Vivek K. Rajwanshi

LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition

Abstract LNA (Locked Nucleic Acids), consisting of 2′- O ,4′- C -methylene bicyclonucleoside monomers, is efficiently synthesized and its nucleic acid recognition potential evaluated for six different nucleobases, namely adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil. Unprecedented increases (+3 to +8 °C per modification) in the thermal stability of duplexes towards both DNA and RNA were obtained when evaluating mixed sequences of partly or fully modified LNA. Studies of mis-matched sequences show that LNA obey the Watson-Crick base pairing rules with generally improved selectivities compared to the corresponding unmodified reference strands.

The conformations of locked nucleic acids (LNA)

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2′‐O, 4′‐C‐methylene bridge. We have investigated a modified 9‐mer single stranded oligonucleotide as well as three 9‐ and 10‐mer modified oligonucleotides hybridized to unmodified DNA. The single‐stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson–Crick base‐pairing with all the nucleotides in anti‐conformation. By use of selective DQF‐COSY spectra we determined the ratio between the N‐type (C3′‐endo) and S‐type (C2′‐endo) sugar conformations of the nucleotides. In contrast to the corresponding single‐stranded DNA (ssDNA), we found that the sugar conformations of the single‐stranded LNA oligonucleotide (ssLNA) cannot be described by a major S‐type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N‐type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N‐type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S‐type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N‐type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases. Copyright

Novel convenient syntheses of LNA [2.2.1]bicyclo nucleosides

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing [2.2.1]bicyclo nucleoside monomers. A novel and significantly improved method for convergent synthesis of LNA [2.2.1]bicyclo nucleosides using a 4-C-tosyloxymethyl-1, 2-di-O-acetyl furanose as a key synthon is described. In addition, an alternative, robust linear approach allowing selective formation of the desired [2.2.1]bicyclo LNA nucleosides via a tricyclic nucleoside intermediate is introduced.

The Eight Stereoisomers of LNA (Locked Nucleic Acid): A Remarkable Family of Strong RNA Binding Molecules

The authors evaluated the RNA binding of all eight possible stereoisomers of LNA. Synthesis of qa-l-xylo-LNAq has been accomplished and the remaining four stereoisomers, all enantiomers of the four synthesized diastereoisomers, are indirectly evaluated by hybridization studies. The remarkable binding affinities and specificities obtained for LNA and a-l-LNA, both in a fully and in a partly modified context, establish these mols. as unique nucleic acid mimics. A strong impetus for this research has been, and continues to be, the therapeutic promises of the antisense strategy. [on SciFinder (R)]

Lna (Locked Nucleic Acid)

Abstract LNA (Locked Nucleic Acid) forms duplexes with complementary DNA, RNA or LNA with unprecedented thermal affinities. CD spectra show that duplexes involving fully modified LNA (especially LNA:RNA) structurally resemble an A-form RNA:RNA duplex. NMR examination of an LNA:DNA duplex confirm the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA is demonstrated suggesting strand invasion by LNA. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has been accomplished.

LNA (locked nucleic acid) and the diastereoisomeric alpha-L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets.

The remarkable binding properties of LNA (Locked Nucleic Acid) and α-L-LNA (the α-L-ribo configured diastereoisomer of LNA) are summarized, and hybridization results for LNA/2′-O-Me-RNA chimera and LNAs with a “dangling” nucleotide are introduced. In addition, results from NMR investigations on the furanose conformations of the individual nucleotide monomers in different duplexes are presented. All these data are discussed with focus on the importance of conformational steering of unmodified nucleotides in partly modified LNA and α-L-LNA sequences in relation to the unprecedented binding properties of LNA and α-L-LNA.

OLIGONUCLEOTIDE ANALOGUE INTERFERENCE WITH THE HIV-1 TAT PROTEIN-TAR RNA INTERACTION

The HIV-1 Tat protein interaction with its RNA recognition sequence TAR is an important drug target and model system for the development of specific RNA-protein inhibitors. 2′-O-methyl oligoribonucleotides complementary to the TAR apical stem-loop effectively block Tat binding in vitro. Substitution by 5-propynylC or 5-methylC LNA monomeric units into a 12-mer 2′-O-methyl oligoribonucleotide leads to stronger inhibition, as does a 12-mer PNA. 10–16 mer 2′-O-methyl oligoribonucleotides give sequence- and dose-dependent inhibition of Tat-dependent transcription of an HIV DNA template in HeLa cell nuclear extract. Inhibition is maintained for the substituted 12-mer analogues but is poorer for PNA and is not correlated with TAR binding strength.

Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2'-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases.

ABSTRACT Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

Synthesis and restricted furanose conformations of three novel bicyclic thymine nucleosides: a xylo-LNA nucleoside, a 3′-O,5′-C-methylene-linked nucleoside, and a 2′-O,5′-C-methylene-linked nucleoside

The xylo-LNA nucleoside 1-(2-O,4-C-methylene-β-D-xylofuranosyl)thymine (9) and the 2′-O,5′-C-methylene linked nucleoside 1-(2,6-anhydro-β-D-altrofuranosyl)thymine (28) were obtained in overall yields of 13% (8 steps) and 31% (7 steps) starting from furanose derivatives 1 and 21, respectively. In the synthesis of 3′-O,5′-C-methylene-linked nucleoside derivatives, cyclization by intramolecular attack from the 6-hydroxy group on the 3-keto functionality to give C-3-hemiketal furanose 11 and its subsequent transformation into nucleoside 15 proved very efficient. It was, however, impossible to isolate the debenzylated 2′-hydroxy, 2′-O-methyl and 2′-O-tert-butyldimethylsilyl derivatives 16, 18 and 20, respectively, in analytically pure form. Solution-phase conformational analysis showed the bicyclic nucleosides 8, 9, 14, 15, 17 and 19 to exist predominantly in an N-type furanose conformation and bicyclic nucleotides 27 and 28 to adopt an S-type conformation.

Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.