WO2007145593A1 - Conformationally constrained 2' -n,4' -c-ethylene-bridged thymidine (aza-ena-t) - Google Patents

Abstract

A compound according to the formula: The 2 ' -deoxy-2 ' -N, 4 ' -C- ethylene -bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step to give a fused piper idino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North- type conformation. The compound is used to make antisense oligonucleotides which are used for the treatment of cancer and viral infections.

Description

Conf ormationally constrained 2 ' -N, 4 ' -C- ethylene -bridged thymididne (Aza-ENA-T)

The present invention relates to novel compounds as disclosed in the description and drawings that follow below, and defined in the appended claims.

In particular the following features are important to the invention:

(1) New 2',4'-piperdino fused aza-ENA-thymine, -guanine, -adenine, -cytosine or -uarcil (Aza-ENA) and fluoro-nucleobase analogs (Aza-ENA, for example, 13, IS or 18 in Scheme 2) nucleosides are conformationally-constrained nucleosides (North- type) . The Aza-ENA block(s) has/have been incorporated into antisense oligonucleotides (AON, See Table 1 for example) and their antisense properties as gene-directed agent has been evaluated in order to selectively arrest translation of mRNA to protein product.

(2) Aza-ENA modified AONs have shown high target affinity to complementary RNA strand (T_m increase of +2.5 to +9 ⁰C per modification, some examples are given in Table 1), depending upon the substitution site in the AON sequence, compared to the native counterpart.

(3) The global helical structure of aza-ENA modified AON/RNA hybrids, as revealed by the CD spectra, has been found to be very similar to the native AON/RNA duplex suggesting that the local conformational perturbations brought about by the North- conformational^ constrained sugar moiety in aza- ENA modifications are not significant enough to be detected by the CD experiment.

(4) AU of the aza-ENA modified AON/RNA hybrid duplexes have been found to be good substrates for the RNase Hl. In these AON/RNA hybrids, a region of 5 to 6 nucleotides in the RNA strand in the 3'-end direction from the site opposite to the aza-ENA modification, was found to be insensitive toward RNase H cleavage presumably owing to the local structural perturbations brought about by the conformationally constrained modifications. These cleavage patterns of the aza-ENA modified AON/RNA hybrids is uniquely different from that of the oxetane modified AONs which had shown found a gap of 5 nucleotides units.

(5) All the aza-ENA modified AONs offered greater protection towards 3' exonucleases compared to the native sequence. In fact, all the modified AONs cleaved at one nucleotide before the modification towards 3'-end and did not degrade any further. These residual AONs have been found to be stable for over 48 h in human serum and for over 24 h with the snake venom phosphodiesterase. This result clearly suggests that a single modification at the second position from the 3 '-end can give even more substantial stability towards 3' exonucleases. (6) This study provides valuable information regarding the optimal design of AONs or small interferring RNAs with chimeric RNAs or/ and in conjunction with its fluorine-modified analogs (siRNA), having completely natural phosphodiester or phosphorothioate backbone, for the therapeutic applications that show high target affinity, high stability towards nucleases in vivo as well as high tissue-specific distribution.

(7) New 2',4'-piperdino fused aza-ENA-thymine, -guanine, -adenine, -cytosine or -uarcil (Aza-ENA) and its derivatives such as mono- di or tri-phosphates and fluoro analogs can be specifically used to inhibit virus- or -tumor specific proteins, and thereby inactivate the pathogen/tumor growth. These analogs have also found to be useful in various diagnostic applications and assays including polymerase chain reaction.

Drawing Figure 1 shows the structure of one compound according to the invention, namely aza-ENA-thymine.

The bicyclonucleoside "aza-ENA", having 2-aza-6-oxabicyclo[3.2.1]octane skeleton, has been synthesized using a key cyclization step involving 2'-αra-trifluoromemylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-ZjZ₁S-OBn protected diastereomerically pure aza-ENAs (12a and 12b) with the chair conformation of the piperidino skeleton, whereas the pentofuranosyl moiety is locked in the North-type conformation (7° < P < 27°, 44°< φ_m < 52°). The origin of the chirality of two diastereomerically pure aza-ENAs was found (by ³JHH analysis) to be due to the axial N-H in 12b and the equatorial N-H in 12a, which is both kinetically and therrnodynamically preferred, E₀ = 25.4 kcal mol^"1, a highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The thymine derivative 5'-<9-DMTr-aza-ENA- 3'-phosphoramidite was employed for solid-phase synthesis to give four different singly-modified 15-

mer antisense oligonucleotides (AON). Their AON/RNA duplex, ₅Lf₍ ⁽G_AAGA_AAAAAI?G_AAGf ₃"

showed a T_m increase of 2.5 to 4 ⁰C per modification, depending upon the modification site (m bold).

The relative rates of the RNase Hl cleavage of the aza-ENA-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 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 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.

Introduction

Modified Oligonucleotides have successfully been used as valuable tools to inhibit the gene expression by utilizing various mechanisms of actions.^1"7 The most matured method is the antisense technology⁶' ⁸ which exploits ability of a single stranded DNA-oligonucleotide to bind to the target messenger RNA (mRNA) via Watson-Crick base pairing in a sequence specific manner. Once bound to the target RNA, antisense agent either sterically blocks the synthesis of ribosomal proteins or induces RNase H mediated degradation of the target mRNA. A revolutionary discovery made recently is the use of the short double stranded RNA duplexes called small interfering RNAs (siRNA)⁷' ^9"π which silence gene expression utilizing naturally occurring mechanism called RNA interference (RNAi). Unlike traditional drug discovery approaches, in which a small ligand specifically binds to an active receptor site of a target protein (which is often difficult to identify), oligonucleotides can be aimed to block one or many part(s) of a target RNA sequence transcribed from, for example, of a disease-causing gene because of straight-forward basepairing rules of nucleic acids. These oligonucleotide based approaches of course have the unique advantage that it has the potential to bypass the chronic resistance problem often encountered with the protein targets. For in vivo applications of the oligonucleotide based approaches, appropriate chemical modifications are warranted to enhance target affinity, specificity, stability towards the endo and exonucleases, as well as tissue-specific delivery in order to improve the overall pharmacokinetic properties. The diversity of chemical modifications in oligonucleotides is continuously growing¹²' ¹³ ever since the antisense technology has opened up the therapeutic horizon¹⁴' ¹⁵ of oligonucleotide-based gene silencing agents, which still remains to be an important unsolved challenge to chemists. It is still very much an open question whether it is possible to come up with an optimally modified monomer blocks with natural phosphate backbone, which can successfully address all the above issues related to the improvement of the pharmacokinetic properties.¹²' ¹⁶

Among various sugar, phosphate and nucleobase modifications reported¹²' ¹³, synthetic oligonucleotides having conformationally constrained furanose fused bicyclic and tricyclic carbohydrate moieties^17"20 or modified pyranose derivative ^{16> 21> 22} in the monomer nucleotide units have been found to be promising in terms of target RNA binding, accessibility and nuclease resistance. The enhanced target binding ability of oligonucleotides modified with Afo/t/z-conformationally constrained sugar units^23"30 can be attributed to the conformational pre-organization by improved stacking between the nearest- neighbors,³¹ thereby minimizing the entropic energy penalty in the free-energy of stabilization for the duplex formation with RNA.

Figure 1: Structures of North-type conformationally-constrained α/β-D/L-pentofuranosyl nucleoside derivatives

Incorporation of the North-type sugar conformationally constrained nucleotides (-1° < P < 34°) into AON has been found to be an excellent antisense strategy to induce several favorable properties to the modified AONs compared to the native counterparts. These nucleotides have allowed highly favored duplex formation with complementary RNA as they had RNA-like (Cy-endό) sugar conformation.²⁶' ^{32> 33} and have shown enhanced stability towards the endo and exonucleases in the blood serum. Notable examples are the 2',4'-bridged nucleosides such as: the 2'-0,4'-C-methylene bridged nucleoside (LNA/BNA) (structure A in Figure 1), reported independently by Wengel et al. and Imanishi et al.³⁴ having furanose fused [2.2.1] five-membered ring. LNA incorporated oligonucleotides showed unprecedented level of affinity towards complementary RNA (AT_m ~ +4° to +8 ⁰C per modification) and a moderate increase of +3 to +5 ⁰C per modification towards complementary DNA.³² Wengel's group has also reported increased affinity towards complementary RNA for other LNA analogs having 2'-thio³⁵ (Δ7j_n ~ +8 ⁰C per modification), 2'-amino³⁶ (structure B in Figure 1, AT_m ~ +6 to +8 ⁰C per modification), xylo-LNA²⁹' ³⁷ (structure C in Figure 1, Δ7"_m ~ -4 ⁰C per modification) and α-L-LNA³⁷' ³⁸ (structure D in Figure 1, AT_m ~ +4 to +5 ⁰C per modification) function. Wang and co workers have reported 2',4'-C-bridged 2'-deoxynucleotides²⁶' ³⁹ (structure E in Figure 1), which showed moderate affinity to RNA (AT_m ~ +1.9 to +3.3 ⁰C per modification). Other bridged nucleosides having 2',4'-C-propylene-bridged 2'-deoxynucleoside¹³ (structure F in Figure 1) have also been reported which showed unfavourable duplex formation with complementary RNA (AT_m — 2.3 ⁰C per modification). We have previously reported, l',2'-bridged oxetane⁴⁰' ⁴¹ (structure G in Figure 1), and azetidine⁴² (structure H in Figure 1), North—type conformational^ constrained nucleotides which have shown sequence-specific target affinity in the following manner: AT_m drop for the AON/RNA duplexes of ~ -5 ⁰C per oxetane-T, ~ -3 ⁰C per oxetane-C and no 7J_n drop for oxetane-A and oxetane-G⁴⁰ modification, and -4 ⁰C for azetidine-T/U and to -2 ⁰C for azetidine-C⁴² modification.

Another TVort/z-constrained sugar modification recently reported by Koizumi et al.²⁷' ³³ is the 2'- O,4'-C-ethylene bridged nucleoside²⁷' ³³ (ENA) (structure I in Figure 1), which showed target affinity with complementary RNA (AT_1n ~ +3.5 to +5.2 ⁰C per modification) as high as that of the isosequential LNA²⁷. The same group reported 2'-0,4'-C-propylene bridged nucleoside (PrNA)³³ (structure J in Figure 1) which on the other hand did not show appreciable T_n, enhancement (AT_m ~ -0.5 ⁰C per modification). Koizumi et al.²¹ have also shown that the ENA modified AONs have approximately 55 times higher stability towards 3'-exonuclease compared to the LNA analog.²⁷ This ability of ENA for efficient target binding and high nuclease resistance have been well exploited to evaluate their antisense, antigene and RNAi^43"47 properties: High target affinity has also been reported⁴⁶ for the triplex formation by ENA monosubstituted oligopyrimidine 2'-deoxynucleotides efficiently binding to dsDNA at physiological pH.⁴⁶ ENA/DNA chimeric AONs have also been used to study the RNase H mediated antisense activity.⁴⁴ More than 90% inhibition of VEGF mRNA production was observed after RT-PCR analysis⁴⁴ when these AONs were used against vascular endothelial growth factor (VEGF) mRNA in A549 lung cancer cells in the presence of a cationic polymer. Incorporation of the ENA residues at 3'- and 5'-ends of AONs that specifically target the genes of rat organic anion transporting polypeptide (oatp) subtype resulted in enhanced inhibitory activity mediated by RNase H with high selectivity⁴³. AONs having T- 0-methyl RNA/ENA chimera were found to be 40 times more effective in exon-19-skipping compared to conventional phosphorothioate AONs associated with human dystrophin gene.⁴⁸ Similarly, the RNA/ENA chimera duplexes were also found to induce skipping of the exon-41 containing the nonsense mutation which suggested that such design of ENA incorporated AONs could be used to promote dystrophin expression in mycocytes of Duchenne muscular dystrophy (DMD) patients.⁴⁹ The RNAi effect of a chemically synthesized siRNA with ENA modification at 3 '-end targeted to mRNA of Jun dimerization protein-2 (JDP2) has also been evaluated.⁴⁷ The 3 '-end modification was found to be recognized by RNA-induced silencing complex (RISC) which resulted in loss of RNAi activity.⁴⁷

These properties of ENA modified AONs, namely high target affinity, sequence selectivity towards ssRNA/dsDNA targets and high nuclease resistance (in vivo and in vitro), prompted us to design the 2'-amino modified ENA analogs (aza-ENA, compound E in Scheme 1), as a new class of conformationally-constrained AON which could potentially be an important analog for effective gene- directed therapeutics and diagnostics. The aza-ENA based AONs may have three clear advantages over the corresponding ENA counterpart: First, the endocyclic amino functionality of the aza-ENA analog could be utilized as a well defined conjugation site⁵⁰ and thereby we can control the hydrophilic, hydrophobic and steric requirements of a minor groove of the duplex. Second, the amine-derivatized

AONs have displayed increased thermal affinities⁵¹' ⁵² towards complementary RNA possibly because of the presence of positively charged moieties at physiological pH, and thus could influence partial neutralization of the negatively charged phosphates in the duplexes.³⁶'⁴² Third, introduction of a fluorescence probe connected to this nitrogen moiety will enable us for real-time in vivo imaging of RNA and can therefore be used for specific detection of nucleic acids while maintaining their hybridization properties.⁵³' ⁵⁴

We report here the synthesis of AONs incorporated with 2'-deoxy-27V,4'C-ethylene bridged nucleoside (aza-ENA) having 2-aza-6-oxabicyclo[3.2.1]octane skeleton (Compound E in Scheme 1). The NMR and computational structural studies of aza-ENA and its analogs are also reported here, showing that the piperidino moiety of the aza-ENA is indeed locked in the chair conformation (with the nitrogen lone-pair in the axial and N-H proton in the equatorial position), whereas the fused sugar is constrained to a North-type conformation similar to that of the 2'-(9,4'-C-ethylene bridged ENA analog.²⁷' ³³ Finally, the aza-ENA nucleotides have been incorporated in 15-mer AON as single modification at four different sites to give four mono aza-ENA substituted AONs #2-5 (sequences shown in Table 1). These AONs have shown an increase in the thermal stability from 2.5 to 4 ⁰C per modification towards complementary RNA depending upon the substitution site. We also show that the relative rates of the RNase Hl promoted cleavage of the aza-ENA-modified AON/RNA heteroduplexes are comparable to that of the native counterpart, and, quite interestingly, the aza-ENA modifications also results in significant increase of AON/RNA resistance to 3'-exonucleases degradation in the blood serum compared to the native counterpart.

