Synthesis of 1-(5-amino-3,5-dideoxy-3-methoxycarbonylmethyl-β-D-ribofuranosyl)thymine

Article abstract of DOI:10.1016/S0008-6215(99)00057-9

In order to synthesize amide-linked antisense oligonucleotides, a general chemical approach was developed for the synthesis of building blocks in which an amino group and a carboxylic group are contained at the 5' and 3' positions of a nucleoside. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00057-9

www.elsevier.nl/locate/carres
Carbohydrate Research 317 (1999) 193–197
Note
Synthesis of
1-(5-amino-3,5-dideoxy-3-methoxycarbonylmethyl-
b-D-ribofuranosyl)thymine
Dong-Mei Lu, Ji-Mei Min, Li-He Zhang *
School of Pharmaceutical Sciences, Beijing Medical Uni6ersity, Beijing 100083, PR China
Received 4 December 1998; accepted 16 February 1999
Abstract
In order to synthesize amide-linked antisense oligonucleotides, a general chemical approach was developed for the
synthesis of building blocks in which an amino group and a carboxylic group are contained at the 5% and 3% positions
of a nucleoside. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Thymidine analogue; Oligonucleotides; Stereoselective synthesis
acid group in the same nucleoside has not
been reported until now.
Antisense oligonucleotides having amide
linkages replacing the phosphodiester linkage
have some advantages over the naturally oc-
curring oligonucleotides [1,2]. The amide unit
is compatible with conditions required for
solid-phase synthesis, and is also more stable
than the phosphodiester linkage under physio-
logical conditions. Furthermore, the lower
overall charge of oligonucleotides containing
neutral amide groups should facilitate pene-
tration of cell membranes. The amide moiety
is readily accessible and achiral.
Amide-linked dimers thus far reported,
which were inserted into oligonucleotides,
were synthesized by combining one deoxynu-
cleoside having an amino group with another
deoxynucleoside having a carboxylic acid
group. Chemical synthesis of a monomer that
has both an amino group and a carboxylic
Recently, a new type of antisense oligonu-
cleotide having a 2%-O-substituted group has
shown significant stability toward enzymes in
vivo and maintains the property of hybridiza-
tion to a target sequence [3]. We are interested
in the synthesis of oligoribonucleotides having
amide linkages, and a general chemical ap-
proach has been developed for the synthesis of
building blocks in which an amino group and
a carboxylic group are contained at the 5% and
3% positions of a nucleoside. In this paper, we
report the synthesis of such a monomer: 1-(5-
amino-3,5-dideoxy-3-methoxycarbonylmethyl-
b-D
-ribofuranosyl)thymine (10).
In order to provide generality for different
bases, -xylose was used as the starting mate-
D
rial. The synthetic route is depicted in Scheme
1.
D-Xylose was treated sequentially with ace-
* Corresponding author. Fax: +86-10-62015584.
E-mail address: zdszlh@mail.bjmu.edu.cn (L.-H. Zhang)
tone and then benzoyl chloride to give 5-O-
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00057-9

194
D.-M. Lu et al. / Carbohydrate Research 317 (1999) 193–197
Scheme 1.
Table 1
H-3 is above the furanosyl ring, and that
compound 6 has the 3-(R)-configuration.
The configuration of 6 was further confirmed
by computer-aided molecular mechanics analy-
sis. The data (Table 1) are consistent with the
NOE interpretation and support the 3-(R)-
configuration.
Deprotection of the 1,2-O-isopropylidene
group in compound 6 was efficiently achieved
by hydrolysis with 1% HCl in MeOH. Com-
pound 7 was obtained from 6 after acetylation
of the 2-OH group. Compound 7 was treated
with silylated thymidine and trimethylsilyl trifl-
uoromethane sulfonate as catalyst to afford 8.
Because of the neighboring group effect of the
2-acetyl group, 8 was obtained as the b anomer
in 75.3% yield [4,5]. For synthesis of compound
9, compound 8 was deprotected under basic
conditions, and then reacted sequentially with
TsCl and NaN3 to afford 9 in 43.7% yield in
three steps. Compound 9 was directly reduced
with hydrogen over 5% palladium–carbon cat-
alyst, and the desired compound 10 was ob-
tained in 65.2% yield (see Scheme 2).
