Kaizhang He

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.

A convenient synthesis of 2′-deoxyribonucleoside 5′-(α-P-borano)triphosphates

Abstract A new class of nucleotides, with one of the two non-bridging oxygens at α-phosphorus replaced by a borane (BH 3 ) group, has shown potential applications in DNA sequencing. We have developed a convenient method for the synthesis of the 2′-deoxy-5′-(α-P-borano)triphosphates of adenosine, guanosine, cytidine, thymidine and uridine ( 6a-e ) that is time and cost effective compared to the previously reported method. The appropriate base/sugar protected nucleoside 1 is phosphitylated with 2-chloro-4 H -1,3,2-benzodioxaphosphorin-4-one to give the corresponding cyclic intermediate, 2-(2′-deoxyribonucleosidyl-5′- O -)-4 H -1,3,2-benzodioxaphosphorin-4-one ( 2 ). The in situ reaction of 2 with pyrophosphate leads to P 2 , P 3 -dioxo- P 1 -(2′-deoxyribonucleosidyl-5′-)cyclotriphosphite ( 3 ). Subsequent reaction of 3 with N , N -diisopropylethylamine-borane complex yields P 1 -borano-(2′-deoxyribonucleosidyl-5′)cyclotriphosphate ( 4 ), which upon hydrolysis under mild conditions gives the base/sugar protected 2′-deoxyribonucleoside-5′-( α - P -borano)triphosphate ( 5 ). Treatment of 5 with ammonia:methanol (2:1 v/v) yields the diastereoisomeric mixture of 2′-deoxyribonucleoside-5′-( α - P -borano)triphosphate ( 6 ). Separation of the two diastereoisomers of 6 is performed by reverse phase HPLC.

Synthesis of Boron-Containing ADP and GDP Analogues: Nucleoside 5′-(P-Boranodiphosphates)

New 5′-(Pα-boronated) analogues of the naturally occurring nucleoside diphosphates ADP and GDP were synthesized in good yields, i.e., adenosine 5′-(Pα-boranodiphosphate) (ADPαB; 5a) and guanosine 5′-(Pα-boranodiphosphate) (GDPαB; 5b). Their diastereoisomers were successfully separated by reversed-phase HPLC, and chemical structures were established via spectroscopic methods. The isoelectronic substitution of borane (BH3) for one of the non-bridging O-atoms in phosphate diesters should impart an increase in lipophilicity and change in polarity in ADPαB and GDPαB. The boronated nucleoside diphosphates could be employed for investigations of the stereochemical course and metal requirements of enzymatic reactions involving ADP and GDP, and as carriers of 10B in boron neutron-capture therapy (BNCT) for the treatment of cancer.

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.