Synthesis of isotopically labeled D-[1′-13C]ribonucleoside phosphoramidites

Article abstract of DOI:10.1016/S0008-6215(00)00327-X

The preparation of fully protected labeled diisopropylamino-β-cyanoethyl-[1′-¹³C]ribonucleoside phosphoramidites with regioisomeric purity is described. We demonstrated in this paper that a regioselective 2′-O-silylation, through a 3′,5′-O-di-tert-butylsilanediyl protection, has been applied for the synthesis of [1′-¹³C]ribonucleoside phosphoramidite units. This method allowed us to obtain only the desired 2′-O-silyl-3′-O-phosphoramidites avoiding the undesired 3′-O-silyl-2′-O-phosphoramidite nucleosides isolated by standard procedures. This is a suitable procedure to RNA precursors with respect to the isotope-containing precursors.

Full text of DOI:10.1016/S0008-6215(00)00327-X

www.elsevier.nl/locate/carres
Carbohydrate Research 331 (2001) 83–90
Note
Synthesis of isotopically labeled
D
-[1%-13C]ribonucleoside
phosphoramidites
Yoshio Saito, Agne`s Nyilas, Luigi A. Agrofoglio *
Institut de Chimie Organique et Analytique, CNRS UMR 6005, Uni6ersite´ d’Orle´ans, BP 6759,
45100 Orle´ans, France
Received 27 October 2000; accepted 20 December 2000
Abstract
The preparation of fully protected labeled diisopropylamino-b-cyanoethyl-[1%-13C]ribonucleoside phosphoramidites
with regioisomeric purity is described. We demonstrated in this paper that a regioselective 2%-O-silylation, through a
3%,5%-O-di-tert-butylsilanediyl protection, has been applied for the synthesis of [1%-13C]ribonucleoside phosphoramidite
units. This method allowed us to obtain only the desired 2%-O-silyl-3%-O-phosphoramidites avoiding the undesired
3%-O-silyl-2%-O-phosphoramidite nucleosides isolated by standard procedures. This is a suitable procedure to RNA
precursors with respect to the isotope-containing precursors. © 2001 Elsevier Science Ltd. All rights reserved.
Keywords: [1-13C]Ribose; Ribonucleosides; Phosphoramidites; Labeled nucleosides
The availability of 13C-labeled ribonu-
cleosides are of special interest since new
NMR techniques have been established which
oxygen to the b-cyanoethyl N,N-diisopropyl-
phosphoramidite. Nevertheless, the need to
protect this additional 2%-hydroxyl group in
RNA (compared to DNA synthesis) has hin-
dered the development of a practical method.
In fact, the principal consideration in the
chemical synthesis of the ribonucleoside phos-
phoramidites is contamination of the desired
2%-O-silyl-3%-O-phosphoramidites with unde-
sired 3%-O-silyl-2%-O-phosphoramidite nucleo-
sides which can yield undesired 5%,2%-inter-
nucleotidic bonds, consuming valuable labeled
ribonucleosides and lowering the yield of de-
sired phosphoramidite units. Moreover, one
of the major problems in the chemical synthe-
sis of 13C enriched nucleoside derivatives is the
price of the starting compound that requires
optimized reactions. Our efforts aimed at re-
producible experimental procedures giving
high-yield products with respect to the iso-
1
allow us to assign overlapping H NMR sig-
nals in spectra of large biomolecules such as
RNA fragments.1,2 The chemical synthesis of
RNA oligoribonucleotides involves the use of
protected ribonucleoside phosphoramidites.3–5
Standard procedures described by Ogilvie et
al.6–8 for preparation of ribonucleoside phos-
phoramidite units used in solid-phase synthe-
sis of RNA oligonucleotides include protec-
tion of the amino groups on the nucleobases,
protection of the 5%- and 2%-hydroxyl groups as
dimethoxytrityl (DMTr) and TBDMS ether,
respectively, and phosphitylation of the 3%-
* Corresponding author. Tel./fax: +33-2-38510728.
E-mail address: luigi.agrofoglio@univ-orleans.fr (L.A. Agro-
foglio).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00327-X

84
Y. Saito et al. / Carbohydrate Research 331 (2001) 83–90
tope-containing precursors. We demonstrate
in this paper that a regioselective 2%-O-silyla-
tion, through the use of di-tert-butyldichloro-
silane, has been applied for the synthesis of
[1%-13C]ribonucleoside phosphoramidite units.
