Synthesis of Stable Isotope Labeled of Deoxynucleosides
J . Org. Chem., Vol. 67, No. 5, 2002 1487
µmol, 61%) of the title compound as a white solid. Mp 183 °C.
[2,5-13C2]2-Deoxy-D-r ibose-5-p h osp h a te (5a ). [1-13C]Di-
hydroxyacetone monophosphate (4a ) (2.5 mmol) was dissolved
in 25 mL water, that further contained 0.1 M TEA, 2 mM
EDTA, 300 units of DERA, 700 units of TPI, and 0.2 M [2-13C]-
acetaldehyde. The solution was stirred for 2 h, after which time
13C NMR indicated complete conversion. BaCl2 dihydrate (1.22
g, 5 mmol) was added, and the suspension was stirred 40 min,
followed by addition of ethanol (50 mL). The mixture was
cooled for 18 h (0 °C), and the precipitate that was obtained
after centrifugation was washed with ethanol and ether. After
treatment with DOWEX H+, the clear colorless solution was
adjusted to neutral pH with 1 M NaOH and lyophilized. The
yield at this point was 2.0 mmol, based on enzymatic assay.
MS (ESI): m/z ) 215 (M - Na).
2
1H NMR (600 MHz, D2O): δ 7.66 (m, H6, 1H), 6.30 (dd, J H-H
2
3
6.7, H1′, 1H), 4.48 (m, H3′, 1H), 4.03 (dd, J C-H 1.0, J C-H 0.7,
1
1
H4′, 1H), 3.85 (d, J C-H 142.6, H5′, 1H), 3.78 (d, J C-H 143.3,
H5′′, 1H), 2.38 (d, 1J C-H 134.6, H2′ and H2′′, 2H), 1.90 (d,4J H-H
1.1, H5, 3H). 13C NMR (600 MHz, D2O): δ 167.1 (C4), 152.3
1
(C2), 138.2 (C6), 112.1 (C5), 87.3 (d, J C-C 41.7, C4′), 85.8 (d,
1J C-C 36.5, C1′), 71.2 (dd, J C-C 35.5, J C-C 3.2, C3′), 62.0
(strong s, C5′), 39.4 (strong s, C2′), 12.4 (5-Me). HRMS (ESI):
calcd for C813C2H14N2O5 267.0868 (M + Na); found 267.0867
(M + Na).
1
2
[1′,2′,5′-13C3]Th ym id in e (1b). According to the above pro-
cedure, 5b (72 mg, 0.3 mmol) and thymine (150 mg, 1.2 mmol)
were reacted to yield 42 mg (0.17 mmol, 57%) of the title
compound. The 1H NMR and the 13C NMR spectra of this
labeled compound were identical to the corresponding spectra
of the unlabeled compound except for the additional signal due
to the presence of 13C isotopes: 1H NMR (600 MHz, D2O): δ
7.66 (d, 3J C-H 2.3, H6, 1H), 6.30 (d, 1J C-H 170.2, H1′, 1H), 4.03
[1,2,5-13C3]2-Deoxy-D-r ibose-5-p h osp h a te (5b) was pre-
pared as described above by using 4a (1 mmol) and [1,2-13C2]-
acetaldehyde (92 mg, 2 mmol) as starting materials.
[3′,4′-13C2]3′,5′-Di-O-a cetyl-2′-d eoxyu r id in e (6a ). Crude
[3′,4′-13C2]-D-2′-deoxyuridine (2b, according to analytical data
the crude compound contains 82 mg, 0.358 mmol of pure 2b)
was dissolved in 5.0 mL of dry pyridine, and acetic anhydride
(3.2 mmol, 0.3 mL) was added. The suspension was stirred
for 2 days at room temperature. The pyridine was removed
under reduced pressure, and the resulting oil was purified by
column chromatography (CH3OH/CH2Cl2 5/95), giving a slightly
yellow oil (102 mg, 0.323 mmol, 90%). 1H NMR (300 MHz,
2
3
1
(dd, J C-H 1.0, J C-H 0.7, H4′, 1H), 3.85 (d, J C-H 142.6, H5′,
1
1
1H), 3.78 (d, J C-H 143.3, H5′′, 1H). 2.38 (d, J C-H 134.6, H2′
and H2′′, 2H). 13C NMR (600 MHz, D2O): δ 87.3 (d, 1J C-C 41.7,
C4′), 85.8 (strong d, 1J C-C 36.5, C1′), 71.2 (dd, 1J C-C 35.5, 2J C-C
1
3.2, C3′), 62.0 (strong s, C5′), 39.4 (strong d, J C-C 36.5, C2′).
HRMS (ESI): calcd for C713C3H14N2O5 268.0899 (M + Na);
found 268.0879 (M + Na).
