Anders Sandström

New stereocontrolled synthesis of isomeric C-branched-β-D-nucleosides by intramolecular free-radical cyclization- opening reactions based on temporary silicon connection

Abstract Silicon-bearing allyl group tethered to a 2′ or 3′-hydroxyl group onto the radical generated at the vicinal 2′ or 3′ center in the free-radical precursors 11, 15, 19 and 23 were used to promote intramolecular stereocontrolled free-radical-cyclization to give 12a + 12b, 16, 20 and 24 in 60–70% yields. The configuration at the 2′ or the 3′ center of the allylsiloxane group dictated the stereochemical outcome of the radical cyclization reaction to give cisfused seven-membered rings in compounds 16, 20 and 24 (from 15, 19 and 23, respectively) due to relatively long SiO bond and large CSiO bond angle leading to exclusive 7-endo cyclization. The only exception to this was found in the radical-cyclization of 11 in which both cis-fused and trans-fused seven-membered rings 12a and 12b were formed as inseparable mixture almost in equal amounts. The seven-membered siloxane ring in the radical-cyclized products 12a + 12b, 16, 20 and 24 were then opened up by a simple oxidation reaction to give different 1,5-diols 13a + 13b, 17, 21 and 25 in high yields. The 5′-O-(4-methoxytrityl) group from 13a + 13b, 17, 21 and 25 was then removed to give pure and isomeric C-branched nucleosides 14a, 14b, 18, 22 and 26, respectively. The acid catalyzed isomerization of the pentofuranose ring in 17 to a pyranose system in 18 has been concluded on the basis of comparative structural analysis of 17 and 18 by 500 MHz1H-NMR spectroscopy. The configurations of triol 18 are C-2′(S), C-4′(R), C-5(R), C-6(S), C-7(S) which are also the configurations of the corresponding chiral centers in the precursors 16 and 17. Note that the acid catalyzed isomerization of furanose in 17 to pyranose ring in 18 has been achieved with full retention of anomeric configuration. The configuration of C-3′ in compounds 14a. 14b, 22, and C-2′ in 26 has been elucidated by 1D differential nOe experiments by 1H-NMR spectroscopy at 500 MHz in D2O solution at 293K. The estimation of the 3JHH coupling constants led us to calculate dihedral angles of 14a, 14b, 17, 18, 22 and 26 using the Karplus-Altona algorithm which have allowed us to define the conformational parameters of their constituent sugar moieties. Molecular mechanics calculations have been subsequently performed on the initial NMR structures of 17 and 18 to give their energy minimized conformations. The structures of 18 has been finally confirmed by estimating proton-proton distances derived from their nOe build-up rates by 2D NOESY experiments at 293K at different mixing times.

Structural studies of anti-HIV 3′-(α-fluorothymidine & 3′-(α-azidothymidine by 500 MHz 1H-NMR spectroscopy & molecular mechanics (MM2) calculations

Abstract NMR studies of anti-HIV 3′-α-fluorothymidine (FLT) ( 1 ) and 3′-α-azidothymidine (AZT) ( 2 ) in aqueous solution gave insight into their conformational dynamics [pseudorotation of the furanose ring rotation around the C4′-C5′ bond (γ) and rotation around the glycosidic C1′-N1 bond (χ)]. The interpretation of scalar proton-proton coupling data for FLT ( 1 ) in solution showed a conformational bias towards a South-type (∼90%) puckered furanose conformation. Energy minimization by molecular mechanics calculations using the MM2 force field gave molecular structure which is in excellent agreement with the NMR data (P = 151° ν m = 34°). This solution structure is grossly similar to the available averaged crystal structures (P = 171° ν m = 34°). The phase angle P = 151° for FLT ( 1 ) in solution indicates that the preferred furanose geometry is an intermediate between the C2′-endo envelope and the C2′-endo/C1′-exo twist conformation while the averaged phase angle in the solid state (P = 171°) indicates an intermediate geometry between the C2′-endo envelope and the C2′-endo/C3′-exo twist structure. 1 H-HMR studies indicated that the North- and South-type pseudorotamer population of the furanose ring in AZT ( 2 ) is approximately 1:1 but it failed to provide any definite estimation of P and ν m from the analysis of J-coupling constant because of identical chemical shifts of H2′ and H2″. Molecular mechanics calculations were used to model both the North and South form. MM2 calculations in the North region converge virtually to a structure with P = 21.6° (unsymmetrical twist with major C3′-endo and minor C4′-exo twist structure),ν m =40° χ = −160.8° and γ= 56.9° while the MM2 calculations in the South region show a predominant puckered geometry represented by P = 160.3° (envelope C2′-endo geometry) ν m = 35.5° χ = −157.8° and γ = 583°. Note that all six known X-ray structures for AZT belong to the South-type geometry. The MM2 calculated South geometry for AZT is grossly similar to one of the two independent molecules found in the asymmetric unit of X-ray crystal structures (P = 175° ν m = 32.3°). This discrepancy between solution and solid state structure may indicate the danger of only using crystal structural data for the formulation of structure-activity relationships for a candidate drug.