In the course of this work, another ap-
proach to this problem has been reported by
Song et al.9 through H-phosphonate deriva-
tives, but through an undesired but separable
mixture of 2%- and 3%-silyl derivatives. The
known procedures, we applied a three step
procedure involving the initial and regioselec-
tive protection of 3%,5%-hydroxyl using di-tert-
butyldichlorosilane.12 Thus treatment of
D-[1%-
13C]ribonucleosides 3a–c with di-tert-butyl-
14
dichlorosilane in the presence of AgNO3 in
DMF afforded the corresponding 3%,5%-O-(di-
tert-butylsilanediyl) derivatives 4a (72%), 4b
(82%), and 4c (97%), respectively. Treatment
with tert-butyldimethylsilyl chloride (TBDM-
SiCl) and silver nitrate in DMF led 2%-O-TB-
DMS ether 5a–c in fairly good yields. The
di-tert-butylsilylene group was then removed
by treatment with pyridinium poly(hydrogen
fluoride) at −20 °C to afford 6a (96%), 6b
(94%), and 6c (95%) yield, respectively. Under
these conditions, no migration of the 2%-O-TB-
DMS group to the 3%-oxygen was ob-
served.12,13 After dimethoxytritylation of 6a–c
to 7a–c, isomerization free synthesis of the
phosphoramidites 8a–c was accomplished us-
ing (N,N-diisopropylamino)(cyanoethyl)phos-
phonamidic chloride as the phosphitylating
agent, in the presence of 2,4,6-collidine and
N-methylimidazole.5 As a result, the desired
RNA phosphoramidite units 8a–c were ob-
tained avoiding byproducts as found by the
classical approach, and with an overall yield
D
-[1-13C]ribose was converted into [1-13C]-1-
O-acetyl-2,3,5-tri-O-benzoyl-b- -ribofuranose
(1) (Scheme 1) according to a procedure used
for the synthesis of unlabeled
isomer.10 Nu-
D
D
cleoside derivatives 2a–c were obtained from
ribosylation of 1 with persilylated nucleobases
(N6-benzoyladenine, uracil, and N4-benzoylcy-
tosine) under Vorbru¨ggen conditions.11 In case
of the synthesis of protected uridine 2c, the
temperatures had to be kept under 45 °C to
avoid undesired side product. The selective
deprotection of O-acyl groups from 2a–c was
achieved with aqueous 1 M NaOH–ethanol–
pyridine (for 3a,b) or NH3–MeOH (for 3c). In
all cases, de-acylated nucleosides 3a–c were
directly used in the next reaction.
To circumvent the problem of undesired
3%-O-silyl-2%-O-phosphoramidite obtained dur-
ing the silylation of 2%-hydroxyl by the largely
for the sequential transformation of
D-[1%-
Scheme 1. Reagents and conditions: (a) silylated heterocycle, 1,2-dichloroethane, TMSOTf; (b) (for 3a and 3b) pyridine–EtOH–1
M NaOH then Dowex-H+ or (for 3c) MeOH–NH3; (c) DTBSiCl2, AgNO3, DMF; (d) TBDMSiCl, DMAP, pyridine; (e) HF-Py,
THF, −20°C; (f) DMTrCl, pyridine; (g) 2,4,6-collidine, CH2Cl2, N-methylimidazole.

Y. Saito et al. / Carbohydrate Research 331 (2001) 83–90
85
13C]ribonucleosides 3a–c to 8a–c of 28% (for
8a), 47% (for 8b) and 63% (for 8c).1
solution of acetate 1 (1.46 g, 2.89 mmol) in
dry DCE (10 mL) and TMSOTf (1.38 mL,
2.89 mmol) at rt. After stirring the reaction
mixture for 30 min at rt, it was heated at
45 °C for 5 h under N2. The reaction solution
was poured into an ice-cold mixture of
CH2Cl2 (50 mL) and satd NaHCO3 solution
(50 mL). The aqueous phase was separated
and extracted with CH2Cl2 (2×50 mL). The
combined organic layer was washed with brine
and dried (MgSO4). The solvents were re-
moved under reduced pressure and the residue
was separated by silica gel column chromatog-
raphy (50:1 CH2Cl2–CH3OH) to give 2a as a
1. Experimental
General methods.—Commercially available
chemicals and solvents were reagent grade and
used as received. Dry THF, pyridine, CH2Cl2,
and 1,2-dichloroethane were obtained by dis-
tillation from CaH2, N,N-dimethylformamide
from BaO. The reactions were monitored by
thin-layer chromatography (TLC) analysis us-
ing silica gel plates (Kieselgel 60 F254, E.
Merck). Compounds were visualized by UV
irradiation and/or spraying with 20% H2SO4
in EtOH, followed by charring at 150 °C.
Column chromatography was performed on
Silica Gel 60 M (0.040–0.063 mm, E. Merck).
NMR spectra were accumulated with a
Bruker AVANCE DPX 250 Fourier Trans-
form 250 spectrometer, with Me4Si as the
internal standard, unless otherwise stated.
Chemical shifts are given in ppm (l). Low-res-
olution mass spectra were obtained on a
Perkin–Elmer SCIEX API-300 (heated nebul-
lizer) spectrometer operating in the ion-spray
(IS) mode. Elemental analyses were performed
by the Service Central de Microanalyse du
CNRS (Vernaison, France).
1
colorless oil (1.70 g, 86%). H NMR (CDCl3)
l 4.70 (dd, 1 H, J5%a,4% 3.0, J5%a,5%b 12.9 Hz,
H-5%a), 4.84 (m, 1 H, H-4%), 4.92 (dd, 1 H,
J5%b,4% 3.3 Hz, H-5%b), 6.25 (dd, 1 H, J2%,1% 4.6,
J2%,3% 5.5 Hz, H-2%), 6.41 (dd, 1 H, J3%,4% 4.3 Hz,
H-3%), 6.50 (dd, 1 H, H-1%, J1%,C?%166 Hz), 7.36–
8.18 (m, 20 H, 4×Bz), 8.70 (s, 1 H, H-2), 8.98
(s, 1 H, H-8). MS: m/z 685 (M+H)+.
Compounds 2b and 2c were synthesized by
a similar methodology as described for the
preparation of compound 2a from compound
1.
