Stereodefined synthesis of O3′-labeled uracil nucleosides. 3′-[17O]-2′-azido-2′-deoxyuridine 5′-diphosphate as a probe for the mechanism of inactivation of ribonucleotide reductases

Article abstract of DOI:10.1021/jo010899i

Thermolysis of a 2′-[¹⁶O]-O-benzoyl-[¹⁷O]-5′-O-(tert- butyldimethylsilyl)-O²,3′-cyclouridine derivative gave the more stable 3′-[¹⁷O]-O-benzoyl-[¹⁶O]-5′-O-(tert- butyldimethylsilyl)-O²,2′-cyclouridine isomer, which was converted into 3′-[¹⁷O]-2′-azido-2′-deoxyuridine by deprotection and nucleophilic ring opening at C2′ with lithium azide. The 5′-diphosphate was prepared by nucleophilic displacement of the 5′-O-tosyl group with tris(tetrabutylammonium) hydrogen pyrophosphate. Model reactions gave ¹⁶O and ¹⁸O isotopomers, and base-promoted hydrolysis of an O²,2′-cyclonucleoside gave stereodefined access to 3′-[¹⁸O]-1-(β-D-arabinofuranosyl)uracil. Inactivation of ribonucleoside diphosphate reductase with 2′-azido-2′-deoxynucleotides results in appearance of EPR signals for a nitrogen-centered radical derived from azide, and 3′-[¹⁷O]-2′-azido-2′-deoxyuridine 5′-diphosphate provides an isotopomer to perturb EPR spectra in a predictable manner.

Full text of DOI:10.1021/jo010899i

1816
J . Org. Chem. 2002, 67, 1816-1819
Ster eod efin ed Syn th esis of O3′-La beled Ur a cil Nu cleosid es.
3′-[17O]-2′-Azid o-2′-d eoxyu r id in e 5′-Dip h osp h a te a s a P r obe for th e
Mech a n ism of In a ctiva tion of Ribon u cleotid e Red u cta ses
Stanislaw F. Wnuk,*,† Saiful M. Chowdhury,† Pedro I. Garcia, J r.,† and Morris J . Robins*,‡
Department of Chemistry, Florida International University, Miami, Florida 33199, and
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602
wnuk@fiu.edu
Received September 4, 2001
Thermolysis of a 2′-[16O]-O-benzoyl-[17O]-5′-O-(tert-butyldimethylsilyl)-O2,3′-cyclouridine derivative
gave the more stable 3′-[17O]-O-benzoyl-[16O]- 5′-O-(tert-butyldimethylsilyl)-O2,2′-cyclouridine isomer,
which was converted into 3′-[17O]-2′-azido-2′-deoxyuridine by deprotection and nucleophilic ring
opening at C2′ with lithium azide. The 5′-diphosphate was prepared by nucleophilic displacement
of the 5′-O-tosyl group with tris(tetrabutylammonium) hydrogen pyrophosphate. Model reactions
gave 16O and 18O isotopomers, and base-promoted hydrolysis of an O2,2′-cyclonucleoside gave
stereodefined access to 3′-[18O]-1-(â-D-arabinofuranosyl)uracil. Inactivation of ribonucleoside diphos-
phate reductase with 2′-azido-2′-deoxynucleotides results in appearance of EPR signals for a
nitrogen-centered radical derived from azide, and 3′-[17O]-2′-azido-2′-deoxyuridine 5′-diphosphate
provides an isotopomer to perturb EPR spectra in a predictable manner.
In tr od u ction
radical intermediates with cytidine 5′-diphosphate and
R1 mutants of RDPR8,9 have appeared. Thelander et al.
found that 2′-azido-2′-deoxyuridine 5′-diphosphate (1a )
(Figure 1) was a potent inactivator of RDPR.10 Sjo¨berg
et al. observed that inactivation of RDPR by 1a was
accompanied by appearance of new EPR signals for a
nitrogen-centered radical and concomitant decay of peaks
for a tyrosyl radical, which was the first direct evidence
for free-radical chemistry with RDPR. The structure of
the elusive nitrogen radical has been investigated
extensively.11-15 Inactivation of RDPR with doubly la-
beled 2′-[15N3]-azido-2′-[13C]-1a resulted in formation of
a nitrogen-centered radical that had no EPR hyperfine
interaction with 13C2′, which was consistent with C2′-
N3 bond cleavage.13 Figure 1 contains mechanistic ratio-
nalization of these results. Abstraction of Ha from 1a by
the Cys439 thiyl radical is followed by loss of azide from
the C3′ radical intermediate in A. An azido species reacts
with the sulfhydryl of Cys225 to generate N2 and the
Ribonucleotide reductases (RNRs) are ubiquitous en-
zymes that execute the only known conversion of 5′-(di
or tri)phosphate esters of ribonucleosides into 2′-deoxy-
nucleotides. Inhibition of RNRs disrupts this primary
source of DNA components and is an appealing target
for rational design of agents against rapidly proliferating
viruses and cancer cells.1 Ribonucleoside diphosphate
reductase (RDPR) from Escherichia coli consists of two
pairs of nonidentical subunits (R1 and R2) whose X-ray
structures have been determined.2 A thiyl radical at
Cys439 initiates nucleotide reduction by abstraction of
H3′ from substrate ribonucleotides.3 Water (O2′) is then
lost from C2′ of the resulting hydrogen-bonded C3′
radical. Siegbahn’s theoretical analysis correlated amino
acid residues identified in X-ray structures with mecha-
nistic processes.4 Generation of radicals at C3′ of adenos-
ine5 and C3 of homoribofuranose model compounds has
been shown to initiate reaction cascades that simulate
processes postulated to occur during enzymatic 2′-deoxy-
genation of ribonucleotides.6
(7) (a) Gerfen, G. J .; van der Donk, W. A.; Yu, G.; McCarthy, J . R.;
J arvi, E. T.; Matthews, D. P.; Farrar, C.; Griffin, R. G.; Stubbe, J . J .