Distances between hydrogens in optimized conformations of
the 3-(S)- and 3-(R)-isomers of 6
,
,
3-(S) (A)
3-(R) (A)
6-CH2···5-CH2 2.64, 3.72, 3.89,
4.33
3-H···5-CH2
6-CH2···2-H
1-H···6-CH2
3.62, 4.60, 5.45, 4.33
2.98, 3.98
3.78, 2.29
4.37, 2.96
3.06, 3.77
2.83, 3.53
4.90, 4.76
benzoyl-1,2-O-isopropylidene-a- -xylofuran-
D
ose (2) in 66.1% yield [4]. Oxidation of com-
pound 2 with chromium trioxide–pyridine–
acetic anhydride in dichloromethane produced
the glycos-3-ulose derivative 3 in 83.5% yield
[5].
5-O-Benzoyl-1,2-O-isopropylidene-a- -
D
erythro-pentofuranos-3-ulose (3) was con-
verted by a Wittig reaction into the isomeric
alkenes 4 and 5, which were stereoselectively
reduced to 6 by hydrogenation in presence of
5% palladium–carbon as catalyst.
The configuration of 6 was determined by 2D
NOE. The enhancement was observed between
H-3 and H-1, H-2, H-4, H-5, indicating that
Scheme 2.

D.-M. Lu et al. / Carbohydrate Research 317 (1999) 193–197
195
H-5b), 4.198 (2 H, q, J 7.0 Hz, –OCH2CH3),
1.480, 1.401 (each 3 H, s, \C(CH3)2), 1.255
(3 H, s, –OCH2CH3); FABMS: m/z 363
([M+1]+). Anal. Calcd for C19H22O7: C,
62.98; H, 6.12. Found: C, 62.77; H, 6.16.
5-O-Benzoyl-3-deoxy-3-ethoxycarbonyl-
1. Experimental
General methods.—Optical rotations were
determined with a Perkin–Elmer 243B polar-
imeter. IR spectra were recorded on a DE-
983G spectrophotometer in KBr pellets. Mass
spectra were obtained on ZAB-HS mass spec-
trometers. NMR spectra were recorded on
VXR-300, INOVA-500 or DPK-400 spec-
trometers with Me4Si as an internal standard.
Exchangeable protons were detected by addi-
tion of D2O. Column chromatography was
performed on silica gel (200–300 mesh) and
GF254 used for TLC was purchased from the
Qingdao Chemical Company, China.
methyl-1,2-O-isopropylidene-h- -ribofuranose
D
(6).—To a solution of the mixture of 4 and 5
(7.50 g, 20.7 mmol) in EtOH (100 mL), 5%
Pd–C (0.75 g) was added. The solution was
stirred under 0.4 MPa hydrogen pressure at
room temperature (rt) until TLC showed the
completion of reaction (ꢀ20 h). The mixture
was filtered to give 6 as the single product,
1
7.30 g (96.8%); [h]D +56.25° (CHCl3); H
NMR (CDCl3): l 8.050 (2 H, m, Ar-H), 7.562
(1 H, m, Ar-H), 7.437 (2 H, m, Ar-H), 5.882
(1 H, d, J 4.0 Hz, H-1), 4.839 (1 H, t, J 4.0
Hz, H-2), 4.576 (1 H, dd, J 2.5, 12.5 Hz,
H-5a), 4.356 (1 H, dd, J 5.0, 12.5 Hz, H-5b),
4.148 (3 H, m, H-4 and –OCH2CH3), 2.760 (1
H, dd, J 10.0, 17.0 Hz, H-6a), 2.507 (1 H, dd,
J 4.5, 17.0 Hz, H-6b), 2.416 (1 H, m, H-3),
1.520, 1.333 (each 3 H, s, \C(CH3)2), 1.254
(3 H, t, J 7.5 Hz, –OCH2CH3); FABMS: m/z
365 ([M+1]+), 387 ([M+Na]+). Anal.
