetylcystine. On dissolving the compound in boiling water, in addition to sulfur and hydrogen sulfide the formation of a substance reacting with phenylhydrazine was observed. It was suspected that the latter substance would prove to be pyruvic acid, the formation of which from cystine was long ago observed by Dewar and Gamgee (I). Examination of the phenylhydrazone quickly disproved this idea for not only were the physical properties of the substance, includ- ing the melting point of about 120°, widely different from pyruvic acid phenylhydrazone but in addition it contained much sulfur. Investigation showed that the hydrazone was mainly a derivative of a neutral ketone approximating in composition the formula C6H100zNS.

The formation of this product seemed sufficiently unusual to warrant further investigation and it was soon found that an analogous reaction took place when most amino acids were acted upon by a mixture of acetic anhydride and pyridine. The ketones derived from tyrosine and phenylalanine were particularly well charact.erized crystalline products while those obtained from other amino acids including leucine, p-trimethylalanine, glycine, alanine, histidine, and glutamic acid were readily converted into crystalline

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derivatives. In the case of proline little or no ketone formation was observed. In every case the evolution of carbon dioxide was noted to accompany ketone formation. An investigation of the structure of the compounds indicated that two CHz.CO groups were present, one being attached to nitrogen in the usual fashion and one attached to carbon in the form of a ketone. The reaction with phenylalanine may be represented as follows:

CsHs . CHz - CHNHz - COOH 4 CO2 + CeH6 - CHz - CH

\NH * CO - CH,

It will be noted that the product represents a substituted acetyl- aminoacetone and the properties of the substance and those of its homologues are in entire accord with this supposition.

It will be convenient to consider first the evidence upon which the structure of these compounds is based and then to consider the possible mechanism of their formation.

(a) In the case of amino acids such as tyrosine, phenylalanine, and leucine, in which the reaction with acetic anhydride and pyri- dine proceeds to virtual completion, it was possible to show that almost exactly 1 molecule of carbon dioxide was evolved from each molecule of amino acid. The estimations were made by aspirating carbon dioxide-free air through the reacting mixture heated on a steam bath. The gases evolved were washed through a tube containing a little borax solution Do remove acetic acid or pyridine and then absorbed in barium hydroxide of known strength. The carbon dioxide was determined by titration, phenolphthalein being used in the usual fashion. The yield of carbon dioxide varied from 85 to 97 per cent of the theoretical amount. (b) The presence of one acetyl group attached to nitrogen was indicated by hydrolysis with boiling 10 per cent sodium hydroxide, followed by steam distillation after acidification with phosphoric acid. (c) The presence of a ketone group was shown by the ready formation of derivatives with phenylhydrazine, semicarbazide, aminoguanidine, and similar bases. (d) E vi d ence that the ketone was a methyl- ketone was obtained by noting t.he strong positive iodoform and nitroprusside reactions together with the formation of benzylidene derivatives on condensation with benzaldehyde in the presence of

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H. D. Dakin and R. West 93

dilute sodium hydroxide. (e) The position of the methylketone group was indicat.ed by the fact that glycine, /I-trimethylala- nine, and phenylaminoacetic acid underwent the reaction in question and in these substances the a-carbon atom of the amino acid is the only possible place of att.achment. (f) The representa- t,ion of the products as derivatives of acetylaminoacetone was shown to be correct by the observation that after removal of the acetyl group attached to nitrogen by hydrolysis w&h acid, by the action of alkali, preferably in the presence of a mild oxidizing agent such as mercuric chloride, they are converted into pyrazine derivatives, just as aminoacetone was shown by Gabriel and Pinkus (2) to give 2,Bdimethylpyrazine. Thus the derivative from alanine gave tetramethylpyrazine. Also like aminoacetone they may be condensed with thiocyanic acid in acid solution but these compounds await further examination. (g) Lastly by the action of p-nitrophenylhydrazine in hot dilute sulfuric acid solu- tion, the new ketones are corrected into the highly characteristic his-nitrophenylhydrazones of glyoxals, just as aminoacetone gives methylglyoxal his-nitrophenylhydrazone. The two last reactions (f> and (g) may be represented as follows in the case of the product from glycine :

