Total synthesis of 2′,3′,4′,5′,5′-2H5- ribonucleosides: The key building blocks for NMR structure elucidation of large RNA

Article abstract of DOI:10.1021/jo010097n

The diastereospecific chemical syntheses of uridine-2′,3′,4′,5′,5″-²H ₅ (21a), adenosine-2′,3′,4′,5′,5″-²H ₅ (21b), cytidine-2′,3′,4′,5′,5″-²H ₅²H₅ (21c), and guanosine-2′,3′,4′,5′,5″-²H ₅ (21d) (>97 atom % ²H at C2′, C3′, C4′, and C5′/C5″) have been achieved for their use in the solution NMR structure determination of oligo-RNA by the Uppsala "NMR-window" concept (refs 4a-c, 5a, 6), in which a small ¹H segment is NMR-visible, while the rest is made NMR-invisible by incorporation of the deuterated blocks 21a-d. The deuterated ribonucleosides 21a-d have been prepared by the condensation of appropriately protected aglycone with 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-α/β-D-ribofuranose- 2,3,4,5,5′-²H₅ (19), which has been obtained via diastereospecific deuterium incorporation at the C2 center of appropriate D-ribose-²H₄ derivatives either through an oxidation-reduction-inversion sequence or a one-step deuterium-proton exchange in high overall yield (44% and 24%, respectively).

Full text of DOI:10.1021/jo010097n

6560
J . Org. Chem. 2001, 66, 6560-6570
Tota l Syn th esis of 2′,3′,4′,5′,5′′-2H5-Ribon u cleosid es: Th e Key
Bu ild in g Block s for NMR Str u ctu r e Elu cid a tion of La r ge RNA
Andra´s Fo¨ldesi,† Anna Trifonova,† Zolta´n Dinya,‡ and J yoti Chattopadhyaya*,†
Department of Bioorganic Chemistry, Box 581, Biomedical Centre, University of Uppsala,
SE-751 23 Uppsala, Sweden, and Department of Organic Chemistry, L. Kossuth University,
H-4010 Debrecen, Hungary
jyoti@bioorgchem.uu.se
Received J anuary 24, 2001
The diastereospecific chemical syntheses of uridine-2′,3′,4′,5′,5′′-2H5 (21a ), adenosine-2′,3′,4′,5′,5′′-
2H5 (21b), cytidine-2′,3′,4′,5′,5′′-2H52H5 (21c), and guanosine-2′,3′,4′,5′,5′′-2H5 (21d ) (>97 atom %
2H at C2′, C3′, C4′, and C5′/C5′′) have been achieved for their use in the solution NMR structure
determination of oligo-RNA by the Uppsala “NMR-window” concept (refs 4a-c, 5a, 6), in which a
1
small H segment is NMR-visible, while the rest is made NMR-invisible by incorporation of the
deuterated blocks 21a -d . The deuterated ribonucleosides 21a -d have been prepared by the
condensation of appropriately protected aglycone with 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-R/â-D-
ribofuranose-2,3,4,5,5′-2H5 (19), which has been obtained via diastereospecific deuterium incorpora-
tion at the C2 center of appropriate D-ribose-2H4 derivatives either through an oxidation-reduction-
inversion sequence or a one-step deuterium-proton exchange in high overall yield (44% and 24%,
respectively).
In tr od u ction
by selective T1 and T2 measurements.7 Site-specific
deuteration has been proven to facilitate the NMR
structure determination of relatively large oligo-RNA,8
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” concept4a,b
NMR is a powerful tool1 to explore specific folding
motifs and dynamics of oligo-DNA or -RNA and their
complexes with various ligands under quasi-physiological
condition at atomic resolution. The severe spectral over-
lap,1 line broadening,1 and the spin diffusion2 encoun-
tered in the conformational study of large biologically
functional oligonucleotides (and their complexes with
various ligands), however, restricts the application of
NMR to study those large molecules with specific function
in natural isotopic abundance.
Various isotope labeling techniques have therefore
been introduced.3 Among these, sequence- and/or site-
specific deuterium labeling of oligo-DNAs has indeed
been proven to simplify the spectral crowding4 consider-
ably, giving coupling information5,6b,c and NOE cross-
peaks with increased intensities with negligible spin-
diffusion,6 as well as allowing us to probe the dynamics
1
in which only a small H segment of the RNA is NMR-
visible, while the rest is made NMR-invisible by incor-
poration of the appropriate deuterated blocks. The level
of deuterium incorporation into each nucleoside building
block (>97 at. % at C2′, C3′, and C5′, ∼35-50 at. % 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
bottleneck4 of our “Uppsala NMR-window” approach was
(5) (a) Fo¨ldesi, A.; Yamakage, S.-i.; Maltseva, T. V.; Nilson, F. P.;
Agback, P.; Chattopadhyaya, J . Tetrahedron 1995, 51, 10065. (b) Yang,
J .; Silks, L.; Wu, R.; Isern, N.; Unkefer, C.; Kennedy, M. A. J . Magn.
Reson. 1997, 129, 212. (c) Yang, J .; McAteer, K.; Silks, L. A.; Wu, R.;
Isern, N. G.; Unkefer, C. J .; Kennedy, M. A. J . Magn. Reson. 2000,
146, 260. (d) Ono, A.; Makita, T.; Tate, S.-i.; Kawashima, E.; Ishido,
Y.; Kainosho, M. Magn. Reson. Chem. 1996, 34, S40.
(6) (a) Agback, P.; Maltseva, T. V.; Yamakage, S.-I.; Nilson, F. P.
R.; Fo¨ldesi, A.; Chattopadhyaya, J . Nucleic Acids Res. 1994, 22, 1404.
(b) Fo¨ldesi, A.; Maltseva, T. V.; Dinya, Z.; Chattopadhyaya, J .
Tetrahedron 1998, 54, 14487. (c) Maltseva, T. V.; Fo¨ldesi, A.; Chatto-
padhyaya, J . Tetrahedron 1998, 54, 14528.
* To whom correspondence should be addressed. Fax: +4618554495.
† University of Uppsala.
‡ L. Kossuth University.
(1) Wuthrich, K. NMR of Proteins and Nucleic Acids Wiley: New
York 1986.
(2) Sattler, M.; Fesik, W. S. Structure 1996, 4, 1245.
(3) (a) Varani, G.; Aboul-ela, F.; Allain, F. H.-T. Prog. NMR
Spectrosc. 1996, 29, 51. (b) Wijmenga, S. S.; van Buuren, B. N. M.
Prog. NMR Spectrosc. 1998, 32, 287. (c) Zimmer, D. P.; Crothers, D.
M.; Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3091. (d) Xu, J .; Lapham,
J .; Crothers, D. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 43. (e) Mer,
G.; Chazin, W. J . J . Am. Chem. Soc. 1998, 120, 607. (f) Louis, J . M.;
Martin, R. G.; Clore, G. M.; Groneborn, A. M. J . Biol. Chem. 1998,
273, 2374. (g) Masse, J . E.; Bortmann, P.; Dieckmann, T.; Feigon, J .
Nucleic Acids Res. 1998, 26, 2618. (h) Brush, C. K.; Stone, M. P.;
Harris, T. M. J . Am. Chem. Soc. 1988, 110, 4405.
(4) (a) Fo¨ldesi, A.; Nilson, F. P. R.; Glemarec, C.; Gioeli, C.;
Chattopadhyaya, J . Tetrahedron 1992, 48, 9033. (b) Fo¨ldesi, A.; Nilson,
F. P. R.; Glemarec, C.; Gioeli, C.; Chattopadhyaya, J . J . Biochem.
Biophys. Methods 1993, 26, 1. (c) Yamakage, S.-I.; Maltseva, T. V.;
Nilson, F. P.; Fo¨ldesi, A.; Chattopadhyaya, J . Nucleic Acids Res. 1993,
21, 5005. (d) Huang, X.; Yu, P.; LeProust, E.; Gao, X. Nucleic Acids
Res. 1997, 25, 4758.
(7) (a) Maltseva, T. V.; Fo¨ldesi, A.; Chattopadhyaya, J . Magn. Reson.
Chem. 1998, 36, 227. (b) Maltseva, T. V.; Fo¨ldesi, A.; Chattopadhyaya,
J . J . Chem. Soc., Perkin Trans. 2 1998, 2689. (c) Maltseva, T. V.;
Fo¨ldesi, A.; Chattopadhyaya, J . Magn. Reson. Chem. 1999, 37, 203.
(d) Maltseva, T. V.; Fo¨ldesi, A.; Ossipov, D.; Chattopadhyaya, J . Magn.
Rason. Chem. 2000, 38, 403.
(8) (a) Fo¨ldesi, A.; Yamakage, S.-I.; Nilson, F. P. R.; Maltseva, T.
V.; Chattopadhyaya, J . Nucleic Acids Res. 1996, 24, 1187. (b) Glemarec,
C.; Kufel, J .; Fo¨ldesi, A.; Maltseva, T.; Sandstro¨m, A.; Kirsebom, L.;
Chattopadhyaya, J . Nucleic Acids Res. 1996, 24, 2022. (c) Maltseva,
T.; Fo¨ldesi, A.; Chattopadhyaya, J . J . Biochem. Biophys. Methods 2000,
42, 153. (d) Cheong, C.; Lee, C. Bull. Korean Chem. Soc. 1995, 16, 383.