Results and Discussion

Synthesis of all 2',4'-bridged nucleosides reported³²' ³³ so far, involve nucleophilic displacement reaction with the nucleophile positioned at C2' and a leaving group at the extended arm of C4' (general structure "A₁" in Scheme 1). We considered an alternative strategy (general structure "A₂" in Scheme 1) for nucleophilic ring closure to give constrained nucleosides: An intermediate such as (B) in Scneme i, can potentially lead us to two types of products depending upon whether we generate a carbanion at the α-carbon to the cyano group (path A in Scheme 1), or successfully reduce the cyano to the primary

amino group without generating a α-carbanion next to the -CN (path B in Scheme 1). We argued that in the former case we might be able to engineer the construction of a cyanomethylene-bridged carbocyclic [2.1.1] system (D), and in the latter could lead us to 2'-deoxy-2'-7V,4'-C-ethylene bridged nucleoside (aza-ENA) having 2-aza-6-oxabicyclo[3.2.1]octane skeleton.

Scheme 1. Structure (A₁): General strategy for 2',4'-cyclization so far reported in the literature involves engineering of a nucleophile (Nu) at C2' and a leaving group (LG) at the extended arm of C4'. Structure (A₂), on the other hand, shows a reverse strategy in which the leaving group LG is placed in the sugar moiety, whereas the nucleophile Nu is engineered at the side-arm. Structure (B): Our strategy involving A₂ (Path "B") for 2',4'-cyclization to give conformationally-constrained 2'- N, 4'-C-cyclization to give [3.2.1]-fused-aza-ENA involves a nucleophile at C4¹ (Nu) and a departing group at C2' (LG). If however a α-carbanion is developed (Path "A") as shown in (C), a ring-closure reaction to give cyanomethylene-bridged carbocyclic [2.1.1] system (D) is formed.

Thus, upon treatment of intermediate (B) (Scheme 1) with NaHMDS in THF, anhydrous THF, r.t., 3 h, we found the formation of a stable fused product (D) (Scheme 1) in 35% yield through the participation of the intermediate (C) (Scheme 1). The structural integrity of this conformationaiiy- constrained product was proven by long-range ¹H, ¹³C NMR correlation (HMBC) experiment [Inset (i) in Figure 2] which showed C2VH6¹ (²Jcn), through bond correlation thereby providing unequivocal evidence for the ring-closure to give 2',4'-cyanomethylene bridged carbocyclic thymine nucleoside having cyclobutane fused [2.1.1] system (See SI Figure S9 for complete spectrum). 1 D difference nOe enhancement (Inset (ii) in Figure 2) of H6 (7.5%), H6' (3.8%), and H2' (4.3%) upon irradiation of H3' shows the steric proximities between H6, H6', H2'and H3' (dπ6-H3_' —2.5 A, duβ_'-m¹ —3.5 A and dm_'-ro¹ ~ 2.8 A) confirming the conformationally-constrained North-type sugar structure. The mass-spectral data by MALDI-TOF also provides evidence for the structural integrity of highly strained carbocyclic nucleoside (see Experimental section for full characterization). However our efforts to remove the benzyl group from compound (D) (Scheme 1) so far has not met with success. The other problem remains to find a mild reducing agent, which can convert the -CN group in compound (D, Scheme 1) to a relatively poorer electron-withdrawing group (for example to an amino function) in order to assess its potential in the strategies concerning gene-directed drug development (reduction by (CFsCO^BH was unsuccessful).

These initial unsuccessful efforts to proceed through path "A" in Scheme 1 has made us to explore the feasibility of structure B (path "B", Scheme 1) which has a masked amino function in the form of a -CN group in the side arm as well as a -CH₂- for one carbon homologation, both of which are necessary for intramolecular cyclization to yield novel aza-ENA nucleoside (compound E, Scheme 1), a 2'-amino analog of ENA (compound I in Figure 1). The complete synthetic strategy which leads us to successful synthesis of aza-ENA is shown in Scheme 2. It is also noteworthy that the aliphatic nitrile (pK_a ~ 29-31)⁵⁵ derivatives (4-11, Scheme 2) were fully compatible with the synthetic strategy containing strongly acidic and basic conditions as described in Scheme 2.

Figure 2. Inset (i): ¹H-¹³C HMBC spectra showing the long range through-bond connectivities between C27H6' (²J_Cn), C37 H6' (³J_cπ), and C5VH6' (³J_CH) for 2',4'-cyanomethylene bridged carbocyclic thymine nucleoside (compound D, Scheme 1), Inset (ii): ID selective NOESY spectra of compound D (Scheme 1) showing enhancements at H6 (7.5%), H6' (3.8%), and H2' (4.3%) upon irradiation at H3'.

(I) Synthesis of Aza-ENA nucleoside

The synthesis of aza-ENA derivative was started with known sugar precursor³² 1, which was converted to 3,5-di-<9-benzyl-4-C- hydroxymethyl-l,2-0-isopropylidene-α-D-ribofuranose 2³². Compound 2 was then converted to the 4- triflyloxymethylene derivative 3 using triflic anhydride in dichloromethane-pyridine mixture (3:1, v/v) at 0 ⁰C. The crude product obtained after aqueous workup was subsequently treated with 3 equivalents of LiCN in DMF, and stirred at r.t. for 3 days, which afforded cyano-sugar 4 in an overall yield of 56% from 2, along with some unidentified minor compounds. Compound 4 was converted to diacetate 5 (l,2-di-0-acetyl-3,5-di-0-benzyl-4-C- cyanomethyl-D-ribofuranose) using a mixture of acetic acid, acetic anhydride and triflic acid by stirring

11

Scheme 2: Reagents and conditions: (i) NaH, BnBr, CH₃CN, -5 ⁰C to r.t, overnight; (ii) Tf₂O, pyridine, CH₂Cl₂, 0 ⁰C, 3 h; (iii) LiCN, DMF, r.t, 3 days; (iv) Acetic acid, Ac₂O, triflic acid, r.t., 3 h; (v) persilylated thymine, TMSOTf, CH₃CN, 80 ⁰C, overnight; (vi) NaOMe, methanol, 3 h; (vii)

MsCl, pyridine, O ⁰C, 6 h; (viii) DBU, CH₃CN, r.t., 1 h; (ix) 0.1 M H₂SO₄, acetone, reflux, overnight; (x) Tf₂O, pyridine, CH₂Cl₂, DMAP, O ⁰C, 2.5 h; (xi) NaBH₄, trifluoroacetic acid, THF, r.t., overnight; (xii) Pd(OH)₂, ammonium formate, methanol, reflux, overnight, followed by 1 M BCl₃ in

CH₂Cl₂, -78 ⁰C, 3 h; (xii) phenoxyacetyl chloride, pyridine, r.t., 3 h; (xiv) DMTr-Cl, pyridine, r.t., 7 h (overnight for 19); (xv)

NC(CH₂)₂OP(Cl)NCPr)₂, DIPEA, THF, r.t., 3 h, (overnight for 20);

Abbreviations: Bn = benzyl, r.t. = room temperature, Ac = acetyl, Tf = trifluoromethylsulfonyl, PAC = phenoxyacetyl, THF = tetrahydrofuran, DBU = l,8-diazabicyclo[5.4.0]undec-7-en, DMTr = 4,4'-dimethoxytrityl, DIPEA = diisopropylethylamine, TFA = trifluoroacetyl.

(A): Major isomer (12a) with N-H_e (B): Minor isomer (12b) with N-H₃

(C): Major isomer with N-H_e (D): Minor isomer with N-H₃

Figure 3. Insets (A) and (B) are the ¹H-¹³C HMBC spectra showing the long range through-bond connectivities between C77H2' and C2'/H7_e' for the two diastereomers 12a and 12b. Insets (C) and (D) are energy-minimized stereochemical representations, showing the ¹H-¹³C HMBC connectivities of the two diastereomers 12a and 12b; R = OCH₂Ph (not shown). for 3 h at r.t. The crude product 5 (4 → 5 was almost quantitative) was subjected to modified Vorbruggen reaction⁴²' ⁵⁶ using in situ silylation of thymine and subsequent trimethylsilyl triflate mediated coupling to give the /3-confϊgured thymine nucleoside 6 in 80% yield. The β configuration of 6 was confirmed by ID differential nOe experiment which showed 8% nOe enhancement of H-6 upon

irradiation of H-2' (CI_H6-_H2' —2.7 A for β-anomer, and dπβ-m' ~4 A for α-anomer). Deacetylation of 6 at C2' was carried out using sodium methoxide in methanol at r.t., and the product (7) was isolated as a crude material (single spot on TLC, for ¹³C NMR see Figure S 12 in SI), which was directly mesylated using mesyl chloride in pyridine at r.t. to afford 8 with an overall yield of 95% in 2-steps from 6. The compound 8 was converted to the 2, 2'-anhydro product 9 using 1.05 equivalent of DBU in acetonitrile in 91% yield. It should be noted that the use of excess base or stronger base such as NaHMDS resulted in instantaneous de-pyrimidation. Opening of 2, 2'-anhydro ring in 9 went smoothly by refluxing with a mixture of 0.1 M aqueous sulfuric acid-acetone (1: 1, v/v) to give the arabino product (10) quantitatively.

Treatment of 10 with triflic anhydride, pyridine, DMAP and anhydrous CH₂Cl₂ at 0 ⁰C gave the desired triflate nucleoside 11 in 84% yield. Reduction of cyano group using trifluoroacetoxy borohydride,⁵⁷ prepared in situ from NaBH₄ and trifluoroacetic acid gave the primary amine which spontaneously resulted in intramolecular cyclization to give a mixture of two diastereomeric aza-ENA 12a and 12b isomers, isolated in 40 and 5% yield, respectively. These diastereomers showed identical masses by MALDI-TOF mass spectroscopy (see Experimental section). That the intramolecular ring- closure reaction has indeed taken place to give the 2'-deoxy-2'-JV, 4'-C-ethylene bridged nucleoside (aza- ENA) having 2-aza-6-oxabicyclo[3.2.1]octane skeleton fused with North-conformationally constrained pentofuranosyl moiety in 12a and 12b was unequivocally proven by long range ¹H₅ ¹³C NMR correlation (HMBC) experiment (Figure 3). The benzyl groups of the diasteromeric 12a/12b were deprotected for characterization using Pd(OH)₂ZNHsCO₂ in methanol and subsequently BCl₃ in dichloromethane at -78 ⁰C to aza-ENA (13) in 60% yield (Scheme 2). (II) NMR Characterization of 12a, 12b and 13

The characterization and conformational analysis of 12a, 12b and 13 has been performed using NMR data (recorded at 500 and 600 MHz in CDCl₃ZDMSO-^₆) obtained by double homodecoupling experiments, ID NOESY⁵⁸' ⁵⁹, ID selective TOCSY⁵⁹ and ¹³C NMR experiments including DEPT⁶⁰ as well as by long-range ¹H-¹³C HMBC correlation (²J_H;C and ³J_HjC )⁶¹ and one-bond HMQC experiment.⁶²

(A) Assignment of ¹H and ¹³C chemical shifts and Evidence for ring closure in 12a and 12b and 13: The COSY and HMQC experiment allowed us to assign the ¹H and ¹³C chemical shifts for 12a, 12b and 13. The observed long-range HMBC correlation (Figure 3) of one of the H7 protons with C2' unequivocally proves the ring-closure reaction (11 → 12a/12b, Scheme 2) and it will be discussed below.

The ID NOESY spectra of both the isomers 12a and 12b (Figure 4) showed nOe enhancements (3.9% and 8.0% in 12a and 12b respectively), for one of the H7 protons (i.e. H7_a) upon irradiation of Hl' in both isomers. That the nOe contact between one of the H7 protons and Hl' has been observed suggests that both the compounds 12a and 12b are in chair conformation and rules out the possibility of boat conformation because estimated distances between those protons in case of the boat conformation would be significantly larger (Hl' - H7_a about 3.7 A; Hl' - H7_e about 4.4 A) compared to the estimated distance between H7_a and Hl' in the case of chair conformation (Hl' - H7_a about 2.3 A).

The COSY and ID selective TOCSY experiments showed coupling between H7_a Z H7_e, and the N-H proton in 12b (Figure S22 in SI) which was used to assign the N-H orienation in the latter (see discussion below). The D₂O exchange experiment and detailed proton spin-spin multiplet simulation (Figure S3-S5 in SI) further confirmed the presence of N-H proton (δ 4.53) in 12b.

(B) Major isomer 12a

The upfield H6_e proton (δl .31) of 12a was distinguished from the FI6_a proton (52.02) by the fact that the former has only a smaller JH₁H coupling of 4.8 Hz beside a geminal coupling of 13 Hz, whereas H6_a proton has a large trans Jn_1H coupling of 11.6 Hz and a cisoid J_H1H coupling of 6.7 Hz. This assignment of H6_a/H6_e protons allowed us to identify immediately the H7_a/H7_e protons (at 53.13 and 53.02) by a

double-decoupling experiment (decoupled simultaneously at 52.02 and 51.31). Similarly, a stepwise decoupling of either H6_e or H6_a proton led us to determine the vicinal coupling constant for H7_e or H7_a proton. The four-line multiplet (doublet of doublet, ³J_H-_7e,_H-_6a ⁼ 6.5 Hz and ²Jn-_7a,n-_7e= 13.3 Hz) at 63.02

was thus assigned to H7_e and the eight-line multiplet at 5 3.13 (doublet of doublet of doublet, ³JH-7a,H-₆a = 11.6 Hz,

4.8 Hz, ²JH_7a;H_7e= 13.3 Hz) was assigned to H7_a. This shows that the dihedral angle between H6_e and H7_e, φ[H7_e-C7-C6-H6_e], is close to 90°. These assignments were further confirmed by detailed proton spin- spin simulation experiments (see Figure S3 -5 in SI) as well by 2D COSY spectra (see Figure S20 in SI). There was no ³JH7_a,H7e coupling observed with the vicinal N-H proton, suggesting that the N-H proton is presumably cisoid with both H7_a/H7_e protons (i.e. N-H proton is most probably equatorial). Furthermore, the HMQC and HMBC spectra allowed us to correlate the ¹H chemical shifts with the ¹³C chemical shifts. The FfMBC spectrum showed that only H7_e had correlations with C2' but not between H7_a and C2'. This suggested that the dihedral angle between H7_e and C2', φ[H7_e-C7-N-C2'],

is close to 180°, whereas H-7_a and C2', φ[H7_a-C7-N-C2'], is about 90°. The presence of long-range HMBC correlation of H7_e with C2' also unequivocally showed that the six-membered piperidino [3.2.1] ring fused with the pentofuranose ring has indeed been formed in the ring-closure reaction (U → 12a/12b, Scheme 2).

