Chemical synthesis of 13C labeled anti-HIV nucleosides as mass-internal standards

Article abstract of DOI:10.1016/S0040-4020(02)01246-2

Synthesis of [¹³C₅]-labeled anti-HIV nucleosides, e.g. d4T, ddI, ddA, is described. The methodology used has been optimized due to the very high cost of the starting compound. The key step of this approach was the stereoselective dehomologation of 1,2:5,6-di-O-isopropylidene-3-oxo-α-D-glucofuranose (2) with periodic acid and sodium borohydride, which gave optically pure ribose derivative as the exclusive product. Nucleoside derivatives 6a-c were obtained from ribosylation of 5 with persilylated nucleobases under Vorbru?ggen conditions. Deoxygenation of 9a-c under Corey-Winter conditions afforded the desired labeled nucleoside analogues 12a-c.

Full text of DOI:10.1016/S0040-4020(02)01246-2

Tetrahedron 58 (2002) 9593–9603
13
Chemical synthesis of C labeled anti-HIV nucleosides as
mass-internal standards
Yoshio Saito,a Thomas A. Zevacob and Luigi A. Agrofoglioa
,
*
a
´
´
´
Institut de Chimie Organique et Analytique, CNRS UMR 6005, Universite d’Orleans, BP 6759, 45100 Orleans, France
bForschungszentrum Karlsruhe GmbH, Inst. Techn. Chemie, Bereich Chemisch-Physikalische Verfahren, D-7601 Karlsruhe, Germany
Received 5 July 2002; revised 6 September 2002; accepted 30 September 2002
Abstract—Synthesis of [13C5]-labeled anti-HIV nucleosides, e.g. d4T, ddI, ddA, is described. The methodology used has been optimized
due to the very high cost of the starting compound. The key step of this approach was the stereoselective dehomologation of 1,2:5,6-di-O-
isopropylidene-3-oxo-a-D-glucofuranose (2) with periodic acid and sodium borohydride, which gave optically pure ribose derivative as the
exclusive product. Nucleoside derivatives 6a–c were obtained from ribosylation of 5 with persilylated nucleobases under Vorbru¨ggen
conditions. Deoxygenation of 9a–c under Corey-Winter conditions afforded the desired labeled nucleoside analogues 12a–c. q 2002
Elsevier Science Ltd. All rights reserved.
1. Introduction
simultaneous MS–MS product ion analysis of both the
analyte and the internal standard, can enhance precision and
accuracy in these low level detections. Therefore, it was of
interest to synthesize the corresponding 13C mass-labeled
(Mþ5) d4T, ddA, and ddI, respectively, in anticipation of
bio-analytical analyses by mass spectrometry. We have
previously reported a preliminary account of 13C labeled
nucleoside as starting material for monomers of DNA or
RNA oligomers.11 In this paper, we report the experimental
details of those hitherto unknown 13C5 labeled d4T, ddA,
and ddI, and we discuss their NMR characteristics. Thus,
our efforts aimed at reproducible experimental procedures
giving high-yield products with respect to the isotope-
containing precursors. One of the major problems in the
chemical syntheses of 13C enriched nucleoside derivatives is
the very high cost of the starting compound that requires
optimized reactions.
Videxw or didanosine0(ddI, 20,30-dideoxyinosine) or one of
its metabolites ddA (2 ,30-dideoxyadenosine) can b0e associ-
ated with stavudine (d4T, 20,30-didehydro-3 -deoxy-
thymidine) in the treatment of HIV therapy.1 – 3
Determining the intracellular levels of a nucleoside and its
anabolites is crucial to understand its mechanism of action,
to describe its pharmacokinetic profile, and also to provide
valuable information about malabsorption, drug–drug
interactions, compliance, therapeutic drug level monitoring.
Quantitative bioanalytical chemistry has been
revolutionized in the past 10 years by the development of
liquid interfaces for tandem mass spectrometry (MS–MS),4
used to study nucleosides.5 The immense selectivity of this
technique stems for MS–MS detection. Others and us have
used that method of choice for quantification of many
nucleoside reverse transcriptase inhibitors in biological
fluid.6–9 To assess the precision (% CV), the accuracy (%
bias), and in general to validate a MS–MS method, the use
of an appropriate internal standard is necessary. Its selection
represents a critical aspect of method development because
it influences repeatability and reproducibility especially
with electrosray mass spectrometry compared to HPLC/UV.
In order to tackle this, synthetic labeled derivatives are the
best choice as they have the same ionization and
fragmentation pattern similar to those of the unlabeled
parent, but with a different molecular weight.10 A unique
mass spectrometric scanning procedure, which allowed
2. Result and discussion
The published methods recently reviewed by Lagoja et al.12a
or Milecki12b and reported by our group for uniformly 13C5-
labeled nucleosides start from the D-[13C6]glucose. Thus,
commercially D-[13C6]glucose was derived into [13C6]-
1,2:5,6-di-O-isopropylidene-D-glucofuranose (1) in 98%
yield from the consumed D-glucose (Scheme 1); the
unreacted glucose was recovered by filtration and re-used.
After treatment of 1 with PDC, the key step of this approach
was the stereoselective dehomologation13 of 1,2:5,6-di-O-
isopropylidene-3-oxo-a-D-glucofuranose (2). This ketone
was submitted through a one-pot sequential transformation
with periodic acid and sodium borohydride, to the optically
Keywords: labeled; HIV; nucleosides.
*
Corresponding author. Tel.: þ33-2-3849-4582; fax: þ33-2-3851-0728;
e-mail: luigi.agrofoglio@univ-orleans.fr
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01246-2

9594
Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
Scheme 1. Reagents: (a) ZnCl2, phosphoric acid, acetone; (b) PDC, Ac2O, CH2Cl2; (c) H5IO6, EtOAc, then NaBH4, EtOH; (d) BzCl, Py; (e) H2SO4, AcOH,
Ac2O; (f) thymine (for 6a), N 6-benzoyladenine (for 6b), or hypoxanthine (for 6c), HMDS, (NH4)2SO4, then TMSOTf, DCE; (g) NH3/MeOH.
pure ribose derivative 314 as the exclusive product in 80%
yield. The stereoselectivity of the reduction step is probably
due to the electronic effect as well as the steric hindrance of
the oxygen of the isopropylidene group, which prevents the
hydride attacking from the same side of the isopropylidene
group. After benzoylation of 3, treatment of 4 with Ac2O/
H2SO4, afforded the 1,2-di-O-acetyl-3,5-di-O-benzoyl-b-
ribo-furanose (5) after recrystallization from 2-propanol-
cyclohexane. Nucleoside derivatives 6a–c were obtained by
condensation of 5 with persilylated nucleobases (thymine,
adenine, hypoxanthine) under Vorbru¨ggen conditions.15
The deprotection of O-acyl groups from 6a–c was achieved
with NH3–MeOH. In all cases, de-acylated nucleosides
7a–c were directly used in the next reaction.
The synthesis of dideoxynucleosides (ddNs) or didehydro-
dideoxynucleosides (d4Ns) from nucleosides has been
extensively reviewed.16 It has been mainly achieved
through a radical r0eaction (Barton deoxygenation),17
a
fragmentation of 2 ,30-cyclic orthoformates (Eastwood
o0lefination)18 or through the reductive elimination of
2 (30)-acetoxy-30(20)-halogeno derivatives (Mattocks reac-
tion).19 We choose to apply a synthetic methodology based
ontheCorey–Winterreactionof20,30-cyclicthionocarbonates
Scheme 2. Reagents: (a) TBDMSCl, imidazole, DMF; (b) TCDI, DCE; (c) triethyl phosphite; (d) H2, Pd/C(10%), TEA(0.5%)–MeOH; (e) TBAF, THF.

Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
9595
Table 2. One bond 1H–13C coupling constants (in Hz, obtained from 1D 1H
NMR spectra) of 13C-labeled sugar derivatives (1–5) (in CDCl3)
Comp.
