Hypoxia-selective DNA interstrand cross-link formation by two modified nucleosides

Article abstract of DOI:10.1002/chem.201201960

The clean crossed code: Two nitroimidazole-modified thymidines 1 a and 1 b were synthesized and incorporated into DNA oligomers. The 350 nm photolysis of 1 a and 1 b generated a 5-(2'-deoxyuridinyl)methyl radical that induced DNA interstrand cross-links (ICL; see scheme). A higher ICL yield was observed under hypoxic conditions than under aerobic conditions. Copyright

Full text of DOI:10.1002/chem.201201960

COMMUNICATION
DOI: 10.1002/chem.201201960
Hypoxia-Selective DNA Interstrand Cross-Link Formation by Two Modified
Nucleosides
Yunyan Kuang,[a, b] Huabing Sun,[a] J. Craig Blain,[c] and Xiaohua Peng*[a]
DNA interstrand cross-links (ICLs) are deleterious to
cells because of their unyielding obstruction to replication
and transcription.[1,2] For example, a single unrepaired ICL
can be sufficient to kill an eukaryotic cell. Many anticancer
agents, such as mitomycin C, nitrogen mustard, and chlor-
ambucil, can kill cancer cells by alkylating DNA opposing
strands. However, these agents are also toxic to healthy
cells. One approach to reduce the toxicity of the cross-link-
ing agents for normal cells is to trigger site-selective cross-
link formation in tumor cells. Hypoxia appears to be a
common and unique property of cells in solid tumors, and is
an important potential mechanism for the tumor-specific ac-
tivation of prodrugs.[3–5] Therefore, it is of great interest to
develop agents that can form DNA ICLs selectively under
hypoxic conditions.
fied pyrimidine nucleosides (1a, 1b) and we determined
their radiosensitivity and hypoxia selectivity with respect to
DNA ICL formation. Our investigations show that 1a and
1b can form ICL selectively under hypoxic condition.
Compounds 1a and 1b were synthesized starting from
thymidine (dT), which was protected in the sugar moiety
with an acetyl group (2; Scheme 1). The bromination of 2
Exogenous molecules have been developed that sensitize
DNA to g-radiolysis under anoxic conditions, such as nitroi-
midazole and nitrophenyl derivatives.[6,7] Coupling of a ni-
troaryl group with active species resulted in hypoxia-target-
ing anticancer drugs.[8] The non-native nucleosides 5-bromo-
and 5-iodo-2’-deoxyuridine are also used as radiosensitiz-
ers.[9] Recently, Greenbergꢀs group has reported that a
phenyl selenide-modified thymidine (dT-SePh) produces
DNA ICLs upon UV photolysis or g-radiolysis via a nucleo-
tide radical.[10–13] The ICL formation is independent of oxy-
gen.[10,12a] We expect that the introduction of a hypoxic radi-
osensitizer to the modified nucleosides will make them
more sensitive to photolysis under hypoxic condition. The
above encourage us to synthesize two nitroimidazole-modi-
Scheme 1. Synthesis of compound 1a and 1b. Reagents: a) i. NBS, ben-
zene, ii. NaH, nitroimidazole, DMF; b) aq. NH3, 1,4-dioxane; c) DMTCl,
pyridine; d) 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite, N,N-
diisopropylethylamine, CH2Cl2.
[a] Dr. Y. Kuang, Dr. H. Sun, Prof. X. Peng
Department of Chemistry and Biochemistry
University of Wisconsin–Milwaukee
3210 N Cramer St., Milwaukee WI 53211 (USA)
Fax : (+1)414-2295530
followed by the introduction of nitroimidazole group result-
ed in 3a and 3b. Deprotection of 3a and 3b yielded 1a and
1b, which were converted to their phosphoramidite building
blocks 5a and 5b. Oligonucleotides (6–13) containing 1a or
1b were synthesized through automated solid-phase synthe-
sis by using 5a or 5b. The coupling yields were more than
98%.
