A Study of Glycine Anhydride

A STUDY OF GLYCINE AKHYDRIDE BY I R E N E H A N S A H S.4XBORh

Yet another study of glycine anhydride may be of interest-a study undertaken, as it were, in the face of much conflicting evidence, but having as its incentive a theory which is constantly accumulating new supporting facts and correlating many old ones. Constructive research into the structure of the proteins started with the almost simultaneous pronouncement by F. Hofmeister and E. Fischer of the -CO NH- linkage as characteristic of proteins. It is not necessary to discuss the well-known peptide theory, or to dwell upon its usefulness. Its relation to the most important criterion in the study of the structure of proteins, namely that furnished by their biological behavior, their reaction with enzymes, is of great import. I t is recognized that the breaking down of proteins by chemical means or by peptic and tryptic enzymes has yielded compounds which are of cyclic structure and not straight polypeptide chains. Such cyclic‘compounds, recognized early in investigations, were 2 , j-dioxopiperazines. These 2 , j-dioxopiperazines are composed of two amino acids, with glycine anhydride in the rBle of their simplest representative. Although the dioxopiperazines were obtained from proteins comparatively early, no systematic experiments were carried out aiming at their establishment as elementary building stones for the protein structure. The polypeptide theory was too well supported. The possibility, however, of the occurrence of preformed dioxopiperazines was not disregarded by either Fischer‘ or Abderhalden.2 Fischer states that the simple amide bond does not represent the only possible linkage within the protein molecule, but on the contrary that the occurrence of piperazine rings is rather probable. . . . By intramolecular formation of anhydrides, the numerous hydroxyl groups of hydroxyamino acids can be transformed into ether and ester groups; the variety increasing still more when one considers that the polyamino acids are probable constituents of proteins.” In a comprehensive article by Klarmann,3 the theories of the cyclic structure of proteins are well discussed and summarized. I t is interesting to note that while he considers the pyrrole theory, the dioxopiperazine theory, the experiments of Waldschmidt-Leitz on enzyme separation and subsequent elucidation of protein structure, the synthetic heterocyclic compounds which might possibly occur in proteins, the iso- and allodioxopiperazines, and the ureide theory, he devotes four times as much space to the dioxopiperazine section as to any other. This may or may not be indicative of any particular Ber., 38, 607 (1905);39, 607 (1906).

* “Lehrbuch physiol. Chemie,” y d ed., 885 (1915). Chem. Reviews, 4, j I (1927).

I 800

IRENE HANNAH SANBORN

aptness of this theory. I t does mean that the n, 5-dioxopiperazine structure is one of considerable importance to many who possess an urge to use it in explaining the behavior of proteins. Not only have homologous dioxopiperazines been repeatedly isolated from the degradation of proteins, but they havealso been theobject of thorough synthetic work. Curtius and Goebe14 prepared them from esters of amino acids; whereas E. Fischer by a similar method, studied the transformation of dioxopiperazines into dipeptides. The primary occurrence of the dioxopiperasines was questioned by Abderhalden and Funk6 and later$ (1923) confirmed by Abderhalden when he first identified a piperazine derivative obtained from a protein. While in some cases, the anhydrides must be present in a preformed state, in other cases the possibility of a secondary formation cannot be ignored. This has been shown clearly by several workers, among them Grave, Marshall, and Eckweiler,” Brig16 and finally Abderhalden and K O ~ ~ The . ~ latter J ~ have 1isted’O the possible tautomeric forms of glycine anhydride as follows: (A)

(B)

(C)

(D)