Am. Chem. Soc. 1998, 120, 3823-3835. (b) van der Donk, W. A.; Gerfen,
G. J .; Stubbe, J . J . Am. Chem. Soc. 1998, 120, 4252-4253.
(8) (a) Persson, A. L.; Eriksson, M.; Katterle, B.; Po¨tsch, S.; Sahlin,
M.; Sjo¨berg, B.-M. J . Biol. Chem. 1997, 272, 31533-31541. (b) Persson,
A. L.; Sahlin, M.; Sjo¨berg, B.-M. J . Biol. Chem. 1998, 273, 31016-
31020.
(9) Lawrence C. C.; Bennati, M.; Obias, H. V.; Bar, G.; Griffin, R.
G.; Stubbe, J . Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8979-8984.
(10) Thelander, L.; Larsson, B.; Hobbs, J .; Eckstein, F. J . Biol. Chem.
1976, 251, 1398-1405.
(11) Sjo¨berg, B.-M.; Gra¨slund, A.; Eckstein, F. J . Biol. Chem. 1983,
258, 8060-8067.
(12) (a) Ator, M.; Salowe, S. P.; Stubbe, J .; Emptage, M. H.; Robins,
M. J . J . Am. Chem. Soc. 1984, 106, 1886-1887. (b) Salowe, S. P.; Ator,
M. A.; Stubbe, J . Biochemistry 1987, 26, 3408-3416.
(13) Salowe, S.; Bollinger, J . M., J r.; Ator, M.; Stubbe, J .; McCracken,
J .; Peisach, J .; Samano, M. C.; Robins, M. J . Biochemistry 1993, 32,
12749-12760.
(14) Behravan, G.; Sen, S.; Rova, U.; Thelander, L.; Eckstein, F.;
Gra¨slund, A. Biochim. Biophys. Acta 1995, 1264, 323-329.
(15) van der Donk, W. A.; Stubbe, J .; Gerfen, G. J .; Bellew, B. F.;
Griffin, R. G. J . Am. Chem. Soc. 1995, 117, 8908-8916.
Direct observation of radical intermediates is problem-
atic, but recent reports of detection of substrate-derived
radicals during inactivation of RDPR7 and evidence for
* To whom correspondence should be addressed.
† Florida International University.
‡ Brigham Young University.
(1) (a) Stubbe, J .; van der Donk, W. A. Chem. Biol. 1995, 2, 793-
801. (b) J ordan, A.; Reichard, P. Annu. Rev. Biochem. 1998, 67, 71-
98. (c) Robins, M. J . Nucleosides Nucleotides 1999, 18, 779-793.
(2) (a) Nordlund, P.; Sjo¨berg, B.-M.; Eklund, H. Nature 1990, 345,
593-598. (b) Uhlin, U.; Eklund, H. Nature 1994, 370, 533-539. (c)
Eriksson, M.; Uhlin, U.; Ramaswamy, S.; Ekberg, M.; Regnstro¨m, K.;
Sjo¨berg, B.-M.; Eklund, H. Structure 1997, 5, 1077-1092.
(3) Mao, S. S.; Yu G. X.; Chalfoun, D.; Stubbe, J . Biochemistry 1992,
31, 9752-9759.
(4) Siegbahn, P. E. M. J . Am. Chem. Soc. 1998, 120, 8417-8429.
(5) Lenz, R.; Giese, B. J . Am. Chem. Soc. 1997, 119, 2784-2794.
(6) (a) Robins, M. J .; Guo, Z.; Samano, M. C.; Wnuk, S. F. J . Am.
Chem. Soc. 1999, 121, 1425-1433. (b) Robins, M. J .; Ewing, G. J . J .
Am. Chem. Soc. 1999, 121, 5823-5824.
