Oleksandr Plashkevych

Unusual radical 6-endo cyclization to carbocyclic-ENA and elucidation of its solution conformation by 600 MHz NMR and ab initio calculations.

In our previous paper (J. Am. Chem. Soc., 2007, 129, 8362), we reported the synthesis of 7-Me-Carba-LNA and 8-Me-Carba-ENA thymidine through 5-hexenyl or 6-heptenyl radical cyclization. Both 5-hexenyl and 6-heptenyl radical cyclized exclusively in the exo form, giving unwanted exocyclic C7-methyl group. In the present study, we showed that the regioselectivity of the 5-hexenyl radical cyclization could be favorably tuned by introduction of a hydroxyl group to the olefinic double bond, yielding about 9% of the 6-endo cyclization product. Possible pathways to give 6-endo cyclization product 9 compared to the intermediates responsible to give the 5-exo cyclization product 5 has been discussed. Based on this unique 6-endo cyclization strategy, a carbocyclic ENA modified thymidine (carba-ENA) has been successfully synthesized, which also enabled us to perform its full solution conformation analysis by using NMR (1H at 600 MHz) observables for the first time.

Oxetane Locked Thymidine in the Dickerson-Drew Dodecamer Causes Local Base Pairing Distortions—An NMR Structure and Hydration Study

Abstract The introduction of a North-type sugar conformation constrained oxetane T block, 1-(1′,3′-O-anhydro-β-D-psicofuranosyl) thymine, at the T 7 position of the self-complementary Dickerson-Drew dodecamer, d[(5′-C1G2C3G4A5A6 T 7T8C9G10C11G12-3′)]2, considerably perturbs the conformation of the four central base pairs, reducing the stability of the structure. UV spectroscopy and ID NMR display a drop in melting temperature of ∼10 °C per modification for the T 7 oxetane modified duplex, where the T 7 block has been introduced in both strands, compared to the native Dickerson-Drew dodecamer. The three dimensional structure has been determined by NMR spectroscopy and has subsequently been compared with the results of 2.4 ns MD simulations of the native and the T 7 oxetane modified duplexes. The modified T 7 residue is found to maintain its constrained sugar- and the related glycosyl torsion conformations in the duplex, resulting in staggered and stretched T 7·A6 and A6·T 7 nonlinear base pairs. The stacking is less perturbed, but there is an increased roll between the two central residues compared to the native counterpart, which is compensated by tilts of the neighboring base steps. The one dimensional melting profile of base protons of the T 7 and T 8 residues reveals that the introduction of the North-type sugar constrained thymine destabilizes the core of the modified duplex, promoting melting to start simultaneously from the center as well as from the ends. Temperature dependent hydration studies by NMR demonstrate that the central T 7·A6/A6·T7 base pairs of the T 7 oxetane modified Dickerson-Drew dodecamer have at least one order of magnitude higher water exchange rates (correlated to the opening rate of the base pair) than the corresponding base pairs in the native duplex.

Diastereomer-Specific Repertoire of 7′R- or 7′S-Me-Carba-Locked Nucleic Acids (cLNAs) in Antisense Oligo/RNA Duplexes and Engineering of Physico-chemical and Enzymological Properties

Modified oligos (AON), with pure 2′, 4′-locked 7′S- or 7′R-Me-cLNA-A, -G, -MeC, and -T (Upadhayaya et al., J Org Chem 76:4408–4431, 2011; Srivastava et al., J Am Chem Soc 129:8362–8379, 2007), show higher RNA affinity and RNA selectivity, highly improved exonuclease (SVPDE), and blood serum stabilities in comparison with native oligos as well as maintained or higher RNase H recruitment capability depending upon the modification site. The AON with 7′S-Me-cLNA-MeC is found to be ~40 times more stable against SVPDE than 7′S- and 7′R-Me-cLNA-T-modified AONs, which are in turn much more stable than 7′S- and 7′R-Me-cLNA-A- and G-modified counterparts. The T m increase of the duplexes is found to be dependent on the AON-sequence context, -modification site, and the cLNA diastereomer type used (7′S or 7′R). MD simulations of the AON/RNA duplexes with 7′S- or 7′R-Me-cLNA have demonstrated that the modifications have only small local effect on the duplex structures, stacking and Watson–Crick base pairing.

