JOURNAL OF POLYMER SCIENCE: Polymer Chemistry Edition

VOL. 15 (1977)

Studies on Reactions of the N-Phosphonium Salts of Pyridines. XVII. Polycondensation of a -Amino Acids and Dipeptides by Means of Phosphites in the Presence of Pyridine

INTRODUCTION

A convenient method for the direct polycondensation of a-amino acids by using diphenyl and triaryl phosphites in pyridine, yielding linear polypeptides from various amino acids and a few dipeptides, has been described. However, polypeptides of high molecular weight could not be obtained by this process.lv2 Factors of retarding the increase in molecular weight may include side reactions, steric hindrance of amino acid residues, and cyclization. The side reactions may be intramolecular and/or intermolecular esterificationsof I with the phenoxide anion in I, and phenol derived by amidation as shown in eq. (1).
HO-P(OC6H5)

R'COOH

PY

/ \W6H5 Py*H+-O
I

I H-P-OCOR'

Q -OGH5
RZNH *

R'CONHR'

+ (HO)2-P(OC&)

C&OH

(1)

The reactions may retard the polycondensation, because the resulting phenyl ester is not reactive enough to be affected by aminolysis under the experimental conditions (4OoCfor 18 hr). Solvents played an important role in the polycondensation, as seen in unexpectedly favorable results in nhexane despite the heterogeneity of the reaction system.' We study here in detail the phosphite-promoted polycondensation of amino acids and dipeptides in terms of the above-mentioned facts which limit the molecular to weight of the polymers formed. EXPERIMENTAL Paper chromatography was carried out on Toyo Roshi No. 51 paper using the solvent system, acetic acid-n-butanol-water (1:8:10). The NMR spectra of dipeptides and polypeptides in trifluoroacetic acid were recorded on a JEOL-JNM4H-100 spectrometer. Polycondensation of Amino Acids in Various Solvents

A mixture of amino acids (25 mmole) and diphenyl phosphite (37.5 mmole) in pyridine (20 ml) or in solvents (20 ml) containing pyridine (75 mmole) was heated at a given temperature for 9 or 18 hr. Polymers from L-leucine,L-isoleucine,DL-valine,and L-phenylalanine were obtained by adding water (20 ml) to the resultant mixture, filtering the precipitates, and washing with boiling 50% aqueous methanol. Polymers from glycine and DL-alanine were precipitated with 20% aqueous methanol and then washed with acetone. Separation of polymers from solvents immiscible with water was carried out by treatment with 20 or 50% methanol as above, after evaporation of the solvents.
Preparation and Polycondensation of Dipeptides To a solution of a-amino acid (16 mmole) and 1.6 g of sodium carbonate in 16 ml of 1N sodium hydroxide and 70 ml of water was added 70 ml of acetonitrile. The system was cooled in an ice-box

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0 1977 by John Wiley & Sons, Inc.

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J. POLYM. SCI.: POLYM. CHEM. ED. VOL. 15 (1977)

TABLE I Polycondensation of L-Leucine in t h e Presence of Hydroxyl Compounds

Additive None
N O I O O H

AdditivelL-leu, mole/mole

Yield, %

'inha

75 43 50

0.1 2 0.15 0.18

1.0

3.0

1.5

43

0.14

a Measured

in dichloroacetic acid a t 30C.

maintained at about -2OOC. A solution of a-amino acid N-carbonic anhydride (20 mmole) in 40 ml of acetonitrile was added to the system and allowed to react for 3 hr in the box with vigorous stirring. Crude crystalline dipeptides were obtained as described in the l i t e r a t ~ r e . The pure di~ peptides were obtained by recrystallization from aqueous ethanol containing a small amount of ether, until each dipeptide gave a single spot of ninhydrin on the paper chromatogram. The absence of diketopiperazineswas examined from the NMR spectra showing the terminal amino group (-NH3+) equivalent to the amide group (-CONH-). The polycondensation of these peptides (5 mmole) was carried out a t 4OoC for 18 hr in pyridine (10ml) by using triphenyl phosphite (5 mmole). The polymers were obtained by precipitating with 20 or 50% aqueous methanol, washing, and drying as above.

