CS217738B1 - Telechelic oligo-and polypeptides and method of preparation thereof - Google Patents

Description

The invention relates to telechelic linear-chain oligo- and polypeptides and to a process for their preparation.

When N-carboxyanhydride (NCA) polymerization is carried out by the action of a primary or secondary diamine or, optionally, a tertiary amine, the resulting oligomeric or polymeric molecules have essentially double end groups; both amino groups and terminal 3-acyl-2,5-dioxo-oxazolidine structures (Bamíord, C. H., Block H., Non-Radical Polymerization, Elsevier 1976, pp. 584-607).

Two types of particles are present at the end of the polymerization regardless of the initiator and under which conditions the NCA polymerization was performed (Goodman M., Arnon U .: J. Amer. Chem. Soo. 1964, 86, 3364. Kricheldorf HR: ). Polymer Sci. Polym. Chem. Ed. 1979, 17, 97. Hashimoto Y., Aoyama A., Imanishii Y, Higashimura T .: Biopolymers 1976, 15, 2407. Hashimoto Y, Imanishi Y .: Biopolymers 1980, 19, 655. Goodman M., Hutchison) J. Amer. Chem. Soc. 1966i, 88, 3627), samples I and II

KFC-R and CO ² · I on ^{HL-HN-C-CO-N-CO-R X} (I / J where R is alkyl or aralkyl, occurring in «-aminokyselinách constituting protein components of living organisms, for example 3 - methylhutyl, benzyl, or 2-carhenzoxyethyl, 2-carboethoxy-ethyl (in the case of a polymer containing glutamic acid units; and Ra is hydrogen or a methyl group. One of these types - particle (I) - terminated with two amino groups is The primary and secondary terminal amino groups are potent nucleophilic reagents, which are widely used in the polymer chemistry of polyamides, polyureas, etc., and can be used to carry out polyaddition reactions even at room temperature, such as β isocyanate, acyl chloride, amide, etc. .

The present invention provides oligo- and polypeptides having a molecular weight of 600-10,000, having a linear chain carrying both primary and secondary amino groups at both ends in an amount of > 90% of all end groups containing single or two amino acid structural units in the chain. multiple amino acids, in which case the individual units may be arranged statistically or in differently arranged sequences and blocks, and having individual amino acid structural units in the D or L configuration, or in the form of an equimolar racemic mixture or a mixture enriched in one of the possible configurations. The oligo- and polypeptides may not be prepared by any known method, nor by isolation from a mixture of particles of formulas I and II.

The telechelic oligo- and polypeptides of the invention can be prepared by a process comprising: an oligoic polypeptide prepared from an N-carboxyanhydride of a--amino acid or a mixture of N-carboxyanhydrides of a «-amino acid with initiation of diamines containing primary or secondary amino groups, respectively; when initiated with tertiary amines or hydrazine, aminolyses using diamines having primary or secondary amino groups, which may be identical to those used as polymerization initiators or may be different, at temperatures below 109 ° C.

As initiators, aliphatic, aliphatic-aromatic or aromatic diamines, diaminoethers, diaminosulfides or oligomeric initiators such as diesters of amino acid and eithylene glycol can be used.

According to what is known about the polymerization of NCA, the proportion of cyclic end group particles is always at least 30 to 50%, and the polymer as such cannot generally be used to prepare a telechelic, i.e., bifunctional, linear prepolymer with reactive amino end groups.

However, our research has shown that when N-carboxyanhydride is converted to N-carboxyanhydride after polymerization (N-carboxyanhydrides are also abbreviated as NCA), the systematic name for the parent compound - 2,5-dioxo-oxazolidine or 1,3 -O'xazolidine-2,5-dione} initiated by primary and secondary diamines or tertiary amines such as hexamethylenediamine, triethylamine and the like, followed by amiinolysis with a bifunctional primary or secondary amine, i.e. preferably with the same diamine used initiation, the diamine reacts with one or two terminal cyclic 3-acyl-2,5-dioxooxazolinedine moieties of formula II to form oligomeric or polymeric linear particles terminated with two reactive amino groups:

_and R _r -CO-C

-WHAT,

R,

HF-C-NH-CO-CO Hz N and ⁿ

R (II) 10-H NRNHz- * H (-NHC-CO-kr NHC-CONHRNH

R.-C-COx 'O

NH-C-CO-b-N-CO 'i

Rf au + H, NR NH _and

-WHAT,

AND

R <

^R i ⁷

H-t-NHC-CO-i-NH 'n + f ^R 11 ^fc

H + - NHC-CO ~ to N-CO? IO

R < RrC-CO &lt; ⁷ &gt;

R,

H -f — NH-C-CO-) -NH + n + 1 r, (II)

This procedure converts a polymer with different end groups into a uniform type of bifunctional telechelic polymer or oligomer. Aminolysis of the end groups can preferably be performed without prior isolation of the polypeptide from the reaction mixture. Carrying out the amiinolytic modification of the end groups does not require any additional equipment or specific conditions - solvent or catalyst, it is sufficient to heat the reaction mixture to a temperature below 10 ° C for several hours. For polypeptides from amino acids containing protected side-chain reactive groups, such as poly (glutamic) or poly (aspartic acid) whose carboxyl group in the β (γ-position is protected by esterification, or the polys (lysine) with a kairbobenzoxylated side amino group) These polypeptides are aminolysed with diamine or hydrazine at a temperature not exceeding 50 ° C to prevent possible side chain aminolysis.The protecting groups can be removed from the prepared telechelic polypeptide by known techniques, and in the Examples, this principle is illustrated on a number of polypeptides, including The only extra operation here is the extraction of the telechelic product after aminolysis to remove the possibly unreacted low molecular weight amine, which can be carried out in various ways, most preferably with water, after isolation of the telechelic polypeptide from the of the reaction solution.

This new type of telechelic linear oligo- and polypeptides, terminated with amino groups, represents a way to prepare block copolymers of type A (BJ _n) , where the advantage is not only the ability to regulate the length of the polypeptide chain and thereby its representation in the polymer, The use of alkali metal or caustic earth metal alcoholates to initiate NCA polymerization in conjunction with subsequent aminolysis leading to telechelic polymers is also possible, but typically occurs here The high molecular weight product and the molecular weight control of the telechelic peptide are very difficult.When using amine catalysts to initiate NCA polymerization and preparation of telechelic polypeptides, the molecular weight of the product can be advantageously influenced by the choice of catalyst concentration. toru.

As can be seen from the examples given by way of illustration, telechelic oligo- and polypeptides contain a small number of carboxyl end groups. So far it has not been investigated how they occur. Based on the knowledge of the mechanism of polymerization of N-carboxylic acid anhydrides, it can be assumed that they arise either through the hydrolysis of the terminal cyclic 3-acyl-2,5-dioxo-oxazolidine moieties under the influence of trace moisture or by the side termination reaction contemplated by Bamford and Blok. However, according to our data, the amount of these undesirable end groups is low, on average about 3% of the total content of all end groups. The mean functionality of the obtained telechelic polypeptides (Iván B., Kennedy JP: Polymer Bull. 2, 3151/1980 / J, expressing the average number of amino groups in the linear particle of a polypeptide of the formula F _n = 2 [- ΝΗζ] / [(—NHz - j ~ | --COOHJ] [where (- ΝΉ2), (—COOH) denotes group concentrations in mol / kg], ranging from 1.945 to 1.996. From these values, the mean degree of chain elongation DE _n can be calculated, which represents the achievable average The number of diblocks n in a block copolymer (A-BJ _n , prepared from a given telechelic polymer, and thus characterizes its quality. For the telechelic polypeptides in the examples herein, DE _n values range from 37-532, demonstrating their very good quality for preparing alternatives block copolymers.

No special precautions are required when storing the telechelic polypeptides of the present invention.

