Contribution to the study of the methods of preparation of amino acids

T H E S I S

SUBMITTED

TO THE GRADUATE SCHOOL OF LAVAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

BY

ROGER GAUDRY

״CONTRIBUTION TO THE STUDY OF THE METHODS

OF PREPARATION OF AMINO ACIDS״

These investigations were carried out in the Dyson Perrins Laboratory, Oxford University, during the tenure of a Rhodes Scholarship and in the Department of C h e ­ mistry, Laval University. The assistance from the Rhodes Trust, as well as from the authorities of Laval University is hereby gratefully acknowledged.

The author wishes to thank Dr. P. 3. King, who proposed and siipervised most of this work and Prof. Paul S. Gagnon for their advices, help and siost useful suggestions.

CONTRIBUTION FO THE STUDY OF THE METHODS

OF PREPARATION OF AMINO ACIDS

BY

ROGER GAUDRY

Although at the present time, at least _{22 amino} acids are recognized by all the chimists as constituents of proteins, and ail have been synthesized, not many are easily accessible in large quantities now. However,

they are very important· Since E* Fisher, they are used for the synthesis of polypeptides_{f and since a few years,} for works dealing with nutrition. In 1906-1907, Wiicock and Hopkins (J. Physiol., 1906, 35, 88) showed that a di&t free from lysine and tryptophane did not permit the growth of mice. In 1914, Osborne and Mendel (J. Biol. Chem., 1914, 17, 342) proved that lysine was an essential

amino acid. These first investigators were followed by many others after whom the indispensability of many amino acids was admitted. W.C. Rose (Physiol. Rev., 1928, 18 # 109) showed that a diet, in which the proteins had been replaced by a mixture of 20 pure amino acids, supported growth very well·, The following amino acids are recog­

nized to-day as indispensable: lysine, tryptophane,

histodine, phenylalanine, leucine, isoleucine, threonine, methionine, valine and arginine.

Amino acids are obtained, either by hydrolysis of׳ suitably chosen proteins, or by direct synthesis.

Although many general methods of synthesis of «*-amino acids are !mown to-day, not one can be used to obtain easily and with a good yield more than a few amino acids and there are still some of these for the synthesis of which no general method has been applied successfully·

The object of this work was the improvement of methods of syntheses of amino acids, in order that they be more easily accessible in larger quantities.

In this paper, the results obtained when apply­ ing the Darapsky method to the synthesis of phenylala­ nine, tyrosine, valine, proline, ornithine, lysine ana aspartic acid will be studied, and a general apprecia­ tion of the method will be given· Then, other methods of syntheses of aspartic acid, proline, c^-phenylpyrro- line, N-benzylpyrrolidine and sarcosine will be discus­

Ourtius (J. Prak. Chem.., 1930, 125, 211) was the first one to apply the reactions of hydrazine to the synthesis of amino acids· He described a general method, of synthesis of *-amino acids from a monosubstituted potassium ethylmalonate, by means of the degradation known by his name and which enables one to replace the ״ester" group by the ״amino״ group.

COOK COOK COOH COOH

R-CH J E g r F a - H g Q Rj H

. HH°g , b. L hydrolyala

J.

I I H C 1 I .

COOC2H5 CO-UH-NHg CO-N3 nh2

One weak point of the method consists in the difficulty of preparing with a good yield the monosubstituted potas­ sium ethylmalonate. Moreover, in the original raei&od, Curtius, instead of transforming the azlde by alcohol into

the corresponding urethane, decomposed it by heat into an isocyanate, which was inclined to give spontaneously a

dike top iperaz i n e ,

COOH COOK CO-NH

R-CK ■ h6at > R-CH - (^ -.mo.1.e s > ) R-CH HO-R + 2C0g

I I \ /

CO-Hg l'J=CO ]:H-CO

or an <*.-carbamino acid anhydride:

COOH COOH

I I

R-CH ---

)

!

CO-K

3

This method has been used by Curtius and his co­ workers to synthesize alanine, glycine, phenylalanine and valine, but it seems that it had only so far a theoretical interest. And although Curtius himself stated that aspartic and glutamic acids, leucine and tyrosine could be synthesized by that method their syn­ thesis has apparently not been published.

In 1936, Darapslcy (J. Prak, Chem·, 1936, 146, 250), former collaborator of Curtius, published an in­ teresting modification of the original method׳of syn­ thesis· He replaced malonic ester by cyanoacetic ester

1'IH / \ -> R-CK CO

and applied the Curtius degradation to a nitrile ester of the form R-CH(CN)-CO2C 2H 5 . By doing this, he did not need to prepare the half ester of a substituted ethylmalonate, and, thus, due to the absence of a free carboxyl group, the degradation was more easily carried out, the possibility of formation of a diketopiperazine

According to the Darapsky method, the reactions leading to the synthesis of an amino acid are as fol-being eliminated« lows: CN R-Br ■f CH2 CO-KH-NHg C O 2C 2H 5 c o2c2h5 (IV.) (III.) (I.) (11.) GOOH CN CN £gg5.0ff.r-c h R-CH KHg N H - C O ^ g H g CO-N3 (VII.) (VI.) (V.)

Cyanoacetic ester is condensed-with an alkyl bromide, in presence of sodium alccholate, and, the substituted ester thus formed reacts at room temperature with hydrazine hydrate to give the corresponding hydrazide. The hydra- zide is treated by nitrous acid, in presence of a large excess of hydrochloric acid, to obtain the azide, which is extracted with ether from the aqueous solution. The ethereal solution is then poured into alcohol, and the ether distilled off. The azide reacts with boiling a l ­ cohol to yield a urethanfe. The urethane, when refluxed for many hours with hydrochloric acid, is hydrolysed together with the nitrile, and, after evaporation of the solution, the residue is a mixture of the amino acid hydrochloride and ammonium chloride, from which the free amino acid can be isolated in many ways.

Darapsky applied this method to the synthesis of leucine, and two other amino acids, o(-amino-n-valeric acid and '*-amino-isoamylacetic acid, which a re not known to be products of hydrolysis of proteins. Although the yields obtained by Darapsky have not been very good, it was believed that the method could have interesting possibilities.

COOH COOH COOH C H 3 -0 -C6H 4-CH2-CH

1

PHENYLALAx!IKE (VIII.). Hot many amino acids have been synthesized more often than phenylalanine. Before des­ cribing the synthesis that was carried out by the Darap- sky method, the syntheses made previously will be simply enumerated.

