Search

The currently known methods of enzymatic β-lactam synthesis, as well as the enzymes and heterogeneous biocatalysts used for this purpose, are presented, and the published reports on advances in the field of enzymatic synthesis of selected antibiotics belonging to the groups of acidic penicillins and acidic cephalosporins are summarized in the present review. The key conditions and parameters of biocatalytic processes, such as the biocatalyst form, concentration of the precursor compounds, solvent type, pH, temperature, etc. are analyzed and compared, and guidelines for further optimization of β-lactam synthesis are given. The present review may be of use for a wide range of readers, as well as to enzymology and biotechnology experts.

The article deals with experimental determination of ionization constants and solubility for the compounds (target products, initial β-lactams, acylating agents and by-products) involved in enzymatic synthesis of some therapeutically used aminopenicillins and aminocephalosporins, namely ampicillin, amoxicillin, cephalexin, cephadroxil, cephaloglycin, cefaclor, cefprozil, cefatrizine. Methodology of investigations and the evaluation of experimental data for the determination of ionization constants and solubility of the different type electrolytes are presented. Applications of the methods based on acid-base potentiometric titration and on determination of solubility-pH dependence of assayed substances are discussed. The original data on ionization constants and solubility of amoxicillin, cefprozil, cefatrizine, cephadroxil and initial β-lactams for production of cefaclor, cefprozil and cefatrizine, as well as solubility of by-product D-(-)-p-hydroxyphenylglycine are presented. Experimentally determined parameters and constants available in the literature for all abovementioned aminopenicillins and aminocephalosporins are collected. These data might be used for choice of the conditions of both processes: the enzymatic synthesis and the isolation of the product from reaction mixture.

The paper presents an analytical review of the literature on enzymatic synthesis of semisynthetic beta-lactam antibiotics. The results of the studies on the thermodynamics and kinetics of beta-lactams synthesis are generalized and the approaches to increasing the efficiency of the biocatalytic processes based on both thermodynamically controlled synthesis (direct) and kinetically controlled synthesis (acyl transfer) are systematized. Characteristic features of the processes for separation of the reaction mass components and recovery of the final products of the biocatalytic synthesis of beta-lactam antibiotics are considered and the pathways to increasing the economic efficiency of biocatalytic processes used in design of the technologies and their introduction to manufacture are discussed.

The methology of the development of new biologically active betalactams is proposed. One of the two ways proposed is specific modification which is peculiar to certain betalactam structure and involves introduction of substitutes changing particular physico-chemical properties of the natural or synthetic analogous. General guidelines for modification of the biologically active compounds are given. The space and depth of the necessary physico-chemical investigation are determined. The alternative way of the new biologically active compounds development is the principle of similarity. The distinctive feature of this approach is the use as building blocks of the substitutes already used in the well-known betalactam antibiotics and "implantation" of this substitutes into other (new) betalactam structures. The ways of the new betalactams synthesis including the methods of enzyme engineering are considered. The possibility to use enzyme engineering processes for production of not only new individual betalactams--hits, but also for synthesis of the groups of betalactams--leads, is shown. More than 6000 new betalactam structures were constructed on the base of the principle of similarity. At least 700 of this compounds demonstrates not only antimicrobial activity but other types of biological activity due to the implementation of additional pharmaceutical units other than betalactams. The constructed compounds are summarized in the tables, the request for the electronic version of the tables can be sent by the address: valan@orc.ru.

Production of cefazolin by acyl transfer enzymatic synthesis with immobilised cefazolin synthetase from Escherichia coli as a biocatalyst acting in accordance with the mechanism including formation of the acyl-enzyme complex was shown possible. The process kinetic parameters and the ratio of the maximum conversion of the key amino acid and the initial concentrations of the substrate and nucleophile were determined. Correlation of the calculated and experimental data on the cefazolin yield in the enzymatic synthesis was good. The main physico-chemical properties of the substrates and the reaction products i.e. dissociation constants and solubility were investigated. The complex of the physico-chemical studies makes it possible to design a highly efficient technological process for production of cefazolin including not only the stage of the enzymatic synthesis but also the stage of separation of the reaction mixture components.

Enzymatic synthesis of cephalexin and cefaclor with the use of immobilized aminocephalosporin synthetase from Xanthomonas sp. as a biocatalyst was studied. The employment of a mathematical model based on the acyl-enzyme mechanism of the biocatalyst action was shown possible for the quantitative description of the antibiotic syntheses. A relationship providing determination of the complex of the physico-chemical investigations required for the enzymatic synthesis design was suggested. Kinetic and thermodynamic parameters of the processes were evaluated and the ratios of the maximum conversion of the key amino acids and the initial concentrations of the substrate and nucleophile were calculated. The regions of the model fit to the experimental data within a wide range of the substrate and nucleophile concentrations were defined. The technology for the enzymatic synthesis of aminocephalosporins exemplified by cephalexin and cefaclor was designed.

Stability of 7-aminocephalosporanic acid (7-ACA) and desacetyl-7-ACA in aqueous solutions at the pH value higher than 6 and wide ranges of the temperature was studied. The kinetic parameters of the hydrolysis of the ester link in the molecule of 7-ACA and the openings of the betalactam link in the molecules of 7-ACA and desacetyl-7-ACA were estimated. It was shown that all the destruction processes were describable by the 1st order equations. A procedure for the calculation of the velocity constants of the above mentioned three processes by changes in the concentration of 7-ACA and desacetyl-7-ACA in the solutions is described.

The kinetics of splitting out the substitute at C3 in the molecule of beta-lactam antibiotics such as cefazolin, cefaclor, cefazedone and others as well as that of splitting out the chlorine atom in the side radical of the cefazedone molecule was studied within wide ranges of pH and temperature. The destruction processes for all the investigated compounds were shown to be described by the 1st order equations. The values of the activation energy, pre-exponential factors and reaction order by the hydrogen ions were measured. This provided the mathematical description of the destruction processes within wide ranges of pH and temperature. An unusual mechanism of synchronous splitting out the substitute at C3 and one of the chlorine atoms in the side radical of the cefazedone molecule was observed.

Benzylpenicillin interaction with amines in the nonaqueous phase was studied by pH dependence of the coefficient of the antibiotic distribution in the system of amine butyl acetate solution--water. A distribution scheme involving interaction of the amine molecular and cationic forms with benzylpenicillin and amine dissociation equilibrium in organic solution is described. On the basis of the experimental data the interaction and dissociation constants were determined for a number of aromatic and aliphatic amines.

Estimation of the equilibrium distribution in the organic solvent-water system provided analysis of efficiency and optimal arrangement of subsequent extraction-reextraction cycles in isolation of various compounds. The analysis was performed with respect to optimization of the total yield and required volumes of the intermediate phases. It was shown that the best arrangement of the subsequent extraction-reextraction cycles depended on the compound distribution coefficients at various stages.