[CONTRIBUTION FROM
THE
FRICKCHEMICALLABORATORY OF PRINCETON UNIVERSITY ]
POLYCONDENSATION OF CERTAIN PEPTIDE ESTERS. I. POLYGLYCINE ESTERS' EUGENE PACSU
AND
E. JUSTIN WILSOK, J R . ~
Received October 22, 1041
The importance of chemical synthesis of protein models, polypeptides of sufficiently high molecular weights, both for enzymatic studies and for investigation of chemical and physico-chemical properties can not be overemphasized. Since the time of the initial preparation of glycylglycine by Fischer and Fourneau (1) only two methods have been developed for the general synthesis of polypeptides, namely, the halogen acyl method of Fischer (2) and the carbobenzoxy method of Bergmann (3). Essentially, Fischer's method consists of utilizing the a-halogen acids; treatment with phosphorus pentachloride yields the ahalogen acyl chloride, which then can be coupled with the free amino group of any amino acid or peptide. Subsequent treatment with excess ammonia under controlled (4) conditions yields the corresponding free peptide. Higher peptides can be prepared by coupling the halide of another molecule of a-halogen acid t o this product, or, according to Fischer, by treating the a-halogen acyl peptide with phosphorus pentachloride and coupling the resulting chloride with either another amino acid or a peptide. The classic example of this method is his synthesis of an octadecapeptide (5). I n the carbobenzoxy method of Bergmann, the amino acid is used for coupling after protection of its amino group b y combination with carbobenzoxy chloride. This is followed by treatment with phosphorus pentachloride to form the carbobenzoxy amino acid chloride, which is then coupled with amino acid esters or peptide esters in neutral solvent. Hydrogenation removes the carbobenzoxy group quantitatively with the formation of carbon dioxide and toluene to yield the free peptide. Use of this procedure implied the addition to a peptide of only a single residue at a time, with yields normally lower than 50% because of the formation of the ester hydrochlorides as a result of liberation of hydrogen chloride from the coupling reaction. We attempted t o avoid such laborious procedure by trying to prepare the acid chloride of carbobenzoxyglycylglycine as a preliminary t o preparing the chlorides of other carbobenzoxy peptides. Our conditions were : slight excess of phosphorus pentachloride in ether at - 15";same at room temperature; excess thionyl chloride at 45-50'; mixing with slight excess of phosphorus pentachloride in the solid state; mixing with large excess in the solid state. I n all cases much carbobenzoxyglycylglycine was recovered. Our products, consisting of a series of yellow oils when phosphorus pentachloride was used, and a reddish brown amorphous solid in the case of thionyl chloride, gave off fumes strongly, and attempts to couple them with leucine to obtain well-defined products failed. Inasmuch as the acid chlorides of several carbobenzoxy amino acids are pure crystalline compounds (3, 6), we attributed our 1 2
This work was supported by a grant from the Rockefeller Foundation. Present address, Kational Institute of Health, Bethesda, Maryland.
117
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EUGENE PACSU AND E. JUSTIN WILSON, JR.