H-1
H-2
H-3
H-4
H-5a
H-5b
H-6a
H-6b
1
2
3
4
5b
185
186
185
184
182
158
161
163
148
163
153
–
oc
oc
147
161
oc
oc
165
153
161
145
oc
–
–
145
oc
158
149
149
–
–
–
149
149
–
–
–
163
153
oc—peak overlapping makes estimation of coupling constants impossible.
for the preparation of ddNs and d4Ns.20 Careful exclusion of
oxygen from the reaction mixture and use of triethyl
phosphite avoided an extensive N-methylation of the base
moiety when applied to the synthesis of d4T. Thus, after
protection of nucleosides 7a–c, the obtained 50-O-silyl
protected nucleosides 8a–c were reacted with 1,10-thio-
carbonyl diimidazole to give the thionocarbonate deriva-
tives 9a–c (Scheme 2). Deoxygenation of 9a–c under
Corey–Winter conditions20b afforded the desired
protected 20,30-didehydronucleosides 10a–c. Deprotection
of 10a gave the labeled [13C5]-d4T (12a), meanwhile the
reduction of 10b,c to 11b,c and subsequent deprotection
afforded [13C5]-ddA (12b) and [13C5]-ddI (12c),
respectively.
Assignment of 1H- and 13C NMR signals were made on the
basis of 1H, 13C, 1H–1H COSY and 1H, 13C-COSY spectra.
The main spectroscopic data are summarized in the
following tables. Tables 1 and 3 contain chemical shift
data, which are closely related to the values for the non-
labeled compounds, meanwhile the Tables 2 and 4 report
the one bond 1H–13C coupling constants for different
13C-labeled nucleosides.
Tables 5 and 6 show 13C–13C coupling constants of various
13C-labeled sugar derivatives, non-observable for the non-
labeled nucleosides. The attribution of the 13C signals for
both the non-labeled- and 13C-labeled fragments of the
molecule is straightforward.
The 1H-spectrum is at first sight a bit more interesting owing
to the 99% 13C-enrichment of the five carbon atoms of the
carbocyclic moiety, the attribution of the non-labeled part of
the molecule (adenosine, 2 protons) being unproblematic.
1
1
1
As it can be taken from the H and H, H-COSY spectra,
1
the J 13C–1H scalar coupling somewhat complicates the
signals’ attribution. An unambiguous attribution 0can be
made for the proton located at a tertiary carbon, H-1 , which
is expected to be found at lower field (Table 7).
d
The attribution can be then made successively along the
carbocyclic backbone: proton H-10shows a strong coupling
with proton H-20 at02.47 ppm which in turn exhibits a strong
coupling with H-3 at 2.11 ppm. Proton H-40 at 4.22 ppm
eventually displays a coupling with protons H-30. The most
interesting spectroscopic feature is found in the non-
equivalence of the geminal protons H-50a and H-50b of
the methylene group belonging to the hydroxylic ‘arm’ of
the carbocycle (2J¼12 Hz, AB coupling pattern) and the
absence of strong 3J coupling with the proton H-40 as it can
1
1
be taken from the H, H-COSY spectrum.

Table 3. 1H NMR chemical shifts (d) of 13C5-labeled nucleosides (6–12)
Comp.
Solvent
CDCl3
H-10
H-20
H-30
H-40
H-50a
H-50b
others
6a
6.60; 5.93
6.05; 5.43
5.84; 5.22
5.07; 4.48
4.87; 4.27
4.79; 4.20
8.22 (s, NH), 8.05–7.37 (m, Ar),
7.17 (s, H-6), 2.23 (AcO)
6.26 (6.26)
6.81; 6.22,
6.51 (6.50)
6.79; 6.23,
6.51 (6.51)
6.10; 5.43,
5.77 (5.78)
6.19; 5.54,
5.87 (5.88)
6.23; 5.59,
5.91 (5.91)
6.29; 5.61,
5.95 (5.96)
6.47; 5.81,
6.14 (6.05)
6.39; 5.72,
6.05 (6.05)
Not isolated
6.89; 6.17,
6.53 (6.52)
6.69; 6.02,
6.35 (6.52)
Not isolated
7.42; 6.76,
7.09 (7.09)
7.38; 6.71,
7.04 (7.09)
6.66; 5.99,
6.32 (6.33)
6.64; 5.96,
6.30 (6.33)
5.74 (5.72)
6.65; 6.05,
6.35 (6.31)
6.67; 6.04,
6.35 (6.33)
4.31; 3.73,
4.02 (4.05)
4.91; 4.33,
4.62 (4.60)
4.81; 4.23,
4.52 (4.52)
4.50; 3.90,
4.20 (4.22)
4.86; 4.26,
4.56 (4.55)
4.83; 4.24,
4.53 (4.55)
5.53 (5.51)
6.53; 5.95,
6.24 (6.22)
6.54; 5.97,
6.25 (6.22)
4.27; 3.67,
3.97 (3.97)
4.43; 3.84,
4.13 (4.14)
4.45; 3.86,
4.16 (4.17)
4.44; 3.85,
4.14 (4.14)
4.73; 4.13,
4.43 (4.36)
4.63; 4.04,
4.33 (4.36)
Not isolated
6.89; 6.17,
6.53 (6.52)
6.49; 5.82,
6.15 (6.15)
Not isolated
6.76; 6.05,
6.40 (6.44)
6.76; 6.08,
6.42 (6.42)
4.77 (4.77)
5.16; 4.67,
4.91 (4.92)
5.18; 4.69,
4.93 (4.92)
4.11; 3.51,
3.81 (3.83)
4.25; 3.66,
3.95 (3.96)
4.26; 3.68,
3.87 (3.98)
4.21; 3.63,
3.92 (3.93)
4.55; 3.95,
4.25 (4.13)
4.42; 3.82,
4.52 (4.50)
4.57 (4.55)
5.11; 4.58,
4.84 (4.85)
5.13; 4.55,
4.84 (4.85)
3.88; 3.32,
3.60 (3.63)
3.92; 3.32,
3.62 (3.67)
3.98; 3.38,
3.68 (3.69)
4.10; 3.55,
3.82 (3.82)
4.26; 3.72,
3.99 (3.99)
4.24; 3.68,
3.96 (3.94)
Not isolated
4.08; 3.56,
3.82 (3.81)
4.09; 3.52,
3.80 (3.80)
Not isolated
4.11; 3.54,
3.82 (3.81)
4.09; 3.52,
3.80 (3.81)
4.27; 3.71,
3.99 (4.00)
4.26; 3.70,
3.98 (4.00)
4.49 (4.48)
4.97; 4.39,
4.68 (4.69)
4.98; 4.41,
4.69 (4.69)
3.84; 3.24,
3.54 (3.55)
3.84; 3.25,
3.55 (3.55)
3.86; 3.27,
3.57 (3.59)
4.06; 3.50,
3.78 (3.80)
4.13; 3.55,
3.84 (3.86)
4.13; 3.57,
3.85 (3.86)
6b
6c
7a
7b
7c
8a
8b
8c
CDCl3
8.95 (s, H-2), 8.42 (s, H-8), 8.25–7.75 (m, Ar),
2.19 (AcO)
CDCl3