(e) Arnold, L.; Pressova´, M.; Saman, D.; Vogtherr, M.; Limmer, S.
Collect. Czech. Chem. Commun. 1996, 61, 389. (f) Sanchez, V.; Redfield,
A. G.; J ohnston, P. D.; Tropp, J . Proc. Natl. Acad. Sci. U.S.A. 1980,
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10.1021/jo010097n CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/08/2001

Total Synthesis of 2′,3′,4′,5′,5′′-2H5-Ribonucleosides
J . Org. Chem., Vol. 66, No. 20, 2001 6561
the residual ∼50-65 at. % proton at C4′. This causes
substantial resonance overlap in the regions where
important aromatic proton to H-2′/3′/4′/5′ and anomeric
proton to H-2′/3′/4′/5′ NOEs appear. Consequently, the
stray NOE cross-peaks from the residual H4′ made the
estimation of desired cross-peak volumes erroneous,
which seriously hampers the quality of the solution
structure determination of larger functional RNAs.
Clearly, we needed to develop a high-yielding synthetic
procedure that gave >97 atom % deuteration at C4′ (as
well as retaining those high deuterium incorporations at
C2′, C3′, and C5′) of all ribonucleosides. We herein report
diastereospecific chemical syntheses of uridine-2′,3′,4′,5′,5′′-
2H5 (21a ), adenosine-2′,3′,4′,5′,5′′-2H5 (21b), cytidine-
2′,3′,4′,5′,5′′-2H5 (21c), and guanosine-2′,3′,4′,5′,5′′-2H5
(21d ) (>97 atom % 2H at C2′, C3′, C4′, and C5′/C5′′)
through the condensation of appropriately protected
aglycone with 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-R/â-D-ri-
bofuranose-2,3,4,5,5′-2H5 (19).
configuration of this center. (v) Alternatively, a large
scale single-step deuterium-proton exchange reaction15
at C2 of 2,3-O-isopropylidene-R,â-D-ribofuranose-2H4 should
be easily achievable, since we have already shown14,16
that 2,3-O-isopropylidene-R,â-D-ribofuranose itself un-
dergoes isotope exchange at C2 (>97 at. % 2H) in a
completely diastereoselective manner during equilibra-
tion in a mixture of 1,4-dioxane/tetrahydrofurane/tri-
ethylamine/2H2O at reflux temperature.
Resu lts a n d Discu ssion
Since 1,2-O-isopropylidene-3-O-benzyl-R-D-ribofuranose-
3,4,5,5′-2H4 (1) (Scheme 1) is an intermediate during the
large-scale synthesis of 2H4-ribonucleosides,10 it has been
chosen as the starting material in the present prepara-
tion of 2H5-ribonucleosides. This compound can easily be
converted either to deuterium labeled methyl 3,5-di-O-
benzyl-R,â-D-ribofuranoside or to 2,3-O-isopropylidene-
R,â-D-ribofuranose described above as appropriate inter-
mediate for deuterium incorporation at C2.
The various deuteration strategies developed so far has
been reviewed by us recently.9 A close scrutiny of these
methods shows that 3′,4′,5′,5′′-2H4-nucleosides10 would be
a plausible choice as a starting material for the synthesis
of the corresponding 2′,3′,4′,5′,5′′-2H5 derivatives, if a
suitable way for deuterium incorporation at C2′ could be
found: (i) One way is to oxidize the 2′-OH of 3′,5′-bis-O-
protected ribonucleosides followed by subsequent reduc-
tion to give predominantly the ara epimer,6b,11 followed
by inversion of the configuration of C2′. This approach,6b
however, becomes complicated owing to the concomitant
loss of the 3′,5′-O-protection, which is usually 1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl group. It has also been
found in this laboratory that this procedure gives a
mixture of products in case of guanosine derivatives.12
(ii) The deuterium incorporation at C2 at the sugar level
followed by nucleoside synthesis is an alternative pro-
cedure since the oxidation of 3,4-O-ispropylidene-â-D-
arabinopyranoside by CrO3/acetic anhydride/pyridine
followed by reduction with LiAl2H4 (LAD) furnishes ribo-
pyranoside derivative with high stereoselectivity.12 (iii)
A similar oxidation-reduction sequence starting with
1,3,5-tri-O-benzoyl-R-D-ribofuranose13 results in deute-
rium enrichment (92-94 at. % 2H), which is insufficient
for high-resolution NMR study. The instability of the
benzoyl protecting groups during the reduction also
complicates this pathway. (iv) In a recent study, the
Swern oxidation of methyl 3,5-di-O-benzyl-R,â-D-ribo-
furanoside14 and subsequently the reduction of the
resulting C2-ketosugar has been reported by us to give
a mixture of predominant ara and minor ribo derivatives,
which could be converted to the 2-O-(4-toluoyl) deriva-
tives and separated by silica gel column chromatography.
The benzyl protection is stable during the synthesis of
the C2 epimeric ribo derivative during inversion of the
Rou te A: In tr od u ction of Deu ter iu m a t C2 a t th e
Su ga r Level Sta r tin g fr om 3-O-Ben zyl-1,2-O-isop r o-
p ylid en e-r-D-r ibose-3,4,5,5′-2H4 (1). The deuterated
precursor 1 is easily available in multigram scale (∼300
mmol) in seven steps from 1,2:5,6-di-O-isopropylidene-
R-D-glucose.17 The fully protected intermediate 2 was
obtained (94%) upon a treatment of 1 with benzyl
bromide in the presence of NaH in dry acetonitrile.
Treatment of this product with concentrated sulfuric acid
in dry methanol furnished the anomeric mixture of block
3 (90%). Crucial in this synthetic strategy (Scheme 1) is
the proper selection of the oxidation-reduction proce-
dures. The back exchange6b at C3 observed upon the
oxidoreduction transformation during the Swern oxida-
tion19a,b and sodium borodeuteride reduction rules out
their application. Hence, compound 3 was subjected to
oxidation with pyridinium dichromate (PDC) and acetic
anhydryde18 in dry dichloromethane at reflux tempera-
ture (the disappearance of the signal at δ 3.32 for the
OCH3 group at the anomeric center confirmed the
completion of the reaction) to obtain ketone 4 (72%). A
further drop of yield (68%) occurred in the subsequent
reduction with LAD in dry diethyl ether. This reduction
step afforded an epimeric mixture of C2-deuterated
arabino- (stereoselectively from the â-anomer of 3) and
ribofuranoside 5 (from the R-anomer) in a ratio of ∼7:3
1
as evidenced by H NMR. The moderate overall yield in
the two-step oxidation-reduction (i.e., 3 f 4 f 5 in 47%
overall yield) indicated that the PDC oxidation of 3 is
suitable for producing the ketone on a large scale (∼11
g). The overall poor yield is most probably owing to
insufficient stability of 3 under the oxidation condition
(reflux). Additionally, it was not entirely possible to
remove the residual oxidizing agent from crude 4 by
simple precipitation procedure, which created further
complication in the reduction step. This prompted us to
employ the Dess-Martin reagent19c for oxidation of
compound 3 in dry dichloromethane at room temperature
(3 mmol scale) to afford the desired ketone 4 in 99% yield.
(9) Fo¨ldesi, A.; Trifonova, A.; Kundu, M. K.; Chattopadhyaya, J .
Nucleosides, Nucleotides Nucleic Acids 2000, 19, 1615.
(10) Trifonova, A.; Fo¨ldesi, A.; Dinya, Z.; Chattopadhyaya, J .
Tetrahedron 1999, 55, 4747.
(11) (a) Hansske, F.; Madej, D.; Robins, M. J . Tetrahedron 1984,
40, 125. (b) Perlman, M. E. Nucleosides Nucleotides 1993, 12, 73. (c)
Robins, M. J .; Samano, V.; J ohnson, M. D. J . Org. Chem. 1990, 55,
410. (d) Robins, M. J .; Sarker, S.; Samano, V.; Wnuk, S. F. Tetrahedron
1997 53, 447.
(12) Wu, J .-C.; Bazin, H.; Chattopadhyaya, J . Tetrahedron 1987, 43,
2355.
(13) Cook, G. P.; Greenberg, M. M. J . Org. Chem. 1994, 59, 4704.
(14) Kundu, M. K.; Trifonova, A.; Dinya, Z.; Fo¨ldesi, A.; Chatto-
padhyaya J . Nucleosides & Nucleotides Nucleic Acids 2001, 20, 1333.
(15) El Nemr, A.; Tsuchiya, T. Tetrahedron Lett. 1998, 39, 3543.
(16) Kundu, M. K.; Fo¨ldesi, A.; Chattopadhyaya, J . Collect. Czech.
Chem. Commun. Symp. Ser. 2 1999, 47.
(17) Fo¨ldesi, A.; Trifonova, A.; Chattopadhyaya, J . Unpublished
results
(18) Andersson, F.; Samuelsson, B. Carbohydr. Res. 1984, C1-C3,
129.

6562 J . Org. Chem., Vol. 66, No. 20, 2001
Fo¨ldesi et al.