RESULTS AND DISCUSSION

Phenyl esterification of terminal carboxyl groups of polymers was investigated by analyzing the showed NMR spectra in trifluoroacetic acid. Absorption bands around 6 = 7.1 ppm (-COOC&Js) that the esterification of the terminal groups actually took place. When the degree of the esterifi6 = 7.5 ppm) cation was calculated from the peak ratio of phenyl groups over amino groups (N J + - 3 , according to the conventional calculation for end group analysis, one-fifth of the groups of poly-Lleucine were esterified. Since the polycondensation was retarded by phenyl esterification as mentioned above, the reaction may be favored by the presence of hydroxyl compounds which are more nucleophilic than phenol and also can form more reactive esters than phenyl ester. Indeed, addition of p-nitrophenol, o hydroxypyridine, and 8-hydroxyquinoline,whose esters were reactive enough to undergo aminolysis under the reaction conditions was effective in producing an increase in molecular weight (Table I). TABLE I1 Relationship between Inherent Viscosity and Molecular Weight Calculated from the NMR Spectra

0.25 0.17 0.12 0.09

47 24 15 9

5217 2664 1665 999

.aMeasured in dichloroacetic acid a t 30C. b Degree of polymerization calculated from t h e NMR spectra.

TABLE I11 Polycondensation of Amino Acids by Using Diphenyl Phosphite in Various Solventsa ~~____---__~~---__ Polypeptides from amino acids
~ ~

___-

____
L-Phe Yield,

GlY Yield,
%
Tinh

D L-Ala

DL-Val Yield
%
Tinh

L-Leu Yield
%
Qinh

L-Ileu Yield,
%
Qinhb

Solvent
45 0.13 (8)

Yield
56
75 0.12 (12) 43

Tinh

%nhb

Pyridine

77

0.09

72

8
46
0.09 0.08 0.09 72 61 75
t/,

0.13 (24) 0.16 0.15 0.14 0.15 0.15 42 45 51 50 79

-

Acetonitrile DMF Dioxane Dichloroethane n-Hexane Benzene

0.09 0.13 69 71 a2 70 a7 87 79 78

56 84 94 91 98 -

0.12 (21 1 0.18 0.16 0.17 0.18 0.14

0.12 (15) 0.16 0.11 0.13 0.17 0.24 0.16 0.09

0.08 0.10

a4 73

aPolycondensation of amino acids (25.0 mmole) was carried out a t 40C for 1 8 h r using diphenyl phosphite (37.5 mmole) in solvents (20 ml) containing pyridine (5 m l ) o r in pyridine (20 ml). b Values in parentheses are t h e degrees of polymerization calculated from t h e NMR spectra.

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J. POLYM. SCI.: POLYM. CHEM. ED. VOL. 15 (1977)

TABLE IV Dipeptides of Various Sequences by t h e NCA Method

Dipeptide L-Val-Gly L-Val-L-Ala L-Val-D L-Ala L -Val-L-Leu L-Val-L -Phe L-Val-L-Ileu L-Leu-Gly L-Leu-DL-Val L-Leu-L-Val
a

Yield, %a

R f va1ueb.c
0.23 0.32 (0.34) 0.32 0.65 (0.68) 0.59 0.62 0.33 0.62 0.69 (0.70)

[&IDb

_ _________________.

62
77

+ 88.0" (+ 93.6" )d + 5.99" (+ 5.89")e

58 51 60 50 68 41 51

82.2" (+ 85.8")d

+ 17.9" (+ 17.7")f

Yield after recrystallization until single spot o n the paper chromatogram.

b Values in t h e parentheses are those in t h e literature.s CSolvent system: acetic acid-n-butanol-water = 1:8:10.
d At C = 2, H,O. e A t C = 5.1, H,O. f A t C = 1, H,O.

The number-average molecular weight of poly-L-leucine,as determined by the end-group analysis by calculatingthe peak ratios of amide groups (--CON&., 6 = 8.0 ppm) over terminal amino groups (-NC3+, 6 = 7.5 ppm) in the NMR spectra, was compared with the inherent viscosity (Table 11). A good linear relationship (eq. 1) was obtained between In 7)inh and In for poly-L-leucine, suggesting that cyclization, which would give relatively smaller viscosity of polymer based on molecular weight because of the unextended conformation of cyclic polymer in solution, did not take place in the polycondensation.
7Jinh =

1.53 x

Steric hindrance of amino acid residues markedly decreases the degree of polymerization, resulting in the following order: glycine > alanine > leucine > isoleucine > valine (Table 111). In order to TABLE V Polycondensation of Various Dipeptides by Means of Triphenyl Phosphite in Pyridinea Yield,

No.
1 2 3 4 5 6 7 8 9 10 1 1 12

Dipeptide
L -Leu-Gly

7)inhb

pn

L-Leu-L-Val L-Leu-D L-Val L-Val-Gly L-Val-L -Ala L-Val-DL-Ala L-Val-L -Leu L-Val-L-Ileu L-Val-L-Phe Gly-Gly d Gly-L-Leud Gly -L-Phed

47 43 38 64 24 35 66 28 61 76 29 26

0.15 0.11 0.12 0.13 0.1 2 0.11 0.11 0.09 0.09 0.12 0.14 0.08

24 14 16 16 16
14

12 8 24 25 =

aConditions: [dipeptide] = [P(OC,H,),] = 0.5 mole/l. in pyridine; temperature 40C; time = 18 hr. bMeasured a t 30C in dichloroacetic acid (0.5 g/dl.) C Degree of polymerization calculated from t h e NMR spectra. d Diphenyl phosphite was used in lieu of triphenyl phosphite.'