E-mail: pengx@uwm.edu
[b] Dr. Y. Kuang
Department of Chemistry, Fudan University
220 Handan Road, Shanghai City, 200433 (P.R. China)
[c] Dr. J. C. Blain
Howard Hughes Medical Institute
Department of Molecular Biology and
The Center for Computational and Integrative Biology
Harvard Medical School, 185 Cambridge St.
Boston, MA 02114 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201960.
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The nitroimidazole slightly decreased the duplex stability
(6 and 10) with a DTm of 3–48C relative to when dT was
present (duplex 14; Table 1). Similar to dT, 1a and 1b show
mismatch discrimination towards dC (DTm =ꢀ10.8 and
ꢀ88C), dG (ꢀ7.7 and ꢀ5.78C), and dT (ꢀ10.4 and ꢀ8.68C).
These data suggest that 1a and 1b form a Watson–Crick
base pair with dA.
Table 1. Melting temperatures [8C] of duplexes 5’-d-AGATGGAT
XTAGGTAC (complementary strand 3’-d-TCTACCTAYATCCATG).
Figure 2. Cross-linking efficiency of compounds 1a and 1b under aerobic
and anaerobic conditions: A) duplexes containing 1a (6–9) were irradiat-
ed with UV for 2 h; B) duplexes containing 1b (10–13) were UV irradiat-
X·Y
Tm (DTm)
X·Y
Tm (DTm)
dT·dA (14)
1a·dA (6)
1a·dC
1a·dG
1a·dT
52.1
48.8
38.0 (ꢀ10.8)
41.1 (ꢀ7.7)
38.4 (ꢀ10.4)
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ed for 2 h [ : aerobic conditions (air); : anaerobic conditions (Ar2)].
1b·dA (10)
1b·dC
1b·dG
47.7
39.7 (ꢀ8.0)
42.0 (ꢀ5.7)
39.1 (ꢀ8.6)
1b·dT
place of 1a was irradiated by UV and less than 0.2% ICL
was observed under both aerobic and anaerobic conditions.
This indicated that the nitroimidazole group in 1a plays an
integral role in ICL formation and hypoxia-selectivity in 6–
9. In order to determine the generality of this property, we
synthesized 2-nitroimidazole-modified thymidine (1b), and
examined its ability for ICL formation. Again, higher cross-
linking yield was observed under hypoxic conditions than
under aerobic conditions when the duplexes containing 1b
(10–13) were irradiated at 350 nm (Figure 2B).
There are two possible mechanisms for the ICL forma-
tion, either through a 5-(2’-deoxyuridinyl)methyl radical
16[12] or via formation of a carbocation derived from the pro-
tonated 1a or 1b.[16] However, the carbocation mechanism is
considered particularly unlikely as the incubation of duplex-
es 6 and 10 at 378C in a buffer of pH 5–9 produced no ICL,
whereas the incubated reaction mixture afforded the cross-
linking adduct after 350 nm photolysis (Supporting Informa-
tion Figure S2). The support for the radical mechanism was
obtained by determining the rate constant for ICL forma-
tion in 6 by measuring the dependence of its formation on
the concentration of thiol (RSH, b-mercaptoethanol).[12,13]
The estimated rate constant (kICL) for ICL formation in-
All DNA duplexes were photo-irradiated at 350 nm by
using a Rayonet photochemical chamber reactor. A wave-
length of 350 nm was chosen because near-UV light (>
300 nm) is either not or only slightly absorbed by most bio-
logical molecules and is compatible with living cells.[14] Both
compounds have their absorbance at 350 nm (A=0.08 for
0.023 mm 1a, and A=0.19 for 0.023 mm 1b; Supporting In-
formation Figure S46). Furthermore, the nitroimidazole de-
rivatives can be excited at 350 nm to form a radical anion
transition state.[15] The UV photolysis of 6 resulted in a new
band the migration of which is severely retarded relative to
unreacted oligonucleotide; this is indicative of interstrand
cross-linked material (Figure 1). The cross-linking yield
under hypoxic conditions (3.6%) is 1.6-times higher than
that under aerobic conditions (2.2%; Figure 1, lane 5 vs. 6).
