4757 3.4. Separation and Spectral Properties of Diastereomers of Borane-Containing Cyclic Monophosphates 4758 4. Nucleoside Boranodiphosphates 4758 4.1. Synthesis of Nucleoside 5′-(R-P-Borano)diphosphates 4.1.1. Synthesis via a Boranophosphoramidate Approach 4.1.2. Synthesis via an Oxathiaphospholane Approach 4.1.3. Synthesis via a Phosphite Approach 4.2. Separation and Configuration Determination of the Diastereomers of NDPRB 4.2.1. Separation of the Diastereomers of NDPRB 4.2.2. Configuration Determination of the Diastereomers of NDPRB 4.3. Substrate Properties of NDPRB Analogues with NDPK, Pyruvate Kinase, and Creatine Kinase 4.3.1. Substrate Properties of NDPRB Analogues with NDPK 4.3.2. Binding Affinity of NDPRB Analogues with Pyruvate Kinase (PK) and Creatine Kinase (CK) 4.4. Synthesis of Nucleoside 5′-(β-P-Borano)diphosphates 5. Nucleoside Boranotriphosphates 5.1. Synthesis of Nucleoside 5′-(R-P-Borano)triphosphates 5.1.1. Synthesis via a Phosphoramidite Approach 5.1.2. Synthesis via a Phosphite Approach 5.2. Separation and Configuration Determination of the Diastereomers of NTPRB 5.2.1. Separation of the Diastereomers of NTPRB 5.2.2. Configuration Determination of the Diastereomers of NTPRB 5.3. Substrate Properties of NTPRB Analogues with Viral RTs and PK 5.3.1. Substrate Properties of dNTPRB Analogues with Viral RTs and DNA Polymerases 5.3.2. Substrate Properties of ddNTPRB Analogues with Viral RTs and DNA Polymerases 5.3.3. Binding Affinity of NTPRB Analogues with Rabbit Muscle PK 5.4. Synthesis of Borane-Containing Novel Triphosphate Analogues 5.4.1. Synthesis of β-P-BH 3 -and γ-P-BH 3 -Modified Triphosphates and Diboranotriphosphates

In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.

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 alpha-P-borano nucleoside triphosphate analogs (e.g., NTPalphaB and dNTPalphaB). The NTPalphaB and dNTPalphaB 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-NTPalphaB 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.

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.

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