[1%-13C]-2%,3%,5%-Tri-O-benzoyl-N4 -benzoyl-
cytidine (2b).—Isolated as a colorless oil
(86%, 2.66 g). [h]2D2 −42° (c2, CHCl3); H
1
1-O-Acetyl-2,3,5-tri-O-benzoyl-i-
oside (1).—Compound 1 was prepared from
-[1-13C]ribose in 54% total yield according to
a procedure for the synthesis of the unlabeled
isomer.10 1H NMR (CDCl3): l 2.01 (s, 3 H,
D-ribofuran-
NMR (CDCl3) l 4.73–4.87 (m, 4 H, H-5,
H-4%, H-5%a, H-5%b), 5.87 (dd, 1 H, J2%,1% 2.8,
J2%,3% 5.7 Hz, H-2%), 5.93 (dd, 1 H, J3%,4% 3.5 Hz,
H-3%), 6.50 (dd, 1 H, J1%,C1% 169 Hz, H-1%),
7.36–8.18 (m, 21 H, H-6, 4×Bz), 8.70 (s, 1 H,
H-2), 8.98 (s, 1 H, H-8). MS: m/z 661 (M+
H)+.
D
D
OAc), 4.52 (dd, 1 H, J5a,4 4.0, J5a,5b 13 Hz,
H-5a), 4.77 (dd, 1 H, H-5b), 4.78 (m, 1 H,
H-4), 5.78 (dd, 1 H, J2,1 1.0, J2,3 5 Hz, H-2),
5.91 (ddd, 1 H, J3,4 6.9, J3,2 5.0, J3,C1 1.5 Hz,
H-3), 6.43 (dd, 1 H, J1,C1 183, H-1), 7.38–8.15
(m, 15 H, 3×Bz).
[1%-13C]-2%,3%,5%-Tri-O-benzoyluridine (2c).—
Isolated as a colorless oil (56%, 0.92 g). [h]D20
1
−37° (c2, CHCl3); H NMR (CDCl3) l 4.69
(dd, 1 H, J5%a,4% 3.7, J5%a,5%b 11.7 Hz, H-5%a), 4.71
(m, 1 H, H-4%), 4.85 (dd, 1 H, J5%b,4% 2.4 Hz,
H-5%b), 5.62 (dd, 1 H, J5,NH 2.2, J5,6 8.1 Hz,
H-5), 5.76 (dd, 1 H, J2%,1% 5.6, J2%,3% 6.0 Hz,
H-2%), 5.89 (dd, 1 H, J3%,4% 4.5 Hz, H-3%), 6.33
(dd, 1 H, J1%,C1% 169 Hz, H-1%), 7.34–8.13 (m,
16 H, H-6, 3×Bz), 8.79 (bs, 1 H, NH). MS:
m/z 558 (M+H)+.
[1%-13C]-2%,3%,5%-Tri-O-benzoyl-N6-benzoyla-
denosine (2a).—A mixture of N6-benzoy-
ladenine (1.30 g, 5.78 mmol) and (NH4)2SO4
(catalytic amount) in hexamethyldisilazane (14
mL) was refluxed for 14 h under N2. The clear
solution obtained was concentrated in vacuo
and the residue was dissolved in dry 1,2-
dichloroethane (DCE, 5 mL) followed by a
[1%-13C]-N6-benzoyladenosine (3a).—To a
solution of 2a (1.41 g, 2.05 mmol) in pyridine
(12 mL) and EtOH (6 mL) at 0 °C was added
1 See Section 2.

86
Y. Saito et al. / Carbohydrate Research 331 (2001) 83–90
(0.58 g, 1.54 mmol) and AgNO3 (0.78 g, 4.6
mmol) in anhyd DMF (10 mL) at 0 °C was
added dropwise di-tert-butyldichlorosilane
(0.5 mL, 2.37 mmol) with vigorous stirring.
The mixture was warmed to rt and subse-
quently stirred for 15 min Et3N (0.65 mL, 4.46
mmol) was added, and the mixture was stirred
for an additional 5 min. After addition of
water (100 mL), the resulting solution was
extracted with EtOAc (2×50 mL). The or-
ganic layer was washed with brine and dried
(MgSO4). The solvents were evaporated to
dryness under reduced pressure. The residue
was purified by silica gel column chromatog-
raphy (25:1 CH2Cl2 –CH3OH) to give 4a
dropwise 1 M NaOH (9.1 mL). The mixture
was stirred at 0 °C for 30 min and neutralized
with Dowex-50WX2-200 (H+). The solution
was filtered and the resin was washed with 1:4
pyridine–water (60 mL). The filtrate was
evaporated and co-evaporated with toluene.
The remaining residue was triturated with
Et2O, leaving a colorless powder of 3a (0.80 g,
\99%). [h]2D0 −50° (c1.3, dimethylsulfoxide);
1H NMR (DMSO-d6) l 3.59 (m, 1 H, H-5%a),
3.70 (m, 1 H, H-5%b), 3.91 (m, 1 H, H-4%), 4.21
(m, 1 H, H-3%), 4.66 (m, 1 H, H-2%), 5.11 (dd,
1 H, C5%OH), 5.22 (d, 1 H, C3%OH), 5.54 (d, 1
H, C2%OH), 6.05 (dd, 1 H, J1%,C1% 166 Hz, H-1%),
7.55–8.05 (m, 5 H, Bz), 8.71 (s, 1 H, H-2) 8.76
1
(s, 1 H, H-8), 11.20 (s, 1 H, NH). MS: m/z 373
(72%, 0.58 g) as a colorless waxy solid. H
13
t
(M+H)+. Anal. Calcd for C C1H17N5O5: C,
NMR (CDCl3) l 1.08 (s, 9 H, Bu), 1.15 (s, 9
16
t
55.10; H, 4.60. Found: C, 55.14; H, 4.62.
