Photochemical generation and reactivity of the 5,6-dihydrouridin-6-yl radical

Article abstract of DOI:10.1021/jo9012805

(Chemical Equation Presented) Nucleobase radicals are the major family of reactive intermediates formed when nucleic acids are exposed to hydroxyl radical, which is produced by γ-radiolysis and Fe·EDTA. Significant advances have been made in understanding the role of nucleobase radicals in oxidative DNA damage by independently generating these species from photochemical precursors. However, this approach has been used much less frequently to study RNA molecules. Norrish type I photocleavage of the tert-butyl ketone (2b) enabled studying the reactivity of 5′-benzoyl-5,6- dihydrouridin-6-yl (1b). High mass balances were observed under aerobic or anaerobic conditions, and O₂ did not affect the photochemical conversion of the ketone (2b) to 1b. Competition studies with O₂ indicate that the radical abstracts hydrogen atoms from β-mercaptoethanol with a bimolecular rate constant = 2.6 ± 0.5 × 10⁶ M^-1s^-1. The major product formed in the presence of O ₂ was 5′-benzoyl-6-hydroxy-5,6-dihydrouridine (6). In contrast, 5-benzoyl-ribonolactone (7), a hypothetical product resulting from C1′-hydrogen atom abstraction by the peroxyl radical, could not be detected. Overall, tert-butyl ketone 2b is a clean source of 5′-benzoyl-5,6-dihydrouridin-6-yl (1b) and should prove useful for studying the reactivity of the respective radical in RNA.

Full text of DOI:10.1021/jo9012805

pubs.acs.org/joc
Photochemical Generation and Reactivity of the
5,6-Dihydrouridin-6-yl Radical
Cory A. Newman, Marino J. E. Resendiz, Jonathan T. Sczepanski, and
Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218
mgreenberg@jhu.edu
Received June 15, 2009
Nucleobase radicals are the major family of reactive intermediates formed when nucleic acids are
exposed to hydroxyl radical, which is produced by γ-radiolysis and Fe EDTA. Significant advances
3
have been made in understanding the role of nucleobase radicals in oxidative DNA damage by
independently generating these species from photochemical precursors. However, this approach has
been used much less frequently to study RNA molecules. Norrish type I photocleavage of the
tert-butyl ketone (2b) enabled studying the reactivity of 50-benzoyl-5,6-dihydrouridin-6-yl (1b). High
mass balances were observed under aerobic or anaerobic conditions, and O2 did not affect the
photochemical conversion of the ketone (2b) to 1b. Competition studies with O2 indicate that the
radical abstracts hydrogen atoms from β-mercaptoethanol with a bimolecular rate constant=2.6(
0.5 ꢀ 106 M-1 -1
s
. The major product formed in the presence of O2 was 50-benzoyl-6-hydroxy-5,6-
dihydrouridine (6). In contrast, 5-benzoyl-ribonolactone (7), a hypothetical product resulting from
C10-hydrogen atom abstraction by the peroxyl radical, could not be detected. Overall, tert-butyl
ketone 2b is a clean source of 50-benzoyl-5,6-dihydrouridin-6-yl (1b) and should prove useful for
studying the reactivity of the respective radical in RNA.
Introduction
come from its use as a tool for studying RNA structure,
folding dynamics, and protein binding.4-8 In this regard
RNA cleavage by hydroxyl radical (HO•) generated by
It is widely accepted that oxidative DNA damage is
associated with a variety of diseases and aging. Because of
its larger copy number and shorter lifetime in many cells,
RNA damage was perceived to be less significant biologi-
cally. This perception has begun to change as RNA oxida-
tion has been posited as playing a role in Alzheimer0s disease,
atherosclerosis, and inflamation.1-3 Although the apprecia-
tion of oxidative RNA damage as a cause and/or effect of
disease is growing, its greatest impact on biology thus far has
Fe EDTA or via pulse radiolysis is the most frequently
3
employed oxidant.9-13 These investigations rely upon the
accessibility of the biopolymer to the extremely reactive
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DOI: 10.1021/jo9012805
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J. Org. Chem. 2009, 74, 7007–7012 7007
2009 American Chemical Society

Newman et al.
