N. Ouwerkerk, J. H. v. Boom, J. Lugtenburg, J. Raap
FULL PAPER
40 °C. Work-up involved the addition of 25 mL of water and 30 mL
of diethyl ether to give two layers. The aqueous layer was separated
from the organic layer and the latter was washed sequentially with
water, dilute aq. sulfuric acid, and 1 NaHCO3 solution. Drying
with MgSO4, filtration, and evaporation of the solvent gave 10 as
Acknowledgments
The authors wish to thank Rob Schoevaart of the TU Delft for a
generous donation of the DERA overproducing organism and for
his continued support with regard to the enzymatic transforma-
tions. Furthermore, we are indebted to Jaap Brouwer and especially
Martina de Ruijter for their help with the overproduction and
isolation of enzymes. We are also grateful to Bertil Hofte for re-
cording the mass spectra, and to Fons Lefeber and Cees Erkelens
for their help in acquiring and analyzing the NMR spectra.
a slightly yellow oil (0.8 g, quant).[14]
–
13C NMR (CDCl3): δ (α,β
1
anomers) ϭ 81.8 and 80.9 (2 d, JC-C ϭ 38.8 and 38.9 Hz), 75.4
1
and 74.6 (2 d, JC-C ϭ 38.9 and 38.9 Hz).
[3,4-13C2]1-Chloro-3,5-toluoyl-2-deoxyribose (11): Glacial acetic
acid (6 mL) was saturated with HCl at 0 °C and then a solution of
10 (1.9 g, 4.9 mmol) in glacial acetic acid (3 mL) was added. A
stream of HCl was continuously passed through this mixture and
after 10 min. white crystals separated. This solid was collected by
filtration to give 11 (720 mg, 1.8 mmol, 39%) as a white powder;
m.p. 110 °C (ref.[14] 109 °C). – 1H NMR (CDCl3): δ ϭ 7.95 (m,
4 H, arom. H), 7.25 (m, 4 H, arom. H), 6.47 (ddd, J ϭ 5.0, J ϭ
[1]
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[2]
1
[3]
7.5 Hz, 1 H, 1-H), 5.56 (m, JC-H ϭ 148.5 Hz, 1 H, 3-H), 4.86 (m,
X.-P. Xu, A. K. Chiu, A.-y. Wing-Lok, C. F. Steve, J. Am.
1JC-H ϭ 148.8 Hz, 1 H, 4-H), 4.68 (m, 1 H, 5’-H), 4.57 (m, 1 H, 5’’-
Chem. Soc. 1998, 120, 4230–4231.
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S. A. M. Nieuwenhuis, R. J. Hulsebosch, J. Raap, J. Lugten-
H), 2.86 (m, 1 H, 2-H), 2.74 (m, 1 H, 2’-H), 2.42 (s, 3 H, CH3),
1
burg, A. J. Hoff, J. Am. Chem. Soc. 1998, 120, 829–830.
2.41 (s, 3 H, CH3). – 13C NMR (CDCl3): δ ϭ 84.7 (d, JC-C
38.6 Hz), 73.5 (d, JC-C ϭ 38.6 Hz).
ϭ
[5]
