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Paper | Regular issue | Vol. 78, No. 7, 2009, pp. 1715-1728
Received, 21st January, 2009, Accepted, 18th March, 2009, Published online, 19th March, 2009.
DOI: 10.3987/COM-09-11660
Synthesis and Structure of New Methylene-Bridged Hexopyranosyl Nucleoside (BHNA)

Chuanzheng Zhou, Oleksandr Plashkevych, Yi Liu, Naresh Badgujar, and Jyoti Chattopadhyaya*

Department of Bioorganic Chemistry, Biomedical Center, Uppsala University, Box 581, S-75123 Uppsala, Sweden

A new member of hexopyranosyl nucleoside family, methylene-bridged hexopyranosyl nucleoside (BHNA), has been synthesized through generation of carbon radical at C6’ in [6’S-Me, 7’S-Me]-carba-LNA T nucleoside, followed by rearrangement to C4’ radical which was quenched by hydrogen atom to give BHNA. The stereoelectronic requirement for this unusual radical rearrangement has been elucidated by chemical model building and ab intio calculations to show that the coplanarity of the single electron occupied p-orbital at C6’ with σ*O4’-C4’ plays an important role for the rearrangement reaction to take place. The solution structure of BHNA has also been studied using NMR as well as by ab initio calculations. The new six-membered pyranosyl ring in BHNA, unlike other known hexopyranosyl nucleosides, adopts a twist conformation, with base moiety occupying the axial position while 3’-hydroxymethyl and 4’-hydroxyl occupying the equatorial position.

The pentose-sugar moiety in natural nucleos(t)ide is very flexible, adopting a dynamic equilibrium of several sugar pseudorotamers.1,2 In nature, sugar moieties in DNA are present mainly in the South-type pseudorotamers which confer B-form global conformation, whereas the predominant North-type sugars in RNA renders it to a A-form global conformation.3 It has been also found that some nucleotides are locked in to either South or North sugar conformation such as in ribozymes,4 or lariat RNAs,5,6 It is very likely that the conformation of the pentose-sugar moiety is a key factor7 that determines the global conformation of nucleic acid, and consequently their physical, chemical and biological properties.
Conformationally-constrained nucleotides have attracted considerable interest in the last two decades
8-11 because they enable us to dictate the sugar to take up a specific conformation that, in turn, gives us a handle to drive the structure and function of nucleic acids. The popular way to constrain the flexible pentose sugar moiety is linking two endocylic sugar carbons by a short covalent linkage.11 Thus, BNA/LNA,12-15 ENA16-19 and their analoges20-23 were obtained by a covalent bond formation between C2′ and C4′ with alkyl or alkyloxyl linker. Replacement of the pentofuranosyl sugar with hexopyranosyl sugar24 however gives another type of conformationally-constrained nucleoside because the activation energy barrier for interconversion of conformers in the later is much higher than in the former. Oligonucleotides with this hexopyranosyl type modification give thermodynamically stable duplexes with the target DNA or RNA.24 It is because, first, the six-membered ring system adopts a rigid chair conformation, requiring a less negative entropy change during the duplex formation; and second, the interstrand phosphate distance in the six-membered pyranosyl-modified system is larger than in the natural nucleic acid duplexes, giving a relatively less interstrand charge repulsion compared to the native counterpart.25 Many different types of hexopyranosyl nucleosides have been so far synthesized by the groups of Herdewijn9 and Eschenmoser.25 These hexopyranosyl nucleosides have also been incorporated in to oligonucleotides, and their affinity toward complementary RNA or DNA, as well as their biochemical features have been studied: oligonucleotides composed of dideoxyglycopyranosyl- 4',6'-nucleoside (homo-DNA, Figure 1) is almost linear, and not able to form duplexes with either RNA or DNA,25,26 while oligonucleotides with 1',5'-anhydrohexitol nucleoside (HNA, Figure 1),27,28 cyclohexanyl nucleoside (CNA, Figure 1)29 or cyclohexenyl nucleoside (CeNA, Figure 1)30-32 modification have improved affinity toward RNA and show improved nuclease resistance compared to the native counterpart. Amongst these, CeNA modified oligonucleotides have attracted more attention because they are able to activate RNase H, resulting in cleavage of the RNA strand in the DNA-RNA heteroduplexes.30

