Independent generation of the 5-hydroxy-5,6-dihydrothymidin-6-yl radical and its reactivity in dinucleoside monophosphates

Article abstract of DOI:10.1021/ja048492t

Hydroxyl radical is a major reactive oxygen species produced by γ-radiolysis of water or Fenton reaction. It attacks pyrimidine bases and gives the 5-hydroxy-5,6-dihydropyrimidin-6-yl radical as the major product. Here we report the synthesis of all four

Full text of DOI:10.1021/ja048492t

Published on Web 09/22/2004
Independent Generation of the
5-Hydroxy-5,6-dihydrothymidin-6-yl Radical and Its Reactivity
in Dinucleoside Monophosphates
Qibin Zhang and Yinsheng Wang*
Contribution from the Department of Chemistry-027, UniVersity of California at RiVerside,
RiVerside, California 92521-0403
Received March 15, 2004; E-mail: yinsheng.wang@ucr.edu
Abstract: Hydroxyl radical is a major reactive oxygen species produced by γ-radiolysis of water or Fenton
reaction. It attacks pyrimidine bases and gives the 5-hydroxy-5,6-dihydropyrimidin-6-yl radical as the major
product. Here we report the synthesis of all four stereoisomers of 5-hydroxy-6-phenylthio-5,6-dihydro-
thymidine (T*), which, upon 254 nm UV irradiation, give rise to the 5-hydroxy-5,6-dihydrothymidin-6-yl radical
(I). We also incorporated the photolabile radical precursors into dinucleoside monophosphates d(GT*) and
d(TT*) and characterized major products resulting from the 254-nm irradiation of these dinucleoside
monophosphates. Our results showed that, under anaerobic conditions, the most abundant product
emanating from the 254-nm irradiation of d(GT*) and d(TT*) is an abasic site lesion. Products with the
thymine portion being modified to thymine glycol and 5-hydroxy-5,6-dihydrothymine were also observed.
In addition, we demonstrated that radical I can attack the C8 carbon atom of its 5′ neighboring guanine
and give rise to a novel cross-link lesion. Moreover, LC-MS/MS results showed that γ-radiation of d(GT)
under anaerobic condition yielded the same type of cross-link lesions.
dihydropyrimidin-6-yl radical,11 and pyrimidin-5-methyl
radical,12-15 or from the deoxyribose ring, such as radicals on
Introduction
Oxidative DNA damage plays an important role in a number
of pathological conditions, including cancer,1 neurodegenera-
tion,2 and aging.3 Hydroxyl radical is one of the major reactive
oxygen species (ROS) formed from Fenton reaction4-6 or the
γ-radiolysis of water.7 It is well-documented that hydroxyl
radical reacts with thymine either by adding to the C5dC6
double bond or by abstracting a hydrogen atom from the methyl
group (Scheme 1a). The addition to the C5 carbon atom,
however, is the major pathway (60%).7 The secondary radicals
formed from the hydroxyl radical attack may further decompose
or react with neighboring nucleoside to form stable diamagnetic
products.
the C1′,16-21 C3′,22 and C4′ 23-29 carbon atoms, have been
synthesized, and their reactivities have been examined. Recently,
we reported the independent generation of the 5-methyl radical
of 5-methyl-2′-deoxycytidine and the facile formation of an
(11) Barvian, M. R.; Barkley, R. M.; Greenberg, M. M. J. Am. Chem. Soc. 1995,
117, 4894-4904.
(12) Anderson, A. S.; Hwang, J. T.; Greenberg, M. M. J. Org. Chem. 2000, 65,
4648-4654.
(13) Bellon, S.; Ravanat, J. L.; Gasparutto, D.; Cadet, J. Chem. Res. Toxicol.
2002, 15, 598-606.
(14) Romieu, A.; Bellon, S.; Gasparutto, D.; Cadet, J. Org. Lett. 2000, 2, 1085-
1088.
(15) Zhang, Q.; Wang, Y. J. Am. Chem. Soc. 2003, 125, 12795-12802.
(16) Gimisis, T.; Chatgilialoglu, C. J. Org. Chem. 1996, 61, 1908-1909.
(17) Goodman, B. K.; Greenberg, M. M. J. Org. Chem. 1996, 61, 2-3.
(18) Chatgilialoglu, C.; Gimisis, T.; Guerra, M.; Ferreri, C.; Emanuel, C. J.;
Horner, J. H.; Newcomb, M.; Lucarini, M.; Pedulli, G. F. Tetrahedron Lett.
1998, 39, 3947-3950.
The independent generation of reactive intermediates of
nucleosides, especially those from their photolabile precursors,
facilitates the examination of the roles of these reactive
intermediates in DNA damage.8 Precursors for a number of
radical intermediates, which are generated either on the pyri-
midine base, such as pyrimidin-6-yl radical,9,10 5-hydroxy-5,6-
(19) Gimisis, T.; Ialongo, G.; Chatgilialoglu, C. Tetrahedron 1998, 54, 573-
592.
(20) Tronche, C.; Goodman, B. K.; Greenberg, M. M. Chem. Biol. 1998, 5,
263-271.
(21) Hwang, J.-T.; Greenberg, M. M. J. Am. Chem. Soc. 1999, 121, 4311-
4315.
(22) Koerner, S.; Bryant-Friedrich, A.; Giese, B. J. Org. Chem. 1999, 64, 1559-
1564.
(1) Dizdaroglu, M. NATO ASI Ser., Ser. A 1999, 302, 67-87.
(2) Rolig, R. L.; McKinnon, P. J. Trends Neurosci. 2000, 23, 417-424.
(3) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239-247.
(4) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine,
2nd ed.; Oxford University Press: New York, 1989.
(5) Halliwell, B.; Gutteridge, J. M. C. FEBS Lett. 1992, 307, 108-112.
(6) Henle, E. S.; Linn, S. J. Biol. Chem. 1997, 272, 19095-19098.
(7) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor &
Francis: London, 1987.
(8) Greenberg, M. M. Chem. Res. Toxicol. 1998, 11, 1235-1248.
(9) Carter, K. N.; Greenberg, M. M. J. Org. Chem. 2003, 68, 4275-4280.
(10) Carter, K. N.; Greenberg, M. M. J. Am. Chem. Soc. 2003, 125, 13376-
13378.
(23) Giese, B.; Dussy, A.; Elie, C.; Erdmann, P.; Schwitter, U. Angew. Chem.,
Int. Ed. Engl. 1994, 33, 1941-1944.
(24) Giese, B.; Beyrich-Graf, X.; Erdmann, P.; Petretta, M.; Schwitter, U. Chem.
Biol. 1995, 2, 367-375.
(25) Gugger, A.; Batra, R.; Rzadek, P.; Rist, G.; Giese, B. J. Am. Chem. Soc.
1997, 119, 8740-8741.
(26) Giese, B.; Dussy, A.; Meggers, E.; Petretta, M.; Schwitter, U. J. Am. Chem.
Soc. 1997, 119, 11130-11131.
(27) Dussy, A.; Meggers, E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 7399-
7403.
(28) Dussy, A.; Giese, B. Nucleosides Nucleotides 1999, 18, 1343-1344.
(29) Strittmatter, H.; Dussy, A.; Schwitter, U.; Giese, B. Angew. Chem., Int.
Ed. 1999, 38, 135-137.
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A R T I C L E S
Scheme 1
Zhang and Wang
Scheme 2
intrastrand cross-link lesion, in which the methyl carbon atom
of 5-methylcytosine and the C8 carbon atom of its adjacent
guanine are covalently bonded, from this reactive intermediate.15
The independent generation of the 5-hydroxy-5,6-dihydro-
thymidin-6-yl radical has been previously realized by Barvian
et al.11 through photoinduced electron transfer from N-methyl-
carbazole (Scheme 1b). The photoirradiation, however, was
carried out in a solvent mixture of acetonitrile and H2O (27:73,
v/v), and the efficiency for the generation of the radical was
very low in the presence of O2 or the absence of the electron
donor.11
We sought to investigate the fate of reactive intermediate I
(Scheme 1) under both aerobic and anaerobic conditions.
Moreover, it is desirable to be able to generate I in pure aqueous
solution for the future investigation of its reactivity in duplex
DNA. With these goals in mind, here we report the synthesis
of all four diastereomers of 5-hydroxy-6-phenylthio-5,6-dihydro-
thymidine (Scheme 2), their incorporation into dinucleoside
monophosphates, and the characterizations of major products
from the 254-nm irradiation of these dinucleoside monophos-
phates in aqueous solution under anaerobic conditions.