We also examined cross-link formation with duplexes 7–9 in
which 1a is flanked by different sequences (Figure 1, lane 8
vs. 9, 11 vs. 12, 14 vs.15). In all cases, hypoxic conditions re-
sulted in higher cross-linking yield than aerobic conditions
(Figure 2A). This further proved the hypoxia-selective
cross-linking formation induced by 1a. In a control experi-
ment, an otherwise identical duplex (14) containing dT in
duced by 1a was 3.5ACHTUNGTRENNUNG
alkyl radical trapping by a thiol (KRSH =1ꢂ107 mꢀ1 sꢀ1) is
used [Eq. (1)] (Figure 3).[12] This is consistent with the rate
Figure 1. Hypoxia-selective interstrand cross-link formation by 1a upon
UV photolysis at 350 nm for 2 h under aerobic and anaerobic conditions.
Phosphorimage autoradiogram of 20% denaturing PAGE analysis of the
ICL product from duplexes containing 1a (6–9). Lanes 1, 4, 7, 10 and 13:
DNA duplexes without UV irradiation; lanes 2, 5, 8, 11 and 14: DNA du-
plexes irradiated under aerobic condition; lanes 3, 6, 9, 12 and 15: DNA
duplexes irradiated under anaerobic condition.
Figure 3. Ratio of ssDNA/ICL produced from 6 as a function of b-mer-
captoethanol (BME) concentration (ssDNA: single stranded DNA; ICL:
interstrand cross-links).
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Hypoxia-Selective DNA Interstrand Cross-Link Formation
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constant for ICL formation from 5-(2’-deoxyuridinyl)methyl
radical 16 (kICL =3.8ꢂ104 sꢀ1) that was observed by Green-
berg et al.[12]
½ssDNAꢂ KRSH
Kx
KICL
ð1Þ
¼
½RSHꢂ þ
KICL
½ICLꢂ
The formation of nucleotide radical 16 was further proved
by the analysis of the product formed by 350 nm photolysis
of the monomer. When 1a was irradiated for 3 h under
aerobic conditions, 5-hydroxymethyl-2’-deoxyuridine (23)
was observed (Scheme 2). Compound 23 was confirmed by
using an authentic sample (Supporting Information Fig-
ure S45). The formation of a hydroxymethyl derivative from
16 was also reported by Greenberg and Cadet.[12,17] All these
indicated that 350 nm photolysis of 1a released nitroimida-
zole, and generated 5-(2’-deoxyuridinyl)methyl radical 16,
which finally produced ICL formation. The details and
mechanism for the ICL formation by 16 have been reported
by Greenberg et al.[11,12] The final ICL product induced by
1a and 1b is stable when heated in phosphate buffer; this is
consistent with the previous report (Supporting Information
Figure S1, lanes 3 and 6).[11,12] However, cleavage bands
were observed upon piperidine treatment (Supporting Infor-
mation Figure S1, lanes 2 and 5). This indicated that apart
from the cross-linking reaction other side reactions took
place.
Scheme 2. Products generated from 1a upon UV photolysis.
Although the reason for the hypoxia selective ICL forma-
tion is unclear, an attractive mechanism appears for the for-
mation of a radical anion 15 through photo-induced single
electron transfer (PSET) from dT8 or other nucleotides in
the duplex DNA to 1a or 1b (Scheme 3).[18] The resulting
anion would spontaneously re-
were observed specially at the positions of the flanking dT8
and the modified nucleosides 1a,b. We believe that the 5-
(2’-deoxyuridinyl)methyl radical in 16 would abstract hydro-
gen from the sugar moiety of the thymidine radical cation
producing a direct strand break at dT8. Greenberg et al.
lease nitroimidazole anion to
generate 5-(2’-deoxyuridinyl)-
methyl radical 16, which direct-
ly produces ICLs (17 and 18).