Recently, our group has reported synthesis of conformationally-constrained carba-LNA and carba-ENA nucleosides through radical cyclization reactions.20,33,34 It is known that carbon radical in the sugar moiety of nucleoside can induce C-O bond scission under anaerobic conditions: For example, C4′ radical leads to scission of C1′-O4′ bond35 followed by rearrangement to C1′ radical (Figure 2A). C1′ may lead to scission of C4′-O4′ bond and subsequent rearrangement to C4′ radical (Figure 2B).36 Accodingly, we argued, if the radical at C6′ in carba-LNA (Figure 2C) were generated under anaerobic conditions, the C6′ radical could be expected (compare with Figure 2A) to induce the scission of C4′-O4′ bond to give rearranged C4′ radical, and subsequent quenching by hydrogen atom. We hypothesized that, if successful, this putative transformation may constitute a new facile route to the synthesis of hexopyranosyl nucleoside.
Here, we report the synthesis of new bicyclo[2. 2.1]-2′,5′-methylene-bridged hexopyranosyl nucleoside
8 (BHNA, Scheme 1) through radical rearrangement under anaerobic conditions. The stereoelectronic requirement for the radical rearrangement from C6′ to C4′ has been elucidated by ab initio calculations, and the structure of BHNA has also been studied by high-resolution NMR.

(A) Synthesis of BHNA 8
The synthesis started from a known compound 1,33,34 6′S-OH, 7′S-Me-carba-LNA T (Scheme 1). Compound 1 was reacted with phenyl chlorothionoformate in pyridine at rt to give radical precursor 2. Treatment of 2 with Bu3SnH in presence of a catalytic amount of AIBN in anhydrous toluene unfortunately gave only C6′ reduced product 3 in a poor yield (17%), and most of the starting material 2 was recovered (46%). This suggested that the in situ generated secondary radical at C6′, under this condition, is not sufficiently reactive to undergo rearrangement to C4′ radical, and therefore was quenched very fast by the surrounding H. We argued that a more stable tertiary C6′ radical could increase the possibility of radical rearrangement from C4′ to C6′.
Therefore, radical precursor
434 was synthesized starting from compound 1. Treatment of compound 4 with Bu3SnH in presence of a catalytic amount of AIBN in anhydrous toluene, under an identical condition as used for compound 3 from 2, gave hithertofore an uncharacterized34 radical rearranged product, protected BHNA 5, in 27% yield along with the known C6′ reduced products 6 (18%) and 7 (15%).34 The protected BHNA 5 was debenzylated using 20% Pd(OH)2/C and ammonium formate under reflux in methanol to give fully deprotected BHNA 8 quantitatively.

