New trends in nucleoside biotechnology.

This review focuses on new trends in nucleoside biotechnology, which have emerged during the last decade. Continuously growing interest in the study of this class of compounds is fueled by a number of factors: ( i ) a growing need for large-scale production of natural 2 ' -deoxy- ß -D-ribonucleosides as well as their analogs with modifications in the carbohydrate and base fragments, which can then be used for the synthesis and study of oligonucleotides, including short-interfering RNA (siRNA), microRNA (miRNA), etc.; ( ii ) a necessity for the development of efficient practical technologies for the production of biologically important analogs of natural nucleosides, including a number of anticancer and antiviral drugs; ( iii ) a need for further study of known and novel enzymatic transformations and their use as tools for the efficient synthesis of new nucloside analogs and derivates with biomedical potential. This article will review all of these aspects and also include a brief retrospect of this field of research.

[Antiviral activity of 2'-deoxy-2'-fluoroguanosine against influenza and herpes simplex viruses in cultured cells]. / Protivovirusnaia aktivnost' 2'-dezoksi-2'-ftorguanozina v otnoshenii virusov grippa i prostogo gerpesa v kul'turakh kletok.

High antiviral activity of 2'-deoxy-2'fluoroguanosine (2'-D-2'-FG) was observed in chicken embryo cells infected with FPV/Rostock/34 (H7N1) influenza virus and herpes simplex virus (HSV) type I (strain 1C). 50% inhibitory concentration (IC50) of 2'-D-2'-FG was 1.44 microM for FPV and 0.093 microM for HSV. IC50 of remantadine hydrochloride, ribavirin (FPV), and acycloguanosine (HSV) were < 0.46, 14.4, and 0.08 microM, respectively. Anti-HSV activity was confirmed in Vero cells: IC50 < 0.34 microM for 2'-D-2'-FG and IC50 < 0.044 microM for acycloguanosine.

Pyrophosphoryl derivatives of 1-(2-deoxy-3-O-phosphono-methyl-beta- and -alpha-D-erythro-pentofuranosyl)thymine: synthesis and substrate properties towards some DNA polymerases.

The synthesis of 1-(2-deoxy-3-O-phosphonomethyl-beta-D-erythropentofuranosyl)thymine (17) and its alpha-anomer 18 is described. Attempts to prepare 1-[2-deoxy-3-O-(pyrophosphoryl)phosphonomethyl-beta-D-erythro-pentofuranosyl]thymine (19) by an activation of the respective phosphonate 17 with 1,1'-carbonyldiimidazole (Im2CO) resulted in the quantitative formation of the corresponding pyrophosphonate derivative 21 (Scheme 2). Activation of inorganic pyrophosphate with Im2CO followed by the condensation with the phosphonates 17 and 18 afforded the desired analogues of nucleoside triphosphate 19 (35%) and its alpha-anomer 20 (27%) along with the respective pyrophosphonate derivatives 21 (37%) and 24 (38%) (Scheme 3). It was found that compounds 19 and 20 display (i) no substrate properties toward calf thymus terminal deoxynucleotidyl transferase (TDT) and AMV reverse transcriptase, and (ii) moderate substrate activity with E. coli DNA polymerase I (Klenow fragment).

Oligonucleotides containing 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine and -guanine: synthesis, hybridization and antisense properties.

Synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (7, ara-A2'F) and -guanine (12, ara-G2'F) was accomplished via the condensation of 2,6-dichloropurine (1) with 2-deoxy-2-fluoro-1,3,5-tri-O-benzoyl-alpha-D-arabinofuranose (2) as a key chemical step. Condensation of silylated N6-benzoyladenine (6) with 2 gave, after deblocking and chromatographic separation, ara-A2'F (7) (14%), it's alpha-anomer 8 (14%) and N7-alpha-isomer 9 (25%). The PSEUROT analysis of N9-betaD-arabinosides 7 and 12 manifested slight preference for the S rotamer (64%) for the former, and an equal population of the N and S rotamers for the latter. The arabinosides 7 and 12 were used for the preparation of the respective phosphoamidite building blocks 13 and 14 for automated oligonucleotide synthesis. Four 15-mer oligonucleotides (ONs) complementary to the initiation codon region of firefly luciferase mRNA were prepared: unmodified 2'-deoxy-ON (AS 1) and containing (i) ara-A2'F instead of the only A (AS2), (ii) ara-G2'F vs. 3-G from the 5'-terminus (AS3), and (iii) both arabinosides at the same positions (AS4). All these ONs display practically the same (i) affinity to both complementary DNA and RNA, and (ii) ability to inhibit a luciferase gene expression in a cell-free transcription-translation system.

