Synthesis of Fluorine-Containing Analogues of Purine Deoxynucleosides: Optimization of Enzymatic Transglycosylation Conditions.

In this work, a comparative analysis of the conditions of transglycosylation reactions catalyzed by E. coli nucleoside phosphorylases was carried out, and the optimal conditions for the formation of various nucleosides were determined. Under the optimized conditions of transglycosylation reaction, fluorine-containing derivatives of N6-benzyl-2'-deoxyadenosine, potential inhibitors of replication of enteroviruses in a cell, were obtained starting from the corresponding ribonucleosides.

Efficiency of recombinant thymosin ß4 in spontaneous mouse model of chronic dermatitis.

The therapeutic efficiency of recombinant thymosin ß4 (rTß4) synthesized by us was studied in vivo on spontaneous CBRB mouse model that is adequate to human chronic dermatitis. Three applications of the drug during a week significantly alleviated symptoms of the disease in female mice, and in complex with subsequent antibacterial and antifungal therapy led to a pronounced and lasting (2 months) therapeutic effect. The results attest to a possibility of using rTß4 in combination with the known treatment protocols for chronic inflammatory diseases of the skin.

Mesenchymal stem cells from human dental pulp: isolation, characteristics, and potencies of targeted differentiation.

We studied cell cultures isolated from the pulp of third molar germ of an adult human and from the skin of a human fetus on gestation day 10. Both cultures expressed similar repertoire of surface markers typical of multipotent mesenchymal cells (CD44, CD90, and CD105). Under in vitro conditions, dental pulp cells were more susceptible to factors inducing their differentiation into adipogenic, chondrogenic, and osteogenic lineage cells.

[Biotechnological production of recombinant analogs of hirudin-1 from Hirudo medicinalis].

Hirudin-1 is a highly selective inhibitor of thrombin secreted by salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in blood coagulation system. Apart from the existing recombinant analog of hirudin-1 (63-desulfatohirudin-1, desirudin) its modified analogs possessing higher activity and stability are of medical value. In this study artificial genes of hirudin and two its analogs (hirudin-1, [Leu1, Thr2]-hirudin-1 and [Leu1, Thr2]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein SspDnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogs were developed. The scheme includes the following stages: isolation of the fusion protein after the desintegration of the cell biomass, refolding of the target peptide within the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the obtained peptides was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu1, Thr2]-desulfatohirudin-1 [Leu1, Thr2]-desulfatohirudin-1/3 allowed to obtain these peptides with high yields (14, 25 and 24 mg per liter of cell culture respectively) and high activity (13423, 33333 and 19802 ATU/mg respectively).

[Recombinant fragment of pigment epithelium-derived factor (44-77) prevents pathological corneal neovascularization].

Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.

Favipiravir and Its Structural Analogs: Antiviral Activity and Synthesis Methods.

1,4-Pyrazine-3-carboxamide-based antiviral compounds have been under intensive study for the last 20 years. One of these compounds, favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide, T-705), is approved for use against the influenza infection in a number of countries. Now, favipiravir is being actively used against COVID-19. This review describes the in vivo metabolism of favipiravir, the mechanism of its antiviral activity, clinical findings, toxic properties, and the chemical synthesis routes for its production. We provide data on the synthesis and antiviral activity of structural analogs of favipiravir, including nucleosides and nucleotides based on them.

[Biotechnological production of acetylated thymosin beta4].

Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.

[Cloning, expression, isolation and properties of thymidine kinase herpes simplex virus, strain L2].

A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.

[Recombinant oxyntomodulin].

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.

A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides.

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

[Using of 4-thiouridine and 4-thiothymidine for pyrimidine nucleoside phosphorylase studing]. / Ispol'zovanie 4-tiouridina i 4-tiotimidina dlia izucheniia pirimidinnukleozidfosforilaz.

4-Thiouridine and 4-thiothymidine were developed as efficient substrates for spectrophotometric determination of uridine phosphorylase and thymidine phosphorylase activity. 4-Thiouridine has maximum absorbance at 330 nm (pH 7.5). The change in extinction coefficient for 4-thiouridine/4-thiouracil, deltaepsilon is 3000 M(-1) x cm(-1). It appeared that 4-thiouridine is a good substrate for uridine phosphorylase with Michaelis-Menten constant 130 microM and kcat 49 s(-1). In the case of 4-thiothymidine/4-thiothymine deltaepsilon is even larger: 5000 M(-1) x cm(-1) at 336 nm.

[Dependence of the level of gene expression in E. coli on the structure of the translation initiation segment (TIR). I. Primary structure of TIR]. / Zavisimost' urovnia ékspressii gena v E. coli ot struktury uchastka initsiiatsii transliatsii (TIR). I. Pervichnaia struktura TIR.

The comparison of expression levels of two genes-interleukin-3 (il3) and epidermal growth factor connected to leader peptide of OmpF (lompegf)-was carried out using specially constructed plasmids contained various structures of translational enhancers. It was shown that besides already known binding sites from mRNA translation initiation region (TIR) to 16S rRNA (SD, UB1 and DB), there is additional binding site of TIR disposed from -30 to -60 nt upstream of start codon AUG (UB2) having significant increasing effect on translation initiation and correspondingly on expression level.