(C) Minor isomer 12b

Analogous to the assignment of the axial and equatorial H6 protons of the piperidino ring, the H6_e proton (51.53) of 12b was distinguished from the H6_a proton (52.04) by the fact that the former has only a smaller ³J_H,_H coupling of 5.3 Hz beside a geminal coupling of 13.4 Hz, whereas H6_a proton has a large trans ³J_H1H coupling of 11.9 Hz and cisoid ³J_H>H coupling of 6.7 Hz. A set of double and single decoupling experiments at the centre of multiplets of H6_a and/or H6_e protons gave the assignment of both the H7_a and H7_e protons. From the geometrical point of view the H7_a is expected to have different

couplings with H6_a (φ[H7_a-C7-C6-H6_a] is close to 180°, hence ³Jn_1H coupling should be large) and H6_e

(φ[H7_a-C7-C6-H6_e] is close to 55° hence ³J_H1H coupling should be small). The ³J_H1H coupling constant

between equatorial H7_e and H6_a is expected to be medium (φ[H7_e-C7-C6-H6_a] is about 40°), while that

between H7_e and H6_e is expected to be zero (φ[H7_e-C7-C6-H6_a] is close to 90°). Thus, the H7_a has been

assigned to the 12-line multiple! (doublet of triplet of doublet) at δ 2.98 because it has three

distinguished vicinal couplings (³JH-7_a,H-_6a - 1 1.9 Hz and ³JH-7_a,H-6e ⁼ 5.2 Hz and ³JH-7a,NHa⁼ H -9 Hz) besides the geminal coupling

14.2 Hz) The H7_e has been unambiguously assigned to the 8-

line niultiplet (doublet of doublet of doublet) at 53.26 because it had only two vicinal couplings (³J_H-7_C,_H-

6a - 6.4 Hz and

⁼ 14.2 Hz). The ³Jπ-7_a coupling observed with the vicinal NH₃ proton of 11.9 Hz and ³J_H-_7e coupling with the vicinal NH_a proton of 3.9 Hz suggested that the NH proton is presumably transoid with respect to H7_a and cisoid with H7_e protons (i.e. NH₃ proton is most probably axial). 2D COSY spectrum (see Figure S25 in SI) for the minor isomer 12b again was used to confirm that the H6_e is only coupled with H-7_a. ID selective TOCSY experiment confirmed the spin system for H7_a, H7_e, H6_a, H6_e, as well as that of the NH₃ proton

at 54.55 ppm (Figure S22 in SI). Furthermore, HMBC spectrum confirmed the assignment of H7_e by

having its correlation with C2' but not between H7₃ and C2' (Figure 3). This furthermore proved that the

dihedral angle between H7_e and C2', φ[H7_e-C7-N-C2'], is «180°, whereas H-7₃ and C2¹, φ[H-7_a-C7-N-

C2'j, is in the region of «90°.

(D) De-protected piperidino [3.2.1] constrained nucleoside 13:

The H6_e proton (51.17) of 13 was distinguished from the H-6_a (51.78) by the fact that the former

has only a smaller ³J_H1H coupling of 4.6 Hz besides a geminal coupling of 12.9 Hz, whereas H6_a proton has a large trans ³J_H1H coupling of 13.0 Hz and cisoid ³J_H1H coupling of 6.8 Hz. A set of double and single decouplings at the centre of multiplets of H6_a and/or H6_e protons gave both the assignment of H7_a (A) : Major isomer (12a) with N-H₆ (B) : Minor isomer (12b) with N-H₃ (C): nOe contacts (D): nOe contacts

H1'

ppm 3.8 3.6 3.4 3.2 ppm ppm 3 . 5 3 . 0 ppm

Figure 4: ID selective NOESY spectra of 12a and 12b. Inset (A) In 12a the irradiation at Hl' shows enhancements at H7_a (3.9%), H2' (1.5%), and H6 (1.8%). Inset (B) In 12b the irradiation at Hl' shows enhancements at H7_a (8.0%), H2' (5.5%), and H6 (5.9%). Insets (C) and (D) show the nOe contacts in the two diastereomers 12a and 12b, respectively; R = OCH₂Ph , which are not shown for clarity of the picture.

and H7_e protons as well as their vicinal couplings. The H7_a was assigned to the six-line multiplet (doublet of triplet) at 52.95 because of two vicinal couplings (³J_H7_a,H6a ⁼ 13 Hz and ³J_H7_a,H6c = 4.8 Hz) besides the geminal coupling (²JH_7a,H7e ⁼ 12.8 Hz) and H7_e was assigned to the four-line multiplet (doublet of doublet) at 52.87 The four lines of H7_e were due to one vicinal coupling (³Jκ-_7e,_H-_6a ⁼ 6.6 Hz) besides geminal coupling (²JH7_a>H7e (gem) = 12.8 Hz). No couplings were observed between H7_e and H6_e confirmed by 2D COSY experiments. This shows that the dihedral angle between H6_e and H7_e, φ[H7_e-

C7-C6-H6_e], is about 90°. HMBC spectrum confirmed the assignment of H7_e by having its correlation with C2' but not between H7_a and C2' (Figure S32 in SI). This furthermore proved that the dihedral angle between H7_e and C2', φ[H7_e-C7-N-C2'], is «180°, whereas H7_a and C2¹, φ[H7_a-C7-N-C2'], is in the

region of «90°. The complete ID and 2D NMR spectral assignments as well as the NMR spectra for 12a, 12b and 13 are available in SI.

(Ill) The ³J_HH simulation and nOe Studies of 12a and 12b and 13

All apparent coupling constants observed and simulated using MestRec software⁶³ (Figures S3-S5 and Table S3 in SI) suggested that the H7_a and the NH are in trans-diaxial position in the minor isomer 12b which is only possible when the NH is axial (NH_a). On the other hand, absence of NH coupling with H7_a or H7_e showed that the dihedral angle, φ[H7_a(H7_e)-C7-NH_e] is in the region of 80-90°, which means that N-H is in equatorial position (NH_e) in the major isomer 12a. This is further evidenced by the fact that the chemical shift of H7_a is more upfϊeld compared to that of H7_e in the minor isomer 12b because H7_a is in the cisoid orientation with the nitrogen lone-pair. Similarly, the situation is reversed in the major isomer 12a by the fact that the nitrogen lone-pair is now cisoid to H7_e, hence it is more upfield shifted than that of H7_a. The absence of Hl' and H2' coupling constant in 12a and 12b indicate that this locked nucleoside has a unique C3'-endo type conformation as observed in LNA and ENA analogs. Strong nOe contacts (10% to 11%) between H6 and H3' for both 12a and 12b further confirms that the sugar is indeed fixed in North conformation as observed for other YVortλ-locked nucleoside such as ENA²⁷' ³³ and LNA³² (8 to 9% nOe between H-6 and H3').

In the fully de-protected nucleoside 13 in DMSO-rf,_?, the H7_e at 52.87 and H7_a at 52.95 showed a

difference in their multiplicities: The multiplet at 52.95 for H7_a appeared as a well-resolved 6-line spectrum with JH7_3i w_e (_gem) = 12.8 Hz, ³JH7_a> H6a ⁼ 13.0 Hz, and ³JH7a, H6e ⁼ 4.8 Hz, while the multiplet at 52.87 for H7e was a 4-line spectrum with ²Jwi&, me (gem) ⁼ 12.8 Hz, and ³JH7_e, H6a ~ 6.8 Hz. On the other

hand, absence of N-H coupling with H7_a or H7_e suggests that the dihedral angle, φ[H7_a(H7_e)-C7-NH_e] is

in the region of 80-90°, which means that N-H is in the equatorial position (NH_e) in the fully de- protected major isomer 13. This is further evidenced by the fact that the chemical shift of H7_e is more upfield compared to that of H7_a because H7_e is in the cisoid orientation with the nitrogen lone-pair. The vicinal and geminal coupling constants and the multiplicities in the ¹H spectra as well as the resemblance of the HMBC spectra of 13 with those of 12a shows that the conformation of fully de- protected major isomer is similar to that of 12a.

(TV) Conversion of Minor Diastereomer (12b) to the Major Diastereomer (12a): Kinetics and Conformational studies of Nitrogen Inversion.

It should be noted that both diastereomers 12a and 12b could be isolated in pure form, and fully characterized by NMR and mass-spectroscopy, suggesting that these isomers were fairly stable in CH₂Cl₂ containing ca 5-10% methanol or in CHCl₃ solution. The piperidino moiety in the major isomer (12a) takes up the chair conformation with N-H equatorial (see Section III) to reduce 1,3-diaxial interaction, whereas the minor isomer (12b) also having piperidino ring in the chair conformation and N-H axial (see Section III) is expected to be relatively less stable because of 1,3-diaxial interaction.

This was further proved by determining the rate of isolated nitrogen inversion for the conversion of the minor (12b) to the major diastereomer (12a) in pyridine-ds (Figure 5) using the first order kinetics. In pyridine-j₅, the minor isomer (12b) was found to have converted almost completely (>99%) to the major isomer (12a) in 33 h at 298 K, and no reverse isomerism was observed starting from the major isomer (12a) under our experimental conditions. We have subsequently determined the rate of inversion at 293 K (k = 1.0 x 10^"5 sec^"1), 298 K (£ = 4.4 x 10^"5 sec^"1), 303 K Qi = 8.0 x 10^"5 sec^"1), 308 K (k = 1.0 x 10^"4 sec^"1), and 318 K (k = 4.0 x 10^"4 sec^"1), respectively using equation of the unimolecular first order rate kinetics.⁶⁴ The populations of 12a and 12b at different time intervals were obtained from the peak integrals of H6 for the two isomers. H6 T (12;a) H1 ' (12a)

S .2 S.1 p pm 7.0 6.Θ β .6 pp m

Figure 5. Non-reversible conversion of 12b (with NH₃) to 12a (with NH_e) in pyridine-Js at 298 K.

Plot of In k Vs 1/T (Arrhenius equation)

0.0031 0.0032 0.0033 0.0034

1/T (K^"1)

Figure 6. Determination of activation energy (E_a) for the nitrogen inversion of 12b to 12a using an Arrhenius plot of In A: vs. 1/T. The Arrhenius plot of In k versus 1/T shows a linear correlation with R = 0.98. The slope gave the E_a = 25.4 kcal mol^"1, whereas the intercept showed the frequency of collision factor A = 1.190 x 10¹⁴ s^"1 (Figure 6). The free energy of activation was also calculated⁶⁵ which was found to be AG^ = 23.4 kcal mol^"1 at 298 K in pyridine-^, hi CDCl₃, the two diastereomers 12a and 12b have very slowly (in 30 days) reached an equilibrium (40:60 of 12a:12b) with equilibrium constant⁶⁴ K_c = 0.67. The ΔG* was found to be 25.4 kcal mol^"1 at 298 K (Figure S38 in SI). The complete conversion in pyridine-t/₅ with 2 kcal mol^"1 lower ΔG* suggests that the conversion of 12b to 12a is base catalyzed.

The factors that influence inversion at pyramidal nitrogen in bicylic amines have been discussed for several decades.⁶⁶ Usual values of nitrogen inversion barriers for alicyclic amines lie in the 5-9 kcal/mol range.⁶⁶' ⁶⁷ However, abnormal barriers (>13 kcal/mol) were found for azanorbornanes, which has been implied to nitrogen inversion-C-N rotation (NIR) the'bicyclic effect'.⁶⁶' ⁶⁸' ⁶⁹ Despite several attempts, the mechanistic reason for this bicyclic effect could not be satisfactory explained in terms of steric interactions or ring strain. Only in crowded systems such as N-^-Bu azanorbornanes steric interactions could be sufficiently strong to play an important factor. The nitrogen inversion-rotation barrier determined amongst a set of various categories of azabicycles using dynamic NMR and MP2/6- 31G*, is found to vary from 6.4 to 13 kcal/mol.⁶⁹ Several interesting conclusions have so far emerged from various works⁶⁷' ⁶⁹ on nitrogen inversion and bicyclic effect in cyclic amines: (1) The NIR barriers increase with a decrease of the ring size in azabicylces. (2) The presence of a 5-membered ring as a component of a rigid nitrogen-bridged bicyclic skeleton increase the NER. barrier by ca 3 kcal/mol per ring, thereby showing a relationship between azacycle geometry and the NIR barrier. (3) The flattening of the nitrogen pyramid, for example through the introduction of a double bond in the 6-membered ring as a component of a rigid nitrogen-bridged bicyclic skeleton decrease the NIR barrier of the ring inversion. (4) It has been evidenced by dynamic NMR at -180 K that the ring inversion in the rigid nitrogen-bridged bicyclic skeleton involves interconversion of conformers with equatorial and axial N- alkyl substituent. (5) Although earlier suggestion that high strain which develops during NIR for the endocyclic CNC angle change from N-pyramid is responsible for the bicyclic effect, no satisfactory correlation has been however found between NIR barriers and the CNC angle for different bicyclic amines. No experimental evidence has been found so far to support the suggestion that the observed high NIR barriers in the constrained amines is caused by the derealization of the N lone pair.

In conclusion, the high AG* found 23.4 kcal mol^"1 at 298 K for the conversion of axial N-H containing isomer 12b to the energetically stable equatorial N-H containing isomer 12a is unique in the long history⁶⁶' ^{68> 69} of search for abnormally high activation barriers for nitrogen inversion. This also constitutes the first example in which both the piperidine isomers with the axial and the equatorial lonepair orientation have been isolated in the pure form and fully characterized by NMR and mass.

(V) Preparation of Aza-ENA phosphoramidite for AON synthesis

We then considered different strategies to prepare appropriately amino protected phosphoramidite blocks from 12a/12b to synthesize aza-ENA incorporated AONs in the following manner.

(i) Why the PAC derivative of Aza-ENA phosphoramidite 17 could not be used in the AON synthesis?

We first considered the employment of diastereomer 12a/12b directly for the preparation of N- phenoxyacetyl (PAC) protected derivative 14. Thus the aza-ENA analog 12a/12b was TV-protected using PAC-Cl in pyridine (we observed conversion of 12b to 12a as discussed earlier which ultimately gave single spot in TLC) which afforded 14 in 70% yield as a mixture of rotamers because of restricted rotation of the amide bond of PAC. The PAC group in 14-17, as such, must take up an equatorial orientation because of its steric bulk. This means that the duplicate NMR resonances that we observe in this piperidino constrained nucleos(t)ides, PAC protected l',2'-azetidine constrained nucleoside(t)es,⁴² as well as for the trifluoroacetyl protected amino-LNAs³⁶ by Wengel's group, is not owing to the chiral nature of the amino-nitrogen. Debenzylation reactions with either Pd(OH)₂/ammonium formate or BCI3 in anhydrous CH₂Cl₂ were sluggish. This problem was circumvented by doing successive reaction with Pd(OH)₂/ammonium formate at refluxing temperature in methanol, overnight and then with BCl₃ in anhydrous CH₂Cl₂ for 3 h to give 15 in 75% yield. Dimethoxytrytilation of 5'-OH in 15 using DMTr-Cl and pyridine followed by phosphytilation of 3'-OH using standard conditions⁴² afforded 16 and 17 in 90% and 86% respectively [³¹P NMR of 17 (CDCl₃): δ 150.7, 150.3, 149.2, and 148.1, see SI: Fig S29]. The phosphoramidite 17 was successfully incorporated into the mixed 15mer sequence (Table 1) but to our surprise the PAC protecting group was very stable and could not be deprotected even in 33% aqueous ammonia and AMA (33% aqueous ammonia/methylamine 1:1 v/v) at 65°C for 2 days, which was clear from the mass measurement using MALDI-TOF mass spectroscopy [expected mass with PAC protection m/z 4624.7 and observed 4624.9]. The PAC protected nucleoside 15, on the other hand, could be deprotected with aqueous ammonia at 55 ⁰C overnight.