Oxygen molecules can reoxid-
ize the one-electron reduced
radical anion 15 to the original
agent 1a or 1b, thereby de-
creasing the ICL yield. This was
further proved by the slower
rate of disappearance of mono-
mers upon 350 nm photolysis in
the presence of air (K1a-air
=
4.9ꢂ10ꢀ6 sꢀ1
;
K1b-air =1.7ꢂ
10ꢀ4 sꢀ1) than that in the pres-
ence of argon (K1a-Ar =6.1ꢂ
10ꢀ6 sꢀ1
;
K
1b-Ar =2.4ꢂ10ꢀ4 sꢀ1).
In addition to the ICL forma-
tion, direct strand breaks
(DSB; 2–7%, Supporting Infor-
mation Figure S1, lanes 1 and
4) and alkaline labile lesions
(10–15%, Supporting Informa-
tion Figure S1, lanes 2 and 5) Scheme 3. Proposed mechanism for the ICL formation.
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have shown that nucleotide radical can abstract C-2ꢀ hydro-
gen from the 5’-adjacent nucleotide.[18] In addition, the thy-
midine radical cation in 16 can react with water to form al-
kaline labile lesions, such as thymine glycol and/or 5-hydrox-
yl-5,6-dihydropyrimidine derivatives (17 and 18), which pro-
duce cleavage bands upon piperidine treatment (Supporting
Information Figure S3A, C, lane 3, and Figure S4A, C,
lane 3).[15,19,20]
In an effort to determine the structure of the cross-linked
product and possible side reactions, we used LC-MS to char-
acterize the materials isolated from denaturing gels. A series
of discrete peaks were observed, whereas the exact mass of
ICL products was not obtained. Many peaks are smaller
than the calculated mass of ICL product having nitroimida-
zole (9872.15), but larger than the calculated mass of ICL
product losing nitroimidazole (9759.49 for 6 and 10; Sup-
porting Information Figures S43 and S44). The differences
between these peaks are usually 17 or 18; this indicates that
a hydroxyl group or a water molecule was added. For exam-
ple, a peak at 9810 corresponds to the addition of three hy-
droxyl groups and 9830 corresponds to the addition of two
water molecules and two hydroxyl groups (Supporting Infor-
mation Figure S43). This further proves the formation of
thymine glycol and/or 5-hydroxyl-5,6-dihydropyrimidine de-
rivatives and other DNA lesions with the addition of the hy-
droxyl group or water molecules. The alkaline labile lesions
were also observed at positions 20–23 of the complementary
strand; this was evidenced by the formation of the cleavage
bands at these positions caused by the piperidine treatment
of the ICL product (Supporting Information, lane 3 in Fig-
ure S3B, D and Figure S4B, D). During LC-MS analysis, the
exact mass of 19 with an aldehyde on the 5-methyl group
was observed (observed m/z 4974.6056, calcd m/z 4974.2792;
Scheme 2 and Supporting Information Figure S42). We be-
lieve that 19 is derived from the peroxyl radical 20, as pro-
posed by Greenberg and Cadet.[12,17]
Hydroxyl radical cleavage of gel-purified cross-linked
DNA was used to determine which nucleotides were cova-
lently bonded to one another.[11] Each oligonucleotide was
either 5’-32P- or 3’-32P-labeled. As expected, cross-linking oc-
curred mainly at the position of 1a or 1b where 16 was gen-
erated (Figure 4A, C). The majority of the cross-links in the
complementary strand involve reaction with dA23. Partial
ICL took place with dA24 (Figure 4B, D, and Supporting
Information Figure S47).
In summary, this work demonstrated that coupling of a
hypoxia-radiosensitizer (e.g., nitroimidazoles) with a pyrimi-
dine nucleoside allows for more efficient DNA interstrand
cross-link formation under hypoxic conditions than that
under aerobic conditions. The mechanism involves the re-
lease of a 5-(2’-deoxyuridinyl)methyl radical from a nucleo-
side radical anion, which is generated by photo-induced
single electron transfer (PSET) within duplex DNA. The
presence of oxygen quenches the radical anion therefore de-
creasing the ICL yield. Work is in progress to determine the
hypoxia-selectivity of these compounds in hypoxic cells, and
their potential as hypoxia-targeting anticancer drugs.
