[COSTRIBUTION FROM
THE
DEPARTMENT OF BIOCHEMISTRY, COLLEQE
OF PHYSICIAN# AND
SURGEONS, COLUMBIA UNIVERSITY]
BENZOYLATION OF AMINO ACIDS ROBERT E. STEIGER Received A p r i l 19, 1944
N-Benzoylamino acids are most conveniently prepared by the action of benzoyl chloride upon amino acids in aqueous solution in the presence of some alkali. Enough alkali, generally sodium hydroxide, is used to neutralize the hydrochloric acid liberated in the reaction, to maintain the benzoyl compound in solution until the end of the operation, and to destroy any excess of benzoyl chloride. For smoothness and rapidity the method leaves nothing to be desired. The material isolated upon the addition of hydrochloric acid to the alkaline solution contains no secondary product save benzoic acid. This method was introduced by Baum (1) and was commonly employed in about its original form (2-10) until six years after Fischer (11) had strongly recommended the use of sodium bicarbonate as a substitute for alkali.‘ Carter and Stevens (13) abandoned Fischer’s bicarbonate procedure after they found it to be conducive to the formation of mixed anhydrides of N-benzoyl-a-amino acids and benzoic acid. As these compounds, when originating from optically active a-amino acids, are racemized under the experimental conditions prior to their hydrolysis in the sodium bicarbonate solution, Carter and Stevens (13) benzoylate optically active amino acids by Baum’s method, in the following way: One mole of amino acid is dissolved in 750 cc. of 2 N sodium hydroxide and 250 cc. of water. The solution, maintained below 30°, is treated with 2 moles (230 cc.) of benzoyl chloride and 2300 cc. of 2 AT sodium hydroxide. It is then cooled in an ice-bath and acidified nith 340 cc. of concentrated hydrochloric acid. The mixture of solids obtained is extracted with hot high-boiling petroleum ether to remove the benzoic acid (1 mole or more). The benzoyl derivative is purified in the appropriate manner. In the course of the last ten years the writer has prepared large quantities of N-benzoylamino acids by Baum’s method, using a technique somewhat different from that adopted by Carter and Stevens. Ice-cold aqueous solutions of one mole of amino acid in one mole of sodium hydroxide mere treated with one mole of benzoyl chloride and one mole of sodium hydroxide: (1)
+ NaOH = NH2-R-COONa + CsHSCQCI+ NaOH =
KH2-R--COC)H
(11) NH2-R--COONa
C6H5CONH-R--C00Na
+ HzO
+ NaCl + H20
The reaction mixtures were then treated with one mole of hydrochloric acid to precipitate the N-benzoylamino acid : (111) C6HSCONH-R-COONa
+ HC1 = CsH5CONH-R-COOH + NaCl
1 However, Fischer (12) invariably used the sodium hydroxide procedure when benzoylating diamino-monocarboxylic acids. No explanation W R S given. 396
BENZOYLATION OF AMINO ACIDS
397
It should be understood that, simultaneously, benzoic acid was liberated from the little sodium benzoate formed as a by-product in the reaction. To convert all unreacted amino acid into the hydrochloride a further quantity of hydrochloric acid was added. The yields of purified benzoyl derivatives were high, 89-98%. The benzoyl compounds prepared in this manner had been obtained by various workers in other ways, but in lower yields and apparently not always in the pure state. EXPERIMENTAL
General procedure for benzoylating amino acids. One mole of amino acid is placed in a three-necked round-bottomed flask, fitted with an efficient stirrer, and dissolved in 1000 cc. of 1 N sodium hydroxide (1 mole). The flask is then almost completely immersed in a large-sized bath of ice and water (temperature about + l o )and the stirrer is now set in very rapid motion. T o the solution is added, dropwise, 116 cc. (1 mole) of pure benzoyl chloride and, a t a rate 4.3 times faster, 500 cc. of 2 N sodium hydroxide (1 mole), also in a continuous flom . The operation may be interrupted a t any time t o refill the graduated analytical burettes through which the two reagents are best delivered. The two burettes are adapted t o the side necks of the flask by means of one-hole rubber stoppers. The middle neck of the flask i3 covered with a round piece of glazed cardboard, thinly coated with Vaseline, through which passes the stirring rod. A shaped stirrer (glass rod bent a t a45" angle or less), nearly touching the