Preparation of 1'-C Deuterated Synthons for RNA Synthesis by H-Phosphonate Method Aiming at Two-Dimensional NMR Secondary Structure Studies

Abstract

1'-C Deuterated H-phosphonate synthons were prepared via a 12-step procedure starting from [1'-2H]ribose. The procedure included nucleosidation of 1'-O-acetyl-2',3',5'-O-tribenzoyl[1'-2H]ribose with appropriately protected nucleobases and preparation of nucleoside-H-phosphonates by slightly modified described procedures. The automated RNA synthesis of 5'-G*C*U*A*U*UUAU-3' and 3'-AC*G*A*U*A*AAGU-5' was performed on a Gene Assembler Plus DNA-synthesizer. These specifically deuterated oligoribonucleotides were subsequently compared with the corresponding non-deuterated sequences using 2D-NMR NOESY spectra. Specific deuterium incorporation resulted in the expected simplification of spectral pattern.

An efficient and economic site-specific deuteration strategy for NMR studies of homologous oligonucleotide repeat sequences

“…Our work has been focused on the efficient syntheses of nucleosides and nucleotides containing 2 H-labeled base residues. As shown in the literature and particularly in this report, these reactions and the subsequent purification are easier to perform and much more cost effective than multi-step chemical and biochemical procedures for sugar deuteration (6,(16)(17)(18). We recognized that in a typical two dimensional (2D) NOESY spectra of oligonucleotides, structural information is primarily contained in the region that connects base with sugar protons ( Figs 1A and 2A).…”

Total Synthesis of 2‘,3‘,4‘,5‘,5‘ ‘-2H5-Ribonucleosides: The Key Building Blocks for NMR Structure Elucidation of Large RNA

“…Among these, sequence- and/or site-specific deuterium labeling of oligo-DNAs has indeed been proven to simplify the spectral crowding 4 considerably, giving coupling information 5,6b,c and NOE cross-peaks with increased intensities with negligible spin-diffusion, as well as allowing us to probe the dynamics by selective T 1 and T 2 measurements . Site-specific deuteration has been proven to facilitate the NMR structure determination of relatively large oligo-RNA, as evident from our study of a 21mer,8a 31mer,8b and recently a 55mer RNA 8c. Most of these studies have been done according to our Uppsala “NMR-window” concept 4a,b in which only a small 1 H segment of the RNA is NMR-visible, while the rest is made NMR-invisible by incorporation of the appropriate deuterated blocks.…”

“…The stepwise chemical synthesis of C2‘, C3‘, C4‘, and C5‘ uniformly deuterated ribonucleosides (>97 atom % 2 H incorporation) reported herein is an improved alternative to the previous Raney-nickel-catalyzed proton−deuterium exchange 4 in that we have achieved a satisfactory level of deuteration at the C4 center. This new total synthesis of uridine-2‘,3‘,4‘,5‘,5‘ ‘- 2 H 5 ( 21a ), adenosine-2‘,3‘,4‘,5‘,5‘ ‘- 2 H 5 ( 21b ), cytidine-2‘,3‘,4‘,5‘,5‘ ‘- 2 H 5 ( 21c ), and guanosine-2‘,3‘,4‘,5‘,5‘ ‘- 2 H 5 ( 21d ), (>97 atom % 2 H at C2‘, C3‘, C4‘, and C5‘/C5‘ ‘), when they are deuterated at the corresponding aglycones, 8a,c and introduced in a nonuniform manner to large oligo-RNA 8c will successfully solve the spectral overcrowding problems, which was not earlier satisfactorily achievable owing to incomplete deuterium exchange at C4‘ by Raney Ni/ 2 H 2 O chemistry …”

“…% at C4‘, ∼0−20 at. % at C1‘) was adequate to perform the sequential assignment of up to a 55nt long oligoRNA,8c but not for quantitative structure determination. The bottleneck 4 of our “Uppsala NMR-window” approach was the residual ∼50−65 at.…”