Carbocyclic C‐C Bond Formation: Intramolecular Radical Ring Closure to Yield Diastereomerically Pure (7′S‐Me‐ or 7′R‐Me‐) Carba‐LNA Nucleotide Analogs

In light of the impressive gene‐silencing properties of carba‐LNA modified oligo DNA and RNA, both in antisense RNA and siRNA approaches, which have been confirmed as proof‐of‐concept for biochemical applications in post‐transcriptional gene silencing, we envision the true potential of carba‐LNA modifications to be revealed soon. Herein we provide detailed protocols for synthesis of carba‐LNA‐A, ‐G, ‐5‐MeC, and ‐T nucleosides on a medium/large scale (gram scale), as well as important guidelines for incorporation of these modified carba‐LNAs into DNA or RNA oligonucleotides. Creation of a stereoselective C‐C bond during the 5‐exo radical intramolecular cyclization involves trapping of a C2′ radical intermediate intramolecularly by the vicinal double bond of a C4′‐tethered ─CH2‐CH═CH2 group. All diastereomers of substituted carba‐LNAs are now available in pure form. The present procedure allows carba‐LNA to be commercialized for medicinal or biotechnological purposes.

Molecular Structure of the Core-Modified siRNA Duplexes Containing Diastereomeric Pair of [C6′(R)-OH]- versus [C6′(S)-OH]-carba-LNAs Suggests a Model for RNAi Action

Molecular structures of native and a pair of modified small interfering RNA–RNA duplexes containing carbocyclic [6 ′-(R)-OH/7 ′-(S)-methyl]- and [6 ′-(S)-OH/7 ′-(S)-methyl]-carba-LNA-thymine nucleotides, which are two diastereomeric analogs of the native T nucleotide, incorporated at position 13 in the antisense (AS) strand of siRNA, have been simulated using molecular mechanics/dynamics techniques. The main aim of the project has been to find a plausible structural explanation of why modification of siRNA at T13 position by the [6 ′(R)-O-(p-Toluoyl)-7 ′(S)-methyl]-carba-LNA-Thymine [IC50 of 3.32 ± 0.17 nM] is ca 24 times more active as an RNA silencing agent against the target HIV-1 TAR RNA than the [6 ′(S)-O-(p-Toluoyl)-7 ′(S)-methyl]-counterpart [IC50 of 79.8 ± 17 nM] [1]. The simulations reveal that introduction of both C6 ′(R)-OH and C6 ′(S)-OH stereoisomers does not lead even to local perturbation of the siRNA–RNA duplex structures compared to the native, and the only significant difference between 6 ′(S)- and 6 ′(R)-diastereomers found is the exposure of the 6 ′-OH group of the 6 ′(R)-diastereoisomer toward the edge of the duplex while the 6 ′-hydroxyl group of the 6 ′(S)-diastereoisomer is somewhat buried in the minor groove of the duplex. This rules out a hypothesis about any possible local distortion by the nature of chemical modification of the siRNA-target the RNA duplex, which might have influenced the formation of the effective RNA silencing complex (RISC) and puts some weight on the hypothesis about the 6 ′-hydroxy group being directly involved with most probably Ago protein, since it is known from exhaustive X-ray studies [2, 3] that the core residues are indeed involved with hydrogen bonding with the internucleotidyl phosphates. Further systematic investigation is in progress to map the position-dependent functional and nonfunctional interactions of the modified [6 ′(R or S)-O-(p-Toluoyl)-7 ′(S)-methyl]-carba-LNA-T with the Ago2 protein of the RISC.