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Using the intramolecular 5-exo-5-hexenyl radical as a key cyclization step, we previously reported an unambiguous synthesis of carba-LNA thymine (cLNA-T), which we subsequently incorporated in antisense oligonudeotides (AON) and investigated their biochemical properties [J. Am. Chem. Soc. 2007, 129 (26), 8362-8379]. These cLNA-T incorporated oligos showed specific RNA affinity of +3.5-5 degrees C/modification for AON:RNA heteroduplexes, which is comparable to what is found for those of LNAs (Locked Nucleic Acids). These modified oligos however showed significantly enhanced nuclease stability (ca. 100 times more) in the blood serum compared to those of the LNA modified counterparts without compromising any RNase H recruitment capability. We herein report the synthesis of 5-methylcytosine-1-yl (C-Me), 9-adeninyl (A), and 9-guaninyl (G) derivatives of cLNA and their oligonucleotides and report their biochemical properties as potential RNA-directed inhibitors. In a series of isosequential carba-LNA modified AONs, we herein show that all the cLNA modified AONs are found to be RNA-selective, but the magnitude of RNA-selectivity of 7'-R-Me-cLNA-G (cLNA-G) (Delta T-m = 2.9 degrees C/modification) and intractable isomeric mixtures of 7'-(S/R)-Me-cLNA-T (cLNA-T, Delta T-m = 2.2 degrees C/modification) was found to be better than diastereomeric mixtures of 7'-(S/R)-Me-cLNA-C-Me with trace of cENA-C-Me (cLNA-C-Me, Delta T-m = 1.8 degrees C/modification) and 7'-R-Me-cLNA-A (cLNA-A, Delta T-m = 0.9 degrees C/modification). cLNA-C-Me modified AONs however exhibited the best nuclease stability, which is 4-, 7-, and 20-fold better, respectively, than cLNA-T, cLNA-A, and cLNA-G modified counterparts, which in turn was more than 100 times stable than that of the native. When the modification sites are appropriately chosen in the AONs, the cLNA-A, -G, and -C-Me modified sites in the AON:RNA hybrids can be easily recognized by RNase H, and the RNA strand of the hybrid is degraded in a s

2',4'-Propylene-bridged thymidine (carba-ENA-T) and five 8'-Me/NH2/OH modified carba-ENA-T analogues have been prepared through intramolecular radical addition to C=N of the tethered oxime-ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleotides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment capability have been investigated in comparison with those of the native and ENA counterparts. These carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization effect for the AON: RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was found that different C8' substituents (at the bottom of the minor groove) on carba-ENA-T only led to rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further modulated by C8' substituent to variable extents depending on not only the chemical nature but also the stereochemical orientation of the C8' substituents: Thus, (1) 8'S-Me on carba-ENA increases the nucleolytic stability but 8'R-Me leads to a decreased effect; (2) 8'R-OH on carba-ENA had little, if any, effect on nuclease resistance hut 8'S-OH resulted in significantly decreased nucleolytic stability; and (3) 8'-NH2 substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase HI with high efficiency, even at twice the rate of those of the native and ENA modified counterpart.

A new class of conformationally constrained nucleosides, alpha-L-ribo-carbocyclic LNA thymidine (alpha-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" alpha-L-ribo-configured tetracyche thymidine (6,7'-methylene-bridged-alpha-L-carba-LNA-T) in which both the sugar puckering and glyeosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These alpha-L-carba-LNA analogues have been subsequently transformed to corresponding phosphoramidites and incorporated into isosequential antisense oligonucleotides (AONs), which have then been examined for the thermal denaturation of their duplexes, nuclease stability, and RNase H recruitment capabilities. Introduction of a single 6',7'-substituted alpha-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T-m reduction by 2-3 degrees C as compared to the native heteroduplex, whereas the parent 2'-oxa-alpha-L-LNA-T modification at the identical position in the AON strand has been found to lead to an increase in the T-m by 3-5 degrees C. This suggests that the 6' and 7' substitutions lead to much reduced thermal stability for the modified heteroduplex, especially the hydrophobic 7'-methyl on alpha-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted alpha-L-earba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-alpha-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted alpha-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the beta-D-LNA-T or beta-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNasc H recruitment capabilities elevate the alpha-L-carba-LNA-modified nu

