Inhibition of hepatitis C viral RNA-dependent RNA polymerase by α-P-boranophosphate nucleotides: exploring a potential strategy for mechanism-based HCV drug design.

Improved treatments for chronic HCV infections remain a challenge, and new chemical strategies are needed to expand the current paradigm. The HCV RNA polymerase (RdR(P)) has been a target for antiviral development. For the first time we show that the boranophosphate (BP) modification increases the substrate efficiency of ATP analogs into HCV NS5BΔ55 RdRP-catalyzed RNA. Boranophosphate nucleotides contain a borane (BH3) group substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics capable of modulating the reading and writing of genetic information. We determine that HCV NS5BΔ55, being a stereospecific enzyme, incorporates the Rp isomer of both ATPαB and the two boranophosphate analogs: 2'-O-methyladenosine 5'-(α-P-borano) triphosphate (2'-OMe ATPαB, 5a) and 3'-deoxyadenosine 5'-(α-P-borano) triphosphate (3'-dATPαB, 5b). The R(p) diastereomer of ATPαB (6), having no ribose modifications, was found to be a slightly better substrate than natural ATP, showing a 42% decrease in the apparent Michaelis-Menten constant (K(m)). The IC50 of both 2'-O-Me and 3'-deoxy ATP was decreased with the boranophosphate modification up to 16-fold. This "borano effect" was further confirmed by determining the steady-state inhibitory constant (K(i)), showing a comparable potency shift (21-fold). These experiments also indicate that the boranophosphate analogs 5a and 5b inhibit HCV NS5B through a competitive mode of inhibition. This evidence, together with previous crystal structure data, further supports the idea that HCV NS5B (in a similar manner to HIV-1 RT) discriminates against the 3'-deoxy modification via lost interactions between the 3'-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3'-OH and the pyrophosphate leaving group during phosphoryl transfer. To our knowledge, these data represent the first time a phosphate modified NTP has been studied as a substrate for HCV NS5B RdRP.

Aptamer-mediated delivery of chemotherapy to pancreatic cancer cells.

Gemcitabine is a nucleoside analog that is currently the best available single-agent chemotherapeutic drug for pancreatic cancer. However, efficacy is limited by our inability to deliver sufficient active metabolite into cancer cells without toxic effects on normal tissues. Targeted delivery of gemcitabine into cancer cells could maximize effectiveness and concurrently minimize toxic side effects by reducing uptake into normal cells. Most pancreatic cancers overexpress epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase. We utilized a nuclease resistant RNA aptamer that binds and is internalized by EGFR on pancreatic cancer cells to deliver gemcitabine-containing polymers into EGFR-expressing cells and inhibit cell proliferation in vitro. This approach to cell type-specific therapy can be adapted to other targets and to other types of therapeutic cargo.

Synthesis and properties of (alpha-P-borano)-nucleoside 5'-triphosphate analogues as potential antiviral agents.

The alpha-P-borano modification, where one of the alpha-phosphate oxygens is replaced by borane, of chain terminating nucleoside triphosphates are currently being tested in cell culture and are showing promise as effective viral polymerase inhibitors. The goal of this project is to combine the alpha-P-borano and Nanogel drug delivery technology to increase the antiviral potency of chain terminating sugar and base modified purine nucleosides versus the Hepatitis C Viral RNA dependent RNA polymerase (HCV RdRp). Here we show the synthesis of Cordycepin and 2'-O-methyl alpha-P-borano triphosphate via a one-pot phosphorochloridite synthesis under mild conditions. These analogues will be used for future structure-activity relationship (SAR) studies.

Boranophosphate siRNA-aptamer chimeras for tumor-specific downregulation of cancer receptors and modulators.

There is a need for novel, effective, and cell- and gene-specific therapeutics for cancer. Modified oligonucleotides can be used to modulate specifically and potently the expression of several genes that are upregulated in breast and prostate cancer and have been found to be causal to the tumor phenotype. Synergistic downregulation of these genes may be a potent therapeutic intervention. We are investigating the use of boranophosphate (BP) analogues of RNA as promising candidates for enhancing the potential of three relatively new, gene-specific, anticancer strategies: (1) Tumor-targeted borane siRNA against a combination of genes that control metabolism and transduction; (2) Tumor-specific modified aptamers against prostate specific membrane antigen (PSMA) and ERB2 in breast cancer as delivery agents; and (3) Cancer cell obliteration by cell-specific radiation therapy: Boron-Neutron-Capture-Therapy.

Stereospecificity, substrate, and inhibitory properties of nucleoside diphosphate analogs for creatine and pyruvate kinases.