JOCArticle
HO• to deduce 3-dimensional structure. Studies on the
effects of ionizing radiation on nucleic acids that employed
RNA as a substrate implicate a variety of possible pathways
by which HO• may produce direct strand breaks. Because of
the poor selectivity of this reactive oxygen species, it is
difficult to unequivocally characterize any one pathway.
We and other research groups have utilized organic chem-
istry to study nucleic acid oxidation by independently gen-
erating reactive intermediates in nucleosides and nucleotides
and at defined sites in oligonucleotides.14-19 This has been a
fruitful approach that has uncovered novel reaction path-
ways and helped resolve mechanistic controversies while
improving our overall understanding of biologically impor-
tant chemistry.20-26 Although most of these studies have
focused on DNA, there has been limited investigation of
RNA radical chemistry.27-29 The growing acceptance of the
importance of RNA oxidation has prompted us to turn our
attention to this nucleic acid molecule.
The primary structural difference between RNA and DNA
is the presence of the 20-hydroxyl group in the former. The 20-
hydroxyl group may have a significant effect on the reactivity
of RNA radicals in several ways. The C20-H bond dissociation
energy is significantly lower in RNA compared to that in
DNA. Computational experiments show that the C20-H bond
is the weakest carbohydrate carbon-hydrogen bond in nu-
cleic acids.30 In addition, the C20-hydroxyl group may facil-
itate heterolytic β-fragmentation from the C20-radical that is
analogous to those observed in C40-radicals in DNA.17,31 The
electronic effects of the 20-hydroxyl group may affect nucleic
acid radical reactivity in other more subtle, indirect ways.27
For instance, the altered conformation of RNA (A-form)
compared to DNA (B-form) may influence radical reactivity
by affecting the proximity of potential reactive centers to one
another. One similarity between RNA and DNA is that
nucleobase radicals resulting from hydroxyl radical addition
to the π-bonds of the Watson-Crick bases are believed to
account for more than 80% of the reactive intermediates
SCHEME 1. Generation of Nucleobase Radicals in RNA via
Hydroxyl Radical Addition
generated.11 Independent generation of 20-deoxypyrimidine
nucleobase radicals has revealed the formation of tandem
lesions via reaction with adjacent nucleotides but few if any
direct strand breaks.14,24,25,32,33 In contrast, analogous pyr-
imidine nucleobase radicals are believed to produce direct
strand breaks via intranucleotidyl and internucleotidyl hydro-
gen atom abstraction from the ribose rings.11-13 Given the
predominance and proposed distinctive reactivity of RNA
pyrimidine nucleobase radicals, we set out to independently
generate one of them in order to begin to gain insight into their
chemical behavior. Characterization of nucleotide radical
reactivity at specific sites in RNA could facilitate extracting
additional structural information from HO• cleavage experi-
ments and shed light on why RNA oxidation is deleterious in
cells. However, product analysis of radical reactions in bio-
polymers is challenging, and it is imperative to demonstrate
that the radical of interest is cleanly generated in solution at
the monomeric level before carrying out such studies.