This result is in sharp contrast with the l',2'-azetidine modified thymine nucleoside⁴² (21b in Scheme 2) where 2'-amino PAC could be deprotected easily both at the monomer as well as at the oligomer level using 33% aqueous ammonia⁴² at room temperature. This difference in chemical reactivities at 2'-amino modified locked nucleosides is due to the fact that the azetidine ring at the l',2' position makes the azetidine ring nitrogen (pK_Α = 6.06 ± 0.03) more electron deficient (hence more acidic) compared to more basic nature of ring-nitrogen (pK_a = 6.66 ± 0.03) of that of aza-ENA analog. This is also evident from δH-2' of 3',5'-benzyl protected azetidine analog 21b⁴² (δ 4.72) compared to that of 3 ',5 '-benzyl aza-ENA modified nucleosides 12a (δ 3.48).

(H) Synthesis ofN-trifluorocaetyl derivative of Aza-ENA phosphoramidite 20 for the synthesis of AONs Since the PAC protection of the amino function of aza-ENA does not work in our hand, we incorporated trifiuoroacetyl protecting group (Scheme 2) where the benzyls were first deprotected using the same condition as for 15 (Scheme T). The deprotected nucleoside 13 was directly treated with excess of ethyl trifiuoro acetate in methanol at r.t, overnight to give the N-COCF₃ protected nucleoside 18 in 45% yield after 3 steps from 12 as a mixure of rotamers. Dimethoxytritylation and phosphitylation were earned out using the same conditions as for 16 and 17 in Scheme 2 to afford 19 in 81% yield and 20 in 61% yield as a mixture of four isomers [³¹P NMR (CDCl₃): δ 150.1, 149.9, 149.8, and 149.2, SI: Fig S36] Phosphoramidite 20 was successfully incorporated at different positions of mixed 15-mer AON sequences (Table 1) using standard phosphoramidite approach⁴² with 10 min coupling time for modified amidite. Deprotection of all the base-labile protecting groups went smoothly with 33% aqueous ammonia at 55 ⁰C as confirmed by MALDI-TOF mass spectroscopy (Table 1). All oligos were purified by PAGE (20% polyacrylamide/7 M urea), extracted with 0.3 M NaOAc and desalted with C18-reverse phase cartridge to give AONs in >99% purity.

Table 1. The aza-ENA modified AONs and the thermal Denaturation Studies of their duplexes with complementary RNA or DNA Targets⁰.

^a T_m values measured as the maximum of the first derivative of the melting curve (A₂₆O vs temperature) recorded in medium salt buffer (60 mM Tris-HCl at pH 7.5, 60 mM KCl, 0.8 mM MgCl₂ and 2 mM DTT) with temperature range 20 to 70 ⁰C using lμM concentrations of the two complementary strands; \T_m = T_m relative to RNA compliment; A.T_m* = T_m relative to DNA compliment. T = aza-ENA monomer; b [M+2H]⁺.

(VI) Thermal Denaturation Studies of Aza-ENA Modified AONs

The thermal stability of duplexes involving aza-ENAs was determined toward the complementary RNA and DNA as shown in Table 1. Single modifications were incorporated at a time at different positions (Table 1) of the of the 15nt AON sequence, 3'-d(CTTCTTTTTTACTTC)-5', to determine the sequence dependency in the target affinity. The results reveal (Table 1) that the single aza-ENA modification enhances the target affinity significantly with complementary RNA (AT_m +2.5 to +4 ⁰C) depending upon the site of the modification in the AON strand. This can be attributed to the site- dependency of the variable conformational pre-organization imparted by the North-fused sugar moiety on the single stranded AON. Even though the thermal stabilities of duplexes containing single ENA modifications were not reported²⁷' ³³ we presume that aza-ENA will have slightly more favorable target affinity towards RNA than the isosteric ENA counterpart (double modification gave +3.5 °C/modification).³³ The 2'-amino function of amino-ENA is almost 50% protonated at the physiological pH considering its pK_a of 6.66 ± 0.03, can have electrostatic interaction with the neighboring phosphate which favors efficient duplex formation as observed in azetidine modified AONs.⁴² On the other hand, with complementary DNA there was significant drop in duplex melting. This can be due to the 2',4'- ethylene bridge which causes steric clash in the minor groove of the AON-DNA duplex.

Figure 7: The CD spectra of aza-ENA modified 15mer AONs (AON 2-5) as duplex with (A) complementary DNA, and (B) with complementary RNA in comparison with the native counterpart (AON 1).

(VII) Circular Dichroism (CD) analysis of the Aza-ENA Modified AON with complementary DNA and RNA:

Since CD spectroscopy is an effective tool to measure the global helical conformation of nucleic acids, we have recorded the CD spectra of the aza-ENA modified 15mer AON/DNA and AON/RNA hybrids (Table 1 and Figure 7). No significant change in ellipticity of the homo or the hetero duplexes was observed in comparison with the native counterpart (duplex with AON 1 + DNA or RNA) (Figure 7). This result is similar to what was observed for doubly modified ENA duplexes.³³ This means that single modifications with North locked aza-ENA nucleoside do not alter the overall global structure compared to that of the native counterpart, whereas the differences in the local conformational changes could easily be identified by RNase H digestion experiment (see next section).

(VIII) RNase H digestion studies of aza-ENA modified AON/RNA heteroduplexes.

Escherichia coli RNase Hl has been used in this work because of two reasons: first, it is commercially available in a pure form, and second its cleavage properties are not very different from those of the mammalian enzyme.⁷⁰ Hence, the antisense properties of aza-ENA modified oligonucleotides duplex with the complementary RNA were compared with the native as well as with the identical oxetane- modified counterparts⁷¹ with Escherichia coli RNase Hl as a model system. Four different aza-ENA containing AON mixmers, each obtained by incorporating single aza-ENA modification at one of the four different positions (AONs 2 -5 in Table 1) of identical DNA sequence, when formed duplex with the complementary RNA, were found to be excellent substrate for RNase Hl, but with varying RNA cleavage sites depending upon the site of modification on the AON strand. We have previously reported the RNase H 1 digestion properties of oxetane modified AON/RNA hybrid duplexes in identical sequence.⁷¹ The RNA cleavage patterns of all aza-ENA modified AONs (AON 2-5 in Table 1) were found to be uniquely different from those of the isosequential oxetane modified AONs (AON 6 - 9; Figure 9). AON 2 showed only one prominent cleavage site at A8 position of the complementary RNA (Figure 8) unlike the oxetane-modified AON 6 which showed cleavages at A7, A8, AlO and UI l with no clear preferences (Figure 9). Comparison of AON 4 versus AON 8 and AON 5 versus AON 9 clearly shows the absence of single RNA cleavage site in aza-ENA modified AONs/RNA duplexes compared to those of the oxetane-modified counterparts (A7 of AON 8 and A9 of AON 9). AON 3 and its isosequential oxetane analog AON 7, on the other hand, showed identical cleavage footprint pattern of 5 nucleotide gap. This shows that the local structures of all aza-ENA modified AONs/RNA duplexes are not the same. RNase H enzyme indeed can finely discriminate these local variations of the stereochemical properties of the microstructure brought about by various type and incorporation site of the North-type modification (aza-ENA versus oxetane modifications) in the AON.

A0N5 A0N4 A0N3 RNA A0N2 A0N1

2' 5' 15' 35' 60' 2' 5' 15' 35' 60' ' 2' 5' 15' 35' 60 2' 5' 15' 35' 60' 2' 5' 15' 35' 60' 120

C D

Figure 8. (A) Autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5'-³²P- labeled target RNA by E.coli RNase Hl in native AON 1/RNA (lane 5) and the aza-ENA modified AONs (2-4)/RNA hybrid duplexes (lane 1 to 4) after 2, 5, 15, 35 and 60 min of incubation. Conditions of cleavage reactions: RNA (0.8 μM) and AONs (4 μM) in buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgC12 and 0.1 mM DTT at 21 ⁰C; 0.08 U of RNase H. Total reaction volume 30 μL. (B) Kinetics of RNase H cleavage. Target RNA remaining is densitometrically evaluated and plotted as a function of time. (C) Pictorial representation of RNase Hl cleavage pattern of AONs 1-5/RNA hybrid duplexes. Vertical arrows show the RNase H cleavage sites, relative length of an arrow shows the relative extend of cleavage at that site and dotted arrows show the partial cleavage at the intial reaction time. Relative percentage cleavage is indicated above the arrow which is taken at 15 min time point from the gel shown above. (D) Quantitative evaluation of the gel picture (shown in SI) of the remaining full length [³²P]-RNA at 1 h as obtained by densitometer. Table 2. Observed cleavage rate and relative rates normalized to the value for control native oligonucleotide for RNase H digestion. Δr_m (⁰C) observed with complementary RNA is also shown.

This shows that the certain specific flexibility of the AON/RNA duplex and accessibility of the RNA strand in the heteroduplex is required for RNase H binding and cleavage. The RNase Hl recruitment capability for LNA modified AONs were reported earlier which showed a minimum gap of 7 to 8 DNA monomers to induce full cleavage activity. This difference in RNase H activity between the LNA, oxetane and aza-ENA-modified AONs/RNA duplexes can be due to the difference in the conformational flexibility as well as resulting hydration pattern in the minor groove imparted by the fused four, five and six membered rings locked to the pentofuranose ring.

Finally, to evaluate cleavage rate, quantification of gels was performed densitometrically and the uncleaved RNA fraction was plotted as a function of the incubation time (Figure 8B). Enzyme digestion experiment was performed at lower enzyme concentration (0.08 U) to observe the cleavage rate (see Figure Sl in SI). Reaction rates were determined by fitting to single exponential decay functions. Recently, Kurreck et α/.⁷⁰ has shown that the RNase H cleavage efficiency of AON can be correlated with its affinity towards target RNA. The relative cleavage rates with aza-ENA modified AON/RNA duplex were however quite comparable to that of the native counterpart (Table 2 and insets B and D in Figure 8). SVPDE AONl AON 7 ^SVPDE AON 8 AON 9 AON 6

Incubation Incubation tune in mm 30' 60' 120' 30' '60 120' tune in mm 30' 60' 120' 30' 60' 120' 30' 120' 5mer

A3 A3

B

3 4 5 6 7 8 9 U 1011l 12 13 14 15 6 7 8 9 K il

5'-r(G A A G A A A AA A U GAA G) 5 '-r (G A A G A A A A A A U G A A G) 3'-d(C T T C T T T T T T A C T T C) 3'-d(C T T C T A C T T C)

AON 6 AON 7 i*

1 2 3 4 5 S 47 8 9 10 11 l; 1 2 3 4 10 11 12 13 14

5' -r (G A A G A A A A A A U G A A G) 5'-r (G A A G A A A A A A ϋ G A A G) 3' -d (C T T C T T T T T T A C T T C) 3'-d(C T T C T A C T T C)

AON 8 AON 9

Figure 9: Insets (A and B) are autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5'-³²P-labeled target RNA by E. coli RNase Hl in native AON 1/RNA and the oxetane modified AONs (6-9)/RNA hybrid duplexes after 30 min, 1 and 2 h of incubation. Conditions of cleavage reactions: RNA (0.8 μM) and AONs (4 μM) in buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgC12 and 0.1 mM DTT at 21⁰C; 0.08 U of RNase H. Total reaction volume 30 μL. (C) Pictorial representation of RNase Hl cleavage pattern of AONs 6-9/RNA hybrid duplexes. Vertical arrows show the RNase H cleavage sites, relative length of an arrow shows the (IX) Stability of Aza-ENA/DNA chimeras in human serum

The stability of AON in cells towards various exo and endonucleases is warranted to fulfill the requirements for an ideal antisense agent.⁶ The stabilities of aza-ENA modified AONs were tested against human serum which mainly comprises of 3'-exonucleases (Figure 10). When compared to native counterpart which was completely degraded after 9 h, AON 3, 4 and 5 (Table 1) were still remaining to

AON l AON 2 AON 3 AON 4 AON 5

9 2 1 30' 15' O _{9 2} 1 30' 15' 0 9 2 1 30' 15' 0 9 2 1 30' 15' 0 9 2 1 30' 15' 0

Figure 10: Autoradiograms of 20% denaturing PAGE, showing the degradation pattern of 5'-³²P-labeled AON 1 to 5 in human serum. Time points are taken after 0, 15, 30 min, 1, 2 and 9 h of incubation. The % of AON remaining after 9 h of incubation: 0% of AON 1, 0% of AON 2, 8% of AON 3, 15% of AON 4, 20% of AON 5.

certain extent (8, 15 and 20 % respectively). It is noteworthy that all modified AONs were cleaved by 3'- exonucleases in the blood serum at the phosphodiester which is one nucleotide before the aza-ENA modification site towards 3'-end, and the residual sequences were found to be stable in human serum for 48 h at 21⁰C (Figure S2 in SI). This is a surprising result in view of the fact that identical AON sequences with North-constrained oxetane⁷² modification are cleaved at the phosphodiester immediately before the modification site under an identical condition. This suggests that the conformational effect of the aza-ENA modification in the AON is transmitted toward the 3 '-end and recognized by 3'- exonucleases, just as in the RNase H cleavage of the AON/RNA duplex, which recognizes the local RNA/RNA type duplex structure, and leaves a footprint at the 5'-end because of the modulation of the structure of the complementary RNA strand by the North-type constrained aza-ENA in the AON strand. The stability studies in human serum with single LNA⁷⁰ nucleotide at 3' and 5' end showed complete degradation in 24 h at 37 ⁰C, while our residual sequences from the aza-ENA modifications were stable over 48 h under our experimental condition (Figure S2 in SI). Even though direct comparison could not be made, but these results clearly show that aza-ENA modification can certainly give substantial stability in human serum which is probably more than that of LNA.