The first one Is that of Erlenmeyer and Lipp (Ber. Ghem. Ges., 1882, 15, 1006; Ann, Chera., 1883, 2 1 9 , 161 and 179), who used the Strecker method; they treated phenylacetaldehyde successively with hydrocyanic acid and ammonia. But most of the satisfactory syntheses of phenylalanine have, as starting material, benzaldehyde or benzyl chloride (or bromide). The most important of all Is that which Is made by condensing benzaldehyde with hippuric acid (Org. Syn., 14, 80). It has been studied

2576; 1887, 20, 2465; Ann. Chem., 1892, 271, 157; 1893, 2 7 5 , 1; Ber. Chem. Ges., 1897, 30, 2896; Ann. Ghem. 1899, 307, 70; 1904, 337, 205 and 265) and gives a total yield of 40Jo ·

Amongst the other methods, the following are those which give satisfactory results; the yields are between 50 and 60“״« The starting materials for these syntheses are: a) Benzyl chloride and the sodium derivative of ethyl phtalimidomalonate (SSrensen, Z. Physiol. Chem., 1905, 4 4 , 448), b) benzyl chloride and malonic ester, followed by a bromination and a treatment with ammonia

(Pisher, Ber. Chem. Ges., 1904, 37, 3062), c) benzal- dehyde and hydantoine (Viheeler and Hoffman, Aner. Chem. J ., 1911, 45, 368), d) benzaldehyde and diketopiperazine

(Sasaki, Ber. Chem* Ges., 1921, 5 4 , 163). The less satis­ factory methods are: reduction of ~oximino-j8-phenyl- propionic acid (Erlenmeyer, Ann. Chem., 1892, 2 7 1 , 137; Ber. Chem. Ges., 1897, 3 0 , 2976; 1898, 3 1 , 2238; Posner, Ber. Chem· Ges., 1903, 36, 4305; Knoop and Hoessli, Ber. Chem. Ges., 1906, 39, 1477; reduction and treatment by ammonia of phenylpyrivic acid (Knoop and Oesterlin, Z.

Physiol. Chem,, 1925, 1 4 3 , 294; 1927, 170, 185); con­

densation of benzaldehyde with 2 - t h i o 3 ־-benzoyl-hydantoi~ ne (Johnson and O ’Brien, J. Biol. Chen., 1912, 12, 205); reaction of potassium benzylmalonate with hydrazine (Cur­ tius and Sieber, 5er, Chem· Ges., 1921, 54, 1430; 1922, 5 5 , 1545); and finally, reaction between benzyl chloride and aminomalonic ester (Cherchez, Bull. Soc. Ghim., 1930,

(4), 47, 1279 and 1331)*

Synthesis of phenylalanine (VIII.) by the Darapsky method ((I.) to (VII.), R - C 0H5 -CH2 *־). It is believed

that the synthesis carried out will be useful for the preparation of this amino acid in large quantities, for the starting materials are easily accessible and the mani­ pulations do not present any special difficulties. Benzyl bromide i3 easily condensed, in presence of sodium ethy- late, with cyanoacetic ester, to give - c y a n o - p h e n y l - propionic ester, which is treated, at room temperature, by one equivalent of hydrazine hydrate. The hydrazide forms a white mass, easily recrystallized. Treated then by nitrous acid, in presence of a large excess of hydro­ chloric acid, the hydrazide is transformed almost in­

stantaneously Into the azide which is extracted with ether; when boiled with alcohol, the a zide is decomposed into the urethane. By complete hydrolysis of the m o l e ­ cule, with 20/6 hydrochloric acid, racemic phenylalanine is obtained, with a 50$ yield, calculated from ^-cyano-

-phenylpropionic ester.

TYROSINE (IX.). Although the constitution of tyrosine approaches that of phenylalanine, its synthesis has always been more difficult to carry out and the yields are generally lower than in the case of analogous syn­

theses of phenylalanineé One of the most ancient syn­ theses is that of Erlenmeyer and Lipp (Ber. Chem. Ges., 1882, 15, 1544; Ann. Chem., 1883, 2 1 9 , 161 and 179), from phenylalanine, by nitration of the phenyl group In para position, reduction into the amine, and treatment by nitrous acid* This synthesis is certainly very important theoretically, for it shows the possibility of diazotiz- ing the amino group of the benzene ring without affecting much the amino g roup of the lateral chain· However, It

is not a practical one, because a secondary product, oc-hydroxy-p-hydroxyphenylpropionic acid, is formed.

Erlenmeyer and Halsey (Ber. Chem. Ge3 ·, 1897, 30, 2981; Ann· Chem♦, 1899, 5 0 7 , 138) synthesized tyrosine from p-hydroxybenzaldehyde and hippuric acid with a 3$ yield. Them, the method was successively improved by Fisher (Ber· Chem· Ges., 1899, 32, 3638) who increased the yield to 47$, calculated from the intermediary pro­ duct, <*-benzo:/lamino-p-hydroxycinnamic acid, and special­ ly by Haring ton and McCartney (Biocher״·. J., 1927, 21,

852), who, after having replaced p-hydroxybenzaldehyde by p-methoxybenzaldehyde, obtained a 60$ yield.

One of the best methods of synthesis that v/e have is that of Wheeler and Hoffman (Amer. Chem* J., 1911,

4 5 , 368), who where successful in preparing tyrosine with a 66$ yield when using anisaldehyde and hydantoine.

Two other syntheses are also known, one b y Sasaki (Ber. Chen. Ges., 1921, 5 4 , 163) by condensation of ani­ saldehyde with glycine anhydride, with a 48$ yield, and the other by Stephen and Welzmann (J. Chem. Soc., 1914, 1 0 5 , 1152), from anizyl bromide and the potassium deri­ vative of ethyl phtalimidomalonate, with a 35$ yield.

Attempt to synthesize tyrosine by the Darapsky method ((I,) to (VII.), R - CH3 ~0 ־־C 6H 4 ־־C H 2-) . When anizyl

chloride is condensed with one equivalent of cyanoacetic ester and ,־one equivalent of sodium, the yield in ¿k-cyano-

<£-(p-methoxyphenyl)-propionic ester is only 26%. There must be an excess of one equivalent of cyanoacetic ester

to increase the yield to 48^. The preparation of the hydrazide does not offer any difficulty; by more mixing, at room temperature, of one equivalent of oi-cyano-jS-

(p-methoxyphenyl)-propionic ester and one equivalent of 100% hydrazine hydrate, the hydrazide is formed and soli­ difies if allowed to stand for many hours in an evacuated desiccator. On the contrary, the preparation of the

azide is difficult· The hydrazide is insoluble in water and in hydrochloric acid; one must place the hydrazide in suspension in a hydrochloric solution, covered with a layer of ether and cooled in a freezing mixture, and add slowly the sodium nitrite solution. The solution must be stirred mechanically to increase the rate of the reaction by preventing the hydrazide to float to the zone of contact of the aqueous solution and ether. Even with rapid stirring and in presence of an excess of nitrous

acid, it is difficult to completely free the solution from solid hydrazide· After having stirred for an

hour, the major part of the hydrazide had reacted. The solution was then extracted thrice with ether and the ethereal solution dried w i t h anhydrous sodium sulphate, filtered and !;oured into 99.5# alcohol. Ether having been distilled off caustiously, and the alcoholic solu­ tion refluxed to transform the azide into the urethane, alcohol was evaporated on a water-cath. The impure urethane is a dark brown viscous liquid. An attempt to hydrolyze it directly into tyrosine by refluxing it with 48% hydrobromic acid, was unsuccessful. The mass was resinifled. On the contrary, the urethane, vihen boiled for a long time with 20% hydrochloric acid is hydrolyzed into the methyl ether of tyrosine. The yield

is 30$ from <* -cyano-i?- (p~methoxyphenyl) -propionic ester. This methyl ether of tyrosine is not easily purified by crystallization· When treated with phenylisocyanate,

<*-phenylureido-j3-(p-methoxyphenyl) -propionic acid was obtained, 'which can be recrystallyzed with difficulty from a mixture of alcohol, ethyl acetate and petroleum ether.