0

0

OH

OH

I/

I

II

HN

I

II

0

P\ CH2

HN

N

NH

HZC

H2C

I

H2C \C/

P\ CH1

CH1

I I N \/ I

I

\c/

I

0

0

H

H diketo

keto-enol

I

N

dienol

I

ACH

HN

I

I

HC \C/

NH

I

0

H diethylene

They prepared several derivatives of glycine anhydride and proved their structures-among them, N, N’ diacetyl-a, 5-diketopiperasine; dibenzoyl2, 5-diketopiperazine; N, N’ dibenzyl-n, 5-diketopiperazine and 0,O’ dibeneoylether-n, 5-dioxydihydroxypiperazine. They conclude: “The acceptance of an acid amide linkage for the albumin building stone in the protein molecule is supported by certain facts. Polypeptides, so long as they are built up from the amino acids occurring in nature, are hydrolyzed with few exceptions by ferments which are present in the pancreas and intestinal juice. In case of separation, amino acids are formed by the absotption of water. One thing is certain, Le., acid amide-like linked amino acids are present in albumin, and if one assumes that piperazines are also present as building Ber., 16,753 (1883);17, 953 (1884). 2.physiol. Chem., 53, 19 (1907). 6 2.physiol. Chem., 129, 143 (1923); 132,238 (1923) 7 J.Am. Chem. Sw., 39, I I Z (1917). * Ber., 56, 1887 (1923). 9 2.physiol. Chem., 134, IZI (1924).

6

102.

physiol. Chem., 139, 188 (1924).

1801

A STUDY OF GLYCINE ANHYDRIDE

stones,-they should be temporary polypeptides-and appear as follows:

dipeptides could

OCHz CHNHz COOH

I

OH

I HC\

C

/\

N

CHz

HC CH3\

+ zHzO+

I

I

N

+

I

2

NHz

I

CH,,CH,COOH

N

HC CH3\

C OCHzCHNHzCOOH

CHz

I

I

N

/

OH

/ C

I

OH

0

OH C

I/

/\

N

I

C

/\

CHz

/

H C N CH1\ / C OH

-

HN F I HC CHI\

0

CHS

1

NH

/

/I + He0 + NHzCHzC.NCH.COOH H &Ha

C

I/

0 So far it is assumed that a special ferment exists for the building up of the anhydride ring. Every assumption of rings stab!e toward alkalies and acids, a8 the piperazine ring, would appear entirely in contradiction with the rapid decomposition of the protein molecule by ferments. It seems then, that the argument of the presence of special ferments would indicate that such ring systems might possibly be in existence. It is not unthinkable that ferments exist which effect the rearrangement, and change one tautomeric form into another whereby in special cases a decomposition of the molecule could be hypothetical. Any acceptance of a definite structure of the proteins must finally stand in harmony with the facts that the fermentation decomposition as likewise that by acids and alkalies leads to amino acids. With these requirements the acceptance of anhydrides easily separable by dilute alkalies and dilute acids stands in complete agreement. It is now only the question whether the anhydrides consisting of two amino acids produce 2,s-diketopiperazine, or another form of the same substance. There is much in favor of the first idea. I t is easy to obtain methyl piperazine by reduction from peptized silk. Other piperazines” have been isolated 2.physiol. Chem., 129, 143 (1923).

I802


which can originate only from diketopiperazine. There remains the possibility that these are formed secondarily. I t is certain that for their origin no dipeptide is to be considered, for among conditions mentioned in the transformation by reduction, no piperazine could be obtained from the polypeptides. Thus, there is probably a tautomeric form. In that case it is possible that the anhydride could occur in relation to amino acids in this form;

OCOCHNHzCHS

I

/c\ I

CHz

N

t

H,C

N

\C/

I OCOCHNHzCHzOH I t would actually be possible under these conditions i.e. in the case of reduction, that the amino acids coupled with the anhydride are separated by simultaneous rearrangement of the enol into the keto form. There is much to substantiate the keto structure.” Much evidence exists to show that dioxopiperazines occur in a preformed state, having been extracted from proteins which were cleaved by enzymes or by concentrated acids. Salaskinl*obtained leucine anhydride by the action of gastric juice on oxyhemoglobin and subsequent extraction with ethyl acetate. Glycyl proline anhydride was isolated from the ether extract of the decomposition product obtained from the action of pancreatin on edestin.13 These are only two of many similar cases. Some of the dioxopiperazines appear to be resistant to acid. E. Fischer14 found that leucine anhydride dissolved in concentrated acids without decomposition, although the ring was split upon prolonged heating. The methods used, aiming at the hydrolysis of the proteins, do not exclude the possibility of a secondary formation of anhydrides; hence mainly interesting from the point of view of dipeptide combinations. Studies of the reduction and oxidation of proteins contributed more substantial results to the conception of a dioxopiperazine structure. Regardless of how dioxopiperazines might combine with each other, or with amino acids and polypeptides, respectively, it is true that a reduction which prevented the splitting of dioxopiperazines produced volatile piperazines which could be driven off by steam distillation and hence identified. Abderhalden and Stix15 reduced silk peptone with metallic sodium and amyl alcohol. The distillate gave typical piperazine reactions although the yield was small. This Z. physiol. Chem., 32, 592 (1901);38, 573 (1903). 2.physiol. Chem., 47, 143 (1906). I‘

Ber., 35, 1164 (1908). Z. physiol. Chern., 132, 238 (1925).