Results and Discussion
1. Synthesis of the Photolabile Precursor of the 5-Hy-
droxy-5,6-dihydrothymidin-6-yl Radical (I). Phenylthio-
substituted nucleosides have been shown to have high efficiency
in generating carbon-centered radicals through homolytic cleav-
age of the C-SPh bond upon 254-nm UV irradiation.13-15 In
addition, the release of the radicals can be achieved in aqueous
solution without adding organic cosolvent. Therefore, we
decided to adopt similar chemistry for the independent genera-
tion of I.
Our synthetic strategy for the preparation of 5-hydroxy-6-
phenylthio-5,6-dihydrothymidine (4a-4d, Scheme 2) was in-
spired by previous work of Harayama et al.,30,31 which showed
that thymidine bromohydrin can react with amines and L-amino
9
13288 J. AM. CHEM. SOC. VOL. 126, NO. 41, 2004

5-Hydroxy-5,6-dihydrothymidin-6-yl Radical
A R T I C L E S
Scheme 3 a
a Reagents. a: NH3/MeOH. b: (i) Dicyanoimidazole/DMF, (ii) t-BuOOH in nonane. c: 80% CH3COOH. d: NH3/MeOH.
acid ethyl esters. Moreover, the reaction was proposed to
proceed through an epoxide intermediate, which gives rise to
thymidine derivatives with a hydroxyl group and the amino
group of amines or L-amino acid ethyl esters being substituted
to the C5 and C6 carbon atoms, respectively.30,31 The incorpora-
tion of the phenylthio group was achieved through in situ
generation of phenylthiolate (PhS-) as described previously.32
The trans (5R,6R) isomer of thymidine bromohydrin (2a) gave
rise to two isomers of 5-hydroxy-6-phenylthio-5,6-dihydro-
thymidine, i.e., cis (5R,6R) (3a) and trans (5R,6S) (3b), with a
The structure of each stereoisomer was determined by 2-D
NMR experiments (spectra shown in the Supporting Informa-
tion). In this regard, 2-D COSY results showed strong correlation
between the OH proton and the H6 proton for the starting
thymidine bromohydrin in DMSO-d6; no correlation, however,
was observed between the H6 proton and the OH proton of the
thymine moiety of all the four isomers of T* (Scheme 2),
demonstrating that the hydroxyl group is substituted to the C5
carbon atom of the thymine portion (data shown in the
Supporting Information). Moreover, 2-D nuclear Overhauser
effect (NOE) spectra of the four isomeric d(GT*) showed strong
correlation between the protons on the phenyl group and the
H1′ proton of the 3′ nucleoside, which furnishes evidence
supporting that the phenylthio group is incorporated to the C6
position of the thymine moiety.
1
ratio of 4:1 as estimated from H NMR spectrum (Scheme 2).
It is worth noting that we attempted but failed to resolve 3a
and 3b by silica gel column chromatography with several
different solvent systems. The chemistry also occurs for
bromohydrin 2b, but we were able to separate 3c and 3d from
each other. Treatment of compounds 3a-3d with methanolic
ammonia gave the desired photolabile precursors 4a-4d
(Scheme 2).
To establish the absolute stereochemistry of the C5 and C6
carbon atoms of the modified thymidine by NMR, we need to
consider first whether the glycosidic bond in the modified
thymidine adopts a syn or anti conformation. In this respect,
substituted pyrimidine nucleosides with a glycosidic bond in
the syn conformation are known to lead to a downfield shift of
the H2′ proton (by ∼0.6 ppm) of the deoxyribose because of
the placement of C2 carbonyl moiety on the top of the
deoxyribose ring.35,36 1H NMR results showed that the H2′
protons of the thymidine portion in d(GT2*) and d(GT3*)
appeared at approximately 2.9 ppm, whereas the chemical shifts
of the respective protons in d(GT1*) and d(GT4*) were slightly
less than 2.4 ppm and very similar to that of the H2′ proton of
the unmodified thymidine. The significant downfield displace-
ment of the H2′ protons of the 3′ nucleoside in d(GT2*) and
d(GT3*) supports that the glycosidic bonds of the thymidine
moiety adopt a syn conformation. The lack of alterations in
chemical shifts of the corresponding protons in d(GT1*) and
d(GT4*), however, shows that the glycosidic bonds of the
modified nucleoside in d(GT1*) and d(GT4*) are in their anti
conformation.
It has been demonstrated previously that the tertiary hydroxyl
group does not have to be protected during solid-phase DNA
synthesis.33,34 These precursors, therefore, were employed
directly for the synthesis of dinucleoside monophosphates
d(GT*) and d(TT*), where T* represents the photolabile radical
precursor, by using solution-phase phosphoramidite chemistry
(Scheme 3 gives an example for the preparation of d(GT3*)).
To prevent the decomposition of saturated thymine portion,
deprotection needs to be carried out under mild conditions.
Therefore, commercially available ultramild dG phosphoramidite
was used for the synthesis of d(GT*) (Scheme 3). We were
able to separate the two isomeric dinucleoside monophosphates
d(GT1*) and d(GT2*) that were initiated from 3a and 3b by
reverse-phase HPLC. On the basis of the peak areas in the HPLC
traces, we estimated that the ratio between these two isomers
was again 4:1 with the cis isomer being more abundant.
(30) Harayama, T.; Yanada, R.; Taga, T.; Machida, K.; Cadet, J.; Yoneda, F. J.
Chem. Soc., Chem. Commun. 1986, 1469-1471.
(31) Harayama, T.; Yanada, R.; Tanaka, M.; Taga, T.; Machida, K.; Yoneda,
F.; Cadet, J. J. Chem. Soc., Perkin Trans. 1 1988, 2555-2562.
(32) Rajanikanth, B.; Ravindranath, B. Indian J. Chem., Sect. B 1984, 23, 1043-
1045.
(35) George, A. L.; Hruska, F. E.; Ogilvie, K. K.; Holy, A. Can. J. Chem. 1978,
56, 1170-1176.
(33) Iwai, S. Angew. Chem., Int. Ed. 2000, 39, 3874-3876.
(34) Iwai, S. Chem.-Eur. J. 2001, 7, 4343-4351.
(36) Cadet, J.; Ducolomb, R.; Hruska, F. E. Biochim. Biophys. Acta 1979, 563,
206-215.
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A R T I C L E S
Zhang and Wang
37
We then employed semiempirical PM3 method
and
optimized the geometry of the four isomeric photolabile
precursors (T1*-T4*) (structures shown in Scheme 2) with the
glycosidic bond in both anti and syn conformations. It turned
out that, in equilibrium geometry, the distances between the
C2′ proton and the phenyl proton that are in closest proximity
are predicted to be 5.8 and 5.0 Å for T1* and T4* in anti
glycosidic conformation, respectively (Figure S27), whereas the
corresponding distance is approximately 2.6 Å for both T2* and
T3* in syn glycosidic conformation (Figure S28). From this
theoretical prediction, we anticipate to observe, in 2-D NOE
spectra, strong correlation between the phenyl protons and the
protons on the C2′ carbon atom of the 3′ nucleoside for d(GT2*)
and d(GT3*), whereas no such correlation should be present
for d(GT1*) and d(GT4*). Indeed, that is exactly what we
observed (spectra are shown in the Supporting Information).
These results, therefore, demonstrate that the C6 carbon atom
of the thymine portion in d(GT2*) and d(GT3*) is in S
configuration, whereas that of d(GT1*) and d(GT4*) is in R
configuration.
Figure 1. HPLC trace for the separation of the 254-nm irradiation mixture
of d(GT1*) under anaerobic condition, in which d(GAbasic), d(GTg), and
d(GTdiH) represent the dinucleoside monophosphate with the thymidine
portion being replaced with an abasic site lesion, thymidine glycol, and
5-hydroxy-5,6-dihydrothymidine, respectively. “XL” designates the cross-
link lesion.