Sch em e 1a
a
Abbreviations: Bn ) benzyl; Tf ) trifluoromethanesulfonyl; Prop ) propionyl; Tol ) 4-toluoyl. Conditions: (i) BnBr and NaH in dry
acetonitrile, rt, 2 h; (ii) concd H2SO4 in dry methanol, reflux, 3 h; (iii) A: pyridinium dichromate, acetic anhydride in dry CH2Cl2, reflux,
3 h; B: Dess-Martin reagent in dry CH2Cl2, rt, overnight; (iv) LiAl2H4 in dry diethyl ether, rt, 6 h; A: ketone from iiiA, B: ketone from
iiiB; (v) TolCl, pyridine, rt; (vi) NaOCH3 in methanol, rt, 30 min; (vii) Tf2O, DMAP, pyridine, CH2Cl2, 0 °C, 3 h; (viii) cesium propionate,
DMF, rt, 36 h; (ix) Pd/C, hydrogen in ethanol, rt, overnight.
The following identical reduction step (iv, Scheme 1)
proceeded also with a high yield (97%). The resulting
mixture of C2-deuterated arabino and ribo compounds 5
were successfully separated after toluoylation to afford
methyl 3,5-di-O-benzyl-2-O-(4-toluoyl)-R-ribofuranoside-
2,3,4,5,5′-2H5 (6) (23%) and methyl 3,5-di-O-benzyl-2-O-
toluoyl-â-arabinofuranoside-2,3,4,5,5′-2H5 (9) (65%). Due
inert atmosphere (80%). The shift of the anomeric proton
resonance at δ4.99 in 11 to δ4.87 in 12 proved that the
SN2 type inversion at C2 has indeed taken place. Also,
the change of the appropriate specific rotations (from
[R]26 -64° (c 0.74, CHCl3) for 11 to [R]27 +14° (c 0.71,
D
D
CHCl3) for compound 1214) corroborated the inversion to
ribo configuration at C2. The 2-O-propionyl group in 12
was cleaved upon treatment with methanolic sodium
methoxide to furnish compound 13 (99%). This was
followed by removal of the benzyl groups from the
combined carbohydrates 7 and 13 in a catalytic hydro-
genation process over 10% Pd/C in ethanol to afford
C2,3,4,5,5′-pentadeuterated 1-O-methyl-ribofuranose 8
(93%). Subsequently, 8 was 4-toluoylated to afford 18
followed by acetylation and crystallization using standard
procedures5a to give the required 1-O-acetyl-2,3,5-tri-O-
toluoyl-R,â-D-ribofuranose-2,3,4,5,5′-2H5 precursor (19).
When the NMR spectrum of the crystalline â-anomer is
compared to the spectrum of the natural counterpart
(Figures 1A,B), the presence of the only singlet in the
sugar region at δ 6.41 ppm establishes the high degree
of deuterium incorporation (>97 at. % 2H) at the remain-
ing carbon centers of the pentofuranose moiety. The
measured specific rotation also proved the identity of this
compound (specific rotation for â-anomer of 19: +62° (c
1.04, CHCl3), for authentic sample: +63°).
1
to the lack of distinctive H NMR signals because of the
deuterated nature of the sugar, the C2 configuration of
these compounds has been corroborated by the appropri-
ate optical rotation values ([R]26 +98° (c 0.67, CHCl3)
D
and [R]26D -74° (c 0.25, CHCl3) for 6 and 9, respectively14).
The 4-toluoyl group from 6 was cleaved by the action of
methanolic sodium methoxide to yield the pure methyl
R-ribofuranoside derivative 7 with >97 at. % deuterium
incorporation at C2.
The inversion of configuration at C2 of the â-arabino
compound 9 was accomplished in four steps involving the
removal of the 2-O-toluoyl group of 9 by a brief treatment
with 1.0 M sodium methoxide in methanol at room
temperature to give the hydroxy block 10 (91%), which
was converted to the 2-O-triflyl derivative 11 (84%).6b,11b
This compound 11 was converted to the 2-O-propionyl
12 via displacement of the triflate leaving group with Cs-
propionate6b,20 in DMF at room temperature under an
Rou te B: In tr od u ction of Deu ter iu m a t C2 a t th e
Su ga r Level Sta r tin g fr om D-Ribose-3,4,5,5′-2H4 (14).
Earlier, we have reported14,16 that it is possible to obtain
>97 at. % diastereospecific exchange of the proton with
(19) (a) Mancuso, A. J .; Huang, S.-L.; Swern, D. J . Org. Chem. 1978,
43, 2480. (b) Mancuso, A. J .; Swern, D. Synthesis 1981, 165. (c) Dess,
D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155.
(20) Kruizinga, W. H.; Strijtveen, B.; Kellog, R. M. J . Org. Chem.
1981, 46, 6, 4321.

Total Synthesis of 2′,3′,4′,5′,5′′-2H5-Ribonucleosides
J . Org. Chem., Vol. 66, No. 20, 2001 6563
F igu r e 1. Expanded regions of the 270 MHz 1D 1H NMR spectra of 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-â-D-ribose-2,3,4,5,5′-2H5
(19â) (Panel A) and its natural abundance counterpart (Panel B), 2′,3′,5′-tri-O-(4-toluoyl)-uridine-2′,3′,4′,5′,5′′-2H5 (20a ) (Panel C)
and its natural abundance counterpart (Panel D), 2′,3′,5′-tri-O-(4-toluoyl)-N6-benzoyladenosine-2′,3′,4′,5′,5′′-2H5 (20b) (Panel E)
and its natural abundance counterpart (Panel F).
deuterium at the C2 center of 2,3-O-ispropylidene-D-
ribose upon equilibration in dioxane/THF/triethylamine/
2H2O mixture at elevated temperature. The reaction
sequence in this route B involves fewer steps (Scheme
2) than in route A (Scheme 1) to obtain the 4-toluoylated
ribose derivative 19. Hence we decided to check the
feasibility of this route B. The starting D-ribose-3,4,5,5′-
2H4 (14) was prepared from 1 as described previously.10
The introduction of the isopropylidene group, however,
proceeded in very moderate yield (20-27%) regardless
of the procedure21 used. The exchange reaction in a
mixture of 1,4-dioxane/THF/2H2O/Et3N took place with
no detectable amount of side product formation to furnish
the deuterated sugar 15 (98% yield) after 5 days of
heating at 80 °C. The deuteration level was found to be
>97 at. % as determined from the 1H NMR spectrum.
The deuterated ribose derivative 15 was converted to
methyl riboside 8 by deprotection of isopropylidene group
(21) (a) Nakata, M.; Arai, M.; Tomooka, K.; Ohsawa, N.; Kinoshita,
M. Bull. Chem. Soc. J pn. 1989, 62, 2618. (b) Kaskar, B.; Heise, G. L.;
Michalak, R. S.; Vishnuvajjala, B. R. Synthesis 1990, 1031. (c) Kiso,
M.; Hasegawa, A. Carbohydr. Res. 1976, 52, 95.

6564 J . Org. Chem., Vol. 66, No. 20, 2001
Fo¨ldesi et al.
Sch em e 2a
a
Conditions: (i) dry acetone, concd H2SO4, 4 °C, overnight; (ii) Dioxane/THF/Triethylamine/2H2O (6/6/3/4 mL, v/v/v/v), 90 °C, 5 days;
(iii) 90% aqueous TFA, rt, 30 min.; (iv) methanol, concd H2SO4, 4 °C, overnight.
Sch em e 3a
a
Abbreviations: Tol ) 4-toluoyl, Ac ) acetyl, G ) guanin-9-yl, A ) adenin-9-yl, C ) cytosin-1-yl, U ) uracil-1-yl, Bz ) benzoyl,
Dpc ) diphenylcarbamoyl. Conditions: (i) TolCl, pyridine, rt, overnight; (ii) Ac2O, AcOH, concd H2SO4, dry CH2Cl2, 0 °C, 15 min.; (iii)
silylated nucleobase, trimethylsilyl trifluoromethanesulfonate, 1,2-dichloroethane or toluene (12 d), heating; (iv) NH3 in methanol, rt.
in 90% aqueous trifluoroacetic acid (98%) followed by
glycosylation in dry methanol in the presence of catalytic
amount of concentrated sulfuric acid to afford compound
8 (95%). This labeled ribose derivative was further
processed as described in route A.
The couplings of compound 19 with persilylated uracil
(U), N6-benzoyladenine (ABz), N4-benzoylcytosine (CBz),
and O6-diphenylcarbamoyl-N2-acetylguanine (GAcDPC) in
dry 1,2-dichloroethane (dry toluene for G)22 applying
trimethylsilyl trifluoromethanesulfonate as a catalyst,
using modified literature procedures, were carried out
following well-established methods23 to give the corre-
sponding fully protected pentadeuterated nucleosides
20a -d . Comparison of their 1H NMR spectra with the
corresponding protected natural nucleosides proves that
no proton/deuterium exchange reaction has taken place
during the coupling process (Figures 1C-F and Figures
2A-D). The protecting groups were removed by treat-
ment with NH3 in methanol to produce the target
ribonucleosides-2′,3′,4′,5′,5′′-2H5 21a -d in 97, 81, 94, 88%
yields, respectively (Scheme 3). The purity and percent-
age of isotope incorporation (>97 atom % 2H at C2′, C3′,
C4′, and C5′) of these compounds are evidenced in
Figures 2E,F and 3. The identity of these nucleosides was
further corroborated by high-resolution mass spectros-
copy and infrared spectroscopy as well as by optical
rotation measurements (see Experimental Section for
details).