Results and Discussion
Synthesis of a Photochemical Precursor to 5,6-Dihydrour-
idin-6-yl (1) and Product Standards. Photochemistry is a
desirable method for independently generating nucleic acid
radicals because this method provides temporal control, is often
compatible with aqueous conditions, and can be carried out
without exposing the biopolymer to conditions or reagents that
may randomly damage it. Hydroxyl radical preferentially adds
to the C5-position of pyrimidines (Scheme 1).11 We chose to
synthesize a C6-radical precursor (2), which generates the
analogous radical (1) resulting from the formal C5-hydrogen
atom addition product instead of HO• addition (eq 1). Hydro-
gen atoms are formed in lower yields by γ-radiolysis but add to
the pyrimidine double bonds and exhibit similar regioselectivity
as hydroxyl radical.34 Ketone 2 was targeted for synthetic
expediency. However, we cannot rule out that the absence of
the C5-hydroxyl group (β-position of the radical) would make
the radical less reactive. The Norrish type I photochemical
cleavage reaction was previously used when studying the role of
pyrimidine nucleobase radicals in DNA oxidation.24,25,32,35-38
Based upon this precedent and synthetic methods developed
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TABLE 1. Product Yields and Mass Balance from Photolysis of 2ba
SCHEME 2. Synthesis of Photochemical Radical Precursors 2a
and 2ba
% Yieldb
mass
balance (%)
conditions
aerobic
anaerobic 50.8 ( 1.2
4
5
6
2b
n.d.
7.1 ( 0.3 37.6 ( 0.1 41.5 ( 1.4 86.5 ( 1.6
n.d. n.d. 42.2 ( 1.1 93.6 ( 2.1
aPhotolyses carried out for 90 min in the presence of 0.5 mM β-
mercaptoethanol. bYields and mass balances are the average of 3 samples.
n.d. = not detected.
aReagents and conditions: (a) 5% Rh/Al2O3, H2 (45 psi), MeOH;
(b) (i) pTSA, dimethoxy propane, DMF, (ii) BzCl; (c) TFA/H2O (1:1).
Photochemical Generation of 5,6-Dihydrouridin-6-yl Radi-
cal (1b). As a prelude to using the tert-butyl ketone (2) to
characterize the reactivity of 5,6-dihydrouridin-6-yl (1) and as
a radical precursor in RNA, we needed to establish the fidelity
of the monomer0s photochemistry. In initial experiments we
characterized the mass balance of photolyses of 2b (e50 μM)
under aerobic and anaerobic conditions in the presence of a
low (0.5 mM) but excess concentration of β-mercaptoethanol
(Table 1). Photolyses were carried out to ∼60% conversion of
the ketone. Two products, the C6-hydrate (6) and uridine (5),
were detected under aerobic conditions in addition to starting
ketone (2b). The average mass balance was greater than 85%.
The mass balance is considerably lower under aerobic condi-
tions in the absence of thiol. A more complex mixture of
products is formed under these conditions, presumably as a
result of reactions between alkyl and peroxyl radicals.
by Tanaka et al., the synthesis of 2 was straightforward
(Scheme 2).39,40 The ribose protecting groups could be removed
under fairly strong acidic conditions due to the greater stability
of the glycosidic bond in the ribonucleoside compared to a 20-
deoxyribonucleoside.
To enhance the detection of the ketone and the products
(4-7) derived from 1 via HPLC using UV-detection, a benzoyl
group was installed at the 50-position (2b). The benzoyl deriva-
tives of the radical precursor (2b), 5,6-dihydrouridine (4), and
uridine (5) were prepared from the free ribonucleosides via a
two-pot procedure that did not require purification of the
intermediates.41 The C6-hydrate (6) was synthesized from 5 via
the respective bromohydrins, which were prepared using a
previously reported method.42,43 Only the major bromohydrin
diastereomer was debrominated using zinc in acetic acid, because
the C6-hydrate epimerizes in water. Finally, 5-benzoyl ribono-
lactone (7) was prepared via the reported procedure.44
The mass balance is even higher (>90%) under anaerobic
conditions, where the only product detected is the dihydrour-
idine (4) formed by hydrogen atom abstraction by 1b. The
requirement of O2 for the formation of the C6-hydrate (6) is
consistent with the formation of this product via the peroxyl
radical (8, Scheme 3) and not direct one-electron oxidation of
1b, followed by trapping by H2O.
In addition to determining the effect of O2 on the mass
balance and product distribution, we examined its effect on
the photochemical conversion of 2b (Figure 1) because dioxy-
gen quenches the excited states of triplet ketones.45 However,
we observed no qualitative difference in the photoconversion
of 2b as a function of time in the presence or absence of O2.