Calcd for C19H24O7: C, 62.63; H, 6.64. Found:
C, 62.39; H, 6.27.
5-O-Benzoyl-3-(Z)-ethoxycarbonylmethyl-
ene-1,2-O-isopropylidene-h- -ribofuranose (4)
and 5-O-benzoyl-3-(E)-ethoxycarbonylmethyl-
ene-1,2-O-isopropylidene-h- -ribofuranose (5).
D
D
—To a stirred solution of 29.0 g (67 mmol) of
Wittig reagent (Ph3P+CH2CO2OEtBr−) in
150 mL of DMF, potassium tert-butoxide 5.6
g (50 mmol) and compound 3 14.32 g (50
mmol) in 50.0 mL of DMF were added at
0 °C. After 10 h, H2O (100 mL) was poured
into the solution then the organic layer was
extracted with CH2Cl2 (3×100 mL). After
drying (Na2SO4) and concentration, the
residue was applied to a column of silica gel
that was eluted with petroleum ether–EtOAc
(30:1–15:1) to give compound 4 1.325 g
(7.3%) and compound 5 10.4 g (57.6%).
Methyl 2-O-acetyl-5-O-benzoyl-3-deoxy-3-
methoxycarbonylmethyl-i- -ribofuranoside
D
(7).—Compound 6 7.30 g (20.1 mmol) was
dissolved in a solution of 1% HCl in MeOH
(100 mL). Stirring was continued for 12 h at
rt, and then pyridine (10 mL) was added to
stop the reaction. After evaporation, the
residue was dissolved in anhyd pyridine (60
mL) and Ac2O (5.0 mL) was added dropwise.