Example 1

2.96 g of N-carboxyanhydride D, L-phenylaniline and 2.96 g of L-leucine N-carboxyanhydride were dissolved in 370 ml of absolute benzene and 0.64 g of hexamethylenediamine in 7.3 ml of benzene were added. The polymerization vessel was fitted with a cap with freshly dried calcium chloride. The reaction solution was stirred with a magnetic stirrer at 215 ° C. After 1.20 hours, 0.32 g of hexamethylenediamine was added and the mixture was heated to 60 ° C under reflux for 4 hours. Then 50 ml of ethanol-free chloroform, water, and acidic impurities were added and the cloudy mixture was stirred for a further 2 hours. The undissolved portion of the reaction mixture was filtered through a glass filter, and the clear filtrate was concentrated and evaporated to dryness on a rotary evaporator under vacuum at a temperature not exceeding 60 ° C. The fields were extracted 5 times 103 ml of distilled water at 40-50 ° C and dried to constant weight at 50 ° C and 0.07 Pa. s in, 48 hours. The white, brittle and brittle product contained 2.55.8 moles of —NH2 groups / kg and 0.040 moles —COOH groups / kg; the mean numerical functionality of this telechelic polymer was 1.969, and the intrinsic viscosity (as measured in dichloroic acid) was 0.1, 12. The yield was 3.18 g, the phenylalanine content of the product was 42.3% by weight. F _n = 1.96.9, DE _n = 65.

Example 2

3.00 g of L-phenylalanine N-carboxyanhydride and 17.00 g of N-carboxyanhydride and 3.00 g of L-leucine N-carboxyanhydride were dissolved in 760 ml of absolute benzene and polymerization was initiated by adding 0.007 g of ethylenidlamine in 0.42 ml of absolute benzene and thorough homogenization. The reaction solution was allowed to react at 35 ° C for 120 hours while shaking on a shaker. Then, 305 ml of chloroform free from protic and acidic impurities was added and the reaction mixture was shaken for a further 12 hours. Finally, 0.050 g of ethyleindiamine was added and the mixture was heated to reflux while excluding air humidity at 60 ° C for 8 hours. The undissolved white fine was filtered off, the solution was concentrated, evaporated to dryness and extracted with water and dried as in the product of Example 1. The off-pale yellow solid product having an intrinsic viscosity of 0.31 (dichloroacetic acid) contained 0.354 mol - NH 2 groups / kg and 0.010 mol - COOH groups / kg. It was soluble in chloroform and dimethylformamide. The yield was 3.69 g, the phenylaniline content of the product was 38.5 weight percent. F _n = 1.945, DE _n = 37.

Example 3

5.78 g of L-phenylalanine N-kairiboxyanhydride was dissolved in 308 ml of dimethylformamide, purified by azeotropic distillation with ethanol, shaken with phosphorus pentoxide and rectified in vacuo. Polymerization was initiated by the addition of 0.33 g

1,4-bis-aminomethylbenzene in 2.8 ml of dimethylformamide of the same quality. The reaction mixture was bubbled through dry nitrogen saturated dimethylformamide during the reaction. After 95 hours, hexamethylenediarine as a 9 wt. solution in benzene and the reaction mixture was heated at 60 ° C for 6 hours. Then the gel fraction was collected by filtration and the product was obtained from the clear solution by reprecipitation into 3 L of diethyl ether. The precipitated telechelic polymer powder was extracted with water in a Soxhlet extractor for 24 hours. It contained 2.424 moles - NHa / kg, and 0.043 moles - COOH / / kg, and the intrinsic viscosity was 0.12 (dichloroacetic acid, 2B ° C), F _n = 1.965, DE _n = 57.