FIGURE 1. Photochemical conversion of 2b as a function of time
under aerobic and anaerobic conditions.
(39) Tanaka, H.; Hayakawa, H.; Miyasaka, T. Tetrahedron 1982, 38,
2635–2642.
(40) Tanaka, H.; Hayakawa, H.; Miyasaka, T. Chem. Pharm. Bull. 1981,
29, 3565–3672.
(41) Kumar, A.; Walker, R. T. Tetrahedron 1990, 46, 3101–3110.
(42) Cadet, J.; Ducolomb, R.; Teoule, R. Tetrahedron 1977, 33, 1603–
1615.
Furthermore, the ketone conversion was linear with respect to
time, even at high conversion. This is consistent with a zero-
order (nonchain) photochemical process.
(43) Carter, K. N.; Greenberg, M. M. Bioorg. Med. Chem. 2001, 9, 2341–
2346.
(44) Sa, M. M.; Silveira, G. P.; Castilho, M. S.; Pavao, F.; Oliva, G.
ARKIVOC 2002, 112–124.
(45) Turro, N. J. Modern Molecular Photochemistry; Benjamin Cum-
mings: Menlo Park, CA, 1978.
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SCHEME 3. Competitive Trapping of 1b by O2 and β-Mercaptoethanol
Reactivity of 50-Benzoyl-5,6-dihydrouridin-6-yl (1b) with
β-Mercaptoethanol. The competition between O2 and β-mer-
captoethanol (BME) was explored as a function of thiol con-
centration (Figure 2). The average slope of this line (6.6 ( 1.3 ꢀ
10-3 M-1) is equal to the ratio of the rate constants for trapping
1b by BME and O2 times the reciprocal of the O2 concentration
(0.2 mM, kRSH/(kO [O2]), eq 2). The rate constant for the
)
was estimated by assuming that the rate constant for O2 trapping
2
reaction of 1b with BME (kRSH = 2.6 ( 0.5 ꢀ 106 M-1 s-1
of the nucleoside radical (kO ) is 2ꢀ 109 M-1 s-1. This is more
2
than 3-fold slower than the respective rate constant measured for
the reaction of the 20-deoxynucleoside analogue of 1 with BME,
although it is not known why.35
FIGURE 2. Ratio of 5,6-dihydrouridine (4) to 6-hydroxy-5,6-dihy-
drouridine (6) as a function of BME concentration.
½4ꢁ kRSH½1bꢁ½BMEꢁ
¼
ð2Þ
½6ꢁ
kO ½1bꢁ½O2ꢁ
2
6 showed that the hydrate is stable under the reaction condi-
tions. Since benzoylated uridine (5) formation was observed
only under aerobic conditions we focused our attention on the
peroxyl radical (8) as its source. Formal elimination of the
hydroperoxyl radical, which deprotonates to superoxide at pH
7.2, from 7 would yield 50-benzoyl uridine. This process has
been observed for other peroxyl radicals but is typically slow
unless the incipient carbocation is especially stabilized.46-49 No
evidence for superoxide elimination was detected using either
epinephrine oxidation or cytochrome c reduction as a means
for indirect detection (data not shown). In addition, uridine
formation was efficiently quenched by β-mercaptoethanol at
concentrations much lower than those used to compete with O2
for 1b. However, the yield varied nonlinearly with thiol con-
centration (Figure 3). This indicated that the thiol did not trap
the peroxyl radical (7) in competition with a first-order process.
The behavior is suggestive of quenching a radical-radical
reaction, but we do not have any additional evidence for such
a process. Overall, these experiments reveal that 50-benzoyl
uridine (5) results from a radical process but its yield is reduced
to 1-2% at even very low concentrations of thiol.