Compounds 3b were synthesized by a simi-
lar methodology as described for the prepara-
tion of compound 3a from compounds 2b.
[1%-13C]-N4-benzoylcytidine (3b).—Isolated
as a colorless powder of 3b (0.12 g, \99%).
[h]2D3 +29° (c0.26, dimethylsulfoxide); 1H
NMR (DMSO-d6) l 3.62 (m, 1 H, H-5%a), 3.76
(m, 1 H, H-5%b), 3.92 (m, 1 H, H-4%), 3.98 (m,
1 H, H-3%), 4.02 (m, 1 H, H-2%), 5.02 (d, 1 H,
J3%,OH 5.5 Hz, C3%OH), 5.15 (dd, 1 H, J5%a,OH
5.0, J5%b,OH 5.0, C5%OH), 5.48 (d, 1 H, J2%,OH 5.0
Hz, C2%OH), 5.82 (dd, 1 H, J1%,C1% 174.5 Hz,
H-1%), 7.32 (dd, 1 H, J5,NH 1.5, J5,6 8.0 Hz,
H-5), 7.52–7.62 (m, 5 H, Bz), 8.48 (dd, 1 H,
J6,C1% 2.5, H-6), 11.20 (s, 1 H, NH). MS: m/z
H, Bu), 4.06–4.17 (m, 2 H, H-5%a, H-5%b),
4.48 (m, 1 H, H-4%), 4.76 (dd, 1 H, J2%,1% 1.0,
J2%,3% 5.0 Hz, H-2%), 4.90 (dd, 1 H, J3%,4% 3.5 Hz,
H-3%), 6.05 (dd, 1 H, J1%,C1% 167 Hz, H-1%),
7.50–8.02 (m, 5 H, Bz), 8.06 (s, 1 H, H-2),
8.78 (s, 1 H, H-8). MS: m/z 513 (M+H)+.
Compounds 4b and 4c were synthesized by
a similar methodology as described for the
preparation of compound 4a from respective
compounds 3b and 3c.
[1%-13C]-3%,5%-O-(Di-tert-butylsilanediyl)-N4-
benzoylcytidine (4b).—Isolated as a colorless
1
waxy solid (82%, 0.12 mg). H NMR (CDCl3)
t
t
l 1.03 (s, 9 H, Bu), 1.04 (s, 9 H, Bu),
3.98–4.23 (m, 4 H, H-3%, H-4%, H-5%a, H-5),
4.39 (dd, 1 H, J2%,1% 1.0, J2%,3% 4.7 Hz, H-2%), 4.55
(dd, 1 H, J5%b,4% 4.7, J5%b,5%a 9.1 Hz, H-5%b), 5.77
(dd, 1 H, J1%,C1% 175 Hz, H-1%), 7.49–7.93 (m, 6
H, H-6, Bz). MS: m/z 489 (M+H)+.
349 (M+H)+. Anal. Calcd for C C1H17-
13
15
N3O6: C, 55.46; H, 4.92. Found: C, 55.51; H,
4.96.
[1%-13C]-Uridine (3c).—A solution of 2c
(0.91 g, 1.63 mmol) in satd methanolic ammo-
nia was stirred at rt for 14 h. The solvent was
then removed under reduced pressure and the
remaining residue triturated with Et2O, leav-
ing a colorless powder of 3c (\98%, 0.39 g).
[1% - 13C] - 3%,5% - O - (Di - tert - butylsilanediyl)-
uridine (4c).—Isolated as a colorless waxy
1
solid (97%, 0.54 g). H NMR (CDCl3) l 1.03
t
t
(s, 9 H, Bu), 1.04 (s, 9 H, Bu), 4.00–4.07 (m,
2 H, H-5%a, H-5%b), 4.18 (m, 1 H, H-4%), 4.39
(dd, 1 H, J3%,2% 4.2, J3%,4% 5.3 Hz, H-3%), 4.47 (dd,
1 H, J2%,1% 1.0 Hz, H-2%), 5.61 (dd, 1 H, J1%,C1%
170 Hz, H-1%), 5.75 (d, 1 H, J5,6 8.3 Hz, H-5),
7.22 (dd, 1 H, J6,NH 3.2 Hz, H-6), 8.70 (bs, 1
H, NH). MS: m/z 386 (M+H)+.
1
[h]2D3 +8° (c2, water); H NMR (D2O) l 3.81
(m, 1 H, H-5%a), 3.92 (m, 1 H, H-5%b), 4.14 (m,
1 H, H-4%), 4.23 (m, 1 H, H-3%), 4.35 (m, 1 H,
H-2%), 5.90 (d, 1 H, J5,6 8.0 Hz, H-5), 5.92 (m,
1 H, H-1%), 7.88 (d, 1 H, H-6). MS: m/z 246
(M+H)+. Anal. Calcd for C183C1H12N2O6: C,
44.49; H, 4.93. Found: C, 44.52; H, 4.96.