10.1021/jo010899i CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/15/2002

Synthesis of O3′-Labeled Uracil Nucleosides
J . Org. Chem., Vol. 67, No. 6, 2002 1817
Sch em e 1a
F igu r e 1. Proposed formation of the nitrogen-centered radical
(C or D) during mechanism-based inhibition of RDPR with
2′-azido-2′-deoxyuridine 5′-diphosphate.
nitrogen-centered radical in B. Abstraction of hydrogen
by C2′ generates the 2′-deoxy-3′-ketonucleoside in B.13,14
EPR studies with 3-[2H]-cysteine-RDPR indicated that
the nitrogen radical hyperfine interaction results from a
â-hydrogen of Cys225.15 An initial nitrogen-centered
radical (undetected by EPR) might react with O3′ or C3′
of the intermediate in B to generate C or D, respec-
tively.15 Chemical arguments favor D because precedents
exist for addition of aminyl radicals to carbonyl or imino
groups.16 Spectroscopic data15 and molecular modeling
favor C, but precedents are lacking. Calculations for
model radicals [R-S-N-(H)•] are in harmony17 with the
hypothesis.15 We targeted 3′-[17O]-2′-azido-2′-deoxyuri-
dine 5′-diphosphate (1c) (Scheme 1) to probe this hy-
pothesis. EPR spectra of 3′-[17O]-C should have one-bond
hyperfine interactions with 17O, whereas two-bond spec-
tral distortions with 3′-[17O]-D should be much smaller.
a
Key: (a) TBDMSCl/imidazole/DMF; (b) PhC[O]Cl/pyridine; (c)
4; (d) (i) NH3/MeOH, (ii) NH4F/MeOH; (e) NaOH/H2O; (f) LiF/
Me3SiN3/TMEDA/DMF; (g) TsCl/pyridine; (h) (Bu4N)3HP2O7/
CH3CN.
Sch em e 2
cleoside 6 into the 3′-O-benzoyl-O2,2′-anhydroarabino
isomer 7. Protection (O5′) of O2,3′-anhydro-1-(â-D-xylo-
furanosyl)uracil21 (4) with TBDMS chloride gave 5 (80%).
Treatment of 5 with [16O, 18O, or 17O]-benzoyl chloride
(3) gave the 2′-O-benzoyl derivatives 6a -c, respectively.
The [18O or 17O]-benzoyl chlorides 3 were prepared by
acid-catalyzed hydrolysis (HCl/[18O or 17O]H2O) of ben-
zonitrile and treatment of the benzoic acids 2 with thionyl
chloride (Scheme 2). Isotopic incorporation (MS) was
calculated22 to be 90% for [2 × 18O]-2a or 53% for [2 ×
17O]-2b beginning with 97.4% (18O) or 74.5% (17O) en-
riched water, respectively. [17O]-Benzoic acid and its
derivatives had been prepared by alkaline hydrolysis of
methyl benzoate23a or by acid-catalyzed exchange (HCl/
BzOH/H217O).23b The mass spectrum of 6c had peaks at
m/z 446 (100%) for [17O]MH+ and m/z 445 (78%) for [16O]-
MH+, which is consistent with 50% 17O enrichment.22
Rearrangement of 6c gave 7c (48% 17O enrichment),
and parallel treatment of 6b gave 7b (81% 18O). Benzoyl
Resu lts a n d Discu ssion
Methods for synthesis of nucleosides with oxygen
isotopes are limited, and especially for sugar isotopo-
mers.18-20 Reversible hydration (HCl/H218O) of a 3′-
ketouridine derivative followed by reduction (NaBH4) and
separation from the xylo epimer (major) gave 3′-[18O]-
uridine in low yield.19a Other [18O]19b,c and [17O]20 isoto-
pomers were prepared from cyclonucleosides.
The key step in our synthesis of 3′-[17O]-2′-azido-2′-
deoxyuridine (9c) (Scheme 1) was the Fox thermal
rearrangement21 of 2′-O-benzoyl-O2,3′-anhydroxylo nu-
(16) (a) Kim, S., J oe, G. H.; Do, J . Y. J . Am. Chem. Soc. 1993, 115,
3328-3329. (b) Kim, S.; J oe, G. H.; Do, J . Y. J . Am. Chem. Soc. 1994,
116, 6, 5521-5522.
(17) Eriksson, L. A. J . Am. Chem. Soc. 1998, 120, 8051-8054.
(18) Follman, H.; Hogencamp, H. P. C. J . Am. Chem. Soc. 1970, 92,
671-677.
(19) (a) Pang, H.; Schram, K. H.; Smith, D. L.; Gupta, S. P.;
Townsend, L. B.; McCloskey, J . A. J . Org. Chem. 1982, 47, 3923-3932.
(b) Solsten, R. T.; McCloskey, J . A.; Schram, K. H. Nucleosides
Nucleotides 1982, 1, 57-64. (c) Schubert, E. M.; Schram, K. H. J .
Labelled Compd. Radiopharm. 1982, 19, 929-935.
(20) (a) Schwartz, H. M.; MacCoss, M.; Danyluk, S. S. J . Am. Chem.
Soc. 1983, 105, 5901-5911. (b) Schwartz, H. M.; MacCoss, M.;
Danyluk, S. S. Magn. Reson. Chem. 1985, 23, 885-894.
(21) Yung, N. C.; Fox, J . J . J . Am. Chem. Soc. 1961, 83, 3060-3066.
(22) (a) The [17O]-labeled percentage was calculated with the equa-
tion 100[(I + 1) - (I + 1)natural]/[I + (I + 1) - (I + 1)natural] with
normalized relative intensities for the (protonated) molecular ion peaks.