(X) 3'-ExonucIease stability assay (Snake venom phosphodiesterase):

The stability of aza-ENA modified AONs 1 to 5 towards 3' exonuclease was investigated using snake venom phosphodiesterase (SVPDE) over a period of 24 h at 21 ⁰C. Time points were taken at O, 1, 2 and 24 h to examine the cleavage pattern (Figure 11). The enzyme digestion pattern was similar to that obtained from the digestion with human serum except the fact that these aza-ENA modified AONs offered resistance to degradation even after 24 h. Note that native AON 1 was completely degraded in 1 h, whereas the full length AONs 2 to 5 were 18%, 20%, 14% and 10% left undegraded respectively after 24 h. It is however noteworthy that similar to the blood serum digestion, all modified AONs were cleaved by SVPDE at the phosphodiester which is one nucleotide before the aza-ENA modification site towards 3 '-end, and the residual sequences were found to be fully stable for 24 h at 21⁰C (Figure 11). This is another proof for future design of aza-ENA modified AONs where only single modification at second position from 3' will offer significant stability towards 3 '-exonucleases. AON 1 AON 2 AOJN 3 AON 4 AON 5

2 1 0 124 2 1 0 124 2 1 0 24 2 1 24 2 1 0

•

Figure 11: Denaturing PAGE analysis of snake venom phosphodiesterase (SVPDE) degradation pattern of 5'-³²P-labeled AON 1 to 5. Time points are taken after O, 1, 2 and 24 h of incubation with enzyme. The % of AON remaining after 24 h of incubation: 0% of AON 1, 18% of AON 2, 20% of AON 3, 14% of AON 4, 10% of AON 5

(XI) Molecular Structure of the aza-ENA derivatives 12a, 12b and 13 based on NMR, ab initio and MD calculations

The experimental coupling constants from 600 MHz spectra of the 3',5'-6Zs-OBn protected (12a, 12b) and fully de-protected (13) aza-ENA compounds have been further analyzed to build up their molecular structures using the following protocol: (i) Derive Initial dihedral angles from the observed J_HH using Haasnoot-de Leeuw-Altona generalized Karplus equation⁷³' ⁷⁴ (Table 3). (ii) Perform NMR constrained molecular dynamics (MD) simulation ( 0.5 ns,10 steps) simulated annealing (SA) followed by 0.5 ns NMR constrained simulations at 298 K using the NMR derived torsional constraints from Step (i) to yield NMR defined molecular structures of 3',5'-bis-OBn protected (12a, 12b) and de-protected aza- ENA (13). The MD simulations were performed using Amber force field (AMBER 7⁷⁵) and explicit TIP3P⁷⁶ aqueous medium (see details in Experimental section), (iii) Acquire 6-31G** Hartree-Fock optimized ab inilio gas phase geometries (by Gaussian 98⁷⁷) in order to compare the NMR derived torsions with the ab initio geometry, (iv) Refine the Karplus parameters with the help of the NMR and ab initio derived torsions, (v) Analyze the full conformational hyperspace using 2 ns NMR/αέ initio constrained MD simulations of compounds 12a, 12b and 13 followed by full relaxation of the constraints. The results of these studies are summarized in Table 3.

(i) Generalized Karplus parameterization

Relevant vicinal proton ⁵JHr_1H2 ¹, ³Jm¹ _My and ³Jm_\m coupling constants have been back-calculated from the corresponding theoretical torsions employing Haasnoot-de Leeuw-Altona generalized Karplus equation ' taking into account β substituent correction in form:

³J= P₁ co/(φ) + P₂ cos(φ) + P₃ + ∑ ( Aχr^np (P₄ +P₅ coJ(& ψ + P₆ |Δχ_i ^Broup| ))

where P₁ = 13.70, P₂ = -0.73, P₃ = 0.00, P₄ = 0.56, P₅ = -2.47, P₆ = 16.90, P₇ = 0.14 (parameters from Ref.⁷³), and Δχ_; ⁸^ = ΔχΛ ^substituent - P₇ Σ Δχ> ^substituent where Δ_Xi are taken as Huggins

electronegativities.⁷⁸ Optimized Δχ; ^⁰"⁹ = 1.305 has been used for N_aze. Least-square optimization

procedure for the Δχi^group of nitrogen atom in the piperidino ring will be reported elsewhere. The other group electronegativities were kept unmodified and their respective standard values were adopted from Altona et aP as follows: Cl' (0.738), 04' (1.244), C3' (0.162), C2' (0.099), 03' (1.3 for OH & 1.244 for OBn), C4' (0.106), C5' (0.218). The correlation between experimental ³JHr₁H_2' and ³JH2_',H3_' vicinal coupling constants of the ENA (compound (I) in Figure 1), aza-ENA (12a, 12b, 13), LNA and 2'-amino LNA analogs (compounds (A) and (B) in Figure 1) are shown in Inset (A) in Figure 12 together with contours of theoretical ³J_Hr_1H2' vs. ³JH2_',_H3' dependencies at fixed sugar puckering amplitudes (from 35° to 65°).

(H) Sugar pucker conformation

Non-observable ³J_Hr,_H2_' and low ³JwZ_My (Table 3, Table S2 in SI) experimental coupling indicate that similar to that in the ENA,³³ LNA³³' ³⁴ and 2'-amino LNA,³⁶ the piperidino modification of the sugar moiety in aza-ENA restricts sugar pucker to the North-type conformation (Tables 3 and 4, Figure 14). This conclusion is corroborated by experimental dihedral angles from the observed ³J_H1H, ab initio and MD simulations (Tables 3 and 4) which show that the sugar moiety is indeed conformationally restricted to the North conformation (pseudorotational phase angle P = 14° ± 7° for 12a and 12b, 19° ± 8° for 13, sugar puckering amplitude φ_m = 48° ± 4° for 12a, 12b, and 13, Table 4). Thus, the sugar pucker in the

aza-ENA is found to be close to that of the ENA and LNA (P = 12-19⁰),³³ with sugar puckering

amplitude, φ_m, lowered by -10° (φ_m « 46°) compared to that of the LNA (φ_m « 56°).³³' ³⁴ ENA and aza-

ENA have also showed very similar conformational dynamics with 5-8° variation of the sugar torsions along the MD trajectories (Table 4), sugar atom positions RMSd is less than 0.1 A (Figure 14).

(Ui) Conformations of the major (12a) and minor (12b) diastereomers of the 3 ',5'-Ms-OBn protected aza-ENA and of the de-protected aza-ENA (13).

Bicyclic [3.2.1] fused sugar and the piperidino heterocycle in both ENA and aza-ENA appeared to interlock each other resulting in very rigid North-type conformation for the sugar ring (Table 4, Figure 12) and chair conformation for the piperidino heterocycle, which is proven by ID NOESY experiment (Figure 4) as well as by simulating ³J_HH versus dihedral angles using generalized Karplus equation⁷³' ⁷⁴ as shown in Figure 13. During the whole period of both the constrained and unconstrained MD simulations the heavy atoms in both the sugar and piperidino rings were displaced from the average position by less then 0.09A (Figure 14). Clearly, because of the restricted degrees of freedom for this fused bicyclic molecular system makes the average NMR-based MD structures very close to that of the optimized ab initio geometry (Tables 3 and 4). Higher dynamics has been observed for the base (RMSd about 0.7 A) in both ENA and aza-ENA, and the most dynamic part appeared to be flanking OBn groups in the 3 ', 5 '-Us-OBn protected aza-ENA compounds 12a and 12b (RMSd 1.3-1.7A, Figure 14). Table 3. Experimental Jn_1H vicinal proton coupling constants, corresponding ab initio and MD (highlighted in blue) ^_H,_H torsions and respective theoretical ³Jn_1H obtained using Haasnoot-de Leeuw- Altona generalized Karplus equation⁷³' ⁷⁴ taking into account β substituent correction (see text). Non- observable constants are marked in red.

Table 4. Sugar torsions (vo - V4 ), pseudorotational phase angle (P),¹⁹ sugar puckering amplitude (φ,,,),¹⁹ backbone (γ,δ) and glycoside bond (χ), as well as selected torsions (C3'-C2'-O2' (NT)-CT) and C3'-C4'- C6'-C7') characterizing six-member piperidino heterocycle in 3',5'-bis-OBn protected and de-protected aza-ENA (12a,12b,13) and ENA³³ (compound (I) in Figure 1). The structural parameters are obtained from the ab initio molecular structures as well as from the last 0.5 ns of unconstrained MD simulations (average values and standard deviations are shown in brackets and highlighted in blue) of the respective nucleosides.

N-H equatorial (12a) N-H axial (12b) N-H equatorial (13)

V₀: C4'-O4'-Cr-C2' 3.38 (4.2 ± 6.0) 3.21 (4.2 ± 5.6) -0.91 (-0.5 ± 6.8) V₁: O4'-C1'-C2'-C3' -31.88 (-32.9 ± 4.9) -31.28 (-32.9 ± 4.6) -28.21 (-29.5 ± 5.5) V₂: C1'-C2'-C3'-C4^T 46.00 (45.2 ± 3.2) 45.07 (45.1 ± 3.2) 43.87 (44.8 ± 3.4) V₃: C2'-C3'-C4'-O4' -45.21 (-45.5 ± 3.8) .43.87 (-45.4 ± 3.8) .45.14 (47.6 ± 4.0) V₄: C3'-C4'-O4'-C1' 26.83 (26.6 ± 5.5) 26.19 (26.5 ± 5.3) 29.65 (30.5 ± 6.1)

P 14.56 (14.1 ± 7.2) 14.38 (14.0 ± 6.8) 19.38 (19.4 ± 8.0)

Φ,,, 47.53 (46.9 ± 3.1) 46.53 (46.8 ± 3.2) 46.50 (48.0 ± 3.4) y: O5'-C5'-C4'-C3' 45.92 (54.9 ± 9.1) 47.44 (57.3 ± 9.3) 64.53 (53.1 ± 10.7) δ: C5'-C4'-C3'-O3' 72.74 (60.0 ± 5.3) 80.31 (69.4 ± 5.4) 75.61 (74.5 ± 5.9)

X : O4'-C1'-N1-C2 -164.31 (-158.6 ± 10.1) -163.21 (-154.9 ± 10.5) -156.21 (-157.2 ± 10.8)

C3'- -C2'-O2' (N2')-C7' 64.31 (66.7 ± 5.0) 58.56 (66.4 ± 5.2) 68.08 (67.0 ± 4.8)

( I3'_C4'-C6'-C7' -59.75 (-57.8 ± 5.1) -59.4 (-57.6 ± 5.4) -57.49 (-55.5 ± 5.2)

Figure 12: Experimental ³JHi _',H_2' and ³JH_2',H3_' vicinal coupling constants of the ENA (compound (I) in Figure 1), aza-ENA (12a,12b,13), LNA and 2'-amino LNA analogs (compounds (A) and (B) in Figure 1) shown together with contours of theoretical ³J_Hr,H2_' vs. ³JH_2',H_3' dependencies at fixed sugar puckering amplitudes (from 35° to 65°) calculated using algorithm and Haasnoot-de Leeuw-Altona generalized Karplus equation reported in Ref.⁷³' ⁷⁴ and using the same parameters as described in the text and in the caption of Table 1.

The experimental ID and 2D NMR spectra unequivocally show (see previous section) that the only significant structural difference between the two chair piperidino conformations (assignment is shown in Inset (B) in Figure 12) of the major (12a) and minor (12b) isomers of the 3',5'-Z)W-OBn protected aza-ENA is that the former has an equatorial orientation of the N-H proton, whereas the latter has an axial orientation of N-H proton in the bicyclic piperidino ring. The molecular structure of the fully-deprotected aza-ENA (13) is however almost identical to that of the major isomer (12a) (Tables 3 and 4). Ab initio gas phase results also show that the N-H equatorial is thermodynamically more stable than the axial counterpart (by 6.92 kcal/mol for the de-protected ENA and by 1.44 kcal/mol for 3',5'-Ws- OBn protected ENA), which is fully consistent with the present ¹H-NMR study. In CDCI₃ the major (12a) and minor (12b) isomers were found to be in dynamic equilibrium with the ratio 12b: 12a = 60:40. Similarly, our unconstrained MD simulations both started from the N-H equatorial and from N-H axial isomers in explicit water resulted in the near 1: 1 distribution of the both isomer (Figure S37 in SI). We can not offer any definite explanation for the irreversibility of transformation of the minor (12b) isomer into the major (12a) observed in pyridine.

For clarity of the view the 3',5'-OBn protective groups in 12a and 12b are not shown.

Figure 14. Superposition of 10 iandomly selected structures duπng last 400 ps of the unconstrained 2 ns MD simulations of 3',5'-bιs-OBn protected (12a,12b) and de-protected (13) aza-ENA Total average RMSd (in A) are shown for all heavy atoms (marked in black) as well as for the heavy atoms in sugar and pipeπdmo moieties (m parentheses marked in red), the base atoms (m parentheses marked in blue), and OBn-groups' heavy atoms (m parentheses marked in gieen)

Conclusions

(1) New 2',4'-piperdmo fused aza-ENA thymine 3',5'-bis-OBn protected (12a,12b) and de-protected (13) nucleosides as well as corresponding aza-ENA nucleotide were synthesized The aza-ENA nucleotide has subsequently been incorporated into 15mer AONs and their antisense properties have been evaluated in vitro.

(2) During the cyclization reaction in Scheme 2 (ll→ 12a and 12b) we observed two diastereomers due to the axial and equatorial orientation of the chiral piperidino-N proton (12a NH_e, 12b NH_a). The non-reversible conversion of 12b to 12a was performed in pyridine-Js at different temperatures, from the rate observed at each temperature, E_a was calculated which was found to be 25.4 kcal mol^"1. The ΔG* at 298 K was found to be 23.4 kcal mol^"1. hi CDCl₃ diastereomers 12a and 12b where found to be in dynamic equilibrium (equilibrium constant K_c = 0.67, ratio 12b:12a = 60:40) with the ΔG* = 25.4 kcal mol^"1 similar to that in pyridine.

(3) The molecular structures of the aza-ENA monomer units have been studied by means of high-field ¹H NMR and theoretical ab initio and MD simulations. The combined experimental and theoretical studies have demonstrated that the piperidino-fused furanose ring is indeed locked in the typical North- type conformation with the pseudorotational phase angle (P) and puckering amplitude (Φ_m) for the ab initio optimized geometries (HF, 6-3 IG**) varying in the ranges 7° < P < 27°, 44°< φ_m < 52°, .respectively.

(4) Aza-ENA modified AONs have shown high target affinity to complementary RNA strand (T_m increase of +2.5 to +4 ⁰C per modification), depending upon the substitution site, compared to the native counterpart, while hybridization with the complementary DNA sequence lead to substantial destabilization of the duplexes (T_m drop of -0.5 to -3°C per modification).

(5) The global helical structure of aza-ENA modified AON/RNA hybrids, as revealed by the CD spectra, has been found to be very similar to the native AON/RNA duplex suggesting that the local conformational perturbations brought about by the North- conformational^ constrained sugar moiety in aza-ENA modifications are not significant enough to be detected by the CD experiment.

(6) All of the aza-ENA modified AON/RNA hybrid duplexes have been found to be good substrates for the E. coli RNase Hl. hi these AON/RNA hybrids, except for one case (AON 2 Figure 8), a region of 5 to 6 nucleotides in the RNA strand in the 3 '-end direction from the site opposite to the aza-ENA modification, was found to be insensitive toward RNase H cleavage presumably owing to the local structural perturbations brought about by the conformationally constrained modifications. These cleavage patterns of the aza-ENA modified AON/RNA hybrids is uniquely different from that of the oxetane modified AONs which had shown found a gap of 5 nucleotides units.

(7) All the aza-ENA modified AONs offered greater protection towards 3' exonucleases compared to the native sequence. In fact, all the modified AONs cleaved at one nucleotide before the modification towards 3 '-end and did not degrade any further. These residual AONs have been found to be stable for over 48 h in human serum and for over 24 h with the snake venom phosphodiesterase. This result clearly suggests that a single modification at the second position from the 3 '-end can give even more substantial stability towards 3' exonucleases.

(8) This study provides valuable information regarding the optimal design of AONs having completely natural phosphodiester backbone for the therapeutic applications that will not only show high target affinity but also high stability towards nucleases in vivo.