8.41 (s, H-2), 8.11 (s, H-8), 8.31–7.72 (m, Ar),
2.23 (AcO)
DMSO-d6
DMSO-d6
DMSO-d6
CDCl3
7.73 (s, H-6), 1.77 (s, CH3), 5.88 –5.76 (m, 20-OH,
30-OH), 5.13 (m 50-OH)
8.34 (s, H-2), 8.13 (s, H-8), 5.54–5.22 (m, 20-OH,
30-OH), 5.11 (t, 50-OH)
8.39 (s, H-2), 8.11 (s, H-8), 5.73–5.34 (m, 20-OH,
30-OH), 5.22 (t, 50-OH)
7.58 (s, H-6), 1.89 (s, CH3), 0.96 (s, tBuSi),
0.01 (s, SiMe2)
8.30 (s, H-2), 8.07 (s, H-8), 0.75 (s, tBuSi),
0.01 (s, SiMe2)
8.30 (s, H-2), 8.04 (s, H-8), 0.92 (s, tBuSi),
0.10 (s, SiMe2)
Not isolated
8.32 (s, H-2), 7.92 (s, H-8), 0.82 (s, tBuSi),
20.03 (s, SiMe2)
8.21 (s, H-2), 8.01 (s, H-8), 0.82 (s, tBuSi),
0.01 (s, SiMe2)
Not isolated
8.38 (s, H-2), 8.09 (s, H-8), 0.88 (s, tBuSi),
0.05 (s, SiMe2)
8.38 (s, H-2), 8.18 (s, H-8), 0.88 (s, tBuSi),
0.05 (s, SiMe2)
8.32 (s, H-2), 8.29 (s, H-8), 0.91 (s, tBuSi),
0.09 (s, SiMe2)
CDCl3
CD3OD
9a
9b
CD3OD
CDCl3
6.74; 6.04,
6.39 (6.37)
6.78; 6.19,
6.48 (6.46)
4.89; 4.28,
4.58 (4.57)
4.23; 3.65,
3.94 (3.94)
4.08; 3.56,
3.82 (3.81)
4.09; 3.52,
3.80 (3.80)
9c
10a
10b
CDCl3
CDCl3
CDCl3
CDCl3
6.39; 5.62,
6.01 (6.01)
6.41; 5.71,
6.06 (6.04)
2.71; 2.19,
2.45 (2.44)
2.79; 2.11,
2.45 (2.45)a 2.67;
1.89 2.28 (2.28)b
5.98; 5.47,
5.72 (5.83)
2.77; 2.23,
2.50 (2.51)
2.66; 2.14,
2.40 (2.43)
5.30; 4.68,
4.99 (4.97)
5.29; 4.69,
4.99 (4.97)
4.52; 3.94,
4.23 (4.25)
4.54; 3.96,
4.25 (4.25)
4.11; 3.54,
3.82 (3.81)
4.09; 3.52,
3.80 (3.81)
4.07; 3.49,
3.78 (3.77)
4.05; 3.49,
3.77 (3.77)
10c
11b
11c
2.41;1.86, 2.13 (2.13)a 2.30;
1.77 2.03 (2.03)b
2.47; 1.79, 2.13 (2.13)a 2.31;
1.62 1.96 (2.00)b
8.32 (s, H-2), 8.11 (s, H-8), 0.92 (s, tBuSi),
0.10 (s, SiMe2)
12a [13C5]-d4T
12b [13C5]-ddA
12c [13C5]-ddI
CDCl3
7.40; 6.90,
7.15 (7.05)
6.61; 5.94,
6.27 (6.28)
6.52; 5.85,
6.18 (6.20)
6.71; 6.19,
6.45 (6.35)
2.41; 1.88, 2.14 (2.15)
5.23; 4.62,
4.92 (4.93)
4.55; 3.96,
4.25 (4.26)
4.46; 3.85,
4.15 (4.15)
4.21; 3.67,
3.94 (3.94)
4.13; 3.58,
3.85 (3.86)
4.01; 3.44,
3.72 (3.74)
4.10; 3.54,
3.82 (3.81)
3.92; 3.39,
3.65 (3.65)
3.82; 3.29,
3.55 (3.57)
7.47 (s, H-6), 1.85 (s, CH3)
8.40 (s, H-2), 8.18 (s, H-8)
8.27 (s, H-2), 7.92 (s, H-8)
CD3OD
CD3OD
2.27; 1.73,
2.00 (2.03)
Observed values, average (12C value).

Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
9597
Table 4. One bond 1H–13C coupling constants (in Hz, obtained from 1D 1H NMR spectra) of 13C-labeled nucleosides (6–12)
Comp.
Solvent
H-10
H-20
H-30
H-40
H-50a
150
153
155
140
147
149
138
135
H-50b
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
10a
10b
10c
11b
CDCl3
CDCl3
CDCl3
DMSO-d6
DMSO-d6
DMSO-d6
CDCl3
167
165
163
167
162
160
169
167
167
155
156
155
144
145
145
150
149
149
155
157
158
151
147
148
147
151
149
148
149
151
148
147
145
145
149
149
148
149
150
150
148
148
140
146
139
CDCl3
CD3OD
138
Not isolated
182
167
Not isolated
168
168
Not isolated
oc
oc
Not isolated
Not isolated
144
142
Not isolated
141
141
141
140
135
140
CD3OD
CDCl3
144
147
oc
145
144
142
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
CD3OD
CD3OD
193
174
132
177
171
154
151
145
147
153
147
153
141
141
146
139
140
140
oc
166
170
123
169
134 H30a 131 H30b
11c
168 H20a oc H20b
oc H30a 171 H30b
12a [13C5]-d4T
12b [13C5]-ddA
12c [13C5]-ddI
128
134
130
130
133
134
169
142
oc—peak overlapping makes estimation of coupling constants impossible.
Table 5. 13C NMR chemical shifts (d), multiplicities and one bond 13C–13C coupling constants (Hz) of 13C-labeled sugar derivatives (1–5) (in CDCl3)
Comp.
C1
C2
C3
C4
C5
C6
1
2
3
4
5b
107.2, d, (33.5)
108.1, d, (36.9)
104.6, d, (33.8)
104.4, d, (33.2)
97.6, d, (44.5)
86.9, dd, (44.1, 33.5)
84.3–81.4, m, (oc)
82.5–80.5, m, (oc)
77.9–75.0, m, (oc)
77.5–75.1, m, (oc)
77.1, dd, (44.1, 36.8)
214.2, t, (36.3)
72.3, dd, (41.1, 40.3)
73.2, t, (41.1)
72.9, t, (41.2)
82.9, dd, (48.1, 36.8)
84.3–81.4, m, (oc)
82.5–80.5, m, (oc)
77.9–75.0, m, (oc)
77.5–75.1, m, (oc)
75.5, dd, (48.1, 34.4)
84.3–81.4, m, (oc)
61.7, d (43.3)
63.17, d, (44.4)
64.10, d, (44.3)
69.5, d, (34.4)
69.6, d, (32.7)
–
–
–
oc—peak overlapping makes estimation of coupling constants impossible.
Fig. 1 (panel A and B) represents the expanded regions of
the sugar of [13C5]-ddA in the 13C (proton decoupled) and
(off-resonance decoupled) are presented. The interpretation
of the 1H, 13C-COSY spectrum corroborates the attribution
made for the different protons.
3. Conclusion
In summary, the syntheses of hitherto unknown [13C5]-
ddA, [13C5]-ddI and [13C5]-d4T have been accomplished
from D-[13C6]-glucose. The nucleosides obtained were
fully characterized by spectroscopic methods and
In Fig. 2, a representation of [13C5]-ddA shows some
calculated and found NMR coupling constants.
especially by NMR. H and 13C NMR spectra of [13C5]-
1
nucleosides as well as other physico-chemical data are
Table 6. 13C NMR chemical shifts (d), multiplicities and one bond 13C–13C coupling constants (Hz) of 13C-labeled nucleosides (6–12)
Comp.