failure to the presence of the reactive peptide link, which in all probability is attacked by the halogenating agents. In view of this result, attention was turned to Fischer’s halogenated peptide chlorides, all of which contain one or more peptide links. An examination of his procedures and analytical data strongly suggested that his chlorides were quite impure. In a series of experiments carried out in this Laboratory3on the simple compound chloroacetylglycylglycine, it has thus far proved impossible to obtain its acyl halide in a state even approximating purity; the reactivity of the peptide link was apparently indicated by the fact that phosphorus was present in the product. Since a survey of the literature revealed little mention of the use of this method of polypeptide synthesis since the time of Fischer, in all probability other observers have encountered the same difficulty. The 18-peptide (and others prepared in a similar manner) thrice involved the use of amorphous peptides and amorphous halides of questionable purity in the same series of reactions to yield an amorphous product, and likewise thrice involved the use of ammonia in a reaction known to yield large amounts of by-products. We are of the opinion that such “polypeptides” are so impure that they are worthless as scientific preparations or protein models, and only by the tedious method of adding one residue a t a time to the amino end of a peptide can reasonably pure products be obtained. Hence, none of the well-established methods of synthesis, which have worked so well in the preparation of a wide variety of simple peptides, is adaptable to the production of protein-like substances. After our failure to obtain pure polypeptides by these methods we turned our attention to the condensation reactions of the amino acids and peptides. These, under certain experimental conditions, lead to interesting products, some of which have been the subject of investigation early in the history of organic chemistry. An important method for the preparation of such products consists in heating the esters of the simple peptides and the amino acids under various condition^.^ We have classified these reactions in four distinct types: 1. Intramolecular removal of one molecule of alcohol from one molecule of an amino acid ester gives a diradical, -NH. CH(R) .CO-, two of which combine in inverted position to yield the corresponding diketopiperazine. (We do not rule out the possibility of a dipeptide ester acting as an intermediate; our classification is for the purpose of system, not an expression of true reaction mechanism.) The heating of the amino acid ester is carried out most conveniently in a sealed tube (9, 10, 11). 2. The ester of a dipeptide loses one molecule of alcohol intramolecularly and the corresponding diketopiperaxine (1, 12, 13) is formed by ring closure of the resulting -NH.CH(R) -CO.NH.CH(R) .CO- diradical. This cyclization proceeds extremely rapidly in the case of glycylglycine ester, the dry crystals of which change into diketopiperaaine even at room temperature in ten days. 3. Conversion of a tripeptide ester into a hexapeptide ester. The condensation is best illustrated by intermolecular elimination of one molecule of alcohol from two molecules of the tripeptide ester and union of the resulting 3 4
Unpublished data of E. Pacsu and Albert F. Chadwick. Preliminary communications on this subject have been published by E. Pacsu (7,8).
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POLYCONDENSATION O F PEPTIDE ESTERS. I.
- -
-
H2N.CH (R) COSNH * CH (R) CO NH CH (R) .CO- and -NH CH (R) CO I\”.CH(R) .CO.NH.CH(R) .COOR’ radicals. Until we undertook the present work, this reaction had been observed only with diglycylglycine methyl ester and Z-alanylglycylglycinemethyl ester (14), which give the corresponding hexapeptide esters and a small quantity of higher condensation products. 4. Tetrapeptide esters do not undergo any condensation at all (14). I n the course of the present work it was discovered that G3M5in water solution suffers partial hydrolysis into methyl alcohol and diglycylglycine, and that simultaneously a considerable portion of G3M changes into GCM and a small quantity of other condensation products. It was also found that GaM is unstable in non-aqueous solvents, since it condenses quite rapidly at room temperature, particularly in methanol solution, into the pure GsM, which then precipitates out. This spontaneous change undoubtedly represents the most convenient method for the preparation of the latter compound in good yield and in excellent purity. Application to the esters of other tripeptides, however, does not give as good results, as will be pointed out in a second paper. The initial purpose of the present work was to ascertain if a hexapeptide ester, when heated, would suffer any condensation and, if it did, to which type the reaction belonged. It is obvious that if, e.g., the GsM molecules were folded hexagonally on account of preformed hydrogen or “cyclol” bonds (I),then the ester could lose one molecule of methyl alcohol intramolecularly as readily as the G2M (11) to give rise to the simplest model of “cyclol-6” postulated by the Wrinch theory (15, 16). On the other hand, the G&q might not suffer any condensation at all, or it might undergo the reaction characteristic for the tripeptide esters. X o prediction could be made as regards these possibilities, since the actual course of the reaction in all probability depends on the shape of the ester molecules, a property about which we have no information at present.
/
CH2-HS
oc/ \
\
CO-CH2
,/
‘“‘C02CH3 Hz X””
\
/
KH-CH2
CH2-CO
\ /”
I
co /””
H2 C
\
COpCH3 H2N I1
\ CH2 /
5 To save space the ti-i-, hexa-, etc. peptide methyl esters of glycine will be designated by the symbols GaM, GeM, etc.
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EUGENE PACSU AND E. JUSTIN WILSON, JR.
