Boris S. Ermolinsky

New syntheses of 2′-C-methylnucleosides starting from d-glucose and d-ribose

Abstract Effective general methods have been developed for the synthesis of 2′-C-methylnucleosides starting from d -glucose and d -ribose. 3-O-benzyl-1,2-O-isopropylidene-3-C-methyl-α- d -allofuranose was prepared in 5 steps from d -glucose and converted into 1,2,3-tri-O-acetyl-2-C-methyl-5-O-p-methylbenzoyl- d -ribofuranose (5), the starting compound for nucleoside synthesis. Compound 5 was also synthesised from 2-C-hydroxymethyl-2,3-O-isopropylidene-5-O-trityl- d -ribofuranose, prepared in 3 steps from d -ribose. Condensation of 5 with the bis-trimethylsilyl derivatives of uracil, N4-benzoylcytosine, and N6-benzoyladenine in the presence of F3CSO3OSiMe3 followed by removal of the protecting acyl groups yielded the corresponding 2′-C-methylnucleosides.

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

Oligonucleotides of a novel type containing 2′‐O‐β‐ribofuranosyl‐cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild‐type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter‐containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein‐nucleic acid interactions.

Substrate properties of C' -methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me‐UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me‐UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force‐field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N‐type conformation.

AFFINITY MODIFICATION OF EcoRII DNA METHYLTRANSFERASE BY THE DIALDEHYDE-SUBSTITUTED DNA DUPLEXES: MAPPING THE ENZYME REGION THAT INTERACTS WITH DNA

ABSTRACT Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly268-Met391 of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII.

Oligodeoxyribonucleosides Containing 1-β-D-Glucopyranosylthymine Synthesis and Substrate Properties

Abstract Regioselective method for 1-β-D-glucopyranosylthymine incorporation into oligonucleotides has been developed and substrate properties of the latters in DNA synthesis and hydrolysis reactions were investigated.

Synthesis and Properties of Novel NTP Derivatives

Abstract Simple method for the preparation of anhydrides of nucleoside-5′-monophosphoric acid and with 1-hydroxyethane-1,1-diylbis(phosphonic acid) has been developed.

[Modified oligonucleotides containing 1-beta-D-galactopyranosylthymine: synthesis and substrate properties].

A convenient method of regioselective introduction of 1-β-D-galactopyranosylthymine into oligonucleotides was developed and the substrate properties of the modified oligonucleotides were investigated in the enzymatic reactions of formation and hydrolysis of internucleotide bonds.

DISACCHARIDE NUCLEOSIDES AND THEIR ENZYMATIC AND CHEMICAL INCORPORATION INTO OLIGONUCLEOTIDES

Abstract A high yield synthesis of different O-ribofuranosylnucleosides has been achieved. Kinetics of the acid-catalysed hydrolysis of disaccharide nucleosides has been studied. Chemical and enzymatic incorporation of 2′-O-ribofuranosyl-nucleoside residue into oligonucleotides was investigated.

Dinucleoside Monophosphates Containing AZT and 1-Methyladenosine or 7-Methylguanosine

Abstract Dinucleoside monophosphates containing AZT and 1-methyladenosine or 7-methylguanosine were synthesized and their in vitro anti-HIV activity was determined.

Oligonucleotides with Reactive Dialdehyde Groups as Novel Affinity Reagents

Abstract The preparation of ON derivatives with regiospecifically incorporated dialdehyde reactive groups and their successful use as affinity labels was described.