[1%-13C]-3%,5%-O-(Di-tert-butylsilanediyl)-N6-
benzoyladenosine (4a).—To a solution of 3a
[1%-13C]-2%-O-tert-Butyldimethylsilyl-3%,5%-O-
(di - tert - butylsilanediyl) - N6 - benzoyladenosine
(5a).—To a solution of 4a (0.41 g, 0.81 mmol)
and DMAP (catalytic amount) in anhyd pyri-
dine (10 mL) was added dropwise tert-

Y. Saito et al. / Carbohydrate Research 331 (2001) 83–90
87
wise to a solution of 5a (0.36 g, 0.58 mmol) in
THF (3 mL) at 0 °C. The mixture was
warmed to rt, stirred for 5 min and then
diluted with pyridine (0.5 mL). Water (5 mL)
was added and the aqueous phase extracted
with CH2Cl2 (3×40 mL). The organic layers
were collected, washed with 5% NaHCO3 (50
mL) and dried (MgSO4). After evaporation
under reduced pressure, the residue was
purified by silica gel column chromatography
(20:1 CH2Cl2–CH3OH) to yield the desired
compound 6a (96%, 0.28 g) as a colorless
butyldimethylsilyl chloride (0.12 g, 0.78
mmol), and the mixture was refluxed
overnight. After evaporation under reduced
pressure, the resulting mixture was diluted
with CH2Cl2 (40 mL), and the organic phase
washed with brine (40 mL) and dried
(MgSO4). After a column chromatography on
silica gel (50:1 CH2Cl2–CH3OH), the desired
product 5a (74%, 0.38 g) was isolated as a
1
colorless waxy solid. H NMR (CDCl3) l 0.18
(s, 3 H, Me), 0.20 (s, 3 H, Me), 0.96 (s, 9 H,
t
t
tBu), 1.07 (s, 9 H, Bu), 1.10 (s, 9 H, Bu), 4.06
(m, 1 H, J5%a,4% 9.2, J5%a,5%b 10.3 Hz, H-5%a), 4.28
(m, 1 H, H-4%), 4.47–4.56 (m, 2 H, H-3%,
H-5%b), 4.65 (d, 1 H, J2%,1% 1.0, J2%,3% 4.0 Hz,
H-2%), 6.11 (dd, 1 H, J1%,C1% 170 Hz, H-1%),
7.51–8.02 (m, 5 H, Bz), 8.05 (s, 1 H, H-2),
8.79 (s, 1 H, H-8). MS: m/z 627 (M+H)+.
Compounds 5b and 5c were synthesized by
a similar methodology as described for the
preparation of compound 5a from respective
compounds 4b and 4c.
1
waxy solid. H NMR (CDCl3) l −0.36 (s, 3
H, Me), −0.13 (s, 3 H, Me), 0.83 (s, 9 H,
tBu), 3.79 (dd, 1 H, J5%a,4% 1.0, J5%a,5%b 13.2 Hz,
H-5%a), 4.03 (dd, 1 H, J5%b,4% 1.6 Hz, H-5%b),
4.37–4.42 (m, 2 H, H-3%, H-4%), 5.17 (dd, 1 H,
J2%,3% 4.7, J2%,1% 7.5 Hz, H-2%), 5.86 (dd, 1 H,
J1%,C1% 163 Hz, H-1%), 7.50–8.02 (m, 5 H, Bz),
8.08 (s, 1 H, H-2), 8.86 (s, 1 H, H-8). MS: m/z
487 (M+H)+.
Compounds 6b and 6c were synthesized by
a similar methodology as described for the
preparation of compound 6a from respective
compounds 5b and 5c.
[1%-13C]-2%-O-tert-Butyldimethylsilyl-3%,5%-O-
(di - tert - butylsilanediyl) - N4 - benzoylcytidine
(5b).—Isolated as a colorless waxy solid (79%,
0.12 g). 1H NMR (CDCl3) l 0.18 (s, 3 H, Me),
[1% - 13C] - 2% - O - tert - Butyldimethylsilyl - N4-
benzoylcytidine (6b).—Isolated as a colorless
t
0.27 (s, 3 H, Me), 0.96 (s, 9 H, Bu), 1.03 (s, 9
1
waxy solid (94%, 0.08 g). H NMR (CDCl3) l
t
t
H, Bu), 1.04 (s, 9 H, Bu), 3.81 (dd, 1 H, J5%a,4%
4.4, J5%a,5%b 9.5 Hz, H-5%a), 4.03 (m, 1 H, H-4%),
4.26–4.32 (m, 2 H, H-3%, H-5), 4.35 (dd, 1 H,
J2%,1% 1.0, J2%,3% 4.4 Hz, H-2%), 4.59 (dd, 1 H, J5%b,4%
4.9, H-5%b), 5.76 (dd, 1 H, J1%,C1% 178 Hz, H-1%),
7.48–7.96 (m, 6 H, H-6, Bz), 8.75 (bs, 1 H,
NH). MS: m/z 603 (M+H)+.
0.10 (s, 3 H, Me), 0.12 (s, 3 H, Me), 0.92 (s, 9
t
H, Bu), 3.64–3.84 (m, 2 H, H-5%a, H-5%b),
4.02 (m, 1 H, H-3%), 4.20 (d, 1 H, J5,6 2.1 Hz,
H-5), 4.27 (m, 1 H, H-4%), 4.83 (dd, 1 H, J2%,3%
4.4, J2%,1% 4.7 Hz, H-2%), 5.54 (dd, 1 H, J1%,C1% 169
Hz, H-1%), 7.49–8.06 (m, 6 H, H-6, Bz), 8.82
(bs, 1 H, NH). MS: m/z 463 (M+H)+.