Although it is fairly easy to transform methyl- tyrosine into tyrosine “by one of the ordinary methods, the transformation was not tried because only a small quantity of amino acid was available, (approximately 1 gram), and also because this method of synthesis is not an improvement of the methods used today. If this

synthesis is compared with the preceding one, that of phenylalanine, it is noted that the presence of a ״me-

thoxy*1 group has a great influence on the reactions of the Curtius degradation. These are not carried out so easily and the yields are lower.

VALINE (XI.). The syntheses of valine are not numerous. The first one is that of Lipp (Ann. Chem., 1880, 205,

1)t by the Strecker method, a reaction of isobutyric

aldehyde with hydrocyanic acid and ammonia. Another, from «*-bromo-isovaleric acid and amaionia, tne most important one today, has firstly been described toy

Clark and Fittig (Ann. Chem., 1866, 159, 199). Later, many investigators (Schlebusch, Ann. Chem., 1867, 1 4 1 ,

322; Schmidt and Sacktleben, Ann. Chem., 1878, 195, 87; Nerberg and Karezag, Biochem. Z., 1909, 18, 435) studied

the method and Improved it. Slimmer *Ber. Chem. Ges., 1902, 35, 400) used it to prepare valine with a 10% yield. The intermediate compound, <*-bromo-isovaleric acid can be prepared from isopropylmalonic ester with a 70-75# yield, and the ester itself, with a 77-85# yield

(Org. Syn., 1 1 , 20). As previously mentioned, valine has also been synthesized by Curtius (J, Prakt. Chem., 1930, 125, 211), by applying the degradation called alter his name, to the monopotassium salt of isopropyl­ malonic ester.

Synthesis of d-1 valine by the Darapsky method ((I.) to (VII.), R * (CH3 )2CH-). Like isopropylmalonic ester, isopropylcyanoacetic ester is prepared with an excellent yield by condensing cyanoacetic ester with isopropyl bro­ mide (Fisher, Ber. Chem. Ges., 1909, 42, 2983), The r e ­ action between isopropylcyanoacetic ester and 100# h y ­ drazine hydrate is exothermic. The hydrazide is a pale yellow viscous liquid. The preparation of the azlde and

of the urethane is not difficult. The urethane is a red­ dish yellow liquid, much purer than those previously m e n ­ tioned. During hydrolysis by hydrochloric acid, a very pale yellow solution is obtained, which, by evaporation

and neutralisation with ammonia, £ives an almost white amino acid, with a 80% yield, calculated from isopropyl- cyanoacetic ester.

This method of synthesis is recommendable Tor the preparation of racemic valine in large quantities.

COOH fm2-(CH2 )3 -CH-I'JH2 (XIV.) COOH CHg— CHg Br-(CH2 )3 -CH ----> C H 2 CH-COOH (XV.) NH, COOH , ״ C H \ / ; CH-CII / \ c h 3 n h 0 (XI.) COOH C 6H 5 0 ־-(CH2 )3 -CH -■ HH, (XIII.) (XII.)

<*-A?3IN0- £-PHEH0XY- VALERIC AC ID (XII.). *-amino-£-phe- noxy-valeric acid, which is not a product of hydrolysis of proteins and has not been isolated from other natural sources, had never been described. It was prepared, due to the interest that it could have as Intermediate pro­ duct for the syntheses of ornithine and proline.

It was thought Io ) that the Darapsky method could be used to prepare fairly large quantities of *-amino-

¿P-phenoxy-valeric acid (XII.) from cyanoacetic ester and /-phenoxy-propyl bromide; 2°) that it would be possible, by treatment of this amino acid by hydrobromic acid, den­ sity 1.5 (48$), to replace the "phenoxy" group by one bromine atom and obtain c*-amino-<T-bromo-valeric acid

(XIII·); 3°) that this acid could easily, either react with ammonia to give ornithine (XIV.), or cyclise to give proline (XV.).

1°) Preparation of c*-amino-<T-phenoxy-valsric acid by the Darapsky method ((I.) to (VII״), R = CgH'sOiCHgJg-) · This synthesis is interesting mainly because it illustra­

tes certain possibilities of the Darapsky method. One molecule of y -phenoxypropyl bromide is condensed with

one molecule of cyanoacetic ester, in presence of sodium alcohola té, and the yield in <*-cyano-<^-phenoxy-valeric ester is 40$. The degradation, through the hydrazide, azide and urethane is easily carried out and the yield in amino acid is 40$, calculated from o<-cyano-<T-phenoxy- valeric ester*

2°) Attempt to replace the ״phenoxy״ group by one bromine atom. The replacement of a ״phenoxy" group by a bromine atom necessitates drastic conditions, that is, long boil­ ing of the substance in 48$ hydrobromlc acid. That is why good results are obtained only with a sufficiently stable compound.

The closest case mentioned in the chemical lite­ rature, is that of the hydrolysis of the ״phenoxy״ group of *-amino-/-phenoxy-butyric acid (Fisher and Blumen- thal, Ber. Chem. Ges·, 1907, 4 0 , 108)· This acid was synthesized by condensation of ^-phenoxy-ethyl bromide with ethyl malonate, then by bromination, simultaneous hydrolysis and decarboxylation, finally by treatment with ammonia. The amino acid, when refluxed for half an hour with 48$ hydrobromlc acid, gives <*-amino-y- hydroxybutyric acid, and not the bromo derivative.

In order to obtain a bromo derivative, instead of an alcohol, t*-amino-^'-phenoxyvaleric acid was boiled many times, with 48$ hydrobromlc acid, the time of boil­

ing being varied. It has been impossible to isolate from the solution another product than the phenol* As it had

already been noted In the case of methyl-tyrosine, amino acids are decomposed by long heating with 48$ hydrobromic acid.

ORNITHINE (XIV.). Although ornithine is not a consti­ tuent of proteins, it is a very Important basic amino acid, because it is found in nature, as dibenzoyl deri­ vative (ornituric acid), and specially because of Its relations vrith arginine and urea in mammals. Since the brilliant investigations of Krebs and his co-workers

(Z. Physiol. Chem., 1932, 2 1 0 , 53; Z. Physiol. Chem., 1933, 217, 191), It is reasonable to believe that argi­ nine plays an important part in the formation of urea in

organism. According to this theory, the essential basic substance Is ornithine;

NHg-(CH2 )3 ־CH-NH2 2£ s J ! J H 3^ N H g״.oO-NH-(CH2) 3-CH-NH2 y

COOH COOH

NHp-C-NH-»(CHp) g-CH-UHo -3J.g^n.a.3e > urea 4· (XIV.) * || ^ «5 | «5 jj^q -7

n h c o o h

(XVIII.)