Antiviral alpha-P-borano substituted NTPs are promising chain terminators targeting HIV reverse transcriptase (RT). Activation of antiviral nucleoside diphosphates (NDPs) to NTPs may be carried out by pyruvate kinase (PK) and creatine kinase (CK). Herein, are presented the effects of nucleobase, ribose, and alpha-phosphate substitutions on substrate specificities of CK and PK. Both enzymes showed two binding modes and negative cooperativity with respect to substrate binding. The stereospecificity and inhibition of ADP phosphorylation by alpha-P-borano substituted NDP (NDPalphaB) stereoisomers were also investigated. The Sp-ADPalphaB isomer was a 70-fold better substrate for CK than the Rp isomer, whereas PK preferred the Rp isomer of NDPalphaBs. For CK, the Sp-ADPalphaB isomer was a competitive inhibitor; for PK, the Rp-ADPalphaB isomer was a poor competitive inhibitor and the Sp-ADPalphaB isomer was a poor non-competitive inhibitor. Taken together, these data suggest that, although the Rp-NDPalphaB isomer would be minimally phosphorylated by CK or PK, it should not inhibit either enzyme.

Synthesis of 9-fluorenemethyl boranophosphonodiphosphate via an H-phosphonate approach.

9-Fluorenemethyl boranophosphonate 6 and its boranophosphonodiphosphate 7 were synthesized via an H-phosphonate approach. The method is efficient for the synthesis of acyclic compounds 6 & 7, and can be explored for the synthesis of nucleoside 5'-deoxy boranophosphonodiphosphate.

Efficient synthesis of thymidine boranophosphoramidates conjugated with amino acids.

An efficient synthesis of a thymidine boranophosphoramidate prodrug was accomplished using a phosphoramidite approach in high yield. This new class of compounds is designed to have improved antiviral and anticancer advantages conferred by combining the boranophosphate and normal nucleoside amino acid phosphoramidate. Compounds were characterized by MS and 31P NMR.

Versatility of borane nucleic acids mimics for coding, decoding and modulating genetic information.

This presentation will focus on the targeted regulation of gene expression, effective siRNA silencing with boranophosphates, and suppression of drugresistant reverse transcriptase by boranophosphate nucleotide analogues.

High potency silencing by single-stranded boranophosphate siRNA.

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.

Synthesis of alpha-P-modified nucleoside diphosphates with ethylenediamine.

This report describes a one-pot synthesis of alpha-P-borano-, alpha-P-thio-, and alpha-P-seleno-modified nucleoside diphosphate analogues that are otherwise difficult to obtain. The key step involves the intramolecular nucleophilic attack by an amino group in 5 to remove the gamma-phosphate. The absolute configurations of P-diastereomers were confirmed by analysis of their 1H NMR. Affinity studies revealed that the nucleoside boranodiphosphates are potentially useful in antiviral research.

Model synthesis of nucleoside boranophosphoramidate with amino acid for prodrug purpose.

A model synthesis of a nucleoside boranophosphoramidate prodrug with (L)-tryptophan methyl ester was accomplished in a one-pot reaction via an H-phosphonate approach. This new type of compound is expected to possess the potent antiviral and anticancer advantages conferred by boranophosphates and normal nucleoside amino acid phosphoramidate.

Incorporation of ribonucleoside 5'-(alpha-P-borano)triphosphates into a 20-mer RNA by T7 RNA polymerase.

The enzymatic synthesis of short boranophosphate RNA was studied by comparing the yield and pattern of abortive products of in vitro transcription at steady-state conditions with that of normal RNA. Boranophosphate short RNA can be readily synthesized by T7 RNA polymerase.

Synthesis of 5-(1-propynyl)-2'-deoxyuridine 5'-(alpha-P-borano)triphosphate and kinetic characterization as a substrate for mmlv reverse transcriptase.

In order to introduce pyrimidine C5-propynyl modification into boranophosphate oligodeoxyribonucleotides (BP- ODNs), 5-(1-propynyl)-2'-deoxyuridine 5'-(alpha-P-borano) triphosphate (d5PUTPalphaB) was synthesized. The two diastereomers were separated by reverse-phase HPLC. Kinetic studies showed that the Rp isomer was a slightly better substrate for MMLV reverse transcriptase than thymidine triphosphate or Rp-thymidine 5'-(alpha-P-borano)triphosphate. Using the Rp isomers of d5PUTPalphaB and the other three 5'-(alpha-P-borano) triphosphates, a DNA primer could be extended to the full length of the template.

The effect of a single boranophosphate substitution with defined configuration on the thermal stability and conformation of a DNA duplex.

Substitution of one non-bridging oxygen in a natural phosphodiester internucleotide linkage with a borano (-BH3) group results in a chiral phosphorus center in boranophosphate. UV thermal melting profiles were recorded for DNA duplexes formed between a DNA 9-mer with either an Sp or a Rp boranophosphate linkage in the middle and the complementary DNA 9-mer, as well as for their unmodified parent duplex. The thermal stability of the DNA duplexes was in the order of normal > Sp borano > Rp borano. CD spectra of all three duplexes exhibited typical B-DNA profile, which closely resembled each other.

Affinity of (alpha-P-borano)-NTP analogs to rabbit muscle pyruvate kinase.

The binding affinity of (alpha-P-borano) and other NTP analogs to rabbit muscle pyruvate kinase (PK) was investigated using a fluorescence quenching approach to obtain structure-activity relationships for substrate specificity of nucleotide analogs.

Synthesis of 5-ethynyl-2'-deoxyuridine-5'-boranomono phosphate as a potential thymidylate synthase inhibitor.

