Peter Agback

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.

A new Synthesis of 1-(2,3-Dideoxy-β-D-Glycero-Pent-2-Enofuranosyl)- Thymine. A Highly Potent and Selective Anti-Hiv Agent

Abstract A high yielding, straightforward synthesis of 1-(2,3-dideoxy-β-D-glyceropent-2-enofuranosyl)thymine (d4T) (10) is reported through a simple oxidation step of key intermediates such as, 1-(5-O-pivaloyl-3-deoxy-3(R)-phenylseleno-β-D-glyceropentofuranosyl)thymine (7) [1 → 3 → 5 → 7 → 9 → 10], or 1-(5-O-(4-methoxytrityl)-3-deoxy-3(R)-phenylseleno-β-D-glycero pentofuranosyl)thymine (6) [1 → 2 → 4 → 6 → 8 → 10]. The scope of this synthesis is also demonstrated by a simple preparation of a potential prodrug of d4T, 1-(2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl)-5-methylcytosine (d4C5me) (15), both from 1-(5-O-MMTr-2,3-dideoxy- β-D-glyceropent-2-enofuranosyl)thymine (8), and 1-(5-O-pivaloyl-2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl)thymine (9)., Furthermore, 3′, 5′-dideoxy-3′, 5′-bis(phenylselenyl)thymidine (19) produced only 2-methylene-5-(R)-(thymin-1-yl)-2,5-dihydrofuran (22) through an oxidation followed by mild alkali treatment.

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.

Solution structure of branched U3′p5′A2′p5′G3′p5′C and its comparison with A2′p5′G3′p5′U by 500 MHz NMR spectroscopy

Abstract In this study, the 1H-1H, 1H-13P and 13C-31P coupling constants of the branched RNA tetramer 2 have been measured at two temperatures to obtain detailed information about its backbone conformation. Evaluation of these coupling constants by Karplus-Altona algorithm shows the decrease of populations of γ+ and βt upon temperature-increase for the branch-point A and 3′-terminal C residues, which have been attributed to a destacking along the U3′→5′A3′→5′C stacked axis in the tetramer 2. In accordance with this observation, it has been clearly established that γ+ and β+ populations of constituent 2′→5′-linked guanosine nucleotide is rather insensitive to temperature-change. The NOEs seen at 270 MHz between AH8 with UH6, and AH2 with CH6 also support that the tetramer 2 stacks along the U3′→5′A3′→5′C axis. The NOEs observed at 270 MHz between CH6 with GH8, and UH6 with GH8 suggest also a spatial proximity between 5′-terminal U and 2′-terminal G, and 3′-terminal C and 2′-terminal G residues. These observations have led us to propose a two-state model for the tetramer 2. On the other hand, detailed temperature-dependent measurements of 1H-1H, 1H-31P and 13C-31P coupling constants and chemical shifts of analogues of the branched trimer 1 in this laboratory and elsewhere have shown that the molecular conformation of the branched trimer 1 is governed by A2′→5′G stack. The introduction of a 5′-terminal uridine residue in trimer 1 to tetramer 2 shifts the molecular conformation from an A2′→5′G stack in the trimer 1 to a A3′→5′C stack in the tetramer 2. This is a new example of 5′-terminal residue promoted conformational transmission.

Synthesis of tetrameric cyclic branched-RNA (lariat) modelling the introns of group II and nuclear pre-mRNA processing reaction (splicing)