Con clu sion s
The stepwise chemical synthesis of C2′, C3′, C4′, and
C5′ uniformly deuterated ribonucleosides (>97 atom %
2H incorporation) reported herein is an improved alterna-
tive to the previous Raney-nickel-catalyzed proton-
deuterium exchange4 in that we have achieved a satis-
factory level of deuteration at the C4 center. This new
total synthesis of uridine-2′,3′,4′,5′,5′′-2H5 (21a ), adeno-
sine-2′,3′,4′,5′,5′′-2H5 (21b), cytidine-2′,3′,4′,5′,5′′-2H5 (21c),
(22) Robins, M. J .; Zou, R. M.; Guo, Z. Q.; Wnuk, S. F. J . Org. Chem.
1996, 61, 9207.
(23) (a) Vorbru¨ggen, H.; Ho¨fle, G. Chem. Ber. 1981, 114, 1256. (b)
Vorbru¨ggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber. 1981, 114,
1234.

Total Synthesis of 2′,3′,4′,5′,5′′-2H5-Ribonucleosides
J . Org. Chem., Vol. 66, No. 20, 2001 6565
1
F igu r e 2. Expanded regions of the 270 MHz 1D H NMR spectra of 2′,3′,5′-tri-O-(4-toluoyl)-N4-benzoylcytidine-2′,3′,4′,5′,5′′-2H5
(20c) (Panel A) and its natural abundance counterpart (Panel B), 2′,3′,5′-tri-O-(4-toluoyl)-O6-diphenylcarbamoyl-N2-acetylguanosine-
2′,3′,4′,5′,5′′-H5 (20d ) (Panel C) and its natural abundance counterpart (Panel D), uridine-2′,3′,4′,5′,5′′-2H5 (21a ) (Panel E) and its
natural abundance counterpart (Panel F).
2
and guanosine-2′,3′,4′,5′,5′′-2H5 (21d ), (>97 atom % 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-RNA8c will successfully
solve the spectral overcrowding problems, which was not
earlier satisfactorily achievable owing to incomplete

6566 J . Org. Chem., Vol. 66, No. 20, 2001
Fo¨ldesi et al.
1
F igu r e 3. Expanded regions of the 270 MHz 1D H NMR spectra of adenosine-2′,3′,4′,5′,5′′-2H5 (21b) (Panel A) and its natural
abundance counterpart (Panel B), cytidine-2′,3′,4′,5′,5′′-2H5 (21c) (Panel C) and its natural abundance counterpart (Panel D),
guanosine-2′,3′,4′,5′,5′′-2H5 (21d ) (Panel E) and its natural abundance counterpart (Panel F).
deuterium exchange at C4′ by Raney Ni/2H2O chemistry.4
This report contains also diastereospecific methods for
the deuterium incorporation at the C2 center of D-ribose-
2H4 derivatives either via an oxidation-reduction-inver-
sion sequence (46% overall yield from 1 to 8) or a one-
step deuterium-proton exchange (∼25% overall yield
from 1 to 8). The 1-O-acetyl-2,3,5-tri-O-(4-toluoyl)-R/â-D-
ribofuranose-2,3,4,5,5′-2H5 (19) has been subsequently
used for the chemical synthesis of partially deuterium
labeled nucleosides which in turn can be used for the

Total Synthesis of 2′,3′,4′,5′,5′′-2H5-Ribonucleosides
J . Org. Chem., Vol. 66, No. 20, 2001 6567
72.6, 71.6 (C-2, 2 × Ph-CH2); 55.5 (OCH3R), 54.9 (OCH3â).
HRMS (FAB+): (M + H)+ calcd for C20H212H4O5: 349.1953,
found 349.1959.
synthesis of RNAs sequence specifically deuterated for
structural studies using our Uppsala “NMR-window”
concept.
1-O-Meth yl-3,5-d i-O-ben zyl-r,â-D-r ibofu r a n ose-2-u lose-
3,4,5,5′-2H4 (4). Meth od A. Pyridinium dichromate (15.64 g;
41.6 mmol) was suspended in the mixture of dry CH2Cl2 (320
mL) and acetic anhydride (11.8 mL; 124.8 mmol). A solution
of compound 3 (11.11 g; 32.0 mmol) in dry CH2Cl2 was added
to the suspension, and the reaction mixture was refluxed at
∼80 °C for 3 h. After the dilution with ethyl acetate a
precipitate was formed, and the solution was filtered through
silica gel using ethyl acetate as eluent. The solvents were
evaporated, and the residue was coevaporated with toluene
to give an oily product (7.93 g; 23.0 mmol; 72%), which was
taken to the next step without further purification. Meth od
B. Ribose derivative 3 (1.04 g, 3 mmol) was dissolved in dry
CH2Cl2 (30 mL). Dess-Martin reagent (7.59 g, 18 mmol) was
added in dry CH2Cl2 (30 mL), and stirring was maintained
overnight at room temperature. Diethyl ether was added, and
the mixture was poured into cold saturated aqueous sodium
bicarbonate containing Na2S2O3. After shaking for a while, the
organic layer was separated and extracted with saturated
aqueous sodium bicarbonate. The organic layer was dried over
MgSO4 and evaporated to give the crude ketone 4 (1.03 g, 2.98
mmol, 99%). Rf: 0.67 (System E). IR νmax (CHCl3): 3018, 2929,
Exp er im en ta l Section
Dichloromethane, 1,2-dichloroethane, and acetonitrile were
refluxed over phosphorus pentoxide followed by distillation
under nitrogen and kept over molecular sieves (3 Å). Pyridine
was stirred with calcium hydride overnight followed by distil-
lation and stored over molecular sieves (3 Å). The chromato-
graphic separations were performed on Merck G60 silica gel.
Thin-layer chromatography was performed on Merck precoated
silica gel 60 F254 glass-backed plates in following systems: (A)
methanol-CH2Cl2 (5:95, v/v), (B) ethyl acetate-cyclohexane
(1:1, v/v), (C) ethyl acetate-cyclohexane (70:30, v/v), (D) ethyl
acetate-propanol-water (30:18:6, v/v/v), (E) ethyl acetate, (F)
methanol-CH2Cl2 (10:90, v/v). (G) petroleum ether-ethyl
acetate (8:2), (H) ethyl acetate-cyclohexane (30:70, v/v). 1H
NMR spectra were recorded with J EOL GX270 spectrometer
at 270 MHz using TMS or acetonitrile (for D2O solutions, set
at δ 2.0 ppm) as internal standards. 13C NMR spectra were
recorded with J EOL GX270 spectrometer at 67.9 MHz using
the central peak of CDCl3 (δ 76.9 ppm), DMSO-d6 (δ39.6) or
CH3CN (δ 1.3 ppm) as reference. Chemical shifts (δ) are
reported in ppm. High-resolution CI(i-butane[i-Bu]) mass
spectra were obtained on a VG-7035 MS mass spectrometer
(VG. Analytical Ltd., Manchester, UK) equipped with an on-
line VG-11-250J data system. The spectra were obtained at
40 eV with an emission current of 200 µA, source temperature
250 °C and the pressure of the chemical ionization reagent
gas, isobutene, was adjusted to optimize the protonation of
methyl stearate. Accurate mass measurements were carried
out at a resolving power of 20000 using perfluorokerosene as
a reference compound. HRMS FAB spectra were obtained on
a VG-70HS spectrometer (VG. Analytical Ltd., Manchester,
UK) at a resolution of R ) 8000. Optical rotation data were
measured on Perkin-Elmer 241 polarimeter. Infrared spectra
were recorded with a Perkin-Elmer 298 spectrometer.
3,5-Di-O-ben zyl-1,2-O-isopr opyliden e-r-D-r ibofu r an ose-
3,4,5,5′-2H4 (2). Compound 1 (10.37 g, 37.5 mmol) was dis-
solved in dry acetonitrile (160 mL). Benzyl bromide (5.35 mL,
45 mmol) and NaH (1.43 g, 47.7 mmol) were added to the
solution at 0 °C, and the reaction mixture was stirred at room
temperature overnight. Methanol was added, and stirring was
maintained for an additional 2 h. The reaction mixture was
partitioned between aqueous sat. NaHCO3 and CH2Cl2. The
organic phase was separated, dried over MgSO4, and then
evaporated. The residue was purified by column chromatog-
raphy to afford compound 2 as yellow syrup (13.3 g, 35.4 mmol,
94%). Rf: 0.51 (System H). [R]28D +85° (c 0.24, CHCl3). 1H NMR
(CDCl3) δ: 7.4-7.2 (m, 10H) 2 × Ph-CH2; 5.75 (d, J H1,H2 ) 3.5
Hz, 1H) H-1; 4.75-4.47 (m, 4H) 2 × Ph-CH2; 4.55 (d, 1H) H-2;
1.59 and 1.36 (2 × s, 6H) CH3. 13C NMR (CDCl3) δ: 138.0,
137.6, 128.3, 128.2, 127.9, 127.8, 127.6, 127.5 (2 × Ph-CH2);
112.8 (1,2-O-C[CH3]2); 104.0 (C-1); 77.2 (C-2); 73.3, 72.1 (2 ×
Ph-CH2); 26.7, 26.4 (2 × CH3). HRMS (FAB+): (M + H)+ calcd
for C20H232H4O5: 351.2110, found 351.2114.