The self-cleavage of lariat-RNA

Abstract Lariat-RNAs 3 and 4 undergo site-specific self-cleavage reaction at the G 3 → C 6 /U 7 phosphodiester bond by the nucleophilic attack of 2′-OH of G 3 sugar moiety to its 3′-phosphate to give 5′-hydroxyl terminal at C 6 or U 7 and 2′,3′-cyclic phosphodiester of G 3 whereas lariat-tetramer 1 , pentamer 2 , the cyclic-A(2′→5′)G-tetramer 5 and the cyclic-A(3′→5′)G-tetramer 6 are completely stable. The lariat-RNAs 3 and 4 are the smallest RNA known to undergo self-cleavage which is reminiscent of the RNA-hammerhead (Ribozyme) activity. The geometry of the cleavage-site in 3 and 4 has been defined by full conformational analysis by NMR and molecular dynamics calculation in water.

The tunable transmission of the aromatic character of the aglycone through the anomeric effect in C-nucleosides drives its own sugar conformation: A thermodynamic study

Abstract The anomeric and the gauche effects are two competing stereoelectronic forces that drive the North (N) (C2′-exo-C3′-endo) ⇆ South (S) (C2′-endo-C3′-exo) pseudorotational equilibrium in nucleosides (ref 1). The quantitation of the energetics of pD dependent N ⇆ S pseudorotational equilibria of the pentofuranose moiety in C-nucleosides 1 – 7 shows that the strength of the anomeric effect of the constituent heterocyclic moiety at C1′ is dependent upon the unique aromatic nature of the nucleobase, which is tuned by the pD of the medium. The force that drives the protonation ⇆ deprotonation equilibrium of the heterocyclic nucleobases in C-nucleosides is transmitted through the anomeric effect to drive the two-state N ⇆ S pseudorotational equilibrium of the constituent furanose (the energy pump), which is supported by the following observations: (i) The enhanced strength (ΔΔG 298 (P-N) of the anomeric effect in the protonated (P) nucleoside compared to the neutral (N) form is experimentally evidenced by the increased preference of N-type sugar conformation with pseudoaxial nucleobase by 2.0 kJ/mol for formycin B ( 1 ), 1.4 kJ/mol for formycin A ( 2 ), 1.4 kJ/mol for 9-deazaadenosine ( 3 ) and 1.9 kJ/mol for Ψ-isocytidine ( 4 ). (ii) In contrast, the S-type sugar conformer, which places the nucleobase in pseudoequatorial orientation, is considerably more preferred in the alkaline medium owing to the weakening of the anomeric effect in the N1 deprotonated (D) formycin B, and N3-deprotonated Ψ-isocytidine, Ψ-uridine and 1-methyl-Ψ-uridine compared to the neutral counterparts by ΔΔG o (N-D) of 0.2 kJ/mol for formycin B ( 1 ), 1.6 kJ/mol for Ψ-isocytidine ( 4 ), 1.7 kJ/mol for Ψ-uridine ( 5 ), 0.8 kJ/mol for 1-methyl-Ψ-uridine ( 6 ). (iii) The quantitation of the pD-dependent drive of N ⇆ S pseudorotational equilibria in C-nucleosides 1 – 6 has allowed us to independently measure the pK a of the constituent heterocyclic nucleobases. (iv) A simple comparison of ΔG N 298 or ΔG P 298 or ΔG D 298 values of all C-nucleosides 1 – 7 (Table 1) with N-nucleosides (ref 1) shows that the C1′ substituent promoted anomeric drive of N ⇆ S equilibrium to N-sugar is weaker in C-nucleosides than in N-nucleosides, but their respective flexibilities from the neutral to the protonated or to the deprotonated state is completely aglycone-dependent.

Conformation of Lariat Structures Formed in the Splicing of Pre-mRNA by NMR Spectroscopy

Abstract Temperature dependent 1H- and 31p-NMR studies have shown that lariat (branched) trimers show a preferential 2′ → 5′ stacking, while the branched tetramers resemble 3′ → 5′ linked linear trimers, reminiscent of a single stranded A-RNA helix.

Determination of the solution conformation of a non-uniformly deuterium labelled (Uppsala 'NMR-window') 21mer RNA hairpin by NMR spectroscopy and computational methods.

Abstract The conformation of a 21mer RNA hairpin, 5′-r(AGCCCGCCUAAUGAG-CGGGCU)-3, was determined by NMR spectroscopy and computer calculations. The ‘Uppsala NMR-window’ approach was used to overcome the problem of spectral overlap.