[1%-13C]-2%-O-tert-Butyldimethylsilyl-3%,5%-O-
(di-tert-butylsilanediyl)uridine (5c).—Isolated
[1%-13C]-2%-O-tert-Butyldimethylsilyluridine
(6c).—Isolated as a colorless waxy solid (95%,
0.42 g). 1H NMR (CDCl3) l 0.09 (s, 3 H, Me),
1
as a colorless waxy solid (90%, 0.62 g). H
t
NMR (CDCl3) l 0.14 (s, 3 H, Me), 0.18 (s, 3
0.11 (s, 3 H, Me), 0.91 (s, 9 H, Bu), 3.79 (dd,
t
t
H, Me), 0.93 (s, 9 H, Bu), 1.02 (s, 9 H, Bu),
1 H, J5%a,4% 6.1, J5%a,5%b 12.2 Hz, H-5%a), 3.95 (dd,
1 H, J5%b,4% 2.9 Hz, H-5%b), 4.15–4.24 (m, 2 H,
H-2%, H-3%), 4.62 (m, 1 H, H-4%), 5.57 (dd, 1 H,
H-1%, J1%,2% 5.4, J1%,C1% 167 Hz, H-1%), 5.75 (d, 1
H, J5,6 8.5 Hz, H-5), 7.55 (dd, 1 H, J6,C1% 3.2,
J5,6 8.5 Hz, H-6), 8.20 (bs, 1 H, NH). MS: m/z
360 (M+H)+.
t
1.05 (s, 9 H, Bu), 3.86 (dd, 1 H, J5%a,4% 4.7,
J5%a,5b% 9.5 Hz, H-5%a), 3.97 (dd, 1 H, J3%,2% 4.7,
J3%,4% 9.1 Hz, H-3%), 4.17 (m, 1 H, H-4%), 4.28
(dd, 1 H, J2%,1% 1.0 Hz, H-2%), 4.51 (dd, 1 H,
J5%b,4% 4.9 Hz, H-5%b), 5.66 (dd, 1 H, J1%,C1% 175
Hz, H-1%), 5.74 (dd, 1 H, J5,NH 2.2, J5,6 8.1 Hz,
H-5), 7.25 (dd, 1 H, J6,C1% 2.5 Hz, H-6), 8.40
(bs, 1 H, NH). MS: m/z 500 (M+H)+.
[1% - 13C] - 5% - O - Dimethoxytrityl - 2% - O - tert-
butyldimethylsilyl-N6-benzoyladenosine (7a).—
To a solution of 6a (0.65 g, 1.32 mmol) in
THF (15 mL) under N2 were added anhyd
pyridine (0.53 mL, 6.60 mmol), AgNO3 (0.27
g, 1.32 mmol) and 4,4%-dimethoxytrityl chlo-
[1% - 13C] - 2% - O - tert - Butyldimethylsilyl - N6-
benzoyladenosine (6a).—HF·pyridine (6 mL,
2.34 mmol) was carefully diluted with anhyd
pyridine (0.31 mmol) and then added drop-

88
Y. Saito et al. / Carbohydrate Research 331 (2001) 83–90
65.48; H, 6.70. Found: C, 65.53; H, 6.74.
[1% - 13C] - 5% - O - Dimethoxytrityl - 2% - O - tert-
butyldimethylsilyl-N6-benzoyladenosine 3%-O-
(2-cyanoethyl-N,N-diisopropylphosphoramidite)
(8a).—A solution of protected nucleoside 7a
(0.75 g, 0.95 mmol) in dry THF (3 mL),
2,4,6-collidine (0.94 mL, 7.10 mmol) and N-
methylimidazole (37.7 ml, 0.437 mmol) was
stirred under N2. N,N-(Diisopropylamino)-
(cyanoethyl)phosphoramidic chloride (0.527
mL, 2.37 mmol) was then added dropwise
over 5 min at rt. The reaction was completed
after 1 h as determined by TLC. The reaction
mixture was diluted with EtOAc (100 mL),
washed with 5% NaHCO3 (150 mL), then with
brine (150 mL). The aqueous layers were back
extracted with EtOAc (2×50 mL) and com-
bined organic phase were dried (MgSO4). The
solvent was removed under reduced pressure
yielding a viscous oil. Co-evaporation with
toluene afforded the crude phosphoramidite
as a white foam. The ribonucleoside phospho-
ramidites were further purified by silica gel
column chromatography (5:4:1 hexane–
EtOAc–Et3N) yielding 8a (75%, 0.71 g) as a
colorless foam and as a mixture of two
ride (0.54 g, 1.58 mmol) successively with
vigorous stirring and exclusion of moisture.