After completion of the reaction, water (50
mL) was added. The mixture was extracted
with CH2Cl2 (3×50 mL), and after drying
and concentration, the residue was applied to
a column of silica gel, and the title compound
was eluted with petroleum ether–acetone
(20:1–10:1) in a yield of 40.6% (2.98 g); [h]D
1
Compound 4: [h]D +260.7° (CHCl3); H
NMR (CDCl3): l 7.947 (2 H, m, Ar-H), 7.562
(1 H, m, Ar-H), 7.442 (2 H, m, Ar-H), 6.209
(1 H, t, J 2.0 Hz, H-1), 6.004 (1 H, d, J 5.0
Hz, H-6), 5.848 (1 H, t, J 2.0 Hz, H-2), 5.159
(1 H, m, H-4), 4.685 (1 H, dd, J 11.5, 3.0 Hz,
H-5a), 4.506 (1 H, dd, J 11.5, 2.5 Hz, H-5b),
4.206 (2 H, m, –OCH2CH3), 1.438, 1.425
(each 3 H, s, \C(CH3)2), 1.313 (3 H, t, J 7.0
Hz, –OCH2CH3); FABMS: m/z 363 ([M+
1]+). Anal. Calcd for C19H22O7: C, 62.98; H,
6.12. Found: C, 63.18; H 6.35.
1
+11.45° (CHCl3); H NMR (CDCl3): l 8.078
1
Compound 5: [h]D +143.2° (CHCl3); H
(2 H, m, Ar-H), 7.567 (1 H, m, Ar-H), 7.445
(2 H, m, Ar-H), 5.226 (1 H, d, J 4.5 Hz, H-2),
4.852 (1 H, s, H-1), 4.548 (1 H, dd, J 3.0, 12.0
Hz, H-5a), 4.316 (1 H, dd, J 5.0, 12.0 Hz,
H-5b), 4.243 (1 H, m, H-4), 3.671 (3 H, s,
–OCH3), 3.336 (3 H, s, –OCH3), 2.954 (1 H,
m, H-3), 2.614 (1 H, dd, J 8.5, 16.0 Hz, H-6a),
NMR (CDCl3): l 7.796 (2 H, m, Ar-H), 7.520
(1 H, m, Ar-H), 7.402 (2 H, m, Ar-H), 5.981
(1 H, t, J 2.0 Hz, H-6), 5.952 (1 H, d, J 4.0
Hz, H-1), 5.751 (1 H, m, J 1.5, 4.5 Hz, H-2),
5.141 (1 H, m, H-4), 4.546 (1 H, dd, J 4.0,
12.0 Hz, H-5a), 4.397 (1 H, dd, J 5.0, 12.0 Hz,

196
D.-M. Lu et al. / Carbohydrate Research 317 (1999) 193–197
DMF (10.0 mL), and the mixture was refluxed
for 12 h at 80–90 °C. The solution was cooled
and H2O (5.0 mL) was added and the organic
layer was extracted by CH2Cl2 (5×10 mL),
EtOAc (3×10 mL). After concentration, the
residue was chromatographed on silica gel and
eluted by petroleum ether–acetone (3:1–2:1)
to give compound 9 in 43.7% yield (87 mg);
IR wmax (cm−1): 3424, 3202, 3062, 2922, 2851,
2.512 (1 H, dd, J 6.0, 16.0 Hz, H-6b), 2.097 (3
H, s, OOCCH3); FABMS: m/z 334 ([M−
CH3OH]+). Anal. Calcd for C18H22O8: C,
59.01; H, 6.05. Found: C, 59.25; H, 5.98.
1-(2-O-Acetyl-5-O-benzoyl-3-deoxy-3-me-
thoxycarbonylmethyl - i -
D
- ribofuranosyl)thy-
mine (8).—A mixture of thymidine (0.15 g, 1.2
mmol) and (NH4)2SO4 (10 mg) in hexamethyl-
disilazane (HMDS) (6.0 mL) was refluxed at
125 °C to give a clear solution. The excess of
HMDS was removed by evaporation at 50–
60 °C. A solution of compound 7 (0.18 g, 0.49
mmol) in anhydrous MeCN (5.0 mL) was
added to the residue. After the addition of
trimethylsilyl trifluoromethanesulfonate (0.25
mL, 1.3 mmol), the solution was refluxed for
2.5 h. The mixture was cooled to rt, CHCl3
(20 mL) was added, and the solution was
poured into saturated NaHCO3 solution. Af-
ter stirring, filtration and concentration, the
residue was applied to a column of silica gel
and compound 8 was eluted by petroleum
ether–acetone (5:1–3:1) to give pure 8 in
75.3% yield (170 mg); [h]D +4.00° (CHCl3);
1H NMR (CDCl3): l 8.813 (1 H, br, H-3),
8.052 (2 H, d, Ar-H), 7.604 (1 H, m, Ar-H),
7.475 (2 H, m, Ar-H), 7.134 (1 H, d, J 1.0 Hz,
H-6), 5.809 (1 H, d, J 2.5 Hz, H-1%), 5.527 (1
H, dd, J 2.5, 7.0 Hz, H-2%), 4.723 (1 H, dd, J
2.0, 12.5 Hz, H-5%a), 4.491 (1 H, dd, J 4.5,
12.5 Hz, H-5%b), 4.259 (1 H, m, H-4%), 3.685 (3
H, s, –OCH3), 3.003 (1 H, m, H-3%), 2.625 (1
H, dd, J 8.5, 17.0 Hz, H-6%a), 2.536 (1 H, m,
H-6%b), 2.176 (3 H, s, OCOCH3), 1.672 (3 H,
s, 5-CH3); FABMS: m/z 461 ([M+1]+), 335
1
2106, 1785, 1689; H NMR (Me2SO-d6): l
11.454 (1 H, s, D2O exchangeable, 3-NH),
7.527 (1 H, s, H-6), 5.881 (1 H, s, H-1%), 5.215
(1 H, d, J 6.9 Hz, H-2%), 4.037 (1 H, br, H-4%),
3.709 (1 H, d, J 13.0 Hz, H-5%a), 3.476 (1 H,
dd, J 6.0, 13.2 Hz, H-5%b), 3.415–3.382 (4 H,
m, H-3% and –OCH3), 3.173 (1 H, dd, J 7.8,
17.0 Hz, H-6%a), 2.817 (1 H, dd, J 9.0, 17.0
Hz, H-6%b), 2.552 (1 H, s, D2O exchangeable,
2%-OH), 1.175 (3 H, s, 5-CH3); FABMS: m/z
340 ([M+1]+). Anal. Calcd for C13H17N5O6:
C, 46.01; H, 5.05; N, 20.63. Found: C, 45.64;
H, 5.10; N 20.05.