Synthesis and biological activity of 2',5'-oligoadenylate trimers containing 5'-terminal 5'-amino-5'-deoxy- and 5'-amino-3',5'-dideoxyadenosine derivatives.

Some new 2',5'-oligonucleotide trimers containing 5'-terminal 5'-amino-5'-deoxy- and 5'-amino-3',5'-dideoxyadenosine derivatives have been synthesized. Some of the trimers showed biological inhibitions of HIV-1 reverse transcriptase (RT), HIV-1 induced syncytia formation and PCR amplification.

Chemical and enzymatic synthesis and antiviral properties of 2'-deoxy-2'-fluoroguanosine.

Chemical and enzymatic methods were employed for the synthesis of the title compound, 2'F-Guo 7. High antiviral activity of 2'F-Guo was established in chick embryo cells infected with influenza virus FPV/Rostock/34 (H7N1) and herpes simplex virus (HSV) type I (1C strain).

[3'-Deoxy-3'-fluoro analogs of core trimers of 2-5A: effect on lytic activity of human NK-lymphocytes]. / 3'-Dezoksi-3'-ftoranalogi korovykh trimerov 2-5A: vliianie na liticheskuiu aktivnost' NK-limfotsitov cheloveka.

The effect of core trimers, (2'-5')-analogues of oligoadenylic acid containing 9-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)adenine (AF) and 3'-deoxy-3'-fluoroadenosine (AF) in various positions of the oligomer chain, on the lytic activity of human natural killer cells (NK cells) was studied in three different ways. The cellular cytotoxicity was determined using a highly sensitive nonradioactive approach employing a chelate europium-diethylenetriamino-pentaacetic acid complex (Eu-DTPA). It was shown that all fluorodeoxyanalogues enhance the lytic activity of intact NK lymphocytes, which follows from the lysis rate constant k2. At the same time, the substitution of either the central adenosine fragment or (to a greater extent) the 5'-terminal residue of (2'-5')A3 with AF causes a decrease in the number of active NK cells, which, unlike the case of the natural core trimer, leads to a loss of the capacity to increase the activity of NK. By contrast, isomeric ribo-analogues. (2'-5')(AF)A2 and (2'-5')A(AF)A, and trimers with the 2'(3')-terminal nucleotide substituted by AF or AF increased the activity of NK cells with an effectiveness close to or higher than the natural trimer (2'-5')A3. Inasmuch as isomeric xylo- and ribo-3'-deoxy-3'-fluoroanalogues of (2'-5')A3 are stereochemically modified oligomers, the data unambiguously suggest that the spatial structure of these trimers affects the increase in the lytic activity of NK cells.

[Dependence of properties of polyclonal antibodies to (2'-5')-oligoadenylates on the method of hapten binding to an immunogen]. / Zavisimost' svoistv poliklonal'nykh antitel k (2'-5') oligoadenilatam ot sposoba prisoedineniia gaptena k immunogenu.

To elucidate the antibody-(2'-5')oligoadenylate relation to the mode of the hapten-immunogen conjugation, a new (2'-5')oligoadenylic acid trimer derivative containing a 2'-terminal N6-(5-carboxypentyl)adenosine and its 125I-labeled immunogenic conjugate were synthesized. The immunization with this conjugate and with a conjugate based on the 2',3'-O-[1-(2-carboxyethyl)]ethylidene derivative of the (2'-5')triadenylic acid gave antisera with different affinities toward modified (2'-5')oligonucleotides. Epitopes involved in the (2'-5')oligomer-binding to different antisera were found.