C
Figure 4. Determination of interstrand cross-linking position via OH
cleavage of UV-treated 6 and 10: A) 5’-[32P]6a, the 4-nitroimidazole-con-
taining strand was labeled; B) 5’-[32P]6b, the complementary strand was
labeled; C) 5’-[32P]10a, the 2-nitroimidazole containing strand was la-
beled; D) 5’-[32P]10b, the complementary strand was labeled.
Experimental Section
General methods: Unless otherwise specified, chemicals were purchased
from Aldrich or Fisher Scientific and were used as received without fur-
ther purification. T4 polynucleotide kinase was obtained from New Eng-
land Biolabs. Oligonucleotides were synthesized by standard automated
DNA synthesis techniques by using an Applied Biosystems model 394 in-
strument in a 1.0 mm scale with commercial 1000 ꢃ CPG-succinyl-nucleo-
side supports. Deprotection of the nucleobases and phosphate moieties
as well as cleavage of the linker were carried out under mild deprotection
conditions with a mixture of 40% aq. MeNH2 and 28% aq. NH3 (1:1) at
room temperature for 2 h. Thermal denaturation temperatures (Tm
values) were measured on a Cary 100 UV/Vis sepectrometer equipped
with a thermoelectrical temperature controller. The temperature was
changed at a rate of 18Cminꢀ1. Radiolabeling was carried out according
to the standard protocols.[21] [g-32P]ATP and [a-32P]ATP were purchased
from Perkin–Elmer Life Sciences. Quantification of radiolabeled oligonu-
cleotides was carried out by using a Molecular Dynamics Phosphorimag-
er equipped with ImageQuant Version 5.2 software. 1H and 13C NMR
spectra were recorded on either a Bruker DRX 300 or DRX 500 MHz
spectrophotometer. High resolution mass spectrometry was performed at
the University of Kansas Mass Spectrometry Laboratory. ESI-MS for oli-
gonucleotides and cross-linking DNA was determined by the Center for
Computational and Integrative Biology of Harvard Medical School.
Sample preparation under hypoxic conditions: For preparing samples
under reduced O2 tension conditions, samples were degassed by using a
high vacuum pump (1 min) to remove the dissolved O2, then purged with
argon (1.0 min; three cycles).
Interstrand cross-link formation and kinetics study with duplex DNA:
The 32P-labeled oligonucleotide (1.0 mm) was annealed with 1.5 equiv of
the complementary strand by heating to 658C for 3 min in buffer (10 mm
potassium phosphate, pH 7.5, and 100 mm NaCl), followed by slow-cool-
ing to room temperature, overnight. The 32P-labeled oligonucleotide
duplex (2 mL, 1.0 mm) was mixed with NaCl (1m, 2 mL), potassium phos-
phate (100 mm, 2 mL, pH 7.5) and an appropriate amount of autoclaved
distilled water to give a final volume of 20 mL. The mixture was degassed
to remove the air and purged with argon, then UV irradiated in a Rayo-
net photochemical chamber reactor (Model RPR-100, sixteen bulbs,
350 nm light wavelength) for 2 h. The reaction was quenched with an
equal volume of 90% formamide stop/loading buffer, then electrophor-
esed on a 20% denaturing polyacrylamide gel at 1200 V for approximate-
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Hypoxia-Selective DNA Interstrand Cross-Link Formation
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ly 4 h. For kinetics studies, aliquots (final concentration: 50 nm 32P-la-
beled oligonucleotide duplex, 100 mm NaCl, 10 mm potassium phosphate)
mixed with appropriate concentration of 2-mercaptoethanol were irradi-
ated under argon for 2 h and immediately quenched with 90% forma-
mide loading buffer, and stored at ꢀ208C until they were subjected to
20% denaturing PAGE analysis.