[1′,2′,5′-13C3]D-2′-Deoxyu r id in e (2a ). According to the
above procedure, 5a (72 mg, 0.3 mmol) and uracil (134 mg,
1.2 mmol) were converted to the title compound in 52% yield
(36 mg, 0.16 mmol). 1H NMR (600 MHz, D2O): δ 7.87 (dd,
3
CDCl3): δ 7.51 (d, 1H, J H-H 8.2 Hz, H6), 6.32-6.27 (dd, 1H,
3
3
3J H-H 8.2 Hz, J H-H 5.8 Hz, H1′), 5.80 (d, 1H, J H-H 8.2 Hz,
1
H5), 5.22 (dm, 1H, J C-H 158.2 Hz, H3′), 4.40-4.29 (m, 2H,
5′-H, H5′′), 4.27 (dm, 1H, J C-H 151.9 Hz, H4′), 2.48-2.59 (m,
1
3J H-H 8.1 Hz, J H-H 2.6 Hz, 1H, H6), 6.29 (dt, J C-H 170.7,
3
1
1H, H2′′), 2.22-2.13 (m, 1H, H2′), 2.11 (s, 3H, Me), 2.12 (s,
3H, Me). 13C NMR (75 MHz, CDCl3): δ 170.2 (CO), 163.3 (C4),
150.3 (C2), 138.8 (C6), 102. 9 (C5), 84.0 (C1′), 82.2 (strong d,
3
3J H-H 6.3 Hz, 1H, H1′), 5.90 (d, J H-H 8.1, 1H, H5), 4.48 (dt,
3J H-H 6.4 Hz, J H-H 4.0 Hz, 1H, H3′), 4.06 (dt, J H-H 5.0 Hz,
3
3
3J H-H 3.8 Hz, 1H, H4′), 3.86 (dd, J H-H 12.6 Hz, J C-H 143.3
3
1
1J C-C 37.5 Hz, C4′), 74.0 (strong d, J C-C 37.5 Hz, C3′), 63.7
1
Hz, 3J H-H 3.6 Hz, 1H, H5′), 3.78 (dd,1J C-H 142.8 Hz, 3J H-H 12.6
1
1
(d, J C-C 42.8 Hz, C5′), 37.7 (d, J C-C 36.6 Hz, C2′), 20.8 (Me),
3
3
3
Hz, J H-H 5.0 Hz, 1H, H5′′), 2.44 (ddd, J H-H 14.3 Hz, J H-H
6.7 Hz, 1J C-H 135.2 Hz, 3J H-H 4.0 Hz, 1H, H2′′), 2.38 (dt, 3J H-H
14.3 Hz,1J C-H 134.1 Hz, 3J H-H 6.7 Hz, 1H, H2′). 13C NMR (151
MHz, D2O): δ 167.1 (C4), 152,4 (C2), 142.8 (C6), 103.2 (C5),
87.6 (C4′), 86.3 (strong d, C1′), 71.3 (C3′), 62.0 (strong s, C5′),
39,6 (C2′, strong d). ESI MS: m/z ) 254 (M + Na).
20.70 (Me). ESI MS: m/z ) 315 (M + H+), 337 (M + Na+).
4-(Tetr a zol-1-yl)-1-(3′,5′-d i-O-a cetyl-2′-[3′,4′-13C2]d eoxy-
â-D-r ibofu r a n osyl)p yr im id in -2-on e (7a ). Compound 6a (102
mg, 0.323 mmol) was dissolved in 0.80 mL of dry pyridine.
Tetrazole (0.45 mmol, 32 mg), diphenyl phosphate (0.27 mmol,
68 mg), and tosyl chloride (0.45 mmol, 86 mg) were added,
and the reaction mixture was stirred at room temperature for
1.5 h. Pyridine was removed under reduced pressure, and the
resulting oil was purified by column chromatography (CH3-
OH/CH2Cl2 3/97), giving 110 mg (0.297 mmol, 92%) of the title
[3′,4′-13C2]D-2′-Deoxyu r id in e (2b) was prepared from 5b
(166 mg, 0.7 mmol) and uracil (336 mg, 3 mmol) according to
the procedure as described above. The crude product was used
to synthesize 6a . ESI MS: m/z ) 253 (M + Na).
