Synthesis of Amino Acid Based Matrix Forms using a Maleic Anhydride with Vinyl Acetate Copolymer

Macromolecular amides were synthesized from copolymer of maleic anhydride with vinyl acetate and amino acids: glycine, leucine, isoleucine and lysine. As shown by NMR spectroscopy and pH-metry methods, amidation of the copolymers in a dioxane solution and mechanical activation results in the formation of semi-amide and cyclic imide units. The reaction conditions in a dioxane solution (synthesis time, the presence of a catalyst, the method of introducing amino acid) have insignificant effect on the ratio of the reaction products. During mechanical reaction, an increase in the energy intensity of the grinder facilitates the formation of maleimide units and a significant reduction of the amidation time. In the reaction of copolymers with amino acids in a homogeneous aqueous medium only maleimide cyclic imide units are formed. Tyurina. Synthesis of Amino Acid Based Matrix Forms using a Maleic Anhydride with Vinyl Acetate Copolymer. Biomed MS.ID.005096.


Introduction
An important direction in the development of modern medicine is targeted drug delivery. Biomedical engineering in this field is rapidly expanding due to the development and synthesis of new multifunctional polymer materials that can be created on the basis of α-amino acids (АА) [1]. Given the complexity of achieving a clear structure and monodispersity of synthetic biomaterials, extensive preparation procedures that should be performed to achieve a high degree of purity, an alternative to them are polymers which contain amino acids in the side chain. Such materials can be obtained by conjugation or modification of polymers with amino acids, however, to date, a limited number of α-amino acid-polymer conjugates are proposed for biomedical use. These include, for example, sustained-release pharmaceutical anxiolytic compositions based on glycine with polylactides, polybutylcyanocrylate [2], poly(Nvinylpyrrolidone) and cellulose derivatives [3]; anabolics based on leucine and isoleucine with polylactide or polybutyl cyanoacrylate [4,5]; immunostimulating gel based on lysine and highly active hydrogenated lecithins [6]. This study deals with the influence exerted on the composition of the amidation products by the conditions of the modification of physiologically active maleic anhydride (MA) with vinyl acetate (VA) copolymer [7,8] with simplest amino acid glycine (Gly), branched-chain amino acids leucine (Leu), isoleucine (Ile) and lysine, amino acid with two amino groups (Lys).