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Hydroxyl radical reaction (Fe·EDTA reaction): The cross-linked DNA
molecules formed in duplexes 6 and 10 were purified by 20% denaturing
PAGE. The band containing cross-linked product was cut, crushed, and
eluted with NaCl (200 mm), EDTA (20 mm, 2.0 mL). The crude product
was further purified by C18 column and eluted with H2O (3ꢂ1.0 mL) fol-
lowed by MeOH/H2O (3:2, 1.0 mL). FeII·EDTA cleavage reactions of
32P-labeled oligonucleotide (0.1 mm) were performed in a buffer contain-
ing (NH4)2FeACHTUNGTRENNUNG(SO4)2 (50 mm), EDTA (100 mm), sodium ascorbate (5 mm),
NaCl (0.5m), sodium phosphate (50 mm, pH 7.5) and H2O2 (1 mm) for
3 min at room temperature (total substrate volume 20 mL), then
quenched with thiourea (100 mm, 10 mL). Samples were lyophilized, dis-
solved in 20 mL H2O/90% formamide loading buffer (1:1) and subjected
to 20% denaturing PAGE analysis.
Kinetic study of monomer reaction upon UV photolysis (350 nm): Com-
pound 1a or 1b (8 mg) was dissolved in
a mixture of [D6]DMSO
(0.93 mL) and D2O (0.07 mL). The solution was equally divided into two
portions, which were placed in two NMR tubes. One sample was de-
gassed to remove air under high vacuum, then purged with Ar2 for 5 min.
Both samples were exposed to UV irradiation at 350 nm. 1H NMR spec-
tra were recorded at different times (2, 6, 12, 18, 24, 36 h for 1a; 5, 10,
30, 60, 90, 120, 150, 180, 240 min for 1b). The reaction yield was deter-
mined by calculating the decrease of the signal at 8.30 ppm, which corre-
sponds to imidazole. The peak at 2.50 ppm (DMSO) was used as a refer-
ence to quantify other peaks.
Stability study of ICL product formed with 6 or 10: After the cross-link
reaction, the reaction mixtures (0.35 mm DNA duplex, 20 mL) were co-
precipitated with calf thymus DNA (2.5 mgmLꢀ1, 5 mL) and NaOAc (3m,
5 mL) in the presence of EtOH (90 mL) at ꢀ808C for 30 min, and then
centrifuged for 5 min at 21890 g. The supernatant was removed, and the
pellet was washed with cold 75% EtOH and lyophilized for 30 min in a
Centrivap concentrator (LABCONCO) at 378C. The dried DNA frag-
ments were dissolved in H2O (30 mL) and divided into three portions.
One portion (10 mL) was incubated with piperidine (2m, 10 mL) at 908C
for 30 min, and the second portion (10 mL) was incubated with NaCl
(0.1m) and potassium phosphate buffer (10 mm, pH 7, 10 mL) under the
same condition, and the third portion was used as a control sample. The
samples were subjected to electrophoresis on a 20% denaturing polya-
crylamide gel.
Acknowledgements
[20] C. von Sonntag, in The Chemical Basis of Radiation Biology, Taylor
& Francis, London, 1987.
[21] T. Maniatis, E. F. Fritsch, J. Sambrook, Molecular Cloning: A Labo-
ratory Manuel, Cold Spring Harbor Laboratory, Cold Spring
Harbor, 1982.
We are grateful for financial support of this research from the National
Cancer Institute (1R15A152914-01) and UWM start-up funds.
Keywords: DNA cross-linking
·
DNA damage
·
Received: June 4, 2012
nucleosides · nucleic acids · UV photolysis
Published online: && &&, 0000
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X. Peng et al.
DNA Damage
Y. Kuang, H. Sun, J. C. Blain,
X. Peng* ....................... &&&& — &&&&
Hypoxia-Selective DNA Interstrand
Cross-Link Formation by Two Modi-
fied Nucleosides
The clean crossed code: Two nitroimi-
dazole modified thymidines 1a and 1b
were synthesized and incorporated
into DNA oligomers. The 350 nm pho-
tolysis of 1a and 1b generated a 5-(2’-
deoxyuridinyl)methyl radical that
induced DNA interstrand cross-links
(ICL; see scheme). A higher ICL yield
was observed under hypoxic conditions
than under aerobic conditions.
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