The resultant pale-yellow solution was stirred
for 1 h and filtered into a 5% NaHCO3 solu-
tion. The filtrate was extracted with CH2Cl2
(50 mL), dried (MgSO4). After evaporation of
the solvent under reduced pressure, the
residue was subjected to a silica gel column
chromatography (19:1 CH2Cl2–CH3OH) to
1
give 7a (72%, 0.75 g) as a colorless foam. H
NMR (CDCl3) l −0.15 (s, 3 H, Me), 0.01 (s,
t
3 H, Me), 0.83 (s, 9 H, Bu), 3.38 (dd, 1 H,
J5%a,4% 3.4, J5%a,5%b 10.5 Hz, H-5%a), 3.54 (dd, 1 H,
J5%b,4% 3.1, H-5%b), 3.77 (s, 6 H, 2×OMe), 4.28
(m, 1 H, H-4%), 4.35 (dd, 1 H, J3%,2% 4.7, J3%,4% 3.5
Hz, H-3%), 5.01 (dd, 1 H, J2%,1% 5.3 Hz, H-2%),
6.09 (dd, 1 H, J1%,C1% 166 Hz, H-1%), 6.75–7.65
(m, 18 H, Bz, DMTr), 8.22 (s, 1 H, H-2), 8.73
(s, 1 H, H-8). MS: m/z 487 (M+H)+. Anal.
13
43
Calcd for C C1H48N5O7Si: C, 67.11; H, 6.13.
Found: C, 67.23; H, 6.15.
Compounds 7b and 7c were synthesized by
a similar methodology as described for the
preparation of compound 7a from respective
compounds 6b and 6c.
[1% - 13C] - 5% - O - Dimethoxytrityl - 2% - O - tert-
butyldimethylsilyl-N4-benzoylcytidine (7b).—
1
diastereoisomers. H NMR (CDCl3) l 0.02 (s,
1
Isolated as a colorless foam (87%, 0.10 g). H
3 H, Me), 0.04 (s, 3 H, Me), 0.75 (s, 9 H, tBu),
NMR (CDCl3) l 0.19 (s, 3 H, Me), 0.31 (s, 3
i
1.19 (m, 12 H, Pr), 2.32 (t, 1 H, J 7.5 Hz,
t
H, Me), 0.97 (s, 9 H, Bu), 3.56–3.59 (m, 2 H,
CH2–CN), 2.63 (t, 1 H, −CH2–CN), 3.29
(m, 1 H, H-5%a), 3.54–3.78 (m, 5 H, H-5%b,
P(O)CH2–, 2×CH(iPr)), 3.78 (s, 6 H, OMe),
4.36 (m, 1 H, H-4%), 4.41 (m, 1 H, H-3%), 5.07
(m, 1 H, H-2%), 6.11 (dd, 1 H, J1%,2% 1.0, J1%,C1%
166 Hz, H-1%), 6.90–8.10 (m, 18 H, DMTr,
Bz), 8.23 (s, 1 H, H-2), 8.70 (s, 1 H, H-8), 9.02
(bs, 1 H, NH) and l 0.02 (s, 3 H, Me), 0.04 (s,
H-5%a, H-5%b), 3.83 (s, 6 H, 2×OMe), 4.11 (m,
1 H, H-4%), 4.29–4.43 (m, 3 H, H-2%, H-3%,
H-5), 5.28 (dd, 1 H, J5,NH 1.5, J5,6 7.1 Hz,
H-5), 5.96 (dd, 1 H, J1%,2% 1.0, J1%,C1% 176 Hz,
H-1%), 6.80–7.72 (m, 18 H, DMTr, Bz), 7.95
(d, 1 H, H-6), 8.62 (bs, 1 H, NH). MS: m/z
765
42
(M+H)+.
Anal.
Calcd
for
13
C C1H49N3O8Si: C, 67.65; H, 6.45. Found: C,
t
3 H, Me), 0.75 (s, 9 H, Bu), 1.19 (m, 12 H,
67.72; H, 6.47.
iPr), 2.32 (t, 1 H, J 7.5 Hz, –CH2–CN), 2.63
(t, 1 H, –CH2–CN), 3.29 (m, 1 H, H-5%a),
3.54–3.75 (m, 5 H, H-5%b, P(O)CH2–, 2×
CH(iPr)), 3.78 (s, 6 H, OMe), 4.36 (m, 1 H,
H-4%), 4.41 (m, 1 H, H-3%), 5.07 (m, 1 H, H-2%),
6.06 (dd, 1 H, J1%,2% 1.0, J1%,C1% 166 Hz, H-1%),
6.90–8.10 (m, 18 H, DMTr, Bz), 8.26 (s, 1 H,
H-2), 8.68 (s, 1 H, H-8), 9.02 (bs, 1 H, NH) in
[1% - 13C] - 5% - O - Dimethoxytrityl - 2% - O - tert-
butyldimethylsilyluridine (7c).—Isolated as a
colorless foam (93%, 0.70 g). 1H NMR
(CDCl3) l 0.08 (s, 3 H, Me), 0.11 (s, 3 H, Me),
t
0.91 (s, 9 H, Bu), 3.49–3.51 (m, 2 H, H-5%a,
H-5%b), 3.80 (s, 6 H, 2×OMe), 4.12 (m, 1 H,
H-4%), 4.34–4.36 (m, 2 H, H-2%, H-3%), 5.28
(dd, 1 H, J5,NH 1.5, J5,6 8.1 Hz, H-5), 5.94 (dd,
1 H, J1%,2% 2.9, J1%,C1% 173 Hz, H-1%), 6.83–7.34
(m, 13 H, DMTr), 7.94 (dd, 1 H, J 2.2 Hz,
agreement with Lit.5 MS: m/z 990 (M+H)+;
13
52
C C1H66N7O8PSi (989.1). Due to the unstable
nature of this compound, an acceptable ele-
mental analysis could not be obtained.