After establishing the absolute stereochemistry of the C6
carbon atom, the stereochemistry of the C5 carbon atom can
be determined since there is no NOE between the phenyl protons
and the methyl protons observed for the cis isomers, d(GT1*)
and d(GT3*). However, strong NOE was found between these
two groups of protons for the two trans isomers, d(GT2*) and
d(GT4*). In addition, our assignment of the absolute stereo-
chemistry of the C5 carbon atom of the photolabile precursors
is in accord with previous observation of the products initiated
from the reaction of thymidine bromohydrin with amines and
L-amino acid esters.30,31
2. Abasic Site Lesion Was the Major Product Initiated
from the 254-nm Irradiation of d(GT*) and d(TT*). To avoid
the complicating effects of molecular oxygen and to mimic the
hypoxic cellular environments, we chose to first examine the
reactivities of I under anaerobic conditions. To this end, the
above dinucleoside monophosphates were purified by HPLC
and irradiated for 15-20 min with 254-nm UV light under
anaerobic conditions. Negative-ion ESI-MS for the irradiation
mixture showed that the starting material is almost undetectable
(data not shown), showing that the release of intermediate I is
efficient. The resulting products were separated again by HPLC
and characterized by mass spectrometry and NMR. Here we
will focus our discussion on the products isolated from the 254-
nm irradiation of d(GT1*).
It turned out that the major product emanating from the
photoirradiation is the dinucleoside monophosphate with the
thymidine portion being degraded to an abasic site (a typical
chromatogram for the separation of the irradiation mixture of
d(GT1*) is shown in Figure 1). Negative-ion ESI-MS for the
17.0-min fraction gave an ion of m/z 462.1, which is the [M -
H]- ion of the lesion (Figure 2a). Exact mass measurement of
the product gave m/z 462.1062, which is consistent with the
calculated m/z of 462.1062 for the [M - H]- ion. Collisional
activation of this ion yields major fragment ions forming from
the eliminations of a H2O molecule (m/z 444.0) or the 3′ remnant
nucleoside (m/z 346.0, spectrum shown in Figure 2b).
Figure 2. Negative ESI-MS (a) and MS/MS (b) of d(GAbasic), and
negative ESI-MS (c) and MS/MS (d) of 2′-deoxyguanosine-3′-monophos-
phate. The latter compound forms from the decomposition of the d(GAbasic)
upon the drying by Speed-vac (see text).
after being dried in a Speed-vac, the major decomposition
product gave an ion of m/z of 346.1 in negative-ion ESI-MS
(Figure 2c). The decomposition product was purified by HPLC;
ESI-MS/MS and 1H NMR results showed its identity as
2′-deoxyguanosine-3′-monophosphate (designated as d(Gp)).
The product-ion spectrum of the ion of m/z 346.1 showed a
dominant fragment ion of m/z 195.1 and an ion of m/z 150.0 of
low abundance. These two fragment ions are induced from the
cleavage of the N-glycosidic bond (Figure 2d). 1H NMR
As reported previously, the abasic site is unstable, and it
decomposes completely upon evaporation to dryness.38 Indeed
(37) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 209-220.
(38) Shishkina, I. G.; Johnson, F. Chem. Res. Toxicol. 2000, 13, 907-912.
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5-Hydroxy-5,6-dihydrothymidin-6-yl Radical
A R T I C L E S
Scheme 4
spectrum showed the presence of only one deoxyribose,
demonstrating that the deoxyribose of the 3′ nucleoside has been
degraded (spectrum shown in the Supporting Information).
For d(TT1*) and all four isomers of d(GT*) that we examined,
the abasic site lesion is the most abundant product that we
observed (Table S1 gives the estimated yields of the formation
of various products initiated from the 254-nm irradiation of these
dinucleoside monophosphates). It is well-documented that the
abasic site lesion can be generated during ionizing radiation.7
Given that reactive intermediate I is the major secondary radical
Alternatively, the spin at the C6 carbon atom can be
transferred to the C1′ carbon atom of the deoxyribose portion
via the elimination of the nucleobase moiety. The resulting C1′
radical is expected to possess reducing property because of the
adjacent oxygen atom on the deoxyribose ring. Similar to radical
I, the C1′ radical may lose an electron to give a carbon cation
at the C1′ position, which can again undergo consecutive
hydration and deprotonation to yield the abasic site lesion
(Scheme 4).
3. Other Lesions Formed from I. In addition to the abasic
site lesion described above, intrastrand cross-link and lesions
with the thymine portion of the dinucleoside monophosphates
being modified to thymine glycol, 5-hydroxy-5,6-dihydro-
thymine, or thymine, have been isolated and characterized
(Scheme 5).
•
induced from OH attack on thymidine and the above finding
that this radical can lead to the facile formation of the abasic
site lesion, our results indicate that radical I may constitute an
important reactive intermediate resulting in the formation of
abasic site lesion.
Several possible mechanistic pathways may give rise to the
abasic site lesion. Reactive intermediate I is recognized as a
reducing radical because of the adjacent N1 nitrogen atom,39
and the reducing property of this radical has been demonstrated
in that it reduces tetranitromethane with a rate constant of ∼109
a. Thymine Glycol and 5-Hydroxy-5,6-dihydrothymine.
The LC fraction eluting at 22.1 min is a product with the
thymine portion being replaced with a thymine glycol (labeled
as d(GTg) in Figure 1). The product-ion spectrum of the ESI-
produced [M - H]- ion (m/z 604) of the thymine glycol-bearing
dinucleoside monophosphate shows a unique elimination of a
143-Da fragment (Figure 3a). Similar fragmentation has been
previously observed in the product-ion spectra of the [M - H]-
ions of thymine glycol-bearing mononucleosides and dinucleo-
tides.42,43 This facile loss was due to the cleavages of the N1-
C2 and N1-C6 bonds of the modified thymine portion.42,43 Our
structure assignment is further supported by 1-D and 2-D NMR
data (spectra shown in the Supporting Information).
M-1 s-1 40
. With that in mind, we reason that reactive intermedi-
ate I can be oxidized to give a C6 carbon cation (Scheme 4),
which can lead to the facile elimination of the thymine portion
with the charge being transferred to the C1′ carbon atom of the
deoxyribose moiety. The latter intermediate can undergo
consecutive hydration and deprotonation to afford the abasic
site lesion. Some precaution, however, should be taken for the
interpretation of the formation of the abasic site lesion from
the 254-nm irradiation of d(GT*). In this regard, previous work
by Kropp et al.41 showed that 254-nm irradiation of phenyl
thioethers leads to the formation of both the carbon-centered
radical and carbon cation. The latter was proposed to form from
electron transfer within the initially formed radical pair cage.41
In the γ-radiation-induced DNA damage, the electron transfer
from radical I may not be as facile as what occurs in the
photolytically generated radical pair cage. In this regard, parallel
LC-MS/MS analyses of the γ-radiation mixture of d(GT) and
the 254-nm irradiation of d(GT1*) showed that this is indeed
the case (vide infra).
We also observed that the thymine portion can be transformed
to 5-hydroxy-5,6-dihydrothymine. Exact mass measurement of
the 23.1-min fraction (labeled as d(GTdiH) in Figure 1) gives
m/z 588.1436 for the [M - H]- ion, which is consistent with
the calculated m/z of 588.1455 for the deprotonated ion of
d(GTdiH). The product-ion spectrum of the [M - H]- ion shows
the facile elimination of a water molecule. In addition, we
observed the cleavages of the N-glycosidic and the 3′ C-O
bonds of the 5′ nucleoside to give ions of m/z 437.1 and 346.1,
respectively (Figure 3b). We also acquired 2-D COSY and
NOESY data for the lesion, which allow us to assign all the
(39) O’Neill, P.; Fielden, E. M. In AdVances in Radiation Biology; Lett, J. T.,
Sinclair, W. K., Eds.; Academic Press: New York, 1993; Vol. 17, pp 53-
121.
(40) Fujita, S.; Steenken, S. J. Am. Chem. Soc. 1981, 103, 2540-2545.
(41) Kropp, P. J.; Fryxell, G. E.; Tubergen, M. W.; Hager, M. W.; Harris, G.
D., Jr.; McDermott, T. P., Jr.; Tornero-Velez, R. J. Am. Chem. Soc. 1991,
113, 7300-7310.
1
protons in H NMR without ambiguity. In the NOE spectrum,
(42) Wang, Y.; Vivekananda, S.; Zhang, K. Anal. Chem. 2002, 74, 4505-4512.
(43) Wang, Y. Chem. Res. Toxicol. 2002, 15, 671-676.
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A R T I C L E S
Scheme 5
Zhang and Wang
two protons at 3.5 ppm, which are the H-6 protons of the
thymine portion, show correlation with the protons on the methyl
group and the C2′ and C3′ carbons of the 3′ nucleoside.
Furthermore, the methyl protons show correlation with protons
on the C2′ carbon atom of the 3′ nucleoside, indicating that the
C5 carbon remains in its S configuration. The above MS and
NMR results support our structural assignment of this lesion.
