Christophe Thibaudeau

How do the energetics of the stereoelectronic gauche and anomeric effects modulate the conformation of nucleos(t)ides

The determination of the energetics of the temperature-dependent two-state N reversible arrow S pseudorotational equilibrium through (3)J(HH) analysis in 36 nucleos(t)ides and 3 abasic sugars has a ...

The interaction of the 2′-OH group with the vicinal phosphate in ribonucleoside 3′-ethylphosphate drives the sugar-phosphate backbone into unique (S,ω−) conformational state

Abstract The analysis of temperature-dependent vicinal proton-proton coupling constants has shown that the North (N)South (S) pseudorotational equilibria of ribonucleoside 3′-ethylphosphates [ApEt (21), GpEt (22), CpEt(23), rTpEt (24) and UpEt (25)], modelling simple diribonucleoside(3′ → 5′)monophosphate without any intramolecular base-base stacking, are driven more towards the South-type sugar (S) by ΔΔ11° = −2.5 kJ mol−1 in the case of purine and by ≈ −3.8 kJ mol−1 in the case of pyrimidine nucleotides compared to the corresponding parent ribonucleosides 1 - 5. In contrast, the S-type sugar conformation in 2′-deoxyribonucleoside 3′-ethylphosphates (ref. 3d) is stabilized by ΔΔH ° ≈ −1.9 kJ mol−1 in both purine and pyrimidine nucleotides compared to the parent 2′-deoxyribonucleosides. The total energetic effect of 2′-OH group due to its interaction with the vicinal phosphate in ribonucleotides in contrast with the corresponding 2′ -deoxynucleotide counterparts can be assessed by subtracting the free-energies of NS pseudorotational equilibria in the ribonucteotide analogs 21 – 25 from the corresponding 2′-deoxynucleotide counterparts 16 – 20: ΔΔG298 ≈ +0.3 kJ mol−1 in ApEt (21), +0.6 kJ mol−1 in GpEt (22), +2.1 kJ mol−1 CpEt (23), +1.1 kJ mol−1 in rTpEt (24) and +1.3 kJ mol−1 in UpEt (25). The additional stabilization of the S-type pseudorotamers in ribonucleoside 3′-ethylphosphates 21 – 25 compared to ribonucleoside 3′-monophosphates 11–15 by ΔΔH ° ≈ -2.0 kJ mol−1 is attributed to the influence of the 2′-OH group in the former. The population of ω1 rotamers increases by 7–13% from 278 to 358K, which corresponds also with an equal increase of the population of N-type pseudorotamers, suggesting a unique cooperativity in the two-state (N,ω1)(S,ω−) conformational equilibria in 21 – 25. These cooperative conformational transitions of (N,ω1)(S,ω−) equilibrium in 21 – 25 have been found to be orchestrated by the interaction of 2′-hydroxyl group with the vicinal phosphate as evident by the non-equivalent methylene protons of the 3′-ethylester function uplo 348K in 21 – 25 compared to the 2′-deoxynucleotide counterparts 16 – 20 (ref. 3d). The intramolecular interaction of the 2′-OH function with the vicinal phosphodiester stabilizes the S and ω− conformers (“On” switch), whereas 2′-OH in a non-interacting stale stabilizes the N and ω1 conformers (“Off switch) in 21 – 25. The strengths of this “On-Off” molecular switch for the preference of (S,ω−) conformational slate over (N,ω1) slate in 21 – 25 are as follows: ΔG298 ≈ −2.8 kJ mol−1 for adenosine 3′-ethylphosphate (21), −2.1 kl mol−1 for guanosine 3′-ethylphosphate (22), ≈ 0.1 kJ mol−1 for cytidine 3′-ethylphosphate (23), −0.9 kJ mol−1 for ribothymidine 3′-ethylphosphate (24) and −0.7 kJ mol−1 for uridine 3′-ethylphosphaie (25).

Solution structure of a let-7 miRNA: lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.

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.

Diels-alder reaction of 2′,3′-unsaturated-3′-nitro-thymidine. First chemical evidence of nitroxide radical formation in the radical-promoted denitration reaction

Abstract Diels-Alder reaction of an appropriately functionalized nucleoside [2′,3′-dideoxy-2′,3′-didehydro-3′-nitrothymidine (1)] has been used for the first time as a substrate to yield various unique fused 2′,3′-dideoxy-2′,3′-bis-substituted nucleoside derivatives (2 – 5, 8 – 16) which are not hithertofore available through any other known routes. First unequivocal evidence of the formation of nitroxide radical during n-Bu3SnH promoted denitration reaction has been also presented through the isolation of fused 4H-5,6-dihydro-1,2-oxazine derivatives of the cycloadducts (6, 7, 18, 19) which were formed due to the trapping of the nitroxide radical with an olefin or a keto function in an intramolecular reaction.

