DNA interstrand cross-linking upon irradiation of aryl halide C-nucleotides

Article abstract of DOI:10.1021/jo4028227

γ-Radiolysis kills cells by damaging DNA via radical processes. Many of the radical pathways are O₂ dependent, which results in a reduction in the cytotoxicity of ionizing radiation in hypoxic tumor cells. Consequently, there is a need for chemical agents that increase DNA damage by ionizing radiation under O₂-deficient conditions. Modified nucleotides that are incorporated in DNA and produce highly reactive σ-radicals are useful as radiosensitizing agents. Aryl halide C-nucleotides (4-6) were incorporated into oligonucleotides by solid-phase synthesis. Duplex DNA containing 4-6 forms interstrand cross-links upon γ-radiolysis under anaerobic conditions or UV irradiation. Deep Vent (exo^-) DNA polymerase accepted the nucleotide triphosphate of C-nucleotide 6 as a substrate and preferentially incorporated it opposite pyrimidines, but no further extension was detected. Incorporation of 6 in extended products by Deep Vent (exo^-) during PCR or by Sequenase during copying of single stranded DNA plasmid was undetectable. Aryl halide nucleotide analogues that produce DNA interstrand cross-links under anaerobic conditions upon irradiation are potentially useful as radiosensitizing agents, but further research is needed to identify molecules that are incorporated by DNA polymerases and do not block further polymerization for this approach to be useful in cells.