I 803


did not indicate that the amount of dioxopiperazines in the protein was small, since direct treatment ,of dioxopiperazine in the same manner also leads to small yields. The dioxopiperazines apparently have low resistances which allow for cleavage in the presence of sodium or sodium alcoholate, whereas only a small portion is reduced. Glycine anhydride and leucyl glycine anhydride were subjected to reduction also and their respective piperazines obtained .I6 Having contented themselves that the results from reduction pointed toward the probability of the primary occurrence of dioxopiperazine, Abderhalden" and his co-workers carried out another series of experiments aiming a t the establishment of their presence by oxidation methods. Their results were corroborated by those of Goldschmidt and Steigerwald.'s All dioxopiperazines yielded oxamide; whereas the dipeptides were decomposed with the exception of glycyl-glycine which also yielded oxamide. With the simplest dioxopiperazine, oxidation takes place thus :

"

e"

\

Ob/

c I

CHz

I

+so+

I

c = o

C =0

HzC

/ c=o

+

2 0 2

+ Hz0

\

\N/

2"

H

Abderhaldenlg and Komm showed that only polypeptides which contain the glycyl-glycine group would yield oxamide. 2"

I

z

CHz CO,NH,CHz COOH

+ 50

----+

/ c=o 1 + z C O ~+ 2H20 c=o \

NH2

The method was used on the proteins with permanganate as the oxidizing agent. Some proteins were difficultly attacked; but oxamide was obtained from blood globulin, egg albumen, gelatin, caseinogen and silk peptone, thus confirming a well-grounded suspicion. A comparative investigation of the dioxopiperazines, peptides, amino acids, and proteins or their cleavage products included the application of color reactions which would be specific for one class and would not be given by other classes. Color reactions of the proteins have been long and extensively 2.physiol. Chem.,139, 16g (1924). 2.physiol. Chem.,140, 92 (1924). 18 Ber., 58, 1346 (1925). 19 2. physiol. Chem.,143, 128 (1925). 17

1804


useful as means of distinguishing them. Abderhalden and his co-workers*Oafter discarding many reagents which showed color with other substances than the dioxopiperazines hit upon a group of reagents, the aromatic nitro compounds, which gave characteristic reactions with dioxopiperazines. Other substances, among them hydantoins, glucose, and malonic ester, gave positive reactions also, but the likelihood of their occurrence in proteins is highly improbable. Picric acid and sodium carbonate will give a positive reaction with all peptones and most of the proteins; whereas, amino acids, and polypeptides do not. m-dinitrobenzene and 3,s-dinitrobenzoic acid give comparable results to those of the picric acid and sodium carbonate. Although these color reactions are not specific, they do constitute another support for the dioxopiperazine theory. The connection of the dioxopiperazines, which in themselves represent only small complexes, with compounds of high molecular weight, has not been neglected by Abderhalden and his workers. A compound consisting of three molecules of l-prolinezl isolated from a native protein is assigned the tentative formula : HzC-CH2

I

CHz-CHz

I

CHz

HzC

I

I

CH.COOH

\/

CH .CO.NH.CHz. C 0 . N

I co I H N/CH-cHz

I

\CH~-CH~ Many compounds of this type could be given as examplesz2to produce an idea as to the assumed nature of combinations of dioxopiperazines and amino acids. One point, that upon the addition of alkali the amount of -NHznitrogen increases, is of interest as it indicates that the ring is opened on one side by this treatment. The problem has also been approached synthetically. Abderhalden and Klarmann**found that when water is excluded a condensation may take place at a higher temperature with the formation of the corresponding dihalogen acyl-dioxopiperazine. /CO-CHe\ C1.CHzCO.N N.CO.CHzC1 \CHz----CO / Z. physiol. Chem., 139,181 (1924);140, 99 (1924). 2. physiol. Chem., 127, 281 (1923);129, 106 (1923). IZ.physiol. Chem., 131,284 (1923);139,169(1924);134, 113(1924);136; 134(1924). Z.physiol. Chem., 135, 199 (1924). zo