The formation of thymidine glycol and 5-hydroxy-5,6-dihydro-
thymidine has also been previously observed by Barvian et al.11
from the independently generated radical I in mononucleoside.
b. Intrastrand Cross-Link Lesion. As observed with the
5-methyl radical of thymine13,14 and 5-methylcytosine,15 reactive
intermediate I in d(GT) can also couple with the C8 carbon
atom of its 5′ neighboring guanine to give a cross-link lesion
(24.4-min fraction, labeled as XL in Figure 1).
The structure of the cross-link lesion was established by
extensive characterizations with MS and NMR. Negative-ion
ESI-MS of the cross-link lesion gives an ion of m/z 586, which
is the [M - H]- ion of the cross-link lesion. Exact mass
measurement of this ion gives m/z 586.1275, which is consistent
with the calculated m/z of 586.1299. The product-ion spectrum
of the ion of m/z 586 does not show the elimination of an
unmodified guanine (Figure 3c), whereas this elimination is the
most facile cleavage resulting from the collisional activation
of the [M - H]- ion of the unmodified d(GT) (data not shown).
Instead, the most abundant fragment ion (m/z 488.0) is due to
the elimination of a 2-deoxyribose (-98 Da). This ion can
further lose [HPO3 + H2O] to yield an ion of m/z 390.1. The
assignments of these two fragment ions are supported by high-
resolution MS/MS acquired on a Fourier transform mass
spectrometer, which gives m/z 488.0908 and 390.1160. The
results are in line with the calculated m/z values of 488.0931
and 390.1162 for these two product ions.
Given the above MS and MS/MS results and that the cross-
link lesion is initiated from a structurally defined radical, we
believe that the cross-link lesion has a covalent bond formed
between the C8 carbon atom of guanine and the C6 carbon atom
1
of the thymine moiety. Indeed, the H NMR spectrum clearly
shows the disappearance of H-8 proton of guanine (Figure 4).
All the proton resonances in 1H NMR were again assigned from
the 2-D COSY and NOESY results (data shown in the
Supporting Information).
It is interesting to note that the yield for the production of
the cross-link lesion depends on the conformation of C5 carbon
atom (LC traces for the separation of the irradiation mixtures
of d(GT2*), d(GT3*), and d(GT4*) are shown in the Supporting
Information). In this respect, d(GT1*) and d(GT2*), which give
rise to the 5S isomer of radical I upon 254-nm irradiation
(Scheme 2), have a yield of approximately 4% (Table S1),
whereas d(GT3*) and d(GT4*), which yield the 5R isomer of
radical I (Scheme 2), show a lower yield for the formation of
the cross-link lesion, that is, approximately 1 to 2% (Table S1).
Figure 3. Product-ion spectra of the ESI-produced [M - H]- ions of
d(GTg) (a), d(GTdiH) (b), and the XL (c).
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5-Hydroxy-5,6-dihydrothymidin-6-yl Radical
A R T I C L E S
Figure 4. 1H NMR spectrum of the cross-link lesion (XL) initiated from the 254-nm irradiation of d(GT1*) (500 MHz, D2O. The peak at 8.5 ppm is due
to impurity.)
The difference in yield for the formation of the lesion is likely
due to different steric hindrance imposed by the C5 carbon atom
of the two isomeric radicals.
min fraction is a guanine oxidation product with 8-oxoguanine
being most likely (spectra shown in Figure S36). The nature of
the product eluting at 33.1 min remains unknown.
We also prepared d(TT1*), irradiated it with 254-nm UV light
under anaerobic condition, and characterized the products by
mass spectrometry. It turned out that d(TT1*) gave very similar
products as d(GT*), that is, d(TAbasic), d(TTdiH), d(TTg), and
d(TT), except that we failed to isolate any cross-link lesion from
the irradiation mixture of d(TT1*) (Figure S29).
4. Identification of the Cross-Link Lesions from the
γ-Irradiation of d(GT) under Anaerobic Conditions. Previ-
ously, Box et al.44 reported a cross-link lesion that was isolated
from the X-ray irradiation of d(GT) under anaerobic conditions,
and they proposed that the cross-link lesion was initiated from
the coupling of the 6-hydroxy-5,6-dihydrothymidin-5-yl radical
(II, Scheme 1a) with the C8 carbon atom of guanine. We,
however, found that the 1H NMR spectrum reported by Box et
al.44 is nearly identical to that for the 5S isomer of the cross-
link lesion that we isolated. This motivated us to propose that
the lesion isolated by Box et al.44 is the 5S isomer of the cross-
link lesion initiated from radical I.
To support such a proposal, we carried out the γ-irradiation
of d(GT) under anaerobic conditions and identified the cross-
link lesions induced under this condition by LC-MS/MS. It
turns out that the 5S isomer is indeed the major cross-link lesion
of this kind formed from γ-irradiation of d(GT). Total-ion
chromatogram for the fragmentation of the ion of m/z 588, which
corresponds to the [M + H]+ ion of the cross-link lesion, in
the positive-ion mode gave four peaks (Figure 5a). The product-
ion spectra of the two earlier eluting fractions gave characteristic
elimination of a 98-Da moiety, which is identical to the
fragmentation of the authentic cross-link lesions that we isolated
from the 254-nm irradiation of d(GT*). The two later eluting
factions at 33.1 and 35.5 min are not cross-link lesions because
the product-ion spectra of the ion of m/z 588 show fragment
ions of m/z 323, which are assigned as the [M + H]+ ion of
thymidine-5′-phosphate. Combining the results from both posi-
tive- and negative-ion MS/MS, we determined that the 35.5-
Selected-ion chromatograms for the m/z 588 f 490 transition,
which corresponds to the elimination of the 2-deoxyribose from
the precursor ion, of the γ-irradiation sample shows that there
are two major peaks at 27.5 and 30.0 min (Figure 5b).
Furthermore, we carried out LC-MS/MS experiments with the
co-injection of the γ-irradiation mixture and the authentic cross-
link lesion that was isolated from the 254-nm irradiation of each
of the four isomeric d(GT*), that is, d(GT1*)-d(GT4*). It turned
out that the 27.5-min eluting fraction comigrates with the cross-
link lesion initiated from the photoirradiation of d(GT1*) or
d(GT2*) (Figure 5c). The fraction eluting at 30.0 min, however,
comigrates with the authentic cross-link lesion that was initiated
from the irradiation of d(GT3*) or d(GT4*) (Figure 5d). These
results demonstrate that, as expected, d(GT1*) and d(GT2*) give
rise to the same cross-link lesion, and the same is true with
d(GT3*) and d(GT4*). Moreover, the yield for the formation of
the 5S isomer of the cross-link lesion from the γ-irradiation
mixture of d(GT) is higher than that for the formation of the
5R isomer, which is consistent with the formation of these two
isomeric cross-link lesions from the two independently generated
isomeric radicals.
The above results demonstrate that γ-irradiation of d(GT)
under anaerobic condition can induce the formation of both
isomers of the cross-link lesions that we have isolated from the
254-nm irradiation of d(GT*). It should be noted that the
analogous 6-hydroxy-5,6-dihydrothymidin-5-yl radical may also
give rise to a cross-link lesion via coupling to the C8 carbon
atom of its adjacent guanine. In this respect, we recently
discovered that γ-irradiation of duplex synthetic oligodeoxy-
nucleotide (ODN) can give rise to a cross-link lesion, with the
C8 carbon atom of guanine and the C5 carbon atom of its 3′
neighboring cytosine covalently bonded.45 This type of lesion
was proposed to form from the dehydration of the coupling
product of the 6-hydroxy-5,6-dihydrocytosin-5-yl radical and
its adjacent guanine.46 The cross-link lesions initiated from the
(44) Box, H. C.; Budzinski, E. E.; Dawidzik, J. B.; Gobey, J. S.; Freund, H. G.
Free Radical Biol. Med. 1997, 23, 1021-1030.
(45) Gu, C.; Wang, Y. Biochemistry 2004, 43, 6745-6750.
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Zhang and Wang
MS/MS experiments were carried out in the negative-ion mode,
and the mass spectrometer was set to monitor alternatively the
fragmentations of the [M - H]- ions of the cross-link lesion
(m/z 586), d(GAbasic) (m/z 462), and d(Gp) (m/z 346).