For the identification of the condensation products of a polypeptide ester with high molecular weight the analytical data for the elements are practically useless in that the calculated values do not differ sufficiently to allow a sharp distinction between the possible reaction products. For example, G6M has C, 41.7, H, 5.9, and N, 22.5; the hypothetical “cyclohexapeptide” of glycine, (NH.CHt.CO)c, contains C, 42.1, H, 5.3, and N, 24.6. For any possible condensation products the corresponding values would fall between these two sets of figures and, hence, well within the limits of error in the analysis. The present work was rendered possible by recognition that quantitative methoxyl determination was the only practical method of analysis.6 From accurate methoxyl values one could draw inferences as to the course of the reaction, since e.g. G6M contains 8.28% methoxyl, whereas a “cyclol-6” should not contain any. TABLE I CONDENSATION OF PENTAGLYCYLGLYCINE METHYLESTERAT DIFFERENT TEMPERATURES
I
T = 102’ *lo
T = 11Zo&l0
Time, hrs.
Methoxyl, %
Time, hrs.
0 1 2 4 8 13 24 48 72 96 168 336
8.28 7.40 6.90 6.28 5.24 4.39 3.45 2.28 1.80 1.64 1.25 0.94
0 1 2 4 7 20 44 68 140
T = 130” &lo Time, hrs.
8.28 6.12 5.40 4.16 3.35 2.02 1.61 1.26 0.97
0.5 1.5 4.5 8 24 144 364
4.91 3.25 2.15 1.50 1.01 0.58 0.54
Experiments were carried out by heating the pure G6M below its melting point in a porcelain or platinum boat contained in a constant temperature oven; samples of the solid were withdrawn for methoxyl determination (17, 18) at known intervals of time. The results of the analyses are shown in Table I for samples heated at 102”, 112”, and 130°, respectively. In similar experiments GsM (MeO, 15.26%) was heated at 80°, 102”, and 105”, respectively; the analytical data are given in Table 11. In order to obtain direct evidence as to the nature of the condensation products, samples of G3M were heated at 10Oo=!=l0for different lengths of time and then analyzed for the products. The results are shown in Table IV. The analysis was carried out as follows. By repeated treatment of the samples, 6 Amino nitrogen determinations were rendered extremely difficult because the insolubility of the products in most cases prevented introduction of samples into the reaction chamber of the Van Slyke apparatus.
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POLYCONDENSATION OF PEPTIDE ESTERS. I.
contained in crucibles with sintered glass bottoms, with hot methyl alcohol the unchanged G3M was removed from the reaction mixtures and its weight determined by the loss in weight of the samples. The washed-out G3M was recovered in crystalline state and identified by methoxyl analysis. By subsequent and repeated treatment of the methanol-insoluble residues with warm (80') water, the weight of the water-soluble condensation product was obtained by the loss in weight of the samples. The material recovered from the cooled filtrate was identified as pure GsM, the result being confirmed by methoxyl analysis. From the methoxyl content of the water-insoluble residues we concluded that, TABLE I1 COXDENSATION OF DIGLYCYLGLYCINE METHYLESTERAT DIFFERENT TEMPERATURES T = 80" *lo Time, hrs.
Methoxyl, %
Time, hrs.
Methoxyl, %
0
15.26 15.01 14.86 14.66 14.48 14.37 14.24 13.46 13.06 12.63 12.09 11.67 11.20 8.89 5.80 4.40 3.55 2.73 2.30 2.16
0.17 0.33 0.50 1 2 4 6 11 23 47 96 192 264 432
15.50 14.10 13.59 13.26 11.33 8.97 7.20 5.67 4.80 3.62 2.42 1.56 1.40 1.14
1 2 3 4 5 8 24 29.5 50
74 98 120 240 480 720 960 1440 2474 2908 5
T
T = 102' +lo
Time, hrs.
0
72 117 360" 492 564
105' &lo
1
Methoxyl, %
14.40 1.93 1.20 0.83 0.64 0.56
After 360 hours the temperature was raised to 112" 1 1 " .
after one hour of heating, the GaM gave rise to almost pure GlzM (found: MeO, 4.39; calc'd: MeO, 4.33) as the highest condensation product. This substance was first obtained in a similar manner by Fischer (14), who concluded from its carbon, hydrogen, and nitrogen content that it could be the GIZM. The gradual decrease in the methoxyl content of the insoluble residues indicated that the GlzMunderwent further condensation during the eight and twenty-four hours the respective samples of G3M had been heated. Confirmation that the GUM undergoes further condensation was received from an experiment in which GlzM prepared by the above method was heated a t 11O0=tlo, samples being withdrawn and analyzed for methoxyl as usual. These data are included in
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EUGENE PACSU AND E. JUSTIN WILSON, JR.