Y. Yamakazi, M. Hatanaka, H. Kandori, J. Sasaki, W. F. J.
1
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[3’,4’-13C2]3’,5’-Di-O-toluoylthymidine (12): To a suspension of
thymine (0.45 g, 3.59 mmol) in 20 mL of hexamethyldisilazane
(HMDS) was added one drop of TMSCl and the mixture was re-
fluxed for 6 h. The HMDS was then distilled off from the clear,
colourless solution under reduced pressure to leave a colourless res-
idue. This silylated thymine was then redissolved in 30 mL of dry,
freshly distilled CHCl3 and the solid α-chloro sugar 11 was added
in a single portion. The resulting solution turned a little cloudy in
the course of 2 h, after which 0.2 mL of methanol was added and
the excess thymine was filtered off. The solvent was removed in
vacuo and the residue was recrystallized from EtOAc to yield
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700 mg (1.45 mmol, 80%) of 12 as a white solid; m.p. 196 °C.[18]
1H NMR (CDCl3): δ ϭ 8.82 (s, 1 H, 3-H), 7.94 (m, 4 H, arom. H),
–
´
Sandström, M. Eden, H. Maisel, A. Sebald, M. H. Levitt,
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3
7.28 (m, 5 H, arom. H and 6-H), 6.47 (dd, JC-H ϭ 8.9, JC-H
ϭ
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N. Bischofberger, H. Waldmann, T. Saito, E. S. Simon, W. Lees,
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3
5.5 Hz, 1 H, 1’-H), 5.63 (dd, JC-H ϭ 158.8, JC-H ϭ 5.6 Hz, 1 H,
3’-H), 4.78 (m, 1 H, 5’-H), 4.65 (m, 1 H, 5’’-H), 4.53 (m, 1 H, 4’-
H), 2.70 (m, 1 H, 2’-H), 2.44 (s, 3 H, CH3), 2.43 (s, 3 H, CH3). 2.31
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(m, 1 H, 2’’-H), 1.61 (d, JC-H ϭ 1.2 Hz, 3 H, 5-H). – 13C NMR
3
Chem. 1995, 34, 412–432; E. J. Toone, E. S. Simon, M. D.
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(CDCl3): δ ϭ 82.8 (d, 1JC-C ϭ 37.2 Hz), 74.8 (d, 1JC-C ϭ 37.2 Hz). –
MS (ESI): m/z ϭ 503 [M ϩ Na], 498 [M ϩ NH4], 481 [M ϩ H].
[3’,4’-13C2]Thymidine (1): The white solid obtained as described
above was added to 30 mL of methanol saturated with NH3 at 0
°C and the suspension was stirred for 2 days. The methanol was
then removed in vacuo, the residue was taken up in water, and this
solution was washed with diethyl ether. Removal of the water gave
thymidine (300 mg, 85% yield) as a white solid. Recrystallization
from water gave a white solid melting at 184 °C.[18] Analysis of the
NMR spectra revealed a 13C isotope enrichment of 96% at the 3’-
position and of 99% at the 4’-position. The remaining 13C atoms
were found to be present in the 5’-position. – 1H NMR (600 MHz,
[13]
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[14]
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[17]
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3
[18]
D2O): δ ϭ 7.66 (m, 1 H, 6-H), 6.30 (dd, JC-H ϭ 6.7 Hz, 1 H, 1’-
T. Gefflaut, M. Lemaire, M.-L. Valentin, J. Bolte, J. Org. Chem.
1
1
H), 4.48 (dm, JC-H ϭ 151.2 Hz, 1 H, 3’-H), 4.03 (dm, JC-H
ϭ
1997, 62, 5920–5922.
[19]
R. P. Hodge, C. K. Brush, C. M. Harris, T. M. Harris, J. Org.
148.8 Hz, 1 H, 4’-H), 3.85 (m, 1 H, 5’-H), 3.78 (m, 1 H, 5’’-H), 2.38
3
Chem. 1991, 56, 1553–1564.
(m, 2 H, 2’- and 2’’-H), 1.90 (d, JC-H ϭ 1.1 Hz, 3 H, 5-H). – 13C
[20]
M. I. Balagopala, A. P. Ollapally, H. Lee, Nucleosides and Nuc-
1
NMR (600 MHz, D2O): δ ϭ 87.3 (d, JC-C ϭ 37.4 Hz), 71.2 (d,
leotides 1958, 15, 899–906.
Received June 28, 1999
[O99386]
1JC-C ϭ 37.4 Hz). – MS (ESI): m/z ϭ 267 [M ϩ Na]. – HRMS
(DIP): calcd. for 13C2C8H14N2O5 244.09698; found 244.101553.
866
Eur. J. Org. Chem. 2000, 861Ϫ866