[CONTBIBWTION FROM WB RESEARCH LABORATORIES OP THE ~ O O L OF PHaBahncy, UNIVEESITY OF MARY LA^]

a-ALKOXIMINO ACIDS]

XENNETH L. WATERS* .4m WALTER H. HARTUNG

Received January 7,1947

The ease with which a-oximino acids may be synthesized and by simple hy- drogenation converted into the corresponding a-amino acids (1, 2, 3) raises the question whether they may also be employed in the formation of the peptide linkage. An ideal intermediate would be the acid halide of the oximino acid, RC(=NOH)COCl, but compounds of such type are not described and attempts to prepare them in these laboratories have been unsuccessful. It was thought, however, that the oximino group might be readily removed to form the a-keto acid, from which the amide with an amino acid might be synthesized, RCOCONHCHR’COOH ; this through reoximination, RC(=NOH) CONH- CHR’COOH, would then 8erve as a suitable intermediate for peptide synthesis. Unfortunately, not all a-ketoximino acids are easily hydrolyzed by methods that work so well for the hydrolysis of simple oximes.

It has been noted (4) that if oximes of the a-keto acids are heated with dilute, mineral acids, they lose carbon dioxide and water, forming nitriles with one less carbon atom. We have verified this observation and have found that the a-oxim- ino acids are also resistant to alkaline hydrolysis. Bouveault and Locquin (5 ) report the hydrolysis of these acids through the use of 85% formic acid and lead- chamber crystals; many variations of this procedure have been reported in the literature. The procedures are cumbersome and not applicable to the hydrolysis of all a-oximino acids (6).

In attempting to obtain a-keto-8-phenylpropionic acid from the corresponding oxime we tried the usual methods described in standard texts and also procedures which recently have been reported to be successful in the hydrolysis of oximes (7 ,8 ,9) ; all of these were unsuccessful when applied to the a-oximino acid. The unsuccessful attempt to reduce an a-oximino acid to an a-imino acid, RC(=NH)COOH, has already been described (10).

Since we were unsuccessful in preparing the a-keto acids, it appeared logical that other derivatives might serve our purposes. In 1938, Adkins and Reeve (1 1) reported an interesting synthesis of threonine by the catalytic reduction of the 0-ethyl ether of oximinoacetoacetic ester. If alkylation should protect t,he oximino group, permitting the synthesis of an acid chloride derivative which could be coupled with an amino acid, subsequent reduction should give the amino group, thus forming a dipeptide. The equations are:

1 Paper No. 7 on syntheses of amino acids. For No. 6, see Barry and Hartung (2). The authors wish to express their appreciation to Dr. George D. Beal, Assistant Director,

Mellon Institute, for granting permission to complete a portion of this work in the Mellon Institute laboratory.

9 Present address: Mellon Institute, Pittsburgh 13, Pa. 469

470 K. L. WATERS AND W. H. HARTUNG

0 f l R”CH(NH2)COOH+ RCCOOH --+ RCCOOH -4 RC-C

II \ NOR‘ C1

II NOR’

/I ?NOH

(1) (11) (111) RCCONHCHR”CO0H --+ RCHC0NHCHR”COOH

a-Alkoximino acids are new compounds, and therefore a method for their synthesis is given and several members of this series, along with derivatives, are described. During the course of this investigation three ethoximino (Type 11, R’ = Et) and two benzyloximino acids (Type 11, R’ = CsH6CH2) were prepared. Three of these acids were converted into their corresponding acid chlorides (Type 111, two where R’ = Et and one where R’ = CsHbCH2). Al- though the preparation of compounds of Type IV was not attempted, the fact that amides will be formed is proved through the preparation of three corre- sponding anilides (two where R’ = Et and one where R’ = CsH&Hz). Reduc- tion studies on compounds of Type I1 have shown that catalytic reduction to the amino group may be accomplished.