(B) NMR characterization of BHNA thymidines 5 and 8
The key intermediates 5, and final product 8 were characterized by 1H, 13C, DEPT, COSY, 1H-13C HMQC, HMBC, 1D-nOe NMR experiments as well as mass spectroscopy.
For compound
5, the formation O1'-C6' bond can not be confirmed by HMBC experiment because the cross peak between H1' and C6' is unfortunately absence, but the structure of 5 is clearly evidenced by several 2D NMR experiments and mass spectroscopy: Although compound 5 has the same mass as that of diastereomer 6 or 7, the comparison of their respective NMR data clearly shows that compound 5 vis-à-vis 6/7 are structurally very different: (1) The COSY spectrum of compound 5 shows that the cross peak between H1' and H2', H2' and H3', H3' and H4' can be observed (Figure S27), which suggests that the covalent linkage along C1'-C2'-C3'-C4' pathway are conserved. (2) The presence of cross peak between H2' and H7' in the COSY spectrum (Figure S27), and the long-range connectivity evidenced by the cross peak between H-6'Me and C7' as well as C4', cross peak between C6' and H7'-Me in HMBC spectrum (Figure S29) suggests that the covalent linkage along C2'-C7'-C6'-C4' pathway is also conserved in compound 5. (3) The obvious difference between compound 5 and 6/7 is that in compound 5 the C4' is a tertiary carbon with H4' attached and C6' is a quaternary carbon (DEPT 45, 90 and 135 spectra in Figure S24), whereas in compound 6/7, the C6' is a tertiary carbon with H6' attached and C4' is a quaternary carbon (DEPT 45, 90 and 135 spectra in Figure S21). (4) That the C4' is tertiary is also further proven by C4'-H4' connectivity in 1H-13C HMQC spectra (Figure S26), which is also solidly backed up H3'-H4' connectivity in the COSY spectrum (Figure S27), which are clearly absent in 6/7 (Figures S15, 16 and 17).
Thus, all the above evidences put together led us argue that quaternary C6' in compound 5 should be attached to O4' beside C4', C7' and 6'-Me, and consequently, the hyper-constrained bicyclo[2.2.1]- 2′,5′-methylene-bridged hexopyranosyl nucleoside, BHNA 5, must have been obtained. This structure of
5 was also supported by 1D-nOe experiment (Figure 3 and S30) as well as by detailed comparison of the experimental and simulated 3JHH (see part D).
The configuration of C4′ and C7′ in protected BHNA
5 was evidenced by 1D nOe (nuclear Overhauser effect) experiments (Figure 3 and S30) and the results are as follows: (1) 7′-Me but not H7′ shows nOe upon irradiation at H1′, suggesting H7′ oriented away from H1′ (Figure S30). (2) Irradiation at H4′ shows more nOe enhancements at H7′ (3.9%) compared to H3′ (1.2%), suggesting H4′ and H3′ are trans orientated while H4′ and H7′ are cis orientated (Figure 3). (3) Similarly, irradiation at H7′ shows enhancements at H4′ (4.1%) and H2′ (2.0%). These results are consistent with the spatial atomic distances (dH4'-H3' = 2.93Å, dH4'-H7' = 2.38Å, dH7'-H2' = 2.65Å) obtained from ab initio geometry optimization (Figure 3).

(C) Mechanism of Radical Rearrangement
Treatment of radical precursor 2 or 4 with Bu3SnH in presence of a catalytic amount of AIBN in anhydrous toluene gives the putative radical intermediate 2a or 4a, which could either be reduced directly by H to give C6′ reduced product (Path “A” in Figure 4), or/and it could induce scission of C4′-O4′ bond