1780, 1495, 1388, 1368, 1217, 1150, 1113, 1085, 1048 cm-1
.
1H NMR (CDCl3) δ: 7.4-7.2 (m, 10H) 2 × Ph-CH2; 4.97-4.47
(m, 4H) 2 × Ph-CH2; 4.83 (s, H-1R); 4.73 (s, H-1â); 3.48 (s,
CH3R), 3.46 (s, CH3â). 13C NMR (CDCl3) δ: 207.8, (CdOR),
207.0 (CdOâ), 137.9, 137.7, 137.5, 137.0, 136.97, 136.8, 128.4,
128.3, 128.3, 128.0, 127.9, 127.7, 127.7, 127.6, 127.5 (benzyl),
98.9 (C-1R), 98.6 (C-1â), 73.5, 73.4, 73.1, 72.5 (CH2-benzyl),
56.0 (OCH3â), 55.7 (OCH3R). HRMS (FAB+): (M + H)+ calcd
for C20H192H4O5: 347.1796, found 347.1804.
1-O-Meth yl-3,5-d i-O-ben zyl-â-D-a r a bin o/r-D-r ibofu r a n -
ose-2,3,4,5,5′-2H5 (5). The crude compound 4 was dissolved
in dry ether (∼100 mL), and LAD (483 mg; 11.5 mmol) was
added at 0 °C. The mixture was stirred for 6 h at room
temperature. Ethyl acetate was added, and the mixture was
partitioned between water and CH2Cl2. The organic phase was
concentrated, and the residue was purified by column chro-
matography to yield the mixture of compound 5 (5.48 g; 15.7
mmol; 68%). Rf: 0.64 (System E). 1H NMR (CDCl3) δ: 7.4-
7.2 (m, 10H) 2 × Ph-CH2; 4.88, 4.85 (2 × s, 1H) H-1 (ribo and
ara); 4.77-4.46 (m, 4H) 2 × Ph-CH2; 3.47, 3.41 (2 × s, 3H)
CH3 (R and â). 13C NMR (CDCl3) δ: 138.0, 137.9, 137.8, 137.7,
128.5, 128.3, 128.25, 127.8, 127.7, 127.6, 127.56 (benzyl), 102.8
(ribo C-1), 102.6 (ara C-1), 73.3, 72.9 (2 × CH2, ribo-benzyl),
73.1, 71.7 (2 × CH2, ara-benzyl), 55.5 (ribo OCH3), 55.3 (ara
OCH3). HRMS (FAB+): (M + H)+ calcd for C20H202H5O5:
350.2016, found 350.2019.
1-O-Meth yl-3,5-d i-O-ben zyl-2-O-(4-tolu oyl)-r-D-r ibofu -
r a n ose-2,3,4,5,5′-2H5 (6) a n d 1-O-Meth yl-3,5-d i-O-ben zyl-
2-O-(4-tolu oyl)-â-D-ar abin ofu r an ose-2,3,4,5,5′-2H5 (9). Com-
pound 5 (4.64 g; 13.3 mmol) was coevaporated with dry
pyridine and dissolved in the same solvent (130 mL). 4-Touoyl
chloride (1.94 mL; 14.65 mmol) was added, and the mixture
was stirred overnight. Saturated sodium bicarbonate solution
was added, and the reaction mixture was stirred for 30 min.
The mixture was extracted with CH2Cl2, and the organic phase
was dried over MgSO4. After removing the solvent under
reduced pressure, the crude product was subjected to column
chromatography to afford sugar derivatives 6 (1.44 g; 3.1
mmol; 23%) and 9 (4.0 g; 8.57 mmol; 65%). Compound 6: Rf:
0.60 (System B). [R]26D +98° (c 0.67, CHCl3). 1H NMR (CDCl3)
δ: 8.04-7.23 (m, 14H) toluoyl, 2 × Ph-CH2; 5.21 (s, 1H) H-1;
4.73-4.43 (m, 4H) 2 × Ph-CH2; 3.46 (s, 3H) OCH3; 2.42 (s,
3H) CH3. 13C NMR (CDCl3) δ: 166.0 (CdO, toluoyl), 143.8,
137.8, 129.9, 129.0, 128.3, 128.2, 128.0, 127.6, 126.8, 126.7
(benzyl, toluoyl), 102.1 (C-1), 73.3 and 72.9 (2 × CH2, benzyl),
55.7 (OCH3), 21.6 (CH3, toluoyl). HRMS (FAB+): (M + H)+
1-O-Meth yl-3,5-d i-O-ben zyl-r,â-D-r ibofu r a n ose-3,4,5,5′-
2H4 (3). Carbohydrate derivative 2 (13.2 g, 35.4 mmol) was
dissolved in dry methanol (∼100 mL). Concd sulfuric acid (10
drops) was added and mixture was heated at reflux for 3h.
Solid NaHCO3 was added for neutralization. The suspension
was filtered and the liquid phase was evaporated to an oil.
This was dissolved in CH2Cl2 and washed with aqueous sat.
NaHCO3. The organic phase was separated, dried over MgSO4
and then evaporated to compound 3 (yellow syrup) (11.4 g, 33.0
mmol, 93%). Rf: 0.64 (System E). 1H NMR (CDCl3) δ: 7.4-
7.2 (m, 10H) 2 × Ph-CH2; 4.88 (d, J H1,H2 ) 4.6 Hz, 0.26H) H-1R;
4.86 (d, J H1,H2 ) 0.9 Hz, 0.74H) H-1â; 4.75-4.42 (m, 4H) 2 ×
Ph-CH2; 4.02 (br s, 0.74H) H-2â; 3.48 (s, 0.78H) CH3R, 3.32
(s, 2.28H) CH3â. 13C NMR (CDCl3) δ: 138.0, 137.8, 137.7,
137.0, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7, 127.6, 127.5
(2 × Ph-CH2); 108.4 (C-1â); 102.8 (C-1R); 73.3, 73.2, 73.1, 72.8,
calcd for C28H262H5O6: 468.2435, found 468.2441. Compound
1
9: Rf: 0.48 (System G). [R]26 -74° (c 0.25, CHCl3). H NMR
D
(CDCl3) δ: 7.93-7.21 (m, 14H) toluoyl, 2 × Ph-CH2; 5.21 (s,
1H) H-1; 4.70-4.59 (m, 4H) 2 × Ph-CH2; 3.28 (s, 3H) OCH3;

6568 J . Org. Chem., Vol. 66, No. 20, 2001
Fo¨ldesi et al.
2.42 (s, 3H) CH3. 13C NMR (CDCl3) δ: 165.9 (CdO, toluoyl),
143.9, 138.0, 137.7, 129.8, 129.0, 128.3, 127.9, 127.6, 126.6
(benzyl, toluoyl), 101.3 (C-1), 73.2, 71.9 (2 × CH2, benzyl), 55.2
(OCH3), 21.6 (CH3, toluoyl). HRMS (FAB+): (M + H)+ calcd
for C28H262H5O6: 468.2435, found 468.2439.
1-O-Meth yl-3,5-di-O-ben zyl-r-D-r ibofu r an oside-2,3,4,5,5′-
2H5 (7). Compound 6 (1.44 g; 3.1 mmol) was treated with 1.0
N sodium methoxide solution in methanol (25 mL) in the same
way as compound 10 to give ribose derivative 7 (0.99 g; 2.84
mmol; 92%). Rf: 0.64 (System E). [R]28D +117° (c 0.18, CHCl3).
1H NMR (CDCl3) δ: 7.4-7.2 (m, 10H) 2 × Ph-CH2; 4.88 (s,
1H) H-1; 4.75-4.42 (m, 4H) 2 × Ph-CH2; 3.48 (s, 3H) OCH3.
13C NMR (CDCl3) δ: 137.8, 137.7, 128.32, 128.28, 127.8, 127.7,
127.6, 127.5 (benzyl), 102.8 (C-1), 73.3, 72.9 (2 × CH2, benzyl),
55.5 (OCH3). HRMS (FAB+): (M + H)+ calcd for C20H202H5O5:
350.2016, found 350.2022.
27.3 (OCH2), 9.0 (CH2CH3). HRMS (FAB+): (M + H)+ calcd
for C23H242H5O6: 406.2278, found 406.2285.
1-O-Meth yl-3,5-di-O-ben zyl-â-D-r ibofu r an oside-2,3,4,5,5′-
2H5 (13). Compound 12 (2.7 g; 6.73 mmol) was treated with
1.0 N sodium methoxide solution in methanol (∼60 mL) as
described for compound 10 to give ribose derivative 13 (2.33
g; 6.69 mmol; 99%). Rf: 0.64 (System E). [R]27 -29° (c 0.71,
D
1
CHCl3). H NMR (CDCl3) δ: 7.37-7.25 (m, 10H) benzyl, 4.86
(s, 1H) H-1; 4.57 (d, 4H) 2 × CH2-benzyl; 3.31 (s, 3H) OCH3.
