Chemical evidence for thiyl radical addition to the C6-position of a pyrimidine nucleoside and its possible relevance to DNA damage amplification

Full text of DOI:10.1021/jo0007433

J . Org. Chem. 2000, 65, 8375-8378
8375
Sch em e 1
Ch em ica l Evid en ce for Th iyl Ra d ica l
Ad d ition to th e C6-P osition of a P yr im id in e
Nu cleosid e a n d Its P ossible Releva n ce to
DNA Da m a ge Am p lifica tion
K. Nolan Carter,† Thomas Taverner,‡
Carl H. Schiesser,*,‡ and Marc M. Greenberg*,†
Department of Chemistry, Colorado State University,
Fort Collins, Colorado 80523, and School of Chemistry,
The University of Melbourne, Victoria 3010, Australia
Sch em e 2
mgreenbe@lamar.colostate.edu
Received May 15, 2000
straction by thiyl radicals has been appreciated for almost
half a century,5a,9 and has been receiving increased
notoriety due to the ability of these species to abstract
hydrogen atoms from carbon-hydrogen bonds during
enzyme mediated processes.5d,10 Rapid and reversible
addition of thiyl radicals to alkenes is probably the most
well studied family of reactions involving thiyl radicals
(Scheme 2).11 Although the equilibrium constants for
these additions are typically less than unity, the â-sulfido
alkyl radicals can be trapped by O2. Of particular
relevance to the current discussion is the isolation of
thiol-pyrimidine nucleobase photoaddition products.12
These nucleoside adducts are believed to result from
predominant addition of the electrophilic thiyl radical to
the more electron rich C5-position of the pyrimidine.
Thiyl radical addition to the C6-position of the pyrim-
idines has been inferred, but the expected 5,6-dihydro-
pyrimidine products resulting from addition to this
position have not been isolated.13 The absence of such
products can be rationalized by extrapolating from the
unstable nature of the respective C6-hydrates which
readily undergo dehydration to reconstitute the nucleo-
bases, and/or fragmentation.1a,14,15
Thiols are potent antioxidants and have been used as
radioprotecting agents on account of their proficiency to
act as hydrogen atom donors in radical reactions and to
participate in electron-transfer processes.1,2 Perhaps less
well appreciated are observations that link physiological
levels of thiols to mutagenicity in bacteria.3,4 Under some
conditions, thiols are believed to enhance DNA damage
through their involvement in the formation of reactive
oxygen species.2 Alternatively, one could consider the
possibility that thiols and/or a species derived from them
react directly with DNA. In this regard, there is chemical
precedent which suggests that under the appropriate
conditions thiyl radicals may abstract hydrogen atoms
from carbon-hydrogen bonds or add to π-bonds.5,6 We
wish to report chemical evidence for thiyl radical addition
to the C6-position of a pyrimidine double bond, resulting
in the formation of an intermediate 5,6-dihydropyrimidin-
5-yl radical. Based upon investigations of the interaction
of DNA with ionizing radiation, where the similar radical,
5,6-dihydrothymidin-5-yl (1, Scheme 1), has been shown
to participate in a novel DNA damage pathway that
involves the formation of a tandem lesion, this result
presents the possibility that thiols may initiate DNA
damage by adding to a pyrimidine double bond.7,8
Resu lts a n d Discu ssion
The involvement of thiyl radicals in reaction processes
is gaining increasing recognition. Hydrogen atom ab-
Syn th esis a n d Ap p lica tion of a P r obe for Th iyl
Ra d ica l Ad d ition to th e C6-P osition of a P yr im i-
d in e. Given the anticipated instability of C6-pyrimidine-
thiyl radical adducts (2), we sought to provide evidence
for this pathway by designing a substrate that would
leave a fingerprint of the initial thiyl radical addition,
regardless of whether a formal addition product could be
isolated (which we considered unlikely). Consequently,
† Colorado State University.
‡ The University of Melbourne.
(1) (a) von Sonntag, C. The Chemical Basis of Radiation Biology;
Taylor & Francis: New York, 1987. (b) Savoye, C.; Swenberg, C.;
Hugot, S.; Sy, D.; Sabattier, R.; Charlier, M.; Spotheim-Maurizot, M.
