C2'-F Stereoconfiguration As a Puckering Switch for Base Stacking at the Dinucleotide Level.

Fluorine configuration at C2' of the bis(2'-fluorothymidine) dinucleotide is demonstrated to drive intramolecular base stacking. 2'-ß F-Configuration drastically reduces stacking compared to the 2'-α series. Hence, base stacking emerges as being tunable by the C2'-F stereoconfiguration through dramatic puckering variations scrutinized by NMR and natural bond orbital analysis. Accordingly, 2'-ß F-isomer photoreactivity is significantly reduced compared to that of the 2'-α F-isomer.

A minute amount of s-puckered sugars is sufficient for (6-4) photoproduct formation at the dinucleotide level.

The di-2'-α-fluoro analogue of thymidylyl(3',5')thymidine, synthesized to probe the effect of a minimum amount of S conformer on the photoreactivity of dinucleotides, is endowed with only 3% and 8% of S sugar conformation at its 5'- and 3'-end, respectively. This analogue gives rise to the (6-4) photoproduct as efficiently as the dithymine dinucleotide (74% and 66% at the 5'- and 3'-end, respectively) under 254 nm. Our results suggest that the 5'-N, 3'-S conformer gives rise to the (6-4) photoproduct.

Free-radical ring closure to conformationally locked α-L-carba-LNAs and synthesis of their oligos: nuclease stability, target RNA specificity, and elicitation of RNase H.

A new class of conformationally constrained nucleosides, α-L-ribo-carbocyclic LNA thymidine (α-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" α-L-ribo-configured tetracyclic thymidine (6,7'-methylene-bridged-α-L-carba-LNA-T) in which both the sugar puckering and glycosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These α-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 α-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T(m) reduction by 2-3 °C as compared to the native heteroduplex, whereas the parent 2'-oxa-α-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 °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 α-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted α-L-carba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-α-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted α-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the ß-D-LNA-T or ß-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNase H recruitment capabilities elevate the α-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.

Synthesis and structure of azole-fused indeno[2,1-c]quinolines and their anti-mycobacterial properties.

Prompted by our discovery of a new class of conformationally-locked indeno[2,1-c]quinolines as anti-mycobacterials, compounds 2a and 3a (Fig. 1; MIC < 0.39 µg mL(-1) and 0.78 µg mL(-1), respectively)(14) with a freely rotating C2-imidazolo substituent, we herein describe the synthesis of pentacyclic azole-fused quinoline derivatives 4 and 5, in which we have restricted the rotation of the C2-imidazolo moiety by fusing it to the adjacent quinoline-nitrogen to give a five-membered fused azole heterocycle. The idea of locking the flexibility of the system by conformational constraint was simply to reduce its entropy, thereby reducing the overall free-energy of its binding to the target receptor. Out of 22 different azole-fused indeno[2,1-c]quinoline derivatives, seven structurally distinct compounds, 9, 15, 17, 25, 27, 28 and 29, have shown 79-99% growth inhibition of Mycobacterium tuberculosis H37Rv at a fixed dose of 6.25 µg mL(-1). The efficacies of these compounds were evaluated in vitro for 8/9 consecutive days using the BACTEC radiometric assay upon administration of single dose on day one. Of these, two compounds, 9 and 28, inhibited growth of M. tuberculosis very effectively at MIC < 0.39 µg mL(-1) (0.89 µM and 1 µM, respectively). These active compounds 9, 15, 17, 25, 27, 28 and 29 were screened for their cytotoxic effect on mammalian cells (human monocytic cell line U937), which showed that the human cell survival is almost unperturbed (100% survival), except for compound 25, hence these new compounds with new scaffolds have been identified as potent anti-mycobacterials, virtually with no toxicity. Thus these "hit" molecules constitute our important "leads" for further optimization by structure-activity relationship against TB.

Double sugar and phosphate backbone-constrained nucleotides: synthesis, structure, stability, and their incorporation into oligodeoxynucleotides.