Mass spectra for 16O isotopomers were measured under identical
experimental conditions to obtain comparable (I + 1)natural values. The
same conditions with (I + 2) values were used for [18O]. (b) Lambert,
J . B.; Shurvell, H. F.; Lightner, D. A.; Cooks, R. G. Organic Structural
Spectroscopy; Prentice Hall: Upper Saddle River, 1998; pp 439-472.
(23) (a) Baltzer, L.; Becker, E. D. J . Am. Chem. Soc. 1983, 105, 5,
5730-5733. (b) Miura, Y.; Shibata, Y.; Kinoshita, M. J . Org. Chem.
1986, 51, 1239-1243.

1818 J . Org. Chem., Vol. 67, No. 6, 2002
Wnuk et al.
(NH3/MeOH) and TBDMS (NH4F/MeOH)24 groups were
removed, and RP-HPLC gave [17O]-8c (65%). Base-
promoted hydrolysis of 8b gave 3′-[18O]-1-(â-D-arabino-
furanosyl)uracil (11b). Treatment of 8 with LiF/TMEDA/
in long pressure tube (Ace glass) and cooled in an ice bath.
HCl (anhydrous) was bubbled in for 3 min, and the HCl
content (∼37%) was calculated from the increased mass.
[2 × 18O]-Ben zoic Acid (2a ). PhCN (1.78 mL, 1.8 g, 17.5
mmol) was suspended in concentrated HCl/H218O in a long
pressure tube, and the mixture was heated for 20 h at 100 °C
with vigorous stirring. The thick suspension was partitioned
between ice-cold NaHCO3/H2O//CH2Cl2, and the organic layer
was quickly extracted with NaHCO3/H2O. The combined
aqueous phase was washed with CH2Cl2 to remove traces of
PhCN and BzNH2. CH2Cl2 was added to the aqueous layer and
the mixture was acidified (dilute HCl/H2O, pH ∼3). The
organic layer was washed (brine) and dried (Na2SO4), and
volatiles were evaporated to give 2a (1.55 g, 70%) as a white
solid: 1H NMR δ 7.49 (t, J ) 7.4 Hz, 2H), 7.65 (t, J ) 7.4 Hz,
1H), 8.15 (d, J ) 7.0 Hz, 2H), 13.00 (br s, 1H); 13C NMR δ
129.0, 129.8, 130.7, 134.3, 173.1; MS m/z 127 (100, [2 × 18O]-
MH+), 125 (9.7, [18/16O]MH+), 123 (1.8, [2 × 16O]MH+).
25a
Me3SiN3 gave 9 in higher yields than with Moffatt’s
LiN3/HMPA procedure.25b The 3′-[17O]-2′-azido-2′-deox-
yuridine (9c) had 47% 17O. Selective tosylation of 9c gave
10c (63%) and small quantities of the 3′,5′-di-O-tosyl
byproduct. The Poulter26 displacement of tosylate with
tris(tetrabutylammonium) hydrogen pyrophosphate, pu-
rification, and conversion to the sodium salt gave 1c (1H
and 31P NMR). No 1H NMR signals for ammonium salts
were observed, but 31P NMR indicated the presence of
inorganic pyrophosphate. The 17O NMR27 spectrum of our
doubly labeled [17O]-benzoic acid (2b) had one signal at
δ 205 (line width 308 Hz) in harmony with reported
data,23a,28 and that of the 3′-[17O]-2,2′-anhydrouridine 8c
had a peak at δ -66 (line width 85 Hz). The 17O shift in
8c is upfield from that of the secondary OH of 2-propanol
(δ 4029) and O2′ of 2′-[17O]-1-(â-D-arabinofuranosyl)uracil
(δ -1120b).
[2 × 17O]-Ben zoic Acid (2b). Treatment of PhCN (2.06 mL,
2.08 g, 20.2 mmol) with HCl/H217O (as described for 2a ) gave
2b (1.54 g, 62%): 1H NMR and 13C NMR peaks were the same
as for 2a except δ 173.0-173.3 (m); MS m/z 125 (100, [2 ×
17O]MH+), 124 (59, [17/16O]MH+), 123 (25, [2 × 16O]MH+).
[
18O]-Ben zoyl Ch lor id e (3a ). [18O]-Benzoic acid (2a , 0.88
In summary, Fox thermolysis of O2,3′-cyclouridine
derivative 6c gave the more stable O2,2′-cyclouridine
isomer with rearrangement of the 2′-O-benzoyl[17O] group
to give the 3′-[17O]-O-benzoyl product 7c. This stereode-
fined rearrangement can be applied to 17O or 18O labeling
of a variety of pyrimidine nucleosides. Deprotection and
anhydro ring opening (LiN3) gave 3′-[17O]-9c, which was
converted into 3′-[17O]-2′-azido-2′-deoxyuridine 5′-diphos-
phate (1c). Studies on inhibition of ribonucleoside diphos-
phate reductase with 1c are in progress.30
g, 6.98 mmol) was placed in a 15-mL round-bottomed flask
and SOCl2 (0.65 mL, 1.05 g, 8.8 mmol) was added dropwise
with stirring. The mixture was heated gently for 1 h at 100
°C (1 mL of SOCl2 was added after 20 min to make a clear
solution), cooled, and then distilled (∼70 °C) at atmospheric
pressure to remove excess SOCl2. A vacuum was applied, and
the bath temperature was slowly increased to ∼130 °C to give
one fraction of 3a (0.85 g, 85%) as a colorless liquid.
