H Y D R O C H L O R I D E M E T H O D FOR THE D E T E R M I N A T I O N OF MONOMER

IN THE P O L Y C O N D E N S A T I O N OF ESTERS OF c~-AMINO A C I D S

T . D. K o z a r e n k o , N. B. N o s k o v a and K. T. P o r o s h i n

N. D. Zelinskii Institute of Organic Chemistry Academy of Sciences USSR

The synthesis of poly-a-amino acids by polycondensation of esters of a-amino acids or their polymers occupies a prominent position among methods of preparing polypeptides [1-3] and is often an important method of synthesizing compounds which may be regarded as fragments of protein molecules, A study of the polycon- densation of esters of a-amino acids showed [4,5] that in the first stages of this process (the degree of completion reaches 80-90 %0) the growth of the linear chains is largely determined by the consumption of monomer molecules, successively added to already formed linear chains. A comparison of the rate of interaction of monomer mo- lecules with polymer molecules (reaction of type A) with the rate of interaction of polymer 1 molecules with similar molecules (reactions of type B) leads to the conclusion that reactions of type A proceed several times faster than reactions of type B. This shows the essential difference in these two processes. From this we may assume that the consumption of monomer in the polycondensation of esters of a-amino acids (in its first stages) may be one of the criteria of the completeness of the process and serve as an additional method of studying the kinetics and chemistry of this reaction andalso of elucidating the mechanism of the formation of peptide chains in the presence of carbon dioxide as the simplest case of a model for biosynthesis.

The consumption of monomer may be determined as the difference between the original amount of mono- mer and the part of it which has not reac ted. The unreacted monomer is readily isolated by extraction with diethyl ether since both polymers and diketopiperazines are insoluble in ether. However, a series of unforeseen difficulties arise in the determination of the amount of monomer in the ether extract. The most important of them is the impossibility of distilling the ether from the monomer without substantial losses (10-25%) of the latter,, which is caused by the volatility of esters of a-amino acids. Determination of the monomer concentra- tion in the ether extract colorimetrically [6] by conversion of the ester of the amino acid into a hydroxamic acid, which gives a colored complex with ferric salts, apart from the cumbersomeness of the method gives variable results (up to 6%o) due to the low stability of complexes of iron with hydroxamie acid under the experimental conditions. It is also impossible to determine the monomer concentration through the amine nitrogen (Van Slyke method). A more convenient method was the one that we developed and used for fixing the monomer in the ether extracts as the hydrochloride, which was readily and quantitatively formed by passing dry, gaseous hydrogen chloride over the surface of the ether solution of the monomer. The hydrochloride of the ester of the a-amino acid precipitated from the solution as a white crystalline powder or an oil, depending on the nature of the amino acid. The convenience of this method lies not only in the fact that the precipitate of monomer hydro- chloride separates quantitatively from the ether (in vacuum with moderate heating), but also in that heating these precipitates (to 100~ which is necessary to dry them completely, does not lead to changes which could give further polyeondensation.

Our experiments showed that this method, which for brevity we call the "hydrochloride method", may be applied successfully to esters of glycine, D, L-alanine, D, L-valine, L-protine and D,L-phenylalanine. The losses of the hydrochloride of the ester of the a-amino acid did not exceed 1-1.5% of the amount of monomer in the ether extract. It should be possible to apply this method successfully to esters of other a-amino acids.

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E X P E R I M E N T A L

The reaction mixture, consisting of unreacted monomer, diketopiperazine, reaction alcohol and peptide esters of various lengths, was treated with 20 times its amount (by volume) of absolute ethyl ether. The mixture was stirred vigorously and cooled in ice-water for 5-6 minutes and after the solid suspension had settled, filtered into a flask with a controllable vacuum. Very slightly reduced pressure (730-740 ram) was sufficient for filtration and this was obtained with a glass apparatus for vacuum control, constructed for this purpose. The use of lower pressures led to uncontrollable losses of monomer, which evaporated together with the ether even with cooling. The solid residue of polymer and diketopiperazine was diluted with absolute ether several times more and the mixture treated as described above.

The ether extracts were combined, diluted with ligroin (a quarter of the volume of the ether) for more complete removal of the dipeptide ester, which partially dissolved in the ether solution, and filtered into a flask of known weight. A stream of dry hydrogen chloride was then passed over the surface of the solution until the formation of turbidity in successive tests ceased. The ethyl ether, ligroiu and reaction alcohol, which also passed into the ether extract from the reaction mass, were removed in vacuum (15-20 ram) at first at 20* so that the monomer hydrochloride would not be carried over and then at 100", The flask with the residual monomer hydro- chloride was weigbed and the amount of monomer which had not undergone polyeondensation determined from the increase in weight.

Ethy)_ester o fgl[ci_ne_: When gaseous hydrogen chloride was passed over the surface of an ether extract of the ethyl ester of glycine, the solution rapidly filled with a white crystalline substance, which precipitated solidly on the walls of the reaction vessel. Removal of the solvent left a crystalline mass with the properties of the hydrochloride of the ethyl ester of glycine; m.p. 143". Literature data [7]: m.p. 144".

That the monomer had been extracted completely from the reaction mixture with ethyl ether was checked by chromatography on paper from Leningrad Factory No.4 with a butanol-acetic acid-water (1 : 2 : 3) system. The ether extract (mixture of ethyl ether and ligroin) showed no spots apart from the one corresponding to the monomer, indicating a comparatively sharp separation of the components of the reaction mixture by ether ex- traction of the un reacted monomer from the mixture of polymers and diketopiperazines.

