Synthetic studies to improve the deuterium labelling in nucleosides for facilitating structural studies of large RNAs by high-field NMR spectroscopy

Article abstract of DOI:10.1081/NCN-100002549

Synthetic studies to prepare ribonucleosides deuterated at C2′ and the application of the developed procedures for the synthesis of ²H₅-ribonucleosides from 1,2-O-isopropylidene-3-O-benzyl-ribofuranose-3,4,5,5′ -²H₄

Full text of DOI:10.1081/NCN-100002549

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1333–1337 (2001)
SYNTHETIC STUDIES TO IMPROVE THE
DEUTERIUM LABELLING IN NUCLEOSIDES
FOR FACILITATING STRUCTURAL STUDIES
OF LARGE RNAS BY HIGH-FIELD
NMR SPECTROSCOPY
Mrinal K. Kundu,1 Anna Trifonova,1 Zolta´n Dinya,2
Andra´s Fo¨ldesi,1,∗ and Jyoti Chattopadhyaya1,∗
1Department of Bioorganic Chemistry, Box 581, Biomedical Center,
University of Uppsala, S-751 23 Uppsala, Sweden
2Department of Organic Chemistry, L. Kossuth University,
H-4010 Debrecen, Hungary
ABSTRACT
Synthetic studies to prepare ribonucleosides deuterated at C2ꢀ and the
application of the developed procedures for the synthesis of 2 H5-ribonucleo-
sides from 1,2-O-isopropylidene-3-O-benzyl-ribofuranose-3,4,5,5ꢀ-2 H4 have
been reported.
INTRODUCTION
Amongst isotope labelling techniques, site-specific deuteration has been
proven to facilitate the NMR structure determination of large RNAs (1) by the
“NMR-window” concept (2) in which only a small segment of the RNA is
∗ Corresponding author. Fax: +46-18-554495; E-mail: andras@bioorgchem.uu.se or jyoti@
bioorg-chem.uu.se
1333
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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NMR-visible. The deuterium incorporation achieved into the nucleoside building
blocks (>97 atom% at C2ꢀ, C3ꢀ and C5ꢀ, ∼35–50 atom% at C4ꢀ, ∼0–20 atom% at
C1ꢀ) was adequate to allow sequential assignment of up to 55nt long oligoRNAs
(1c). The residual ∼50 atom% proton at C4ꢀ causes substantial resonance overlap
in important nOe regions hampering the determination of the solution structure
of long oligomers. This prompted us to seek for appropriate synthetic ways for a
reliable high level deuterium incorporation at C4ꢀ.
We envisioned that the synthesis of 3ꢀ,4ꢀ,5ꢀ,5ꢀꢀ-2 H4-nucleosides (3) could
be extended to 2ꢀ,3ꢀ,4ꢀ,5ꢀ,5ꢀꢀ-2 H5 derivatives provided a suitable method for deu-
terium incorporation at C2ꢀ could be found. We here report the preparation of 2ꢀ-
2 H1-nucleoside block (4) by introducing deuterium right at the sugar level be-
cause it is problematic to introduce the 2ꢀ-2 H at the nucleoside level due to partial
loss of the 3ꢀ,5ꢀ-O-protecting group (6b) (which is commonly 1,1,3,3-tetraisopro-
pyldisiloxane-1,3-diyl).
The scale-up of equilibration of 1 (5) (Scheme 1) to ∼22 mmol has been
achieved with excellent level of isotope incorporation (>97 atom%). The procedure
is easy to carry out and the deuteronucleoside precursor 3 can be obtained in only
3 steps.
The second procedure (Scheme 2), based on the oxidation and subsequent
reduction of C2-OH of compound 4, afforded a mixture of D-arabinose-2 H1 and D-
ribose-2 H1 derivatives 5. Protection of the hydroxyl with 4-toluoyl group has made
the separation of epimers 6 and 7 feasible. The major arabino derivative 7 has sub-
sequently been converted to 1-O-methyl-β-D-ribofuranose-2 H1 (3) (>97 atom%)
via inversion of the configuration at C2 (6) using the displacement of the 2ꢀ-triflate
leaving group in compound 9 with cesium propionate. Compound 3 has been further
converted to the 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-α/β-D-ribofuranose-2 H1 (13),
which has been used in the coupling reaction with the protected persilylated nucle-
obases to obtain fully protected 2ꢀ-deuterated nucleosides 14a–d. The subsequent
deprotection in methanolic ammonia gave the final nucleosides-2 H1 (15a–d).
Finally both methods have been used for the synthesis of 2ꢀ,3ꢀ,4ꢀ,5ꢀ,5ꢀꢀ-2 H5-
ribonucleosides taking the previously described 3ꢀ,4ꢀ,5ꢀ,5ꢀꢀ-2 H4 analogue of 4 as
starting material. The quality of the deuterium substitution is exemplified in Figure 1
for the appropriate cytidine derivatives.
2
=
Scheme 1. Abbreviation: Tol 4-toluoyl. Conditions: (i) dioxane/THF/triethylamine/ H2O (24/24/
12/16 mL, v/v/v/v), 90◦C, 5 days; (ii) acetic acid, 90◦C, 3 days; (iii) methanol, conc. H2SO4, 4◦C,
12 h.