to give C4′ radical, which upon quenching by H could give hexopyranosyl nucleoside (Path “B” in Figure 4). Actually, radical 2a only followed the Path “A” to give C6′ reduced product 3. Whereas radical 4a gave both diastereomeric mixture of C6′ reduced compounds 6(6'S) and 7(6'R) by Path “A”, and the diastereomerically pure rearranged product 5 through radical 4b. The diastereospecific nature of 5 (4′R, 6′S) suggests that the H atom quenches the C4' radical in 4b exclusively from below the plane of the sugar-ring. These different fates of radical 2a and 4a suggest that a stable tertiary radical is indeed a prerequisite to ensure the rearrangement to take place through path “B”, in competition with path “A” giving the reduced product.
In contradistinction, generation of a radical at the C6′ center of the 6-membered carbocyclic nucleoside
9 (Figure 4C), under an identical condition, gave only the C6′ reduced products, 10 (6′R, 75%) and 11 (6'S, 16%), without any trace of the rearranged product.34
Crich et al. have shown that the C4' radical of 2',3'-dideoxythymidine is known to rearrange to C1' radical by scission of the C1'-O4' bond to give the acyclic product, but this reaction does not take place with 2'-deoxy-3'-methoxythymidine.
35 They suggested that different stereoelectronic effect in these two radical intermediates leads to their different fates. Accordingly, the structures of tertiary radicals 4a and 9a were investigated to show how the nature of different stereoelectronic effects causes their different fates.
Molecular structure (from
ab initio HF/6-31G** geometry optimization) of the radical 4a revealed (Table S1) that the radical formation expectedly changes sp3 character of C6' to the sp2 leading to formation of near-planar [C4'-C6'(Me)-C7'] geometry with the unpaired electron located on the p-orbital perpendicular to the plane. The [O4'-C4'-C6'-C(Me]) torsion in 4a is 80.8º (Figure 5C and Table S2) and the single electron occupied p-orbital of C6' is nearly coplanar (φ = 5º) with the σ*O4'-C4' orbital. This coplanarity favors the rupture of C4'-O4' bond and formation of new C6'-O4'.37 In contradistinction, the sp2 configured radical at C6' of the 6-membered carba-ENA radical 9a is also formed with the [O4'-C4'-C6'-C(Me)] torsion of 154.5º (Figure 5F) which results in a dihedral angle of 51º between the single electron occupied p-orbital at C6' and the σ*O4'-C4' orbital. Energy calculations utilizing DFT B3LYP/6-311+G(d, p) (as implemented in GAUSSIAN98) show that the activation energy from 9a to 9b (Figure 4C) is 2.7 kcal/mol, which is somewhat higher than the activation energy from 4a to 4b (Figure 4B) [0.2 kcal/mol, Table S1], thereby showing that the formation of 4b from 4a is energetically more favored than that of the rearrangement of 9a to 9b. We thus demonstrate that stereoelectronic requirement of coplanarity of single electron occupied p-orbital at C6 with σ* orbital of O4'-C4' bond is a prerequisite for the rupture O4'-C4' bond, and the energy penalty involved in that process seems to be a key determinant for the rearrangement reaction to take place in 4a to 4b, but not in 9a to 9b.

(D) Structure of BHNA
In order to elucidate the solution structure of BHNA 8, we have utilized vicinal proton coupling constants from homodecoupling 1H NMR experiments (Figure S35) and the results from the theoretical simulations. Theoretical vicinal proton coupling constants have been back-calculated using Haasnoot-de Leeuw-Altona generalized Karplus equation38 from the corresponding torsional angles of the ab initio optimized molecular structures from HF/6-31G* or B3LYP/6-31++G* geometry optimization by GAUSSIAN 98.39 As shown in Table 1, the experimental vicinal coupling constants of BHNA 8 are well reproduced by this theoretical approach. This indicates that the BHNA 8 is indeed in rigid locked conformation and its average molecular structure observed experimentally is close to that of the minimized theoretical structure.

The molecular structure of BHNA 8 obtained from ab initio geometry optimization (HF/6-31G**) is shown in Figure 3. A comparison of the different hexopyranosyl nucleosides shows that BHNA is very similar to cyclohexanyl nucleoside (CNA, Figure 1) in that C4′-hydroxymethyl and C3′-hydroxyl groups are trans orientated in both cases. CNA adopts chair conformation,40 and its base moiety is orientated equatorially while 3′-hydroxymethyl and 4′-hydroxyl are orientated axially.40 It may be noted that when CNA was incorporated into an oligonucleotides, a chair inversion occurred and as a result, the base moiety took up the axial position.29 This suggested that the chair like six-membered ring in CNA is relatively flexible. In contradistinction, the additional methylene bridge between C2′ and C6′ in BHNA makes the six-membered ring completely rigid and it adopts a twist conformation (Figure 3). The base moiety occupies the axial position while 3′-hydroxymethyl and 4′-hydroxyl take up the equatorial position.
8 has also been transformed to the corresponding phosphoramidite and incorporated to oligonucleotides.41 In view of the rigidity of the bicyclic system in BHNA, it should adopt the same conformation as that in the corresponding nucleoside, with base moiety taking the axial position while both 3′-hydroxymethyl and 4′-hydroxyl taking the equatorial position. Just like CNA, BHNA modified oligodeoxynucleotides are also RNA selective. But unfortunately, the BHNA destabilized both DNA/DNA and DNA/RNA duplexes, unlike CNA which stabilizes the thermal stability of DNA/DNA as well as DNA/RNA.29 Clearly, our preliminary study41 on this BHNA modified oligo did not show any improvement on the thermal stability of the DNA/DNA or DNA/RNA duplex, we are however exploring other avenues to reveal some useful applications in the antisense, siRNA or triplexing technologies.