[3′,4′-13C2]2′-Deoxycytid in e (3a ). Compound 8a (58.0 mg,
0.185 mmol) was dissolved in methanol (4 mL). Benzylamine
(0.534 mmol, 59 µL) was added, and the reaction mixture was
stirred at room temperature for 3 days. Methanol was removed
in vacuo, water (15 mL) was added, and the solution was
extracted three times with dichloromethane (15 mL). The
water layer was lyophilized, leaving a white powder (44 mg,
0.178 mmol, 96% in the monohydrate form). 1H NMR (600
MHz, D2O): δ 7.79 (d, 1H, 3J H-H7.6 Hz, H6), 6.23 (t, 1H, 3J H-H
1
compound. H NMR (300 MHz, CDCl3): δ 9.64 (s, 1H, tetra-
3
3
zole-H), 8.51 (d, 1H, J H-H 7.2 Hz, 6-H), 7.27 (d, 1H, J H-H 7.2
3
1
Hz, H5), 6.26 (t, 1H, J H-H 6.5 Hz, H 1′), 5.27 (dm, 1H, J C-H
2
1
157.7 Hz, J CH)2.5 Hz, H3′), 4.48 (dm, 1H, J C-H 152.6 Hz,
H4′), 4.48-4.36 (m, 2H, 5′-H, H5′′), 3.02-2.92 (m, 1H, H2′′),
2.30-2.17 (m, 1H, H2′), 2.15 (s, 3H, Me). 2.11 (s, 3H, Me). 13
C
NMR (75 MHz, CDCl3): δ 170.2 (CO), 170.0 (CO), 157.2 (C4),
153.5 (C2), 147.0 (tetrazole-C), 140.5 (C6), 94.8 (C5), 83.5
1
1
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(strong d, J C-C 37.9 Hz, C4′), 88.3 (C1′), 73.7 (strong d, J C-C
37.9 Hz, C3′), 63.3 (d, J C-C 42.5 Hz, C5′), 38.9 (d, J C-C 36.4
Hz, C2′), 20.7 (Me).
6.7 Hz, H1′), 6.00 (d, 1H, J H-H 7.6 Hz, H5), 4.39 (dm, 1H,
1
1
1
2
1J C-H 151.2 Hz, H3′), 4.01 (dm, 1H, J C-H 148.9 Hz, J C-H 4.7
2
Hz, H4′), 3.82-3.78 (dm, 1H, J H-H 12.5 Hz, H5′), 3.73-3.70
Di-O-a cetyl-[3′,4′-13C2]2′-d eoxycytid in e (8a ). Compound
7a (68.0 mg, 0.192 mmol) was dissolved in 2.0 mL of dioxane.
Ammonium hydroxide (0.22 mmol, 0.034 mL) was added, and
the solution was stirred at room temperature for 3 days. The
solvent was evaporated, and the residue was absorbed onto
silica and purified by column chromatography (CH3OH/CH2-
Cl2 10/90). The yield of the title compound was 58.0 mg (0.185
mmol, 96%). 1H NMR (300 MHz, CD3OD): δ 7.71 (d, 1H, 3J H-H
2
(dm, 1H, J H-H 12.5 Hz, H5′′), 2.41-2.36 (m, 1H, H2′′), 2.28-
2.23 (m, 1H, H2′). 13C NMR (150 MHz, D2O): δ 166.9 (C4),
158.32 (C2), 142.3 (C6), 97.0 (C5), 89.3 (C1′), 87.3 (strong d,
1
1J C-C 37.5 Hz, C4′), 71.3 (strong d, J C-C 37.5 Hz, C3′), 62.0
1
1
(d, J C-C 41.8 Hz, C5′), 40.0 (d, J C-C 35.5 Hz, C2′). ESI MS:
m/z)252 (M + Na+).
[1-13C]Dih yd r oxya ceton e m on op h osp h a te (4a ). [1-13C]-
Dibenzyl-3-benzyloxypropanone phosphate (10a ) (1.2 g, 2.7
mmol) was dissolved in dioxane (32 mL), 2-propanol (16 mL),
and water (8 mL). After addition of Pd/C (500 mg), hydrogen
pressure was applied with a balloon, and the mixture was
stirred (300 rpm) overnight. Conversion was checked with TLC
(n-BuOH/acetone/water 5/3/2), and the mixture was filtered
to give a clear, colorless solution. One equivalent of NaOH as
a 1 M solution was added, and the solvent was evaporated in
vacuo. The residue was used without further purification. This
residue contained 2.5 mmol of the title compound (94%; based
on enzymatic assay). ESI MS: m/z ) 170 (M - H).
3
3
7.5 Hz, H6), 6.24-6.20 (dd, 1H, J H-H 8.1 Hz, J H-H 5.8 Hz,
H1′), 5.93 (d, 1H, 3J H-H 7.7 Hz, H5), 5.23 (dm, 1H, 1J C-H 160.7
Hz, H3′), 4.38-4.25 (m, 2H, 5′-H, H5′′), 4.27 (dm, 1H, J C-H
1
152.0 Hz, H4′), 2.58-2.47 (m, 1H, H2′′), 2.29-2.17 (m, 1H,
H2′), 2.08 (s, 3H, Me), 2.06 (s, 3H, Me). 13C NMR (75 MHz,
CD3OD): δ 172.2 (CO), 172.1 (CO), 167.6 (C4), 158.0 (C2),
141.7 (C6), 96.4 (C5): 86.2 (C1′), 83.7 (strong d, 1J C-C 36.9 Hz,
1
1
C4′), 76.0 (strong d, J C-C 37.1 Hz, C3′), 65.1 (d, J C-C 42.6
1
Hz, C5′), 38.8 (d, J C-C 36.5 Hz, C2′), 20.8 (Me), 20.70 (Me).
ESI MS: m/z ) 314 (M + H+), 336 (M + Na+).