13C NMR (CDCl3) δ: 138.1, 137.0, 128.5, 128.2, 128.1, 127.8,
127.5, 108.4 (C-1), 73.1, 72.6 (2 × CH2-benzyl), 54.9 (OCH3).
HRMS (FAB+): (M + H)+ calcd for C20H202H5O5: 350.2016,
found 350.2024.
2,3-O-Isopr opyliden e-r/â-D-r ibofu r an ose-3,4,5,5′-2H4 (15).
The 0.1% solution of H2SO4 in dry acetone was added to
compound 14 (12.02 g, 61.9 mmol) (HRMS (CI-iBu): (M + H)+
calcd for C5H72H4O5: 155.0858, found 155.0867), and the
mixture was kept in a refrigerator at 4 °C for 3 d, followed by
stirring the reaction mixture at room temperature for 24 h.
Solid Na2CO3 was added to the solution for 2 h after which
the residual solid material was filtered away. The acetone was
evaporated, and the crude mixture was subjected to silica gel
chromatography to obtain compound 15 (3.2 g, 16.7 mmol,
27%). Rf: 0.53 (System F). 1H NMR (CDCl3) δ: â-anomer: 5.41
(s, 1H) H-1; 4.58 (s, 1H) H-2; 1.49, 1.33 (2 × s, 6H) 2 × CH3;
R-anomer: 5.42 (d) H-1; 4.65 (d, J H1,H2 ) 4.2 Hz) H-2; 1.58,
1.40 (2 × s) 2 × CH3. 13C NMR (CDCl3) δ: â-anomer: 112.0
(C[CH3]2); 102.7 (C-1); 86.6 (C-2); 26.2 and 24.6 (2 × CH3);
R-anomer: 114.1 (C[CH3]2); 96.8 (C-1); 79.4 (C-2); 26.0 and 24.6
(2 × CH3). HRMS (CI-iBu): (M + H)+ calcd for C8H112H4O5:
195.1171, found 195.1184.
Meth yl r/â-D-r ibofu r a n osid e-2,3,4,5,5′-2H5 (8). The ben-
zyl groups of the combined sugars 7 and 13 (3.32 g; 9.53 mmol)
were cleaved using Pd/C-H2 (721 mg) in ethanol (50 mL)
overnight at room temperature. The reagent was filtered
through Celite, and the filtrate was evaporated to dryness to
afford methyl ribofuranoside derivative 8 (1.50 g; 8.87 mmol,
93%). Rf: 0.58 and 0.42 (System D). 1H NMR (D2O) δ: 4.92 (s,
0.25H) H-1R, 4.83 (s, 0.75H) H-1â; 3.36 (s, 0.75H) OCH3R, 3.33
(s, 2.25H) OCH3â. 13C NMR (CD3OD) δ: 110.2 (C-1â), 104.9
(C-1R) 55.9 (OCH3R), 55.7 (OCH3â). HRMS (CI-iBu): (M + H)+
calcd for C6H82H5O5: 170.1077, found 170.1085.
1-O-Meth yl-O-3,5-d i-O-ben zyl-â-D-a r a bin ofu r a n ose-2,-
3,4,5,5′-2H5 (10). The sugar 9 (4.0 g, 8.57 mmol) was dissolved
in 1.0 N sodium methoxide in methanol (75 mL), and the
solution was stirred at room temperature for 30 min. The
mixture was neutralized by addition of concd acetic acid and
worked up using saturated NaHCO3 solution in the usual way.
Removal of solvents and purification on silica gel column gave
compound 10 (2.7 g; 7.75 mmol; 91%). Rf: 0.64 (System E).
2,3-O-Isop r op ylid en e-r/â-D-r ibofu r a n ose-2,3,4,5,5′-2H5
(16). The deuterated 2,3-O-isopro-pylidene-R/â-D-ribose (15)
(2.6 g; 13.5 mmol) was coevaporated with 2H2O and dissolved
in the system dioxane/THF/triethylamine/2H2O (16/16/8/11 mL,
v/v/v/v). The solution was heated at 90 °C for 5 days, and then
volatile matters were evaporated to give compound 16 (2.55
g; 13.3 mmol; 98%) as brown oil. Rf: 0.53 (System F). 1H NMR
(CDCl3) δ: â-anomer: 5.42 (s, 1H) H-1; 1.49, 1.32 (2 × s) 2 ×
CH3; R-anomer: 5.43 (s) H-1; 1.58, 1.40 (2 × s) 2 × CH3. 13C
NMR (CDCl3) δ: â-anomer: 112.0 (C[CH3]2); 102.6 (C-1); 26.2,
24.6 (2 × CH3); R-anomer: 114.1 (C[CH3]2); 96.8 (C-1); 26.0,
[R]26 -42° (c 0.71, CHCl3) δ: 1H NMR (CDCl3): 7.34-7.25
D
(m, 10H) benzyl; 4.85 (s, 1H) H-1; 4.77-4.56 (m, 4H) CH2-
benzyl 3.41 (s, 3H) OCH3. 13C NMR (CDCl3) δ: 137.93, 137.87,
128.2, 127.8, 127.60, 127.53 (benzyl), 102.5 (C-1), 73.1, 71.7
(2 × CH2), 55.3 (OCH3). HRMS (FAB+): (M + H)+ calcd for
C
20H202H5O5: 350.2016, found 350.2022.
1-O-Met h yl-3,5-d i-O-b en zyl-2-O-t r iflu or om et h a n esu l-
fon yl-â-D-a r a bin ofu r a n osid e-2,3, 4,5,5′-2H5 (11). The sugar
derivative 10 (3.52 g; 10.1 mmol) was coevaporated with dry
pyridine, and it was dissolved in dry CH2Cl2 (∼75 mL) followed
by the addition of 4-N,N-(dimethylamino)pyridine (4.32 g;
35.36 mmol) and pyridine (7.5 mL). The mixture was cooled
to 0 °C, triflic anhydride (2.4 mL; 14.24 mmol) was added
dropwise, and the resultant mixture was stirred at the same
temperature for 3 h. The reaction mixture was poured into
cold sat. sodium bicarbonate solution and extracted by CH2-
Cl2. The combined organic extract was dried over MgSO4 and
concentrated. The residue was purified by column chromatog-
raphy to give the triflyl derivative 11 (4.06 g; 8.45 mmol; 84%).
24.6 (2 × CH3). HRMS (CI-iBu): (M + H)+ calcd for C8H10
-
2H5O5: 196.1232, found 196.1239.
Meth yl r/â-D-r ibofu r a n osid e-2,3,4,5,5′-2H5 (8). Meth od
A. The benzyl groups of the combined sugars 7 and 13 (3.32
g; 9.53 mmol) were cleaved using Pd/C-H2 (721 mg) in ethanol
(50 mL) overnight at room temperature. The reagent was
filtered through Celite, and the filtrate was evaporated to
dryness to afford the deuterated methyl ribofuranoside deriva-
tive 8 (1.50 g; 8.87 mmol, 93%) as an oil. Meth od B. The
deuterated ribose derivative 16 (1.72 g; 9.05 mmol) was
dissolved in 90% aqueous trifluoroacetic acid (27 mL) and
stirred at room temperature for 1 h. The aqueous phase was
evaporated, and the residual acid was removed by repeated
coevaporations with water. The obtained crude compound 17
(1.41 g, 8.92 mmol, 98%) (HRMS (CI-iBu): (M + H)+ calcd for
C5H62H5O5: 156.0920, found 156.0928) was dissolved in dry
methanol (30 mL) and a few drops of concd H2SO4 were added
at 0 °C. The solution was kept in a refrigerator at 4 °C
overnight and then neutralized passing through an Amberlist
A-21 column (OH- form) using methanol as an eluant. The
methanol was evaporated to give compound 8 (1.46 g; 8.47
mmol; 95%) as a thick light yellow syrup. Rf: 0.59 and 0.42 (â
and R, System D). 1H NMR (D2O) δ: 4.92 (s, 0.3H) R-H-1; 4.83
(s, 1H) â-H-1; 3.36 (s, 0.9H) CH3R; 3.33 (s, 3H) CH3â. 13C NMR
(D2O) δ: â-anomer: 108.1 (C-1); 55.3 (OCH3); R-anomer: 103.3
(C-1); 55.6 (OCH3).
Rf: 0.59 (System G). [R]27 -64° (c 0.74, CHCl3). 1H NMR
D
(CDCl3) δ: 7.38-7.20 (m, 10H) benzyl; 4.99 (s, 1H) H-1; 4.65-
4.45 (m, 4H) CH2-benzyl; 3.38 (s, 3H) OCH3. 13C NMR (CDCl3)
δ: 137.6, 136.8, 128.4, 128.3, 128.0, 127.9, 127.7, 127.6,
(benzyl), 118.4 (q, J CF ) 319.7 Hz, CF3), 100.3 (C-1), 73.3 and
72.5 (2 × CH2, benzyl), 55.4 (OCH3). HRMS (FAB+): (M + H)+
calcd for C21H192H5F3O7S: 482.1509, found 482.1514.