Int. J . Radiat. Biol. 1997, 71, 193. (c) Prise, K. M.; Gillies, N. E.;
Whelan, A.; Newton, G. L.; Fahey, R. C.; Michael, B. D. Int. J . Radiat.
Biol. 1995, 67, 393.
(2) (a) Becker, D.; Summerfield, S.; Gillich, S.; Sevilla, M. D. Int. J .
Radiat. Biol. 1994, 65, 537. (b) Pru¨tz, W. A.; Mo¨nig, H. Int. J . Radiat.
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Lawrence, C. C.; Stubbe, J . Curr. Opin. Chem. Biol. 1998, 2, 650.
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W. Biochemistry 1994, 33, 9875.
(6) (a) Breen, A. P.; Murphy, J . A. J . Chem. Soc., Perkin Trans. 1
1993, 2979. (b) Chatgilialoglu, C.; Guerra, M. In Supplement S: The
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Matray, T. J .; Tronche, C.; Venkatesan, H. J . Am. Chem. Soc. 1997,
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10.1021/jo0007433 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/28/2000

8376 J . Org. Chem., Vol. 65, No. 24, 2000
Notes
Sch em e 3
we sought to take advantage of the rapid cyclopropyl
carbinyl radical rearrangement (Scheme 3).16 We antici-
pated that even if the initial product (5) derived from the
C6-thiyl adduct (4) was unstable, the cleaved cyclopro-
pane ring would serve as an indication of radical addition
to the C6-position of the pyrimidine ring. A phenylcyclo-
propane was chosen as a trigger following consideration
of previous measurements made on reactions of alkenes
with thiyl radicals. Using these reports as a guide, we
assumed that the rate of elimination of a thiyl radical
Sch em e 4a
from 4 would be e108 s-1 11
A phenyl-substituted cyclo-
.
propane was incorporated into the radical substrate in
order to maximize the probability that the reverse
reaction (k-1, Scheme 3) would not compete with ring
opening (k3). The trans-phenylcyclopropyl carbinyl radical
rearrangement occurs with a rate constant equal to 3 ×
1011 s-1 at 25 °C.16 Although conflicting reports on the
effects of substituents on the rate of cyclopropyl carbinyl
radical ring opening have appeared, any retardation was
expected to be significantly less than 103 s-1. Hence,
regardless of the effect (if any) of the substituents on the
rate of cyclopropyl carbinyl radical ring opening of 4, we
were confident that k3 would be significantly larger than
17
k-1
.
The cyclopropane substrate (3) was prepared as a
single diastereomer from 5-trans-styrenyl-2′-deoxy-
uridine.18 Following bis-silylation, cyclopropanation (60%)
was carried out using a modified Simmons-Smith pro-
cedure.19 The bis-silylated nucleoside (3) was employed
as a substrate in order to facilitate isolation of potential
products. Extended reaction of 3 with â-mercaptoethanol
(BME) in the presence of azo initiator (VAZO-52) in
a
Reagents: (a) s-BuLi, DMPU, hydrocinnamoyl chloride, THF,
-78 °C; (b) PhSeCl, pyridine, CH2Cl2; (c) H2O2, CH2Cl2; (d) NaBH4,
CeCl3‚7H2O, MeOH; (e) (i) MsCl, Et3N, CH2Cl2; (ii) BME, K2CO3,
DMF.
lowing reduction of 9 under Luche conditions, the respec-
tive crude mesylate was displaced by BME to afford a
diastereomeric mixture of 6.