Solvent
C10
C20
C30
C40
C50
6a
6b
6c
7a
7b
7c
8a
8b
CDCl3
CDCl3
CDCl3
DMSO-d6
DMSO-d6
DMSO-d6
CD3OD
CDCl3
87.6, d, (42.8)
88.1, d, (42.3)
86.8, d, (42.6)
87.4, d, (43.0)
87.9, d, (42.1)
87.3, d, (42.0)
89.6, d, (43.0)
89.2, d, (41.8)
90.0, d, (42.5)
Not isolated
74.5, dd, (42.8, 38.1)
73.7, dd, (42.3, 38.5)
74.6, dd, (42.6, 37.5)
73.2, dd, (43.0, 37.5)
73.4, dd, (42.1, 37.5)
74.1, dd, (42.0, 37.5)
75.7, dd, (43.0, 38.1)
75.6, dd, (41.8, 36.9)
76.6, dd, (42.5, 37.4)
68.7, dd, (38.1, 37.5)
71.2, dd, (38.5, 36.3)
70.5, dd, (37.5, 36.3)
69.8, dd, (38.1, 37.5)
70.6, dd, (37.5, 36.0)
70.3, dd, (37.5, 36.0)
71.7, dd, (38.8, 38.1)
70.4, dd, (40.0, 36.9)
71.4, dd, (39.0, 37.4)
Not isolated
84.3, dd, (42.0, 37.5)
84.5, dd, (41.7, 36.3)
85.9, dd, (41.5, 36.3)
84.7, dd, (41.8, 38.1)
85.9, dd, (41.1, 36.0)
85.5, dd, (41.2, 36.0)
86.4, dd, (43.0, 38.8)
85.7, dd, (43.0, 40.0)
86.5, dd, (43.1, 39.0)
61.1, d, (42.0)
62.2, d, (41.7)
62.1, d, (41.5)
60.9, d, (41.8)
61.7, d, (41.1)
61.7, d, (41.2)
64.3, d, (43.0)
62.8, d, (43.0)
63.9, d, (43.1)
Not isolated
8c
9a
CD3OD
9b
9c
10a
10b
10c
11b
11c
CDCl3
CDCl3
90.2, d, (42.0)
90.4, d, (40.0)
Not isolated
89.8, d, (40.0)
88.6, d, (44.2)
86.8, d, (35.7)
85.6, d, (35.7)
89.8, d, (39.4)
87.6, d, (36.0)
87.3, d, (35.6)
88.6–86.8, m, (oc)
88.5–86.1, m, (oc)
88.6–86.8, m, (oc)
88.5–86.1, m, (oc)
Not isolated
126.3, dd, (69.0, 43.0)
125.2, dd, (69.0, 41.7)
25.7, dd, (33.3, 31.5)
25.0, dd, (33.3, 32.1)
126.2, dd, (69.6, 41.2)
26.7, dd, (35.0, 31.0)
26.3, dd, (33.5, 32.2)
88.6–86.8, m, (oc)
88.5–86.1, m, (oc)
62.2, d, (42.5)
62.5, d, (44.2)
Not isolated
66.0, d, (42.4)
64.9, d, (42.4)
65.1, d, (41.8)
64.0, d, (43.6)
63.3, d, (41.2)
64.7, d, (41.0)
64.2, d, (42.3)
CDCl3
CDCl3
CD3OD
CDCl3
CDCl3
CD3OD
CD3OD
135.7, dd, (69.0, 40.0)
135.1, dd, (69.0, 44.2)
34.0, dd, (35.7, 31.5)
33.7, dd, (35.7, 32.1)
134.7, dd, (69.6, 39.4)
33.8, dd, (36.0, 31.0)
33.9, dd, (35.6, 32.2)
89.6, dd, (43.0, 42.4)
88.1, dd, (42.4, 41.7)
83.8, dd, (41.8, 33.3)
82.4, dd, (43.6, 33.3)
87.2, dd, (41.2, 41.2)
83.7, dd, (41.0, 35.0)
83.8, dd, (42.3, 33.5)
12a [13C5]-d4T
12b [13C5]-ddA
12c [13C5]-ddI
oc—peak overlapping makes estimation of coupling constants impossible.

9598
Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
Table 7. 1H- and 13C NMR analysis of [13C5]-ddA
Location
H-10
H-20
33.8
H-30
26.7
H-40
83.7
H-50a and H-50b
64.7
dC (ppm)
1JC–C (Hz)
1JC–H (Hz)
dH (Hz)a
Mult.
87.6
0
J1 –2 : 36
169
6.25
d
0
0
0
J2 –3 : 31
0
0
J3 –4 : 35
0
0
J4 –5 : 41
134
2.47
m
133
2.11
m
147
4.22
d
–
–
140
3.82/3.29
ddd
2J (Hz)
–
–
–
–
–
0
J5 a–5 b: 12
0
3J (Hz)
0
0
0
0
J5 a–4 : 4 J5 b–4 : 7
4J (Hz)
–
0
J5 a–Cx: 2 J5 b–Cx: 3
0
The 1H- and 13C NMR spectra were recorded on a Varian InovaUnity 400 spectrometer (1H: 399.81 MHz, 13C: 100.54 MHz) in (D4)-methanol, shift values d in
ppm relative to SiMe4 as internal reference; J in Hz.
a
Average.
Figure 1. The expanded regions of the sugar moiety of [13C5]-ddA in the 13C-proton decoupled (panel A) and in the 13C-off resonance decoupled (panel B).
identical with those of authentic samples except for
the expected additional signal multiplicity arising
from 13C–1H and 13C–13C couplings, respectively.
Labeled d4T and ddI have been used as internal
standard for the validation of the quantification procedure,
on cell extract, by MS–MS. Data will be reported
elsewhere.
4. Experimental
4.1. General
Commercially available chemicals and solvents were
reagent grade and used as received. Dry tetrahydrofuran,
pyridine and dichloromethane were obtained from

Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
9599
(CDCl3) [see Table 5], others d 114.3 (C(Me)2), 111.6
(C(Me)2), 28.7 (CH3), 28.7 (CH3), 28.1 (CH3), 27.1 (CH3);
MS: m/z 267 [MþH]þ. Anal. calcd for (13C6)C6H20O6: C,
56.38; H, 7.57. Found: C, 56.41; H, 7.58.
4.1.2. [13C6]-1,2:5,6-Di-O-isopropylidene-3-oxo-a-D-glu-
cofuranose (2). To a stirred solution of protected sugar 1
(2.50 g, 9.6 mmol) in anhydrous CH2Cl2 (20 mL) were
added pyridinium dichromate (PDC, 2.17 g, 5.7 mmol) and
acetic anhydride (3 mL, 31.7 mmol), the mixture was then
refluxed for 1.5 h. The solvent was evaporated under
reduced pressure and EtOAc (50 mL) was added. The
mixture was applied to a silica gel pad and eluted with
EtOAc. The combined eluant was concentrated under
reduced pressure and co-evaporated with toluene
(2£25 mL) to give 2 as a colorless solid23 (2.33 g, 94%);
Rf 0.65 (1:9 hexanes–EtOAc); mp 127–1298C; IR (neat)
1728 cm21 (CvO); 13C NMR (CDCl3) [see Table 5], others
d 135.5 (C(Me)2), 132.6 (C(Me)2), 27.9 (CH3), 27.3 (CH3),
25.6 (CH3), 25.5 (CH3); MS: m/z 265 [MþH]þ. Anal. calcd
for (13C6)C6H18O6: C, 56.81; H, 6.86. Found: C, 56.72; H,
6.89.
Figure 2. Some calculated and found NMR coupling constants for [13C5]-
ddA. An optimal configuration for [13C5]-ddA was simulated using the
semi-empirical method AM1 (Software package Chemdraw ultra 6.0/Chem
3D Pro 5.0). Note that the assignments have been represented by Cx for
H-30a and b, D for H-40, E1 and E2 for H-50a and H-50b, respectively.
Atoms: gray¼C, pale blue¼H, dark blue¼N, red¼O.
distillation over CaH2 or Na, N,N-dimethylformamide over
BaO. The reactions were monitored by thin-layer chroma-
tography (TLC) analysis using 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 1508C. Column chromatography
was performed on Silica Gel 60 M (0.040–0.063 mm,
E. Merck). Melting point were recorded on a Bu¨chi (Dr
4.1.3. [13C5]-1,2-O-Isopropylidene-a-D-ribofuranose (3).
A solution of the dried ketonic compound 2 (2.65 g,
10 mmol) and H5IO6 (2.75 g, 12 mmol) in anhydrous
EtOAc (28 mL) was stirred at room temperature for 2 h.
The reaction mixture was filtered and the filtrate evaporated
under reduced pressure. The residue was dissolved in EtOH
(abs., 20 mL), and NaBH4 (1 g, 26.4 mmol) was added in
small portions with vigorous stirring. Stirring was continued
for 30 min then 10% acetic acid was added for neutralization,
and volatiles were evaporated. The residue was dissolved in
EtOAc, and the organic phase was washed with H2O, dried
over MgSO4 and volatiles were removed under reduced
pressure. The residue was purified by silica gel column
chromatography (9:1 CH2Cl2–MeOH) to give 314,20a,22
(1.52 g, 80%) as a colorless solid; Rf 0.35 (1:9 hexanes–
EtOAc); mp 86–878C lit.24 mp 85.5–868C; IR (neat)
3300 cm21 (HO); [a]D25¼2128 (c 2.0, CHCl3); 13C NMR
(CDCl3) [see Table 5], others d 113.5 (C(Me)2), 27.4 (CH3),
27.2 (CH3); MS: m/z 196 [MþH]þ. Anal. calcd for
(13C5)C3H14O5: C, 51.79; H, 7.23. Found: C, 51.82; H, 7.20.