To determine the losses which could occur during the treatment of the ether extract described, a series of experiments were carried out with a known amount of monomer. The data obtained for the ethyl ester of glycine are presented in Table 1.

TABLE 1

Amount of ethyl ester of glycine hy- drochloride i ng

0.4832 0.1180

Amount of ethyl ester of glycine hydrochloride in g

0.6422 0.1581

Ethyl ester of glycine determined

in %

98.5 99.3

Amount of ethyl ester of glycine hy- drochloride in g

0.1387 0.2056

Amount of ethyl ester of glycine hydrochlo- ride in g

0.1387 0.2768

Ethyl ester of glycine determined in %

98.7 99.3

The losses (up to 1.5%) could be reduced by careful washing during filtration of the ether solution.

Ethyl ester of D,L-phenylalanine. When an ether solution containing the ethyl ester of D,L-phenylalanine was saturated with hydrogen chloride, as in the case of the ethyl ester of glycine, a crystalline precipitate of the ethyl ester of phenylalanine hydrochloride formed immediately; the m.p. was 126". Literature data [8]:. m.p. 127".

Table 2 gives results for the ethyl ester of D,L-phenylalanine, obtained by the above procedure for de- termining the monomer.

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T A B L E 2

..... Amount of

ethyl ester ofphenyl- alanine taken in g

0.3483 0.2606

Amount of ethyl ester of phenyl- alanine hydro- chloride in g

0.4121

0.3100

Ethyl ester of phenyl- alanine de- termined in %

99.6 100.3

TABLE 3

Amount of ethyl ester of D,L- alanine taken in g

0.3930

0.3012

Amount of ethyl ester of D,L- alanine hydro- chloride in g

0.5099

0.3893

Ethyl ester of alanine de- termined in %

98.9 98.5

TABLE 4

Amount of ethyl ester of D,L- valine taken in

0.0763

0.1243

0.3000

Amount of ethyl ester of D,L.- valine hydro-

chloride in g

0.0945 0.1563 0.3702

Ethyl ester of D,L-val~ne de- termined in %

99.0 100.3 98.8

Ethyl ester of D,L-alanine. The hydrochloride of the ethyl ester of D,L-alanine, formed by the passage of a stream of dry hydrogen chloride over an ether solution of the ethyl ester of D,L-alanine, precipitated as an oil, which crystallized readily after evaporation of the solvent and removal of traces of moisture with benzene in vacuum. The melting point before recrystallization of the substance was 69-72*. According to literature data [9] the m.p. is 69 -72* and after recrystallization the substance had m.p.81-83*. According to literature data [10]: m.p. 86-87 ~

Table 3 gives the results obtained for the ethyl ester of D,L-alanine by the procedure described above for determining the monomer.

Ethyl_ester of D,_L-valine_. When a stream of dry hydrogen chloride was passed for a comparatively long period (15-20 minutes) over an ethyl solution of the ethyl ester of D,L-valine, a white crystalline precipitate formed and this could change into a solution with a further excess of hydrogen chloride. However, this excess of hydrogen chloride did not affect the determination of the amount of the ethyl ester of D,L-valine; removal of the solvent from the ether solution by the method described above yielded the crystalline ethyl ester of D,L- valine hydrochloride with m.p. 101-104". Literature data [11]: m.p. 76". Results obtained with a known amount of the ethyl ester of D,L-valine in ether are presented in Table 4.

A chromatogram of the substance obtained in the butanol-acetic acid-water system showed only one spot. The ethyl ester of valine hydrochloride obtained by Fischer's method [12] also melted at 101-104 ~ A mixed melting point of the substances was not depressed. Many recrystallizations of the substance from ethyl alcohol and from chloroform did not change the melting point. Found: C 46.30; H 8.82; N 7.84%. Calculated : C 46.28, H 8.82; N 7.72~ tool. weight 181.5.

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Ethyl ester of L-proltne. The hydrochloride of this substance rapidly separated as an oil in ethyl ether when gaseous hydrogen chloride was passed over the surface of the solution. Removal of the solvent in vacuum left a thick sirup, which gave an analysis corresponding to the ethyl ester of L-prollne hydrochloride. Found: C 46.62; H 7.67; N 7.62~ CTHI4OgNC1. Calculated: C 46.8; H 7.8; N 7.8%.

SUMMARY

A method is proposed for determining the monomer in the polyeondensation of esters of a -amino acids. The accuracy of the method was to within 1-1.5~

[I] E. Fischer, Ber, 39, 2893 (1906).

[2]

[3i [4]

1500. *

[5] 642. *

[6]

[7]

[8]

[9]

[10]

[11]

[12]

L I T E R A T U R E C I T E D

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K. T. Poroshin, T. D. Kozarenko and Yu. I. Khurgin, Bull. Acad. Eei. USER, Div. Chem. 8ci., 1957,

R. T. Hall and W.E. Schaffer, Organ.Analysis 2,56 (1954).

Th. Curtius and F. Gobel, J. prakt. Chem. (2), 37, 160 (1880).

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B. Johnson and A. Ticknor, J. Amer, Chem. Eoc. 4, 642 (1918).

Th. Curtius, J. prakt. Chem. 125,254 (1930).

E. Fischer, Ber. 34, 433 (1901).

Received November 30, 1957.

* Original Russian pagination. See C. B. Translation,

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