[
17O]-Ben zoyl Ch lor id e (3b). Treatment of [17O]-benzoic
acid (2b, 1.4 g, 11.3 mmol) with SOCl2 (1.03 mL, 1.67 g, 14.1
mmol) (as described for 3a ) gave [17O]-benzoyl chloride (3b,
1.3 g, 80%).
Exp er im en ta l Section
1-[2,3′-An h yd r o-5′-O-(ter t-bu tyld im eth ylsilyl)-â-D-xylo-
fu r a n osyl]u r a cil (5). TBDMSCl (0.9 g, 6.0 mmol) was added
to a stirred suspension of imidazole (0.725 g, 12.5 mmol) and
1-(2,3′-anhydro-â-D-xylofuranosyl)uracil21 (4, 1.13 g, 5 mmol)
in dried DMF (30 mL) at ambient temperature under N2. After
18 h, volatiles were evaporated, and the residue was chro-
matographed (EtOAc f 60% S1/EtOAc) and recrystallized
(EtOH/EtOAc) to give 5 (1.36 g, 80%) as needles: mp 246-
247 °C; UV max 250 (sh), 232 nm (ꢀ 7400, 9600), min 241 (sh),
216 nm (ꢀ 7900, 6300); 1H NMR (DMSO-d6) δ 0.02 (s, 6H), 0.85
(s, 9H), 3.71 (dd, J ) 11.0, 6.4 Hz, 1H), 3.81 (dd, J ) 11.0, 6.0
Hz, 1H), 4.41 (“dt”, J ) 6.2, 2.5 Hz, 1H), 4.74 (s, 1H), 4.92 (s,
1H), 5.64 (s, 1H), 5.80 (d, J ) 7.4 Hz, 1H), 6.45 (br s, 1H), 7.68
(d, J ) 7.4 Hz, 1H); 13C NMR (DMSO-d6) δ -5.00, 18.3, 26.0,
61.4, 69.7, 79.5, 83.6, 89.6, 108.2, 141.3, 153.7, 170.6; MS (CI)
m/z 341 (100, MH+), 283 (30, M+ - t-Bu); HRMS (CI) m/z
341.1538 (MH+ ) 341.1532). Anal. Calcd for C15H24N2O5Si
(340.45): C, 52.92; H, 7.11; N, 8.23. Found: C, 52.43; H, 7.26;
N, 8.11.
1-[2,3′-An h yd r o-2′-O-ben zoyl-5′-O-(ter t-bu tyld im eth yl-
silyl)-â-D-xylofu r a n osyl]u r a cil (6a ). BzCl (50 µL, 60.6 mg,
0.43 mmol) was added to 5 (20 mg, 0.05 mmol) in dried
pyridine (1 mL), and the solution was stirred overnight at
ambient temperature and then for 2 h at 40 °C. Volatiles were
evaporated in vacuo, and toluene was added. Volatiles were
evaporated, and the residue was chromatographed (90%
EtOAc/hexanes f EtOAc f 25% S1/EtOAc) to give 6a (10 mg,
50%) as a white solid: mp 238-240 °C; UV max 233 nm (ꢀ
25 800), min 212 nm (ꢀ 9400); 1H NMR δ 0.02 (s, 6H), 0.85 (s,
9H), 3.89 (dd, J ) 10.7, 7.3 Hz, 1H), 3.93 (dd, J ) 10.8, 6.0
Hz, 1H), 4.70 (“dt”, J ) 7.4, 2.5 Hz, 1H), 5.22 (s, 1H), 5.55 (s,
1H), 5.70 (s, 1H), 6.13 (d, J ) 7.3 Hz, 1H), 7.21 (d, J ) 7.4 Hz,
1H), 7.52 (t, J ) 7.9 Hz, 2H), 7.70 (t, J ) 7.4 Hz, 1H), 8.06 (d,
J ) 7.1 Hz, 2H); 13C NMR δ -0.5, 18.8, 26.2, 61.1, 72.6, 77.6,
85.2, 88.8, 110.5, 128.1, 129.3, 130.5, 134.9, 139.8, 153.5, 165.3,
171.4; MS m/z 445 (100, MH+), 446 (29.6, MH+ + 1), 447 (8.7,
MH+ + 2). Anal. Calcd for C22H28N2O6Si (444.56): C, 59.44;
H, 6.35; N, 6.30. Found: C, 59.03; H, 6.57; N, 6.30.
Uncorrected melting points were determined with a capil-
lary tube apparatus. UV spectra were recorded with solutions
in MeOH. 1H (Me4Si) NMR spectra were determined with
solutions in CDCl3 at 400 MHz, 13C (Me4Si) at 100.6 MHz, and
31P (H3PO4) at 161.9 MHz unless otherwise noted, and 17O
spectra (H217O external) were recorded at 54.245 MHz and a
90° pulse angle with a Bru¨ker DPX-400 spectrometer equipped
with a 5-mm broad-band probe at ambient temperature. Mass
spectra (MS) were obtained with atmospheric pressure chemi-
cal ionization (APCI) except when CI (CH4) is noted. Labeled
water (97.4% 18O or 74.5% 17O enrichment) was purchased
from Isotec. Reagent grade chemicals were used and solvents
were dried by reflux over and distillation from CaH2 under
an argon atmosphere. TLC was performed on silica plates with
MeOH/CHCl3 (1:9), MeOH/EtOAc (1:19), or EtOAc/i-PrOH/H2O
(4:1:2, upper layer; S1). Merck kieselgel 60 (230-400 mesh)
was used for column chromatography. Preparative reversed-
phase (RP)-HPLC was performed with a Supelcosil LC-18S
column.