1 - (5 - Amino - 5,3 - dideoxy - 3 - methoxycarb-
onylmethyl-i- -ribofuranosyl)thymine (10).—
D
Compound 9 (25 mg, 0.074 mmol) was added
to MeOH (10.0 mL) which was acidified to
pH 5–6 with acetic acid. Hydrogenation was
carried out in the presence of 5% Pd–C (10
mg) as catalyst under 0.3 MPa hydrogen pres-
sure for 30 min. After filtration and concen-
tration, the residue was chromatographed by
PTLC eluted by CH2Cl2–CH3OH (10:1) to
afford the title compound (15 mg, 65.2%); [h]D
1
−11.05° (CH3OH); H NMR (Me2SO-d6): l
7.623 (1 H, s, H-6), 5.874 (1 H, s, H-1%), 5.182
(1 H, d, J 7.0 Hz, H-2%), 4.480 (2 H, br,
5%-NH2), 3.851 (1 H, m, H-4%), 3.387 (4 H,
overlapped, –OCH3 and H-5%a), 3.169 (1 H,
dd, J 7.0, 17.0 Hz, H-5%b), 3.001 (1 H, dd, J
3.5, 14.0 Hz, H-6%a), 2.921 (1 H, dd, J 7.5,
13.5 Hz, H-6%b), 2.858 (1 H, q, J 8.5 Hz, H-3%),
1.785 (3 H, s, 5-CH3); High-resolution
FABMS: m/z Calcd. for C13H20N3O6 ([M+
1]+): 314.13521; Found: 314.13465. Calcd. for
([M+1−basyl]+).
Anal.
Calcd
for
C22H24N2O9: C, 57.39; H, 5.25; N, 6.08.
Found: C, 57.55; H, 5.53; N, 5.72.
1-(5-Azido-3,5-dideoxy-3-methoxycarbonyl-
methyl-i- -ribofuranosyl)thymine (9).—Com-
D
pound 8 (270 mg, 0.58 mmol) was dissolved
into a mixture of MeOH (10.0 mL) and
K2CO3 (30 mg). The solution was stirred for
26 h at rt. Acetic acid was added dropwise to
the solution to keep the pH at 7. After con-
centration, the residue and TsCl (550 mg, 2.88
mmol) were dissolved in anhydrous pyridine
(5.0 mL). After stirring for 36 h, MeOH (3.0
mL) was added to decompose the excess of
TsCl. After concentration, the residue was
mixed with NaN3 (300 mg, 4.62 mmol) in
C13H19N3O6Na
Found: 336.11578.
([M+Na]+):
336.11716;
Acknowledgements
This project was supported by the Natural
Sciences Foundation of China.

D.-M. Lu et al. / Carbohydrate Research 317 (1999) 193–197
197
[3] T.H. Keller, R. Haner, Nucleic Acids Res., 21 (1993)
4499–4505.
References
[4] C. Mathe, G. Gosselin, M.-C. Bergogne, A.-M. Aubertin,
G. Obert, A. Kirn, J.-L. Imbach, Nucleosides Nucleotides,
14 (1995) 549–550.
[5] D.H.R. Barton, S.D. Gero, B. Quiclet-Sire, M. Samadi,
Tetrahedron Lett., 30 (1989) 4969–4972.
[1] J. Lebreton, A. Waldner, V. Fritsch, R.M. Wolf, A.D.
Mesmaeker, Tetrahedron Lett., 35 (1994) 5225–5228.
[2] A.D. Mesmaeker, A. Waldner, J. Lebreton, P. Hoffmann,
V. Fritsch, R.M. Wolf, S.M. Freier, Angew. Chem., Int.
Ed. Engl., 33 (1994) 226–229.
.