Table 111. Had "cyclol-6" been formed in the condensation reaction of GaM as well as GeM, it could have been represented only by the insoluble residues, and the fact that the latter substances contained methoxyl ruled out this possibility. In addition, the insoluble residues could not have consisted of a mixTABLE I11 CONDENSATION OF HENDECAGLYCYLGLYCINE METHYLESTERAT 110" fl o T-,
i
HPS.
LIETHOXYL,
%
4.39 3.47 3.07 2.69 2.39 2.07 1.89 1.61
0 3 6.4 12 24 48 96 192 226" 394 1052
0.77 0.54
Temp. raised to 130" *lo; subsequent values a t this temp. TABLE IV ANALYSES~ OF SAMPLES OF DIGLYCYLGLYCINE METHYLESTERHEATEDAT 100" il" FOR DIFFERENT LENGTHS OF TIME LOSS METHYL ALCOHOL,
70 GsM, %
TIME, MIN.
obs'd
calc'd
0 10
30 45 hrs. 1 2 3 8 24
0.87 1.73 2.26 2.64 6.01 7.77 9.03 9.24
1.52 2.67 2.33 5.60 7.45
GzM
AND HIGHER., % '
METHOXYL OF GMMAND HIGHER, %
-
98.94 91.96 81.05 76.96
1.06 7.26 15.72 19.33
0.78 3.22 3.71
71.10 32.95 11.68 0.74 0.38
23.40 54.66 69.80 73.03 67.08
5.50 12.39 18.72 26.23 32.54
4.30 4.39 2.72 2.64
The figures represent average values for several runs starting with initial samples of about 0.4 g. of G3M. I n each run the figures for G3M and GsM percentage represented the constant values obtained after three to five washings with hot methanol and 80" water, respectively.
ture of GgM and G12M, because the GgM with 5.68% methoxyl could not lower the methoxyl content (4.3377,) of the G12M. We therefore interpret our data as indicating that the GaM underwent a series of successive condensation reactions yielding G6M, G12M,G=M, GdsM, and G9sM with theoretical methoxyl contents of 8.28, 4.33, 2.21, 1.12, and 0.56%, respectively. Since there was no
POLYCONDENSATION OF PEPTIDE ESTERS. I.
123
appreciable difference in the experiments starting with G3M, GsM, or GlzM between the methoxyl values of the last two samples taken at long interval of time, it appears very likely that the condensation reaction ends a t the 96-peptide stage, yielding a nearly uniform product with a methoxyl content of 0.5%, and with the empirical formula C193H292N9609z and molecular weight of 5504. From the nature of this type of condensation reaction, it follows that a t the intermediate stages the figures of the methoxyl estimations represent the overall methoxyl content of the reaction products, which necessarily consist of mixtures of polypeptide esters. When the G3M was heated at 80” the condensation reaction was so slow that after a total of four months heating the methoxyl reached only the value for G24M (found: MeO, 2.16; calc’d: MeO, 2.21). An amino nitrogen determination on the product, carried out under the obvious difficulty of introducing a large solid sample into the reaction chamber of the Van Slyke apparatus, gave a value of 0.680 mg. of nitrogen gas. The calculated value for the sample on the basis of the methoxyl content was 0.707 mg., showing excellent agreement. Accordingly, any possibility of a partial decomposition of G6Rf or GlzM into methanol and the corresponding free peptide, a reaction that might be thought to have caused the gradual decrease of the methoxyl content, is definitely ruled out, since the amino nitrogen value has suffered the same relative drop as the methoxyl content and the reaction must be tied up with the disappearance of free amino groups. The absence of G9M among the analyzed condensation products of G3M is surprising. It would indicate that the reaction is not a random condensation between all of the peptides present, e.g., G3M and GsRI to yield GJV, etc., but it proceeds according to a pattern which can be expressed by the simple formula 3 x 2”, where n = 1, 2,3,4, and 5. Whether or not such selective condensation has anything to do with the presumably different shapes of the polypeptide ester molecules present, or with the oriented structure of the crystalline starting material, or with some unknown factors that may in general control the niechanism of reactions occurring in the solid state, a t present we are unable to say. However, it does not seem to us unlikely that the rapid condensation of the amino acids in the living cell by the catalytic action of the enzymes takes place according to such economic pattern instead of random Condensation. All the polypeptide esters of glycine obtained in the course of this investigation were almost colorless substances, amorphous in appearance, and insoluble in alcohol but slightly (0.1-0.57c) soluble in water. They all give very strong biuret reaction and dissolve completely in cold conc’d hydrochloric acid, though only partly in dilute alkali solution. They are strongly reminiscent of denatured proteins, and like many of the latter substances are soluble in concentrated urea solution. Although the inability of G&f to form the simplest “cyclol-6” molecule by this method would seem to favor strongly the conception of a more or less openchain structure, it should be pointed out that the tetrapeptide esters do not appear to undergo any type of condensation a t all. There is thus an indication of some fundamental difference between the shape of the molecules of the di-,