Experimental section

General Experimental Methods:

Chromatographic separations were performed on Merck G60 silica gel. Thin layer chromatography (TLC) was performed on Merck pre-coated silica gel 60 F₂₅₄ glass-backed plates. ¹H NMR spectra were recorded at 270.1 MHz, 500 MHz and 600 MHz respectively, using TMS (0.0 ppm) or methanol (3.4 ppm) as internal standards. ¹³C NMR spectra were recorded at 67.9 MHz, 125.7 MHz and 150.9 MHz respectively, using the central peak of CDCl₃ (76.9 ppm) as an internal standard. ³¹P NMR spectra were recorded at 109.4 MHz using 85% phosphoric acid as external standard. Chemical shifts are reported in ppm (S scale). Compound names for the bicyclic structures are given according to the von Baeyer nomenclature. MALDI-TOF mass spectra were recorded in positive ion mode for oligonucleotides and for other compounds as indicated. The mass spectrometer was externally calibrated with a peptide mixture using alpha-cyano-4-hydroxycirmamic acids as matrix. Thermal denaturation experiments were performed on a PC-computer interfaced UV/VIS spectrophotometer with Peltier temperature controller.

Compound (D) in Scheme 1 [(lR,3R,4R,6S)-l-Benzyloxymethyl-6-benzyloxy-5-carbonitrile-3- (thymin-l-yl)-2-oxabicydo[2.1.1]hexane]. The nucleoside 11 (2.5 g, 4.1 mmol) was dissolved in 45 mL of dry THF, cooled in icebath and IM NaHMDS (8.2 mL) was added dropwise. Reaction was warmed slowly to r.t. and stirred for 3 h under nitrogen atmosphere. The reaction was quenched by adding water and extracted with CH₂Cl₂ (3 times). The organic phase was dried over anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (0-3% methanol in dichloromethane, v/v) afforded D with traces of another diastereomer as shown by NMR (655 mg, 1.4 mmol, 35%). R_f = 0.40 (CH₂C1₂/CH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 460.9, calcd 459.1; ¹H NMR (500 MHz, CDCl₃) δ ppm 8.93 (s, IH, NH, Thymine), 7.45 - 7.12 (m, 1OH, Bn), 6.92 (q, J = 1.2 Hz, IH, H-6), 5.81 (s,lH, H6'), 5.08 (d, J = 2.5 Hz , IH, Hl¹), 4.58 (d, J = 13.4 Hz, IH, CH₂Ph), 4.50 - 4.46 (m, 5H, CH₂Ph), 3.96 (dd, J = 7.7, 1.5 Hz, IH, H3'), 3.25 (dd, J = 7.7, 2.5 Hz, IH, H2'), 1.50 (d, J = 1.2 Hz, CH₃, Thymine). ¹³C NMR (125.7 MHz, CDCl₃): 163.2 (C-4), 159.9 (C-4¹), 150.2 (C-2), 136.6, 135.7, 134.4 (C-6), 128.6 128.5, 128.4, 128.2, 128.0, 127.8, 115.1 (CN), 110.3 (C-5), 99.3 (C-6¹), 73.2(CH₂Ph), 71.7 (CH₂Ph), 70.7 (C-5¹), 68.3 (C-3¹), 63.6 (C-I'), 58.8 (C-2¹), 11.9 (CH₃, thymine).

3,5-Di-0~benzyl-4-C-hydroxymethyI-l,2-0-isopropylidene-a-D-ribofuranose (2). To a stirred suspension of 1 (12.3 g, 39.54 mmol) in anhydrous acetonitrile (400 mL) at -5 ⁰C was added NaH (1.81 g, 1.15 mmol) in four portions during 1.5 h. Benzyl bromide (5.4 mL, 1.15 mmol) was added dropwise and stirred at r.t. overnight under nitrogen atmosphere. The reaction was quenched with water and extracted with dichloromethane. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (0-20% ethyl acetate in cyclohexane v/v) which afforded 2 (10.6 g, 26.5 mmol, 67%). All analytical data were identical to those previously reported.³²

S^-Di-O-benzyM-C-cyanomethyl-l^-O-isopropylidene-α-D-ribofuranose (4). The sugar 2 (10.6 g, 26.5 mmol) was dissolved in anhydrous dichloromethane-pyridine mixture (250 mL, 3:1, v/v) and cooled in icebath. To this solution triflic anhydride (5.3 mL, 31.8 mmol) was added dropwise and stirred for 3 h under nitrogen atmosphere. The reaction was quenched with cold saturated aqueous NaHCO₃ and extracted with dichloromethane. The organic phase was dried over anhydrous MgS O₄, evaporated under reduced pressure followed by co evaporation with toluene 3 times and dichloromethane 3 times. The crude reaction product was dissolved in 150 mL dry DMF and 80 mL of IM LiCN in DMF was added and stirred for 3 days at r.t. Solvent was carefully evaporated and the residue was dissolved in dichloromethane, saturated aqueous NaHCO₃ was added and extracted with dichloromethane (3 times). The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (0-20% ethylacetate in cyclohexane v/v) which afforded 4 (6.06 g, 14.8 mmol, 56%). R_f = 0.61 (cyclohexane/ethylacetate 60:40 v/v); MALDI- TOF m/z [M + H]⁺ found 410.0, calcd 409.1; ¹H NMR (270 MHz, CDCl₃): 7.32-7.2 (m, 1OH, benzyl), 5.71 (d, J_H-i_{, H}-₂= 3.71 Hz, IH, H-I), 4.74 (d, J_gem= 12 Hz, IH, CH₂Ph), 4.59-4.52 (m, 4H, H-2, CH₂Ph, 2xCH₂Ph), 4.06 (d, J_H-2, _H-₃= 4.95 Hz, IH, H-3), 3.5 (ABq, J_gem - 10.39 Hz, 2H, H-5', H-5"), 3.15 (d, Jg_em = 17.07 Hz, IH, H-6'), 2.86 (d, IH, H-6"), 1.58 (s, 3H, CH₃, isopropyl), 1.32 (s, 3H, CH₃, isopropyl); ¹³C NMR (67.9 MHz, CDCl₃): 137.3, 137, 128.3, 128.2, 127.7, 127.5, 117.0 (CN), 113.4 (q, isopropyl), 103.9 (C-I), 83.2 (q, C-4), 78.4 (C-2), 78 (C-3), 73.6 (CH₂Ph), 72.4 (CH₂Ph, C-5), 26.5 (CH₃, isopropyl), 25.6 (CH₃, isopropyl), 22.1 (C-6). l-[2-0-Acetyl-3,5-di-0-benzyl-4-C-cyanomethyl-j3-D-ribofuranosyl]thymine (6). Triflic acid (0.065 mL, 0.74 mmol) was added dropwise to a stirred solution of 4 (6.06 g, 14.8 mmol) in acetic acid (89 mL) and acetic anhydride (16.7 mL, 177.6 mmol). The solution was stirred for 3 h at r.t. and then poured into cold NaHCO₃ solution. The mixture was extacted with dichloromethane and the organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure. The crude product was co-evaporated several times with dry toluene till the product solidifies to give 5 (more than 90% pure by NMR). The crude product was dissolved in 150 mL of anhydrous CH₃CN, thymine (2.24 g, 17.7 mmol) and N,O-bis(trimethylsilyl)acetamide (10.2 mL, 41.4 mmol) was added and stirred at 80 ⁰C for 1 h under nitrogen atmosphere. The reaction mixture was cooled to r.t, TMSOTf (3.48 mL, 19.24 mmol) was added and again warmed to 80 ⁰C and stirred overnight under nitrogen atmosphere. The reaction was quenched with saturated aqueous NaHCO₃ and extracted with dichloroniethane. The organic phase was dried over anhydrous MgSO₄, evaporated under reduced pressure and purified by silica gel column chromatography (0-3% methanol in dichloromethane, v/v) to afford 6 (6.14 g, 11.8 mmol, 80%). Rf = 0.56 (CH₂C1₂/CH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 520.0, calcd 519.2; ¹H NMR (270 MHz, CDCl₃): 8.8 (br s, IH, NH), 7.35-7.24 (m, HH, benzyl, H-6), 6.1 (d, J_H-r, _H-_2'= 4.8 Hz, IH, H-I'), 5.50 (app t, J= 5.32 Hz, IH, H-2'), 4.62 (d, J_gem = 11.13 Hz, CH₂Ph), 4.53-4.44 (m, 4H, H-3', CH₂Ph, 2xCH₂Ph), 3.80 (d, J_gem = 10.14 Hz, IH, H-5'), 3.63 (d, IH, H-5"), 2.75 (ABq, J_gem = 17.07 Hz, 2H, H- 6', H-6"), 2.10 (s, 3H, OAc), 1.60 (s, 3H, CH₃, thymine); ¹³C NMR (67.9 MHz, CDCl₃): 169.7 (CO), 163.3 (C-4), 150.1 (C-2), 136.6, 135.8 (C-6),128.6, 128.4, 128.1, 128.0, 116.3 (CN), 111.5 (q, C-5), 87.9 (C-I'), 84.7 (q, C-4'), 77.1 (C-3¹), 74.7 (CH₂Ph), 74.3 (C-2¹), 73.7 (C-5'), 22.2(C-6'), 20.5 (CH₃, OAc), 11.9 (CH₃, thymine). l-^jS-Di-O-benzyM-C-cyanomethyl^-O-methanesulfonyl-jS-D-ribofuranosyllthymine (8).