bottom of the flask, will most effectively disperse the benzoyl chloride in the alkaline so!ution. Stirring should be effected a t a speed such that the liquid is forcibly projected against the walls of the vessel. The time for the introductior- of the reagents is about one hour for runs on 0.2 mole of amino acid and three hours for runs on 1.S8molesof amino acid. The final volume of the solution should not exceed one-half of the capacity of the reaction flask. In runs on more than one mole of amino acid, alkali of a higher concentration than 1 N sodium hydroxide may be used t o dissolve the amino acid (e.g., 2 iV sodium hydroxide for a-aminoisobutyric acid), provided the sodium salt of the benzoyl compound is sufficiently soluble a t 0" to remain in solution. When crystallization of the sodium salt of the benzoylamino acid occurs before all the benzoyl chloride has been added, the benLoyl compound is obtained in a lower yield than usual. T o prevent this crystallization the amino acid is dissolved in alkali of a suitably lower concentration than 1 A: sodium hydroxide (e.g., 0.2 N sodium hydroxide for anthranilic acid). When the reagents have been added, the solution is stirrcd for an additional fifteen minutes, and, if necessary, is treated with h'orit at room temperature and filtered with suction. The clear solution is made strongly acid t o Congo red by the addition of 5 S hydrochloric acid (200 cc. of this is theoretically required). The hydrochloric acid used in excess will dissolve the unreacted portion of those amino acids which are only sparingly soluble in water. The hydrochloric acid is added very slowly with continuous stirring and outside cooling in ice and water The well cooled suspension is filtered and the crystals are washed with ice-cold water. in t3mall portions. The filtrate should be concentrated under reduced pressure, if containing a quantity of benzoyl compound sufficient to warrant its isolation. When precipitated a t low temperature, certain benzoyl compounds are of a crystalline texture such that filtration of the suspension is extremely slow and thorough washing of the material is well-nigh impossible. Precipitation, then, may be effected as follows: T o the solution is added 5 .\' hydrochloric acid, a t room temperature, until the appearance of the first crystals. The mixture is then heated on a steam-bath and the remainder of the theoretical amount of 5 LV hydrochloric acid is added with continuous stirring. The suspension is immediately cooled in an ice and water mixture and made strongly acid t o Congo red by the addition of a further quantity of 5 .V hydrochloric acid. Lbying of substances. The crude products, as obtained by precipitation with 5 iV hydrochloric acid, were dried in a vacuum desiccator over phosphorus pentoxide and sodium hydroxide, the latter being used t o remove any hydrochloric acid that may have been
--
398
ROBERT E. STEIGER
retained by the wet crystals. The precaution is all the more necessary if the product is t o be recrystallized from an alcohol. All substances were eventually dried t o constant weight in a vacuum desiccator over phosphorus pentoxide alone. The weights given refer t o materials dried in this way. The final substances were preserved over phosphorus pentoxide. Melting points. These were taken in the melting point apparatus described by Steiger (14) with total immersion precision thermometers. Unless otherwise stated, the bath was brought t o a temperature about 5" lower than the melting point, before the capillary tube was introduced into the apparatus; the temperature of the bath was then raised very SI owly. ,V-Benzoyl-dl-a-aminophenylacetic acid (N-benzoyl-dl-phenylglycine). A solution of 30.22 g. (0.2 mole) of dl-a-aminophenylacetic acid (15) in 200 cc. of 1 N sodium hydroxide (0.2 mole) was treated with 23.2 cc. (0.2 mole) of benzoyl chloride and 100 cc. of 2 N sodium hydroxide (0.2 mole) in the way described. The solid benzoylation product was washed with 1 liter of ice-cold water in small portions, then freely with benzene, which dissolves only the contaminating benzoic acid. The yield was 49.50 g. (97.0%). The product was suspended i n benzene, the mixture was boiled under a reflux condenser and filtered while hot; the residue was washed on the funnel with hot