The quantitation of the competing energetics of the stereoelectronic and steric effects of the 3′-OH and the aglycone in the α-versus & by 1H-NMR

By comparative NMR study of 2′,3′-dideoxynucleosides (see ref 1) with or , we have been able to quantify for the first time the competing medium-dependent influences of the 3′-OH promoted gauche and the aglycone-configuration dependent anomeric effects that result in the overall drive of the sugar conformation in 2′-deoxynucleosides. It has been shown that although the pKas of the nucleobases in α- and are identical, the transmission of the free-energy of protonation-deprotonation equilibria to steer the sugar conformation is not the same, indeed it is finely tuned by the balance between the 3′-gauche and anomeric effect. It has emerged that the counteracting 3′-OH gauche effect reduces the influence of the pH-dependent anomeric effect, thereby limiting the conformational flexibility of with respect to the corresponding .

The first experimental evidence for a larger medium-dependent flexibility of natural β--nucleosides compared to the α--nucleosides

Abstract A pairwise comparison of the free-energy of the two-state N (North, C2′-exo-C3′-endo, 0° -2′,3′-dideoxynucleosides (ddNs) 2, 4, 5, 7 and 9 with their α- -counterparts 1, 3, 6 and 8 shows that the conformation of the pentofuranose moiety is efficiently driven towards the N-type sugars in the β- - series and to the S-type conformation in the α- - anomers. The extent of the free-energy of stabilization of the N-type sugars in β- -ddNs is however 2–3 times greater than the corresponding S-type sugar stabilization in α- -ddNs. The estimation of ΔH° contribution to the free-energy shows that the intrinsic flexibility of the sugar moiety in β- -ddNs is 2–4 times larger than in α- -ddNs as a result of change of either temperature or pD of the medium. This enhanced modulation of conformation by the change of temperature or pD of the solution in β- -ddNs compared to α- -ddNs is the result of the more efficient transmission of the anomeric effect [ n(O 4′)→σ C1′N ∗ orbital interactions between O4′ lonepair of sugar with the σ ∗ of C1′N bond] to drive the sugar conformation in the former compared to the latter. An important implication of this finding is that the evolutionary forces of Nature have opted for molecules that can adopt multiple conformations owing to the unique internal flexibility as a result of interaction with various ligands.

The Discovery of Intramolecular Stereoelectronic Forces That Drive The Sugar Conformation in Nucleosides and Nucleotides

Abstract This report summarizes our results8 on how the determination of the thermodynamics of the two-state North (N, C2′-exo-C3′-endo) ⇄ South (S,C2′-endo-C3′-exo) pseudorotational equilibrium in aqueous solution (pD 0.6 - 12.0) basing on vicinal 3JHH extracted from 1H-NMR spectra measured at 500 MHz from 278K to 358K yields an experimental energy inventory of the unique stereoelectronic forces that dictate the conformation of the sugar moiety in β-D-ribonucleosides (rNs), β-D-nucleotides, in the mirror-image β-D- versus β-L-2′-deoxynucleosides (dNs) as well as in α-D- or L- versus β-D- or L-2′-dNs. Our work shows for the first time that the free-energies of the inherent internal flexibilities of β-D- versus β-L-2′-dNs and α-D- versus α-L-2′-dNs are identical, whereas the aglycone promoted tunability of the constituent sugar conformation is grossly affected in the α-nucleosides compared to the β-counterparts.

Determination of the Group Electronegativity of CF3 Group in 3′-O-CF3-Thymidine by 1-NMR

Abstract The interplay of enthalpy of the gauche effect (ΔH°GE) of the [X3′-C3′-C4′-O4′] fragment in various 3′-substituted (X) 2′,3′-dideoxythymidine derivatives 1–7 and the inherent anomeric effect drives the two-state North ⇄ South equilibrium in the constituent sugar moiety. The group electronegativity of 3′-OCF3 substituent in Marriotts, Inamotos and Mullays scales has been determined from simple calibration graphs correlating the group electronegativity of various 3′-substituents (X) in 2′,3′-dideoxythymidine derivatives 1–7 with the experimental strength (ΔH°GE) of the [X3′-C3′-C4′-O4′] gauche effect. ΔH°GE has been experimentally determined from pseudorotational analyses of temperature-dependent 3JHH coupling constants, and can be used as an unambiguous tool for direct experimental estimation of the group electronegativity of a specific substituent covalently attached to 3′-carbon of 2′,3′-dideoxythymidine, which can be compared, in turn, with the theoretical estimation carried out according to...

The Information Transmission from the Nucleobase Drives the Sugar-Phosphate Backbone Conformation in the Nucleotide Wire

Abstract We herein present our results showing that a simple mononucleotide block actually acts as a wire. This is evidenced by the fact that any change of the electronic character of the nucleobase is transmitted to drive the sugar conformation, which in turn dictates cooperatively the preferred orientation of the phosphate backbone.