Search for C10-Hydrogen Atom Abstraction by the C6-
Peroxyl Radical (8). 2-Deoxyribonolactone was produced in
low yield by the 20-deoxynucleoside analogue under aerobic
conditions.35 The postulated mechanism involved C10-hydrogen
atom abstraction by the respective peroxyl radical and subse-
quent transformation of the C10-radical via a known O2-depen-
dent mechanism to the lactone.46,47 Analysis for ribonolactone
(7) formation was carried out using aqueous (unbuffered)
acetonitrile (10% acetonitrile) because it was unstable in aqu-
eous buffer. We were unable to detect 5-benzoyl ribonolactone
(7) upon photolysis of 2b (85 μM) under aerobic conditions.
Using independently synthesized 7, we established the limit of
detection of the lactone in these experiments to be 5%. We
speculate that C10-hydrogen atom abstraction by the electro-
philic peroxyl radical (7) is even slower than the analogous
reaction in the deoxyribonucleoside system, due to the inductive
effect of the 20-hydroxyl group (Scheme 4).
Formation of 50-Benzoyl-uridine (5) from 5,6-Dihydrouri-
din-6-yl (1b). 50-Benzoyl-uridine (5) is observed under aero-
bic but not anaerobic conditions (Table 1), and its yield is as
high as ∼20% at low concentrations of BME (<1 mM).
Dehydration of the C6-hydrate was a possible source of this
product; as such molecules are reported to undergo this reac-
tion.11 However, examination of independently synthesized
Conclusions
These studies establish that 5,6-dihydrouridin-6-yl can
be generated cleanly via Norrish type I photocleavage.
The mass balances are high, and under anaerobic conditions
(46) Tallman, K. A.; Tronche, C.; Yoo, D. J.; Greenberg, M. M. J. Am.
Chem. Soc. 1998, 120, 4903–4909.
(47) Emanuel, C. J.; Newcomb, M.; Ferreri, C.; Chatgilialoglu, C. J. Am.
Chem. Soc. 1999, 121, 2927–2928.
(48) von Sonntag, C.; Schuchmann, H.-P. Angew. Chem., Int. Ed. 1991,
30, 1229–1253.
(49) Schuchmann, M. N.; Von Sonntag, C. Z. Naturforsch. B 1987, 42,
495–502.
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2.80-2.85 (m, 1H), 3.14-3.20 (m, 1H), 3.66-3.87 (m, 3H),
3.97-4.00 (m, maj) and 4.10-4.14 (m, min) (2H) and 5.27-5.30
(m,1H), 5.53-5.54 (d, J = 4.0 Hz, min) and 5.68-5.69 (m, maj)
(1H); 13C NMR (CDCl3) δ 26.4, 26.5, 33.2, 33.7, 42.9, 43.1, 52.8,
54.0, 60.9, 61.1, 69.6, 70.1, 71.4, 73.4, 83.5, 84.2, 89.0, 90.3,
153.8, 154.6, 168.35, 168.4, 211.4, 212.5. IR (neat) 1023.7 m,
1164.2 m, 1106.4 m, 1225.0 m, 1289.6 w, 1372.2, m, 1463.3 m,
1682.7 s, 1694.3 s, 1713.2 s, 2802.5 w, 2842.9 w, 2966.9 m, 3422.7
br s cm-1; HR-FABMS calculated for C14H23N2O7 (M þ Hþ)
331.1517, found 331.1526.
General Procedure for Preparation of 50-Benzoylated Com-
pounds. To a flame-dried round-bottom flask equipped with a
magnetic stir bar were added the ribonucleoside (1.0 equiv),
˚
p-toluenesulfonic acid monohydrate (0.23 equiv), and 4 A M.S.
The contents were diluted with DMF (2.9 mL/mmol) and
dimethoxy propane (3.9 equiv). The flask was heated to 45 °C
for 2 h, at which time the reaction mixture was cooled to room
temperature and Amberlyst A-21 resin (0.05 g/mmol) was added
to neutralize the acid. The mixture was stirred for 20 min and
then filtered through a pad of Celite. The Celite pad was washed
with methanol, and then the combined solvents were removed
under reduced pressure. The crude oil was used for the next step
without purification.