Con cen tr a ted HCl in [18O or 17O]-H2O. H2O (97.4% 18O,
1.05 g, 52.5 mmol; or 74.5% 17O, 1.08 g, 57.7 mmol) was placed
(24) Zhang, W.; Robins, M. J . Tetrahedron Lett. 1992, 33, 1177-
1180.
(25) (a) Kirschenheuter, G. P.; Zhai, Y.; Pieken, W. A. Tetrahedron
Lett. 1994, 35, 8517-8520. (b) Verheyden, J . P. H.; Wagner, D.; Moffatt,
J . G. J . Org. Chem. 1971, 36, 250-254.
(26) Davisson, V. J .; Davis, D. R.; Dixit, V. M.; Poulter, C. D. J . Org.
Chem. 1987, 52, 1794-1801.
(27) (a) Boykin, D. W.; Baumstark, A. L. Tetrahedron 1989, 45,
3613-3651. (b) 17O NMR Spectroscopy in Organic Chemistry; Boykin,
D. W., Ed.; CRC Press: Boca Raton, 1990.
(28) (a) Delseth, C.; Nguyen, T. T.-T.; Kintzinger, J .-P. Helv. Chim.
Acta 1980, 63, 498-503. (b) Baumstark, A. L.; Balakrishnan, P.;
Dotrong, M.; McCloskey, C. J .; Oakley, M. G.; Boykin, D. W. J . Am.
Chem. Soc. 1987, 109, 1059-1062.
(29) Crandall, J . K.; Centeno M. A. J . Org. Chem. 1979, 44, 1183-
1184.
(30) In collaboration with Professor J . Stubbe at Massachusetts
Institute of Technology.

Synthesis of O3′-Labeled Uracil Nucleosides
J . Org. Chem., Vol. 67, No. 6, 2002 1819
1-[2,3′-An h yd r o-2′-O-b en zoyl[18O]-5′-O-(ter t-b u t yld i-
m eth ylsilyl)-â-D-xylofu r a n osyl]u r a cil (6b). Treatment of
5 (200 mg, 0.59 mmol) with [18O]-BzCl (3a , 140 µL, 170 mg,
1.19 mmol) (as described for 6a ) gave 6b (180 mg, 68%) with
identical physical and spectral properties except MS m/z 447
(100, [18O]MH+), 445 (15.6, [16O]MH+).
1-[2,3′-An h yd r o-2′-O-b en zoyl[17O]-5′-O-(ter t-b u t yld i-
m eth ylsilyl)-â-D-xylofu r a n osyl]u r a cil (6c). Treatment of
5 (1.15 g, 3.3 mmol) with [17O]-BzCl (3b, 500 µL, 606 mg, 4.28
mmol) (as described for 6a ) gave 6c (0.45 g, 30%) with identical
physical and spectral properties except MS m/z 446 (100, [17O]-
MH+), 445 (77.6, [16O]MH+).
were added. Stirring was continued for 30 min, 8a (0.226 g,
1.0 mmol) was added, and the mixture was heated with
stirring for 48 h at 110 °C (oil bath). Volatiles were evaporated
in vacuo, and MeOH was added to the residue and evaporated
(3 ×). The oily residue was dissolved in MeOH (1 mL), and
EtOAc (4 mL) was added to precipitate salts and residual
starting material. The mixture was filtered and the filtrate
was chromatographed (MeOH/EtOAc, 1:9) to give 9a (0.22 g,
82%) as a slightly yellow foam. This material was purified (RP-
HPLC, 15% CH3CN/H2O) to give 9a (140 mg, 52%) as an off-
white foam with spectral data25 as reported: MS m/z 270 (100,
MH+), 271 (12, MH+ + 1).
3′-[17O]-2′-Azid o-2′-d eoxyu r id in e (9c). Treatment of 8c
(50 mg, 0.22 mmol) (as described for 9a ) gave 9c (33 mg, 56%)
as a slightly yellow foam that was purified (RP-HPLC) to give
9c (24 mg, 40%) as an off-white foam with identical spectral
data except MS m/z 271 (98.5, [17O]MH+), 270 (100, [16O]MH+).
2′-Azid o-2′-d eoxy-5′-O-(p-tolu ylsu fon yl)u r id in e (10a ).
