IE63973B1 - Process for preparing a copolymer of two alpha-amino acids - Google Patents

Description

Process of preparing a copolymer of two «f-amino acids The present invention relates to a new process of synthesizing a copolymer of two ef-amino acids, which comprises the preparation of a mixture of Ncarboxyanhydride of a first efc-amino acid and the Ncarboxyanhydride of a second Λ-araino acid, followed by the copolymerization of this mixture. It also relates to a novel use of the copolymer obtained by this process.

In known processes of this type, the mixture of the Ncarboxyanhydride of the first and second cfc-amino acids is obtained (i) by preparing separately the Ncarboxyanhydride of the first 0(-amino acid and the Ncarboxyanhydride of the second d-amino acid by separate reaction of the phosgene on the first amino acid and on the second aminated acid, then (ii) by mixing, possibly after purification, the two N-carboxyanhydrides thus obtained. Because of the separate preparation of the two N-carboxyanhydrides, these known processes require the use of two parallel production lines, each comprising a reactor with different accessories (agitator, temperature measuring means, reflux device, intakes for the reagents, means for adjusting the phosgene flowrate, means for discharging the reaction mixture), means for purifying the N-carboxyanhydride and means for transferring said Ncarboxyanhydride to the copolymerization reactor. The use of these processes requires then necessarily complex and therefore costly equipment. Furthermore, it is often difficult to programme all the operations carried out with this equipment for there to be synchronism between the two parallel production lines. Such a difficulty results from the complexity of the equipment used but also from the fact that the reaction of one of the Ci-amino acids with the phosgene may require a longer time than the other oiamino acid. It thus often happens, with the above described known processes that the operations relative to one of the N-carboxyanhydrides are finished before those relative to the other N-carboxyanhydride, so that the Ncarboxyanhydride obtained first must be stored for a more or less long period before being used for copolymerization. Now, it has been observed that these Ncarboxyanhydrides do not keep well and are sensitive to different agents, particularly humidity, which means that precautions must be taken which further complicate the equipment and the synthesis of said N-carboxyanhydrides.

Furthermore, copolymers of leucine and glutamic acid and copolymers of lysine and alanine are known from G. Ebert et al., Angew. Makromol. Chem. 40/41 (1974).

The purpose of the present invention is to overcome the above drawbacks and for this it provides a process according to the one defined in the first paragraph ot this description which is characterized in that the mixture of the N-carboxyanhydrides of the first and second a-amino acids is obtained by action of the phosgene on a mixture of a first and a second Ο-amino acids having substantially the same reaction rate with the phosgene. It will in fact be understood that by appropriately selecting the starting α-amino acids, namely those having substantially the same reaction rate with phosgene, it becomes possible to react them simultaneously with the phosgene in a single reactor and a single production line is then sufficient for obtaining the desired N-carboxyanhydrides which not only greatly simplifies the equipment to be provided for synthesis of the desired copolymer but further avoids any risk of degradation of the Ncarboxyanhydrides since they are obtained simultaneously and the temporary storage of one or the other is no longer necessary .

The first and second X-amino acids are formed by 1 eucine, particularly L-leucine, and by glutamic acid or glutamic acid whose carboxyl function the furthest away from the amine function is esterified by a methyl or benzyl group, particularly L-glutamic acid or the methylic or benzylic ester of L-glutaraic acid.

The mixture of the first and second Λ-amino acids to be reacted with the phosgene advantageously comprises from 40 to 60 mole % of one of these amino acids and 60 to 40 mole % of the other.

The reaction of the c^-araino acids may be carried out in an organic solvent and dioxane or tetrahydrofurane may be mentioned by way of examples.

Before copolymerization of the resultant Ncarboxyanhydrides, it is desirable to subject them to an appropriate treatment for reducing their chlorinated derivative content, preferably to a value less than 0.1%, these derivatives being produced during^ the reaction of the phosgene with the 06-aroino acids. Such treatment is described hereafter within the scope of preparations given by way of illustration of the invention.