Rin g-Op en ed Nu cleosid e P r od u ct 6 Is P r od u ced
via Th iyl Ad d ition to th e C6-P osition of 3. Ring-
opened product 6 is the formal product resulting from
direct attack of the thiyl radical derived from â-mercap-
toethanol at C1 of the cyclopropane, followed by thiol
trapping of the benzyl radical (eq 1). High-level compu-
benzene (50 °C) provided a single isolated product in 46%
yield. Spectral analysis of this product indicated that it
was a mixture of diastereomers of 6. The assignment of
the isolated product as 6 was confirmed via its indepen-
dent synthesis starting from the bis-5′,3′-O-tert-butyldi-
methylsilyloxy ether of 5,6-dihydro-2′-deoxyuridine (7,
Scheme 4). The dianion of 7 was acylated with hydro-
cinnamoyl chloride.20 The readily epimerizable center in
8 was then quaternized and the resulting phenyl selenide
was subjected to oxidative elimination conditions. Fol-
tational studies indicate that release of cyclopropane
strain energy may facilitate thiyl attack on a carbon-
carbon bond.21 However, low product yields and poor
regioselectivity observed in reactions of phenylcyclopro-
pane and trans-2-methyl-1-phenylcyclopropane indicate
(16) (a) Newcomb, M. Tetrahedron 1993, 49, 1151. (b) Newcomb,
M.; J ohnson, C. C.; Manek, M. B.; Varick, T. R. J . Am. Chem. Soc.
1992, 114, 10915.
(17) (a) Horner, J . H.; Tanaka, N.; Newcomb, M. J . Am. Chem. Soc.
1998, 120, 10379. (b) Beckwith, A. L. J .; Bowry, V. W. J . Am. Chem.
Soc. 1994, 116, 2710.
(18) (a) Selvidge S. D., III; J ang, J .; Ray, P. S. Nucleosides Nucle-
otides 1997, 16, 2019. (b) Bigge, C. F.; Kalaritis, P.; Deck, J . R.; Mertes,
M. P. J . Am. Chem. Soc. 1980, 102, 2033.
(19) Yang, Z.; Lorenz, J . C.; Shi, Y. Tetrahedron Lett. 1998, 39, 8621.
(20) Tanaka, H.; Hayakawa, H.; Ijima, S.; Haraguchi, K.; Miyasaka,
T. Tetrahedron 1985, 5, 861.
(21) QCISD/aug-cc-pVDZ//MP2/aug-cc-pVDZ calculations reveal that
homolytic substitution by methylthiyl radical at the carbon atom in
cyclopropane is predicted to be exothermic and to have an associated
energy barrier of about 90 kJ mol-1. In contrast, reaction between
methylthiyl radical and ethane is calculated to be endothermic at the
same level of theory and to have an associated energy barrier of about
215 kJ mol-1 higher.

Notes
J . Org. Chem., Vol. 65, No. 24, 2000 8377
temperature and stirred overnight. The reaction was diluted
with EtOAc, washed with H2O and brine, dried over Na2SO4,
and concentrated in vacuo. The crude reaction mixture was
purified via silica gel flash chromatography (10-40% EtOAc/
CH2Cl2) to afford a diastereomeric mixture of the selenylated
ketone (276 mg, 47%) as a yellow foam. The phenyl selenide (50
mg, 0.0670 mmol) was dissolved in CH2Cl2 (1 mL) and cooled to
0 °C. A 30% solution of H2O2 (7 µL) in H2O was added, and the
reaction was allowed to warm to ambient temperature overnight.
The reaction was taken up in Et2O, washed with saturated
NaHCO3, H2O, brine, dried over Na2SO4, and concentrated in
vacuo. The crude reaction mixture was purified via silica gel
flash chromatography (10-30% EtOAc/Hexanes) to afford 9 (36
mg, 91%) as a white foam: 1H NMR (CDCl3) δ 8.84 (s, 1 H),
7.28-7.38 (m, 5 H), 6.24 (dd, J ) 7.8, 7.6 Hz, 1 H), 4.44-4.42
(m, 1 H), 4.07-4.05 (m, 1 H), 3.84-3.81 (m, 2 H), 3.36 (t, J )
6.9 Hz, 2 H), 2.97 (t, J ) 7.8 Hz, 2 H), 2.42-2.40 (m, 1 H), 2.09-
2.07 (m, 1 H), 0.92-0.86 (m, 18 H), 0.12-0.10 (m, 12 H); IR (film)
that it is unlikely that homolytic substitution of the
cyclopropane in 3 yields the observed product 6.22 Fur-
thermore, thiyl radicals typically add into the alkene
group of vinyl cyclopropanes, as opposed to attacking the
cyclopropane component.6 Another mechanistic alterna-
tive to explain the formation of 6, nucleophilic addition
of the thiolate to 3 can be ruled out on the basis that no
reaction occurs between 3 and BME in the absence of
the azo initiator under these conditions.23 We propose
that 6 is derived from 5, and that its formation is driven
by reconstitution of the uracil base. Although we have
no evidence for the mechanism of the transformation of
5 into 6 at this time, we envision 3 possibilities. Rear-
rangement could occur stepwise by an ionic pathway in
which thiolate is initially eliminated to form the reso-
nance stabilized carbocation. This pathway is analogous
to that postulated for the decomposition of pyrimidine
C6-hydrates and C6-hydroperoxides, and under the
relatively elevated temperature (50 °C) would be expected
to occur at a reasonable rate.14,15 Alternatively, 6 can be
produced from 3 in a single step by either a SN2′ pathway,
or its radical equivalent.