The preparation of the a-ethoximino acids was accomplished by ethylation with diethyl sulfate in a slightly alkaline medium in the presence of acetone. The three acids which were prepared were low-melting solids which could be purified by distillation under diminished pressure. Preliminary hydrogenation studies on these acids indicated that they would be somewhat difficult to reduce at room temperature and a t atmospheric pressure (conditions which would be very desirable if the compounds are to be of value in the preparation of optically active peptides). Since reductive debenzylation of certain types of compounds is very successful, it was thought that it might be preferable to prepare the a-benzyloximino acids. It was found that essentially the same procedure as that employed in the ethylation of the a-oximino acids could be used in the benzylation, benzyl chloride being used as the alkylating agent. Two a-benzyl- oximino acids were prepared in yields of 42% and 56% respectively; it is believed that the yields will approach the yield of the a-ethoximino acids if the mode of isolation is improved. Preliminary hydrogenation studies have shown that the a-benzyloximino acids may be reduced at room temperature and a t two atmos- pheres pressure.

EXPERIMENTAL

Preparation of the a-oximino acids. The a-oximino acids used in these experiments were prepared by either of two procedures: (a) From the substituted acetoacetic ester in 85% sulfuric acid by the action of alkyl nitrite as described by Hamlin and Hartung (1); (b) from the substituted malonic ester by the action of alkyl nitrite and hydrogen chloride using ether as the solvent as described by Barry (2).

8-Phenyl-a-etlwximinopropionic acid. One-tent.h mole, 17.9 g., of p-phenyl-a-oximino-

a-ALKOXIMINO ACIDS 47 1

propionic acid in a 500-ml. three-neck flask was dissolved in 100 ml. of 5% aqueous sodium hydroxide and 50 ml. of acetone. The flask was equipped with a mechanical stirrer and two burettes so arranged that a solution of sodium hydroxide and diethyl sulfate could be added simultaneously; a total of 100 ml. of diethyl sulfate and 100 ml. of sodium hydroxide solu- tion (30.5 g. per 100 ml. of solution) was added. The rate of addition after the initial addi- tion of 10 ml. of the sodium hydroxide was such that one drop of alkali was added per drop of diethyl sulfate. Since the concentration of the alkali solution was such that one ml. of alkali would neutralize the acid formed during the hydrolysis of one ml. of diethyl sulfate, the mixture was slightly alkaline throughout the ethylation. The addition of the ethylat- ing reagent required about thirty minutes and during this time the temperature of the water-bath was raised to 70". As a general rule five moles of diethyl sulfate were added per mole of a-oximino acid.

When the addition waa completed, the burettes were replaced by a reflux condenser and the temperature of the bath was raised to boiling. The mixture was refluxed half an hour, after which the reflux condenser was removed and the heating continued about an hour, or until most of the acetone had evaporated, the last traces being removed by suction. The reaction mixture was cooled, made definitely acid to Congo Red with hydrochloric acid, and extracted completely with ether. The combined extracts were washed with several small portions of water, Ntered, and dried over sodium sulfate. The solvent was removed and the resulting material distilled a t 1 to 2 mm. pressure. The fraction distilling a t 115- 120' was collected; a yield of 16.3 g., or 79% of 8-phenyl-a-ethoximinopropionic acid, was obtained. The colorless crystals melted a t 58.5-59".8

Anal. Calc'd for CllHl~N08: N, 6.76; Neutr. equiv. (phenolphthalein), 207.2. Found: N, 6.59; Neutr. equiv., 209.5.

a-Ethoximinobutyric acid. The ethylation was performed as described above. From 11.7 g. (0.1 mole) of a-oximinobutyric acid, 8.6 g., or 60% of a-ethoximinobutyric acid waa obtained. When cooled, the product crystallized in long, colorless needles which melted a t 61.0-61.5'.

Anal Calc'd for C6H11N03: N, 9.65; Neutr. equiv. (phenolphthalein), 145.2. Found: N, 9.58; Neutr. equiv., 146.5.

a-Ethoximinohexanoic acid. The ethylation was performed as described above. From 29 g. (0.2 mole) of a-oximinohexanoic acid, 28.1 g., or SOYo yield of a-ethoximinohexanoic acid was obtained. The fraction distilling a t 83-88" under 1 to 3 mm. pressure was collected. The product was a clear liquid which on standing in an ice-bath solidified to long, colorless needles melting a t 23".