Example 4 Polymerization 4.00: g of L-leucine N-carboxyanhydride in a solution of a mixture of 170 ml of anhydrous dioxane and dimethylacetamide (1: 1 by volume) was initiated with 0.045 g of hexamethylenediamine with shaking for 72 hours at 30 ° C. Aminolysis was performed after the addition of 0.070 g hydrazine monohydrate for 3 hours / 80 ° C. After filtration, the bifunctional polymer solution was precipitated into 1.7 L of diethyl ether and then twice more from 100 ml of a 1: 1 by volume mixture of benzene dimethylacetamide to 1000 ml of diethyl ether as in the first precipitation. Finally, the product was extracted 3 times with 50 ml of water at 50 ° C for 2 hours each and dried to constant weight. The telechelic polymer (1.5 g) contained 2.656 mol — NH2 / kg, 0.005 mol — COOH / kg, intrinsic viscosity (dichloroacetic acid, 25 ° C) 0.10. F _n = 1.996, Ε _η = 532.

Example 5

Polymerization of 6.50 g of N-carboxyanhydride χ-benzyl-L-glutaimic acid in a solution of a mixture of 75 ml of absolute 1,4-dioxane and 75 ml of de-acidic and traces of water was carried out at 25 ° C in the presence of 0.491 g octamethylenediamine initiator with the exclusion of air humidity. The polymerization mixture was after 40 hours added 0.5 g of the same diamine and subsequent aminolysis at 30 ^Q C was performed for 25 hours. The undissolved portion of the reaction mixture was separated by centrifugation at 2000 rpm, and the telechoic polymer was isolated by precipitation into 1200 ml of methanol, washed on a frit with methanol, dried under vacuum, extracted with anhydrous diethyl ether in a Soxhlet extractor for 8 hours and finally 2 times with water at 40 ° C and dried at the same temperature in vacuo. Yield 4.1 g, intrinsic viscosity 0.21 (dichloroacetic acid, 25 ° C), amino content 1.015 mol / kg, carboxyl content 0.007 mol / kg, F _n = 1.986, DE _n = 143. Mean molecular weight average 2020 (vapor phase osnometry (VPO), dimethylformamide solution (DMIF))

Example · 6

3.2 g of N-cariboxyanhydride χ-benzyl-L-glutarate was dissolved in 95 ml of dimethylformamide under exclusion of air humidity and polymerization was initiated at 25 ° C by addition of 0.20 g of freshly prepared α-L-phenylalanine ethyl ester glycol diester. The reaction mixture was continuously bubbled through the agrgon for 24 hours, when all the monomer was half-measuring. The polymerization was then continued after addition of 1.25 g of L-alainine N-carboxylainhydride for another 24 hours. After the addition of 0.1 g of hexamethylenediamine, the reaction solution was heated to 30 ° C for 20 hours. The bifunctional prepolymer was isolated by reprecipitation after addition of an equal volume of 3 M hydrochloric acid. The precipitate was filtered off, washed with water until neutral, stirred for 24 hours at 25 ° C with 500 ml of 2% NaHCO 3 solution, extracted 4 times with 200 ml of water for 4 hours and dried by lyophilization. The amino group content was 0.33.2 mol / kg, the carboxyl group content was 0.009 mol / kg, molecular weight (os217738 miometry in VPO vapor phase) 6400, intrinsic viscosity 0.44 (25 ° C, dlchloroacetic acid). The L-alanine content of the polymer was 20.8% by weight. Polymer yield 3.30 g. F _n = 1.947, DE _n = 38.

Claims (2)

1. Telechelic oligo- and polypeptides having a molecular weight of 600 to 10,000, with a linear chain, carrying at both ends terminal primary or secondary amino groups in an amount of> 90% of all terminal groups, containing in the chain structural units of one α-amino acid or two or more In these cases, the individual units may be arranged statistically or in differently arranged sequences and blocks, and having individual amino acid structural units in the D or L configuration, or in the form of an equimolar racemic mixture or a mixture enriched in one of the possible configurations. OF THE INVENTION 2. A process according to claim 1, wherein the oligo- or polypeptide is prepared from N-carboxyanhydride or amino acid N-carboxyanhydride mixtures by initiation with diamines containing primary or secular amino groups and with tertiary initiation, respectively. amines or hydrazine, aminolyses using diamines having primary or secondary amino groups, which may be the same as those used as polymerization initiators or may be different, at temperatures below 100 ° C.