[Dependence of the level of gene expression in E. coli on the structure of the translation initiation segment (TIR)]. / Zavisimost' urovnia ékspressii gena v E. coli ot struktury uchastka initsiatsii transliatsii (TIR).

The expression levels of genes that are transcribed to give mRNAs with identical leader sequences and even with identical extended coding regions may differ considerably. In order to determine the mechanism of this phenomenon, secondary structures of some mRNAs synthesized from a series of expression plasmids were studied. It was shown that the effect of the mRNA secondary structure in the translation initiation region on the initiation efficiency is due not only to the hairpin formation in this region but also to long-range interactions. When complementary structures tighter than those resulted from the interaction of regions SD, UB1, UB2, and DB with 16S rRNA are formed, the efficiency of the translation initiation and, consequently, the expression level decrease.

[Construction of artificial genes by PCR methods using the synthetic template]. / Poluchenie nekotorykh iskusstvennykh genov metodom PTsR na sintetiche sko'i matritse.

Artificial genes were synthesized by the PCR method. Single-stranded DNA contained in an unpurified mixture of oligodeoxynucleotides after automated synthesis was used as a template. The features of this approach were studied.

[The dependence of the E.coli gene expression level on the structure of the translation initiation region (TIR). IV. Distal complementary TIR interactions with the mRNA coding region]. / Zavisimost' urovnia ékspressii gena v E.coli ot struktury uchastka initsiatsii transliatsii (TIR). IV. Dal'nie komplementarnye vzaimodeistviia TIR s kodiruiushchei chast'iu mRNK.

To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA. A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation. The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.

[Relation between the level of E. coli gene expression and the structure of translation initiation region (TIR). III. Sites of complementary interaction of TIR with 16S rRNA]. / Zavisimost' urovnia ékspressii gena v E.coli ot struktury uchastka initsiatsii transliatsii (TIR) III. Saity komplementarnogo vzaimodeistviia TIR s 16S rRNK.

Potential sites of the complementary interaction of the translation initiation region (TIR) with 16S rRNA are revealed, and the role of these sites in the gene expression level is studied. The high expression level of a gene depends not only on the complementary interaction of TIR with 16S rRNA in sites proximal to the start codon [anti-Shine-Dalgarno (ASD) (delta G > -8 to -10 kcal/mol) and downstream box (DB)] and located at the -15 to +20 mRNA region but also on complementary interactions in distal sites of the untranslated branch of TIR (mTIR). Among them, the UB (upsteam box) 1 site, complementarily interacting with the exposed 452-490 segment of the 440-490 loop of 16S rRNA, may be located in the -15 to -50 mTIR segment. In the -50 to -70 mTIR segment may be located UB2 and UB3 sites, which interact with the exposed segment 478-488 of the 440-490 loop and segment 520-532 of the 520-540 loop of 16S rRNA, the UB3 site being much more efficacious. The high expression level requires that the total free energy of complementary interactions of UB1, UB2, and UB3 sites with 16S rRNA exceeds -20 kcal/mol.

[Recombinant thymosin alpha1]. / Rekombinantnyi timozin alpha1.

An artificial gene encoding thymosin alpha1 was obtained by the chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin alpha1 and the Saccharomyces cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin alpha1 were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

[Chemical and chemico-enzymatic synthesis of alpha-thiotriphosphate nucleosides]. / Khimicheskii i khimiko-fermentativnyi sintez alpha-tiotrifosfatov nukleozidov.

New methods of chemical and chemoenzymatic synthesis of nucleoside 5'-thiophosphates and 5'-alpha-thiotriphosphates are developed. The 5'-alpha-thiotriphosphates are used as substrates both in template-dependent enzymatic PCR synthesis and in a T7-RNA transcription polymerase system. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Mechanism of action of the plant hormone jasmonate. 1. Jasomonate-interacting proteins that regulate transcription of the p. pinII potato gene]. / Mekhanizm deistviia rastitel'nogo gormona--zhasmonata. 1. Belki-reguliatory transkriptsii gena p. pinII kartofelia, vzaimodeistviushchie s zhasmonatom.

A fragment containing the regulatory region of the p. pinII gene was isolated from potato DNA by polymerase chain reaction. Interactions of this DNA region with jasmonate determines the transcriptional activation. The isolated DNA fragment was cloned into the pTE2pb plasmid, which was used for preparing an affinity sorbent. Using this sorbent, four proteins were isolated from the total protein capable of desorption at physiological concentration of jasmonate. These proteins are likely to be subunits of two transcription repressors, whereas jasmonate serves as an inducer. Three sequences of the regulatory regions (boxes G, I, and III) are binding sites for repressors; similar sequences were found in various plant genes activated by jasmonate.

[Methods of preparation of recombinant proteins-cytokines. IV. Renaturation of recombinant human interleukin-3]. / Metody polucheniia recombinantnykh belkov-tsitokinov IV. renaturatsiia rekombinantnogo chelovecheskogo interleikina-3.

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for denaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 x 10(7) U/mg.