Anal. Calc'd for CsHlsN03: N, 8.09; Neutr. equiv. (phenolphthalein), 173.2. Found: N, 7.88; Neutr. equiv., 174.0.

8-Phenyl-a-benzyloximinopropionic acid. The benzylation of 8-phenyl-a-oximino- propionic acid was accomplished in a manner similar to that described for the ethylation. Nine grams of 8-phenyl-a-oximinopropionic acid was treated with five equivalents of benzyl chloride; the sodium hydroxide solution used for the neutralization of the liberated hydro- chloric acid had a concentration of 34.7 g. per 100 ml. After the removal of the last traces of acetone, the alkaline benzylating mixture was extracted with ether CO remove the benzyl alcohol. However, the mixture of ether and benzyl alcohol was found to contain the sodium salt of 8-phenyl-a-benzyloximinopropionic acid. On evaporation, the mixture left an oily residue of benzyl alcohol and the salt; the benzyl alcohol waa removed by adding small portions of cold absolute ether and decanting the solvent. When dried, 10.3 g. of the sodium salt of 8-phenyl-a-benayloximinopropionic acid remained. A solution of the salt in water was acidified and the precipitated acid waa filtered and recrystallized from ethyl alcohol. A yield of 7.5 g. or 56% of 8-phenyl-a-benzyloximinopropionic acid, was obtained as colorless crystals melting a t 79-80'.

The fraction distilling a t 89-95' under 12-14 mm. pressure was collected.

~~ ~~ -~~ 3 All melting points are corrected.

472 K. L. WATERS AND W. H. HARTUNG

Anal. Calc'd for CltH~~NOs: N, 5.20; Neutr. equiv. (phenolphthalein), 269. Found: N, 5.13; Neutr. equiv., 272.

a-Benzylozimimhemnoic acid. The benzylation was performed and the product isolated 88 described above. From 7.2 g. of a-oximinohexanoic acid, 4.8 g., or 42% of a-benzyl- oximinohexanoic acid was obtained. The colorless crystals melted a t 61.0-61.5O.

Anal. Calc'd for ClaHI7NOa: N, 5.95; Neutr. equiv. (phenolphthalein), 235.3. Found: N, 5.87; Neutr. equiv., 236.

8-Phenyl-a-ethozimimpropionyl chloride. To 6.6 g. of @-phenyla-ethoximinopropionic acid was added 6.6 g. (4 ml.) of thionyl chloride. The mixture waa refluxed for half an hour; during this time the condenser waa protected from moisture by a calcium chloride tube. To remove the excess thionyl chloride and the liberated hydrogen chloride, two SO-ml. portions of dry benzene were added and each removed under reduced pressure. The product was distilled under 1 to 2 mm. pressure and the fraction distilling a t 95-97' was collected. A yield of 6.0 g., or 85% of 8-phenyl-a-ethoximinopropionyl chloride was obtained.

Ana.!. Calc'd for CtlH&lNOe: N, 6.21; C1, 15.72. Found: N, 6.04; C1, 15.61.

a-Ethoziminohezanoyl chloride. To 10 g. of a-ethoximinohexanoic acid dissolved in 25 ml. of dry benzene waa added 9.8 g. (6.0 ml.) of thionyl chloride dissolved in 15 ml. of dry benzene. The mixture was allowed to stand overnight, protected from atmospheric mois- ture, and then refluxed on the steam-bath for about 20 minutes or until the mixture started to darken. The excess thionyl chloride and liberated hydrogen chloride were removed by the addition of two portions of dry benzene with subsequent distillation under reduced pressure. The product was distilled under 1 to 2 mm. pressure. Seven and eight-tenths grams of a-ethoximinohexanoyl chloride was obtained which distilled between 58" and 63' a t 1 to 2 mm. An additional 1.2 g. of material distilling within the range of the original a-ethoximinohexanoic acid (78-83") was also collected. Allowing for the recovered acid, a yield of 81% of a-ethoximinohexanoyl chloride was obtained.