1
Tottoli) and were uncorrected. H and 13C NMR spectra
were recorded on a Bruker AVANCE DPX 250 Fourier
1
Transform spectrometer at 250 MHz for H and 62.9 MHz
for 13C, respectively, using tetramethylsilane as the internal
standard; signals are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet). Mass spectra were
recorded on Perkin–Elmer SCIEX API-300 (heated neb-
ullizer) spectrometer. HRMS were performed by the
CRMPO, University of Rennes 1–Fr.
1
The characteristic H, 13C data for compounds 1–12 are
collected and shown in Tables 1–7. References in the
experimental part refer to the unlabeled homologues except
for compound 3.
4.1.1. [13C6]-1,2:5,6-Di-O-isopropylidene-a-D-glucofura-
nose (1). To a well-stirred suspension of anhydrous [13C6]-
a-D-glucose (5.0 g, 26.9 mmol) in anhydrous acetone
(50 mL) was added pulverized anhydrous zinc chloride
(4.0 g) followed by 0.25 g of 85% phosphoric acid. This
mixture was stirred 30 h at room temperature, and the
unreacted sugar was collected and washed with a little
acetone. The filtrate and washings were cooled and made
slightly alkaline with 2.5N NaOH. The insoluble inorganic
material was removed by filtration and washed with
acetone. The almost colorless filtrate and washings were
concentrated under reduced pressure and the residue was
diluted with water (10 mL), extracted with CH2Cl2
(3£50 mL), dried over MgSO4 and concentrated under
reduced pressure. Crystallization of the residue from hexane
gave the desired compound as colorless needles (4.14 g,
58%). The recovered unreacted sugar was converted to the
title compound by the same way. Combined yield gave 81%
(5.72 g) of the protected glucofuranose (1)21; Rf 0.45 (1:1
hexanes–EtOAc); [a]2D5¼2128 (c 1.0, CHCl3), lit.22
[a]D25¼2138 (c 5.0, CHCl3); mp 106–1078C; 13C NMR
4.1.4. [13C5]-3,5-Di-O-benzoyl-1,2-O-isopropylidene-a-
D-ribofuranose (4). To a stirred solution of diol 3 (1.45 g,
7.4 mmol) in anhydrous pyridine (10 mL) at 08C was added
dropwise benzoyl chloride (3.0 mL, 26.12 mmol) and the
mixture was stirred at room temperature for 3 h. The solvent
was evaporated under reduced pressure and the residue
extracted with EtOAc, washed with saturated NaHCO3, and
dried over Na2SO4. After concentration under vacuum, the
residue was purified by silica gel column chromatography
(7:3 hexanes–EtOAc) to give 4 (2.79 g, 95%) as a colorless
oil. Analytical quality was obtained by recrystallization of 4
from ether to a colorless solid; Rf 0.60 (8:2 hexanes–
EtOAc); mp 82–848C dec; [a]2D5¼þ122.48 (c 0.85, CHCl3)
[lit.26 mp 83–858C; [a]D25¼2122.48 (c 0.9, CHCl3) for the
(2)-12C-enantiomer]; 13C NMR (CDCl3) [see Table 5],
others d 166.2 (CvO), 133.5 (CH), 129.7 (quat C), 129.1
(CH), 128.3 (CH), 100.9 (C(Me)2), 26.0 (C(Me)2); MS: m/z
404 [MþH]þ. Anal. calcd for (13C5)C17H22O7: C, 66.74; H,
5.49. Found: C, 66.82; H, 5.51.

9600
Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
4.1.5. [13C5]-1,2-Di-O-acetyl-3,5-di-O-benzoyl-b-D-ribo-
furanose (5). Sulfuric acid (1.5 mL) was added dropwise to
a stirred solution of isopropylidene sugar 4 (4.23 g,
10.5 mmol) in AcOH (40 mL) and Ac2O (15 mL). After
storage overnight at room temperature, the mixture was
poured into ice-water, extracted with CHCl3, washed with
saturated NaHCO3 and dried over Na2SO4. The solvent was
removed under reduced pressure and the residue was
purified by silica gel column chromatography (7:3
hexanes–EtOAc) to give 5 (4.08 g, 87%) as needles after
recrystallization from 2-propanol-cyclohexane. Rf 0.52
(75:25 hexanes–EtOAc); mp 127–1288C, lit.25 mp 126–
1278C; 13C NMR (CDCl3) [see Table 5], others d 171.1
(CvO), 170.5 (CvO), 168.4 (CvO), 168.2 (CvO), 133.5
(CH), 130.1 (quat C), 127.8 (CH), 127.3 (CH), 21.2 (CH3),
20.9 (CH3); MS: m/z 448 [MþH]þ. Anal. calcd for
(13C5)C18H22O9: C, 62.86; H, 4.96. Found: C, 62.78; H,
5.01.
for de-O-acylation. A solution of 6a (255 mg, 0.497 mmol)
in saturated methanolic ammonia (5.0 mL) was stirred at
room temperature for 48 h. The flask was opened and left
under the hood for 5 h to allow ammonia gas to escape. The
solution was carefully evaporated, the remaining solid was
triturated with ethyl ether, leaving a colorless powder of 7a
(110 mg, 84%); Rf 0.30 (4:1 CH2Cl2–MeOH); UV (H2O)
l
max 273.0 nm; 13C NMR (DMSO-d6) [see Table 6], others
d 165.2 (CvO), 158.4 (CvO), 135.1 (C5), 108.4 (quat C),
15.9 (CH3); MS: m/z 264 [MþH]þ. Anal. calcd for
(13C5)C5H14N2O6·0.5H2O: C, 45.96; H, 5.55; N, 10.29.
Found: C, 45.94; H, 5.58; N, 10.32.
4.1.10. D-[13C5]-Adenosine (7b). A solution of 6b (202 mg,
0.323 mmol) in saturated methanolic ammonia (7.5 mL)
was stirred at room temperature for 48 h. After concen-
tration, 7b was obtained as a colorless solid (98.6 mg,
.98%); Rf 0.10 (10:1 CH2Cl2–MeOH); UV (H2O) lmax
261.0 nm; 13C NMR (DMSO-d6) [see Table 6], others d
156.8 (quat C), 153.5 (C2), 149.8 (quat C), 142.5 (C8),
120.5 (quat C); MS: m/z 273 [MþH]þ. Anal. calcd for
(13C5)C5H13N5O4·0.3H2O: C, 45.89; H, 4.93; N, 25.23.
Found: C, 45.93; H, 4.85; N, 25.26.
4.1.6. [13C5]-1-(2-O-Acetyl-3,5-di-O-benzoyl-b-D-ribo-
furanosyl)-5-methyluracil (6a). General Procedure for
coupling. Dry thymine (704 mg, 5.60 mmol) and
(NH4)2SO4 (0.1 g) were stirred in refluxing HMDS
(30 mL) under nitrogen until homogeneous solution and
was then evaporated under vacuum to give the silylated
thymine. A solution of protected sugar 5 (1.0 g, 2.24 mmol)
in anhydrous dichloroethane (DCE, 8 mL) was added to the
silylated thymine, followed by the dropwise addition of
TMSOTf (1.05 mL, 5.43 mmol) and the resulting solution
was refluxed for 3.5 h. It was quenched with saturated
NaHCO3, extracted with CH2Cl2 (2£50 mL), washed
successively with saturated NaHCO3 and brine, dried over
Na2SO4. The solvent was removed under reduced pressure
and the residue was purified by silica gel column
chromatography (20:1 CH2Cl2–MeOH) to yield 6a
(1.15 g, 89%) as a colorless oil. Rf 0.53 (10:1 CH2Cl2–
MeOH); [a]2D5¼þ328 (c 1.0, CHCl3); MS: m/z 514
[MþH]þ. Anal. calcd for (13C5)C21H24N2O9: C, 61.80; H,
4.71; N, 5.45. Found: C, 62.11; H, 4.68; N, 5.73.