Abstract Convergent synthesis of cyclic branched tetraribonucleotide 9, modelling the lariat of pre-mRNA processing reaction (Splicing), is reported. The first key step in the present strategy involves the condensation of the appropriately protected 5′-O-levulinyl-G3′p5′ U3′ phosphodiester block 4 with the 3′, 5′-dihydroxy-6-N-benzoyl-2′-O-pixyl(9-phenylxanthen-9-yl) adenosine 5,in presence of an activating agent, to give 6a (53%). Chemospecific phosphorylation of 3′-OH of 6a afforded the intermediate 6b (88%) which was treated with mid acid to achieve a regiospecific removal of the 2′-O-pixyl group to give compound 6c (99%). The second key step involved the introduction of biscyanoethylphosphotriester moiety to the 2′-OH of the branch-point adenosine in 6c, in one single step using (biscyanoethoxy)-(diisopropylamino)phosphine to give the crucial branch-point building block 6d (68%), with two dissimilar vicinal phosphates at 2′- and 3′-of the branch-point, 6d was then condensed with N-4-benzoyl-2′,3′-di-O-acetylcytidine then specifically removed from 7a by hydrazine hydrate to give the intermediate 7b (83%) which was subsequently treated with a bulky tertiary amine to give the branch-point-2′-cyanoethylphosphodiester block 7c (74%). A unique intramolecular 5′ → 2′ cyclization was then brought about in 7c by treatment with an activating agent under a high dilution condition to afford the fully protected cyclic branched tetramer 8 (61%). This oligomer was then deprotected in the usual manner and purified to give the cyclic branched tetramer 9 in 30% isolated yield, the first synthetic lariat known to date. Detailed 500 MHz 1H-NMR and 202.4 MHz 31P-NMR studies on 9 have unequivocally established its purity. Detailed spectroscopic studies such as COSY, HOHAHA, ROESY & 31P-1H-NMR shift correlation spectroscopy have clearly established the structural integrity of the tetrameric lariat RNA.

Synthesis of Heptameric Lariat-RNA Modelling the Lariat Introns of Group II and Nuclear Pre-mRNA Processing Reaction (Splicing)

Abstract A new convergent synthetic procedure has been developed for preparation of lariat heptaribonucleotide 19 , modelling the lariat formed in Group II and nuclear pre-mRNA processing reaction (Splicing). The first three steps in this strategy involves the condensation of the appropriately protected 5′-O-levulinylated-cytidylyl(3′→5′)uridine-3′-phosphodiester 4 with the 3′, 5′-dihydroxy-6-N-(4-anisoyl)-2′-O-pixyl(9-phenylxanthen-9-yl)adenosine 14 , in presence of an activating agent, to give 15a (49%). Chemospecific phosphorylation of 3′-OH of 15a afforded the intermediate 15b (92%) which was treated with mild acid to achieve a regiospecific removal of the 2′-O-pixyl group to give 15c (91%). The fourth step is the introduction of the (2-cyanoethyl)-(2-(4-nitrophenyl)ethyl)phosphotriester moiety to the 2′-OH of the branch-point adenosine in 15c in a single step, by using (2-cyanoethoxy)-(2-(4-nitrophenyl)ethoxy)-(diisopropylamino)phosphine, to give the crucial branch-point building block 15d (58%), with two dissimilar vicinal phosphates at 2′- and 3′- of the branch-point. 15d was then condensed with the appropriately protected 5′-hydroxy-uridylyl(3′→5′)-(2′,3′-di-O-acetylcytidine) 11 to afford the fully protected intermediate 16a (57%). Regiospecific deblocking of 2-cyanoethyl group from 16a afforded the 2′-(2-(4-nitrophenyl)ethylphosphodiester 16b (94%), which was condensed with the dimeric 5′-hydroxyguaninylyl(3′→5′)uridine-3′phosphotriester 13 to afford the fully protected 17a (59%). The 5′-O-levulinyl and the 2-cyanoethyl groups were regiospecifically removed from 17a successively to afford first 17b (88%) and then the 5′-hydroxy-3′-phosphodiester block 17c (68%). 17c was allowed to undergo intramolecular phosphorylation, in presence of an activating agent, under a condition of high dilution to afford the fully protected lariat-RNA 18 (66%) which was then deprotected in four steps and purified to give the fully deprotected lariat-RNA 19 (29%). Detailed 500 MHz 1 H-NMR and 202.4 MHz 31 P-NMR studies, using Clean-TOCSY, DQF-COSY, NOESY & 31 P- 1 H-NMR shift correlation techniques, have unequivocally established the purity and the structural integrity of lariat 19 .