[Interaction of 3'-fluoro-3'-deoxyanalogs of a trimer of (2'-5')oligoadenylic acid with (2'-5')A3-antibodies: stereochemical aspect]. / Vzaimodeistvie 3'-ftor-3'-dezoksianalogov trimera (2'-5')oligoadenilovoi kisloty s (2'-5')A3-antitelami: stereokhimicheskii aspekt.

Mouse antibodies to (2'-5')oligoadenylates were obtained by the immunization of animals with the (2'-5')oligoadenylic acid trimer conjugated with bovine serum albumin through a 2',3'-levulinic acid residue. Using radioimmunoassay, the reactivity of mouse polyclonal antibodies to the (2'-5')oligoadenylic acid trimer was studied for the trimer analogues containing 9-(3-deoxy-3-fluro-beta-D- xylofuranosyl)adenine and 3'-deoxy-3'-fluoro-adenosine in various positions of the chain. It was found that (a) the three-dimensional structure of short oligonucleotides is an important factor in the antibody recognition; (b) antibodies are more sensitive to modifications of the 5'-terminal and central ribose fragments of the (2'-5')oligoadenylic acid trimer; (c) the 3'-hydroxyl group plays a secondary role in the formation of the antigen determinant.

Unusual nucleoside triphosphate donors for nucleoside kinases: 3'-deoxyadenosine-2'-triphosphate and 2'-deoxyadenosine-3'-triphosphate.

Two non-conventional analogues of ATP, 3'-deoxyadenosine-2'-triphosphate (3'-d-2'-ATP) and 2'-deoxyadenosine-3'-triphosphate (2'-d-3'-ATP), the syntheses of which are described, were examined as potential phosphate donors for the nucleoside kinases: 2'-deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2). The reactions were monitored by means of a mixture of [gamma-32P]ATP and cold analogue, and/or with the use of 3H-labelled acceptors and cold donor. With dCK, using equimolar mixtures of ATP with each analogue, and dC as acceptor, phosphate transfer from 3'-d-2'-ATP and 2'-d-3'-ATP amounted to 34% and 14%, respectively. With each analogue used alone (each at concentration of 100 microM), phosphate transfer from 3'-d-2'-ATP was 55% that from ATP, and from 2'-d-3'-ATP 16%. With human TK2, and equimolar mixtures of [gamma-32P]ATP with each of the analogues, and 1 microM dT as acceptor, there was no detectable transfer from either analogue. But, when each analogue was used alone, phosphate transfer attained 11% and 5%, respectively, that for ATP alone. With the low affinity form of human TK1, and dT as acceptor, only low phosphate transfer occurred with either analogue used alone. Both compounds exhibited Michaelis-Menten kinetics (with significantly lower Vmax than ATP), while ATP exhibited cooperative kinetics with all three kinases.

Dissection of the roles of adenine ring nitrogen (N-1) and exocyclic amino (N-6) moieties in the interaction of 2-5A with RNase L.

To elucidate further the roles played by the adenine bases in the interaction of RNase L (EC 3.1.2.6) with the 2',5'-oligoadenylate 2-5A, p5'A2'(p5'A2')np5' A, a series of sequence-specific 1-deazaadenosine (c1A)-substituted analogues were synthesized and evaluated for their ability to bind to and activate human RNase L in comparison to earlier reported inosine-substituted congeners of 2-5A. Substitution of only the 5'-terminal adenosine of p5'A2'p5'A2 p5 A with c1A afforded an analogue with strongly diminished RNase L binding and activation ability, while replacement of the second or middle adenosine of p5 A2' p5'A2'p5' A had only a modest effect. In distinct contrast to p5'A2'p5'A2'p5'I, the c1A analogue with the third or 2'-terminal adenosine replacement approached parent p5' A2'p5'A2'p5' A in RNase L activation ability. These results permitted a further dissection of the role of various nucleotidic functional groups in the interaction of 2-5A with RNase L: specifically, that the 5'-terminal adenosine purine N-1 moiety is key for binding to RNase L, while the 2'-terminal adenosine N-6 exocyclic amino group is critical for RNase L activation.

