Dmitri Sergueev

Reading, Writing, and Modulating Genetic Information with Boranophosphate Mimics of Nucleotides, DNA, and RNA

Abstract: The P‐boranophosphates are efficient and near perfect mimics of natural nucleic acids in permitting reading and writing of genetic information with high yield and accuracy. Substitution of a borane (−BH3) group for oxygen in the phosphate ester bond creates an isoelectronic and isosteric mimic of natural nucleotide phosphate esters found in mononucleotides, i.e., AMP and ATP, and in RNA and DNA polynucleotides. Compared to natural nucleic acids, the boranophosphate RNA and DNA analogs demonstrate increased lipophilicity and resistance to endo‐ and exonucleases, yet they retain negative charge and similar spatial geometry. Borane groups can readily be introduced into the NTP and dNTP nucleic acid monomer precursors to produce α‐P‐borano nucleoside triphosphate analogs (e.g., NTPαB and dNTPαB). The NTPαB and dNTPαB are, in fact, good to excellent substrates for RNA and DNA polymerases, respectively, and allow ready enzymatic synthesis of RNA and DNA with P‐boranophosphate linkages. Further, boranophosphate polymer products are good templates for replication, transcription, and gene expression; boronated RNA products are also suitable for reverse transcription to cDNA. Fully substituted boranophosphate DNA can activate the RNase H cleavage of RNA in RNA:DNA hybrids. Moreover, certain dideoxy‐NTPαB analogs appear to be better substrates for viral reverse transcriptases than the regular ddNTPs, and may offer promising prodrug alternatives in antiviral therapy. These properties make boranophosphates promising candidates for diagnostics; aptamer selection; gene therapy; and antiviral, antisense, and RNAi therapeutics. The boranophosphates constitute a versatile family of phosphate mimics for processing genetic information and modulating gene function.

Boranophosphate backbone: a mimic of phosphodiesters, phosphorothioates, and methyl phosphonates.

Nucleoside boranophosphates are distinctive in that one of the non-bridging oxygens in the phosphate diester 1 is replaced by a borane moiety (BH3). Although they retain the same net charge, BH3(-)-ODN have unique chemical and biochemical characteristics relative to other analogs. The change in polarity, lipophilicity, nuclease resistance, and the activation of RNase H cleavage of RNA in RNA: boranophosphate hybrids make boranophosphates very attractive for applications in enzymology and molecular biology and as potential antisense agents.

IN VITRO TRANSPORT AND DELIVERY OF ANTISENSE OLIGONUCLEOTIDES

A variety of techniques are currently available to enhance the cellular uptake and pharmacological effectiveness of antisense oligonucleotides in the in vitro setting. The choice of technique will depend on the context of investigation, the likelihood of cytotoxity due to the delivery agents, and the ease and convenience of the approach. The considerations for the delivery of antisense molecules in the in vivo setting are likely to be quite different from the cell culture situation emphasized in this article.

Boranophosphate Oligonucleotides: New Synthetic Approaches

Abstract Recently our laboratory reported a new backbone-modified class of oligonucleotides, with a borane (B33−) group replacing one of the non-bridging oxygen atoms. Here we present two new approaches to synthesize the boranophosphate oligonucleotides. All-stereoregular boranophosphate oligonucleotides can be prepared by enzymatic template extension reactions using nucleoside a-boranotriphosphates, which are good substrates for a number of polymerases. Larger scale synthesis of boranophosphate oligonucleotides can be carried out by effective chemical synthesis using the H-phosphonate approach, instead of previously used phosphoramidite methodology. The main advantage of H-phosphonate methodology is the ability to carry out one boronation reaction, after oligonucleotide chain elongation has been completed, using mild conditions without base damage and producing the desired boranophosphate oligonucleotides in high yield.

BORANE-AMINE COMPLEXES - VERSATILE REAGENTS IN THE CHEMISTRY OF NUCLEIC ACIDS AND THEIR ANALOGS

A new method for synthesis of N-alkylated nucleosides was developed. Exceptionally mild and selective conversion of N-acyl to the corresponding N-alkyl nucleosides was achieved by reduction with borane-amine complexes. The borane-amine complexes were also used as efficient scavengers of a 4,4′-dimethoxytrityl (DMT) cation. Neutralization of the cation eliminated the boranophosphate group degradation during acidic DMT deprotection and allowed milder acidic conditions for the deprotection.

Synthesis of dithymidine boranophosphates via stereospecific boronation of H-phosphonate diesters and assignment of their configuration

Abstract The absolute configurations of the two dithymidine boranophosphate isomers were established by chemical correlation with the H-phosphonate isomers. All chemical transformation leading from H-phosphonate diesters to boranophosphate diesters were found to be stereospecific with retention of configuration around phosphorus. The data verify our previous assignment of Rp and Sp isomers of dithymidine boranophosphate made on the basis of NMR and enzymatic hydrolysis.

Rapid and Selective Reduction of Amide Group by Borane-Amine Complexes in Acyl Protected Nucleosides

Abstract Borane-amine complexes provide an unusually fast and selective reduction of a deoxynucleoside N-acyl group to a corresponding N-alkyl group. Three different nucleosides (dG, dA, and dC) each having one of three N-protecting groups (benzoyl, isobutyryl, or acetyl) were used to prepare N-alkylated nucleosides in good yields under mild conditions. Deoxyribose O-acyl protecting groups remain intact at the conditions of N-acyl group reduction.

RNase H Activation by Stereoregular Boranophosphate Oligonucleotide

Abstract A stereoregular all-(S p)-boranophosphate oligodeoxyribonucleotide (BH3 −-ODN) 15-mer was synthesized using an enzymatic approach. The BH3 −-ODN formed a hybrid with the complementary RNA 15-mer and induced RNase H hydrolysis of the RNA strand at ODN concentrations as low as 10 nM at 37°C, but with a lower efficiency than that of its natural phosphodiester analogue.

SYNTHESIS OF DIURIDINE 3', 5'-BORANOPHOSPHATE : H-PHOSPHONATE APPROACH

Abstract Diuriine 3′,5′-boranophosphate, the RNA analogue of boranophosphate nucleic acids, was synthesized by a new approach via the H-phosphonate. Two diastereomers of diuridine 3′,5′-boranophosphate were separated by reverse phase HPLC.

BORANOPHOSPHATE NUCLEIC ACIDS : A VERSATILE DNA BACKBONE

Important chemical and biochemical properties of boranophosphate DNA and RNA oligonucleotides are reviewed. Stereoregular boranophosphate oligomers can be synthesized enzymatically and form stable duplexes with DNA. Fully boronated, non-stereoregular oligothymidylates, synthesized chemically, form hybrids with poly(A) that have lower melting points than oligothymidylate:poly(A), yet they nevertheless can support the RNase H mediated cleavage of RNA.