Synthesis of Methylene-bridged hexopyranosyl nucleoside (BHNA) represents a new C6′ radical rearrangement (intermediate 4a) to C4′ accompanied by scission of C4′-O1′ bond. The stereoelectronic configuration of the tertiary radical intermediate in the 5-membered system gives a slight energetic edge to drive the rearrangement of the C6' tertiary radical in 4a compared to that of the corresponding 6-membered counterpart. The structure of BHNA has been studied using NMR as well as by theoretical ab initio and Karplus empirical approaches. The six-membered pyranose ring in BHNA is hyper-constrained by the C2′ to C6′ methylene linker, and adopts a twist conformation, with base moiety occupying the axial position while 3′-hydroxymethyl and 4′-hydroxyl occupying the equatorial position. Though the structure of BHNA is similar to another member of hexopyranosyl nucleoside family, but its synthetic rout is novel; BHNA, unlike CNA, does not improve the thermal stability of DNA/DNA and DNA/RNA duplex when it was incorporated into DNA strand, but future work may reveal new applications in the design of gene-directed therapeutics.

Experimental procedures for the synthesis of compounds 4, described in the present synthetic Scheme (Scheme 1), have been reported in our earlier JOC paper.34

(1R,3R,4R,5S,6S,7S)-7-benzyloxy-1-benzyloxmethyl-5-methyl-6-O-phenoxythiocarbonyl-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane (2). Compound 1 (175 mg, 0.37mmol) was coevaporated thrice with anhydrous pyridine and dissolved in the same solvent. The solution was cooled to –5 °C, and phenyl chlorothionoformate (0.172 mL, 0.55 mmol) was added dropwise. Then the reaction mixture was allowed to warm to rt and kept at this temperature for 2 h. After recovery of pyridine under reduced pressure, reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with CH2Cl2. The organic layer was dried over MgSO4, concentrated, and chromatographed over silica gel (0-2 % MeOH in CH2Cl2, v/v) to give compound 2 as colorless oil (0.108 mg, 50 %). 1H NMR (600 MHz, CDCl3): δ 8.63 (1H, bs, NH), 7.71 (1H, s, H6), 7.10-7.43 (15H, m, aromatic), 5.83 (1H, s, H1′), 5.76 (1H, d, J = 9.60 Hz, H6′), 4.47-4.63 (4H, m, CH2Bn), 4.12 (1H, s, H2′), 3.91 (1H, d, Jgem = 10.8 Hz, H5′), 3.86 (1H, d, Jgem = 10.8 Hz, H5′′), 3.05 (1H, m, H7′), 2.66 (1H, d, JH2′, H7′ = 4.2 Hz, 2′H), 1.50 (3H, s, T-CH3). 1.25 (3H, d, J 7′-CH3, H7′ = 6.9 Hz, 7′CH3). 13C NMR (150 MHz): δ 195.4 (C=S),164.43 (C4), 150.1 (C2), 137.5 (C6), 136.8, 135.8, 129.5, 128.5, 128.6, 128.1, 127.8, 126.7, 126.1, 121.8 (aromatic), 109.5 (C5), 88.3 (C4′), 88.0 (C1′), 83.6 (C6′), 78.2 (C3′), 73.9 (CH2Bn), 72.3 (CH2Bn), 66.1 (C5′), 47.9 (C2′), 12.0 (T-CH3), 8.75 (7′-CH3). MALDI-TOF m/z: [M+H]+ found 615.19, calcd 615.21.