Antiviral effect of trimeric 2',5'-oligoadenylic acid and some of its analogues.

The antiviral effect of 2',5'-trioligoadenylate (2',5'-A3) and some of its analogues was studied using several model cell culture systems and viruses: mice L929 fibroblast cells inoculated with vaccine virus, testicular piglet cells inoculated with Aueszki disease virus (strain BUK-628), and the same culture inoculated with a reference strain of transmissible gastroenteritis virus, strain Purdue-115. Our results suggest that both 2',5'-trioligoadenylate and its analogues are promising antiviral substances against DNA- and RNA-containing viruses.

The effect of trimeric 2',5'-oligoadenylic acid and its epoxy-derivative on human immunodeficiency virus (HIV-1) reproduction and retroviruses reverse transcriptase activity.

The effect of trimeric 2',5'-oligoadenylic acid (2',5' A3) and its epoxy-derivative (2',5'-A2 (RAA) on human immunodeficiency virus (HIV-1) reproduction was studied. An HIV-1 infectivity titer decrease was shown on the model of limphoblastoid cells when the substances under study were used. The interferonogenic effect of both substances was discovered. 2',5'-A2 (RAA) inhibited the activity of retrovirus reverse transcriptase (a C-type).

Synthesis and antiviral activity of 3'-C-branched-3'-deoxy analogues of adenosine.

The synthesis of some 3'-C-branched-3'-deoxy adenine nucleosides is described. Starting from the known 3-deoxy-3-C-(hydroxymethyl)-1,2;5,6-di-O-isopropylidene-alpha-D-allof uranose (1), a versatile glycosylating agent, namely 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-C-(mesyloxymethyl)-beta-D-ri bofuranose (6), was prepared in three steps. Condensation of the latter with bis(trimethysilylated) N6-benzoyladenine in the presence of tin(IV) chloride gave the N9-beta-nucleoside 7. Compound 7 was converted into (i) 9-[3-C-(azidomethyl)-3-deoxy-beta-D-ribofuranosyl]adenine (10), (ii) 9-[3-C-(azidomethyl)-2,3-dideoxy-beta-D-glycero-pent-2-enofuran osyl]adenine (14) and 9-[2-azido-3-C-(azidomethyl)-2,3-dideoxy-beta-D-arabinofuranosyl]a denine (15), and (iii) 9-[3-deoxy-2-O,3-C-(methylene)-beta-D-ribofuranosyl]adenine (16). None of the tested nucleosides showed marked cytostatic or antiviral activity in vitro.

Conformation analysis of 3'-fluorinated A(2'-5')A(2'-5')A fragments. Relation between conformation and biological activity.

A one- and two-dimensional NMR study has been performed on seven A(2'-5')A(2'-5')A fragments containing 9-(3'-fluoro-3'-deoxy-beta-D-xylofuranosyl)-adenine (AF) or 3'-fluoro-3'-deoxyadenosine (AF) residues at different positions, and on the corresponding monomers. A(2'-5')A(2'-5')A served as a reference compound. The fluoro substituent governs the conformation of the sugar ring: an AF residue displays mainly N-type sugar and the ring is considerably flattened (phi N approximately 30 degrees) compared to AF residues (phi S approximately 40 degrees), which exhibit almost pure S-type conformation. Moreover, in AF moieties the rotamer distribution around torsion angle gamma (O5'-C5'-C4'-C3') and the base orientation are influenced to a large extent by the presence of the fluorine substituent. The sugar rings of nonfluorinated residues in the trimers appear rather flexible. A possible correlation between the conformational characteristics of the fluorinated fragments and their biological activity has been found: the fragments that meet the prerequisites for binding to RNase L indeed show enhanced binding to this endonuclease. Furthermore, substitution of the 3'-OH group of the second residue by hydrogen or of the 3'-OH group of the 2'-terminal residue by fluorine or hydrogen results in increased resistance towards 2'-5'-phosphodiesterase.

