T. V. Maltseva

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2′-α-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of α-hydroxy moieties. 2′-Deoxy-2′-β-fluoro-4′-azidocytidine (RO-0622) and 2′-deoxy-2′-β-hydroxy-4′-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC50 = 171 ± 12 nm and 24 ± 3 nm for RO-9187 and RO-0622, respectively; CC50 >1 mm for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4′-azidocytidine) or 2′-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC50 values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2′-α-deoxy-4′-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.

2'-deoxy-4'-azido nucleoside analogs are highly potent inhibitors of HCV replication despite the lack of 2'-alpha hydroxyl groups

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2′-α-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of α-hydroxy moieties. 2′-Deoxy-2′-β-fluoro-4′-azidocytidine (RO-0622) and 2′-deoxy-2′-β-hydroxy-4′-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC50 = 171 ± 12 nm and 24 ± 3 nm for RO-9187 and RO-0622, respectively; CC50 >1 mm for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4′-azidocytidine) or 2′-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC50 values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2′-α-deoxy-4′-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.

THE FIRST EXAMPLE OF SEQUENCE-SPECIFIC NON-UNIFORMLY 13C5 LABELLED RNA : SYNTHESIS OF THE 29MER HIV-1 TAR RNA WITH 13C RELAXATION WINDOW

Abstract A complete synthetic protocol as well as 1H- and 13C-NMR data on the monomer building blocks used for the solid-phase synthesis of specifically 13C-labelled (99 atom % 13C) stem (27A and 43G), bulge (24C) and loop (31U) regions of 29mer HIV-1 TAR RNA hairpin starting from the 13C6- D -glucose are presented. The complex NMR spectra of 13C-labelled monomer building blocks, due to the interaction of various 13C and 1H spins, have been assigned. It has been demonstrated by heteronuclear 2D NMR that the non-uniform labelling of the HIV-1 TAR 29mer RNA achieved herein by chemical synthesis provides an optimal opportunity to perform full T1 and T2 relaxation measurements (the “NMR Relaxation Window”) of each type of sugar-carbons for all four strategically placed 13C-labelled residues in a unique and unprecedented manner because of minimal overlap of 13C resonances compared to uniformly labelled oligo-RNA.

T1 AND T2 RELAXATIONS OF THE 13C NUCLEI OF DEUTERIUM-LABELED NUCLEOSIDES

The effect of 2H on 13C longitudinal (T1) and transverse (T2) relaxation parameters was determined for the first time for diastereospecifically deuterium‐labeled nucleosides, which are used as the building blocks for non‐uniform isotope labeling for the solution NMR structure determination of the large biologically functional oligo‐DNA and ‐RNA (‘NMR window’ approach, ref. 7). It emerged that the T1 and T2 of the deuterated methine carbon in the diastereospecifically deuterium‐labeled nucleoside 9 could be used as the correction term to give the monoexponential decay of 13C longitudinal and transverse magnetization of the constituent 1H–13C–2H group. The correlation time derived from this corrected T1 of the methylene carbon corresponds well with the correlation time obtained from deuterium relaxation study. The extreme narrowing limit (ωτc≪1) where dipole–dipole (DD) relaxation of 13C and quadrupole (Q) relaxation of 2H are related by T1DD/T2DD≈1 and T1Q/T2Q≈1 was used to demonstrate the above conclusion. The difference in the observable T1 and T2 in various methylene and methine‐type carbons with either fully protonated or diastereospecifically deuterated nucleosides 1–14 allowed the estimation of the contribution of the alternative relaxation pathways other than DD relaxation. It was found by comparison of the T1 relaxation of the quaternary carbon with the methine carbon (13C–2H) or (13C–1H) in compound 2 that the contribution of the intermolecular and intramolecular relaxations of 13C with protons that are two bonds away is larger than DD(13C–2H), and the sum of all these contributions define the T1 of the methine carbon (13C–2H). The observed difference between the experimental T1 and T2 of the methine carbon is attributed to the cross‐correlation between DD(13C–2H) and Q(2H) relaxation, which is consistent with recent theoretical predictions. For T2 measurement, the decoupling of deuterium with 0.6–2.5 kHz power during the echo period by WALTZ does not effectively eliminate the DD(13C–2H)–Q(2H) cross‐correlation for the methine carbon. The suppression of this DD(13C–2H)–Q(2H) cross‐correlation was, however, more effective by applying a 180° deuterium pulse in the middle of the short (0.5 ms) echo period (compare T2 of 3.91s and 0.3s, respectively, at 294 K using these two different decoupling procedures). The comparison of the observed T1 and T2 relaxations of the methylene carbon shows that they are indeed very close. The various contributions of the methine carbon relaxation such as DD(13C–2H), intermolecular and cross‐correlation, DD(13C–1H)–Q(2H), to the relaxation of the methylene carbon were ca. 15% in T1 and ca. 25% in T2.

Synthesis of partially-deuterated 2'-deoxyribonucleoside blocks and their incorporation into an oligo-DNA for simplification of overcrowding and selective enhancement of resolution and sensitivity in the 1H-NMR spectra

Abstract The chemical synthesis of appropriately protected partially-deuterated 2′( R / S ),3′,5′( R / S )- 2 H 3 -2′-deoxyribonucleoside blocks [∼43 atom % 2H at C5′( R ), ∼57 atom % 2H at C5′( S ); ∼15 atom % 2H at C2′( R ), ∼85 atom % 2H at C2′( S ) and >99 atom % 2H at C3′] is reported. The availability of these deuterium labelled blocks on large scale has enabled the chemical assemblage of the deuterio isotopomeric 12mer DNA duplex by standard solid-phase synthesis protocol in order to demonstrate the usefulness of the new “NMR-window III” approach (see the following paper).