*'

I805


At the same time, Bergmann prepared compounds which probably have the structure -CH* and are resistant to ammonia.*‘ >-CO-

-CO The structural formula of the dioxopiperazine nucleus allows the assump t i m of the following tautomeric structures:25 R

HN

/CO-CH\ \CH-CO/ /

NH

OH

R

I

I

//C--CH\

OH R /C=C\

N

N

\CH-C//

R

”

\C=C/

I

I

OH

R I.

”

11.

I

/

OH

R

111.

Compounds possessing a markedly unsaturated character were obtained under certain conditions.zB When glycine anhydride was heated with glycerol in the presence of tyrosine, a compound was isolated which had the empirical composition of glycine anhydride, but in contrast to it immediately decolorized permanganate, gave a positive xanthoprotein reaction and readily allowed the introduction of methyl groups via diazo methane. Abderhalden and SchwaWBassume that formula I11 is the most probable for this compound. In the absence of tyrosine the compound obtained loses its unsaturated nature during the course of purification. The rBle of tyrosine is not quite clear. Rearrangement of the enol form of 2,s-dioxopiperazine into its keto form took place by heating in aqueous solution to g o - ~ o o ~ . * ~ Dioxopiperazines were obtained in the enol form by heating the respective dipeptides with diphenylamine.28 Several enolic anhydrides, among them, d,l-leucyl-glycine, d,l-leucyl-d,l-valine, were prepared in excellent yields. Glycyl-glycine and glycylalanine behaved differently from the other dipeptides. The first gave a difficultly soluble compound, probably a polymer, with composition of glycine anhydride. The dioxopiperazines in the keto form are not changed into the enol form by heating with glycerol or diphenylamine, but the change is effected by heating with aniline. A proof for the assumed structure (I) is given by preparing the anhydride from alpha-aminoisobutyryl-alpha-aminoisobutyricacid. The anhydride cannot exist in the enol form on account of its particular structure. l4

Z. physiol. Chem., 140, 128 (1924); 143, 108(1925);146,247 (1925);152, 189 (1926).

Z.physiol. Chem., 139,64 (1924);Naturwieaenschaften, 13,99 (1925). *e 2. physiol. Chem., 149, 100 (1925). *’Z.physiol. Chem,. 152,90 (1926). 15

Z. physiol. Chem., 152, 125 (1926).

I 806

IRENE HANNAH BANBORN

OH R

CH3

I 1 c=c

/

\

iTH

HN

\ / c=c

I

I

R

oc-c /

\/

HN

\

NH

\ / c--co CH3' 'CH3

OH I.

11.

The product gave all anhydride reactions, but unlike other anhydrides prepared by the same method, did not give the xanthoprotein reaction or decolorize permanganate. Since it was possible to obtain sarcosine anhydride in the unsaturated form,29 the existence of the -C = C- linkage in enolic dioxopiperazines seems close to establishment. Methods have been devised to distinguish between these tautomer compounds in a physical way. Abderhalden and Haas have found that several amino acid anhydrides give a characteristic absorption in the ultraviolet, the enol form showing a more pronounced absorption than the keto form. Some proteins also absorb in the ultraviolet. The absorption spectrum of an amino acid depends on the method of its preparation. In one case, the enol-keto rearrangement could be observed spectroscopically in d, 1-leucyl-glycine anhydride. The rearrangement was complete after eight hours.30 Abderhalden continued this particular bit of his work by examinations via polarized light of the copper salts of optically active amino acids and their polypeptides.3' Moreover his conclusion based upon this and further studies of absorption in the ultraviolet region by polypeptide^,^? is that when treated with alkali, amino acids assume an inner anhydride structure; whereas, in the case of polypeptides where the inner anhydride structure is improbable, no absorption is shown. Such studies were continued33and the absorption spectra for several aromatic amino acids and their derivatives were determined, among them, 3,s-diiodotyrosine anhydride. Shibatas4and Asahima in their investigations of desmotropy of dioxopiperazines prepared glycine anhydride, alanine anhydride, and sarcosine anhydride by heating the corresponding amino acids in glycerol according to Balbian03~and Maillard.36 None of the compounds showed an absorption spectrum; hence the conclusion that they existed in the keto form only. There would appear to be a disagreement between these observations and those of Abderhalden and Haas. Other interesting observations, capable of utilization for structural investigations of the dioxopiperazines are: I ) the refractive index of the enol 2Q