N

H& - C’k II I

:H

CHz-NH.CO-CH, HC kf”’ CH3 I

CHz - NH2 - COOH --$ CO 7

I L &Ha CH : N - NH * GHn - NOz

I c: N - NH - CsH4.NOz I

CH,

Assuming that the foregoing evidence adequately justifies the structure, R.CH.(CO*CHS)+NH.C0.C!H3, assigned to these derivatives, the next question is how to account for their formation for it will be generally conceded t.hat the substitution of hydrogen attached to carbon in an aliphatic chain by a CH,.CO group is sufficiently unusual to warrant further consideration. At first the

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General Reaction of Amino Acids

possibility was considered that a diacetyl derivative of the amino acid was initially formed and that as the result of intramolecular rearrangement one acetyl group migrated from nitrogen to carbon. The resulting product would be a p-ketonic acid, which would be expected to lose carbon dioxide, giving the products in question:

R - CHNHz . COOH -+ R . CHN(COCH& - COOH -+

l-t - CCC0 * CH,) . (NH. COCH,) . COOH +

R . CH . (COCHI) . NH - CO - CH3 + CO1

Such a change involving the migration of a CH,. CO group from nitrogen to carbon while sufficiently unusual would bear a certain resemblance to the formation of acylaminoketones from diacyl- anilides as observed by Chattaway (3). The likelihood of the pre- ceding hypothesis was reduced almost to the vanishing point by the following observations. In the first place no similar reaction involving ketone formation takes place when a base such as benzylamine or @-phenylethylamine is heated with acetic anhy- dride and pyridine, although conditions should favor the initial production of diacetylated bases. Furthermore the reaction involving ketone formation from amino acids is to a certain extent specific to pyridine and its homologues including lutidine and collidine, while other bases such as dimethylaniline and quinoline, while certainly not interfering with acetylation do not bring about the formation of significant amounts of ketones, at least at temperatures which are effective with pyridine, namely about 80-90”. An attempt to settle the question whether diacetylation of the amino group was a necessary preliminary to ketone formation was made by using N-methyl- and N-dimethylamino- phenylacetic acids. Obviously in the case of these substances diacetylation could not take place. When heated with acetic anhydride and pyridine carbon dioxide was not evolved and the final products of the reaction appeared to be almost entirely the monoacetyl derivative. Sarcosine gave similar results and though a little carbon dioxide was evolved no ketone could be identified in either case, A much more definite answer to the problem in question was obtained by the observation that the reaction with acetic anhydride and pyridine was not restricted to amino acids and that an analogous reaction-though oft,en complicated by side

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H. D. Dakin and R. West

changes-took place with acids containing no nitrogen and in- capable of acetylation. Thus while hydroxy acids appeared not to react, acids as different as chloroacetic, phenylbromoacetic, and ol-bromostearic acids, all underwent at varying speeds the reaction typical of the amino acids with evolution of carbon dioxide. Hip- puric acid behaved like glycine, the benzyl group being replaced by acetyl. Even an unsubstituted acid such as phenylacetic acid on being heated with pyridine and acetic anhydride was converted into methylbenzylketone. The change may be represented as follows :

CsH6. CHz . COOH + C&H5 . CH . (CO . CH,) . COOH ---f CsHs . CHz . CO . CH,

In the light of the foregoing evidence it may be taken as certain that migration of an acetyl group from nitrogen to carbon is not involved in the formation of these ketones from amino acids and that diacetylation if it occurs at least is not essential.

On the other hand the reaction bears a close resemblance to certain changes observed over 40 years ago by IV. H. Perkin, senior (4). Acetone for example resulted from heating at 180-200’ acetic anhydride with sodium acetate in sealed tubes, and pro- prione and butyrone were similarly obt.ained. Perkin interpreted the action along lines analogous to those adopted by him to repre- sent his cinnamic acid synthesis but Fittig (5) 10 years later without further experimental data indicated the probability that p-ketonic acids were first formed and then decomposed to the ketone and carbon dioxide.

R . CHz . CO \ /

O+CH~COOH-+R~CH~~CO~CH~~COOH+R~CH~~COOH R . CH2 . CO

SO far as we are aware little or no attention has been directed during recent years to these early experiments of Perkin.