benzene and dried. Verylittle substance was lost in this operation, designed to remove any benzoic acid not previously eliminated. The benzoyl compound was dissolved in boiling acetone and the solution filtered while hot, after addition of Norit. The hot filtrate was treated with warm water until turbidity was just produced. The mixture was stirred while allowed t o cool very slowly to room temperature. The crystals were washed first with water containing a little acetone, later with wster, and dried. The substance melted sharply a t 178.0-178.5' (corr.) with decomposition. It melted a t the same temperature after recrystallization from acetone and water. Prepared previously by: Kossel (16),m.p. 174"; Bayer and Co. (3), m.p. 175.5'; Baum (17), m.p. not given; Shemin and Herbst (18), n1.p. 175" (corr.). N-Benzoyl-dl-a-amino-@-phenylpropionic acid (AV-benzoyl-dl-p-phenylalanine).A solution of 33.02 g. (0.2 mole) of dl-a-amino-,%phenylpropionic acid in 200 cc. of 1 hTsodiuni hydroxide (0.2 mole) was treated with 23.2 cc. (0.2 mole) of benzoyl chloride and 100 cc. of 2 rV sodium hydroxide (0.2 mole) in the way described. The solid benzoylation product was washed with 1liter of ice-cold water, in small portions, and dried. T o this was added the material isolated from the filtrate upon its concentration t o a small volume under strongly reduced pressure. The crude product weighed 51.10 g. (94.9% yield, assuming no contaminating benzoic acid present). It was suspended in 6 times its weight of heptane, the mixture was boiled under reflux, and filtered while hot; the residue was freely washed on the funnel with hot heptane and dried. The yield m-as 47.90 g. (89.0%). This material was dissolved in 13 times its xeight of boiling 50Ye (by weight) ethanol. The solution was treated with liorit and filtered with suction, as hot as possible. Crystallization set in immediately. The suspension was eventually cooled in ice and water and filtered. The crystals were washed first with ice-cold 50y0 ethanol, later with water, and dried. They melted a t 186" (corr.). After recrystallization from acetone (a very suitable solvent), the substance melted at 187.0' (corr.). Other authors: Erlenmeyer (19), m.p. 182-183"; Fischer and Mouneyrat (20), m.p. 187-188" (corr.); Mohr and Stroschein (21), m.p. 182-183'; Waser (22), m.p. 184"; Lamb and Robson (23), m.p. 184-185'. N-Benzoyl-dl-B-amino-@-phenylpropionicacid. A solution of 33.02 g. (0.2 mole) of dl-j3-amino-p-phenylpropionic acid (24) in 200 cc. of 1 N sodium hydroxide (0.2 mole) was treated with 23.2 cc. (0.2 mole) of benzoyl chloride and 100 cc. of 2 N sodium hydroxide (0.2 mole) in the way described. The solid benzoylation product precipitating upon the addition of an excess of 5 AThydrochloric acid to the ice-cold reaction mixture was so voluminous t h a t the suspension had t o be diluted several times with water to facilitate stirring. The product was washed with 1 liter of ice-cold water, in small portions, and then with alcohol-free ethyl ether t o remove benzoic acid. The filtrate was concentrated under strongly reduced pressure; i t yielded only a small quantity of benzoyl compound. The yield was 52.71 g . (97.9%). The entire material was dissolved in 8 times its weight of
BENZOYLATION OF AMINO ACIDS
399
boiling 96% ethsnol. The clear solution obtained was cooled t o room temperature and continuously stkred. The crystals deposited were washed first with some ice-cold 95% ethanol, later with water, and dried. They weighed 44.0 g. Most of the benzoyl conipound contained in the mother liquor of crystallization may be recovered by heating and adding a large volume of water; i t crystallizes on cooling. The substance was crystallized once more from 8 times its weight of boiling 9570 ethanol. It then melted sharply a t 199.5' (corr.). Posner (10) reported the melting point 194-196". .Y-Ben;:oyZ-a-aminoisobutyric acid. A solution of 154.6 g. (1.5 moles) of a-aniinoisobutyric acid in 750 cc. of 2 N sodium hydroxide (1.5 moles) was treated with 174 cc. (1.5 moles) of benzoyl chloride and 750 cc. of 2 iV sodium hydroxide (1.5 moles) in the way described. The solid benzoylation product was washed with ice-cold water, in small portions, and then with ether t o remove benzoic acid. Upon concentration under strongly