1-O-Met h yl-3,5-d i-O-b en zyl-2-O-p r op ion yl-â-D-r ib ofu -
r a n ose-2,3,4,5,5′-2H5 (12). Cesium propionate (2.26 g; 11.0
mmol) was added to a solution of compound 11 (4.06 g; 8.45
mmol) in dry DMF (50 mL) and stirred for 36 h at room
temperature. The DMF was removed under reduced pressure,
water was added, and the compound was extracted with CH2-
Cl2. After removal of volatile matters, the residue was purified
on a silica gel column to give ribofuranose derivative 12 (2.7
1-O-Met h yl-2,3,5-t r i-O-(4-t olu oyl)-r/â-D-r ib ofu r a n ose-
2,3,4,5,5′-2H5 (18). Compound 8 (1.50 g; 8.87 mmol) was
coevaporated with dry pyridine and dissolved in the same
solvent (80 mL). 4-Toluoyl chloride (4.6 mL, 34.6 mmol) was
added under stirring. The mixture was kept overnight at
ambient temperature. Saturated sodium bicarbonate solution
was added, and stirring was maintained for 3 h. The compound
g; 6.73 mmol; 80%). Rf: 0.54 (System G). [R]27 +14° (c 0.71,
D
1
CHCl3). H NMR (CDCl3) δ: 7.35-7.22 (m, 10H) benzyl; 4.87
(s, 1H) H-1; 4.61-4.38 (m, 4H) 2 × CH2-benzyl; 3.33 (s, 3H)
OCH3; 2.40 (q, 2H) OCH2; 1.14 (t, 3H) CH3. 13C NMR (CDCl3)
δ: 173.5 (CdO), 138.1, 137.5, 128.2, 127.8, 127.7, 127.5, 126.7
(benzyl), 106.2 (C-1), 73.1 and 72.9 (2 × CH2); 54.9 (OCH3),

Total Synthesis of 2′,3′,4′,5′,5′′-2H5-Ribonucleosides
J . Org. Chem., Vol. 66, No. 20, 2001 6569
was extracted with CH2Cl2 from water. Volatile matters were
removed under reduced pressure to give chromatographically
homogeneous compound 18 (5.01 g; 8.87 mmol; 99%). Rf: 0.81
2′,3′,5′-Tr i-O-(4-tolu oyl)-N4-ben zoylcytid in e-2′,3′,4′,5′,5′′-
2H5 (20c). N4-Benzoylcytosine (280 mg; 1.3 mmol) and deu-
terated sugar 19 (550 mg, 1.0 mmol) were condensed following
the above procedure at 70 °C overnight. After usual workup
and purification, compound 20c (526 mg; 0.75 mmol; 75%) was
1
(System C). H NMR (CDCl3) δ: 7.98-7.10 (m, 12H) toluoyl;
5.36 and 5.13 (2 × s, 1H) H-1; 3.47 and 3.40 (2 × s, 3H) OCH3;
2.41, 2.39, 2.36 (3 × s, 9H) CH3. 13C NMR (CDCl3) δ: 166.3,
166.2, 166.0, 165.50, 165,3, 165.2 (CdO, toluoyl), 144.4, 144.1,
144.0, 143.9, 143.7, 130.1, 129.9, 129.8, 129.74, 129.70, 129.1,
128.9, 127.0, 126.82, 126.8, 126.5, 126.4, 126.2, 106.4 (C-1â),
101.9 (C-1R), 55.6 (OCH3R), 55.2 (OCH3â), 21.6 (CH3R+â,
toluoyl). HRMS (FAB+): (M + H)+ calcd for C30H262H5O8:
524.2333, found 524.2337.
isolated as a white foam. Rf: 0.72 (System E). [R]26 -68° (c
D
0.43, CHCl3). IR νmax (KBr): 3060, 3035, 2920, 1722, 1669,
1625, 1610, 1550, 1480, 1279, 1246, 1179, 1109, 1091, 1019
1
cm-1. H NMR (CDCl3) δ: 8.78 (br s, 1H), NH; 8.02-7.83 (m,
9H) H-6, toluoyl, benzoyl; 7.64-7.48 (m, 3H) benzoyl; 7.32-
7.15 (m, 7H) H-5, toluoyl, benzoyl; 6.49 (s, 1H) H-1′; 2.44, 2.40,
2.38 (3 × s, 9H) CH3, toluoyl. 13C NMR (CDCl3) δ: 166.1, 165.3,
165.2 (CdO, toluoyl), 162.2 (C-4), 153.8 (C-2); 144.44, 144.4,
144.38 (toluoyl), 144.32 (C-6), 133.2, 129.9, 129.8, 129.6, 129.4,
129.1, 128.9, 127.6, 126.4, 125.8, 125.7 (toluoyl, benzoyl), 97.3
(C-5), 88.9 (C-1′), 21.6 (CH3, toluoyl). HRMS (FAB+): (M +
H)+ calcd for C40H312H5N3O9: 707.2765, found 707.2771.
1-O-Acet yl-2,3,5-t r i-O-(4-t olu oyl)-r/â-D-r ib ofu r a n ose-
2′,3′,4′,5′,5′′-2H5 (19). A cold mixture of acetic anhydride (5.0
mL), acetic acid (4.0 mL), and concd sulfuric acid (0.8 mL) was
added to a solution of compound 18 (5.01 g; 8.87 mmol) in dry
CH2Cl2 (25 mL) at 0 °C and stirred for 15 min. The reaction
mixture was slowly poured into cold saturated NaHCO3
solution, and stirring was maintained for 3 h. The acetyl
derivative was extracted with CH2Cl2 from the water phase
and dried over magnesium sulfate. The solvent was evapo-
rated, and coevaporation with toluene furnished compound 19
(4.84 g, 8.78 mmol; 99%). The â-anomer was crystallized from
methanol as a white solid (2.92 g; 5.3 mmol; 60%). Rf: 0.75
(System C). [R]26D +62° (c 1.04, CHCl3). For natural [R]28D +63°.
IR νmax (KBr): 3062, 3039, 3015, 2920, 2848, 1750, 1758, 1738,
1620, 1410, 1370, 1285, 1274, 1220, 1177, 1110, 1092, 1070,
973 cm-1. 1H NMR (CDCl3) δ: 7.98-7.10 (m, 12H) toluoyl; 6.41
(s, 1H) H-1; 2.41, 2.40, 2.37 (3 × s, 9H) 3 × CH3 toluoyl; 2.01
(s, 3H) CH3 acetyl. 13C NMR (CDCl3) δ: 169.0 (CdO, acetyl),
166.0, 165.3, 164.9 (CdO, toluoyl), 144.3, 144.2, 143.8, 129.8,
129.7, 129.1, 129.0, 126.8, 126.0, 125.9 (toluoyl), 98.4 (C-1),
21.59, 21.54 (CH3, toluoyl), 20.8 (CH3, acetyl). HRMS (FAB+):
(M + H)+ calcd for C31H262H5O9: 552.2282, found 552.2287.
2′,3′,5′-Tr i-O-(4-tolu oyl)-N2-a cetyl-O6-d ip h en ylca r ba m -
oylgu a n osin e-2′,3′,4′,5′,5′′-2H5 (20d ). N2-Acetyl-O6-diphenyl-
carbamoyl-guanine (378 mg; 0.98 mmol) was suspended in dry
1,2-dichloroethane (6.0 mL), and bis(trimethylsilyl)acetamide
(0.32 mL) was added. The mixture was heated at ∼90 °C under
nitrogen for 1 h. The volatile materials were evaporated, and
after a coevaporation with dry toluene the residue was kept
on an oil pump for 20 min. Sugar derivative 19 (414 mg; 0.75
mmol) was added in dry toluene (11.0 mL) to the persilylated
nucleobase followed by trimethylsilyl trifluoromethanesulfonate
(0.2 mL). The reaction was kept at ∼83 °C overnight. After
NaHCO3 workup, crude nucleoside derivative was subjected
to column chromatography to yield pure compound 20d (426
mg; 0.47 mmol; 63%) as white foam. Rf: 0.75 (System E). [R]26
D
-36° (c 1.03, CHCl3). IR νmax (KBr): 3061, 3039, 2920, 1725,
1610, 1589, 1508, 1489, 1450, 1409, 1370, 1278, 1210, 1178,
1093, 1058, 1018, 979 cm-1 1H NMR (CDCl3) δ: 8.10 (br s,
.
1H) N-H; 8.06 (s, 1H) H-8; 7.93-7.13 (m, 22 H) phenyl,
toluoyl; 6.33 (s, 1H) H-1′; 2.47 (s, 3H) N2-C(O)CH3; 2.41, 2.37
(2 × s, 9H) 3 × CH3 (toluoyl). 13C NMR (CDCl3) δ: 170.0 (C(O)-
CH3); 166.1, 165.2, 165.0 (3 × CdO, toluoyl); 156.3 (C-6); 154.3
(C-4); 152.2 (C-2); 150.1 (DPC); 144.6, 144.4, 144.1 (toluoyl);
142.1 (C-8); 141.6 (DPC); 129.7, 129.6, 129.2, 129.1; 126.8,
126.4, 125.9, 125.5 (DPC, toluoyl); 121.1 (C-5); 87.1 (C-1′); 25.0
(C(O)CH3); 21.6 (CH3, toluoyl). HRMS (FAB+): (M + H)+ calcd
for C49H382H5N6O10: 880.3354, found 880.3359.