FIGURE 3. Dependence of uridine yield on β-mercaptoethanol con-
centration formed upon photolysis of 2b under aerobic conditions.
SCHEME 4. Hypothetical Formation of 5-Benzoyl Ribonol-
actone (7) from 8
Assuming 100% conversion in the first step, the crude
acetonide was diluted with pyridine (9.8 mL/mmol), and then
benzoyl chloride (1.5 equiv) was added dropwise over 5 min. The
reaction mixture was stirred for 17 h at room temperature, at
which time it was evaporated under reduced pressure. To ensure
the pyridine was removed, toluene (1 mL/mmol) was added and
removed under vacuum (ꢀ2). The material was then diluted
with dichloromethane and transferred to a separatory funnel
containing water. The layers were separated, and the dichloro-
methane was dried over magnesium sulfate, filtered, and con-
centrated under reduced pressure. The benzoylated acetonide
was used for the next step without further purification.
Again assuming100% conversion to the product, a 1:1mixture
of trifluoroacetic acid/water (10.8 mL/mmol) was added to the
crude benzoylated acetonide, and the mixture was stirred at room
temperature for 21 h. The water and TFA were removed under
reduced pressure. The final products were purified via flash
column chromatography (8% MeOH in chloroform, unless
specified otherwise) and isolated as white foams. When the
reaction was carried out using 5.0 g (20.4 mmol) of uridine,
5.16 g (72% yield) of the desired, previously reported product
50-benzoyl uridine (5) was obtained.41
in the presence of mM thiol 1-2% of uridine is produced via
an uncertain mechanism that involves the alkyl radical. The
reactivity of 5,6-dihydrouridin-6-yl radical is similar to that of
the 20-deoxyribonucleoside analogue. However, it is slightly
less reactive with thiol and intramolecular hydrogen atom
abstraction from the C10-position could not be detected. The
C20-position of RNA is expected to be more susceptible to
hydrogenatom abstraction thanitisinDNA, andit ispossible
some of the unaccounted for material (7-14%) in these
experiments is consumed by this or other pathways involving
intramolecular hydrogen atom abstraction.
Experimental Section
General Procedure for the Photolysis of 2b. Solutions for all
experiments were prepared in Eppendorf tubes to have the same
concentration of 2b (50 μM) in a 10% CH3CN/phosphate buffer
(10 mM, pH 7.2) mixture. The internal standard (dU) was added
to yield a final concentration of 17 μM. The solutions were then
vortexed and transferred into Pyrex tubes. Anaerobic photo-
lyses were carried out in sealed tubes that were subjected to
3 freeze-pump-thaw degas cycles. All tubes were placed at a
similar distance (ca. 5 cm) from the lamps and rotated during
irradiation (350 nm). Experiments were carried out in multiple
replicates (2 or 3). All photolysis samples were kept at room
temperature.
Preparation of 4. The reaction was carried out using 1.0 g
(4.06 mmol) of dihydrouridine, and 0.487 g (34% yield) of the
desired product was isolated. 1H NMR (CD3OD) δ 2.51-2.55
(m, 2H), 3.35-3.46 (m, 2H), 4.14-4.17 (m, 3H), 4.42-4.46 (dd,
1H, J = 4.8, 12 Hz), 4.60-4.64 (dd, 1H, J = 3.2, 12 Hz),
5.86-5.87 (d, 1H, J = 4.8 Hz), 7.49-7.53 (m, 2H), 7.61-7.66
(m, 1H), 8.04-8.05 (m, 2H); 13C NMR δ 30.5, 36.8, 64.2, 70.6,
71.2, 80.8, 89.0, 128.5, 129.3, 129.9, 133.2, 153.8, 166.4, 171.3;
IR 599 w, 713 m, 762 w, 906 w, 1047 m, 1118 m, 1179 w, 1276 s,
1316 w, 1376 m, 1377 m, 1602 w, 1699 br s, 1713 br s, 2986 w,
2999 w, 3116 w, 3258 br m cm-1; HR-FABMS calculated for
C16H19N2O7 (M þ Hþ) 351.1204, found 351.1182.