(1R,3R,4R,5R,7S)-7-benzyloxy-1-benzyloxmethyl-5,6-dimethyl-3-(thymin-1-yl)-2-oxabicyclo[2.2.1]heptane (3). Compound 2 (85 mg, 0.138 mmol) was dissolved in dry toluene (10 mL) and nitrogen was purged for 30 minutes. Bu3SnH (0.037 mL in 5 mL dry toluene) and AIBN (10 mg in 5 mL dry toluene) was added. The above mixture was heated to 80 ˚C and stirred at this temperature for 30 minutes. Then another portion of Bu3SnH (0.037 mL in 5 mL dry toluene) and AIBN (10 mg in 5 mL dry toluene) were added over 30 min and continued reaction for further 1 h. Solvent was evaporated and the obtained residue was applyed to short silica column chromatography (CH2Cl2/MeOH, 99:1) to give compound 3 (10 mg, and recover 40 mg of substrate). 1H NMR (500 MHz, CDCl3): δ 8.09 (1H, brs, NH), 7.72 (1H, s, H6), 7.32-7.24 (10H, m, aromatic), 5.75 (1H, s, H1′), 4.59-4.44 (4H, m, CH2Bn), 4.01 (1H, s, H3′), 3.79 (1H, d, Jgem = 10.5 Hz, H5′), 3.69 (1H, d, Jgem = 10.5 Hz, H5′′), 2.60 (2H, broad, H7′, H2′), 2.06 (1H, t, JH6, H7 = 11.6 Hz, JH6, 6H = 12.3 Hz, H6′), 1.54 (3H, s, T-CH3), 1.26 (3H, d, J7-Me, H7 = 6.0 Hz, 7′-CH3), 1.15 (1H, dd, JH6, H7 = 4.0 Hz, JH6, 6H = 12.9 Hz, H6''). 13C NMR (125 MHz, CDCl3): δ 163.7 (C4), 149.7 (C2), 137.8, 137.5, 136.4 (C6), 128.5-127.5 (aromatic), 109.1 (C5), 88.9 (C4′), 84.2 (C1′), 78.7 (C3′), 73.7, 71.8 (CH2Bn), 67.6 (C5′), 48.0 (C2′), 37.7 (C6′), 28.8 (C7′), 15.5 (7′Me), 12.1 (T-CH3), 10.1 (7′-CH3), 8.0 (6′-CH3). MALDI-TOF m/z: [M+H]+ found 463.1, calcd 463.2.

(1S,3R,4R,5S,6R,7S)-5-benzyloxy-6-benzyloxmethyl-1,7-dimethyl-3-(thymin-1-yl)-2-oxabicyclo[2.2.1]heptane (5). Compound 4 (210 mg, 0.36 mmol) was dissolved in dry toluene (10 mL) and purged with dry nitrogen for 30 min. AIBN (30 mg, 0.18 mmol) and Bu3SnH (0.29 mL, 1.08 mmol) were added to the mixture and heated to 80 ˚C for 0.5 h. The reaction was cooled to rt and evaporated. The residue was chromatographed over silica gel (20-35% EtOAc in cyclohexane, v/v) to obtain earlier reported 6 and 7 as a mixture (57 mg, 33.3%, 6/7 = 4/3)34 along with a new hithertofore unidentified compound 5 (46 mg, 27%). Compound 5 : 1H NMR (500MHz, CDCl3): δ 8.23 (1H, brs, H3), 7.35-7.25 (m, 11H), 5.52 (d, J1, 2 = 3.1 Hz, H1′), 4.57-4.45 (m, 4H, BnCH2), 3.58 (dd, J5, 4 = 6.9 Hz, Jgem = 9.5 Hz, 1H, H5′), 3.36 (s, 1H, H2′), 3.31 (dd, J5, 4 = 6.9 Hz, Jgem = 9.5 Hz, 1H, H5′′), 3.28 (d, J3, 4 = 3.2 Hz, 1H, H3′), 2.47 (m, 1H, H7′′), 2.19 (m, 1H, H4′), 1.78 (s, 3H, T-Me), 1.37 (s, 3H, 6′-Me), 1.12 (d, J7, 7Me = 6.9 Hz, 3H, 7′-Me). 13C-NMR (125 MHz, CDCl3): δ 163.5 (C4), 149.7 (C2), 138.03, 138.01, 136.9 (C6), 128.6-127.3 (aromatic), 109.4 (C5), 89.7 (C6′), 88.0 (C1′), 77.2 (C3′), 73.1, 70.8 (CH2Bn), 68.0 (C5′), 56.6 (C4′), 49.8 (C2′), 45.5 (C7′), 15.4 (6′-Me), 12.4 (T-Me), 8.8 (7′-Me). MALDI-TOF m/z: [M+H]+ found 477.5, calcd 477.3.