The mixture of the N-carboxyanhydrides is then subjected to the copolymerization reaction, advantageously at moderate temperatures, in an appropriate solvent such as dioxane and in the presence of a copolymerization initiator such as a tertiary amine (triethylamine, tributylamine, triethanolamine etc..), possibly in the presence of a lithium salt (chloride or nitrate for example) or a strong base such as an alkaline metal alcoholate (sodium methoxide for example). The ratio of the sura of the number of moles of the N-carboxyanhydrides to the number of moles of the copolymerization initiator depends on the initiator used and on the viscosity desired for the copolymer. Thus, in the case where the initiator is formed by tributylamine, the ratio in the range from 25 to 200 leads to a copolymer having an intrinsic viscosity appropriate to the uses described hereafter for which this copolymer is intended. Similarly, when the initiator is formed by sodium methoxide , a ratio in the range from 100 to 1000 leads to an appropriate copolymer.

The copolymer obtained with the above process is isolated by flowing the reaction mixture in a great excess of water. By proceeding thus, the copolymer precipitates in the form of a very hydrophobic and very bulky fibrous product.

This copolymer finds essentially its application as a means for covering skin wounds, skin zones being repaired, burns and zones from which grafts have been taken, which covering means promotes cicatrization, while ensuring, until healing or cicatrization has taken place, the protection of these wounds and zones against external aggressions, whether they be of a mechanical or microbial kind.

With a view to this use, the copolymer is applied in a form appropriate to its application in a thin layer on the part to be protected. Thus, this copolymer may for example be applied in the form of a thin film or a powder or incorporated in a gel. From this copolymer a thin film may more particularly be formed using the well known technique of phase inversion as will be described hereafter with greater detail. Such a film is generally translucent, flexible and non porous, it has a thickness preferably between 300 and 750 Jim and it is permeable to oxygen, to water vapour and to certain disinfectant solutions which it might possibly be desirable to apply through this film. To obtain a film with these characteristics, it is desirable for the copolymer to have an intrinsic viscosity at 30°C in dichloroacetic acid between 0.5 and 3 dl/g. After healing or cicatrization, the film, the powder or the gel is removed or is spontaneously eliminated from the zone on which it was applied.

The following preparations are given by way of illustration of the invention.

Preparation of methyl L-glutamate (L-Glu(OMe)) 1.25 litre of methanol is placed in a 2 litre reactor. 100 ml of acetyl chloride are then poured in at a temperature less than 20°C. With the pouring finished and under the same temperature conditions, 148 g of L-glutamic acid is added in one go and is agitated at ambient temperature for 24 hours. Then 125 ml of pyridine are poured into the mixture obtained at a temperature less than 20°C. A precipitate forms and agitation is again carried out for 48 hours at ambient temperature. The precipitate is separated by filtration, washed twice with ethanol and once with ether, then it is dried in a vacuum which leads to 174 g (yield : 86.56Z) of the expected ester, which is in the form of a white powder.

Elementary analysis Found (%) Calculated (%) C : 45.22 - 45.23 44.72 H : 7.14 - 7.16 6.88 N : 8.46 - 8.46 8.69 TLC : eluant : BuOH/CH^ COOH/^O : 4/1/1 Preparation of the mixture of the N-carboxyanhydrides of L-leucine and methyl L-glutamate 65.5 g of L-leucine (0.5 mole), 80.5 g of methyl Lglutaraate (0.5 mole) and 1600 ml of anhydrous dioxane passed previously over alumina to free it of the peroxides which it may contain are placed in a 2 litre reactor having a 2 litre non return system, a gas intake permitting a high phosgene and argon flow, a ball refrigerant, a NaOH receiver and an active carbon column.

The suspension obtained is purged with argon for mins. Then a rapid flow of phosgene is passed while stirring the mixture energetically. The temperature inside the reactor is brought to 40°C. After six hours, the reaction mixture becomes homogeneous. The solution is then swept with argon for at least 48 hours at 40°C so as to eliminate a maximum of the chlorinated derivatives dissolved in the solution. Then it is evaporated in a vacuum and the residue is exhausted with 20 litres of anhydrous dioxane per 2 litre portions.lt is verified that the amount of chlorinated derivatives in the distillate decreases progressively.

The solution in the dioxane is evaporated in a vacuum and the residue is taken up with 800 ml of dry hexane. There is crystallization of a product on which, after vacuum drying, a potentiometric quantity determination of the chloride ions is carried out. In the case where the chloride ion content is greater than 0.4%, the dioxane exhaustion should be taken up again. In the opposite case, a final treatment with the theoretical amount of Ag20 in solution in the dioxane should lower the chloride ion content to less than 0.1%. This is the condition required for obtaining, in the following step, a copolymer suitable for the formation of a thin film. About 140 g (yield 81.39%) of the expected mixture ready for copolymerization is finally obtained.