Z. physiol. Chem., 153, 83 (1926).

Z.physiol. Chem., 155, 202 (1926). Z.physiol. Chem., 164,37 (1927). 32 Z.physiol. Chem., 164, I (1927). 33 Z.physiol. Chem., 166,78 (1927). 30

31

A STUDY O F GLYCINE ANHYDRIDE

1807

form is found to be higher than the keto form;34 2) the optical rotation of solutions of dioxopiperazines decreases under the influence of x-rays and ultraviolet rays, while that of the corresponding peptides remains unchanged. The formation of ozone under irradiation may cause oxidation. Abderhalden3' and Schwab have evidence that the cleavage of enolic dioxopiperazines leads to unsaturated peptides. Glycyl-glycine prepared from enolic glycine anhydride behaves differently from that obtained from the keto form in that it decolorizes permanganate, gives the xanthoprotein reaction] no anhydride reaction, and dissolves in aqueous sodium hydroxide, giving solutions bright yellow in color. The color disappears upon heating. d,l-leucyl-glycine shows a similar behavior. The two forms of leucyl-glycine behave differently toward alkalies, the saturated form being unchanged while the unsaturated form is split quantitatively. Anhydride rings such as glycyl-d-alanine, which may undergo cleavage due to action of alkali, are undoubtedly of use in enzymic digestion. Glycine1-tyrosine anhydride, on the other hand, is rendered more susceptible to alkali cleavage by the action of enzymes. Abderhalden and SchnitzleP studied this by the formation of copper salts. Open chain structures with a -COOH group form copper salts immediately; otherwise the anhydride ring must be broken by an alkali before salt formation can occur. The change was detected by changes in rotation using a quartz-mercury lamp. In the further preparation of diketopiperazines, an unsaturated anhydride from d-1-leucyl glycine anhydride is mentioned3g although no comments are made concerning its structure. It is resistant to hydrolysis by normal alkalies, adds bromine, gives an intense red picric acid reaction, shows strong absorption in the ultraviolet, and hydrolyzes with dilute sulphuric acid to yield glycine, ammonia, and alpha-keto caproic acid. Another paper40 indicated other combinations of diketopiperazine with amino acids, i.e. leucyl glycine anhydride with glycine anhydride gives a crystalline substance. A still later report of Abderhalden and l\lahn41 concerning the action of alkali, acid, and enzymes on proteins, polypeptides and 2 j-diketopiperazines decides that acids and alkalies act in the same manner t,hough at different velocities, their action alone furnishing no insight into the behavior of the diketopiperazine groupings which are probably present in these proteinoids. According to Karrer,4*the double bonds are not in the position assumed by Abderhalden and Schwab. Karrer states that the dibenzyl compound is derived from an enolic form for which he suggests the following formula: Bull. Chem. SOC. Japan, 1, 7 1 (1926). Ber., 33, 2323 (1900);34, 1501 (1901). 36 Compt. rend., 153, 1078 (1911);Ann. Chim., (9) 1, 519, 2 2 1 0 ( 1 9 1 4 ) . 37 Z. physiol. Chem., 154, 99 (1926). 38 2. physiol. Chem., 164, 159 (1927). 38 2. physiol. Chem., 163, 149 (1927). 4 0 Z. physiol. Chem., 164, 274 (1927). 4 1 Z. physiol. Chem., 174, 47 (1928). 4 p Helv. Chim. Acta, 6, 1108 (1923). j4