By taking into consideration the different ionization and
fragmentation efficiencies of the three compounds, we estimated
that the yield for the formation of the abasic site lesion, that is,
d(GAbasic), from the 254-nm irradiation of d(GT1*) is approxi-
mately eight times as high as that of the cross-link lesion, which
is in reasonable agreement with what we obtained from the LC
trace as monitored by UV absorbance at 260 nm (Table S1).
The decomposition product of the abasic site lesion, d(Gp), was
undetectable under these UV irradiation conditions (LC-MS/
MS traces and the relevant calculations are shown in Figures
S33-35 and page S40 in the Supporting Information).
The distribution of the three products from the γ-irradiation
of d(GT), however, is very different from that of the UV
irradiation of the d(GT1*). In this regard, the formation of
d(GAbasic) from the γ-irradiation is approximately 11 times
less efficient than the formation of the 5S isomer of the cross-
link lesion, though the decomposition product of d(GAbasic),
that is, d(Gp), is produced at a similar efficiency as the 5S isomer
of the cross-link lesion. Therefore, the relative yield for the
formation of the abasic site lesion over that of the cross-link
lesion is much higher from the 254-nm UV irradiation of
d(GT1*) than from the γ-irradiation of d(GT). Moreover, the
d(GAbasic) and d(Gp) may also be initiated from deoxyribose
radicals and other types of nucleobase radicals under γ-irradia-
tion.7 Therefore, the C6 carbon cation, which forms from the
oxidation of the reactive intermediate I and is produced more
readily from the 254-nm irradiation of d(GT1*) than from the
γ-radiation of d(GT), makes a significant contribution to the
formation of the abasic site lesion.
Figure 5. LC-MS/MS of the γ-irradiation mixture of d(GT) under
anaerobic condition. The mass spectrometer was set at monitoring the
fragmentation of the ion of m/z 588 in the positive-ion mode: (a) Total ion
chromatogram for the fragmentation of the m/z 588 ion with the injection
of 4-nmol γ-irradiation mixture of d(GT). (b) Selected-ion chromatogram
for the m/z 588 f m/z 490 transition for the same LC-MS/MS analysis as
in (a). (c) Selected-ion chromatogram for the m/z 588 f m/z 490 transition
with the co-injection of the 1.3-nmol γ-irradiation mixture of d(GT) and 5
pmol of the XL lesion initiated from d(GT1*). (d) Selected-ion chromato-
gram for the m/z 588 f m/z 490 transition with the co-injection of the
0.8-nmol γ-irradiation mixture of d(GT) and 3 pmol of the XL lesion
initiated from d(GT3*). Shown in the insets are the product-ion spectra for
the ion of m/z 588 for the two fractions containing the cross-link lesion.
Experimental Section
Materials. Common reagents for solid-phase DNA synthesis were
obtained from Glen Research Co. (Sterling, VA). Silica gel and TLC
plates were purchased from EM Science (Gibbstown, NJ). All chemicals
unless otherwise specified were from Sigma-Aldrich (St. Louis, MO).
Mass Spectrometry and NMR. ESI-MS and MS/MS experiments
were carried out on an LCQ Deca XP ion-trap mass spectrometer
(ThermoFinnigan, San Jose, CA). An equal-volume solvent mixture
of acetonitrile and water was used as solvent for electrospray, and a
2-µL aliquot of a ∼5 µM sample solution was injected in each run.
The spray voltages were 4.0 and 3.4 kV for experiments in the positive-
and negative-ion modes, respectively. The capillary temperature was
maintained at 150 °C. An IonSpec HiResESI external ion source FTICR
mass spectrometer (IonSpec Co., Lake Forest, CA) equipped with a
4.7 T unshielded superconducting magnet was used for high-resolution
MS and MS/MS measurements as described previously.15 Dinucleoside
monophosphate ApA and an oligodeoxynucleotide d(AATTGA) were
used as internal standards for mass calibration.
6-hydroxy-5,6-dihydrothymidin-5-yl radical (II, Scheme 1a) and
radical I have the same molecular weight, and the two lesions
are expected to have similar ionization efficiency. Thus, the
above LC-MS/MS studies show that the cross-link lesion
initiated from radical I is more abundant than that formed from
radical II, if the cross-link lesion can be initiated from the latter
radical.
To examine to what extent the abasic site lesion was formed
from electron transfer within the initial radical pair cage as
alluded to in the foregoing discussion, we employed LC-MS/
MS and determined the relative yields of formation of the abasic
site lesion over cross-link lesion from the 254-nm irradiation
of d(GT1*) and the γ-irradiation of d(GT) under anaerobic
conditions. In the course of this study, we noticed that the abasic
site lesion d(GAbasic) and its decomposition product d(Gp) do
not ionize well in positive-ion ESI-MS. Thus, the LC-ESI-
All NMR spectra were recorded on a Varian Unity Inova 500 MHz
instrument (Palo Alto, CA). The residual proton signal of the solvent
served as internal reference.
UVC Irradiation. An unbuffered aqueous solution of dinucleoside
monophosphate d(GT*) or d(TT*) with AU260 of 0.4 in a quartz tube
was degassed by three cycles of freeze-pump-thaw. The solution was
irradiated for 15-20 min with 254-nm light generated by a Rayonet
photochemical reactor that was equipped with 16 light tubes (The
Southern New England Ultraviolet Company, CT). The resulting
solution was lyophilized to dryness.
(46) Box, H. C.; Budzinski, E. E.; Dawidzik, J. B.; Wallace, J. C.; Iijima, H.
Radiat. Res. 1998, 149, 433-439.
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5-Hydroxy-5,6-dihydrothymidin-6-yl Radical
A R T I C L E S
HPLC. The HPLC separation was performed on a system composed
of a Hitachi L-6200A pump (Hitachi Ltd., Tokyo, Japan), a HP-1050
UV detector (Agilent Technologies, Palo Alto, CA), and a Peak Simple
Chromatography Data System (SRI Instruments Inc., Las Vegas, NV).
A 4.6 × 250 mm Apollo C18 column (5 µm in particle size and 300
Å in pore size, Alltech Associates Inc., Deerfield, IL) was used for the
separation.
A solution of 50 mM ammonium formate (solution A) and a mixture
of 50 mM ammonium formate and methanol (60/40, v/v) (solution B)
were used as the mobile phases. For the purification of d(GT*) and
d(TT*), a gradient of 20-min 0-100% B followed by 10-min 100% B
was used. Appropriate fractions were pooled, evaporated to dryness,
and redissolved in doubly distilled water for photoirradiation. For the
separation of the 254-nm irradiation products of d(GT*) and d(TT*),
a gradient of 5-min 0-15% B, 30-min 15-55% B, and 5-min 55-
100% B was used. The flow rate was 1.0 mL/min, and the effluents
were monitored by UV detection at 260 nm. The HPLC condition for
LC-MS experiments was different and described below.
γ-Irradiation of d(GT) and LC-MS/MS. A 1.5-mL solution of
d(GT) (80 nmol) in a 35-mL Pyrex tube was degassed by three cycles
of freeze-pump-thaw. The tube was filled with high-purity argon and
exposed to a Mark I 137Cs Irradiator (JL Shepherd and Associates, San
Fernando, CA) at a dose rate of 2.0 Gy/min for 4 h (total dose: 480
Gy). The resulting solution was dried by the Speed-vac, redissolved in
40 µL water, and subjected directly to LC-MS/MS analysis.
A 0.32 × 250 mm C18 column with a particle size of 5 µm and a
pore size of 300 Å (Micro-Tech Scientific, Vista, CA) was employed
for the separation. A homemade precolumn splitter was incorporated
between the HPLC pump and the injector, the flow rate was 4-6 µL/
min after splitting, and a 100-min gradient of 0-35% acetonitrile in
10 mM ammonium acetate was used. The flow from HPLC column
was coupled directly to the LCQ Deca XP ion-trap mass spectrometer.
The mass spectrometer was set up to monitor the fragmentation of the
[M + H]+ ion (m/z 588) of the cross-link lesions, and 4 nmol of the
above γ-irradiated d(GT) was injected in each run. In comigration
experiments, standard 5S or 5R isomer of the cross-link lesion was
mixed with the above γ-irradiation sample and injected to the column
with identical experimental setup as for the γ-irradiation mixture.
Slightly different conditions were employed for the determination
of the relative yields of the formation of different products from 254-
nm irradiation of d(GT*) and the γ-irradiation of d(GT). In this regard,
the mass spectrometer was run in the negative-ion mode with the
monitoring of the fragmentation of the ions of m/z 346, 462, and 586,
which correspond to the deprotonated ions of 2′-deoxyguanosine-5′-
monophosphate, d(GAbasic), and the cross-link lesion, respectively,
in an alternative manner. In addition, the flow splitter was placed after
the injector. Therefore, the retention times are different from those LC-
MS/MS experiments performed in the positive-ion mode.