Ornithine (XIV.) adds up carbonic anhydride and ammonia to give citrulline (XVI.) which reacts with another

molecule of ammonia to give arginine (XVII.). Arginase, an anzyme of liver, hydrolyses arginine into urea and ornithine and the cycle starts again·

Many syntheses of ornithine are known. Fisher (Ber. Chem. Ges., 1901, 34, 434) prepared it from

y-phtalimidopropylmalonic ester, by bromination, hydro­ lysis and decarboxylation, and treatment by ammonia. He isolated it as ornithuric acid (dibenzoyl derivative).

y-Phtalimidopropyl-malonic ester had already been pre­ pared with a 80% yield by Gabriel and Aachan (Ber. Chem. Ges., 1891, 2 4 , 1364) from malonic ester, trimethylene bromide and potassium phtalimide. Riesser (Z. Physiol. Chem., 1906, 4 9 , 210), has prepared ornithine as plcrate, by the SiJrensen method (Chem. Zentr. 1903, II, 35), from ethyl phtalimidomalonate and y-bromopropylphtalimide.

Fisher must also have credit for two other syntheses: one (Fisher and Haske, Ber. Chem. Ges., 1905, 38, 3607), by treating ^-vinylacrylic acid with ammonia, during 20 hours, at 150°; the other (Fisher and Zemplen, Ber. Chem. Ges., 1909, 4 2 , 1022), by oxidation of benzoyl- piperidine into <T-henzoylamino~n-valeric acid by potas­

sium permanganate, followed by bromination and treatment with ammonia* Mere recentl?/, Keimatsu and Sugasawa (J. Pharm. Soc. Japan., 1928,

₄₁₃

,

It was thought interesting to try a double degra­ dation on c*-cyanoadipic ester. This compound was not described in the literature. A method of preparation wa3 to treat monobromadipic ester by potassium cyanide, but this method can not be applied in practice because of the difficulty of preparing pure monobromadipic ester, free from the dibromo derivative. The method that was used consists in the condensation of y-bromobutyric ester with cyanoacetic ester, in presence of sodium ethylate.

It is possible to prepare y-bromobutyric ester In fairly large quantities by condensing ethylene oxide with the sodium derivative of malonic ester. The carbethoxybuty- rolactone'is obtained, which, hydrolyzed by barium hy- droxyde and decarboxylated, gives the butyrolactone, with a 87$ yield. By opening the ring of the lactone by means of dry hydrobromic acid in absolute alcohol, the ethyl ester of ,y-bromobutyric acid is directly obtained.

Ba(OH)2 CHg-CH-COgC 2H 5 CHg CO ^ O 7 CHg^CHg COgCgHg

I

)

(II.)

HHg-NH-CO(GHg)3 - G H (C N )CO-NH-NHg

(XXV.)

When oC-cyanoadix:ic ester is treated by two molecules of hydrazine hydrate, the dihydrazide is formed with evolution of heat and solidifies slowly if allowed to stand in a desiccator. By reaction with nitrous acid, in presence of an excess of hydrochloric acid, the hy~ drazide gives the azide, which, by boiling with alcohol, is transformed into the urethane, nitrogen being abun­ dantly evolved# Likely, hydrolysis of the urethane by hydrochloric acid is accompanied by an abundant evolu­

tion of carbonic anhydride. Prom the product of hydro­ lysis, it was tried unsuccesfully to isolate ornithine as ornithuric acid (dibenzoyl derivative) or to preci­ pitate it either as paosphotungstate, or as dipicrate.

It is hard to understand why ornithine is not in present the solution, because all twe stages of the de­ gradation seem to proceed normally*

kh2 -(ch2 )4 -ch(cooh)-nh2 CH2 (C00H)-CH(C00H)-NH2

LYSINE (XXVII♦). Lysine is a fairly general constituent of proteins. The first synthesis is that of Fisher and Weigert (Ber. Chen. Ges., 1902, 35, 3772), who condensed

the nitrile of y-chlorobutyric acid with malonic ester, and treated the ester of y-cyanopropylmalonic acid thus formed by ethyl nitrite to obtain the ester of ־*-oxixnido-

8 -cyanovaleric acid. The latter was reduced into lysine

by sodium and alcohol. The yield, of the reduction, is only 32$. It is the weak point of this method.

Before 1939, all the other syntheses of lysine had as intermediate product 6 -benzoylamino-n-caproic acid, which was firstly brominated in presence of phos­ phorous and then treated with ammonia and hydrolysed by hydrochloric acid into lysine dihydrochloride. Von Braum

(Ber. Chem, Ges,, 1909, 4 2 , 839), was the first one to describe this method of synthesis and prepare 6-benzoyl- amino-n-c&proic acid from benzoylpiperidine. Later,

Sugasawa (J. Pharm. Soc, Japan, 1927, 5 5 0 , 1044) obtained the acid, from acrolein. Finally, Eck and Marvel (J.

Biol. Chem,, 1934, 106, 387) prepared it in a very satis­ factory way from commercial cyclohexanone. By using this

latter method, lysine can be synthesized with a total yield of 23$.

The last published synthesis (Adamson, J. Chem* Soc., 1939, 1564) is much better than the preceding ones.

It consists in the treatment of the ethyl ester of cyclo- hexanone-2-carboxylic acid by hydrazoic acid, in pre­

sence of concentrated sulphuric acid and the yield is 60$,

Although it was impossible to prepare ornithine by a double Curtius degradation from c^-cyanoadipic ester,

it was thought interesting to try to apply a similar de­ gradation to o<y-cyanopi2relic ester* There are many exam­ ples inhere the only presence of a -CHg- group more or less in a chain greatly affects the reactions; thus, Pisher h a d met with many difficulties in trying to pre­ pare lysine from potassium phtalimide and tetramethylene bromide, by the method that he had used for the obtention of ornithine. Likely, SiJrensen has not been able to apply successfully to lysine his method of synthesis which was suitable for ornithine. <X-Cyanoplmolic ester was not

described in the chemical literature. It was prepared by condensing cyanoacetic ester with £-bromovaleric ester, in presence of sodium and alcohol, ¿‘-bromova- leric acid i3 easily prepared, in large quantities, by hydrolysis by 4:8% hydrobromic acid of the y-phenoxy-propylmalonic ester. ?0 2C 2H5 ) Br-(CHg)4 -C00H C6H5>0(CH2)3-CH COgCgHs (XXIX.) (XXVIII.) CN C2H5OH ^ Br-(0Hg)4-C0gCgH5 4־ CHß NaOG2H5 ^ (II.) (XXX.) HH2־N H 2 »HgO C0 2C 2H 5 ־ (CH2 U ־CH G 0 2G 2H5

CN

n h2-m h-o o-(c h2 )4-c h-c o-iih-n h2 (XXXII.)

?/hen <*-cyanopimelic ester is treated by two molecules of 100$ hydrazine hydrate, the dihydrazide

solidifies more rapidly and is more easily crystallized than in the case of «*־cyanoadipic dihydrazide. The Curtius degradation was applied to it, transforming it

successively into the azide and urethane. However, it was impossible to extract lysine from the product of hydrolysis of the urethane by hydrochloric acid.