NOTES

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TABLE V I Polycondensation of L-Leucine in Various Hydrocarbons Containing Pyridine" Solvent n-Hexane n-Hexane Cyclohexane Benzene n-Heptane n-Heptane n-Octane n-Octane n-Decane Temp, "C Time, hr Yield, 76
Vin h

40 69 (reflux) 81 (reflux) 40 40 98 (reflux) 40 98 (reflux) 40

18 9 9 18 18 9 18 9 18

79 68 61 78 75 61 57 64 57

0.24 0.25 0.21 0.16 0.30 0.37 0.28 0.34 0.25

a Polycondensation of L-leucine (25 mmole) was carried o u t using diphenyl phosphite (37.5 mmole) in solvents (20 ml) containing pyridine ( 5 ml).

obtain more precise information about steric hindrance of the C- and N-terminal residues, the polycondensation of dipeptides of various combinations and sequences was carried out with the use of triphenyl phosphite in pyridine. The dipeptides were prepared by the reaction between amino acids and amino acid N-carbonic anhydrides in aqueous acetonitrile4 [eq. (2)].

R'
NH,-CH-COOH

R2

I + CH-C/O I 0 '
NH-C'
O \

R2

R'

NH,-CH-CONH-CH-COOH

(2)

The polymer yield was not always good enough, because the polymers were difficult to separate completely from the reaction mixtures. Absence of intramolecular cyclization in the polycondensation of dipeptides was also shown by the fact that glycylglycinegave in high yield a polymer whose infrared spectrum was identical with that of polyglycine.' A lower degree of polymerization of Lleucyl-L-valinethan that of L-leucylglycine,and the absence of substantial differences in the degree of polymerization between glycylglycine and glycyl-L-leucine suggested that alkyl groups branched a t the &position such as in L-valine strongly depressed the degree of polymerization, and those a t the y-position such as in L-leucine did so less markedly. A similar steric effect of the N-terminal amino acids upon the reaction was observed in the polycondensation of L-valylglycine, L-leucylglycine, and glycylglycine. Although bulkiness of both the C- and N-terminal residues affected the polycondensation, it is difficult to determine which is more influential. There seemed no practical changes in molecular weight of polymers from optical isomers (see Table V). The effect of solvent was investigated in the polycondensation of various amino acids (Table 111). The reaction of L-leucine was facilitated in n-hexane, despite the heterogeneity of the reaction system. A very small increase in the viscosity can be also seen in the polycondensation of L-phenylalanine in benzene. On the other hand, no noticeable solvent effects were observed in the reaction of other amino acids. The molecular weight of poly-L-leucinewas also enhanced in other aliphatic hydrocarbons (Table VI). The viscosity of the polymer appeared to depend on the chain length of the hydrocarbons, the highest value being obtained in n-heptane a t reflux temperature (ca. 98C). Cyclic and aromatic hydrocarbons such as cyclohexane and benzene did not give as large an increase in viscosity as aliphatic ones. The peculiar increase of molecular weight in aliphatic hydrocarbons was seen only in the polycondensation of L-leucine, but of no other amino acids. The reaction system in these solvents was separated into two layers, a solution and an insoluble, viscous portion, since L-leucine, its polymer, and the N-phosphonium salt (I) are insoluble. These facts suggest that the polycondensation takes place in an interfacial phase, where thebobutyl group of L-leucine interacts with the hydrocarbon layer.

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J. POLYM. SCI.: POLYM. CHEM. ED. VOL. 15 (1977)

The authors are indebted to Drs. T. Komoto and S. Nakahama in their Institute and Dr. R. Katakai of Gunma University for the preparation of dipeptides.

References
1. N. Yamazaki, J. Kawabata, and F. Higashi, Makromol. Chem., 175,1825 (1974). 2. N. Yamazaki, J. Kawabata, and F. Higashi, J . Polym. Sci. Polym. Chem. Ed., 12, 2149 (1974). 3. N. Yamazaki and F. Higashi, Tetrahedron, 30,1323 (1974). 4. R. Katakai, M. Oya, K. Uno, and Y. Iwakura, J . Org. Chem., 37,327 (1972). 5. R. Katakai and M. Oya, Yuki Gosei Kagaku Kyokai Shi, 30,490 (1972).

NOBORU YAMAZAKI

JUHEIJI KAWABATA
FUKUJI HIGASHI Department of Polymer Science Tokyo Institute of Technology Ookayama, Meguro-ku, Tokyo 152, Japan Received May 29,1975 Revised July 15,1976