The 5-ethynyl-2'-deoxyuridine nucleoside and the 5'-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2'-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.

Use of phosphorus ligand NMR probes to investigate electronic and second-sphere solvent effects in ligand substitution reactions at manganese(II) and manganese(III).

Manganese/ligand association dynamics were studied using a series of structurally related anionic phosphorus ester ligand probes [CH(3)OP(O)(X)(Y)(-), where X = CH(3)O, CH(3)CH(2), or H and Y = O, S, or BH(3)]. Reactions of the probe ions with Mn(H(2)O)(6)(2+) and a manganese(III) porphyrin (Mn(III)TMPyP(5+)) were studied in aqueous solution by paramagnetic (31)P NMR line-broadening techniques. A satisfactory linear free energy relationship for reactions of the probe ions with Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) required consideration of both the basicity and solvent affinity of the probe ligands: log(k(app)) = log(k(0)) + alpha pK(a) + beta log(K(ext)), where k(0), alpha, and beta are metal complex dependent parameters and pK(a) and K(ext) represent the measured Bronsted acidity and water/n-butanol extraction constant for the probe anions, respectively. Reactions of Mn(H(2)O)(6)(2+) were relatively insensitive to changes in ligand basicity (alpha = -0.04) and favored the more hydrophilic anions (beta = -0.54). These observations are consistent with a dissociative ligand exchange mechanism wherein the outer-sphere complex is stabilized by hydrogen bonding between Mn(H(2)O)(6)(2+) and the incoming ligand. In contrast, reactions with Mn(III)TMPyP(5+) are accelerated by decreases in both the basicity (alpha = -0.43) and the hydrophilicity (beta = +0.97) of the probe. We conclude that reactions of Mn(III)TMPyP(5+) are also dissociative but that the aromatic groups of the porphyrin provide a hydrophobic environment surrounding the ligand binding site in Mn(III)TMPyP(5+). Thus, the probe/water solvent interactions must be significantly weakened in order to form the outer-sphere complex that leads to ligand substitution. This work demonstrates the utility of phosphorus relaxation enhancement (PhoRE) techniques for characterizing the second coordination sphere environment of metal complexes leading to ligation and will allow comparison of the second coordination spheres of Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) to those of other metal complexes.

Synthesis of nucleoside boranophosphoramidate prodrugs conjugated with amino acids.

[structure: see text] Nucleoside boranophosphates and nucleoside amino acid phosphoramidates have been shown to be potent antiviral and anticancer agents with the potential to act as nucleoside prodrugs. A combination of these two types of compounds results in a boranophosphoramidate linkage between the nucleoside and amino acid. This new class of potential prodrugs is expected to possess advantages conferred by both types of parent compounds. Two approaches, specifically the H-phosphonate and oxathiaphospholane approaches, are described here to synthesize nucleoside boranophosphoramidate prodrugs conjugated with amino acids. The H-phosphonate approach involves a key intermediate, silylated nucleoside amino acid phosphoramidite 6, prepared from a series of reactions starting from nucleoside H-phosphonate in the presence of condensing reagent DPCP. Due to the lengthy procedure and the difficulties in removing DPCP from the final products, we switched to the oxathiaphospholane approach in which the DBU-assisted oxathiaphospholane ring-opening process constituted a key step for the generation of nucleoside amino acid boranophosphoramidates 24. We demonstrate that this key step did not cause any measurable C-racemization of boranophosphorylated amino acids 22. Diastereomers of compounds 24a-f were separated by RP-HPLC. An "adjacent"-type mechanism is proposed to explain the diastereomer ratio in the final products obtained via the oxathiaphospholane approach. A tentative assignment of configuration for the diastereomers was carried out based on the mechanism, molecular modeling, and (1)H NMR. Conclusively, the oxathiaphospholane methodology proved to be more facile and efficient than H-phosphonate chemistry in the preparation of the nucleoside amino acid boranophosphoramidate analogues that are promising as a new type of antiviral prodrugs.

Convenient synthesis of nucleoside borane diphosphate analogues: deoxy- and ribonucleoside 5'-P(alpha)-boranodiphosphates.

The nucleoside boranophosphates, having one of the nonbridging phosphate oxygens substituted with a borane (BH(3)) group, have shown potential therapeutical applications as aptamers, antisense agents, and antiviral prodrugs. An oxathiaphospholane approach, which does not require exocyclic amine protection of the nucleobase, has been successfully developed to efficiently synthesize 5'-P(alpha)-boranodiphosphates of 2'-deoxythymidine, adenosine, guanosine, and uridine. The approach involves a key intermediate, the borane complex of nucleoside 5'-O-1,3,2-oxathiaphospholane 16, that undergoes a ring-opening reaction catalyzed by 1,4-diazabicyclo[5.4.0]-undec-7-ene to form the protected nucleoside 5'-P(alpha)-boranodiphosphate 18. Treatment of 18 with ammonium hydroxide yielded diastereoisomeric mixtures of nucleoside 5'-P(alpha)-boranodiphosphates 5. This oxathiaphospholane approach ensures the availability of nucleoside 5'-P(alpha)-boranodiphosphate analogues needed for antiviral drug research.