Anal. Calc'd for CsHl&lNOn: N, 7.31; C1, 18.51. Found: N, 7.30; C1, 16.1.4

P-Phenyl-a-benzyloziminopropionyl chloride. To 5 g. of 8-phenyl-a-benzyloximino- propionic acid in 25 ml. of dry benzene was added 6.6 g. (4 ml.) of thionyl chloride in 15 ml. of dry benzene. The mixture was refluxed for two hours with protection from moisture. To remove the exces8 thionyl chloride and liberated hydrogen chloride, two 50-ml. portions of dry benzene were added and each removed under diminished pressure. The product was distilled under 1 to 2 mm. pressure and the fraction distilling a t 170-175" was collected. A yield of 4.7 g., or 88% of 8-phenyl-a-benzyloximinopropionyl chloride was obtained.

Anal. Calc'd for ClSHlaClN02: N, 4.87; C1, 12.31. Found: N, 4.82; C1, 12.13.

Ethyl ,9-phenyl-cY-ethoximinopropionate. About 3 g. of 6-phenyl-a-ethoximinopropionyl chloride was added to 10 ml. of absolute ethyl alcohol and the mixture refluxed on the steam- bath for half an hour. The excess alcohol was removed under diminished pressure and the product was dissolved in ether. The ethereal solution was washed with several small por- tions of saturated sodium chloride solution, then dried over sodium sulfate, and filtered into a distilling flask. After removal of the solvent, the product was distilled a t 1 to 3 mm. pressure and the fraction distilling a t 118-120° was collected, The ethyl 8-phenyl-a- ethoximinopropionate so prepared was a clear, colorless liquid.

Anal. Calc'd for C13H17NO~: N, 5.95; sapon. value, 235.3. Found: N, 5.79; sapon. value, 232.

Ethyl a-ethoximinohexanoate. To 4 g. of a-ethoximinohexanoyl chloride was added 25 ml. of absolute ethyl alcohol and the mixture refluxed on the steam-bath for one hour. The excess alcohol was removed under diminished pressure and the residue dissolved in ether.

4 Chlorine analysis unsatisfactory; highest result of several determinations reported.

a-ALKOXIMINO ACIDS 473

The ethereal solution was washed with two small portions of saturated sodium chloride solution, dried over sodium sulfate, and filtered into a distilling flask. After the removal of the solvent the ester was distilled at 1 to 3 mm. pressure and the fraction distilling a t 60-65" was collected. A yield of 3.7 g., or 88% of ethyla-ethoximinohexanoate was obtained as a clear colorless liquid.

Anal. Calc'd for CIOHIONOI: N, 6.96; sapon. value, 201.3. Found: N, 6.82; sapon. value, 201.

6-Phenyl-a-ethoximinopropionanilide. To 9.2 g. of crude p-phenyl-a-ethoximinopro- pionyl chloride (product not distilled but excess thionyl chloride removed under diminished pressure), dissolved in 25 ml. of dry benzene, was added 9.2 g. (9 ml.) of aniline dissolved in 25 ml. of dry benzene. The mixture was refluxed on the steam-bath for half an hour, then cooled, and the precipitated aniline hydrochloride was filtered. The benzene was removed by distillation under reduced pressure and the residue taken up with 20 ml. of absolute ether. To this solution was added 80 ml. of petroleum ether; a light yellow pre- cipitate deposited which was a t first thought to be the desired anilide. This product weighed 3.2 g. and on further purification (dissolving in absolute ether and precipitating with petroleum ether) had the constant melting point 88-89'. Nitrogen analysis, neutrali- zation equivalent, and examination of hydrolysis products indicated that i t was a salt of aniline and 0-phenyl-a-ethoximinopropionic acid which probably resulted through the use of the crude starting material.

p-Phenyl-a-ethoximinopropionanilide was isolated from the ethereal filtrates by evapo- rating to dryness and purifying the residue by dissolving in ethyl alcohol and precipitating by adding water. A total of 5.8 g. of the anilide was obtained. The colorless crystals melted a t 59-60'.