The accomplishment of a similar reaction to that of Perkin at relatively low temperatures, with amino acids as one component and pyridine as a kind of catalyst, is somewhat surprising. As- suming that the analogy between Perkin’s reaction and the change under discussion is well founded, the sequence of events leading to

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96 General Reaction of Amino Acids

an acetylated aminoketone from phenylalanine may be repre- sented thus :l

CsHs . CHz. CHNH? . COOH + CsH5. CH, . CHNH(CQ . CHI) . COOH ---f

C$Hs. CHB . C(C0 . CH,) . NH(C0 . CHI) . COOH +

CoH5. CHz + CH(COCHa)NH . CO . CH, + COz

It may be noted that in conformity with the above scheme it was found that a-aminohydratropic acid, CsHS.C(NH2)CH3.COOH, which contains no unsubst.ituted hydrogen atom in the (Y position, failed to undergo the reaction typical of the ordinary a-amino acids. The homologues of acetic anhydride are much less reactive than the latter substance. Acid chlorides such as acetyl chloride react vigorously but less smoothly than the anhydrides.

Reference may be made at this point to a paper that recently appeared by Levene and Steiger (6) on the action of acetic anhy- chide, acetone, and pyridine on tyrosine and phenylaminoacetic acid. Two of the tyrosine derivatives described by these authors have the same elementary composition as those described in the present communicat,ion but the product QH1704N differs in melt- ing point by some 30”. Structural formulae were suggested by Levene and Steiger for these compounds containing isopropylidene groups which,for reasons that do not seem clear, were supposed to arise from acetone derived from or present in the acet,ic anhydride or deliberately added. The possible identity of Levene and Stei- ger’s compounds with the ketones from tyrosine described by us remains open. The properties of our tyrosine derivatives are certainly not those indicated by the provisional form&e suggested by Levene and Steiger, although the ketone from phenylamino- acetic acid is probably identical with their product and has one of the alternative formuhe proposed by them.

r It will be recalled that Victor Meyer (Ber. &em. Ges., 1893, xxvi, 1365) showed that in certain cases the (OH) group in the carboxyl group of amino acids was capable of acetylation with formation of what is vir- tually a mixed anhydride. Thus p-dimethylaminobeneoic acid gave

C&Ha /N(CHJt

\ It is possible that an analogous reaction repre-

CO.OCO.CH, sents a step in the change under discussion but it is certainly not a necessary assumption with the evidence at present available.

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H. D. Dakin and R. West 97

It may be observed that the acetylaminoketones described in this paper contain an asymmetric carbon atom, but when made from optically active amino acids, they were invariably inactive. Their mode of formation would lead one to suppose that racemiza- tion might readily occur.

A word may be said as to possible biological analogies with the reaction with which we are concerned. The synthesis of p-ketonic acids--i.e. the reverse of p-oxidation-at reasonably low tempera- tures, through the catalytic action of a base, is suggestive and will be further explored. The observation was made that the catalytic action of pyridine in this reaction could also be exercised by nico- tinic acid-pyridine-p-carboxylic acid-a substance closely asso- ciated with Funk’s vitamin preparations from rice polishings and yeast. Possibly this association is purely fortuitous and in any case we refrain from further speculation at this time. A reaction involving the removal of carbon dioxide from a-amino acids with the formation of amine derivatives which in some cases are related to the pressor amines, is also significant and should lead to the preparation of pharmacologically interesting substances. As was to be expected the acetylated aminoketone derived from tyrosine showed little or no pressor effect but on reduction with sodium and alcohol a derivative was obtained, presumably C,H,OH. CH2. CH . (CHOH . CH,) . NH2, which showed some activity. Some histidine derivatives were much more active and will be studied further. We are indebted to Dr. Lieb for making these prelimi- nary pharmacological tests. Lastly the ready conversion of amino acids into pyrazine derivatives, some of which are found in fuse1 oil, is noteworthy.

EXPERIMENTAL.