124
EUGENE PACSU AND E. JUSTIN WILSON, JR.
tri-, and 3 X 2"-peptide esters on the one hand, and that of the tetra- and probably penta-, hepta-, etc. peptides on the other. In addition, a 96-peptide is one-third of the Svedberg unit of 288; whether this has any significance we are not in a position to state a t the present time. EXPERIMENTAL
Preparation of diglycylglycine methyl ester. Fischer's procedure (11) was followed closely for the conversion of glycine ethyl ester hydrochloride into 2,ti-diketopiperazine and for the preparation of chloroacetylglyclglycine from chloroacetyl chloride and the piperazine derivative. The exchange of the chlorine atom for an amino group in chloroacetylglycylglycine was carried out according to Abderhalden and Fodor (19), and the methyl ester hydrochloride of the resulting diglycylglycine was obtained by Fischer's method (11). For the preparation of the free tripeptide ester i t became necessary to modify slightly Fischer's original procedure (ll),which gave an unsatisfactory yield, due to rapid condensation of the liberated ester during the evaporation of the dilute methyl alcohol solution. The following procedure gave a pure crystalline product in nearly quantitative yield. To 4.8 g. (1/50 mole) of diglycylglycine methyl ester hydrochloride, suspended in 10 cc. of icecold methanol in a distilling flask, was added, drop by drop and under stirring a t 0"' an approximately 2% methyl alcoholic sodium methoxide solution containing 3% less than the theoretical quantity of sodium. The reaction mixture was immediately concentrated in vacuo a t 30" to a solid mass which was repeatedly extracted a t 40" with 10 cc. portions of dry chloroform. The filtered extracts were united, and absolute ether or petroleum ether was added to the cold solution until turbidity developed. On standing a t low temperature, the solution rapidly deposited the tripeptide methyl ester in groups of long needles; yield, nearly quantitative; m.p. and other physical properties agree with the data given by Fischer. Anal. Calc'd for C~HlaN30,:OCH3, 15.26. Found: OCHa, 15.40. The methyl ester of diglycylglycine is soluble in cold water, giving a clear solution strongly alkaline to litmus. However, the solution rapidly develops turbidity, indicating the formation of water-insoluble condensation products. I n a special experiment, 1 g. of pure ester was dissolved in 7 cc. of distilled water, and the clear solution was kept at room temperature for two days; heavy precipitation occurred during this time. The filtered substance was dried in vacuo over solid sodium hydroxide a t room temperature; yield, 0.4 g. Although its methoxyl content (4.1%) corresponded to that of pure dodecapeptide methyl ester of glycine, the substance was a mixture of pentaglycylglycine and its methyl ester, both of which are soluble in warm water but only slightly soluble in cold. Addition of three volumes of absolute alcohol to the original filtrate of this substance gave crystalline diglycylglycine ; yield, 0.3 g. This experiment showed that diglycylglycine methyl ester in water solution suffered partial hydrolysis into the tripeptide and partial condensation into the hexapeptide ester, the latter substance in turn being partly hydrolyzed to the free hexapeptide. I n similar experiments carried out in boiling water, further condensation occurred, yielding substances insoluble in hot water and containing little methoxyl, besides large quantities of diglycylglycine. Preparation of pentaglycylglycine methyl ester. The condensation of diglycylglycine methyl ester in non-aqueous solvents proceeded fairly rapidly, and the only product of the reaction was pure hexapeptide ester of glycine, which precipitated out immediately after i t had been formed. Three grams of crystalline tripeptide ester was dissolved in 30 cc. of cold absolute methanol and the solution was filtered rapidly with activated carbon. Heavy precipitation was obtained by keeping the clear filtrate a t room temperature for three days. The substance was filtered in a crucible with sintered glass bottom and washed with absolute methanol; yield, 1 g. of pure hexapeptide ester. From the filtrate, on standing for several days, a further quantity (0.7 9.) of the same substance was obtained.