ASPARTIC ACID (XXVII.). The first synthesis of aspartic acid was made in 1850, when Dessaignes (Compt. Rend. Acad., 1850, 5 0 , 324) obtained a few crystals by heating ammo­

nium acid malate in acid solution. In 1887, Piretti (Gazz. Chitf¡♦ Ital., 1887, 17, 518) synthesized it from the oxime o f oxaloacetic ester, and In 1909 Schmidt and Widman (Ber. Chen!· Ges., 1909, 42, 497) obtained it from

acetylsuccinic ester. Using more general methods, Kei- maku and Kato (J. Pharm. Soc. Japan, 1929, 49, 731) pre­ pared it from chloracetic ester, aminomalonic ester and

sodium alcoholate with a 55$ yield. However, the sepa­ ration of the intermediate compound ethane-<*-amino-<*-o<~j9-.

tricarboxylic ester is very tedious, Dunn and Smart (J. Biol. C hem., 1950, 89, 41) have prepared aspartic acid with a total yield of 27$, starting with chloracetic ester and the sodium derivative of ethylphtalimidomalo- nate. The best method, giving a 59$ yield, is that of Dunn and Pox (J. Biol. Chem., 1933, 1 0 1 , 493) which

consists in the treatment of ethyl fumaiate by alcoholic ammonia. 3-6-diacetamido-2-5-diketopiperazine is firstly obtained and hydrolysed with sodium hydroxide. Aspartic acid is isolated through its copper salt which needs only to be decomposed by hydrogen sulphide to obtain the free amino acid.

Attempt to synthesize aspartic acid by the Darapslcy method. When ethylene glycol nitrile is condensed with cyano-

acetic ester, ot-jff-dicyanopropionic ester is obtained with a 80$ yield. To prepare aspartic acid, one would need only to apply a Curtius degradation to the ester group with a final hydro-ysis of the two nitrile groups.

so

It Is known that hy&razine can react with a nitrile group to give one compound of the form -C (MI) - H H - M g , This reaction usually needs heating. But in the Darap- sky method, one molecule of hydrazine is treated at room temperature with one molecule of the form R-CH(CN)-GOgCgHs and the corresponding hydrazide R-CH(Cli) -CO-NH-NHg is ob­

tained, hydrazine reacting preferably with the ester group. In order to obtain ¿-,/3-dicyanopropionic hydra­ zide, d-^-dicyanopropionic ester was treated with hydra­ zine .hydrate . It has been impossible to obtain the de­ sired compound. When the ester and hydrazine are mixed together without solvent, a vigourous reaction takes place, and resines only are formed. When efforts are made to control the reaction by cooling, and operating

in presence of a solvent, the reaction is slow, but the final product is the same. The two nitrile groups on the two neighboring carbon atoms are so active that h y ­ drazine does not react any more preferably on the ester group. An attempted degradation of the resinous product was unsuccessful.

APPRECIATION OF TIIE DARAPSaY M E THOD. After the investi­

by the Darapsky method, It can be reasonably concluded that this method is mainly applicable to substituted cyanoacetic esters bavin¿ an inactive lateral chain, for valine and phenylalanine were prepared with good yields, whereas ornithine, lysine or aspartic acid could not be synthesized. However, it is believed that the method can be used successfully for the synthesis of

isoleucine, norleucine and methionine. It has been applied to leucine by Darapsky himself, and undoubtedly could be applied successfully in many other cases.

ATTEMPT TO SYNTHESIZE ASPARTIC ACID BY THE PKTALIMIDO- MALQNIC ESTER METHOD. The synthesis of aspartic acid made by Dunn a n d Smart, starting with phtalimidomalonic

ester and chloracetic ester gives a yield of 27^ only· It was thought possible to replace chloracetic ester by ethylene glycol nitrile, or by bromo״:alonic ester. The sodium derivative of ethyl phtalimidomalonate was pre­ pared, and an attempt was made to condense It with gly­

col nitrile in alcoholic solution, or with ethyl bromo- malonate by simply heating the mixture without solvent. The condensation products were immediately hydrolysed by hydrochloric acid. The products of hydrolyses con­ tained no aspartic acid.

ATTEMPTS TO PREPARE PROLINE (XV.). Among the amino acids which are known to be constituents of proteins, two of them, proline and hydroxyproline, are pyrrolidine deri­ vatives. Although proline is a very important consti­

hy-drolysates is particularly difficult. And, although it has been synthesized many times, by widely different m e ­ thods, there is no really practical method by which this amino acid can be prepared in large quantities.

An attempt was made to work up a practical synthe­ sis of proline by studying first of all the preparation of compounds containing a pyrrolidine ring.

1°) Starting with benzyl-J3-aminoproplonlc acetal (XXXIII.). In 1901, Wohl (Eer. Chem. Ges., 1901, 34 1922), by drop­ ping this acetal into cold dilute hydrochloric acid, ob­

tained a substance which, he claims, has the formula (XXXIV,), without giving any conclusive evidence in fa­ vour of it. CHo-CH HC1 I I' C 6H 5 ־CH2-NH-CH2-CH2-CH(0C2H 5 )2 — — --- > CK2 C-GqBs NH.EC1 (XXXIII.) (XXXIV.)

W o h l ‘s work was repeated, and a compound obtained which melts at 239-240°. But the results of many analy­ ses indicate that the formula proposed by Wohl is not the

right one. The analyses indicate that there is one more molecule of ?,־ator than required by Y'ohl·s formula. The right formula would then be C 10H 14ONCI and not CioHigKOl. It is very unlikely that the substance is hydrate, b e ­ cause the analyses give the same results when the sub­ stance has been dried at 150° in a vacuum, over phospho­ rus pentoxide. In order to explain that result, the sub­

stance was made to react with Schiff's reagent, with 2-4-dinitrophenylhydrazone and with an amnoniacal sil­ ver solution. All these tests being negative, the ob­ vious conclusion is that the substance contains no free aldehyde group. These results are in accordance with

those obtained by Wohl.

When the substance is boiled for 4-5 hours with 10^ hydrochloric acid, and the solution evaporated to dryness, a liquid residue remains which, taken up with chloroform, yields a small amount of a white crystalline solid, melting at 242°, the analyses of which corresponds perfectly with the formula C10H 14ONCI, i.e., the same as

that of the starting material. However, these two sub­ stances are different. The starting material is inso­ luble in alcohol, whereas the other one is soluble in

alcohol. Furtherinore, the melting point of a mixture of tho two was 228-229°, that is much lower than the melting point of each of them* They are prohably isomers, Tho

one that was obtained first will be called first isomer, and the one obtained from the first will bo called second

The isomers are not very stable, When the first isomer is boiled with hydrochloric acid, the yield of the second isomer is only 10$ of the starting material, most of it being resinified, As it was possible to detect a small amount of the second isomer in the residue obtained when benzyl-j3-aminopropionic acetal has been dropped into cold hydrochloric acid, boiling with hydrochloric acid is not

essential to its formation. The isomers are also unsta­ ble in alkaline solution. It is practically impossible isomer.