H-6), 8.26 (bs, 1 H, NH). MS: m/z 663 (M+
13
H)+. Anal. Calcd for C C1H44N2O8Si: C,
35

Y. Saito et al. / Carbohydrate Research 331 (2001) 83–90
89
[1% - 13C] - 5% - O - Dimethoxytrityl - 2% - O - tert-
butyldimethylsilyl-N4-benzoylcytidine 3%-O-(2-
cyanoethyl - N,N - diisopropylphosphoramidite)
(8b).—The cytidine phosphoramidite was ob-
tained as described for 8a. From 7b (0.04 g,
0.05 mmol), phosphoramidite 8b5 (89%, 0.05
g) was obtained after silica gel column chro-
matography (55:45:3 cyclohexane–EtOAc–
Et3N). Isolated as a colorless foam (89%, 0.05
g). 1H NMR (CDCl3) l 0.15 (s, 3 H, Me), 0.24
1.16 (d, 6 H, J 6.8 Hz, Me), 2.64 (t, 2 H, J 6.1
Hz, –CH2–CN), 3.36–3.73 (m, 4 H, H-5%a,
H-5%b, 2×CH(iPr)), 3.79 (s, 6 H, 2×OMe),
3.92 (m, 2 H, –CH2–O–P), 4.22 (m, 1 H,
H-4%), 4.28–4.45 (m, 2 H, H-2%, H-3%), 5.27 (d,
1 H, J5,6 8.1 Hz, H-5), 5.89 (dd, 1 H, J1%,2% 4.9,
J1%,C1% 173 Hz, H-1%), 6.81–6.87 (m, 4 H,
DMTr), 7.24–7.42 (m, 9 H, DMTr), 7.92 (d, 1
H, H-6) in agreement with Lit.5 MS: m/z 862
13
(M+H)+; C C1H61N4O9PSi (861.2). Due to
44
t
the unstable nature of this compound, an
acceptable elemental analysis could not be
obtained.
(s, 3 H, Me), 0.93 (s, 9 H, Bu), 1.02 (d, 6 H,
J 6.7 Hz, 2×Me), 1.16 (d, 6 H, J 6.8 Hz,
2×Me), 2.58 (t, 2 H, J 5.9 Hz, –CH2–CN),
3.81 (m, 2 H, –CH2–O–P), 3.61–3.67 (m, 4
H, H-5%a, H-5%b, 2×CH(iPr)), 4.23–4.46 (m,
4 H, H-5, H-2%, H-3%, H-4%), 5.99 (dd, 1 H, J1%,2%
1.0, J1%,C1% 176 Hz, H-1%), 6.85–7.90 (m, 18 H,
DMTr, Bz), 8.58 (d, 1 H, J5,6 6.1 Hz, H-6) and
l 0.15 (s, 3 H, Me), 0.24 (s, 3 H, Me), 0.91 (s,
2. Supplementary material
All new compounds gave satisfactory spec-
tral and analytical data. Crystallographic data
for the structural analysis have been deposited
with the Cambridge Crystallographic Data
Centre, CCDC nos. 140767–140771. Copies
of this information may be obtained free of
charge from The Director, CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (fax: +44-
1223-336033; e-mail: deposit@ccdc.cam.ac.uk
or www: http://www.ccdc.cam.ac.uk).
t
9 H, Bu), 1.01 (d, 6 H, J 6.4 Hz, 2×Me),
1.12 (d, 6 H, J 6.8 Hz, 2×Me), 2.38 (t, 2 H,
J 6.1 Hz, –CH2–CN), 3.80 (m, 2 H, CH2–O–
P), 3.49–3.58 (m, 4 H, H-5%a, H-5%b, 2×
CH(iPr)), 4.23–4.46 (m, 4 H, H-5, H-2%, H-3%,
H-4%), 5.89 (dd, 1 H, J1%,2% 1.0, J1%,C1% 177 Hz,
H-1%), 6.85–7.90 (m, 18 H, DMTr, Bz), 7.85
(d, 1 H, J5,6 7.3 Hz, H-6) in agreement with
13
Lit.5 MS: m/z 965 (M)+; C C1H66N5O9PSi
51
(965.5). Due to the unstable nature of this
compound, an acceptable elemental analysis
could not be obtained.
Acknowledgements
[1% - 13C] - 5% - O - Dimethoxytrityl - 2% - O - tert-
butyldimethylsilyl-uridine 3%-O-(2-cyanoethyl-
N,N-diisopropylphosphoramidite) (8c).—The
uridine phosphoramidite was obtained as de-
scribed for 8a. From 7c (0.06 g, 0.09 mmol),
phosphoramidite 8c5 (82%, 0.06 g) was ob-
tained after silica gel column chromatography
This work was supported by a research
grant from the CNRS (Grant ACC Physique
Chimie du Vivant). We gratefully acknowl-
edge the Region Centre and the Studium for a
postdoctoral fellowship.
(60:40:3
cyclohexane–EtOAc–Et3N).
1H
NMR (CDCl3) l 0.12 (s, 3 H, Me), 0.14 (s, 3
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H, Me), 0.91 (s, 9 H, Bu), 1.02 (d, 6 H, J 6.7
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