4.1.11. D-[13C5]-Inosine (7c). A solution of 6c (175 mg,
0.334 mmol) in saturated methanolic ammonia (5.0 mL)
was stirred at room temperature for 48 h. After concen-
tration, 7c was obtained as a colorless solid (93.6 mg,
.98%); Rf 0.10 (10:1 CH2Cl2–MeOH); UV (H2O) lmax
250.0 nm; 13C NMR (DMSO-d6) [see Table 6], others d
156.9 (CvO), 148.5 (quat C), 147.3 (C2), 139.1 (C8), 124.8
(quat C); MS: m/z 274 [MþH]þ. Anal. calcd for
(13C5)C5H12N4O5: C, 45.79; H, 4.43; N, 20.50. Found: C,
45.82; H, 4.38; N, 20.63.
4.1.12. D-[13C5]-5-Methyl 50-O-tert-butyldimethylsilyl-
uridine (8a). To a solution of 7a (580 mg, 1.54 mmol)
and imidazole (34.2 mg, 1.05 mmol) in anhydrous DMF
(2.0 mL) at 08C was added tert-butyldimethylsilyl chloride
(75.9 mg, 0.503 mmol) with stirring. The mixture was
warmed to room temperature and subsequently stirred for
17 h. After evaporation of the solvent, the residue was
purified by silica gel column chromatography (10:1
CH2Cl2–MeOH) to give 8a20b (103 mg, 76%) as a colorless
waxy solid. Rf 0.53 (10:1 CH2Cl2–MeOH); 13C NMR
(CDCl3) [see Table 6], others d 166.3 (CvO), 152.6
(CvO), 137.4 (C6), 111.5 (quat C), 26.5 ((CH3)3CSi), 19.3
(CH3), 12.6 (CH3Si), 25.2 (CH3Si); MS: m/z 378 [MþH]þ.
4.1.13. D-[13C5]-50-O-tert-Butyldimethylsilyl adenosine
(8b). To a solution of 7b (87.9 mg, 0.323 mmol) and
imidazole (52.9 mg, 0.777 mmol) in anhydrous DMF
(2.0 mL) at 08C was added tert-butyldimethylsilyl-chloride
(58.4 mg, 0.388 mmol) with stirring. The mixture was
warmed to room temperature and subsequently stirred for
14 h. After evaporation of the solvent, the residue was
purified by silica gel column chromatography (5:1 CH2Cl2–
MeOH) to give 8b27 (105 mg, 85%) as a colorless solid; Rf
0.30 (15:1 CH2Cl2–MeOH); mp 184–1868C, lit.27 mp
185–1868C; 13C NMR (CDCl3) [see Table 6], others d
156.9 (quat C), 154.0 (C2), 150.8 (quat C), 140.3 (C8),
121.0 (quat C), 27.2 ((CH3)3CSi), 10.8 (CH3Si), 24.7
(CH3Si); MS: m/z 387 [MþH]þ.
4.1.7. [13C5]-1-(2-O-Acetyl-3,5-di-O-benzoyl-b-D-ribo-
furanosyl)adenine (6b).
A mixture of 5 (690 mg,
1.54 mmol), silylated adenine (1.44 g, 4.63 mmol) and
TMSOTf (725 mL, 3.75 mmol) in DCE (10 mL) was
refluxed for 3.5 h under Ar. After workup and silica gel
column chromatography (20:1 CH2Cl2–MeOH), 6b was
obtained as a colorless oil (655 mg, 68%). Rf 0.62 (20:1
CH2Cl2–MeOH); MS: m/z 523 [MþH]þ. Anal. calcd for
(13C5)C21H23N5O7: C, 60.72; H, 4.43; N, 13.40. Found: C,
60.84; H, 4.31; N, 13.75.
4.1.8. [13C5]-1-(2-O-Acetyl-3,5-di-O-benzoyl-b-D-ribo-
furanosyl)hypoxanthine (6c). A mixture of 5 (285 mg,
0.638 mmol), silylated hypoxanthine (448 mg, 1.59 mmol)
and TMSOTf (309 mL, 1.59 mmol) in DCE (7.0 mL) was
refluxed for 2.5 h under Ar. After workup and silica gel
column chromatography (15:1 CH2Cl2–MeOH), 6c was
obtained as a colorless solid (288 mg, 86%). Rf 0.61 (15:1
CH2Cl2–MeOH); MS: m/z 523 [MþH]þ. Anal. calcd for
(13C5)C21H22N4O8: C, 60.61; H, 4.23; N, 10.70. Found: C,
60.83; H, 4.18; N, 11.05.
4.1.9. D-[13C5]-5-Methyluridine (7a). General procedure

Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
9601
4.1.14. D-[13C5]-50-O-tert-Butyldimethylsilyl inosine (8c).
To a solution of inosine 7c (87.9 mg, 0.323 mmol) and
imidazole (52.9 mg, 0.777 mmol) in anhydrous DMF
(2.0 mL) at 08C was added tert-butyldimethylsilyl
chloride (58.4 mg, 0.388 mmol) with stirring. The
mixture was warmed to room temperature and subsequently
stirred for 14 h. After evaporation of the solvent, the residue
was purified by silica gel column chromatography (5:1
CH2Cl2–MeOH) to give 8c20b (105 mg, 85%) as a colorless
waxy solid; Rf 0.35 (15:1 CH2Cl2–MeOH); 13C NMR
(CD3OD) [see Table 6], others d 152.3 (CvO), 148.9 (quat
C), 143.9 (C2), 137.3 (C8), 125.7 (quat C), 23.2
((CH3)3CSi), 13.3 (CH3Si), 23.8 (CH3Si); ESIMS m/z
388 [MþH]þ.
4.1.19. D-[13C5]-(20,30-Didehydro-20,30-dideoxy-50-O-tert-
butyldimethylsilyladenosine (10b). A solution of 9b
(208 mg, 0.488 mmol) in triethyl phosphite (6.0 mL) was
refluxed for 3 h. After completion of the reaction, the excess
triethyl phosphite was removed in vacuo. The residue was
purified by silica gel column chromatography (30:1
CH2Cl2–MeOH) to afford 10b20b (145 mg, 85%) as a
colorless solid; Rf 0.49 (8:1 CH2Cl2–MeOH); mp 118–
1208C, lit.20b mp 117–1218C; 13C NMR (CDCl3) [see Table
6], others d 153.7 (C2), 142.5 (C8), 133.2 (quat C), 26.2
((CH3)3CSi), 12.3 (CH3Si), 24.9 (CH3Si); MS: m/z 353
[MþH]þ.
4.1.20. D-[13C5]-(20,30-Didehydro-20,30-dideoxy-50-O-tert-
butyldimethylsilylinosine (10c).
A
solution of 9c
4.1.15. D-[13C5]-5-Methyl-20,30-O-thiocarbonylene-50-O-
tert-butyldimethylsilyluridine (9a). A mixture of 8a
(103 mg, 0.273 mmol) and 1,10-thiocarbonyl diimidazole
(72.9 mg, 0.409 mmol) in 1,2-dichloroethane (4.0 mL) was
refluxed for 3 h. After evaporation of the solvent, the residue
was purified by silica gel column chromatography (20:1
CH2Cl2–MeOH) to afford 9a20b (80.0 mg, 70%) as a
colorless glassy material. This product was engaged directly
into the next step without any further characterization. Rf
0.70 (10:1 CH2Cl2–MeOH).
(63.3 mg, 0.148 mmol) in triethyl phosphite (4.5 mL) was
refluxed for 3 h. After completion of the reaction, the excess
triethyl phosphite was removed in vacuo. The residue was
purified by silica gel column chromatography (20:1
CH2Cl2–MeOH) to afford 10c20b (30.1 mg, 57%) as a
colorless waxy solid; Rf 0.38 (10:1 CH2Cl2–MeOH); 13C
NMR (CDCl3) [seeTable 6], others d 157.4 (CvO), 146.9
(C2), 143.1 (C8), 24.1 ((CH3)3CSi), 13.8 (CH3Si), 24.9
(CH3Si); MS: m/z 354 [MþH]þ.