reduced pressure, the filtrate yielded a further quantity of benzoyl compound. The crude product weighed 284.0 g. (91.4% yield, assuming no contaminating benzoic acid present). It was suspended in 500 g. of ether, the mixture was boiled under a reflux condenser, and filtered after cooling; the residue u'as washed on the funnel with ether and dried. The yield was 274.7 g. (88.4%). After two crystallizations from 8 times its weight of boiling acetone. the sutbstance melted and decomposed a t 202' (corr.). Previously prepared by: Mohr and GeiR (25), m.p. 198" (softening a t 195"); Gabriel (26), m.p. not given; Heller and Lauth (27), In.]).199'. Y-Beni!oyl-2-aminobenzoic acid (N-benzoylanthranilic acid). A solution of 27.41 g . (O.!? mole) of anthranilic acid in 1 liter of 0.2 N sodium hydroxide (0.2 mole) is treated with 23.2 cc. (0.2 mole) of benzoyl chloride and 100 cc. of 2 N sodium hydroxide (0.2 mole) in the way described. A little of the sodium salt of N-benzoylanthranilic acid separates in the course of the benzoylation. When afterwards the reaction mixture is stirred for some time a t room temperature a clear solution is obtained. This, if colored, should be decolorized with Norit a t room temperature and filtered with suction. The solution is placed in a two-liter beaker and treated with a little 5 N hydrochloric acid until crystallization sets in. The mixture is heated in a steam-bath as high as possible, kept hot, and vigorously stirred while there is added, drop by drop, the remainder of the 40 cc. of 5 N hydrochloric acid theoretically required t o set free the benzoylaniino acid. Immediately thereafter the suspension is cooled in an ice- and water-bath and treated with 10 cc. of 5 N hydrochloric acid with stirring. After thorough cooling, the suspension is filtered and the crystals are washed with 1 liter of ice-cold water in small portions, then sucked as dry as possible, and dried. The yield is 46.24 g. (95.970). This material melts a t 181-182' (corr.) with decomposition. For purification, there is added t o this material somewhat less 0.1 N sodium hydroxide than theoretically required t o form the sodium salt. The mixture, which then contains a little undissolved benzoyl compound, is treated with Xorit a t room temperature and filtered with suction. T o the colorless solution, which is continuously heated in a steam-bath and vigorous1.y stirred, is added in a thin stream just enough 0.5 N hydrochloric acid t o liberate the benzoyl compound. The suspension is immediately cooled in ice and water and now made strongly acid t o Congo red with 0.5 N hydrochloric acid. The crystals are exhaustively waiehed on the funnel with water a t room temperature. The purification is repeated in exactl:? the same manner. N-Benzoylanthranilic acid melts and decomposes a t 182" (corr.), themelting point tube being introduced into the bath a t 176' (corr.). This compound has been prepared twice previously by benzoylation of anthranilic acid: Briickner (28), using a procedure which was not described, gave the melting point 182'. Bamberger and Sternitzki (4), using Baum's method, but giving no details, found the melting point 177". N-Benzoylanthranilic acid has been obtained very often in other ways, but only exceptionally has i t been found to melt a t a temperature higher than 177'. Confirniation of the high melting point, 182", reported by Bruckner, has hitherto been lacking. It would appear that the compound exists in two modifications having different melting points, or t h a t i t obstinately retains traces of organic solvents, causing a lowering of the melting point. The issue is very much obscured by the fact that in the molten state the
400
ROBERT E. STEIGER
substance undergoes dehydration t o its azlactone. At any event, N-benzoylanthranilic acid should not be purified by crystallization from organic solvents. Unlike the material precipitated from solutions of its sodium salt with hydrochloric acid, the products obtained on crystallization from organic solvents will melt almost instantaneously if the capillary tube is introduced in the bath a t 176' (corr.) ; they then solidify more or less completely t o melt again at a higher temperature, always below 182" (corr.), because a t the first fusion a certain amount of azlactone is formed.
