Partially-deuterated nucleotide residues in large DNA duplex simplify the spectral overlap and provide both the J-coupling and nOe informations by the “NMR-window” approach
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The nucleobase-prolecled parlially-deuteraled 5′-O-DMTr-2′#,2″#,3′,4′#, 5′,5″-2H5-2′-deoxyribonucleoside 3′-phosphoramidite derivatives 17a,b – 20a,b. containing C2′ isolopomeric mixture of deuterons and protons 1~15 atom % 2H at C2′(R), ~85 atom % 2H at C2′(S); 65 atom % 2H at C4′ (#), >97 atom % 2H at C3′ and C5′, i.e. Blocks B and C in Fig. 11, have been site specifically incorporated into a self-complementary 12-mer [d(C5G6C7*G8A9*A10T11 T'12*C13G14*C15G16)]2 (I) and a 20-mer [d(C1G2C3G4C5*G6*C7*G8*A9*A 10*T11*T12*C13*G14*C15*G16* C17G18C19G20)]22 (II) DNA duplex (N* indicates the partially-deuterated blocks B and C in Fig. 1) by the solid phase synthesis methodology to develop the “NMR window II” concept (for our “NMR window I” concept see refs 5–8). The present “NMR window II” concept simplifies spectral crowding as well as allows the retrieval of both J-coupling and nOe informations from the partially-deuterated nucleotide residues, whereas our older “NMR-window I” concept helped to suppress the unwanted proton resonances by substituting with deuterium but the sensitivity of the proton resonances in the “NMR-window” was poorer in a 20-mer DNA duplex because of the line-broadening. The overall spectral simplification of the spectral crowding in “NMR window II” concept, owing to ≥97% suppression of the proton resonances from C3′ and C5′ has resulted in an enhancement of the spectral resolution, and thereby following structural information could be obtained in an unambiguous manner: (i) The partial deuteralion ofC2′ along with full suppression of H3′ rersonance by deuleration creating an C2′-isotopomeric mixture has given us an unprecedented possibility for the extraction of the 3JH1′,H2′ and 3JH1′,H2″coupling constant information easily and unambiguously from DQF-COSY or other double quantum experiments for the 20 base pair long DNA duplex with high accuracy as a consequence of the increased intensity of the crosspeaks because of the elimination of J2′2″, J2″3″ and J2′3′ couplings in these partiallydeuterated blocks [i.e. N* residues in duplex (I) and (II) tj. (ii) It is also noteworthy that the T2 relaxation for the H2″ protons of partially-deuteraied residues in deuterated duplex (I) has increased by −1.5 to 2 fold compared to the nondeuterated residues (see Table 1). (iii) Because of the suppression of the proton resonances completely from C3′ and C5′ as well as owing to the fact that there is only −15 atom % residual 1H at C2′(S) in the β-face, we observe only interresidual I(H2″)i−1 - (Ar)i, (H1′)i,-(Ar)J and intraresidual [(H2″-Ar)i, (Ar-H1′)i, (H1′-H2″), (H4′-H1′)i, (H4′-H2″)i, (H4′-Ar)i] nOes using HAL-NOESY experiment, allowing the filtration of all proton resonances belonging to the nondeuterated nucleotides. The comparison of the relative nOe intensities, as judged by comparison of crosspeak to its own diagonal peak at the same mixing lime both in deulerated and its non-deuterated counterparts, obtained in HAL-NOESY experiment with that of a standard NOESY experiment, shows that the errors in the nOe volume estimation are quite similar, and thereby allowing the extraction of quantitative interproton distance information in “NMR-window II” concept. (iv) The elimination of proton resonances completely from C3′ and C5′ and only ~15 atom % residual 1H at C2′ in the β-face has made it possible to eliminate spin-diffusion taking place through the H2′-Ar, H1′-H2′, H3′-Ar, H1′-H3′, H2′-H3′ and H2′-H2″ pathways in the NOESY spectra, (v) The present HAL-NOESY experiment allows an unambiguous extraction of the H1′-H4′, H4′-H2″ nOe volumes for large duplex, which are known to be quite sensitive to the sugar conformation.
Ämnesklassifikation (UKÄ) – OBLIGATORISK
|Sidor (från-till)||10065 - 10092|
|Status||Published - 1995|
|Peer review utförd||Ja|