Preparation of 2a. To a high pressure flask equipped with a
magnetic stir bar was added a solution of 339 (1.4 g, 4.3 mmol) in
methanol (65 mL). Rh/Al2O3 (1.74 g, 0.85 mmol Rh) was then
added to the flask. The flask was placed under 45 psi H2
atmosphere and vented to release the pressure. This was re-
peated ꢀ3, and the fourth time, the flask was kept at 45 psi H2.
The reaction was allowed to stir at room temperature for 3 h.
The mixture was filtered through Celite and washed ꢀ3 with
methanol (∼300 mL). The solvent was removed under reduced
pressure, and purification was accomplished via flash column
chromatography (5% methanol in dichloromethane) to give
0.84 g (60% yield) of a 3.4:1 mixture of diastereomers of 2a as a
Preparation of 2b. The reaction was carried out using 100 mg
(0.303 mmol) of 2a, and 62.2 mg (70% yield) of the desired
product was isolated as a 5.9:1 mixture of diastereomers.
Purification was carried out via flash chromatography using
1
4% MeOH in chloroform. H NMR (CD3OD) δ 1.10 (s, min)
and 1.16 (s, maj) (1H), 2.60-2.67 (m, 1H), 3.05-3.15 (m, 1H),
4.02-4.21 (m, 3H), 4.39-4.45 (m, 1H), 4.55-4.58 (m, 1H),
4.91-4.93 (d, J = 7.6 Hz, min) and 4.98-5.00 (d, J = 7.6 Hz,
maj) (1H), 5.36-5.37 (d, J = 4.0 Hz, maj) and 5.46 (s, min) (1H),
7.46-7.53 (m, 2H), 7.59-7.66 (m, 1H), 8.03-8.05 (d, 2H, J =
7.2 Hz); 13C NMR (CD3OD) δ 27.2, 27.7, 35.2, 44.7, 56.7, 65.9,
1
white foam. H NMR (CD3OD) δ 1.24 (s) and 1.26 (s) (9H),
J. Org. Chem. Vol. 74, No. 18, 2009 7011

Newman et al.
JOCArticle
71.9, 72.9, 81.6, 94.9, 129.8, 129.9, 130.7, 130.8, 131.3, 134.6,
155.0, 167.9, 169.9, 170.1, 213.3; IR (neat) 584 w, 714 m, 763 w,
908 w, 969 w, 1026 m, 1069 m, 1109 m, 1179 w, 128 s, 1316 w,
1281 m, 1317 w, 1385 w, 1455 m, 1464 m, 1602 w, 1683 s, 1694 s,
1699 s, 1712 s, 1722 s, 2526 br m, 3409 br s cm-1
.
To a round-bottom flaskequipped witha magnetic stir bar were
added the less polar diastereomer of the bromohydrin (359 mg,
0.81 mmol) and a 3:1 mixture of THF/water (8 mL). The solution
was cooled to 0 °C, at which time Zn dust (75.8 mg, 1.16 mmol)
and acetic acid (85 μL, 1.47 mmol) were added. The ice bath was
removed, andthe reactionwas allowedtostiratroom temperature
for 22 h. The mixture was then filtered through a pad of Celite and
washed with methanol. After removing the solvent under reduced
pressure 6 (81 mg, 28%) was purified via flash column chromato-
graphy (7% MeOH in chloroform). 1H NMR (CD3OD) δ
2.53-2.57 (d, 1H, J = 16.8 Hz), 2.85-2.90 (m, 1H), 4.21-4.29
(m, 3H), 4.53-4.69 (m, 2H), 5.33 (s, 1H), 8.05-8.07 (d, J =
7.6 Hz), 7.49-7.51 (t, 2H, J = 7.6 Hz), 7.62-7.66 (m, 1H),
8.05-8.07 (d, 2H, J = 7.8 Hz); 13C NMR (CD3OD) δ 39.9,
65.7, 71.7, 74.4, 75.3, 82.5, 92.1, 129.8, 130.7, 131.2, 134.7, 154.2,
168.0, 171.4; IR (neat) 712 m, 760 w, 892 w, 1026 m, 1061 m, 1120
m, 1276 s, 1316 w, 1384 m, 1474 m, 1601 w, 1701 br s, 3295 v br m
cm-1; HR-FABMS calculated for C16H18N2O8 349.1063, found
(M - OHþ) 349.1029.