Acknowledgment. The author wishes to thank Professor H. T. Clarke for his interest in the work and the facilities accorded. SUMMARY
1. N-Benzoylamino acids may be obtained, in high yields, by the action a t about +lo of only one mole of benzoyl chloride upon the amino acids, in the presence of the required amount of aqueous sodium hydroxide. Little benzbic acid is formed as a by-product under the conditions of the procedure used. 2. Five N-benzoylamino acids were prepared and purified in the appropriate manner. The highest melting points recorded in the literature for these compounds have either been confirmed or found to be definitely too low. NEW YORK,N. Y. REFERENCES (1) BAUM,2. physiol. Chem., 9,465 (1885); Ber., 19,502 (1886). (2) SCHOTTEN, Ber., 21, 2238 (1888). (3) German Patent 55,026 (1890) (to Farbenfabriken, vorm. Friedr. Bayer and Co.); Frdl., 2, 529 (1887-1890). (4) BAMBERGER AND STERNITZKI, Ber., 26,1305 (1893). (5) KLEBS,Z . physiol. Chem., 19, 331 (1894). (6) SENFTER AND TAFEL,Ber., 27, 2313 (1894). (7) WALLACH, Ann., 312, 180, 198 (1900). (8) SCHULTZE, Z . physiol. Chem., 29, 467 (1900). (9) HOLM,Arch. Pharm., 242, 611 (1904). (10) POSNER, Ber., 38, 2322 (1905). (11) FISCHER, Ber., 32, 2453 (1899). (12) FISCHER, Ber., 34, 462, 2905 (1901); FISCHER AND WEIGERT,Ber., 35, 3776 (1902). (13) CARTERA N D STEVENS, J . B i d . Chem., 138, 628 (1941). (14) STEIGER,Bull. S O C . chin., (5), 2, 284, 1488 (1935). (15) STEIGER,Org. Syntheses, 22, 23 (1942). (16) KOSSEL,Ber., 24, 4151 (1891). (17) BAUM, Ber., 37, 2961 (1904). (18) SHEhlIN AND HERBST,J.Am. Chem. S O C . , 60,1956 (1938). JR.,Ann., 276, 15 (1893). (19) ERLENMEYER, (20) FISCHER A N D MOUNEYRAT, Ber., 33, 2383 (1900). J. prakt. Chem., (2), 82,326 (1910). (21) MOHRA N D STROSCHEIN, (22) WASER,HeEv. Chim. Acta, 8, 122 (1925). (23) LAMB AND ROBSON, BiOChem. J.,26, 1234 (1931). (24) STEIGER, Org. Syntheses, 22, 26 (1942). (25) MOHRAND GEIS, Ber., 41,798 (1908); J. prakt. Chem., (2), 81.56 (1910). (26) GABRIEL,Ber., 46, 1352 (1913). (27) HELLERA N D LAUTH,Ber., 62, 2302 (1919). (28) BRUCKNER, Ann., 206, 132 (1880).