4.1.21. D-[13C5]-20,30-Dideoxy-50-O-tert-butyldimethyl-
silyladenosine (11b). A mixture of 10b (145 mg,
4.1.16. D-[13C5]-20,30-O-Thiocarbonylene-50-O-tert-butyl-
dimethylsilyl adenosine (9b). A mixture of 8b (193 mg,
0.50 mmol) and 1,10-thiocarbonyl diimidazole (178 mg,
1.00 mmol) in 1,2-dichloroethane (5.5 mL) was refluxed
for 3 h. After evaporation of the solvent, the residue was
purified by silica gel column chromatography (10:1
CH2Cl2–MeOH) to afford 9b20b (208 mg, 97%) as a
colorless solid; Rf 0.37 (15:1 CH2Cl2–MeOH); mp 198–
2008C, lit.20b mp 198–1998C; 13C NMR (CD3OD) [see
Table 6], others d 189.9 (CvS), 157.7 (quat C), 152.3 (C2),
148.3 (quat C), 140.1 (C8), 118.9 (quat C), 24.7
((CH3)3CSi), 17.3 (CH3Si), 23.8 (CH3Si); MS: m/z 429
[MþH]þ.
0.415 mmol) and 10% Pd/C (15.0 mg) in 0.5% triethyl-
amine–MeOH (15 mL) was stirred under hydrogen atmos-
phere for 3 h. The reaction mixture was filtered through a
celite pad and washed with MeOH (10 mL£2), the filtrate
and the washings were combined. After evaporation of the
solvent, the residue was purified by silica gel column
chromatography (10:1 CH2Cl2–MeOH) to afford 11b
(141 mg, 97%) as a colorless waxy solid; Rf 0.71 (5:1
CH2Cl2–MeOH); 13C NMR (MeOD-d3) [see Table 6],
others d 156.9 (CvO), 154.2 (C2), 140.7 (quat C), 137.2
(C8), 118.6 (quat C), 22.3 ((CH3)3CSi), 13.5 (CH3Si), 23.7
(CH3Si); MS m/z 355 [MþH]þ.
4.1.17. D-[13C5]-20,30-O-Thiocarbonylene-50-O-tert-butyl-
dimethylsilyl inosine (9c). A mixture of 8c (78.2 mg,
0.202 mmol) and 1,10-thiocarbonyl diimidazole (54.0 mg,
0.303 mmol) in 1,2-dichloroethane (5.0 mL) was refluxed
for 3 h. After evaporation of the solvent, the residue was
purified by silica gel column chromatography (20:1
CH2Cl2–MeOH) to afford 9c20b (74.5 mg, 86%) as a
colorless waxy solid; Rf 0.47 (15:1 CH2Cl2–MeOH); 13C
NMR (CDCl3) [see Table 6], others d 190 (CvS), 159.4
(CvO), 155.8 (quat C), 148.7 (quat C), 146.6 (C2), 138.3
(C8), 126.2 (quat C), 26.5 ((CH3)3CSi), 12.6 (CH3Si), 25.2
(CH3Si); MS: m/z 430 [MþH]þ.
4.1.18. D-[13C5]-(20,30-Didehydro-20,30-dideoxy-50-O-tert-
butyldimethylsilylthymidine (10a). A solution of 9a
(74.2 mg, 0.177 mmol) in triethyl phosphite (3.0 mL) was
refluxed for 3 h. After completion of the reaction, the excess
triethyl phosphite was removed in vacuo. The residue was
purified by silica gel column chromatography (10:1
CH2Cl2–MeOH) to afford 10a20b (31.7 mg, 52%) as a
colorless waxy solid. This product was engaged directly into
the next step without any further characterization. Rf 0.47
(8:1 CH2Cl2–MeOH).
4.1.22. D-[13C5]-20,30-Dideoxy-50-O-tert-butyldimethyl-
silylinosine (11c).
A
mixture of 10c (29.9 mg,
0.0846 mmol) and 10% Pd/C (10.0 mg) in 0.5% triethyl-
amine–MeOH (7.5 mL) was stirred under hydrogen
atmosphere for 3 h. The reaction mixture was filtered
through a celite pad and washed with MeOH (10 mL£2), the
filtrate and the washings were combined. After evaporation
of the solvent, the residue was purified by silica gel column
chromatography (10:1 CH2Cl2–MeOH) to afford 11c
(28.4 mg, 95%) as a colorless waxy solid; Rf 0.35 (10:1
CH2Cl2–MeOH); 13C NMR (CDCl3) [see Table 6], others d
161.8 (CvO), 149.7 (quat C), 147.2 (C2), 141.4 (C8), 126.2
(quat C); 23.4 ((CH3)3CSi), 11.9 (CH3Si), 25.1 (CH3Si);
MS: m/z 356 [MþH]þ.
4.1.23. D-[13C5]-20,30-Didehydro-20,30-dideoxythymidine
(d4T, 12a). To a solution of 10a (31.7 mg, 0.092 mmol)
in THF (5.0 mL) at 08C was added TBAF (1.0 M solution in
THF, 184 mL, 0.184 mmol) with stirring. The mixture was
warmed to room temperature and subsequently stirred for
40 min. After evaporation of the solvent, the residue was
purified by silica gel column chromatography (10:1
CH2Cl2–MeOH) to give 12a28 (15.0 mg, 71%) as a

9602
Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
Benech, H. Rapid Commun. Mass Spectrom. 2001, 15,
1401–1408. (b) Becher, F.; Pruvost, A.; Goujard, C.;
Guerreiro, C.; Delfraissy, J.-F.; Grassi, J.; Benech, H. Rapid
Commun. Mass Spectrom. 2002, 16, 555–565.
colorless powder; Rf 0.23 (10:1 CH2Cl2–MeOH); mp 164–
1668C (lit.28 mp 165–1668C); 13C NMR (CDCl3) [see Table
6], others d 163.9 (CvO), 150.6 (CvO), 136.8 (C6), 110.7
(quat C), 12.3 (CH3); HRESIMS m/z 252.0863 calcd for
(13C5)C5H12N2O4Na [MþNa]þ, found 252.0865.
4.1.24. D-[13C5]-20,30-Dideoxyadenosine (ddA, 12b). To a
solution of 11b (183 mg, 0.52 mmol) in THF (15 mL) at 08C
was added TBAF (1.0 M solution in THF, 1.04 mL,
1.04 mmol) with stirring. The mixture was warmed to
room temperature and subsequently stirred for 40 min. After
evaporation of the solvent, the residue was purified by silica
gel column chromatography (10:1 CH2Cl2–MeOH) to give
12b29 (118 mg, 95%) as a colorless powder; Rf 0.57 (5:1
CH2Cl2–MeOH); mp 186–1888C, lit.29 mp 185–1878C;
13C NMR (CD3OD) [see Table 6], others d 157.3 (quat C),
153.5 (C2), 149.8 (quat C), 141.0 (C8), 120.5 (quat C);
HRESIMS m/z 263.1135 calcd for (13C5)C5H13N5O2Na
[MþNa]þ, found 263.1134; Rf 0.57 (5:1 CH2Cl2–MeOH).
7. (a) Font, E.; Rosario, O.; Santana, J.; Garcia, H.; Sommadossi,
J.-P.; Rodriguez, J. F. Antimicrob. Agents Chemother. 1999,
43, 2964–2968. (b) Rodriguez, J. F.; Rodriguez, J. L.;
Santana, J.; Garcia, H.; Rosario, O. Antimicrob. Agents
Chemother. 2000, 44, 3097–3100.
8. (a) Cahours, X.; Dessans, H.; Morin, Ph.; Dreux, M.;
Agrofoglio, L. A. J. Chromatogr., A 2000, 895, 101–109.
(b) Cahours, X.; Tran, T. T.; Mesplet, N.; Kieda, C.; Morin,
Ph.; Agrofoglio, L. A. J. Pharm. Biomed. Anal. 2001, 26,
819–827. (c) Cahours, X.; Morin, Ph.; Dessans, H.;
Agrofoglio, L. A. Electrophoresis 2002, 23, 88–92.
9. (a) St Claire, R. L. Rapid Commun. Mass Spectrom. 2000, 14,
1625–1634. (b) Fung, E. N.; Cai, Z.; Burnette, T. C.;
Sinhababu, A. K. J. Chromatogr., B: Biomed. Sci. Appl.
2001, 754, 285–295.
10. (a) La Franc¸ois, C. J.; Fujimoto, J.; Sowers, L. C. Chem. Res.