Synthesis and Characterization of Compounds. Preparation of
Bromohydrins 2a and 2b (Scheme 2). Compound 1 (1.63 g, 5 mmol)
and 200 mL of water were added to a 500-mL flask, and the mixture
was vigorously stirred for 30 min until compound 1 was completely
dissolved. The flask was then immersed in an ice bath for 15 min, and
bromine (282 µL, 1.1 equiv) was slowly added to the vigorously stirred
solution over 15 min. After being stirred for another 15 min, the volume
of the solution was reduced to 30 mL by rotavap. The same procedures
were repeated for another three batches of reaction. The concentrated
reaction mixtures were combined and extracted with chloroform twice
(120 mL each). The organic layer was washed with brine and dried
with anhydrous Na2SO4, and the solvent was evaporated off to give a
white foam.
Compound 2a: Rf: 0.48 (CH2Cl2/EtOAC, 5/6, v/v). ESI-MS: m/z
1
445.1, 447.1 [M + Na]+. H NMR (DMSO-d6): 10.86 (s, 1H, NH),
6.82 (br, s, 1H, 6-OH), 6.04 (dd, 1H, H-1′, J ) 8.7, 5.9 Hz), 5.19 (s,
1H, H-6), 5.14 (m, 1H, H-3′), 4.18 (d, 2H, J ) 4.8 Hz, H-5′ and H-5′′),
4.11-4.07 (m, 1H, H-4′), 2.48-2.41 (m, 1H, H-2′), 2.15-2.09 (m,
1H, H-2′′), 2.06 (s, 6H, COCH3), 1.82 (s, 3H, CH3).
Compound 2b: Rf: 0.33 (CH2Cl2/EtOAC, 5/6, v/v). ESI-MS: m/z
1
445.1, 447.1 [M + Na]+. H NMR (DMSO-d6): 10.88 (s, 1H, NH),
7.14 (br, s, 1H, 6-OH), 6.06 (dd, 1H, J ) 9.1, 5.5 Hz, H-1′), 5.13 (m,
1H, H-3′), 5.08 (s, 1H, 6-H), 4.24-4.15 (m, 2H, H-5′ and H-5′′), 4.07
(m, 1H, H-4′), 2.48-2.40 (m, 1H, H-2′), 2.14-2.08 (m, 1H, H-2′′),
2.07 (s, 6H, COCH3), 1.81 (s, 3H, CH3).
Preparation of Compounds 3a and 3b (Scheme 2). The incorpora-
tion of the phenylthio group was achieved by in situ generation of
phenylthiolate (PhS-) as described previously.32 To a 50-mL flask,
which was equipped with a dean-stark tube and a condenser, was added
benzene (20 mL), ZnO (211.7 mg, 0.55 equiv), and PhSH (0.534 mL,
1.1 equiv). The solution was refluxed for 4 h and cooled, and to the
cooled solution was added compound 2a (2.0 g, 4.73 mmol) and
anhydrous pyridine (765 µL, 2 equiv). The mixture was refluxed for
overnight and cooled to room temperature. Some white precipitates
formed during the reaction, and they were removed by filtration. The
resulting filtrate was washed with HCl (0.05 M), 1% NaHCO3, and
brine. The benzene layer was further dried with anhydrous Na2SO4
and evaporated to dryness under reduced pressure to give a light orange
oil. The oily product was loaded onto a silica gel column and eluted
with 0-4% MeOH in CH2Cl2. The appropriate fractions were pooled,
and the solvent was evaporated to give slight yellowish oil. This
yellowish oil was loaded to a silica gel column and eluted with hexane/
EtOAc (1/2, v/v). The appropriate fractions were combined, and the
solvent was evaporated off to afford a mixture of 3a and 3b as white
foam (1.11 g, total yield of 3a and 3b: 51.8%). The ratio of 3a to 3b
was 4:1 as established by 1H NMR. Compounds 3a and 3b: ESI-MS:
m/z 450.9 [M - H]-, MS/MS 341.1 [M - H - PhSH]-; 452.9 (weak)
[M + H]+, 475.1 (strong) [M + Na]+.
1H NMR of compound 3a (DMSO-d6): 10.62 (s, 1H, NH), 7.54-
7.49 (m, 2H, H-PhS), 7.33-7.29 (m, 3H, H-PhS), 6.19 (s, 1H, 5-OH),
5.39 (dd, 1H, J ) 7.1, 6.7 Hz, H-1′), 5.10 (s, 1H, H-6), 4.99 (m, 1H,
H-3′), 3.96-3.86 (m, 3H, H-4′, H-5′ and H-5′′), 2.30-2.23 (m, 1H,
H-2′), 2.00 (s, 3H, COCH3), 1.94 (s, 3H, COCH3), 1.65-1.60 (m, 1H,
H-2′′), 1.40 (s, 3H, CH3).
1H NMR of compound 3b (DMSO-d6): 10.28 (s, 1H, NH), 7.47-
7.44 (m, 2H, H-PhS), 7.38-7.33 (m, 3H, H-PhS), 6.24 (s, 1H, 5-OH),
5.92 (dd, J ) 6.3, 5.9 Hz, 1H, H-1′), 5.13-5.11 (m, 1H, H-3′), 4.92
(s, 1H, H-6), 4.20-4.15 (m, 3H, H-4′, H-5′ and H-5′′), 2.67-2.60 (m,
1H, H-2′), 2.05-1.97 (m, overlap with COCH3 signal, 1H, H-2′′), 2.05
(s, 3H, COCH3), 2.03 (s, 3H, COCH3), 1.94 (s, 3H, CH3).
Synthesis of Compounds 3c and 3d (Scheme 2). Compounds 3c
and 3d were prepared according to the same procedures as described
for the syntheses of compounds 3a and 3b. These two compounds,
however, could be separated from each other by silica gel column
chromatography, and they were obtained as colorless glass.
Compound 3c (yield: 34.6%): Rf ) 0.44 (hexane/EtOAc, 1/2, v/v).
ESI-MS: m/z 450.9 [M - H]-, MS/MS 341.1 [M - H - PhSH]-;
452.8 (weak) [M + H]+, 475.1 (strong) [M + Na]+. 1H NMR (DMSO-
d6): 10.67 (s, 1H, NH), 7.52-7.49 (m, 2H, H-PhS), 7.37-7.33 (m,
3H, H-PhS), 6.28 (s, 1H, 5-OH), 6.01-5.98 (m, 1H, H-1′), 4.97 (m,
1H, H-3′), 4.83 (s, 1H, H-6), 4.08 (m, 2H, H-5′ and H-5′′), 3.99 (m,
1H, H-4′), 2.24-2.18 (m, 1H, H-2′), 2.02 (s, 3H, COCH3), 1.93 (s,
3H, COCH3), 1.56-1.52 (m, 1H, H-2′′), 1.32 (s, 3H, CH3).
Compound 3d (yield: 26.0%): Rf ) 0.28 (hexane/EtOAc, 1/2, v/v).
ESI-MS: m/z 450.9 [M - H]-, MS/MS 341.2 [M - H - PhSH]-;
452.7 (weak) [M + H]+, 475.1 (strong) [M + Na]+. 1H NMR (DMSO-
d6): 10.48 (s, 1H, NH), 7.50-7.47 (m, 2H, H-PhS), 7.35-7.30 (m,
3H, H-PhS), 6.42 (s, 1H, 5-OH), 5.81 (t, J ) 5.9 Hz, 1H, H-1′), 5.04-
5.01 (m, 1H, H-3′), 4.97 (s, 1H, H-6), 4.06-4.04 (m, 1H, H-4′), 3.99-
The white foam was loaded onto a silica gel column and eluted with
CH2Cl2/EtOAC (5/6, v/v). Appropriate fractions were combined, and
the solvent was evaporated off under reduced pressure to afford
compounds 2a (5.27 g, 62.3%) and 2b (2.07 g, 24.4%) as colorless
crystal and amorphous white powder, respectively.
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A R T I C L E S
Zhang and Wang
3.97 (m, 2H, H-5′ and H-5′′), 2.37-2.31 (m, 1H, H-2′), 2.04 (s, 3H,
COCH3), 1.96 (s, 3H, COCH3), 1.95-1.93 (m, 1H, H-2′′), 1.42 (s, 3H,
CH3).
of 4-15% MeOH in EtOAc. Appropriate fractions were pooled, and
the solvent was evaporated to afford white foam (210 mg, 53.8%).