Nucleoside 6 (6.14 g, 11.8 mmol) was dissolved in methanol 60 mL, 18 mL of 1 M sodium methoxide was added and stirred at r.t. for 3 h. The solvent was partially evaporated under reduced pressure and extracted with dichloromethane. The combined organic phase was dried over anhydrous MgSθ₄, evaporated to give 7 (more than 90% pure by NMR) as a white solid. The crude product was co- evaporated with dry pyridine 3 times to remove traces of moisture and dissolved in 60 mL of the same solvent. Reaction was cooled in ice bath; methanesulfonyl chloride (1.8 ml, 23.6 mmol) was added drop wise and stirred at 0 ⁰C for 6 h. The reaction was quenched with saturated aqueous NaHCO₃ and extracted with dichloromethane. The organic phase was dried over anhydrous MgSO₄, evaporated under reduced pressure and co-evaporated 3 times with toluene and 3 times with dichloromethane. The product was purified using silica gel column chromatography (0-3% methanol in dichloromethane, v/v) to afford 8 (6.2 g, 11.2 mmol, 95%). Rf = 0.41 (CH₂C1₂/CH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 556.0, calcd 555.1; ¹H NMR (270 MHz, CDCl₃): 10.1 (br s, IH, NH), 7.46 (s, H-6), 7.33-7.2 (m, 1OH, benzyl), 6.05 (d, J_H-r, _H-₂< = 2.72 Hz, IH, H-I'), 5.39 (m, IH, H-2'), 4.84 (d, J_gem = 11.38 Hz, IH, CH₂Ph), 4.53-4.40 (m, 4H, H-3', CH₂Ph, 2xCH₂Ph), 3.88 (d, J_661n= 10.27 Hz, IH, H-5¹), 3.56 (d, IH, H- 5"), 3.15 (s, 3H, OMs), 2.92 (d, J_gem = 17.32 Hz, IH, H-6'), 2.71 (IH, H-6"), 1.45 (s, 3H, CH_3, thymine); ¹³C NMR (67.9 MHz, CDCl₃): 163.7 (C-4), 150.5 (C-2), 136.3 (C-6), 136.2, 135.3, 128.7, 128.4, 128.3, 128.1, 128.0, 116.1 (CN), 111.1 (q, C-5), 85.6 (C-I'), 84.7 (q, C-4'), 79.2 (C-2'),75.3 (C-3¹), 73.7 (CH₂Ph),73.4 (CH₂Ph), 70.8 (C-5¹), 38.6 (OMs), 22.1(C-6'), 11.6 (CH₃, thymine). l^'-Anhydro-iPjS-di-O-benzyl-ΦC-cyanomethyl-jS-D-ribofuranosylJthymine (9). Nucleoside 8 (6.2 g, 11.2 mmol) was dissolved in 70 mL of anhydrous CH₃CN; DBU (1.75 mL, 11.76 mmol) was added drop wise and stirred at r.t. for 1 h. The reaction was quenched with water and extracted with dichloromethane. Combined organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (0-4% methanol in dichloromethane, v/v) to afford 9 (4.7 g, 10.2 mmol, 91%). R_f = 0.21 (CH₂C1₂/CH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 460.0, calcd 459.1; ¹H NMR (270 MHz, CDCl₃): 7.35-7.12 (m, 1 IH, benzyl, H-6), 6.25 (br d, J_{H-r, H-2'}= 3.8 Hz, IH, H-I'), 5.32 (br d, IH, J_H-2. _H-r= 3.6 Hz, H-2'), 4.77 (d, Jgem = 11.63 Hz, IH, CH₂Ph), 4.62 (d, IH, CH₂Ph), 4.43-4.31 (m, 3H, H-3', 2xCH₂Ph), 3.36 (d, J_gera = 10.02 Hz, IH, H-5'), 3.23 (d, IH, H-5"), 2.80 (d, J_gem = 16.70 Hz, IH, H-6'), 2.7 (d, IH, H-6"), 1.96 (s, 3H, CH₃, thymine); ¹³C NMR (67.9 MHz, CDCl₃): 171.9 (C-4), 159.0 (C-2), 136., 135.4, 129.7 (C-6), 128.7, 128.6, 128.4, 128.1, 127.8, 119.4 (q, C-5), 116.4 (CN), 89.6 (C-I'), 88.1 (q, C-4'), 85.7 (C- 20,83.4 (C-3¹), 73.7 (CH₂Ph),73.3 (CH₂Ph), 71.1 (C-5'), 22.2 (C-6'), 13.9 (CH₃, thymine). l-[3,5-Di-0-benzyl-4-C-cyanomethyl-/3-D-arabinofuranosyl]thymine (10). To a solution of 9 (4.7 g, 10.2 mmol) in 200 mL of acetone 204 mL of 0.1M H₂SO₄ was added and refluxed overnight with stirring. The solvent was partially evaporated; saturated aqueous NaHCO₃ was added and extracted with dichloromethane. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure to give 10 quantitatively. R_f = 0.35 (CH₂CyCH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 478.1, calcd 477.1; ¹H NMR (270 MHz, CDCl₃): 11.1 (br s, IH, NH), 7.38-7.23 (m, HH, benzyl, H-6), 6.18 (d, JH-r, H-2_'= 3.09 Hz, IH, H-I'), 5.22 (d, J= 4.45 Hz, IH, 2'-0H) 4.89 (m, IH, H-2'), 4.66- 4.52 (m, 3H, CH₂Ph, 2xCH₂Ph) 4.41 (d, J_gem = 11.5 Hz, IH, CH₂Ph), 4.05 (s, IH, H-3'), 3.80 (ABq, J_gem = 9.5 Hz, 2H, H-5', H-5"), 2.90 (d, J_gem = 16.70 Hz, IH, H-6'), 2.73 (d, IH, H-6"), 1.62 (s, 3H, CH_3> thymine); ¹³C NMR (67.9 MHz, CDCl₃): 165.9 (C-4), 150.3 (C-2), 138.7 (C-6), 137.1., 136.6, 128.3 128.1, 127.9, 127.8, 127.5, 117.1 (CN), 107.6 (q, C-5), 87.3 (C-I'), 84.5 (q, C-4'), 83.5 (C-3¹), 73.5 (CH₂Ph), 72.9 (C-2'), 71.9 (CH₂Ph), 70.8 (C-5'), 21.6 (C-6'), 12.1 (CH₃, thymine). l-PjS-Di-O-benzyM-C-cyanomethyl-l-O-trifluoromethanesulfonyl-jS-D- arabinofuranosyl] thymine (11). Nucleoside 10 (4.8 g, 10.2 mmol) was co-evaporated 3 times with dry pyridine to remove traces of moisture and dissolved in a mixture of anhydrous dichloromethane (40 mL) and anhydrous pyridine (10 mL). To this solution DMAP (5 g, 40.8 mmol) was added, cooled in ice bath and trifluoromethanesulfonic anhydride was added drop wise and stirred under nitrogen atmosphere for 2.5 h. The reaction was quenched with cold saturated aqueous NaHCO₃ and extracted with dichloromethane. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure and co-evaporated with toluene (3 times) and dichloromethane (3 times). The crude product was purified by silica gel column chromatography (0-2% methanol in dichloromethane, v/v) to afford 11 (5.2 g, 8.5 mmol, 84%). R_f= 0.58 (CH₂C1₂/CH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 609.99, calcd 609.1; ¹H NMR (270 MHz, CDCl₃): 9.47 (br s, IH, NH), 7.38-7.20 (m, 1OH, benzyl), 7.15 (d, J_n. 6,H-_CH₃ = L l IHz, IH, H-6), 6.33 (d, J_H-i_{\ H}-₂- = 3.59 Hz, IH, H-I'), 5.47 (br d, J = 2.85 Hz, IH, H-2'), 4.81 (d, J_gem = 11.63 Hz, IH, CH₂Ph), 4.59-4.48 (m, 3H, CH₂Ph, 2xCH₂Ph) 4.42 (s, IH, H-3'), 3.73 (d, Jg_em = 9.65 Hz, IH, H-5'), 3.57 (d, IH, H-5"), 2.90 (d, J_gem = 16.95 Hz, IH, H-6'), 2.76 (d, IH, H-6"), 1.85 (d, JH-6,H-CH3 = 1.1Hz, 3H, CH₃, thymine); ¹³C NMR (67.9 MHz, CDCl₃): 163.2 (C-4), 149.8 (C-2), 136.4 (C-6), 135.1., 134.9, 128.6 128.5, 128.2, 128.1, 127.6, 118 (q, J= 320.2, CF₃), 116.3 (CN), 111.2 (C-5), 85.4 (C-2'), 83.9 (C-I'), 83.6 (C-4'), 82.2 (C-3'), 73.66 (CH₂Ph), 73.60 (CH₂Ph), 70.7 (C-5'), 21.7 (C-6¹), 12.2 (CH₃, thymine). (lR,5R,7R,8S)-5-Benzyloxymethyl-8-benzyIoxy-7-(thymin-l-yl)-2-aza-6-oxabicycIo[3.2.1]octane (12a and 12b). To a solution of 11 (5.2 g, 8.5 mmol) in 120 mL of dry THF, NaBH₄ (965 mg, 25.5 mmol) was added. To this suspension trifluoroacetic acid (1.3 mL, 17 mmol) was added drop wise over a period of 30 min. under nitrogen atmosphere and stirred overnight at r.t. After complete conversion, excess NaBH₄ was hydrolyzed carefully with water, stirred at r.t. for 2 h and extracted with dichloromethane. Note that if reaction is worked up after 30 min after hydrolyzing NaBH₄ gives substantial amount of minor isomer indicating that this isomer is the kinetic product which convert to the major isomer in presence of NaOH formed during hydrolyzing NaBH₄. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure. Purification by silica gel column chromatography (0-6% methanol in dichloromethane, v/v) afforded 12a (1.6 g, 3.4 mmol, 40%) along with the other diasteriomer 12b as a minor product (190 mg, 0.4mmol, 5%). (Major diastereomer): Rf= 0.42 (CH₂CVCH₃OH 90:10 v/v); MALDI-TOF m/z [M + H]⁺ found 464.1, calcd 463.2; ¹H NMR (500 MHz, CDCl₃): 7.97 (q, J= 1.3 Hz, H6, Thymine), 7.37 - 7.24 (m, 1OH, benzyl), 5.94 (s, IH, H-I'), 4.68 (d, J = 11.8 Hz, IH, CH₂Ph), 4.57 (d, J = 11.5 Hz, IH, CH₂Ph), 4.53 (d, J = 11.8 Hz, IH, CH₂Ph), 4.51 (d, J= 11.5 Hz, IH, CH₂Ph), 3.98 (d, J= 3.9 Hz, IH, H-3'), 3.71 (d, J = 10.8 Hz, IH, H5'), 3.58 (d, J = 10.8 Hz, IH, H-5"), 3.52 (d, J = 3.9 Hz, IH, H2'), 3.13 (ddd, J= 13.3, 11.6, 4.9 Hz, IH, H-7"), 3.02 (dd, J = 13.3, 6.5 Hz, lH,H-7'), 2.03 (ddd, J = 13.1, 11.6, 6.7 Hz, IH, H-6'), 1.43 (d, J = 1.3 Hz, IH, CH₃, Thymine), 1.31 (dd, J = 13.1, 4.8 Hz, IH, H6"); ¹³C NMR (125.7 MHz, CDCl₃): 163.9 (C-4), 150.0 (C- 2), 137.4, 137.2, 135.7 (C-6), 128.5 128.3, 128.1, 128.0, 127.8, 109.4 (C-5), 87.0 (C-F), 84.4 (C-4'), 73.4 (CH₂Ph), 71.8 (CH₂Ph), 71.7 (C-3¹), 70.3 (C-5¹), 59.1 (C-2¹), 38.4 (C-7'), 27.5 (C-6¹), 11.7 (CH₃, thymine).

(Minor diastereomer): R_f= 0.71 (CH₂CyCH₃OH 92:8 v/v); MALDI-TOF m/z [M + H]⁺ found 464.0, calcd 463.2; ¹H NMR (600 MHz, CDCl₃) δ ppm 7.95 (s, IH, NH, Thymine), 7.77 (q, J = 1.2 Hz, IH, H6, Thymine), 7.44-7.24 (m, 10 H, Bn), 6.28 (s, IH, H-F), 4.64 (d, J = 11.6 Hz, IH, CH₂Ph), 4.58 (d, J = 11.5 Hz, IH, CH₂Ph), 4.56 (d, J = 11.6 Hz, IH, CH₂Ph), 4.53 (dd, J = 11.92, 3.91 Hz, IH, NH), 4.50 (d, J = 11.5 Hz, IH, CH₂Ph), 4.33 (d, J = 4.1 Hz, IH, H3'), 3.73 (d, J = 11.0 Hz, IH, H5¹), 3.58 (d, J = 4.1 Hz, IH, H2^r), 3.52 (d, J = 11.0 Hz, IH, H5"), 3.26 (ddd, J = 14.2, 6.4, 3.9 Hz, IH, H7'), 2.98 (dtd, J = 14.2, 11.9, 11.9, 5.2 Hz, IH, H7"), 2.04 (ddd, J = 13.4, 11.9, 6.4 Hz, IH₉ Ho¹), 1-53 (dd, J = 13.4, 5.2 Hz, IH, H6"), 1.50 (d, J = 1.2 Hz₅CH₃, Thymine). ¹³C NMR (125.7 MHz, CDCl₃): 163.5 (C-4), 149.1 (C-2), 136.8, 136.0, 135.6 (C-6), 128.6 128.3, 128.2, 128.0, 127.9, 110.3 (C-5), 82.6 (C-I'), 82.5 (C-4¹), 73.68 (CH₂Ph), 73.64 (CH₂Ph), 72.0 (C-3¹), 69.5 (C-5¹), 64.2 (C-2'), 45.9 (C-7¹), 26.8 (C-6¹), 11.9 (CH₃, thymine).

(lR,5R,7R,8S)-8-Hydroxy-5-hydroxymethyI-7-(thymin-l-yl)-2-aza-6-oxabicycIo[3.2.1]octane (13). Nucleoside 12a/12b (1.6 g, 3.4 mmol) was dissolved in 15 mL methanol, 20% Pd(OH)₂ on charcoal (615 mg) was added followed by ammonium formate (2.5g, 40 mmol) and refluxed for 12 h. The catalyst was filtered off through celite bed and the filtrate evaporated under reduced pressure and co- evaporated with dichloromethane to remove traces to methanol. The crude material was dissolved in 20 mL of anhydrous dichloromethane, cooled to -78 ⁰C and 1 M BCl₃ (27 mL) was added and stirred under nitrogen atmosphere for 3 h. Solvent and volatile materials were removed under reduced pressure to give 13 which was purified for characterization using silica gel column chromatography (0-20% methanol in dichloromethane, v/v) to give 13 in 60% yield. R_f = 0.18 (CH₂Cl₂ZCH₃OH 80:20 v/v) MALDI-TOF m/z [M + H]⁺ found 284.2, calcd 284.1; ¹H NMR of 13 (600 MHz, DMSO _d6) δ ppm 11.30 [s, IH, NH(Thymine)], 8.27 (s, IH, H6), 5.84 (s, IH, H-I'), 5.39 (t, J = 5.0, 5.0 Hz, IH, OH(5'), 5.16 (br, IH, OH(3'), 3.99 (dd, J = 3.9, 4.6 Hz , IH, H3'), 3.57 (dd, J = 12.2, 5.2 Hz, IH, H5'), 3.50 (dd, J = 12.2, 5.2 Hz, IH, H5"), 3.25 (d, J = 3.19 Hz, IH, H2'), 2.95 (dt, J = 12.8, 13.0, 4.8 Hz, IH, H7"), 2.87 (dd, J = 12.8, 6.6 Hz, IH, H7'), 1.78 (dt, J = 12.9, 13.0, 6.8 Hz, IH, H6'), 1.78 (s, 3H, CH₃, Thymine), 1.17 (dd, J = 12.9, 4.6 Hz, IH, H6"). ¹³C NMR (600 MHz, DMSO_d6): 164.9 (C-4), 151.0 (C-2), 137.0 (C-6), 108.5 (C-5), 86.3 (C-4¹), 86.2 (C-I'), 64.5 (C-3¹), 62.3 (C-5'), 62.0 (C-2'), 38.9 (C-7^r), 27.2 (C-6'), 13.3 (CH₃, Thymine). (lR,5R,7R,8S)-5-BenzyloxymethyI-8-benzyloxy-2-phenoxyacetyl-7-(thymin-l-yl)-2-aza-6- oxabicyclo[3.2.1]octane (14). To a solution of 12a (1.6 g, 3.4 mmol) in pyridine was added phenoxyacetyl chloride (0.6 mL, 4.4 mmol) was added dropwise and stirred under nitrogen atmosphere for 2 h. Reaction was quenched with cold saturated aqueous NaHCO₃ and extracted with dichloromethane. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure and co-evaporated with toluene (3 times) and dichloromethane (3 times). The crude product was purified by silica gel column chromatography (0-3% methanol in dichloromethane, v/v) to afford 14 (Note that 12b reacted very slowly as it converted to 12a first and then to 14 in 24 h) (1.4 g, 2.4 mmol, 70%). Rf = 0.35 (CH₂CVCH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 598.2, calcd 597.2; ¹³C NMR (67.9 MHz, CDCl₃): 168.6, 166.8, 163.6, 163.5, 157.9, 157.6, 150.0, 149.5, 137.0, 136.9, 135.1, 135.0, 129.5, 129.4, 128.5, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 127.4, 121.6, 121.4, 114.6, 114.5, 110.0, 109.8, 87.1, 85.9, 84.7, 84.6, 73.5, 73.4, 72.5, 72.4, 72.5, 72.4, 71.0, 69.6, 69.4, 67.7, 59.5, 55.8, 53.3, 39.1, 36.6, 27.0, 25.6, 11.7

(lR,5R,7R,8S)-8-Hydroxy-5-hydroxymethyI-2-phenoxyacetyl-7-(thymin-l-yI)-2-aza-6- oxabicyclo [3.2.1] octane (15). Nucleoside 14 (1.4 g, 2.4 mmol) was dissolved in 15 mL methanol, 20% Pd(OH)₂ on charcoal (430 mg) was added followed by ammonium formate (1.8 Ig, 28.8 mmol) and refluxed for 12 h. The catalyst was filtered off through celite bed and the filtrate evaporated under reduced pressure and co-evaporated with dichloromethane to remove traces to methanol. The crude material was dissolved in 25 mL of anhydrous dichloromethane, cooled to -78 ⁰C and 1 M BCl₃ (19 mL) was added and stirred under nitrogen atmosphere for 3 h. Solvent and volatile materials were removed under reduced pressure and co-evaporated with methanol. The residue was purified by silica gel column chromatography (0-4% methanol in dichloromethane, v/v) to give 15 (750 mg, 1.8 mmol, 75%). Rf = 0.38 (CH₂C1₂/CH₃OH 90: 10 v/v); MALDI-TOF m/z [M + H]⁺ found 418.2, calcd 417.1; ¹³C NMR (67.9 MHz, CD₃OD): 170.7, 166.9, 159.8, 152.4, 137.9, 137.7, 130.7, 130.6, 122.7, 122.5, 116.2, 116.0, 110.6, 87.8, 87.5, 87.3, 67.9, 66.9, 65.7, 65.4, 63.1, 62.9, 60.4, 37.8, 27.2, 26.5, 12.9. (lR,5R,7R,8S)-5-(4,4'-DimethoxytrityIoxymethyl)-8-hydroxy-2-phenoxyacetyl-7-(thymin-l-yI)-2- aza-6-oxabicyclo [3.2.1] octane (16). Nucleoside 15 (750 mg, 1.8 mmol) was co-evaporated with anhydrous pyridine to remove traces of water and dissolved in 15 mL of the same solvent, 4,4'- dimethoxytrityl chloride was added and stirred at r.t. for 7 h. Reaction was quenched using cold saturated aqueous NaHCO₃ and extracted with dichloromethane. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure and co-evaporated with toluene (2 times) to remove pyridine partially. The crude product was purified by silica gel column chromatography (0-3% methanol in dichloromethane, v/v containing 1% pyridine) to afford 16 (1.16 g, 1.6 mmol, 90%). Rf = 0.30 (CH₂CVCH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 720.1, calcd 719.2;¹³C NMR (67.9 MHz, CDCl₃ + DABCO): 168.4, 163.6, 158.6, 157.1, 150.0, 144.1, 135.2, 135.0, 134.6, 129.9, 129.7, 129.5, 128.9, 128.0, 127.7, 127.0, 121.9, 121.6, 114.6, 114.4, 113.2, 110.5, 86.6, 86.0, 67.4, 65.3, 62.2, 62.2, 55.1, 46.7, 36.4, 25.6, 11.9.