Toxicol. 1998, 11, 75–83. (b) Dizdaroglu, M. FEBS Lett.
1993, 315, 1–6. (c) Hamberg, M.; Zhang, L. Y. Anal.
Biochem. 1995, 229, 336–344. (d) Polson, A. G.; Crain, P. F.;
Pomerantz, S. C.; McCloshey, J. A.; Bass, B. L. Biochemistry
1991, 30, 11507–11514. (e) Schwandt, H. J.; Wu, A. T.; Finn,
C.; Sadee, W. Nucleic Acid Chem. 1978, 2, 901–915.
11. Agrofoglio, L. A.; Jacquinet, J.-C.; Lancelot, G. Tetrahedron
Lett. 1997, 38, 1411–1412.
4.1.25. D-[13C5]-20,30-Dideoxyinosine (ddI, 12c). To a
solution of 11c (27.1 mg, 0.0763 mmol) in THF (4.5 mL)
at 08C was added TBAF (1.0 M solution in THF, 153 mL,
0.153 mmol) with stirring. The mixture was warmed to
room temperature and subsequently stirred for 40 min. After
evaporation of the solvent, the residue was purified by silica
gel column chromatography (6:1 CH2Cl2–MeOH) to give
12c30 (15.4 mg, 84%) as a colorless powder; Rf 0.53 (5:1
CH2Cl2–MeOH); mp 183–1858C, lit.30 mp 184–1868C;
13C NMR (CD3OD) [see Table 6], others d 156.7 (CvO),
148.3 (quat C), 144.3 (C2), 138.1 (C8), 123.5 (quat C);
HRESIMS m/z 264.0975 calcd for (13C5)C5H12N4O3Na
[MþNa]þ, found 264.0973.
12. (a) Lagoja, I. M.; Herdewijn, P. Synthesis 2002, 3, 301–314,
and references cited therein. (b) Milecki, J. J. Label. Compd.
Radiopharm. 2002, 45, 307–337, and references cited therein.
13. Xie, M.; Berges, D. A.; Robins, M. J. Org. Chem. 1996, 61,
5178–5179.
¨
14. Milecki, J.; Zamaratski, E.; Maltseva, T. V.; Foldesi, A.;
Adamiak, R. W.; Chattopadhyaya, J. Tetrahedron 1999, 55,
6603–6622.
Acknowledgements
15. Niedballa, U.; Vorbru¨ggen, H. Angew. Chem. Int. Ed. Engl.
1970, 9, 461–462.
This work was supported in part grant by the Agence
Nationale de Recherches sur le SIDA (ANRS), and by the
SIDACTION-ECS.
16. Huryn, D. M.; Okabe, M. Chem. Rev. 1992, 92, 1745–1768,
and references cited therein.
17. (a) Prisbe, E. J.; Martin, J. C. Synth. Commun. 1985, 15,
401–409. (b) Kim, C.-H.; Marquez, V. E.; Broder, S.;
Mitsuya, H.; Driscoll, J. S. J. Med. Chem. 1987, 30,
862–866. (c) Herdewijn, P.; Balzarini, J.; De Clercq, E.;
Pauwels, R.; Baba, M.; Broder, S.; Vanderhaeghe, H. J. Med.
Chem. 1987, 30, 1270–1278. (d) Sekine, M.; Nakanishi, T.
J. Org. Chem. 1990, 55, 924–928.
References
1. Furman, P. A.; Fyfe, J. A.; St. Clair, M. H.; Weinhold, K.;
Rideout, J. L.; Freeman, G. A.; Nusinoff Lehrman, S.;
Bolegnesi, D. P.; Broder, S.; Mitsuya, H.; Barry, D. W.
Proc. Natl Acad. Sci. USA 1986, 83, 8333–8337.
18. (a) Crank, G.; Eastwood, F. W. Aust. J. Chem. 1964, 17,
1392–1398. (b) Josan, J. S.; Eastwood, F. W. Aust. J. Chem.
1968, 21, 2013–2020.
2. Preey, C. M.; Noble, S. Drugs 1999, 58, 1099–1135, and
references cited therein.
19. Mattocks, A. R. J. Chem. Soc. 1964, 1918–1930.
20. (a) Corey, E. J.; Winter, R. A. E. J. Am. Chem. Soc. 1963, 85,
2677–2678. (b) Chu, C. K.; Bhadti, V. S.; Doboszewski, B.;
Gu, Z. P.; Kosugi, Y.; Pullaiah, K. C.; Van Roey, P. J. Org.
Chem. 1989, 54, 2217–2225.
3. Molina, J.-M.; Chene, G.; Ferchal, F.; Journot, V.; Pellegrin,
I.; Sombardier, M.-N.; Rancinan, C.; Cotte, L.; Madelaine, I.;
Debord, T.; Decazes, J.-M. J. Inf. Dis. 1999, 180, 351–358.
4. (a) Gelpi, E. J. Chromatogr., A 1995, 703, 59–80. (b) Maurer,
H. H. J. Chromatogr., B: Biomed. Sci. Appl. 1998, 713, 3–25.
5. (a) Font, E.; Lasanta, S.; Rosario, O.; Rodriguez, J. F.
Nucleosides Nucleotides 1998, 17, 845–853. (b) Jajoo, H. K.;
Bennett, S. M.; Kornhauser, D. M. J. Chromatogr. 1992, 577,
¨
21. (a) Quant, S.; Wechselberger, R. W.; Wolter, M. A.; Worner,
H.-H.; Schell, P.; Engels, J. W.; Griesinger, C.; Schwalbe, H.
Tetrahedron Lett. 1994, 35, 6649–6652. (b) Glen, W. L.;
Myers, G. S.; Grant, G. R. J. Chem. Soc. 1951, 2568–2572.
(c) Stevens, J. D. Methods Carbohydr. Chem. 1972, 6,
123–128.
`
299–304. (c) Esmans, E. L.; Broes, D.; Hoes, I.; Lemiere, F.;
Vanhoutte, K. J. Chromatogr., A 1998, 794, 109–127.
6. (a) Pruvost, A.; Becher, F.; Bardouille, P.; Guerrero, C.;
Creminon, C.; Delfraissy, J.-F.; Goujard, C.; Grassi, J.;
22. Murray, D. H.; Prokop, J. In Synthetic Procedures in Nucleic

Y. Saito et al. / Tetrahedron 58 (2002) 9593–9603
9603
Acid Chemistry. Zorbach, W., Tipson, S., Eds.; Interscience:
New York, 1968; p 193.
27. (a) Beaton, G.; Jones, A. S.; Walker, R. T. Tetrahedron 1988,
44, 6419–6428. (b) Robins, M. J.; Lewandowska, E.; Wnuk,
S. F. J. Org. Chem. 1988, 63, 7375–7381.
23. Baker, D. C.; Horton, D. R.; Tindall, C. G. Carbohydr. Res.
1972, 24, 192–197.
28. Mansuri, M. M.; Starrett, J. E.; Ghazzouli, I.; Hitchcock, M. J.;
Sterzycki, R. Z.; Brankovan, V.; Lin, T.-S.; August, E. M.;
Prusoff, W. H.; Sommadossi, J.-P.; Martic, J. C. J. Med. Chem.
1989, 32, 461–466.
24. Sinhababu, A. K.; Bartel, R. L.; Pochopin, N.; Borchardt, R. T.
J. Am. Chem. Soc. 1985, 107, 7628–7632.
25. Tsutsumi, H.; Kawai, Y.; Ishido, Y. Carbohydr. Res. 1979, 73,
293–296.
29. Robins, M. J.; Hansske, F.; Low, N. H.; Park, J. I. Tetrahedron
Lett. 1984, 25, 367–370.
26. Ma, T.; Pai, S. B.; Zhu, Y. L.; Lin, J. S.; Shanmuganathan, K.;
Du, J.; Wang, C.; Kim, H.; Newton, M. G.; Cheng, Y. C.; Chu,
C. K. J. Med. Chem. 1996, 39, 2835–2843.
30. Saito, I.; Ikehira, H.; Kasatami, R.; Watanabe, M.; Matsuura,
T. J. Am. Chem. Soc. 1986, 108, 3115–3117.