TsCl (21 mg, 0.11 mmol) was added to 9a (20 mg, 0.074 mmol)
in dried pyridine (1 mL) and the solution was stirred for 14 h
at ambient temperature. Volatiles were evaporated and the
residue was partitioned (0.1 M HCl/H2O//CHCl3). The organic
layer was washed (NaHCO3/H2O, brine), dried (MgSO4), and
volatiles were evaporated. The residue was chromatographed
(CHCl3 f 5% MeOH/CHCl3) to give a less polar byproduct
tentatively assigned (1H NMR, MS) as 2′-azido-2′-deoxy-3′,5′-
di-O-(p-toluylsufonyl)uridine (7 mg, 17%) and then 10a (21 mg,
67%): 1H NMR δ 4.15-4.20 (m, 2H), 4.30 (d, J ) 11.1 Hz,
1H), 4.38 (d, J ) 11.4 Hz, 1H), 4.46 (t, J ) 5.7 Hz, 1H), 5.75
(d, J ) 8.0 Hz, 1H), 5.88 (d, J ) 2.8 Hz, 1H), 7.41 (d, J ) 7.6
Hz, 2H), 7.51 (d, J ) 7.7 Hz, 1H), 7.82 (d, J ) 7.4 Hz, 2H),
9.24 (br s, 1H); 13C NMR δ 22.2, 66.3, 67.9, 70.2, 81.7, 88.7,
103.3, 128.3, 130.7, 132.4, 139.7, 145.5, 150.5, 163.5; MS m/z
424 (100, MH+), 425 (17.5, MH+ + 1). Anal. Calcd for
1-[2,2′-An h yd r o-3′-O-ben zoyl-5′-O-(ter t-bu tyld im eth yl-
silyl)-â-D-a r a bin ofu r a n osyl]u r a cil (7a ). Compound 6a (100
mg, 0.22 mmol) was placed in a 25 mL round-bottomed flask
under argon and heated (Bunsen burner) for 1 min until the
solid melted and became brown. After cooling, the solid was
chromatographed (EtOAc f 15% S1/EtOAc) to give 7a (65 mg,
65%) as a white solid: mp 224-226 °C; UV max 254 (sh), 230
1
nm (ꢀ 9200, 24 000), min 211 nm (ꢀ 9000); H NMR δ 0.02 (s,
6H), 0.90 (s, 9H), 3.67 (dd, J ) 11.1, 4.4 Hz, 1H), 3.81 (dd, J
) 11.2, 5.9 Hz, 1H), 4.50 (“dt”, J ) 4.4, 2.6 Hz, 1H), 5.60 (d, J
) 5.5 Hz, 1H), 5.70 (s, 1H), 6.08 (d, J ) 7.4 Hz, 1H), 6.42 (d,
J ) 5.7 Hz, 1H), 7.42 (d, J ) 7.4 Hz, 1H), 7.50 (t, J ) 7.9 Hz,
2H), 7.65 (t, J ) 7.4 Hz, 1H), 8.05 (d, J ) 7.2 Hz, 2H); 13C
NMR δ -0.5, 19.0, 26.4, 63.6, 78.3, 87.4, 88.8, 91.2, 110.6,
128.8, 129.1, 130.3, 134.4, 135.4, 160.1, 165.8, 172.4; MS m/z
445 (100, MH+), 446 (29.6, MH+ + 1), 447 (8.8, MH+ + 2). Anal.
Calcd for C22H28N2O6Si (444.56): C, 59.44; H, 6.35; N, 6.30.
Found: C, 59.80; H, 6.42; N, 6.32.
1-[2,2′-An h yd r o-3′-[18O]-O-b en zoyl-5′-O-(ter t-b u t yld i-
m eth ylsilyl)-â-D-a r a bin ofu r a n osyl]u r a cil (7b). Thermoly-
sis of 6b (70 mg, 0.15 mmol) (as described for 7a ) gave 7b (44
mg, 63%) with identical physical and spectral properties except
MS m/z 447 (100, [18O]MH+), 445 (24.0, [16O]MH+).
C
16H17N5O7S (423.40): C, 45.39; H, 4.05; N, 16.54. Found: C,
45.71; H, 3.89; N, 16.78.
1-[2,2′-An h yd r o-3′-[17O]-O-b en zoyl-5′-O-(ter t-b u t yld i-
m eth ylsilyl)-â-D-a r a bin ofu r a n osyl]u r a cil (7c). Thermoly-
sis of 6c (50 mg, 0.11 mmol) (as described for 7a ) gave 7c (27
mg, 54%) with identical physical and spectral properties except
MS m/z 446 (100, [17O]MH+), 445 (81.9, [16O]MH+).
3′-[17O]-2′-Azid o-2′-d e oxy-5′-O-(p -t olu ylsu fon yl)u r i-
d in e (10c). Treatment of 9c (17 mg, 0.063 mmol) with TsCl
(as described for 10a ) gave 10c (17 mg, 64%) with identical
spectral data except MS m/z 425 (100, [17O]MH+), 424 (98.7,
[16O]MH+).