Two diastereomerically pure carba-LNA dioxaphosphorinane nucleotides [(S(p))- or (R(p))-D(2)-CNA], simultaneously conformationally locked at the sugar and the phosphate backbone, have been designed and synthesized. Structural studies by NMR as well as by ab initio calculations showed that in (S(p))- and (R(p))-D(2)-CNA the following occur: (i) the sugar is locked in extreme North-type conformation with P = 11 degrees and Phi(m) = 54 degrees ; (ii) the six-membered 1,3,2-dioxaphosphorinane ring adopts a half-chair conformation; (iii) the fixed phosphate backbone delta, epsilon, and zeta torsions were found to be delta [gauch(+)], epsilon (cis), zeta [anticlinal(+)] for (S(p))-D(2)-CNA, and delta [gauche(+)], epsilon (cis), zeta [anticlinal(-)] for (R(p))-D(2)-CNA. It has been found that F(-) ion can catalyze the isomerization of pure (S(p))-D(2)-CNA or (R(p))-D(2)-CNA to give an equilibrium mixture (K = 1.94). It turned out that at equilibrium concentration the (S(p))-D(2)-CNA isomer is preferred over the (R(p))-D(2)-CNA isomer by 0.39 kcal/mol. The chemical reactivity of the six-membered dioxaphosphorinane ring in D(2)-CNA was found to be dependent on the internucleotidic phosphate stereochemistry. Thus, both (S(p))- and (R(p))-D(2)-CNA dimers (17a and 17b) were very labile toward nucleophile attack in concentrated aqueous ammonia [t(1/2) = 12 and 6 min, respectively] to give carba-LNA-6',5'-phosphodiester (21) approximately 70-90%, carba-LNA-3',5'-phosphodiester (22) approximately 10%, and carba-LNA-6',3'-phosphodiester (23) <10%. In contrast, the (S(p))-D(2)-CNA was about 2 times more stable than (R(p))-D(2)-CNA under hydrazine hydrate/pyridine/AcOH (pH = 5.6) [t(1/2) = 178 and 99 h, respectively], which was exploited in the deprotection of pure (S(p))-D(2)-CNA-incorporated antisense oligodeoxynucleotides (AON). Thus, after removal of the solid supports from the (S(p))-D(2)-CNA-modified AONs by BDU/MeCN, they were treated with hydrazine hydrate in pyridine/AcOH to give pure AONs in 35-40% yield, which was unequivocally characterized by MALDI-TOF to show that they have an intact six-membered dioxaphosphorinane ring. The effect of pure (S(p))-D(2)-CNA modification in the AONs was estimated by complexing to the complementary RNA and DNA strands by the thermal denaturation studies. This showed that this cyclic phosphotriester modification destabilizes the AON/DNA and AON/RNA duplex by about -6 to -9 degrees C/modification. Treatment of (S(p))-D(2)-CNA-modified AON with concentrated aqueous ammonia gave carba-LNA-6',5'-phosphodiester modified AON ( approximately 80%) plus a small amount of carba-LNA-3',5'-phosphodiester-modified AON ( approximately 20%). It is noteworthy that Carba-LNA-3',5'-phosphodiester modification stabilized the AON/RNA duplex by +4 degrees C/modification (J. Org. Chem. 2009, 74, 118), whereas carba-LNA-6', 5'-phosphodiester modification destabilizes both AON/RNA and AON/DNA significantly (by -10 to -19 degrees C/modification), which, as shown in our comparative CD studies, that the cyclic phosphotriester modified AONs as well as carba-LNA-6',5'-phosphodiester modified AONs are much more weakly stacked than carba-LNA-3',5'-phosphodiester-modified AONs.

Fine tuning of electrostatics around the internucleotidic phosphate through incorporation of modified 2',4'-carbocyclic-LNAs and -ENAs leads to significant modulation of antisense properties.

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 internucleotidic 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 part 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 -CH(3)) 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 resistance without loss 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/RNA hybrid duplex, but the cleavage rate depended on the modification site in the AON sequence. If the original preferred cleavage site by RNase H was included in the 4-5nt stretch from the 3'-end of the modification site in the AON, decreased cleavage rate was observed. Upon screening of 52 modified AONs, containing 13 differently modified derivatives at C6' and C7' (or C8') of the carba-LNAs and -ENAs, two excellent modifications in the carba-LNA series were identified, which synergistically gave outstanding antisense properties such as the target RNA affinity, nuclease resistance, and RNase H activity and were deemed to be ideal candidates as potential antisense or siRNA therapeutic agents.

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.

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-Me C, 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. © 2017 by John Wiley & Sons, Inc.

How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?

A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pKa 9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg2+ ion availability at the cleavage site causing sequence-specific hydrolysis of the phosphodiester bond of scissile phosphate. The enhancement of the total rate of cleavage of the complementary mRNA strand by up to 25%, presented in this work, provides opportunities to engineer a single modification site in appropriately substituted AONs to design an effective antisense strategy based on the nucleolytic stability of the AON strand versus RNase H capability to cleave the complementary RNA strand.

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 T(13) position by the [6 '(R)-O-(p-Toluoyl)-7 '(S)-methyl]-carba-LNA-Thymine [IC(50) 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 [IC(50) 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.

Chemical and structural implications of 1',2'- versus 2',4'- conformational constraints in the sugar moiety of modified thymine nucleosides.