The First Example of a Hoogsteen Basepaired DNA Duplex in Dynamic Equilibrium with a Watson-Crick Basepaired Duplex—A Structural (NMR), Kinetic and Thermodynamic Study

Abstract A single-point substitution of the O4′ oxygen by a CH2 group at the sugar residue of A 6 (i.e. 2′-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5′- d(C1G2C3G4A5A6T7T8C9G10C11G12)2 −3, has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A 6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)→←(1B): Keq = k1/k-1 = 0.56±0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9± 0.8 sec−1; δH°‡ = 164±14 kJ/mol;-TδS°‡ (298K) = −92 kJ/mol giving a δG298°‡ of 72 kJ/mol. Ea (k1) = 167±14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0±0.6 sec-1, δH°‡ = 153±13 kJ/mol;-TδS°‡ (298K) = −82 kJ/mol giving a δG298°‡ of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of δG298°‡ of the forward (k1) and backward (k-1) conversions, (1A)→←(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A 6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A 6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2′- deoxyaristeromycin moiety, A 6, as a result of substitution of the endocyclic 4′-oxygen in the natural sugar with a methylene group in A 6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.

Synthesis of tetrameric branched RNA-DNA conjugate & branched-RNA analogue & their comparative conformational studies by 500 MHz NMR spectroscopy

Abstract We report herein the unambigous synthesis of pure tetrameric branched oligonucleotides A3′p5′G2′p5′[dC]3′p5′C (13) found naturally in gram-negative bacterium Stigmatella aurantiaca, and corresponding branched RNA analogue A3′p5′G2′p5′C3′p5′C (14). The conformational features of branched tetramers 13 and 14 have been elucidated and compared by assesing temperature- and concentration-dependent 1H and 31P chemical shifts, (C2′-exo and C3′-endo) and (C2′-endo, C3′-exo) equilibrium, and equilibrium amongst staggered γ and β rotamers using various 2D homo- and heteronuclear correlation, NOESY and ROESY experiments by 500 MHz NMR spectroscopy. Subsequently the conformational features of 13 and 14 have been compared with those of A2′p5′G3′p5′C (ref. 19) and U3′p5′A2′p5′G3′p5′C (ref. 24) found as the branch-point in the lariat formed in the pre-mRNA processing reaction (Splicing). These studies have clearly shown that (1) the intramolecular geometries of both 13 and 14 are dominated by stacking along the axis A3′→5′G2′→5′dC(C), but the RNA-DNA conjugate 13 has a more defined tertiary structure than that of 14, (2) these branched tetramers tend to associate intermolecularly above ∼2 mM concentration producing an aggregate which is vertically stacked along the axis A3′→5′G2′→5′dC(C), (3) the G2′→5′dC(C) stacking and the predominant S conformation of branch-point G found in 13 and 14 suggest that their structures are quite different from the ones found for U3′p5′A2′p5′G3′p5′C (ref. 24). Note however that the structures found for 13 and 14 are reminiscent of A2′→5′G stacking found in the branched trimer A2′p5′G3′p5′C (ref. 19).

The diastereospecific synthesis of new 2′,3′-Cis-α-fused carbocyclic nucleosides

Abstract The first diastereospecific synthesis of [3.3.0]- and [3.4.0]-α-cis-fused-carbocyclic nucleosides 10, 12 and 20, starting directly from 2′-O-(TBDMS) or 3′-O-(TBDMS) derivatives of 5′-O-MMTr-2′,3′-seco-ribo-thymidines, 1 and 13 (ref. 4), have been reported. The key steps involve the unsymmetrical modification of the 2′- and 3′-hydroxyls in seconucleosides 1 and 13 and their diastereospecific recyclisation to the furanose-fused carbocyclic rings using either radical cyclization [ 1 → 2 → 3 (72%) → 4 (90%) → 5 (83%) → 6 → 7 (77%) → 8 (61%) → 9 (41%) and 11 (42%); 9 → 10 (87%) & 11 → 12 (84%) ] or Diels-Alder reaction [13 → 14 → 15 (91%) → 16 (80%) → 17 → 18 (36%) → 19 (69%) → 20 (84%)].

Solution and Solid State Structure of 2′,5′-BIS-(O-Trityl)-3′-Oximinouridine

Abstract Comparison of the solution (in CDCl3 at 500 MHz1H NMR) and X-ray crystal studies of 3′-oximinouridine 1 shows in general good agreement with the high anti glycosidic angle and in the conformation about C4′-C5′. The sugar pucker (C2′-endo) is qualititatively identical in both cases. This is the first example of a conformationally sugar-rigid nucleoside in which the rigidity arises from the sp2 character of an endocyclic carbon (i.e. C3′), not from the strain due to the ring fusion (see ref. 7 for conformationally strained nucleosides).