3061, 2953, 2856, 1702,1471 cm-1
.
â-Mer ca p toeth a n ol Nu cleosid e Ad d u ct (6). To a solution
of 9 (36 mg, 0.061 mmol) and CeCl3‚7H2O in MeOH (0.6 mL)
was added NaBH4 (2.5 mg, 0.067 mmol). The reaction was
stirred for 15 min, taken up in Et2O, washed with saturated
NH4Cl, H2O, and brine, dried over Na2SO4, and concentrated
in vacuo. The crude reaction mixture was purified via silica gel
flash chromatography (15-30% EtOAc/Hexanes) to afford the
alcohol (21 mg, 58%) as a clear oil: 1H NMR (300 MHz, CDCl3)
δ 8.19 (s, 1 H), 7.51 (d, J ) 5.1 Hz), 7.27-7.16 (m, 6 H), 6.30-
6.25 (m, 1 H), 4.45-4.39 (m, 2 H), 3.95-3.94 (m, 1 H), 3.79-
3.72 (m, 2 H), 2.88-2.68 (m, 3 H), 2.29-1.94 (m, 4 H), 0.93-
0.91 (m, 18 H), 0.11-0.083 (m, 6 H); IR (Film) 3456, 3181, 3027,
2857, 1693, 1471 cm-1. The alcohol (150 mg, 0.254 mmol) was
dissolved in CH2Cl2 (2 mL) and cooled to O °C. Triethylamine
(41 mg, 0.40 mmol), and then MsCl (44 mg, 0.38 mmol) was
added. The reaction was stirred for 2.5 h at 0 °C, quenched with
saturated NaHCO3. The crude muxture was taken up in Et2O,
washed with H2O and brine, dried over Na2SO4, and concen-
trated in vacuo. The crude product was immediately dissolved
in DMF (2 mL) with K2CO3 (62 mg, 0.44 mmol) and â-mercap-
toethanol (34.5 mg, 0.44 mmol) and stirred at room-temperature
overnight. The reaction was taken up in taken up in Et2O,
washed with H2O, brine, dried over Na2SO4, and concentrated
in vacuo. The crude reaction mixture was purified via silica gel
flash chromatography (10-40% EtOAc/Hexanes) to afford 6 (51
mg, 31%) as a clear oil: 1H NMR (CDCl3) δ 9.48-9.46 (m, 1 H),
7.51 (s, 1 H), 7.30-7.18 (m, 5 H), 6.34-6.30 (m, 1 H), 4.43-4.42
(m, 1 H), 4.01-3.97 (m, 2 H), 3.81-3.77 (m, 4 H), 2.81-2.70 (m,
4 H), 2.32-2.30 (m, 1 H), 2.17-2.01 (m, 3 H), 0.91-0.89 (m, 18
H), 0.13-0.097 (m, 12 H); 13C NMR (CDCl3) 163.2, 163.0, 149.7,
141.0, 137.2, 136.9, 128.6, 128.5, 126.3, 116.3, 116.1, 88.2, 88.1,
85.8, 85.6, 72.8, 72.6, 63.5, 63.4, 61.5, 61.4, 41.3, 41.1, 40.8, 40.2,
37.0, 36.9, 35.4, 35.2, 34.1, 26.3, 26.0, 18.7, 18.3, -4.3, -4.5,
Su m m a r y
These studies support the proposal that a thiyl radical
can add to the C6-position of a pyrimidine nucleoside.