In order to understand how the chemical nature of the conformational constraint of the sugar moiety in ON/RNA(DNA) dictates the duplex structure and reactivity, we have determined molecular structures and dynamics of the conformationally constrained 1',2'-azetidine- and 1',2'-oxetane-fused thymidines, as well as their 2',4'-fused thymine (T) counterparts such as LNA-T, 2'-amino LNA-T, ENA-T, and aza-ENA-T by NMR, ab initio (HF/6-31G** and B3LYP/6-31++G**), and molecular dynamics simulations (2 ns in the explicit aqueous medium). It has been found that, depending upon whether the modification leads to a bicyclic 1',2'-fused or a tricyclic 2',4'-fused system, they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and the backbone torsions as well as by characteristic North-East type sugar conformation (P = 37 degrees +/- 27 degrees , phi(m) = 25 degrees +/- 18 degrees ) of the 1',2'-fused systems, and (ii) pure North type (P = 19 degrees +/- 8 degrees , phi(m) = 48 degrees +/- 4 degrees ) for the 2',4'-fused nucleosides. Each group has different conformational hyperspace accessible, despite the overall similarity of the North-type conformational constraints imposed by the 1',2'- or 2',4'-linked modification. The comparison of pK(a)s of the 1-thyminyl aglycon as well as that of endocyclic sugar-nitrogen obtained by theoretical and experimental measurements showed that the nature of the sugar conformational constraints steer the physicochemical property (pK(a)) of the constituent 1-thyminyl moiety, which in turn can play a part in tuning the strength of hydrogen bonding in the basepairing.

Five- and six-membered conformationally locked 2',4'-carbocyclic ribo-thymidines: synthesis, structure, and biochemical studies.

Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range 1H-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 degrees C and 1.5 degrees C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA ( approximately 85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.

Synthesis and structure of novel conformationally constrained 1',2'-azetidine-fused bicyclic pyrimidine nucleosides: their incorporation into oligo-DNAs and thermal stability of the heteroduplexes.

[structures: see text] The synthesis of novel 1',2'-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) "azetidine" pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks (20, 39, and 42) for automated solid-phase oligonucleotide synthesis is reported. The novel bicyclonucleoside "azetidine" monomers were synthesized by two different strategies starting from the known sugar intermediate 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose. Conformational analysis performed by molecular modeling (ab initio and MD simulations) and NMR showed that the azetidine-fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in the range of 44.5-53.8 degrees and sugar puckering amplitude (phi(m)) of 29.3-32.6 degrees for the azetidine-modified T, U, C, and 5-Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes show that the azetidine-fused nucleosides display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues.

Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides.

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn-protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.

The chemical nature of the 2'-substituent in the pentose-sugar dictates the pseudoaromatic character of the nucleobase (pKa) in DNA/RNA.

We here show that the pKa (error limit: 0.01 to 0.03 pKa unit) of a nucleobase in a nucleotide can be modulated by the chemical nature of the 2'-substituent at the sugar moiety. This has been evidenced by the measurement of nucleobase pKa in 47 different model nucleoside 3',5'-bis- and 3'-mono-ethylphosphates. The fact that the electronic character of each of the 2'-substituents (Fig. 1) alters the chemical shift of the H2' sugar proton, and also alters the pKa of the nucleobase in the nucleotides has been evidenced by a correlation plot of pKa of N3 of pyrimidine (T/C/U) or pKa of N7 of 9-guaninyl with the corresponding deltaH2' chemical shifts at the neutral pH, which shows linear correlation with high Pearson's correlation coefficients (R = 0.85-0.97). That this modulation of the pKa of the nucleobase by a 2'-substituent is a through-bond as well as through-space effect has been proven by ab initio determined pKa estimation. Interestingly, experimental pKas of nucleobases from NMR titration and the calculated pKas (by ab initio calculations utilizing closed shell HF 6-31G** basis set) are linearly correlated with R = 0.98. It has also been observed that the difference of ground and protonated/de-protonated HOMO orbital energies (DeltaHOMO, a.u.) for the nucleobases (A/G/C/T/U) are well correlated with their pK(a)s in different 2'-substituted 3',5'-bis-ethylphosphate analogs suggesting that only the orbital energy of HOMO can be successfully used to predict the modulation of the chemical reactivity of the nucleobase by the 2'-substituent. It has also been demonstrated that pKa values of nucleobases in 3',5'-bis-ethylphosphates (Table 1) are well correlated with the change in dipole moment for the respective nucleobases after protonation or de-protonation. This work thus unambiguously shows that alteration of the thermodynamic stability (Tm) of the donor-acceptor complexes [ref. 20], as found with various 2'-modified duplexes in the antisense, siRNA or in triplexes by many workers in the field, is a result of alteration of the pseudoaromatic character of the nucleobases engineered by alteration of the chemical nature of the 2'-substitution.

Synthesis, physicochemical and biochemical studies of 1',2'-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogues.

We have earlier reported the synthesis and antisense properties of the conformationally constrained oxetane-C and -T containing oligonucleotides, which have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells. Here we report on the straightforward syntheses of the oxetane-A and oxetane-G nucleosides as well as their incorporations into antisense oligonucleotides (AONs), and compare their structural and antisense properties with those of the T and C modified AONs (including the thermostability and RNase H recruitment capability of the AON/RNA hybrid duplex by Michaelis-Menten kinetic analyses, their resistance in the human serum, as well as in the presence of exo and endonucleases).