Phenylalanine.-This amino acid was converted into benzyl- acetylaminoacetone as follows: The synthetic acid (3 gm.) was heated on a steam bath with pyridine (10 cc.) and acetic anhydride (15 cc.) for 5 hours. Carbon dioxide was evolved freely after about 10 minutes and most of the reaction was over in an hour. The mixture was distilled in steam until about 150 cc. of distillate had collected. Toward the end of the distillation an oil begins to separate out from the residue. The mixture was cooled, neutra- lized with sodium bicarbonate, and extracted four times with

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ether. On evaporation of the ether the residue which weighed 3.1 gm. readily solidified in the form of clumps of waxy prisms. The crude product when dried on tile melted at 96”. It is very soluble in alcohol, acetone, and ethyl acetate, sparingly soluble in water. It is best crystallized from xylene, separating in clusters of long thin needles melting sharply at 98-99”. An aqueous solu- tion gives an immediate precipitate of iodoform on adding iodine and sodium hydroxide. With sodium nitroprusside and sodium hydroxide an intense dark cherry-red color is produced which assumes a slightly violet tinge on acidifying with acetic acid. Ammoniacal silver nitrate, Fehling’s solution, and mercuric salts in the presence of alkali are all reduced on boiling. On heating with zinc dust it gives a strong pyrrole reaction. Small amounts may be distilled unchanged in a high vacuum.

Analysis. C12H1502N. Calculated. C 70.2, H 7.32, N 6.83. Found. “ 70.5, “ 7.18, “ 6.80.

The semicarbaxone was prepared by dissolving 0.5 gm. of the ketone in a little alcohol and adding semicarbazide hydrochloride (0.3 gm.) and potassium acetate (0.3 gm.) dissolved in water. The semicarbazone separates out as an oil at first and is rather hard to crystallize. It crystallizes from alcohol in needles melting at 103-104”.

Analysis. C13H1802N4. Calculated. C 59.5, H 6.87. Found. “ 59.7, “ 6.95.

The phenylhydrazone was prepared by dissolving the ketone in a little 50 per cent acetic acid and adding a solution of phenylhy- drazine in dilute acetic acid. An oily hydrazone separates at once. It is sparingly soluble in hot water, readily soluble in alco- hol. It was not analyzed. The aminoguanidine derivative crystallizes from water in stout prisms in which it is sparingly soluble and melts at MO-181’ with evolution of gas.

Tyrosine.-The optically active amino acid was used. The product, namely p-hydroxybenzyl-acetylaminoacetone was readily obtained as follows: Tyrosine (6 gm.) was heated on the steam bath with pyridine (20 cc.) and acetic anhydride (20 cc.) for about 6 hours. The tyrosine dissolves slowly and the evolution of carbon

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H. D. Dakin and R. West 99

dioxide was noticeable after half an hour or less. The bulk of the acetic acid and pyridine was removed by a short steam distillation. The residue contains the 0-acetyl derivative of the desired product and this may be crystallized out directly by concentrating the solution. It is very soluble in water and in most organic solvents. It crystallizes best from dilute acetic acid on slow evaporation and melts at 123” with some slight softening.

Analysis. C14H1104N. Calculated. C 63.8, H 6.51, N 5.32. Found. “ 63.7, “ 6.70, “ 5.35.

In order to obtain the free hydroxybenzyl-acetylaminoacetone the separation of the preceding compound is neither necessary nor desirable. A moderate excess of sodium carbonate is added to the contents of the distillation flask and steam is blown through for about 20 minutes. In this way the acetyl group attached to the phenolic hydroxyl is removed and at the same time residual pyri- dine is got rid of. The solution is filtered, if necessary, to remove a trace of oily matter and then extracted four times by shaking with butyl alcohol. The ketone is readily extracted and on removal of the solvent it crystallizes easily. It is best recryst,allized from water with a little charcoal, and separates in the form of stout prisms which melt at 135-136”. The yield is almost equal to that of the tyrosine from which it is prepared. The substance even when pure frequently separates out as an oil which readily solidifies on standing. It is sparingly soluble in ether or chloroform, readily soluble in alcohol and ethyl acetate. Its aqueous solution gives a neutral reaction to litmus, and gives the iodoform and cherry-red nitroprusside reactions. It gives a strong pyrrole reaction on distillation with zinc dust. It yields no amino nitrogen on treat- ment with nitrous acid.

Analysis. CISHE,O~N. Calculated. C 65.1, H 6.84, N 6.33. Found. “ 65.0, “ 6.80, “ 6.20.

