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 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 penicillin acids and cephalosporin acids 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 review describes two major groups of α-amino acid ester hydrolases (AEHs)—enzymes with a similar active center structure, which determines their unique specificity to esters containing an amino group in the α position to the carbonyl. The first group comprises microbial AEHs of the β-lactam acylase type. Technical biocatalysts based on this group of enzymes are used for the production of semi-synthetic amino-β-lactam antibiotics. The second AEH group includes eukaryotic valacyclovirases, which activate in vivo a number of antiviral and anticancer prodrugs. The directed activity of these enzymes is used for the development of target pharmaceutical preparations for the therapy of viral and oncological diseases. The review summarizes and compares the available data on the structure and properties, substrate specificity, and the kinetic parameters of enzymes of these two groups. Experiments identifying the AEH active site and providing the molecular basis for the unique specificity of these enzymes are discussed. The data from the available scientific and patent publications concerning the aminopenicillin and aminocephalosporin synthesis catalyzed by β-lactam acylase AEHs are reviewed and systematized.

In order to use hydroxyapatite and other biocompatible calcium phosphates as carriers for local prolonged-release drug, it is necessary to have an active component capable of retaining the preparation deposited onto the carrier surface for a long time (at least, for a few days). The grafting of the layer of active functional groups onto the carrier surface is employed to affect kinetic adsorption-desorption characteristics of calcium phosphates. The method of chemical modification of biocompatible calcium phosphates that uses B(OC4H9)3, SiCl4, POCl3, PCl5, and SnCl4 is developed. The effect of the modification of calcium phosphate’s surface on the kinetics of hydrolytic desorption of octadecylamine and tetraethylenepentamine modeling the hydrophobic and hydrophilic drugs, respectively, as well as gentamicin, an antibiotic that is widely used in clinical practice to prevent and cure inflammation processes upon the damage of bony tissue, is studied. It is shown that the rate of desorption of these substances from calcium phosphates into the aqueous phase is significantly retarded when the carrier surface is preliminarily modified. By means of ESR and IR Fourier spectroscopy, the interaction of octadecylamine and tetraethylenepentamine with calcium phosphate’s surface is investigated. A conclusion about the existence of two mechanisms of adsorption of amines on modified calcium phosphates is drawn. The procedure for the preparation of biocompatible calcium phosphates with hemisorbed gentamicin is developed.

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.

An optimal way to design an enzymatic process for the production of betalactam antibiotics based on thermodynamic and kinetic studies is described. The study was performed on model reactions involving synthesis of cephalosporin-acids (cephalotin, cefazolin, cefoxitin) using immobilised cephalosporin-acid synthetase from Escherichia coli as biocatalyst, and aminocephalosporins (cephalexin) using immobilised cells of Xanthomonas rubrilineans containing the aminocephalosporin synthetase. The possibility of direct synthesis of cephalotin and cefoxitin was shown, the main equilibrium parameters were determined and the operation conditions were evaluated. The maximum key amino acid conversion to product of approximately 90% for cefoxitin and cephalotin was achieved using initial concentrations of the corresponding key amino acids of 0.05 u M and, respectively, 2-fold and 4-fold molar excess of the carboxylic acids. Cefazolin and cephalexin production by enzymatic synthesis with using of corresponding biocatalyst with a mechanism of action involving the acylenzyme intermediate was shown possible. The kinetic parameters of the process were estimated and the relationship between the maximum antibiotic yield and the initial concentrations of the substrate and nucleophile in the kinetically controlled synthesis was determined. The technologies for cefazolin and cephalexin enzymatic synthesis were designed and the cefazolin technology was optimised. Maximum yields of cefazolin and cephalexin of more than 90% were predicted by the kinetic model using 4-6-fold molar excess of the acylating agents and maximum yields of approximately 85% were achieved in experiments.

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.

The methodology for the discovery of new biologically active betalactams is proposed. The one of the two ways proposed is specific modification, which is peculiar to any betalactam structure and involves introduction of substitutes changing particular physico-chemical properties of the natural or synthetic analogous. The principle of similarity is discussed as an alternative of the specific modification in the design of new biologically active compounds. The distinctive feature of this approach is usage as building blocks substitutes favourable for the well-known in medical practice betalactam antibiotics and their “implantation” into other (new) betalactam structures. The paths of new betalactam synthesis, including the methods of enzyme engineering are considered. The possibility of usage of enzyme engineering processes for production of not only new individual betalactams— hits, but also for synthesis of the complex betalactams—leads, is shown. More than 4500 new penicillins, cephalosporins and monobactams are constructed with accordance of the principle of similarity. More than halves of them can be produced by enzymatic synthesis or combination of chemical and enzymatic synthesis. The constructed compounds are enumerated in the tables, the request for the electronic version of which can be sent to the address: davidnys@writeme.com.

in the design of new biologically active compounds. The distinctive feature of this approach is usage as building blocks substitutes favourable for the well-known in medical practice betalactam antibiotics and their \implantation" into other (new) betalactam structures. The paths of new betalactam synthesis, including the methods of enzyme engineering are considered. The possibility of usage of enzyme engineering processes for production of not only new individual betalactams| hits, but also for synthesis of the complex betalactams|leads, is shown. More than 4500 new penicillins, cephalosporins and monobactams are constructed with accordance of the principle of similarity. More than halves of them can be produced by enzymatic synthesis or combination of chemical and enzymatic synthesis. The constructed compounds are enumerated in the tables, the request for the electronic version of which can be sent to the address: davidnys@writeme.com.

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.

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.

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.