In this study, 12 different native or LNA, carba-LNA-modified dinucleoside phosphates were designed as simple chemical models to study how carba-LNA modifications improve the 3'-exonuclease (SVPDE in this study) resistance Of internucleotidic phosphate compared to those exhibited by LNA-modified and the native counterparts. Michaelis-Menten kinetic studies for dimers 3 - 7, in which the LNA or carba-LNA modifications are located at the 5'-end, showed that (i) increased 3'-exonuclease resistance of (5')[LNA-T](p)T (3) compared to the native (TpT)-T-5' (1) was mainly attributed to steric hindrance imposed by the LNA modification that retards the nuclease binding (K-M) and (ii) digestion of (5')[carba-LNA-dT](p)T (4) and (5')[LNA-T](p)T (3), however, exhibit similar K(M)s, whereas the former shows a 100x decrease in K-cat and is hence more stable than latter. By studying the correlation between log k(cat) and pK(a) of the departing 3'(or 6')-OHs for 3-7, we found the pK(a) of 3'-OH of carba-LNA-T was 1.4 pK(a) units higher than that of LNA-T, and this relatively less acidic character of the 3'-OH in the former leads to the 100x decrease in the catalytic efficiency for the digestion Of (5')[Carba-LNA-T](p)T (4). In contrast, Michaelis-Menten kinetic studies for dimers 9-12, with the LNA or carba-LNA modifications at the 3'-end, showed that the digestion of T-5'(p)[LNA-T] (9) exhibited similar K-M but k(cat) decreased around 40 times compared to that of the native (TpT)-T-5' (1). Similar k(cat) values have been observed for digestion of T-5'(p)[carba-LNA-T] (10) and T-5'(p)[LNA-T] (9). The higher stability of carba-LNA modified dimer 10 compared with LNA modified dimer 9 comes solely from the increased K-M.

This review highlights progress made during the past 2 to 3 years in the field of therapeutic locked nucleos(t)ides. Synthetic strategies to construct the conformationally locked nucleos(t)ides, their properties and potential therapeutic applications as antiviral compounds, and modified oligonucleotides to modulate antisense and RNAi properties are described.

In the antisense (AS) and RNA interference (RNAi) technologies, the native single-stranded 2'-deoxyoligonucleotides (for AS) or double-stranded RNA (for RNAi) are chemically modified to bind to the target RNA in order to give improved downregulation of gene expression through inhibition of RNA translation. It is shown here how the fine adjustment of the electrostatic interaction by alteration of the substituents as well as their stereochemical environment around the intemucleotidic phosphodiester moiety near the edge of the minor grove of the antisense oligonucleotides (AON)-RNA heteroduplex can lead to the modulation of the antisense properties. This was demonstrated through the synthesis of various modified carbocyclic-locked nucleic acids (LNAs) and -ethylene-bridged nucleic acids (ENAs) with hydroxyl and/or methyl substituents attached at the carbocyclic pan and their integration into AONs by solid-phase DNA synthesis. The target affinity toward the complementary RNA and DNA, nuclease resistance, and RNase H elicitation by these modified AONs showed that both the nature of the modification (-OH versus -CH3) and their respective stereochemical orientations vis-a-vis vicinal phosphate play a very important role in modulating the AON properties. Whereas the affinity to the target RNA and the enzymatic stability of AONs were not favored by the hydrophobic and sterically bulky modifications in the center of the minor groove, their positioning at the edge of the minor groove near the phosphate linkage resulted in significantly improved nuclease resisitance without of target affinity. On the other hand, hydrophilic modification, such as a hydroxyl group, close to the phosphate linkage made the internucleotidic phosphodiester especially nucleolytically unstable, and hence was not recommended. The substitutions on the carbocyclic moiety of the carba-LNA and -ENA did not affect significantly the choice of the cleavage sites of RNase H mediated RNA cleavage in the AON

The hyper-constrained nucleoside, methylene-bridged hexopyranosyl nucleoside (BHNA) was incorporated into the antisnese oligonucleotides (AON), which show more preference for binding toward the complementary RNA (T-m loss by ca 5 degrees C) than that with the complementary DNA (T-m loss by 10 C), vis-a-vis corresponding native duplex. The origin of reduction of T-m of the duplexes formed by the BHNA incorporated AON and the complementary RNA or DNA was further investigated by thermal denaturation study with the single-mismatched DNA or RNA, CD spectroscopy, RNase H digestion study, as well as by molecular model building. These studies showed that the introduction of BHNA causes only a limited local conformational perturbation in the AON/RNA heteroduplex, whereas it affects the global conformation in the AON-DNA duplex. BHNA incorporated AONs also show improved stability in the human blood serum, which may prove to have some potential therapeutic application.