The molecular weight was determined by Barger’s method. A + N cane sugar solution was in equilibrium with a 1.90 per cent aqueous solution of the substance, indicating a molecular weight of 211 as against a theoretical value of 221.

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100 General Reaction of Amino Acids

An estimation of the acetyl group was made by boiling 0.5 gm. with 15 cc. of 10 per cent sodium hydroxide for 3 hours. On acidifying with phosphoric acid and distilling the filt,rate with steam, acetic acid equivalent to 20.6 per cent was obtained, com- pared with a calculated value of 19.5.

The semicarbaxone was prepared by warming an aqueous 10 per cent solution of the substance with semicarbazide (1 mol) and sodium bicarbonate (1 mol). Crystals promptly separate which are very sparingly soluble even in boiling water. The yieldis quantitat,ive. On recrystallization from water, stout cubic prisms are obtained melting at 230-231’ with evolution of gas.

Analysis. C13H1803N4. Calculated. C 56.1, H 6.48. Found. “ 56.4, “ 6.95.

A beaut.ifully crystalline picrate is obtained by dissolving 0.5 gm. of p-hydroxybenzyl-acetylaminoacetone in hot water and adding picric acid (0.6 gm.) also dissolved in hot water. The picrate separates in the form of large thin golden plates with a curious silvery luster. It is readily soluble in hot water and moderately so in cold. The recrystallized substance melts at 125-126”.

The phenylhydrazone is best prepared by adding phenylhydrazine dissolved in dilute acetic acid to a cold aqueous solution of the ketone. It is precipitated as a compact white powder which turns slightly yellow on long exposure. It is very soluble in alcohol, moderately soluble in benzene, and insoluble in petroleum ether, and is difficult to crystallize. The crude substance melts in the neighborhood of 100”. On dissolving the hydrazone in warm 10 per cent hydrochloric acid, a bulky mass of fine needles of the hydrochloride separates on cooling. The hydrochloride melts at 195-197” with evolution of gas with some previous sintering.

Analysis. C18H2102N3. Calculated. C 69.4, H 6.75, N 13.5. Found. “ 69.6, “ 6.90, “ 13.4.

The benzylidene derivative of p-hydroxybenzyl-acetylamino- acetone was prepared as follows : The ketone (1 gm .) was dissolved in 10 cc. of 0.5 N sodium hydroxide. Benzaldehyde (0.6 gm.)

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H. D. Dakin and R. West 101

together wit,h a little alcohol was then added and after shaking allowed to stand overnight. A trace of unchanged aldehyde was removed by filtration and carbon dioxide was then passed into the filtrate. The benzylidene derivative separated at once as a white precipitate very sparingly soluble in water, very soluble in alcohol and acetic acid. It is difficult to recrystallize from organic sol- vents but may be purified by solution in a little sodium hydroxide and reprecipitation with carbon dioxide. The yield is about 80 per cent of the theoretical amount. The substance has no sharp melting point but begins to decompose above 150” but does not melt completely until between 170-180”.

The positive catalytic action of other substances such as iso- quinoline, lutidine, collidine, and nicotinic acid was established by heating them with acetic anhydride and tyrosine exactly as described in the case of pyridine, noting the evolution of carbon dioxide, and isolating the ketone. The failure of quinoline and dimethyianiline to induce the reaction was shown by absence of carbon dioxide evolut,ion and the formation of no ketones.

Phenylaminoacetic A&.-The preparation of a-phenyl-a- acetaminoacetone from this amino acid was carried out under precisely the same conditions as those used for phenylalanine. The ketone is freely soluble in alcohol, less so in ether, and moder- ately soluble in water. It crystallizes in glistening plates melting at 103-104” and shows the usual ketone reactions of its homologues. The yield is almost the theoretical amount.

Analysis. CllH1302N. Calculated. C 69.1, H 6.85, N 7.33. Found. “ 69.3, “ 6.97, “ 7.21.

On treat.ing the substance even in the cold with sodium hydrox- ide, ammonia is slowly evolved owing to deep seated decomposition, while on warming on the steam bath with 5 parts of 20 per cent hydrochloric acid for an hour and then concentrating, hydrolysis proceeds normally and the hydrochloride of a-phenyl-a-aminoace- tone is obtained in excellent yield. This substance has already been obtained by Kolb (7) and the products a,gree in every particu- lar. It turns red at about 200” and melts at 204-208” according to the rate of heating. On treating the hydrochloride wibh sodium hydroxide it is rapidly converted into 3,6-dimethyl-2,5- diphenylpyrazine.