POLYCONDENSATION O F PEPTIDE ESTERS. I.
125
Anal. Calc’d for CI3HZ2KC07:OCH3, 8.28. Found: OCHB,8.33.
We are indebted to Dr. S. M. Trister and Mr. R. E. Kobilak of this Department for their assistance in the analyses reported in this paper. SUMMARY
A study of the condensation reactions which take place on heating the amino acid esters and simple polypeptide esters has been made, and the reactions have been classified into four distinct types. It has been concluded that instead of cyclization to give the simplest model of a “cyclol-6” postulated by the Wrinch theory, the hexapeptide methyl ester of glycine, on being heated, undergoes the type of condensation characteristic for the tripeptide esters, in a series of successive reactions yielding 12-, 24-, 48-, and 96-peptide esters of glycine. The course of the reaction has been followed by methoxyl estimation on the samples withdrawn at certain intervals of time. Similarly, the condensation reactions of the tripeptide and dodecapeptide esters of glycine proceed according to the simple formula 3 X 2”, where n represents whole numbers, to give 96 as the final stage of condensation. From the results of the analyses of the condensation products it has been concluded that neither “cyclol-6” nor, in all probability, nonapeptide is formed when the tripeptide ester of glycine is heated, The polypeptides obtained are reminiscent of denatured proteins and give strong biuret reactions. An improved procedure for the preparation of diglycylglycine methyl ester, and a new method for the preparation of pentaglycylglycine methyl ester have been given. PRINCETON, N. J REFERENCES (1) FISCHER A N D FOURNEAU, Ber., 34,2868 (1901). (2) FISCHER, “Untersuchungen iiber Aminosauren, Polypeptide und Proteine”, Berlin, Vol. I (1906), Vol. I1 (1923). (3) BERGMANN AND ZERVAS, Ber., 66, 1192 (1932). (4) CHADWICK -4ND Pacsu, J . Am. Chem. Soc., 63, 2427 (1941). Ber., 40, 1754 (1907). (5) FISCHER, AKD SCHLEICH, 2.physiol. Chem., 224, 23 (1934). (6) BERGMANN, ZERVAS, SALZMANN, (7) PACSU, Nature, 144, 551 (1939). (8) PACSU, J.Franklin Inst., 230, 132 (1940). (9) FISCHER, Ber., 34, 433 (1901). (10) FISCHER AND SUZUKI, Ber., 38, 4173 (1905). (11) FISCHER, Ber., 39, 453 (1906). (12) FISCHER, Ann., 340, 126 (1905). (13) FISCHER, Ber., 39, 557 (1906). (14) FISCHER, Ber., 39, 2893 (1906). (15) WRINCH,Yature, 137, 411 (1936), et seq. (16) WRINCH,Proc. Roy. Soc., A, 160, 59 (1937). (17) VIEBOCKA N D SCHWAPPACH, Ber., 63,2818 (1930). (18) CLARK,J. Assoc. Ojicial Agr. Chem., 16,136 (1932). (19) ABDERHALDEN A N D FODOR, Ber., 49,564 (1916).