Materials and Methods
Monomers, initiator and solvents were purified by known methods [9,10] (up to 99.98 % of the basic substance). The copolymerization of MA with VA, isolation and purification of the copolymer was carried out according to [11]. Amino acids (manufactured by Snanghai Synnad Fine Chemical Co) were used without further purification. The modification in 1,4-dioxane (DO) or water was performed at an equimolar ratio of MA units and AА without catalyst and in the presence of triethylamine (TEA). A copolymer (0.185 g) was dissolved in 5 ml of solvent, AА was added to the solution (once before heating, or 4 times in equal proportions every 0.5 h), TEA was added to the reaction mixture as solution in DO (1.26•10 -6 mol/L) and the mixture was refluxed for 2 or 4 h at The final precipitate, MA-VA/AA, was then collected by centrifugation at 3000 rpm for 15min [12]. After removal of the liquid phase, the precipitate was dried/purified in a vacuum incubator at 50 °C for 24 h. The modification products were in the copolymer samples, its modification products, and amino acids was determined by alkalimetric potentiometric titration; in an aqueous medium and a mixed solvent DMFA-water, used as an titrant aqueous 0.1 М NaOH and KOH in IPA respectively. The volume corresponding to the equivalence point was found from the intersection of the second derivative curve (d 2 pH/dV 2 ) with the abscissa. The NMR spectra of copolymer and its modification products were recorded with a Bruker Avance II device (400 or 100 MHz when recording 1 H or 13 C NMR spectra) at 25 °С using DMSO-d 6 or D 2 O as a solvent.
Evidence that the amino acid enters the side chain of the polymer is a broader view of the signals related to above protons ( Figure 1).
At the same time, the broadened waveform does not allow one to evaluate whether signals 1-3 in the 1 H NMR spectrum ( Figure 1a) belong to units with semi-amide (SA) or cyclic imide groups (CI), which can be formed upon the interaction of amino acids amino groups with carboxyl groups copolymer, as shown оn the Scheme The titration curves of the modification products unambiguously confirm the binding of the carboxyl groups of the polymer chain to the amino group of the added AA, since they have one inflection corresponding to a lower alkali volume compared to the titration curves of the initial hydrolyzed copolymer. The presence of one inflection is due to the insufficiently large difference in the dissociation constants of two carboxyl groups in the SA unit (pK 1 of succinic acid 4.21 [15], pK 1 of glycine, leucine, isoleucine, lysine 2.18-2.32 [16]), therefore both the groups are titrated together, in contrast to the hydrolyzed MA-VA copolymer, for which the pK of neighboring carboxyl groups in the structural unit differ by almost 4 orders (4.90 and 8.44 -our data; 4.85 and 8.70 [17]). To enhance their acidic properties, titration in a protophilic polar organic solvent with pronounced differentiating properties, such as DMF, is necessary.
In such an medium one maximum is observed on the isoleucine  (1) 1 st -order differential curves of potentiometric titration of isoleucine, (2) MA-VA copolymer, where 45 is the molecular mass of the COOH group, g/mol; 2 -the number of COOH groups in the initial hydrolyzed copolymer; n MA is the content of MA units in the initial copolymer, wt %; M uSA and M uCI -molecular mass of a copolymer unit modified to SA and CI, respectively.
Transforming Equation (1), we obtain an expression for calculating the fraction of semi-amide units:  the establishment of equilibrium [18]. The use of water as a solvent ensures a homogeneous medium; in this case, only maleimide units are formed. However, in this case it was not possible to achieve complete isolation of the products from the solution. 1)The content of carboxy groups in the initial copolymer was calculated for the 50:50 mol % composition, and that in the modified copolymers, for the 100 % conversion of anhydride units to semi-amide ones.
2)The ratio of the unit of MA:TEA = 1:0.6 mol/mol.
3)AA was introduced into the reaction system in equal proportions after 0.5 h.
When using ethyl alcohol as a precipitant, as suggested in [12], the maximum yield of conjugates was 21 %, and when precipitated in diethyl ether, it was 50 %. However, in the latter case, the modification product contains substantial amount of water, the conjugate is a sticky mass, which makes it very difficult to work with. In order to increase the yield of the modified copolymer, to eliminate the stages of its isolation, purification and drying, the possibility of VA-MA copolymer amidation by amino acids by the mechanochemical method was studied. This method is widely used in pharmaceutical practice, in particular, to modify the properties of pharmaceutical substances and obtain dosage forms with improved therapeutic properties [19]. Initially, in the mechanochemical reactor, an interaction was carried out between succinic anhydride as a low molecular weight model of the MA unit in the copolymer and leucine. It was found that the degree of conversion of SAn to semi-amide during the reaction in a ball mill after 1 h exceeds 80 wt %, the maximum value (92 wt %) is reached after 2 h of mechanical activation. In this case a cyclic imide is also formed, its amount increases from 9 to 20 wt % with an (increase in the of the reagents treatment duration from 1 to 6 h).
Modification of SAn by leucine in a vibration grinder under the action of a load by ⁓20 times larger than in a ball mill proceeds similarly: the main reaction product is succinic acid semi-amide, but only 1 min of the reagent's mixture exposure in the vibration grinder is enough for the formation of 86 wt % SA. The results of potentiometric titration, namely, the presence of succinic semiamide, initial glycine and succinic anhydride in the mechanical activation products was confirmed by the 13 C NMR spectrum of the product (Figure 3). The residue of succinic acid CH 2 CH 2 -in the semiamide (fragment 2 in Figure 4) corresponds to signals 2 IV , 2 V at 28.3 and 29.1 ppm; group -СО-NН-(fragment 3 in Figure 4) -signal 2 II at 173.4 ppm; CH 2 -glycine (fragment 4 in Figure 4) -signal 2 III at 40.7 ppm. The acid groups of the residues of succinic acid and AA in the semi-amide (fragments 1 and 5 in Figure 4) belong to signals   The results obtained during the mechanical activation of succinic anhydride and AA were taken into account as the basic conditions for the mechanochemical reaction between amino acids and MA-VA copolymer. During the treatment of the copolymer and leucine in a ball mill for 4 h it was found that the degree of the mixture conversion extremely depends on the magnitude of the applied load. This may be due to a decrease in the specific surface area of the particles and, correspondingly, in the amount of reaction sites, and also to a decrease in the rate of the anhydride diffusion through the amine layer with an additional increase in the pressure [20,21]. Subsequently, the mechanochemical reaction between the copolymer and amino acids in a ball mill was carried out at a load of 20 N, which corresponded to the formation of the maximum amount of semi-amide units. The results of determining of the carboxyl groups content and calculating of the amidated copolymers composition are given in Table 2. As we can see, the content of carboxyl groups in the composition of the copolymer -AА mixture after mechanical activation decreases compared to their amount in the initial sample, which indicates the occurrence of an amidation reaction ( Table 2).