ESI-MS: m/z 1227.2 [M - H]-.
Synthesis of (5R,6R)-cis-5-Hydroxy-6-phenylthio-5,6-dihydro-
thymidine (4a) and (5R,6S)-trans-5-Hydroxy-6-phenylthio-5,6-di-
hydrothymidine) (4b) (Scheme 2). The above 3a/3b mixture (0.500
g, 1.11 mmol) was dissolved in 100 mL of saturated methanolic
ammonia and stirred at room temperature for 9 h. The solvent was
removed under reduced pressure. The dried residue was redissolved in
1 mL of MeOH/EtOAc (1/80, v/v), loaded onto a silica gel column,
and eluted with 4% MeOH in EtOAc. Appropriate fractions were
pooled, and the solvent was removed to afford colorless glass (349
mg, 86.1%). ESI-MS: 367.0 [M - H]-, MS/MS 257.1 [M - H -
PhSH]-; 368.9 [M + H]+. Compounds 4a and 4b again could not be
resolved from each other by silica gel column chromatography, and
the proton resonances in 1H NMR were assigned based on 2-D COSY
and NOESY experiments.
1H NMR of compound 4a (DMSO-d6): 10.48 (s, br, 1H, NH), 7.53-
7.49 (m, 2H, H-PhS), 7.31-7.28 (m, 3H, H-PhS), 6.00 (s, 1H, 5-OH),
5.38 (t, J ) 6.3 Hz, 1H, H-1′), 5.25 (s, 1H, H-6), 4.99 (d, J ) 4.3 Hz,
1H, 3′-OH), 4.68 (t, J ) 5.1 Hz, 1H, 5′-OH), 4.05-4.01 (m, 1H, H-3′),
3.61-3.57 (m, 1H, H-4′), 3.26 (m, 2H, H-5′ and H-5′′), 2.06-1.99
(m, 1H, H-2′), 1.74-1.69 (m, 1H, H-2′′), 1.38 (s, 3H, CH3).
1H NMR of compound 4b (DMSO-d6): 10.38 (s, br, 1H, NH), 7.48-
7.44 (m, 2H, H-PhS), 7.37-7.32 (m, 3H, H-PhS), 6.07 (s, 1H, 5-OH),
6.01 (t, overlapped with 5-OH of compound 4a, 1H, H-1′), 5.20 (s,
1H, H-6), 5.10 (d, J ) 4.0 Hz, 1H, 3′-OH), 4.89 (dd, J ) 5.5, 5.1 Hz,
1H, 5′-OH), 4.15-4.11 (m, 1H, H-3′), 3.65 (m, 1H, H-4′), 3.48 (m,
2H, H-5′ and H-5′′), 2.38-2.32 (m, 1H, H-2′), 1.78-1.74 (m, 1H,
H-2′′), 1.29 (s, 3H, CH3).
The above foam was deprotected with 7 N methanolic ammonia
(100 mL) for 12 h at room temperature, and the solvent was removed
to give colorless solid. ESI-MS: m/z 998.3 [M - H]-, MS/MS 888.1
[M - H - PhSH]-. The resulting solid was detritylated with 80% acetic
acid at room temperature for 6 h. The solvent was removed under
reduced pressure. The dried residue was dissolved in water and extracted
with ether. The aqueous layer was dried by Speed-vac to yield light
yellowish powder. ESI-MS: m/z 696.1 [M - H]-, MS/MS 586.1 [M
- H - PhSH]-.
The above mixture was separated by HPLC as described in an earlier
section. The ratio between d(GT1*) and d(GT2*) was estimated to be
approximately 4:1 from the peak areas in the HPLC trace, which is
consistent with the ratios of compounds 3a/3b and 4a/4b.
Compound d(GT1*): ESI-MS: m/z 696.2 [M - H]-. HRMS (ESI-
1
FTICR) calcd 696.1489 [M - H]-, found 696.1501. H NMR (D2O):
7.96 (s, 1H, H-8 of dG), 7.45-7.37 (m, 3H, H-PhS), 7.28-7.23 (m,
2H, H-PhS), 6.24 (t, J ) 6.4 Hz, 1H, H-1′ of dG), 6.01 (t, J ) 6.9 Hz,
1H, H-1′ of dT*), 5.05 (s, 1H, H-6 of dT*), 4.97 (m, 1H, H-3′ of dG),
4.49 (m, 1H, H-3′ of dT*), 4.26 (m, 1H, H-4′ of dG), 4.21-4.15 (m,
1H, H-4′ of dT*), 4.14-4.10 (m, 2H, H-5′ and H-5′′ of dT*), 3.89-
3.80 (m, 2H, H-5′ and H-5′′ of dG), 2.89-2.83 (m, 1H, H-2′ of dG),
2.79-2.74 (m, 1H, H-2′′ of dG), 2.41-2.35 (m, 1H, H-2′ of dT*),
2.21-2.16 (m, 1H, H-2′′ of dT*), 1.52 (s, 3H, CH3).
Compound d(GT2*): ESI-MS: m/z 696.3 [M - H]-. HRMS (ESI-
1
FTICR) calcd 696.1489 [M - H]-, found 696.1482. H NMR (D2O):
8.00 (s, 1H, H-8 of dG), 7.49-7.46 (t, J ) 7.3 Hz, 1H, H-PhS), 7.37-
7.31 (m, 4H, H-PhS), 6.33 (dd, J ) 7.7, 6.4 Hz, 1H, H-1′ of dG), 6.14
(dd, J ) 7.7, 6.9 Hz, 1H, H-1′ of dT*), 5.03 (s, 1H, H-6 of dT*), 5.02
(m, 1H, H-3′ of dG), 4.62 (m, 1H, H-3′ of dT*), 4.33 (m, 1H, H-4′ of
dG), 4.18-4.13 (m, 3H, H-4′ and H-5′, H-5′′ of dT*), 3.90-3.83 (m,
2H, H-5′ and H-5′′ of dG), 2.94-2.86 (m, 2H, H-2′ of dG and H-2′ of
dT*), 2.82-2.77 (m, 1H, H-2′′ of dG), 2.29-2.24 (m, 1H, H-2′′ of
dT*), 1.72 (s, 3H, CH3).
Synthesis of d(GT3*) (Scheme 3). Compound d(GT3*) was prepared
by using the same procedures as described for the synthesis of d(GT1*)/
d(GT2*), and it was obtained as white powder (158 mg, 56.2%). ESI-
MS: m/z 696.3 [M - H]-. HRMS (ESI-FTICR) calcd 696.1489 [M -
H]-, found 696.1489. 1H NMR (D2O): 8.02 (s, 1H, H-8 of dG), 7.49-
7.42 (m, 3H, H-PhS), 7.35-7.32 (dd, J1 ) 7.3 Hz, J2 ) 7.7 Hz, 2H,
H-PhS), 6.35 (m, 1H, H-1′ of dG), 6.18 (m, 1H, H-1′ of dT*), 5.04 (s,
1H, H-6 of dT*), 4.99 (m, 1H, H-3′ of dG), 4.55 (m, 1H, H-3′ of dT*),
4.33 (m, 1H, H-4′ of dG), 4.12 (m, 3H, H-4′ and H-5′, H-5′′ of dT*),
3.89-3.82 (m, 2H, H-5′ and H-5′′ of dG), 2.90-2.75 (m, 3H, H-2′,
H-2′′ of dG and H-2′ of dT*), 2.19-2.14 (m, 1H, H-2′′ of dT*), 1.54
(s, 3H, CH3).
Synthesis of d(GT4*). The title compound was prepared by using
the same procedures as described for the synthesis of d(GT3*), and it
was obtained as white powder (180 mg, 57.5%). ESI-MS: m/z 696.3
[M - H]-. HRMS (ESI-FTICR) calcd 696.1489 [M - H]-, found
696.1494. 1H NMR (D2O): 7.95 (s, 1H, H-8 of dG), 7.40 (t, 1H,
H-PhS), 7.32 (d, 2H, H-PhS), 7.25 (t, 2H, H-PhS), 6.22 (dd, J ) 6.4,
6.0 Hz, 1H, H-1′ of dG), 6.15 (dd, J ) 6.9, 6.4 Hz, 1H, H-1′ of dT*),
5.02 (s, 1H, H-6 of dT*), 5.01-4.97 (m, 1H, H-3′ of dG), 4.52 (m,
1H, H-3′ of dT*), 4.25-4.14 (m, 4H, H-4′ of dG, H-4′, H-5′, and H-5′′
of dT*), 3.92-3.83 (m, 2H, H-5′ and H-5′′ of dG), 2.93-2.87 (m, 1H,
H-2′ of dG), 2.81-2.76 (m, 1H, H-2′′ of dG), 2.42-2.36 (m, 1H, H-2′
of dT*), 2.21-2.17 (m, 1H, H-2′′ of dT*), 1.66 (s, 3H, CH3).