(lR,5R,7R,8S)-8-(2-(Cyanoethoxy(diisopropylamino)-phosphinoxy)-5-(4,4'- dimethoxytrityIoxymethyl)-2-phenoxyacetyl-7-(thyrain-l-yl)-2-aza-6-oxabicyclo[3.2.1] octane (17). Compound 16 (1.16 g, 1.6 mmol) was dissolved in 15 mL of dry THF, diisopropylethylamine (1.4 mL, 8 mmol) was added at 0 ⁰C followed by 2-cyanoethyl N, N- diisopropylphosphoramidochloridite (0.71 mL, 3.2 mmol). After 30 min. reaction was warmed to room temperature and stirred overnight. MeOH (0.5 mL) was added and stirring was continued for 5 min. thereafter saturated aqueous NaHCO₃ was added and extracted with freshly distilled CH₂Cl₂ (3 times). The organic phase was dried over MgSθ₄, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (40-70% CH₂Cl₂ in cyclohexane containing 1% Et₃N) afforded 17 (1.26 g, 1.37 mmol, 86%) as a mixture of four isomers. R_f = 0.40 (CH₂C1₂/CH₃OH 96:4 v/v); MALDI-TOF m/z [M + H]⁺ found 920.2, calcd 919.3; ³¹P NMR (67.9 MHz, CDCl₃): 150.7, 150.3, 149.2, 148.1. (lR,5R,7R,8S)-8-Hydroxy-5-hydroxymethyl-2-trifluoroacetyl-7-(thymiii-l-yl)-2-aza-6- oxabicyclo[3.2.1]octane (18). The nucleoside 12a (1.6 g, 3.4 mmol) was deprotected as it was done for 13 and the crude material was dissolved in methanol (20 mL), DMAP (415 mg, 3.4mmol) and ethyl trifluoroacetate (4 mL, 34 mmol) was added and stirred at r.t. overnight. The solvent was removed under reduced pressure and purified by silica gel column chromatography (0-6% methanol in dichloromethane, v/v) to give 18 (580 mg, 1.5 mmol, 45%) with slight contamination of DMAP. Compound 18 Rf = 0.43 (CH₂C1₂/CH₃OH 90:10 v/v); MALDI-TOF m/z [M + H]⁺ found 380.1, calcd 379.0; ¹³C NMR (67.9 MHz, CD₃OD): 166.8, 157.7, 152.1, 145.4, 145.2, 138.4, 137.7, 110.8, 110.7, 108.2, 87.3, 87.2, 86.8, 65.5, 65.2, 62.6, 62.5, 61.8, 41.5, 39.9, 39.0, 27.4, 26.7, 12.9.

(lR,5R,7R,8S)-5-(4,4'-Dimethoxytrityloxymethyl)-8-hydroxy-2-trifluoroacetyI-7-(thymin-l-yl)- 2-aza-6-oxabicyclo[3.2.1]octane (19). Nucleoside 18 (580 mg, 1.5 mmol) was co-evaporated with anhydrous pyridine to remove traces of water and dissolved in 15 mL of the same solvent, 4,4' dimethoxytrityl chloride was added and stirred at r.t. overnight. Reaction was quenched using cold saturated aqueous NaHCO₃ and extracted with dichloromethane. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure and co-evaporated with toluene (2 times) to remove pyridine partially. The crude product was purified by silica gel column chromatography (0-3% methanol in dichloromethane, v/v containing 1% pyridine) to afford 19 (827 mg, 1.2 mmol, 81%). Rf = 0.31 (CH₂C1₂/CH₃OH 96:4 v/v); MALDI-TOF m/z [M + Na]⁺ found 704.2, calcd 681.2;¹³C NMR (67.9 MHz, CDCl₃ + DABCO): 164.02, 163.5, 158.6, 150.0, 149.7, 144.0, 143.9, 134.5, 129.9, 127.9, 127.2, 127.0, 113.3, 110.6, 86.7, 85.4, 85.3, 65.8, 65.4, 63.0, 62.9, 60.1, 55.1, 46.0, 40.1, 37.3, 29.5, 27.0, 26.1, 11.9, 11.8.

(lR,5R,7R,8S)-8-(2-(Cyanoethoxy(diisopropylamino)-phosphinoxy)-5-(4,4'- dimethoxytrityloxymethyl)-2-trlfluoroacetyl-7-(thymin-l-yI)-2-aza-6-oxabicycIo[3.2.1]octane (20). Compound 19 (827 mg, 1.2 mmol) was dissolved in 12 mL of dry THF, diisopropylethylamine (1.05 mL, 6 mmol) was added at 0 ⁰C followed by 2-cyanoethyl N, N- diisopropylphosphoramidochloridite (0.53 mL, 2.4mmol). After 30 min. reaction was warmed to room temperature and stirred overnight. MeOH (0.5 mL) was added and stirring was continued for 5 min. thereafter saturated aqueous NaHCO₃ was added and extracted with freshly distilled CH₂Cl₂ (3 times). The organic phase was dried over anhydrous MgSO₄, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (40-100% CH₂Cl₂ in cyclohexane containing 1% Et₃N) afforded 20 (645 mg, 0.73 mmol, 61%) as a mixture of four isomers. R_f= 0.44 (CH₂Cl₂ZCH₃OH 96:4 v/v); MALDI- TOF m/z [M + H]⁺ found 882.2, calcd 881.3; ³¹P NMR (109.4 MHz, CDC13): 150.1, 149.9, 149.8, 149.2.

Synthesis, Deprotection and Purification of Oligonucleotides.

All oligonucleotides were synthesized using an automated DNA/RNA synthesizer by Applied Biosystems, model 392. For modified AONs containing aza-ENA units, fast deprotecting phosphoramidites (nucleobases were protected using the following groups: Ac for C, ¹Pr-PAC for G, and PAC for A) were used. The AONs were deprotected at room temperature by aqueous NH₃ treatment for 24 h. All AONs and the target RNA were purified by 20% polyacrylamide/7M urea) PAGE, extracted with 0.3 M NaOAc, desalted with C18-reverse phase catridges and their purity (greater than 95%) was confirmed by PAGE.

UV Melting Experiments.

Determination of the T_m of the AON/RNA hybrids was carried out in the following buffer: 57 mM Tris-HCl (pH 7.5), 57 mM KCl, 1 mM MgCl₂. Absorbance was monitored at 260 run in the temperature range from 20 ⁰C to 70 ⁰C using UV spectrophotometer equipped with Peltier temperature programmer with the heating rate of 1 ⁰C per minute. Prior to measurements, samples (1 μM of AON and 1 μM RNA mixture) were preannealed by heating to 80 ⁰C for 5 min followed by slow cooling to 4 ⁰C and 30 min equilibration at this temperature.

CD Experiments. CD spectra were recorded from 300 to 200 run in 0.2 cm path length cuvettes. Spectra were obtained with a AON/RNA duplex concentration of 5 μM in 57 mM Tris-HCl (pH 7.5), 57 mM KCl, 1 mM MgCl₂. All the spectra were measured at 25 ⁰C and each spectrum is an average of 5 experiments from which CD spectrum of the buffer was subtracted.

³²P Labeling of Oligonucleotides. The oligoribonucleotide, oligodeoxyribonucleotides were 5'-end labeled with P using T4 polynucleotide kinase and [γ-³²P] ATP by standard procedure. Labeled AONs and RNA were purified by 20% denaturing PAGE and specific activities were measured using Beckman LS 3801 counter.

RNase H Hydrolysis Experiment.

The source of RNase Hl (obtained from Amersham Bioscience) was Escherichia coli containing clone of RNase H gene. The solutions of 15mer AON (l→ 5)/RNA duplex: [AON] = 10^"6 M, [RNA] = 10^~7 M in a buffer, containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA and 0.1 mM dithiothreitol (DTT) at 21 ⁰C in 30 μL of the total reaction volume have been used. The percentage of RNA cleavage was monitored by gel electrophoreses as a function of time (0-60 min), using 0.08 U and 0.12 U of RNase H.

Exonuclease Degradation Studies.

Stability of the AONs toward 3'-exonucleases was tested using snake venom phosphodiesterase from Crotalus adamanteus. All reactions were performed at 3 μM DNA concentration (5'-end ³²P labeled with specific activity 50 000 cpm) in 56 mM Tris-HCl (pH 7.9) and 4.4 mM MgCl₂ at 21 ⁰C. Exonuclease concentration of 17 ng/μL was used for digestion of oligonucleotides. Total reaction volume was 14 μL. Aliquots (3 μL) were taken at 1, 2, 4, and 24 h and quenched by addition to 7 μL volume of 50 mM EDTA in 80% formamide. Reaction progress was monitored by 20% denaturing (7 M urea) PAGE and autoradiography.

Stability studies in human serum.

AONs (6 μL) at 2 μM concentration (5'-end ³²P labeled with specific activity 90 000 cpm) were incubated in 26 μL of human serum (male AB) at 21 ⁰C (total reaction volume was 36 μL). Aliquots (3 μL) were taken at 0, 15 and 30 min, 1, 2 and 9 h, and quenched with 7 μL quenching solution containing 50 mM EDTA in 80% formamide, resolved in 20% polyacrylamide denaturing (7 M urea) gel electrophoresis and visualized by autoradiography. Theoretical calculations.

2',4' conformationally constrained aza-ENA (3',5'-bis-OBn protected compounds 12a and 12b and de- protected 13) nucleosides have theoretically simulated to build up their molecular structures using the following protocol: (i) Derive Initial dihedral angles from the observed ³J_H1H using Haasnoot-de Leeuw- Altona generalized Karplus equation⁷³' ⁷⁴ (ii) Perform NMR constrained molecular dynamics (MD) simulation ( 0.5 ns,10 steps) simulated annealing (SA) followed by 0.5 ns NMR constrained simulations at 298 K using the NMR derived torsional constraints from Step (i) to yield NMR defined molecular structures of 3',5'-bis-OBn protected (12a, 12b) and de-protected aza-ENA (13). (iii) Acquire 6-3 IG** Hartree-Fock optimized ab initio gas phase geometries in order to compare the experimentally derived torsions with the ab initio geometry, (iv) Refine the Karplus parameters with the help of the NMR- derived and ab initio derived torsions, (v) Analyze the full conformational hyperspace using 2 ns ΗMRJab initio constrained MD simulations of compounds 12a, 12b and 13 followed by full relaxation of the constraints. The geometry optimizations of the modified nucleosides have been carried out by GAUSSIAN 98 program package⁷⁷ at the Hartree-Fock level using 6-3 IG** basis set. The atomic charges and optimized geometries of compounds 12a, 12b, and 13 were then used as AMBER⁷⁵ force field parameters employed in the MD simulations. The protocol of the MD simulations is based on Cheathan-Kollman's procedure employing modified version of Amber 1994 force field as it is implemented in AMBER 7 program package.⁷⁵ Periodic boxes containing 1394, 1449, 1076, and 715, TIP3P⁷⁶ water molecules to model explicit solvent around the 3\5'-bis-OBn protected (N-H axial and N- H equatorial), and the de-protected (N-H axial and N-H equatorial) aza-ENAs, respectively, were generated using xleap extending 12.0 A from these molecules in three dimensions in both the NMR constrained and unconstrained MD simulations. SA protocol included ten repeats of 25 ps heating steps from 298 K to 400 K followed by fast 25 ps cooling steps from 400 K to 298 K. During these SA and NMR constrained MD simulations torsional constrains of 50 kcal mol^" rad^" were applied. The constrains were derived from the experimental ³JHr₁H₂ ¹, ³JH2_',H3_' ^and available ³JH7a/H7b,NH coupling constants using Haasnoot-de Leeuw-Altona generalized Karplus equation⁷³' ⁷⁴ and parameters discussed in the text. Ten SA repeats were followed by 0.5 ns MD run at constant 298 K temperature applying the same NMR constrains.

Acknowledgement.

Generous financial support from the Swedish Natural Science Research Council (Vetenskapsradet), the Swedish Foundation for Strategic Research (Stiftelsen for Strategisk Forskning) and the EU-FP6 funded RIGHT project (Project no. LSHB-CT-2004-005276) is gratefully acknowledged.

Supporting Information Available.

¹³C NMR spectra of compounds 4, 6-11, 14-16, 18 and 19; ³¹P NMR spectra of compounds 17 and 20; ¹³C, ID NOE, HSQC, HMBC NMR spectra of compound D (Scheme 1); ¹³C, HSQC, HMBC and COSY NMR spectra of compounds 12a, 12b and 13; RNase H digestion profile of AONs at 0.08 U enzyme concentration; denaturing PAGE picture in human serum and tables of ¹H and ¹³C chemical shifts and coupling constants of compounds 12a, 12b and 13; Complete Reference 75 and 77 are available free of charge via the Internet at http://pubs.acs.org.

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Table of Contents Graphic

3 ' -d (CTTCTTTTTTACTTC) -5 ' 5 ^τ -r (GAΑGAAAAΑAUGAA.G) -3 '

* Single at the time aza-ENA (T) substitution in AON strand.

* T_m increase by 2.5° to 4°C per T modification vis-a-vis Native.

* KNase H recruitment as efficient as the Native.

* Stable in the blood serum (> 48 h) compared to the Native (full degradation in 4 h).

13 15 18

From Scheme 2

Claims

CLAIMS:

1. A compound according to the formula:

2. Compound according to claim 1, modified by replacing the thymin moiety with adenine, guanine, cytosine or urazil.

3. Antisense oligo-nucleotides comprising a compound according to claim 1 or 2.

3. A compound according to claim 1 or 2 for use as a medicament.

4. A compound according to claim 1 or 2 for use as a medicament for the treatment of viral infections by inhibiting virus specific proteins, thereby inactivating the pathogen growth.

5. A compound according to claim 1 or 2 for use as a medicament for the treatment of cancer by inhibiting tumor specific proteins, thereby inactivating the tumor growth.

6. A substance as claimed in claim 1 or 2 for use in therapy.

7. A method of making a compound as claimed in claim 1 or 2, by the process according to Scheme 2.

8. Use of a compound as claimed in claim 1 or 2 for the manufacture of a medicament for the treatment of viral infections by inhibiting virus specific proteins, thereby inactivating the pathogen growth.

9. Use of a compound as claimed in claim 1 or 2 for the manufacture of a medicament for the treatment of cancer by inhibiting tumor specific proteins, thereby inactivating the tumor growth.

10. Compositions and/or formulations comprising a compound according to claim 1 or 2 or pharmaceutically acceptable modifications thereof, together with pharmaceutically compatible carriers and/ or excipients, for use as medicaments.

11. Use of a compound as claimed in claim 1 or 2 for diagnostic purposes.

12. Use according to claim 11 in a diagnostic method involving polymerase chain reaction.

13. A diagnostic kit comprising a compound as claimed in claim 1 or 2.