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=
=
=
=
Scheme 2. Abbreviations: Bn
benzyl; Tf
trifluoromethanesulfonyl; Pr
propionyl; Tol
=
=
=
=
=
=
4-toluoyl; Ac acetyl; G guanin-9-yl, A adenin-9-yl, C cytidin-1-yl, U uracil-1-yl, Bz
◦
=
benzoyl, Dpc diphenylcarbamoyl. Conditions: (i) oxalyl chloride, DMSO in DCM, −70 C; (ii)
LiAl2H4 in dry diethylether or NaB2H4 in ◦ethanol, r.t; (iii) TolCl, pyridine, r.t.; (iv) NH3 in methanol,
r.t.; (v) Tf2O, DMAP, pyridine, DCM 0 C, 3 h.; (vi) cesium propionate, DMF, r.t.; (vii) NH3 in
methanol, r.t.; (viii) Pd/C, hydrogen in ethanol, r.t.; (ix) TolCl, pyridine, r.t.; (x) Ac2O, AcOH, conc.
H2SO4, DCM, 0 ◦C, 15 min.; (xi) silylated base, TMS-Tf, 1,2-dichloroethane or toluene (14d), heating;
(xii) NH3 in methanol, r.t.
Some selected relevant data: Compound 3. [α]2D6: −38 (c 0.15, H2O);
HRMS (Ei+): (M+) calcd. for C6H11DO5: 165.0747, found 165.0748. Compound
6. [α]2D6: +98 (c 0.67, CHCl3); HRMS (Ei+): (M+) calcd. for C28H29DO6: 463.2106,
found 463.2109. Compound 7. [α]2D6:−74 (c 0.25, CHCl3); HRMS (Ei+): (M+)
calcd. forC28H29DO6:463.2106, found463.2110. Compound8. [α]2D6:−42(c0.71,
CHCl3); HRMS (Ei+): (M+) calcd. for C20H23DO5: 345.1687, found 345.1695.
Compound 9. [α]2D7: −64 (c 0.74, CHCl3); HRMS (Ei+): (M+) calcd. for C21H22
DF3O7S: 477.1179, found 477.1184. Compound 10. [α]2D7: +14 (c 0.71, CHCl3);
HRMS (Ei+): (M+) calcd. for C23H27DO6: 401.1949, found 401.1955. Com-
pound 12. [α]2D6: +75 (c 0.17, CHCl3); HRMS (Ei+): (M+) calcd. for C30H29DO8:
519.2004, found 519.2009. Compound 13. [α]2D6: +62 (c 1.04, CHCl3); for nat-
ural [α]2D8: +63; HRMS (Ei+): (M+) calcd. for C31−H29DO9: 547.1953, found
547.1960. Compound 15a. [α]2D6 +9 (c 0.2, H2O); [α]2D6 for natural uridine +10;
HRMS (Ei+): (M+) calcd. for C9H11DN2O6: 245.0758, found 245.0759. Com-
pound 15b. [α]2D6: −53 (c 0.17, H2O). For natural [α]2D6: −60; HRMS (Ei+): (M+)
calcd. for C10H12DN5O4: 268.1030, found 268.1036. Compound 15c. [α]2D6: +32
(c 0.08, H2O). For natural [α]2D7: +33; HRMS (Ei+): (M+) calcd. for C9H12DN3O5:

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1
Figure 1. Expanded regions of the 270 MHz 1D H NMR spectra of 2ꢀ-2 H1-cytidine (Panel A),
2ꢀ,3ꢀ,4ꢀ,5ꢀ,5ꢀꢀ-2 H5-cytidine (Panel B), their natural counterpart (Panel C) and the 1ꢀ#,2ꢀ,3ꢀ,4ꢀ#,5ꢀ,5ꢀꢀ-
2 H6-cytidine2 (Panel D).
244.0918, found 244.0922. Compound 15d. [α]2D6 −36 (c 0.04, H2O); [α]2D6 for
natural guanosine −37; HRMS (Ei+): (M+) calcd. for C10H12DN5O5: 284.0979,
found 284.0983.
ACKNOWLEDGMENTS
Authors thank the Swedish Board for Technical Development (NUTEK)
(to JC), the Swedish Natural Science Research Council (NFR contract # K-KU
12067-300 to AF & K-AA/Ku04626-321 to JC), the Swedish Research Council for
Engineering Sciences (TFR) (to JC) and Carl Tryggers Stiftelse (CTS) (to AF).

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