Synthesis and antiviral and cytostatic properties of 3'-deoxy-3'-fluoro- and 2'-azido-3'-fluoro-2',3'-dideoxy-D-ribofuranosides of natural heterocyclic bases.

A series of 3'-deoxy-3'-fluoro- and 2'-azido-2',3'-dideoxy-3'-fluoro-D-ribofuranosides of natural heterocyclic bases have been synthesized with the use of universal carbohydrate precursors, viz., 1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-3-fluoro-D-ribofuranose and methyl 2-azido-5-O-benzoyl-2,3-dideoxy-3-fluoro-beta-D-ribofuranoside, respectively. The cytostatic and antiviral activity of the compounds was evaluated against a variety of tumor cell lines and DNA/RNA viruses, respectively. As the most active compound, from both a cytostatic and antiviral activity viewpoint, emerged 3'-deoxy-3'-fluoroadenosine. It inhibited the proliferation of some tumor cell lines (i.e. murine leukemia L1210 and human T-lymphocyte MT-4) at a concentration of 0.2-2 micrograms/mL, and proved inhibitory to the replication of positive-stranded RNA viruses (i.e. polio, Coxsackie, Sindbis, Semliki forest), double-stranded RNA viruses (i.e. reo), and some DNA viruses (i.e. vaccinia) at a concentration of 1-4 micrograms/mL, which is well below the cytotoxicity threshold (40 micrograms/mL).

Specificities of rabbit anti-(2',5')oligoadenylate antibodies towards phosphorothioate and seco analogs of oligoadenylates.

Rabbit antibodies to 2',5'-linked triadenylate were prepared by immunization with (2',5')A3 conjugated via the 2'3'-levulinic group, (2'5')A3-Lev, to BSA. New radioimmunoassay for (2',5')oligoadenylates was developed using 125I thyrosine labeled derivative of (2',5')A3-Lev. Reactivity of antibodies with phosphorothioate and seco analogs of oligoadenylates was studied. It was found that (i) stereospecific substitution of the diastereotopic oxygens with sulfur in the internucleotide phosphodiester linkages changes the immunoreactivity of such analogs; (ii) the seco analogs of oligoadenylates display in some cases a rather high reactivity.

Synthesis of 9-(beta-D-arabinofuranosyl)guanine using whole cells of Escherichia coli.

Synthesis of 9-(beta-D-arabinofuranosyl)guanine (ara-G) from 1-(beta-D-arabinofuranosyl)cytosine (ara-C) and guanine, guanosine or 2'-deoxyguanosine (dG) by glutaraldehyde-treated Escherichia coli BM-11 cells is described. It is shown that the concentration of phosphate ions, molar ratio of substrates and pH of the reaction medium are factors affecting product yield. Under optimum conditions ara-G was produced in the reaction mixture in a yield of 63%-65% based on dG as the best source of guanine base. The yield of isolated ara-G was 48%-53%.

3'-Fluoro-3'-deoxy analogs of 2-5A 5'-monophosphate: binding to 2-5A-dependent endoribonuclease and susceptibility to (2'-5')phosphodiesterase degradation.

Analogs of 2-5A trimer 5'-monophosphate (2'-5')pA3,p5'A2'p5'A2'p5'A containing 9-(3-fluoro-3-deoxy-c-D-xylofuranosyl)adenine (AF) or 3'-fluoro-3'- deoxyadenosine (AF) at different positions of the chain have been synthesized. All of them were compared with (2'-5')pA3 and (2'-5')pA2 (3'dA) by (i) their ability to bind to 2-5A-dependent endoribonuclease(RNase L) of mouse L cells and of rabbit reticulocyte lysates and (ii) their susceptibility to the degradation by the (2'-5')phosphodiesterase activity. The results of this study suggest that the oligonucleotide conformation is important for its biochemical properties.