1808


OCHzCeHs

I

O.CHz.CeH5 The enolic form, moreover, must possess a higher reactivity, since it was previously found that glycine anhydride must be heated with chloroacetyl chloride in the presence of nitrobenzene to 160’ in order to effect substitution; and the dichloroacetyl compound results on heating the dibenzyl dioxopiperazine with chloroacetyl chloride on a water bath. Maillard4* in his study of compounds formed from glycine anhydride (2,s diketopiperazine) states that 2 , s diketopiperazine does not possess a sufficiently basic character to form with acids, salts stable in the presence of water or alcohol. Fischer and F ~ u r n e a uhave ~ ~ shown the same thing to be true. They showed that the “chlorhydrate of glycine anhydride” obtained by Th. Curtius and Fr. G ~ e b e by l ~ ~boiling the anhydride with concentrated hydrochloric acid and recrystallizing from alcohol is none other than the chlorhydrate of glycyl-glycine: HCI~NH2CHsCO-NHCHz,COOH. The action of the hot acid has broken down the ring structure of the piperazine. Maillard confirmed this work by repetition. Moreover, being interested in an investigation of crystalline compounds which would permit preparation and differentiation by microscopic observation, he sought to differentiate between the chloroplatinates of cyclo glycyl-glycine, glycyl-glycine, tri glycyl-glycine and glycine anhydride. All his efforts furnished exactly the same substance quite regardless of whether his initial reactant was cyclo glycyl-glycine or glycyl-glycine,-analysis indicating the following formula-(HC1~NHzCHz. CO.NHCH&OOH)Z P t C14.zH20. This substance is evidently identical with that described by Th. Curtius and Fr. Goebel to which they attributed the formula (NHCHz~CO)4(HC1)z~PtC14 3Hz0. When Maillard tried to prepare the chloroplatinate of z,s-diketopiperazine, he states that the piperazine ring is opened at once as in the case of the chlorhydrate. Another sidetrack was investigated by Zelinski and Gavrilof16 in their work on the anhydride nature of the proteins. They find the proportion of glycine anhydride to vary in proteins, being highest in gelatin and lowest in sturine. Their method is to carry out acid hydrolysis by autoclaving dipeptides and anhydrides. Hydrolysis of aqueous solutions of dipeptides results in the formation of anhydrides which increases with the concentration of the dipeptide solution. Since the synthesis of anhydrides is inhibited by weak

+

Ann. Chim., (9)1, 542 (1914). Ber., 34, 2868 (1901). 46 J. prakt. Chem., (N.F.) 37, 178 (1888). ‘6 Biochem. Z.,182, I I (1927).

44


I 809

acid and suppressed by I molar acid, their formation in autoclave hydrolysis appears impossible. Goldschmidt and c o - ~ o r k e r sreport ~ ~ that upon treating diketopiperazines with hypobromic acid, no decomposition occurs in neutral solution, but only a normal bromination of the ring. Morel and Preceptis's find that picric acid hydrolyzes z ,5-diketopiperazine to glycyl-glycine; while Levene and cow o r k e r ~ *find ~ the rate of hydrolysis of diketopiperazine much lower when a methyl group is substituted on the a! carbon. Yaichnikovso reports the hydrolysis of 2,s-diketopiperazines and dipeptides as monomolecular reactions, and OlandeP finds the basic dissociation constant of diketopiperazines in 0.01 N NaOH too small to be measured electrometrically, and the acid dissociation constant to be 7 x 10-l~. Stiasny and SCotti,s2in their study of the acid-alkali binding power of peptides, have run a series of titration curves to determine whether the peptide unions play a part in the acid and alkali binding power of peptides. They used glycine, and its mono, di, and tri homologues in order to have an increasing number of peptide unions along with one -NH2 and one -COOH group. At the same time they ran glycine anhydride. Their results showed glycine to behave like a weak monacidic base and a weak monobasic acid. The addition of hydrochloric acid at first greatly diminished the pH value of its solution and then slowly decreased the pH value of the resulting buffer. After the addition of one equivalent of hydrochloric acid, the curve coincided with that of hydrochloric acid itself. The curve for glycine anhydride indicated that there was no acid binding and only a very slight binding of the alkali. Another interesting sidelight upon the structure of the enol form of diketo~~ piperazine is found in some work of Richardson, Welch, and C a l ~ e r t .They found that diketopiperazine did not condense with aromatic aldehydes upon fusion. The Perkin reaction (sodium acetate and acetic anhydride) seemed to be the only method to prepare derivatives of diketopiperazine without acetylation of amino nitrogen. Even so the reaction was not general; aliphatic aldehydes did not form simple 3,6 derivatives, and though generally applicable to the aromatic series, 0.hydroxy-subaldehyde gives poor yields. By this reaction condensations of vanillin, cinnamaldehyde, piperonal, salicylaldehyde, tolualdehyde and 0-chlorobenzaldehyde with glycine anhydride were effected. Diketopiperazine failed to condense with benzylidene aniline on fusion. Likewise, all attempts to condense diketopiperazine with substituted amidines have been unsuccessful. There would appear to be five possible enol forms:

*'

Ann., 456, I (1927),Proteins IV.

Compt. rend., 187, 236 (1928). I s J. Biol. Chem., 81, 697 (1929). sa Biochem. Z., 190,114(1927). s1 2.physiol. Chem., 134, 381 (1928). s2 Ber., 63B,2977 (1930). J. Am. Chem. Soc., 51, 3074 (1929). l8


IS10

H

H N

N

A HC COH II I1

HOC

CH

\./

N

/\

HzC

COH

I

1

HOC

CHZ

/”\

HzC

I

H N COH

/I

HOC

CH

\/

\/

11.

111.

N

N

H I.

N

/4 H & COH I I/ O=C CH \/

enol forme

N H

/\ HzC COH I 1

O=C

CHz

\/

IV.

N H

V. half enol forma

The proof of the presence of hydroxyl groups in the enol form of diketopiperazine is based mainly upon color tests, i.e. positive xanthoproteic and a negative picric acid reaction. Definite confirmation of their presence is that two moles of a naphthyl isocyanate condense easily with one mole of diketopiperazine. Probably there is only one enol form present in appreciable quantity. Concerning the structure of that one we know that there are two hydroxy groups by its behaviour with isocyanate. I n order to determine the predominant enol form, a condensation with m-nitrobenzaldehyde was tried. If compound I-no condensation, therefore no -CHzgroups. If compound 11-condensation, with two aldehyde groups. If compound 111-condensation with one aldehyde group. Upon condensation, the product showed a nitrogen content of I4.73%, an amount identical with that obtained from condensation of glycine anhydride and m-nitro benzaldehyde. If enol form (11) had resulted, the condensation product would have 11.70% nitrogen, and if enol form (III), 13.21% nitrogen. The probable explanation seems to be that during the course of the reaction the isocyanate first hydrolyzed, forming enol-diketopiperazine, which reverted t o a more stable keto form and then condensed with the aldehyde. One would expect (11) to condense without hydrolysis, but (I) must hydrolyze before condensing. Analysis showed that nearly complete hydrolysis has taken place, therefore (I) must be the ordinary form of diketopiperazine. To confirm this, another condensation was made with tolualdehyde. The resulting compound has a nitrogen content of 8.81%~while the condensation product of the diketopiperazine derivative of (Y naphthyl isocyanate has 8.92%. Hence, the probability of -C = C- is supported, since (Y naphthyl isocyanate derivative condenses with aldehydes only after hydrolysis. If one is justified in making deductions based upon researches into the polysaccharides, it can be assumed that associations of elementary complexes are present in the proteins. Although only a working hypothesis, the conception has seemed so convincing that the existence of specifically acting disaggregating enzymes was assumed by O ~ p e n h e i m e r . ~Pepsin ~ was relegated to the r61e of a non-hydrolyzing ferment whose disaggregating mechan\

54

“Die Fermente und ihre Wirkungen,” 2, 811, r z d (1926).

1811


ism was to dissolve the subordinate valences which held together the elementary complexes. Delightful though this theory might be, it could not be corroborated by experiment. Certain experiments have been adduced in favor of the existence of associated compounds. Pfeiffer and Wittkab5 showed that certain dioxopiperazines, among them glycine anhydride, are capable of forming molecular combinations with various salts, amino acids and organic compounds. The following are described as well-crystallized compounds: CHz-CO

>NH, ~ H ~ O

CaClz, NH< CO -CHI CH2-CO zLic1, NH

A Study of Glycine Anhydride

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