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Leucine.-This amino acid was readily converted into a-isobutyl- a-acetaminoacetone. 3 gm. were warmed on the steam bath with pyridine (10 cc.) and acetic anhydride (10 cc.) for 6 hours. On steam distillation a trace of ketone was found in the distillat,e but almost all remained in solution in the residue. This was saturated with sodium bicarbonate and extracted four times with ether. On evaporation of the ether solution the ketone (3.0 gm.) remains as a clear slightly yellow oil which did not crystallize even in a freezing mixture or after long keeping. It is fairly soluble in water and freely soluble in most organic solvents except petroleum. It gives intense iodoform and nitroprusside reactions. The substance was dried in vacua for analysis.

Analysis. C~H~TOZN. Calculated. C 63.2, H 9.94, N 8.18. Found. “ 62.9, “ 10.20, “ 8.20.

The aminoguanidine derivative is the most attractive derivative and is readily obtained by warming an aqueous solution of t.he ketone and aminoguanidine in equivalent amounts. It crystal- lizes readily from boiling water in the form of stout prisms melting at 182-183”. It is sparingly soluble in cold water.

Analysis. CbH210N~. Calculated. C 52.9, II 9.25. Found. “ 52.9, (‘ 9.37.

The semicarbaxone prepared in the usual way, separates as an oil which slowly solidifies. It is very soluble in water and alcohol and moderately soluble in chloroform. It is best crystallized by dissolving in a little absolute alcohol and adding petroleum ether, when it separates as well formed prisms. The substance does not melt sharply but undergoes some change around 170”, again turns solid, and finally melts at 215-218”. The 4,4-diphenylsemicar- basone has similar properties and does not crystallize more readily.

The phenylhydraxone is obtained as a slowly solidifying oil when aqueous solutions are used but is better prepared by mixing equivalent amounts of the ketone and phenylhydrazine in a little dry ether. It forms glistening almost colorless prisms, slowly turning yellow on exposure, and melting at 110-113°.

AZaninz.-The amino acid (4 gm.) was heated with pyridine (10 cc.) and acetic anhydride (25 cc.). Carbon dioxide was

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H. D. Dakin and R. West 103

evolved steadily but the reaction was slower than with the higher homologues. Heating was continued for 6 hours and then the mixture was distilled with steam. Only a trace of ketone was in the distillate. A slight excess of sodium carbonate was then added and a little residual pyridine removed by a further short steam distillation. The residue was then extracted with butyl alcohol repeated five times. On removing the solvent 3 gm. of clear colorless oil were obtained which did not crystallize. It gave all the ketone reactions in typical fashion. The most convenient crystalline derivative was that obtained from aminoguanidine which crystallizes in stout prisms melting at 183-184”. It is readily soluble in hot water, sparingly soluble in cold.

Analysis. C1H150Ns. Calculated. C 45.4, H 8.11. Found. “ 45.7, “ 8.23.

The constitution of the alanine derivative was proved to be a-methyl-a-acetaminoacetone by two very convincing reactions. In the first place on boiling the ketone with phenylhydrazine in 2 per cent sulfuric acid, the very characteristic bis-phenylhydrazone of diacetyl quickly separated out. It melted at 240”, and was identical with the product previously obt,ained by Pechmann (8) and others.

Secondly, on heating the ketone for an hour with 5 parts of 20 per cent hydrochloric acid and then concentrating, a syrupy residue of the hydrochloride of oL-methyl-a-aminoacetone was obtained. On distilling this residue with potash and mercuric chloride according to the conditions described by Kiinne (9) an excellent yield of the highly characteristic crystalline hydrate of tetramethylpyrazine was obtained, melting at 74-75”.