The potential significance of this observation with regard
to naturally occurring DNA damage and the design of
agents to do so which utilize this pathway remains to be
explored.
Exp er im en ta l Section
All reactions were carried out in oven-dried glassware under
an atmosphere of argon or nitrogen unless otherwise noted. THF
was freshly distilled from Na0/benzophenone ketyl. Pyridine,
methylene chloride, DMF, benzene, methanol, triethylamine,
and methane sulfonyl chloride were distilled from CaH2. â-Mer-
captoethanol was distilled from itself.
Dih yd r oa r yl Keton e 8. To a solution of TBDMS-protected
5,6-dihyrodo-2′-deoxyuridine (7, 2.00 g, 4.36 mmol) in THF at
-78 °C was added 10.0 mL (3.0 equiv) of a 1.3 M solution of
s-BuLi in cyclohexane. The reaction was stirred 1 h at -78 °C,
and DMPU (2.78 g, 21.8 mmol) was added. After 15 min,
hydrocinnamoyl chloride (1.46 g, 8.72 mmol) was added. The
reaction was stirred overnight and allowed to warm to ambient
temperature. The reaction was quenched with saturated aqueous
NH4Cl, taken up in EtOAc, washed with H2O and brine, dried
over Na2SO4, and concentrated in vacuo. The crude product was
purified via silica gel flash chromatography (20-50% EtOAc/
hexanes) to afford 8 (1.94 g, 75%) as a tautomeric/diastereomeric
-4.9, -5.0; IR (film) 3418, 3186, 2857, 1713, 1682, 1496 cm-1
HRMS (FAB) calcd 651.3319 (M + H), found 651.3301.
;
P h en ylcyclop r op a n e Nu cleosid e Su bstr a te (3). A solution
of diethylzinc (8.98 mL, 1.0 M, 5.0 equiv) in hexanes was added
to CH2Cl2 (9 mL) and the resulting solution cooled to 0 °C. A
solution of TFA (1.02 g, 8.98 mmol) in CH2Cl2 (4 mL) was added
very slowly via syringe. After 20 min of stirring, a solution of
CH2I2 (2.40 mL, 8.98 mmol) in CH2Cl2 (4 mL) was added. After
an additional 20 min of stirring, a solution of 5′,3′-bis-O-tert-
butyldimethylsilyloxy-5-styrenyldeoxyuridine18 (1.00 g, 1.80 mmol)
in CH2Cl2 (4 mL) was added. The reaction was allowed to warm
to ambient temperature and, after 20 h, quenched with saturated
NH4Cl and diluted with hexanes. The aqueous layer was
extracted with hexanes, and the combined organic extracts were
washed with brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was purified via silica gel flash
chromatography (5-50% EtOAc/Hex) to yield 3 (611 mg, 60%)
as a white foam: 1H NMR (CDCl3) δ 8.26 (s, 1 H), 7.39-7.16
(m, 6 H), 6.31 (m, 1 H), 4.44-4.41 (m, 1 H), 3.98-3.96 (m, 1 H),
3.82-3.79 (m, 2 H), 2.29-2.26 (m, 2 H), 2.04-1.92 (2, H), 1.45-
1.42, (m, 1 H), 1.28-1.22 (m, 1 H), 0.91 (s, 18 H), 0.089 (s, 12
H); 13C NMR (CDCl3) δ 163.1, 163.0, 150.0, 141.9, 134.8, 128.5,
126.5, 126.3, 126.0, 115.4, 88.2, 87.9, 85.5, 85.3, 72.8, 72.4, 63.4,
63.2, 41.5, 41.3, 26.2, 26.0, 24.5, 24.2, 21.0, 20.8, 18.7, 18.3, 15.0,
1
mixture:; H NMR (CDCl3) δ 7.67 (s, 1 H), 7.32-7.17 (m, 5 H),
4.42-3.36 (m, 1 H), 3.91-3.45 (m, 5 H), 3.10-2.90 (m, 3 H),
2.65-2.50 (m, 1 H), 2.19-2.16 (m, 1 H), 1.98-1.84 (m, 2 H), 0.91
(s, 18 H), 0.09-0.01 (m, 12 H); IR (film) 3208, 3064, 2857, 1713,
1471, 1454, 1362 cm-1
.