The First Example of a Hoogsteen Basepaired DNA Duplex in Dynamic Equilibrium with a Watson-Crick Basepaired Duplex—A Structural (NMR), Kinetic and Thermodynamic Study

Abstract A single-point substitution of the O4′ oxygen by a CH2 group at the sugar residue of A 6 (i.e. 2′-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5′- d(C1G2C3G4A5A6T7T8C9G10C11G12)2 −3, has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A 6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)→←(1B): Keq = k1/k-1 = 0.56±0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9± 0.8 sec−1; δH°‡ = 164±14 kJ/mol;-TδS°‡ (298K) = −92 kJ/mol giving a δG298°‡ of 72 kJ/mol. Ea (k1) = 167±14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0±0.6 sec-1, δH°‡ = 153±13 kJ/mol;-TδS°‡ (298K) = −82 kJ/mol giving a δG298°‡ of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of δG298°‡ of the forward (k1) and backward (k-1) conversions, (1A)→←(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A 6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A 6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2′- deoxyaristeromycin moiety, A 6, as a result of substitution of the endocyclic 4′-oxygen in the natural sugar with a methylene group in A 6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.

Measurement of the deuterium relaxation times in double-labeled (13C/2H) thymidine and 2′-deoxyadenosine and in the selectively labeled DNA duplex5′d(1C2G3A4T5T6A7A8T9C10G)23′

The T1 and T1ρ of deuterium in 13C/2H double‐labeled 2′(R/S),5′(R/ S)‐2H2 ‐1′,2′,3′,4′,5′‐ 13C5 ‐2′‐deoxyadenosine and the corresponding thymidine derivative as well as in the non‐uniformly labeled (shown in bold and underlined) DNA duplex, d5′(1C2G3A4T5T6A7A8T9C10G)23′, have been determined for the first time. These double‐labelled nucleoside blocks have a special feature in that the geminal 2′–2″ and 5′–5″ proton–proton couplings are eliminated by replacement with diastereomeric deuterium at C‐2′ and C‐5′ centers. This uniquely enables us to perform deuterium relaxation measurement experiments through selective polarization transfer, 1H–13C– 2H–13C–1H at C‐2′ and C‐5′ centers, thereby allowing filtration of all other naturally abundant methylene‐ and methine‐13C as well as enriched methine‐ 13C fragments. Comparison of T1 and T 1ρ of 2H in double‐labeled ( 13C/2H) 2′‐deoxyadenosine and thymidine with that of the non‐uniformly labeled DNA duplex, d5′(1C2G3 A4T5T6A7A8T9C10G)23′, shows that the dynamics of various nucleotide residues are indeed non‐uniform. Copyright

Synthesis of 2H,13C-Labelled 2′-Deoxynucleosides and Their Site Specific Incorporation into Oligo-DNA for Structural Studies via Relaxation Time Measurements

Abstract We have recently shown1 the usefulness of 2H, 13C-labelled 2′-deoxynu-cleoside building blocks for structural studies via relaxation time measurements. The synthesis of phosphoramidite blocks 11 and 12 for their site-specific incorporation (indicated by underlines) into the d5′(1C2G3 A 4 T 5 T 6 A 7 A 8 T 9C10G)2 3′ is briefly described for studying the T1 and T1[sgrave] relaxations of 2H and 13C at specific deuterated carbons in a large molecule.

Partially-deuterated oligo-DNA reduces overcrowding, enhances resolution and sensitivity and provide improved NMR constraints for structure elucidation of oligo-DNA

Abstract The usefulness of the “NMR-window III” approach (see ref. 2e) has been demonstrated through the use of the deuterio isotopomeric 12mer oligo-DNA duplex obtained from protected partially deuterated 2′ ( R / S ), 3′, 5′ ( R / S )-2H3- 2′-deoxyribonucleoside blocks [see preceding paper). The 2D-NMR spectra of this deuterio isotopomeric DNA duplex I were compared with those of the natural counterpart (duplex II) and deuterium-labelled 12mer duplex III (prepared earlier by the “NMR-window II” approach, ref. 2e), and the results can be summarised as follows: (i) The simplification of the crosspeak pattern in H1′–H2′/2″ and H4′–H5′/5″ areas in the DQF-COSY spectra reduces the spectral crowding, thereby allowing a precise extraction of the coupling constants. (ii) The deuterio isotopomeric mixture of DNA duplex I provide hitherto unavailable nOe data sets which are sensitive to the conformational changes of the sugar moiety or the backbone. (iii) The extractable number of nOe constraints based on fully resolved crosspeaks and free from spin diffusion for deuterio isotopomeric dodecamer I are 308, whereas they are only 188 for the natural counterpart, suggesting the usefulness of the nOe crosspeaks in the semi-quantitative approach for the NMR-constrained structure refinement.

The NMR conformation study of the complexes of deoxycytidine kinase (dCK) and 2'-deoxycytidine/2'-deoxyadenosine.

The structures of the bound 13C/2H double-labelled 2′(R/S), 5′(R/S)-2H2-1′,2′,3′,4′,5′-13C5-2′-deoxyadenosine and the corresponding 2′-deoxycytidine moieties in the complexes with human deoxycytidine kinase (dCK) have been characterized for the first time by the solution NMR spectroscopy, using Transferred Dipole-Dipole Cross-correlated Relaxation and Transferred nOe experiments. It has been shown that the ligand adopts a South-type sugar conformation when bound to dCK.