Synthesis of d(TT1*). This compound was prepared according to
the same procedure as for the preparation of d(GT1*) except that dT-
CE phosphoramidite was used in place of the iPr-PAC-dG-CE
phosphoramidite. Yield for the coupling step: 83.0%. The resulting
mixture of d(TT1*) and d(TT2*) was again separated by HPLC.
d(TT1*): ESI-MS: m/z 671.1 [M - H]-, MS/MS: 561.1 [M - H -
Synthesis of (5S,6S)-cis-5-Hydroxy-6-phenylthio-5,6-dihydro-
thymidine (4c) (Scheme 2). Similar to the synthesis of compounds 4a
and 4b, compound 3c (364 mg) was deacetylated with methanolic
ammonia to afford compound 4c as glass (242 mg, 81.7%). Rf ) 0.48
(MeOH/EtOAc, 1/25, v/v). ESI-MS: 367.0 [M - H]-, MS/MS 257.1
[M - H - PhSH]-; 368.9 [M + H]+. 1H NMR (DMSO-d6): 10.58 (s,
br, 1H, NH), 7.46-7.42 (m, 2H, H-PhS), 7.33-7.29 (m, 3H, H-PhS),
6.07 (s, 1H, 5-OH), 6.04 (t, J ) 5.5 Hz, 1H, H-1′), 5.15 (s, 1H, H-6),
5.04 (d, J ) 4.0 Hz, 1H, 3′-OH), 4.83 (t, 1H, 5′-OH), 4.01 (m, 1H,
H-3′), 3.61 (m, 1H, H-4′), 3.40-3.35 (m, 1H, H-5′), 3.29 (m, 1H, H-5′′),
2.06-2.00 (m, 1H, H-2′), 1.42-1.39 (m, 1H, H-2′′), 1.38 (s, 3H, CH3).
Synthesis of (5S,6R)-trans-5-Hydroxy-6-phenylthio-5,6-dihydro-
thymidine (4d) (Scheme 2). Compound 3d (278 mg) was deacetylated
with methanolic ammonia and separated on silica gel column with
MeOH/EtOAc (1/10, v/v) to afford compound 4d as glass (168 mg,
74.2%). Rf ) 0.42 (MeOH/EtOAc, 1/10, v/v). ESI-MS: 367.1 [M -
1
H]-, MS/MS 257.1 [M - H - PhSH]-; 368.9 [M + H]+. H NMR
(DMSO-d6): 10.34 (s, 1H, NH), 7.53-7.51 (m, 2H, H-PhS), 7.34-
7.32 (m, 3H, H-PhS), 6.27 (s, 1H, 5-OH), 5.77 (dd, J ) 7.1, 5.9 Hz,
1H, H-1′), 5.12 (s, 1H, H-6), 5.04 (d, J ) 4.3 Hz, 1H, 3′-OH), 4.74 (t,
J ) 5.1 Hz, 1H, 5′-OH), 4.10-4.07 (m, 1H, H-3′), 3.68-3.66 (m, 1H,
H-4′), 3.38-3.35 (m, 2H, H-5′ and H-5′′), 2.15-2.10 (m, 1H, H-2′),
1.86-1.81 (m, 1H, H-2′′), 1.35 (s, 3H, CH3).
Synthesis of d(GT1*) and d(GT2*) (T1* and T2* Represent 4a
and 4b, Respectively). To a 25-mL flask, which contained a mixture
of compounds 4a and 4b (117 mg), was added 4,5-dicyanoimmidazole
(100 mg) and DMF (0.8 mL). The flask was kept under argon
atmosphere, and to the stirred solution was added dropwise iPr-PAC-
dG-CE phosphoramidite (0.25 g in 0.8 mL of DMF) over 15 min. The
solution was stirred for another 15 min, to which solution t-BuOOH
(100 µL, 5 M solution in nonane) was added. The solution was stirred
for still another 25 min, and the reaction mixture was extracted with
EtOAc. The organic layer was washed with 5% NaHCO3 solution,
followed by saturated NaCl solution, and dried with anhydrous Na2-
SO4. The solvent was removed by evaporation under reduced pressure.
The dried residue was separated on a silica gel column with a gradient
9
13296 J. AM. CHEM. SOC. VOL. 126, NO. 41, 2004

5-Hydroxy-5,6-dihydrothymidin-6-yl Radical
A R T I C L E S
PhSH]-, 435.1 [M - H - PhSH - T]- (T is thymine). 1H NMR
(D2O): 7.57 (s, 2H, H-PhS), 7.56 (s, 1H, H-6 of dT), 7.48 (t, J ) 7.3
Hz, 1H, H-PhS), 7.37 (m, 2H, H-PhS), 6.25 (dd, J ) 6.9, 6.4 Hz, 1H,
H-1′ of dT*), 5.96 (dd, J ) 6.8, 6.4 Hz, 1H, H-1′ of dT), 5.15 (s, 1H,
H-6 of dT*), 4.83 (m, 1H, H-3′ of dT), 4.47 (m, 1H, H-3′ of dT*),
4.16 (m, 1H, H-4′ of dT*), 4.11-4.04 (m, 3H, H-4′ of dT, H-5′ and
H-5′′ of dT*), 3.89-3.79 (m, 2H, H-5′ and H-5′′ of dT), 2.65-2.60
(m, 1H, H-2′ of dT), 2.52-2.47 (m, 1H, H-2′′ of dT), 2.42-2.36 (m,
1H, H-2′ of dT*), 2.22-2.17 (m, 1H, H-2′′ of dT*), 1.84 (s, 3H, CH3
of dT), 1.58 (s, 3H, CH3 of dT*).
6-hydroxy-5,6-dihydrothymidin-5-yl radical as was originally
proposed.44
We also found that the major lesion initiated from the 254-
nm irradiation of the dinucleoside monophosphates d(GT*) and
d(TT*) is the dinucleoside monophosphate with the thymidine
portion being degraded to an abasic site. However, the relative
yield for the formation of the abasic site lesion over the cross-
link lesion is much higher from the 254-nm irradiation of
d(GT1*) than that from the γ-radiation of d(GT). This result
demonstrates that the C6 carbon cation, which forms from the
oxidation of the reactive intermediate I and is generated more
readily from the 254-nm irradiation of d(GT1*) than from the
γ-radiation of d(GT), contributes to a significant extent to the
formation of the abasic site lesion.
The realization of the independent generation of this radical
also sets the stage for the examination of the reactivity of this
radical in double-stranded DNA. In this respect, we are in the
process of synthesizing the phosphoramidite building block for
the precursor.
Conclusions
We synthesized photolabile precursors for the generation of
both the 5R and 5S isomers of 5-hydroxy-5,6-dihydrothymidin-
6-yl radical (I) and examined their reactivities in dinucleoside
monophosphates through rigorous product analyses.
We observed that, under anaerobic conditions, radical I can
attack the C8 carbon atom of its 5′ neighboring guanine and
give rise to a cross-link lesion. The yield for the formation of
this lesion is affected by the stereochemistry of the C5 carbon.
The 5S radical forms the cross-link lesion more efficiently than
the 5R radical. Moreover, we showed by LC-MS/MS that
γ-irradiation of d(GT) under anaerobic condition can also lead
to the formation of the two isomeric cross-link lesions again
with the formation of the 5S isomer being more facile than that
of the 5R isomer. Our study also demonstrated that the cross-
link lesion previously isolated by Box et al.44 from the X-ray
irradiation mixture of d(GT) under anoxic condition is the lesion
that is initiated from the 5S isomer of radical I, not from the
Acknowledgment. We want to thank the National Institute
of Health (R01 CA101864) for supporting this research.
Supporting Information Available: NMR spectra of synthetic
compounds, LC traces, LC-MS/MS data, and spectroscopic
characterizations of products isolated from the irradiation
mixtures. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA048492T
9
J. AM. CHEM. SOC. VOL. 126, NO. 41, 2004 13297