Ar yl Keton e 9. To a solution of phenylselenyl chloride (164
mg, 0.857 mmol) in CH2Cl2 (7 mL) at O °C was added pyridine
(68 mg, 0.857 mmol). The reaction was stirred at 0 °C for 15
min, and a solution of 8 (460 mg, 0.779 mmol) in CH2Cl2 (3 mL)
was added. The reaction was allowed to warm to ambient
(22) Reaction of the BME thiyl radical (2-hydroxyethylthiyl) with
phenylcyclopropane and trans-2-methyl-1-phenylcyclopropane produces
ring opened products in low yields (<1%). Furthermore, the regio-
selectivity for attack at C2/C3 in and trans-2-methyl-1-phenylcyclo-
propane (2:1) is much lower than that observed for reaction with 3.
(23) For a postulated example of such a process, see: Vega, E.; Rood,
G. A.; de Waard, E. R.; Pandit, U. K. Tetrahedron 1991, 47, 4361.

8378 J . Org. Chem., Vol. 65, No. 24, 2000
Notes
14.4, - 4.3, - 4.5, -5.1; IR (film) 3184, 2856, 2359, 1686 cm-1
HRMS (FAB) calcd. 573.3180 (M + H), found 573.3168.
;
85.6, 72.8, 72.6, 63.6, 63.5, 61.5, 61.3, 41.3, 41.1, 40.9, 40.4, 37.0,
36.8, 35.5, 35.4, 34.1, 26.3, 26.2, 26.0, 18.7, 18.3, -4.4, -4.5,
-4.9,-5.0; IR (film) 3410, 3189, 2857, 1713, 1681, 1496 cm-1
HRMS (FAB) calcd 651.3319 (M + H), found 651.3324.
;
â-Mer ca p toeth a n ol Nu cleosid e Ad d u ct (6) via Th er -
m olysis of 3. Phenylcyclopropane 3 (50 mg, 0.088 mmol), BME
(137 mg, 1.75 mmol), and Vazo 52 (2.2 mg, 0.0088 mmol) were
dissolved in benzene (1 mL), sparged with N2 for 20 min, and
heated to 50 °C. Additional Vazo 52 (2.2 mg, 0.0088 mmol) and
BME (137 mg, 1.75 mmol) were added after 24 h. The reaction
was stirred for an additional 24 h, cooled to ambient tempera-
ture, taken up in Et2O, washed with H2O and brine, dried over
Na2SO4, and concentrated. The crude product was purified via
silica gel flash chromatography (10-50% EtOAc/hexanes) to
yield unreacted 3 (42 mg, 84%) and 6 (4.2 mg, 46% based upon
unrecovered starting material) as a clear residue: 1H NMR
(CDCl3) δ 8.35 (s, 1 H), 7.58 (s, 1 H), 7.30-7.15 (m, 5 H), 6.27 (t,
J ) 6 Hz, 1 H), 4.40-4.38 (m, 1 H), 3.96-3.91 (m, 2 H), 3.75-
3.73 (m, 4 H), 2.81-2.66 (m, 5 H), 2.30-1.93 (m, 3 H), 0.91 (m,
18 H), 0.91 (m, 12 H); 13C NMR (CDCl3) 162.8, 162.7, 149.6,
141.1, 137.3, 137.0, 128.7, 128.6, 126.4, 115.9, 88.3, 88.1, 85.8,
Ack n ow led gm en t. Financial support of this work
from the National Institutes of Health (GM-54996) and
the Melbourne Advanced Research Computing Center
is appreciated. M.M.G. is grateful for a fellowship from
the Alfred P. Sloan foundation.
Su p p or tin g In for m a tion Ava ila ble: Gaussian archive
1
entries for all optimizations in this study and H NMR spectra
of compounds 3, 6, 8, and 9. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O0007433