A formula which might have explained the experi­ mental datas so far obta.ned is forinula (XXXV.).

CH 0 ._{\ 2/} cn-CsH s_{־< 5}

NH MH.HC1

(XXXVI.) (XXXV.)

to isolate til© free base3.

In ordor to know if the molecule contained an alcoholic group, an attempt was made to prepare some de­ rivatives which might be analysed. An acetyl derivative of the first isomer and a picrate of the second isomer were obtained, but their analyses correspond neither

with K o h l ’s formula nor with formula (XXXV.). The acetyl determination gave: 21,7%, which obviously shows that there is not more than one acetyl group per molecule. Wohl formula requires 23% and formula (XXXV.) requires

37% for two acetyl groups per molecule. This proves the

absence of an alcoholic group in the molecule.

This result is confirmed by thè fact that the first isomer does not dissolve when heated with PCI5 in POCI3 . The reduction product of the first isomer by hy~ driodic acid and red phosphorus gave a picrate melting at 186-107°, which is not identical with the picrate of

c*-phenylpyrrolidine (XXXVI.), which melts at 149°. Oxi­ dation of the first isomer with potassium permanganate

Although it is difficult to draw definite con­ clusions from the results of these experiments, it seems probable that the substance first obtained is a polymer of some sort, tlie molecular weigth of which is higher than the one required by Y.'ohl1 s formula. Moreover, it is unlikely that a -CH2- group next to a phenyl group might be sufficiently active to condense with the alde­ hyde group. Also, the instability of the substance in alkaline solution seems to be incompatible with a pyrro- line or a pyrrolidine ring.

2°) N-benzylpyrrolidine (XXXIX.). K-benzylpyrrolidine is a known compound. It has been synthesized by Schlinch

(Ber. Cher»* Ges., 1899, 32, 952) by treating pyrrolidine with benzyl chloride. A new synthesis has been made as follows: y-aminobutyric acetal was condensed with

benzaldehyde, to give benzylidene-/־־aminobutyric acetal with an almost theoretical yield.

HP 1

C 6H 5 -CH2 -KH - (C H 2 ) 3־CH (OC gil5 ) 2 ־>

c h2-c 5h5 (XXXIX.) 0H2 -06H5

(XXXVIII.)

It was reduced by sodium and alcohol to give

y -benzylaminobutyric acetal which ״·as dropped into cold hydrochloric acid. The pyrroline ring thus formed was reduced by tin and hydrochloric acid to yield N-benzyl- pyrrolidine·

3°) Attempt to prepare acetonitril-jQ-amlnoproplonic ace- tal(XXXX.)« It was stated previously that a -CH2- group next to a phenyl group ·would not be reactive enough to condense with an aldehyde group. It was believed that if the phenyl group could be replaced by a nitril group,

the -CHg- group next to it would definitely condense with an aldehyde group, and that if thi3 replacement

could be made in benzyl-j3-amino-propionic acetal, the new compound would easily cyclise and proline might be prepared.

H CII

5

0

5

1 nci 1 1

CH2 CHo-CIT — } CII2 C-CII

----\ / 7 \ / ^

NH

im

(XXXX.) (XXXXI.)

CHp— CH CHo— CHp

I I H I I

CHp C — COOH — £2:---- * CHo CH-COOH

\V

\V

Mi NH

(XXXXII.) (XV.)

The main difficulty consisted In the preparation of acetonitrile-j^-aminopropionic acetal. Ethylene gly­ col nitrile reacts with primary amines to give two sorts compounds, -NH-CHg-CK and ~N-(CH2-CN)2 · 75:16 reaction is not easily stopped at the first stage, except when car­ ried on in presence of a large excess of amine. Although it is easy to prepare glycol nitrile in large quantities,

^-aminopropionic acetal can not he prepared in large quantities at the time. One has to treat, in an auto­ clave, j3-chloropropionic acetal with a large excess of a saturated solution of alcoholic ammonia. An attempt

was made to condense the amine with the nitrile, by drop­ ping the nitrile into an alcoholic solution of the amine,

in such a *way as to have always an excess of araine pre­ sent in the solution. It ,was hoped that, in these con­ ditions, only a small amount of dinitrile would be formed, which could easily be separated by fractional distilla­ tion. Unfortunately, that did not hapjjen. The condensa­ tion product was obviously a mixture of the mono and the dinitrile, and it was found impossible to separate them by fractional distillation, for at no. time, during the distillation, could a definite boiling point be detected. All attempts to prepare a derivative for analysis having been unsuccessful, the work, was not carried further·

4°) Attempt to synthesize proline (XV.). Keimatsu and Sugasawa (J♦ Pharm. Soc. Japan. 1928, 48, 24) have des­ cribed one synthesis of ornithuric acid by applying the Strecker method (reaction of hydrocyanic acid and ammo­ nia on an aldehyde) to y-benzoylaminobutyric aldehyde.

It was believed that proline could be prepared by having y-aminobutyric aldehyde (XXXXIII.) to react with hydro­ cyanic acid to obtain firstly the cyanhydrine (XXXIV.), and then by treating the latter with hydrochloric acid·

Tir» I I | — --) Clip CH-COOH C H g — CH2 \ V NII ch2 cii(oh)~cn \ h 2 (XV.) (XXXXIV.) (XXXXIII.)

y -aminobutyric acetal \va3 treated by hydrocyanic acid in hydrochloric solution at room temperature and then heated to boiling. The solution did not contain proline; cycli­ sation took place before the formation of the cyanhydrine.

sists in the addition of an alkyl, aryl or alkaryl halide on a compound containing the group R ! R 2®C-N-R3 to givo a quaternary salt which is very easily hydrolyzed into the corresponding aldehyde or ketone and into monoalkylated

It was thought that bromoacetic ester would p e r ­ haps react in a similar way to give finally R-NH(HBr)- C H 2-C00H. Benzylidene-methylamine was then prepared by

condensing benzyldehyde with monomethylamine (Zaunschirm, Ann. Chern., 1888, 2 4 5 , 279), and allowing the product to react with bromoacetic ester to prepare sarcosine hydro-ATTEMPT TO SYNTHESIZE SARCOSINE, The Decker reaction

bromide CH3-KH(HBr)-CHg-COOH.

A very hygroscopic crystalline substance was ob­ tained, which, hydrolysed merely by boiling with water, gave, not sarcosine hydrobromide, but monomcthylamine

hydrobromide· This reaction is very difficult to explain, since hydrobromic acid has been taken off from bromoace- tic ester, the only compound containing bromine amongst the reagents.