And. Calc'd for C1,H18N1O2: N, 9.92. Found: N, 9.84. a-Ethoximinohexanilide. To 4.2 g. of a-ethoximinohexanoyl chloride, dissolved in 25

ml. of dry benzene, was added 4.2 g. (4.1 ml.) of aniline, dissolved in 15 ml. of dry benzene; the aniline hydrochloride, which formed as a white precipitate, was filtered and the solvent removed under diminished pressure. The product was distilled a t 1 to 3 mm. pressure and the fraction distilling at 133-138' was collected. A yield of 4.3 g. (77%) of a-ethoximino- hexanilide, was obtained as a slightly yellow, somewhat viscous, liquid, which failed to crystallize on prolonged standing a t room temperature.

And. Calc'd for C14H2oN~02: N, 11.28. Found: N, 11.18. p-Phenyl-a-benzyloximinopropionanilide. To 1.1 g. of p-phenyl-a-benzyloximinopro-

pionyl chloride, dissolved in 25 ml. of absolute ether, was added 0.8 g. of aniline, dissolved in 10 nil. of absolute ether. The mixture was allowed to stand and the precipitated aniline hydrochloride filtered. The solvent was removed under diminished pressure. The product was purified by dissolving in warm 95% ethyl alcohol and precipitating with water. A yield of 1.2 g. (90yo) of 0-phenyl-a-benzyloximinopropionanilide was obtained as a colorless crystalline substance which melted at 73.5-74'.

A n d . Calc'd for CzzHmN202: N, 8.14. Found: N, 8.03. Hydrogenolysis of the a-alkoximino acids. A thorough investigation of the hydrogenoly-

sis and reduction of the a-alkoximino acids will be made later; however, preliminary experi- ments show that this class of compounds may be catalytically reduced. Using a prepared palladium charcoal catalyst (12) and acidified ethyl alcohol for the solvent, the reduction of 8-phenyl-a-ethoximinopropionic acid was very slow a t room temperature and two atmos- pheres pressure; only about 10% being reduced after about three hours. Raney nickel catalyst was no more satisfactory than the palladium catalyst. The use of Adams' catalyst in methyl and in ethyl alcohol under 2 atmospheres of pressure and a t room temperature did not effect a rapid reduction of a-ethoximinohexanoic acid. Several experiments in the cataly7,ic reduction of 8-phenyl-a-benzyloximinopropionic acid indicated this compound is more easily reduced than is its ethoxy analog. A fifty per cent yield of amino acid was obtained using a palladium catalyst in glacial acetic acid. It is believed that a systematic

474 K. L. WATERS AND W. H. HARTCTNG

study of conditions of hydrogenation will show that theor-alkoximino acids may be catalyt- ically reduced a t room temperature and a t atmospheric pressure.

SUMMARY

1. A method for the alkylation of the a-oximino acids has been devised and employed successfully.

2. It has been shown that, if the oximino group of the a-oximino acids is protected by alkylation, the corresponding acid chlorides may be prepared in good yields.

3. The acid chlorides of the a-alkoximino acids have been shown to react as characteristic acid chlorides. 4. Preliminary reduction studies indicate that the a-alkoximino acids may be

reduced to the corresponding amino acid. 5. It is proposed that the a-alkoximino acids may serve as convenient inter-

mediates in the synthesis of peptides.

BALTIMORE, MD.

REFERENCES (1) HAMLIN AND HARTUNG, J . Biol. Chem., 146,349 (1942). (2) BARRY AND HARTUNG, J . Org. Chem., 12, 460 (1947). (3) MATTOCKS, Thesis, University of Maryland, 1945. (4) BOTJVEAULT AND LOCQUIN, Bull. SOC. chim., [3] 31,1142 (1904). (5) BOTJVEATJLT AND LOCQUIN, Compl. rend., 136, 179 (1902). (6) HALL, HYNES, AND LAPWORTH, J . Chem. Soc., 107,132 (1915). (7) KARRER AND HOFFMAN, Helv. Chim. Acta, 22,654, (1939). (8) KEAGLE AND HARTUNG, J . Am. Chem. Soc., 88, 1608 (1946). (9) STJMERFORD AND DALTON, J . Am. Chem. SOC., 88, 1330 (1944). (10) WATERS AND HARTUNG, J . Org. Chem., 10,524 (1945). (11) ADKINS AND REEVE, J . Am. Chem. SOC., 60, 1328 (1938). (12) HARTUNG, J . Am. Chem. SOC., 60, 3370 (1928).