Preparation of the L-leucine and methyl L-glutamate copolymer. 137 g of the mixture prepared as described above and 2000 ml of anhydrous dioxane are introduced into a 2 litre reactor having a powerful agitator. The solution obtained is brought, in an argon atmosphere, to a temperature of 40°C. Then, in a single go, 16 ml of a 0.1 M solution of sodium methoxide in dioxane or 8 ml of a IM solution of tributylamine in dioxane is introduced. After 4 hours agitation, the polymerization reaction starts and the solution becomes more and more viscous. It is left under agitation for 3 days while checking the disappearance of the characteristic band of the N-carboxyanhydrides at 1785 cm“l in IR. The polymerization time depends on the amount of chlorinated derivatives present in the medium and the amount of polymerization initiator used. For a chlorinated derivative content less than 0.1%, a mole ratio of the Ncarboxyanhydride mixture to the initiator of 100 in the case of using tributylamine or 500 in the case of the use of sodium methoxide is particularly suitable for obtaining a copolymer which can be formed as a film.

The viscous solution of the copolymer in the dioxane is then poured slowly into a container containing 10 litres of water. The copolymer precipitates in contact with the water, the precipitate is separated by filtration, it is dried in air on paper and the drying is finished in a vacuum. Thus, 85 g of a white product is obtained having the following characteristics : . intrinsic viscosity at 30°C in dichloroacetic acid : 1.79 dl/g . elementary analysis : Found (%) 0:56.04-56.17 N: 7.99- 7.96 N:10.48-10.42 Calculated 56.23 7.83 .93 (The percentages calculated are determined for an equimolar amount of L-leucine and methyl L-glutamate). . rotatory power : =-44° (c=l% in dichloroacetic acid) at 20°C for the D spectral ray of sodium.

. High pressure liquid chromatography : amount of Lleucine and L-glutamic acid after 48 hours hydrolysis in concentrated HCl at 100°C : L-leucine : 44% L-glutamic acid : 56% Manufacture of a thin film from the L-leucine and methyl L-glutamate copolymer A collodion of the copolymer is first of all formed in a very polar organic solvent such as N-methyl pyrrolidone, N-dimethylacetamide, N-dimethylformamide, dimethylsulfoxide or difluoro- or trifluoroacetic acid. After pouring the collodion obtained, to the desired thickness (500 /dm for example), on a glass plate, the plate supporting the copolymer is immersed in several successive water baths to coagulate the collodion and wash the resulting film. This film is removed from the glass plate and it is immersed in a bath containing 75% of PEG 600 and 25% of physiological serum so as to impregnate the 10 film with it for conservation thereof. After being placed in an impermeable bag, the whole is sterilized by gamma ray radiation.

Claims (4)

1. Process of synthesizing a copolymer of two α-amino acids which 4 comprises the preparation of a mixture of the N-carboxyanhydride of a first aamino acid and the N-carboxyanhydride of a second α-amino acid, followed 5 by the copolymerization of this mixture, characterized in that said mixture is obtained by action of phosgene on a mixture of a first <χ-amino acid constituted by leucine and a second α-amino acid constituted by glutamic acid or glutamic acid whose carboxyl function the furthest away from the amine function is esterified by a methyl or benzyl group. 10 2. Process according to claim 1, characterized in that before the copolymerization of said mixture, if necessary, the latter is subjected to a treatment for reducing its chlorinated derivative content to a value less than 0.1 %. 3. Process according to claim 2, characterized in that said treatment 15 comprises the reaction of said mixture with Ag,0. 4. Process according to one of the preceding claims, characterized in that said mixture of the first and second α-amino acids comprises from 40 to 60 mole % of one of these amino acids and 60 to 40 mole % of the other. 5. Agent for covering skin wounds, skin zones being repaired, bums

2. O and zones from which grafts have been taken, characterized in that it comprises the copolymer obtained by the process according to any one of claims 1 to 4. 6. Agent according to claim 5, characterized in that it is in the form ί of a film having a thickness comprised between 300 and 750 pm, a powder or - a gel. 7. A process according to claim 1, of synthesizing a copolymer of two α-amino acids, substantially as hereinbefore described. 8. A copolymer of two α-amino acids, whenever

3. 5 synthesized by a process claimed in a preceding claim.

4. 9. An agent according to claim 5, substantially as hereinbefore described.