EXPEB !?¿SîITAL PART

d l -PHEKYLALAN 1115 (VIII.).— (1) -cyano-jff-phenylpropio­ nic ethyl ester. To a solution of sodium (5.7 g,) in absolute alcohol (125 c.c.), cyanoacetic ester (65 g., 2 mois.) and benzyl bromide (50 g., one ־mol.) were added. The mixture was heated on a water-bath for 2 hours. After cooling, it was poured into 4 or 5 times its volume of cold water; the oily layer which separated was extracted with ether, the ethereal solution was dried over anhy­ drous sodium sulphate, and the ether evaporated on a wa­ ter-bath. The residue was distilled under reduced pres­ sure. After separating the first portion which consisted mainly of cyanoacetic ester, the fraction distilling bet­

ween 160-188°/15 mm. was collected. This last portion was refractionated and the liquid, b.p, 165-173°/l5 ram., was collected. Yield, 26 g. (44$ calculated from benzyl bromide). Continuing the distillation above 188°, the disubstituted derivative (10 g.) was obtained, c׳.p, 1S0- 200°/l5 mm·.

(2) -Cyano-j3-pheny !propionic hydrazide. The ester (26 g.) was mixed with 100^ hydrazine hydrate (6.5 g.) and stirred with a glass rod. After 5 or 10 minutes the reaction took placo with evolution of heat. On cooling

in an evacuated desiccator, the hydrazide solidified. It was rscrystallized from ethyl alcohol, from ?;hich It separated in plates, m.p. 123-124°. Yield, 21 g. (SQ%). (Pound: C, 63.S3; H, 5*32. C 10H 11OX3 requires C, 63.5; H, 5.8/5) .

The hydrazide of the disubstituted ester was pre­ pared in the same way, and a white product, easily crys­

tallized from ethyl alcohol, was obtained, m.p. 235-237°. (Pound: C, 72.89; H, 6.01, C17H 17ON3 requires C,

73.1; H, 6,1%).

(3) ct-Cyano-J3-pheny!propionic a zide. c*-Cyano-j3-phenyl- propionic hydrazide (10 g.) was dissolved in a mixture of ice water (50 c.c.) and concentrated hydrochloric

acid (75 c,c.). The solution was covered with ether (100 c.c.) and placed in a cooling mixture; with constant

stirring, a concentrated aqueous solution of sodium ni­ trite (8 g.) was added. The azide formed almost instant­ ly dissolved in the ether. The yellowish green ether

solution was separated, and the aqueous solution was extracted twice with ether; the combined ether solutions were dried over anhydrous s odium s ulphate.

(4) Benzyl-cyanomothyl-ure thane « The ether solution of the azide v.as poured into absolute alcohol (100 c.c.) and the mixture was heated in such a way as to distill off the ether and keep the alcohol. This was accomplished by connecting to the flask a long distilling column. When all the ether had been removed, the alcohol solution was refluxed until the evolution of nitrogen had ceased, which require about 1 hour. The alcohol was then evaporated on a water-bath. The residue was a reddish brown viscous

»t

liquid which did not solidify on cooling, a n d which did not distill, even under a high vacuum. Yield, 11 g.

(5) dl-Phénylalanine. The urethane was boiled for about 48 hours with 20% hydrochloric acid (200 c.c.). After that time, most of the urethane had dissolved. The solu­ tion was filtered, evaporated to dryness under reduced p r e s s u r e , 'taken up with water and boiled with bone-black

to decolorize the solution as completely as possible and again evaporated to dryness to remove the last traces of

hydrochloric acid* The residue was dissolved in water (10 c.c·), and ammonia added until the pH was 5.9, iso­ electric point of phenylalanine. An equal volume of alcohol was added, and the mixture w a s cooled. The phe­ nylalanine precipitated abundantly almost colourless and was filtered and air dried, m.p, 265° with decomposition. Yield, 4 g. (50$).

(S) r-o< (J? -phenylureldo) -jS-phenylproplonic a c i d . Phenyl­ alanine (0.1 g.) was dissolved in as little concentrated sodium hydroxide solution as possible, and stirred vigo­ rously with an equivalent amount of phenylisocyanate. The mixture was filtered and the filtrate acidified with dilute hydrochloric acid, giving a white precipitate which crystallized on cooling. It was filtered and r e ­ crystallized from dilute alcohol, m.p* 168-170° with evo­ lution of gaz. (Pound: C, 67,92; H, 5.58. GigHigOgNg requires G, 67.6; H, 5.6$).

dl - METHYL-TYRO S IKE (X.).— (1) Ani3yl chloride. In a 1 liter round bottom flask, chloroform (120 c,c.) and thio- nyl chloride (45 c.c.) were mixed. The flask is equipped

with a separating funnel, a condenser and a calcium chloride tube. Over a period of 2 hours, a solution of anisic alcohol (57 g·) in dimethylaniline (SO c.c.) was added drop by drop. The mixture was shaken after each addition and, the flask was cooled in running v/ater. When the addition was completed, the mixture was heated on a water-bath f o r ,20 minutes. After cooling, the so­ lution was extracted 3 times with hydrochloric acid and

twice with v/ater. It v/as then dried over anhydrous so­ dium sulphate, the chloroform distilled off and the r e ­ sidue fractionated under reduced pressure. The anisyl chloride bolls at 120°/13 mm. Yield, 55 g, (85$).

(2) o¿-Cyano-j3-(p-raethoxy-phenyl-)-propionic ethyl ester. Sodium (8,1 g.) was dissolved in absolute alcohol (150 c.c.). To the cooled solution, cyanoacetic e 3ter (80 g., 2 m o l 3 .) and anisyl chloride (55 g., 1 mol.) were added. The mixture was refluxed on the water-bath for 3 hours, cooled, and poured into 4 times its volume of cold water; the mixture v/as slightly acidified and the oily layer which separated was extracted with ether. The ether so­ lution was dried over anhydrous sodium sulphate, the

ether distilled off and the residue fractionated under reduced pressure, b.p. 165-170°/0.2 mm.. Yield, 39 g.

(46#). '״hen using only 1 mol. of cyanoacetic ester, the yield of the monosubstituted ester was only 25#,

(3) -Cyano-fl-(p-me thox:~phenyl) -propionic hydrazide. d-Cyano-j3-(p~methoxyphenyl) -propionic ester (39 g.) was mixed with 100# hydrazine hydrate (8.4 g.). After stir­ ring for a few minutes the temperature of the mixture reached 40Q and the hydrazine completely dissolved in the ester. After standing over-night in an evacuated desiccator, the hydrazide solidified into a very hard white mass which was recrystallized from ethyl alcohol, m.p. 119°. Yield, 36 g. (97#). (Found: N, 19.49.

C 11H 13O2N3 requires

₁₇

,

(4 ) ek-Cy&no-JS-(p-methoxyphenyl)-propionic a zide« The hydrazide (4 g.) in suspension in concentrated hydrochlo ric acid (50 c.c*) was covered with ether (50 c.c.) and cooled in an ice-bath. To t h e mixture a solution of so­ dium nitrite (2 g.) in water (10 c.c.) «as added. The

formation of the azide seems to be difficult. After 1 hour of mechanical stirring, there still remained some undissolved hydrazide. The mixture was diluted with

water (50 c.c.), extracted with ether, and the ether solution dried over anhydrous s odium sulphate, then p o u ­ red into absolute alcohol (50 c.c.)·

(5) p - Me thoxybenzy 1 - cyanome thyl -ur e than e . The azide dis­ solved in the ether alcohol mixture was heated in a flask fitted with an ascending condenser connected with another condenser, in order to remove the ether but keep the a l ­ cohol. When all the ether had distilled the alcohol so­ lution of the azide was refluxed until the evolution of nitrogen had ceased (about 1 hour). The alcohol was completely r e m o v e d ·under reduced pressure. The Impure urethane remained as a dark brown viscous residue.