1-(2,2′-An h yd r o-â-D-a r a bin ofu r a n osyl)u r a cil (8a ). NH3/
MeOH (5 mL, saturated at ∼0 °C) was added to 7a (22 mg,
0.05 mmol) in MeOH (2 mL), and the solution was stirred for
2 h at ∼0 °C. Volatiles were evaporated, NH4F (28 mg, 0.75
mmol) and dried MeOH (5 mL) were added to the white
residue, and the solution was stirred overnight at ambient
temperature. Volatiles were evaporated, and the residue was
purified by RP-HPLC (8% CH3CN/H2O, tR ) 25 min) to give
8a (8 mg, 71%) as off-white crystals: mp 242-245 °C (lit.31
mp 238-244 °C); UV max 250, 224 nm (ꢀ 7900, 8900), min
3′-[17O]-2′-Azid o-2′-d eoxyu r id in e 5′-Dip h osp h a te (1c).
(Bu4NH)3HP2O726 (54 mg, 0.06 mmol) was added (one portion)
to 10c (17 mg, 0.04 mmol) in dried CH3CN (0.5 mL), and the
solution was stirred for 48 h at ambient temperature. Volatiles
were evaporated, and the residue was dissolved (H2O) and
purified by ion-exchange chromatography (DEAE Sephadex
A-25, Et3NHHCO3/H2O, 0.05 f 0.50 M). Evaporation of
appropriate fractions and coevaporation (H2O and MeOH, 5
×) gave 1c (14 mg, ∼50%) as a triethylammonium salt (∼1
equiv, 1H NMR). This material was dissolved (H2O) and passed
through a column of Dowex 50 × 8(Na+) (H2O) to give the
trisodium salt of 1c (8 mg, 40%), RP-HPLC (3% H2O/CH3CN,
tR 5.6 min), provided 1c (5.5 mg, 30%) with spectral data as
reported13 for 1a : 1H NMR (D2O) δ 4.05-4.17 (m, 2H), 4.26
(t, J ) 5.3 Hz, 1H) 4.49 (t, J ) 5.0 Hz, 1H), 5.84 (d, J ) 8.1
1
237, 212 nm (ꢀ 6800, 6300); H NMR (DMSO-d6) δ 3.19 (“dt”,
J ) 11.3, 5.5 Hz, 1H), 3.28 (“dt”, J ) 11.2, 6.0 Hz, 1H), 4.07 (t,
J ) 4.5 Hz, 1H), 4.38 (d, J ) 3.4 Hz, 1H), 4.99 (t, J ) 5.2 Hz,
1H), 5.20 (d, J ) 5.6 Hz, 1H), 5.85 (d, J ) 7.4 Hz, 1H), 5.90 (d,
J ) 4.3 Hz, 1H), 6.31 (d, J ) 5.7 Hz, 1H), 7.85 (d, J ) 7.4 Hz,
1H); 13C NMR (DMSO-d6) δ 61.7, 75.8, 89.6, 90.1, 90.9, 109.5,
137.7, 160.7, 172.1; MS m/z 227 (100, MH+), 228 (10.6, MH+
+ 1), 229 (1.5, MH+ + 2).
Hz, 1H), 5.95 (d, J ) 5.2 Hz, 1H), 7.85 (d, J ) 8.0 Hz, 1H); 31
P
NMR (D2O) δ -10.17 (d, J ) 21.1 Hz, PR), -9.24 (d, J ) 21.1
Hz, Pâ) [inorganic pyrophosphate impurity (-9.10 ppm, ∼50%)].
3′-[18O]-1-(â-D-Ar a bin ofu r a n osyl)u r a cil (11b). NaOH/
H2O (1 M, 1 mL, 1 mmol) was added to 8b (23 mg, 0.1 mmol)
in MeOH/H2O (1:1, 1 mL), and the solution was stirred
overnight and neutralized (AcOH, pH ∼7). Volatiles were
evaporated, and the residue was chromatographed (EtOAc f
8% MeOH/EtOAc) to give 11b (17 mg, 69%) with data as
reported19c,20b except MS m/z 247 (100, [18O]MH+), 245 (29,
[16O]MH+).
3′-[18O]-1-(2,2′-An h ydr o-â-D-ar abin ofu r an osyl)u r acil (8b).
Deprotection of 7b (8 mg, 0.034 mmol) (as described for 7a )
gave 8b (3 mg, 73%) with identical physical and spectral
properties except MS m/z 229 (100, [18O]MH+), 227 (27, [16O]-
MH+).
3′-[17O]-1-(2,2′-An h ydr o-â-D-ar abin ofu r an osyl)u r acil (8c).
Deprotection of 7c (22 mg, 0.05 mmol) (as described for 8a )
gave 8c (7.4 mg, 65%) with identical physical and spectral
properties except MS m/z 228 (92.5, [17O]MH+), 227 (100, [16O]-
MH+).
Ack n ow led gm en t . Support from the American
Cancer Society (M.J .R.) and a MBRS RISE program
grant (NIH/NIGMS, R25 GM61347) (P.I.G.) is acknowl-
edged. We thank Alberto J . Sabucedo (FIU) for mass
spectra.
2′-Azid o-2′-d eoxyu r id in e (9a ). LiF (47 mg, 1.8 mmol) was
added to dried DMF (2 mL), the stirred suspension was heated
(during 10 min) to ∼105 °C, and N,N,N,N-tetramethylethyl-
enediamine (2 mL) and TMS-N3 (240 µL, 210 mg, 1.8 mmol)
(31) Hampton, A.; Nichol, A. W. Biochemistry 1966, 5, 2076-2082.
J O010899I