(1S,3R,4R,5S,6R,7S)-5,6-dihydroxyl-1,7-dimethyl-3-(thymin-1-yl)-2-oxabicyclo[2.2.1]heptane (8). To a solution of compound 5 (53 mg, 0.11 mmol) in dry MeOH (3 mL) was added 20% Pd(OH)2/C (168 mg) and ammonium formate (423 mg, 6.7 mmol) and reflux for 1.5 h. The suspension was filtered over Celite bar and organic phase was evaporated to give the compound 11 (33 mg, 100%). 1H-NMR (500 MHz, D2O): δ 7.64 (s, 1H, H6), 5.58 (d, J1, 2 = 2.8 Hz, 1H, H1′), 3.77 (dd, J5, 4 = 6.8 Hz, Jgem = 11.5 Hz, 1H, H5′), 3.58 (d, J3,4 = 3.4 Hz, 1H, H3′), 3.51 (dd, J5, 4 = 7.9 Hz, Jgem = 11.5 Hz, 1H, H5′′), 2.84 (m, 1H, H2′), 2.39 (dt, J2,7 = 1.6 Hz, J7,7Me = 6.9 Hz, 1H, H7′), 1.89 (m, 1H, H4′), 1.86 (s, 3H, T-Me), 1.30 (s, 3H, 6′-Me), 1.03 (d, J7, 7Me = 6.9 Hz, 3H, 7′-Me). 13C-NMR (125 MHz, CDCl3): δ 164.1 (C4), 149.6 (C2), 138.3 (C6), 110.2 (C5), 90.9 (C6′), 88.1 (C1′), 69.4 (C3′), 59.8 (C5′), 59.5 (C4′), 52.3 (C2′), 44.9(C7′), 14.2(6′-Me), 11.6 (T-Me), 7.9 (7′-Me). MALDI-TOF M/S: [M+Na]+ found 318.9, calcd 319.1.

Theoretical calculations. The geometry optimizations have been carried out by GAUSSIAN 98 program package39 at the Hartree-Fock level using HF/6-31G** and B3LYP/6-31++G**. The experimental torsion angles have been back-calculated from experimental vicinal proton 3JH,H coupling constants employing Haasnoot-de Leeuw-Altona generalized Karplus equation38 taking into account β substituent correction in form:
3J = P1 cos2(φ) + P2 cos(φ) + P3 + Σ ( Δχigroup (P4 +P5 cos2i φ + P6 |Δχigroup| ))
where Δχ
i group = Δχi α substituent - P7 Σ Δχiβ- substituent, Δχi are taken as Huggins electronegativities42 and P1 = 13.70, P2 = -0.73, P3 = 0.00, P4 = 0.56, P5 = -2.47, P6 = 16.90, P7 = 0.14 (parameters from38).

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


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