(6 ) dl-Methyl- tyrosine. To the urethane 20% hydrochloric acid (100 c.c.) was added and the mixture refluxed for 40 hours. After this period, the oily layer no longer dissolved. After cooling and filtering, the solution was boiled with bone-black to decolorize it as much a 3 p o s ­

sible, and then evaporated to dryness under reduced pres­ sure. The residue was taken up with a little water and ammonia added until the pH was a_:out 6.0. The amino acid precipitated, was filtered and air dried. Yield, 1.1 g#

(7) Attempted hydrolysis of the urethane into tyrosine. The urethane (5 g.) was refluxed for 15 hours with 48$ hydrobromic acid (100 c.c.). The solution was evaporated

to dryness under reduced pressure, taken up with water and filtered« A large amount of resin remained on the filter. The solution was decolorized with bone-black, evaporated to a small volume, neutralized with ammonia and treated with an equaL volume of alcohol and cooled. Eo precipitate was formed. It seemed that the amino acid h a d completely been destroyed by 48$ hydrobromic acid*

(8) r-oi(J3-phenylureido) -jg-(p-methoxyphenyl)-propionic a c i d . Methyl-tyrosine (1 g.) was dissolved in the least possible amount of concentrated sodium hydroxide solu­

tion, and stirred with one equivalent of phenylisocyanate, !,he solution was filtered and strongly a cidified. A whit© mass precipitated and crystallized by cooling on ice» In

order to recrystallize it, the acid was dissolved in a very small volume of alcohol and an equal volume of ethyl acetate and petroleum ether added. Crystals were formed when the solvents evaporated. (Pound: N, 9.13. C1 7 H 1.8 O4N 2

requires N, 8 .88$).

dl-VALINE (XI.),— (1) Isopropylcyanoacetic ethyl eater (Fisher, Eer. Chem* Ges*, 1S09, 4 2 , 2983). To a solu­ tion of sodium (18*8 g*) in absolute alcohol (280 c.c*), cyanoacetic ester (80 g*) and isopropyl bromide (88 g.) were added* The mixture was refluxed on a water-bath

for 15 hours, cooled, filtered, and a large part of the alcohol removed on the water-bath* The residual liquid was poured into cold water (500 c.c.) and extracted with ether; the ether solution was dried over anhydrous a o- dium sulphate, the ether evaporated and the residue fractionated under reduced pressure. The major part boiled at 107-1080/ 1 4 1 5 ־־ A fraction b.p. 104-114°/ 14-15 mm* was collected* ^ield, 92 g. (76$).

(2) Isopropylcyanoacetic hydrazide. Isopropylcyanoacetic ester (92 g·) was mixed with 100$ hydrazine hydrate (24 g*). The mixture rapidly wanned up and became perfectly clear* The hydrazide was a pale yellow liquid which did not soli­ dify even after long standing in a desiccator*

(3) Isopropylcyanoacetic azide. The hydrazide (46 g.) was dissolved in 25$ hydrochloric a old (400 c.c.). The solution was covered with ether (200 c.c.), and with m e ­ chanical stirring and cooling, treated with a solution of sodium nitrite (34 g.) in water (50 c.c.)· The ether

solution was separated, dried over anhydrous sodium sul­ phate and poured into absolute alcohol (300 c.c.).

(4) Isopropyl-cyanomethyl-urethane. The solution was placed in a flask equipped with a long distilling column and heated on a wator-bath until ether was removed. It was then boiled for'l hour to complete the evolution of nitrogen and the alcohol was then distilled off under reduced pressure. The residue was a reddish yellow viscous liquid which could not be distilled.

(5) dl-Valine. The urethane was refluxed with 20$ h y ­ drochloric acid (300 c.c.) for 48 hours. The pale yellow solution was filtered and evaporated to dryness in a v a ­ cuum. The residue was dissolved in the least possible amount of cold water; neutralized with ammonia, snequal volume of 95$ alcohol added, and the mixture left over­ night in the cold♦ A white precipitate of dl-valine was

filtered and air dried. Yield, 22-23 g. (80$ calculated from isopropylcyanoacetic ester). The amino acid was recrystallized from water.

(6 ) r-<* -(^-phenylureido)-isobutyric acid, dl-Valine (1 g.) was dissolved in water containing one equiva­ lent of sodium hydroxide, and the solution was stirred for 2 or 3 minutes with an equivalent of phenylisocyanate filtered, cooled on ice and acidified with hydrochloric acid. The white precipitate which formed was filtered and recrystallized from dilute alcohol, m.p. 149°,

(Found: IT, 11.95. Gl2^1603N2 requires k, 12.0$).

dl-«-AI,ii;:0-S -PliEH OX Y VALERIC ACID (XII.).— (1) ■*-Cyano- ¿‘-phenoxyvaleric ethyl ester. The trimethylene bromide needed for this preparation was obtained by refluxing for 5 or 6 hours, a mixture of trimethylene glycol (228 g 48$ hydrobromic acid (125 g.) and concentrated sulphuric acid (375 g.) (Org. Syn., JT, 18). It was found that steam distillation was preferable for the extraction of the trimethylene bromide. Yield, 550 g.· From this bro­ mide y -phenoxypropyl bromide was prepared (Org. Syn.,

and phenol (185 g.) were placed in a 3 liter round bottom flask fitted with a n ascending condenser, a mechanical stirrer and a dropping funnel. The mixture was stirred and heated to boiling. A solution of sodium hydroxide

(75 g.) in water (250 c.c,) was slowly added over a p e ­ riod of 1 hour. The reaction was completed by boiling for 5 or 6 hours. After cooling, the upper aqueous layer was discarded. " The lower layer which contained an excess . of trimethylene bromide, ^׳-phenoxypropyl bromide and

the diphenoxypropane, was reactionated under reduced

pressure. The first portion, which distilled up to 136°/ 20 rum., contained water, the excess of trimethylene.bro­ mide and some y-phenoxypropyl bromide. The next portion,

boiling between 136-142°/20 mm·, consisted of pure y*phe­ noxypropyl bromide. Yield, 250 to 300 g. This was con­ densed with cyanoacetic ester. Cyanoacetic ester (57 g.)

and y -phenoxypropyl bromide (108 g.) were added to a

solution of sodium (11.5 g.) dissolved in absolute alco­ hol (300 c.c.). The mixture was refluxed on a water bath for 1 hour and after cooling, v/as poured into 4 times its volume of cold v/ater. The oily layer was extracted with ether, the ether solution v/as then dried over anhydrous sodium sulphate, and after evaporation of the ether, the