Glycine and Hippuric A&.-The reaction of glycine with acetic anhydride and pyridine does not proceed as smoothly as with the other amino acids. The yield of carbon dioxide under the condi- tions used for other amino acids was only about 35 to 40 per cent of the theoretical amount and considerable darkening of the mixture takes place on heating. On steam distillation little or no ketone was in the distillate, while the residue showed all the typical reactions. It was not found possible to iso1at.e the pure acetaminoacetone but its presence was established by using the

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two reactions previously described in the case of the ket.one from alanine. In the first place the aqueous solution was boiled with excess of p-nitrophenylhydrazine in 5 per cent sulfuric acid. For a few moments no obvious reaction occurs and then the separa- tion of the characteristic scarlet bis-p-nitrophenylhydrazone of methylglyoxal begins. The yield of product amounts to about half the weight of glycine used. It was crystallized from nitro- benzene and melted at 300-302”, and gave the characteristic blue color with alcoholic sodium hydroxide (10). It contained 24.5 per cent of nitrogen, compared with a calculated value of 24.6.

Confirmatory evidence of the presence of acetaminoacetone was obtained by acid hydrolysis, followed by distillation with mercuric chloride and alkali as described for the ketone from alanine. A good yield of 2,5-dimethylpyrazine was obt,ained which was identified by both the gold salt m.p. 152-153” and pierate m.p. 157”.

The behavior of hippuric acid when heated with acetic anhydride and pyridine exactly paralleled that of glycine with the single difference that steam distillation revealed the presence of much benzoic acid, resulting from the replacement of the benzoyl group by acetyl. The aqueous solution after steam distillation was treated as just described and gave the methylglyoxal and 2,5- dimethylpyrazine derivatives.

The behavior of other amino acids toward acetic anhydride and pyridine is the subject of further investigation.

SUMMARY

A reaction which appea,rs general to a-amino acids is de- scribed. On warming amino acids with acetic anhydride and pyridine, carbon dioxide is evolved and two acetyl groups are introduced, one attached to nitrogen and one to carbon. The compounds have the general formula R * CH . (NH. COCH,) * CO. CH3 and are derivatives of acetylaminoacetone.

Proof of the constitution of the products is adduced and various derivatives described as well as their conversion into glyoxal and pyrazine derivatives.

Proline and alkylamino acids do not react analogously but undergo simple acetylation. The same is t,rue of cu-aminohy-

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dratropic acid which contains no unsubstituted hydrogen in the a: position.

The reaction between a-amino acids, pyridine, and acetic anhy- dride has certain analogies to a change observed long ago by W. H. Perkin, senior, who obtained acetone by heating acetic anhydride and sodium acetate at high temperat,ures. It is believed that p-ketonic acids represent intermediate stages of the reaction, ketones being formed by loss of carbon dioxide.

The function of the pyridine in the reaction appears to be catalytic and is not shared by dimethylaniline or quinohne. On the other hand pyridine derivat.ives such as lutidine, collidine, and nicotinic acid were effective.

Reference is made to the fact that the reaction is not limited to amino acids but is shared by a-halogen acids and some unsubsti- tuted acids such as phenylacetic acid which gives methylbenzyl- ketone. The possible biological significance of the reaction is discussed toget,her with reference to the possible uses of alkyl acetaminoacetones for the preparation of pharmacologically active substances.

BIBLIOGRAPHY.

1. Dewar, J., and Gamgee, A., J. Anat. and Physiol., 1870, vii, 142. 2. Gabriel, S., and Pinkus, G., Ber. &em. Ges., 1893, xxvi, 2797. 3. Chattaway, F. D., J. Chem. Xoc., 1904, lxxxv, 386. 4. Perkin, W. H., J. Chem. Xoc., 1886, xlix, 317. 5. Fittig, R., Ber. them. Ges., 1897, 2150. xxx, 6. Levene, P. A., and Steiger, R. E., J. Biol. Chem., 1927, Ixxiv, 689. 7. Kolb, A., Ann. Chem., 1896, ccxci, 276. 8. Pechmann, H., Ber. them. Ges., 1887, 3164. xx, 9. Kiinne, H., Ber. &em. Ges., 1895, xxviii, 2040.

10. Dakin, H. D., and Dudley, H. W., J. Biol. Chem., 1913, xv, 132.

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H. D. Dakin and Randolph WestACIDS

A GENERAL REACTION OF AMINO

1928, 78:91-104.J. Biol. Chem. 

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