12372 m, 1463 m, 1602 w, 1716 br s, 2971 m, 3247 br m cm-1
;
HR-FABMS calculated for C21H27N2O8 (M þ Hþ) 435.1697,
found 435.1759.
Preparation of 6. To a round-bottom flask equipped with a
magnetic stir bar was added the benzoylated uridine (1.0 g,
2.87 mmol) and a 1:1 mixture of THF/water (80 mL). The mixture
was cooled to 0 °C, at which time NBS (0.612 g, 3.44 mmol) and
calcium carbonate (0.43 g, 4.3 mmol) were added. The ice bath was
removed, and the reaction was allowed to stir at room temperature
for 4 h. The crude reaction mixture was then filtered through a pad
of Celite, washed with methanol (∼200 mL), and concentrated
under reduced pressure. Purification was done via flash column
chromatography using a solvent gradient from 2% MeOH in
chloroform to 5% MeOH in chloroform to give 578 mg (45% yield)
of the less polar diastereomer and 155 mg (12% yield) of the more
polar diastereomer. Less polar diastereomer: 1H NMR (CD3OD) δ
4.22-4.39 (m, 4H), 4.59-4.70 (m, 2H), 5.36 (s, 1H), 5.84 (s, 1H),
7.51-7.58 (m, 2H), 7.65-7.69 (m, 1H), 8.08-8.15 (m, 2H); 13C
NMR (CD3OD) δ 40.3, 64.0, 70.3, 73.9, 77.0, 80.9, 89.8, 128.4,
129.2, 129.8, 133.2, 151.1, 166.45, 166.59; IR (neat) 567 w, 714 m,
763 w, 905 w, 1072 m, 1118 m, 1179 w, 1281 m, 1317 w, 1385 w,
1455 m, 1464 m, 1602 w, 1683 s, 1695 s, 1699 s, 1714 s, 1733 s, 2534 br
m, 3440 br s cm-1; HR-FABMS calculated for C16H17BrN2O8
427.0168, found (M - OH) 427.0144. More polar diastereomer: 1H
NMR (CD3OD) δ 4.20-4.36 (m, 3H), 4.40-4.42 (m, 1H),
4.54-4.58 (dd, 1H, J = 5.2, 12 Hz), 4.69-4.72 (dd, 2H, J = 2.8,
6.8 Hz), 5.33 (d, 1H, J = 2.4 Hz), 5.85-5.86 (d, 1H, J = 3.6 Hz),
7.52-7.56 (m, 2H), 7.64-7.69 (m, 1H), 8.10-8.12 (d, 1H, J =
7.2 Hz); 13CNMR(CD3OD) δ41.3, 65.3, 71.4, 74.4, 78.4, 79.5, 81.6,
91.4, 129.7, 130.1, 130.69, 130.74, 130.8, 131.3, 134.6, 152.2, 167.9,
168.0; IR (neat) 554 w, 713 m, 762 m, 906 w, 1027 m, 1118 m, 1179 w,
Acknowledgment. We are grateful for support of this
research from the National Institute of General Medical
Sciences (GM-054996). J.T.S. thanks The Johns Hopkins
University for a Sonneborn fellowship. M.J.E.R. thanks the
NIGMS for a Research Supplement to Promote Diversity in
Health-Related Research.
Supporting Information Available: General experimental
methods, NMR spectra of previously unreported molecules
and 7, and a table of HPLC response factors. This material
is available free of charge via the Internet at http://pubs.acs.org.
7012 J. Org. Chem. Vol. 74, No. 18, 2009