2′,3′,5′-Tr i-O-(4-t olu oyl)u r id in e-2′,3′,4′,5′,5′′-2H 5 (20a ).
Uracil (146 mg; 1.30 mmol) was suspended in hexamethyldi-
silazane (2.4 mL), and trimethylchlorosilane (0.25 mL) was
added. The reaction mixture was stirred at 120 °C in nitrogen
atmosphere for 4 h. The volatile materials were evaporated,
and the residue was kept on oil pump for 20 min. Sugar 19
(552 mg; 1.0 mmol) was dissolved in dry 1,2-dichloroethane
(12 mL), and this solution and trimethylsilyl trifluoromethane-
sulfonate (0.25 mL) were added to the persilylated nucleobase.
The reaction was kept overnight at 32 °C in nitrogen atmo-
sphere. Workup by saturated sodium bicarbonate solution and
separation on silica gel column gave compound 20a (534 mg;
Ur id in e-2′,3′,4′,5′,5′′-2H5 (21a ). Nucleoside 20a (500 mg;
0.87 mmol) was dissolved in methanolic ammonia (50 mL) and
stirred at room temperature for 3 days. Solvent was evapo-
rated, and the residue was dissolved in water and extracted
two times with CH2Cl2 and then with diethyl ether. Evapora-
tion of aqueous phase gave uridine 21a (215 mg; 0.86 mmol;
99%). [R]26 +9° (c 0.2, H2O); [R]26 for natural uridine +10°.
0.89 mmol; 89%) as a white foam. Rf: 0.74 (System E). [R]26
D
-74° (c 0.75, CHCl3). IR νmax (KBr): 3028, 2920, 1728, 1690,
1609, 1451, 1376, 1280, 1210, 1179, 1108, 1092, 1019 cm-1
.
D
D
1H NMR (CDCl3) δ: 8.47 (br d, 1H) N-H; 8.00-7.15 (m, 12H)
toluoyl; 7.41 (d, 1H) H-6; 6.34 (s, 1H) H-1′; 2.43, 2.41, 2.38
(3 × s, 9H) 3 × CH3 (toluoyl). 13C NMR (CDCl3) δ: 166.0, 165.3,
165.2 (3 × CdO); 162.1 (C-4); 149.8 (C-2); 144.60, 144.51, 144.5
(toluoyl); 139.3 (C-6); 129.9, 129.8, 129.6, 129.4, 129.1, 126.3,
125.8 125.5 (toluoyl); 103.2 (C-5); 87.4 (C-1′); 21.6 (CH3 toluoyl).
HRMS (FAB+): (M + H)+ calcd for C33H262H5N2O9: 604.2343,
found 604.2349.
IR νmax (KBr): 3346, 3103, 2918, 2798, 1776, 1670, 1467, 1418,
1390, 1358, 1320, 1263, 1212, 1178, 1150, 1126, 1085, 1060,
1038, 975, 961, 950, 900, 825, 766 cm-1. 1H NMR (D2O) δ: 7.79
(d, J H5,H6 ) 8.1 Hz, 1H) H-6; 5.83 (s, 2H) H-1′, H-5. 13C NMR
(D2O) δ: 166.4 (C-4); 151.9 (C-2); 142.0 (C-6); 102.5 (C-5); 89.6
(C-1′). HRMS (FAB+): (M + H)+ calcd for C9H82H5N2O6:
250.1087, found 250.1091.
Ad en osin e-2′,3′,4′,5′,5′′-2H5 (21b). Nucleoside 21b (112 mg;
0.41 mmol; 82%) was obtained as white powder after depro-
tection of 20b (365 mg; 0.5 mmol) in methanolic ammonia.
[R]26 -58° (c 0.2, H2O). For natural [R]26 -60°. IR νmax
2′,3′,5′-Tr i-O-(4-tolu oyl)-N6-ben zoyladen osin e-2′,3′,4′,5′,5′′-
2H5 (20b). N6-Benzoyladenine (232 mg; 0.98 mmol) was
condensed with sugar 19 (414 mg; 0.75 mmol) as described
for compound 20a to give 20b as white foam (417 mg; 0.57
D
D
(KBr): 3422, 3160, 2798, 1678, 1659, 1603, 1573, 1474, 1417,
1379, 1338, 1292, 1245, 1208, 1177, 1160, 1130, 1111, 1073,
992, 970, 843, 819, 792, 752, 730 cm-1. 1H NMR (D2O) δ: 8.32
(s, 1H) H-8; 8.24 (s, 1H) H-2; 6.06 (s, 1H) H-1′. 13C NMR
(DMSO-d6) δ: 156.2 (C-6), 152.4 (C-2), 149.1 (C-4), 140.0 (C-
8), 119.4 (C-5), 87.9 (C-1′). HRMS (FAB+): (M + H)+ calcd for
mmol; 76%). Rf: 0.68 (System E). [R]26 -97° (c 0.27, CHCl3).
D
IR νmax (KBr): 3060, 3038, 2920, 1718, 1609, 1580, 1508, 1480,
1
1451, 1409, 1284, 1179, 1093, 1019 cm-1. H NMR (CDCl3) δ:
9.15 (s, 1H) NH, 8.71 (s, 1H) H-8; 8.19 (s, 1H) H-2; 8.04-7.15
(m, 17H) toluoyl, benzoyl; 6.50 (s, 1H) H-1′; 2.42, 2.41, 2.38
(3 × s, 9H) CH3, toluoyl. 13C NMR (CDCl3) δ: 166.1, 165.3,
165.1 (3 × CdO, toluoyl), 164.4 (CdO, benzoyl), 152.6 (C-2),
151.7 (C-6), 149.5 (C-4), 144.6, 144.5, 144.2 (toluoyl), 141.5 (C-
8), 133.4, 132.7 (benzoyl), 129.8, 129.7, 129.3, 129.2, 129.1,
128.8, 127.8, 127.3, 126.5, 125.9, 125.5 (toluoyl, benzoyl), 123.3
(C-5), 86.7 (C-1′), 21.6 (CH3, toluoyl). HRMS (FAB+): (M +
H)+ calcd for C41H312H5N5O8: 731.2878, found 731.2885.
C
10H92H5N5O4: 273.1359, found 273.1363.
Cytid in e-2′,3′,4′,5′,5′′-2H5 (21c). Compound 20c (493 mg;
0.7 mmol) was stirred for 3 days in methanolic ammonia
followed by removal of methanol. The residue was dissolved
in water and extracted with CH2Cl2 and diethyl ether to afford
21c (156.mg; 0.63 mmol; 90%). [R]26 +32° (c 0.08, H2O). For
D
natural [R]27 +33°. IR νmax (KBr): 3332, 3200, 2920, 2780,
D

6570 J . Org. Chem., Vol. 66, No. 20, 2001
Fo¨ldesi et al.
1
1645, 1602, 1525, 1488, 1400, 1369, 1289, 1208, 1132, 1050,
681 cm-1. H NMR (DMSO-d6/D2O) δ: 8.11 (s, 1H) H-8; 5.99
(s, 1H) H-1′. 13C NMR (DMSO-d6) δ: 156.8 (C-6); 153.7 (C-2);
151.4 (C-4); 135.6 (C-8); 116.8 (C-5); 86.4 (C-1′). HRMS
(FAB+): (M + H)+ calcd for C10H92H5N5O5: 289.1309, found
289.1314.
1
969, 784 cm-1. H NMR (D2O) δ: 7.76 (d, J H5,H6 ) 7.6 Hz, 1H)
H-6; 5.97 (d, 1H) H-5; 5.81 (s, 1H) H-1′. 13C NMR (D2O) δ:
166.4 (C-4), 157.8 (C-2), 141.9 (C-6), 96.4 (C-5), 90.5 (C-1′).
HRMS (FAB+): (M + H)+ calcd for C9H92H5N3O5: 249.1247,
found 249.1252.
Gu a n osin e-2′,3′,4′,5′,5′′-2H5 (21d ). Compound 20d (363 mg,
0.4 mmol) was deprotected upon stirring in methanolic am-
monia (50 mL) for 3 days at room temperature. After evapora-
tion of the methanol, the residue was dissolved in water and
extracted with CH2Cl2 (2×) and then with diethyl ether.
Evaporation of the aqueous phase gave compound 21d as white
Ack n ow led gm en t. Thanks are due to the Swedish
Board for Technical Development (NUTEK) to J .C., the
Swedish Natural Science Research Council (NFR con-
tract # K-KU 12067-300 to A.F. and K-AA/Ku04626-321
to J .C.), the Swedish Research Council for Engineering
Sciences (TFR) to J .C., and Carl Tryggers Styftelse to
A.F. for generous financial support.
solid (99 mg; 0.34 mmol; 85%). [R]26D -36° (c 0.04, H2O); [R]26
D
for natural guanosine -37°. IR νmax (KBr): 3470, 3320, 3203,
2850, 2730, 1738, 1690, 1620, 1531, 1482, 1421, 1383, 1330,
1241, 1180, 1138, 1079, 1058, 1018, 982, 970, 910, 887, 820,
J O010097N