Full text of DOI:10.1021/jo4028227

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DNA Interstrand Cross-Linking upon Irradiation of Aryl Halide
C‑Nucleotides
Dianjie Hou and Marc M. Greenberg*
Department of Chemistry Johns Hopkins University 3400 North Charles Street, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: γ-Radiolysis kills cells by damaging DNA via
radical processes. Many of the radical pathways are O2
dependent, which results in a reduction in the cytotoxicity of
ionizing radiation in hypoxic tumor cells. Consequently, there
is a need for chemical agents that increase DNA damage by
ionizing radiation under O2-deficient conditions. Modified
nucleotides that are incorporated in DNA and produce highly reactive σ-radicals are useful as radiosensitizing agents. Aryl halide
C-nucleotides (4−6) were incorporated into oligonucleotides by solid-phase synthesis. Duplex DNA containing 4−6 forms
interstrand cross-links upon γ-radiolysis under anaerobic conditions or UV irradiation. Deep Vent (exo−) DNA polymerase
accepted the nucleotide triphosphate of C-nucleotide 6 as a substrate and preferentially incorporated it opposite pyrimidines, but
no further extension was detected. Incorporation of 6 in extended products by Deep Vent (exo−) during PCR or by Sequenase
during copying of single stranded DNA plasmid was undetectable. Aryl halide nucleotide analogues that produce DNA
interstrand cross-links under anaerobic conditions upon irradiation are potentially useful as radiosensitizing agents, but further
research is needed to identify molecules that are incorporated by DNA polymerases and do not block further polymerization for
this approach to be useful in cells.
repaired by nucleotide excision repair (NER). The possible
importance of cross-linking by 1 is magnified by recent
examples in which ICLs are converted (“misrepaired”) during
NER to double strand breaks, the most deleterious form of
DNA damage.12−14 These observations inspired us to design
radiosensitizing agents that produce ICLs in base paired regions
of DNA.
INTRODUCTION
■
Radical-mediated DNA damage is the source of the cytotoxic
effects of ionizing radiation. Ionizing radiation’s effects are
enhanced by O2, which competes with thiols that can restore
DNA to its native structure. Some tumors are deficient in O2
(hypoxic), resulting in a decrease in radiation efficiency.
Radiosensitizing agents have been developed to overcome the
limitations imposed by hypoxia. Some of the most well studied
radiosensitizing agents are nucleotides that are incorporated
into cellular DNA by polymerases. 5-Bromo- (BrdU) and 5-
iodo-2′-deoxyuridine (IdU) are incorporated in DNA in place
of thymidine and sensitize the biopolymer to ionizing radiation
by scavenging solvated electrons produced from the ionization
of water and/or that are released from other portions of the
DNA and producing a highly reactive σ-radical (1, Scheme
1).1,2 The σ-radical abstracts hydrogen atoms from adjacent
nucleotides producing strand breaks and alkali-labile lesions.3−8
Recently, it was discovered that 1 also yields interstrand cross-
links (ICLs) but only in nonbase paired regions of duplex
DNA.9−11 ICLs are a very deleterious form of DNA damage
that are absolute blocks to replication and transcription and are
Based upon cross-linking resulting from the exposure of
DNA containing BrdU and other nucleotides to ionizing
radiation (or UV irradiation), we rationalized that the rotational
barrier around the glycosidic bond, coupled with the high
reactivity of 1, prevented it from producing ICls in base-paired
regions.11,15−17 We hypothesized that non-hydrogen-bonding
nucleotide analogues would be well suited for producing ICLs
because the absence of stabilizing interactions with the opposite
strand would reduce barriers for adopting a conformation that
is conducive to cross-link formation. In choosing molecules that
might be expected to display this reactivity, we benefited from
the significant advances over the past two decades in
developing nonnative nucleotides to probe polymerase
mechanism and to expand the genetic code. These molecules
avoid hydrogen bonding during selective recognition of native
and other nonnative nucleotides.18−22 Our objective is less
challenging in this regard because nonselective incorporation
opposite native nucleotides is desirable, provided cross-linking
is inducible. Furthermore, high incorporation levels are
unnecessary due to the high impact that DNA interstrand
Scheme 1
Received: December 19, 2013
Published: February 24, 2014
© 2014 American Chemical Society
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cross-links have on biochemical processes. Using the work of
Kool as a guide, a series of oligonucleotides containing aryl
iodide C-nucleotides (2, 3, Scheme 2) were synthesized by
formation upon UV photolysis and γ-radiolysis. Compound 4
was previously reported by Romesberg and is most closely
related structurally to BrdU and Idu.29 Aryl halides 5 and 6
were conceived on the basis of the successful cross-linking by
3.26 (Note that for simplicity the aryl halides are identified to by
the same numerical descriptor whether they are present as the
monomer or as a component within an oligonucleotide.)
Synthesis of C-Aryl Halide Nucleosides and Their
Incorporation into Oligonucleotides. Compound 4 was
previously incorporated into oligonucleotides via its respective
phosphoramidite.29 This synthesis was repeated as described,
and the general approach was used to prepare the requisite
phosphoramidites (10a,b) for synthesizing oligonucleotides
containing 5 and 6 (Scheme 3). Consequently, 5-bromo-2-
Scheme 2
a
Scheme 3
solid-phase synthesis.23−26 The molecules produced ICLs in
duplexes containing any of the four native nucleotides opposite
the nucleotide analogues when exposed to UV irradiation. O2
had little effect on UV-induced cross-linking. Cross-links were
formed with the opposing nucleotide and to varying extents
with flanking thymidines depending upon the nucleotide
opposite the radical precursor. ICLs were also produced
when the duplexes were exposed to γ-radiolysis under
anaerobic conditions. The presence of a hydroxyl radical
quencher (t-BuOH) had no effect on ICL formation, ruling out
the involvement of this reactive oxygen species. In contrast to
UV irradiation, O2 quenched cross-linking when DNA was
exposed to γ-radiolysis, suggesting that the nucleotides would
selectively sensitize hypoxic cells. The dioxygen effect also
suggested that solvated electrons, which are scavenged by O2,
react with the aryl iodide C-nucleotide analogues to produce σ-
radicals that are directly responsible for cross-linking. These
experiments established that halogenated aromatic nucleotide
analogues could produce ICLs, but the respective nucleotide
triphosphates of these first-generation molecules were not
expected to be good substrates for DNA polymerases. Herein,
we describe our efforts to design nucleotide analogues that
selectively cross-link the opposing strand of DNA when
exposed to ionizing radiation under O2-deficient conditions
but whose nucleotide triphosphates are also accepted as
substrates by DNA polymerase(s).
a
Key: (a) Ph3As, Pd(OAc)2, DMF; (b) Bu4NF, THF; (c) NaB-
(OAc)3H, AcOH, CH3CN; (d) DMTrCl, pyridine; (e) 2-cyanoethyl-
N,N′-diisopropylphosphoramidic chloride, diisopropylethylamine
CH2Cl2; (f) 11, CuI, NaI, pentan-1-ol.
iodoanisole (8) was coupled with 7, and the 3′-ketonucleoside
analogue (9) was partially purified following desilylation. The
nucleoside (5) was obtained via directed reduction and carried
on to phosphoramidite 10a using standard methods. The
iodine analogue (6) was prepared from the 5 by displacing the
bromide using a mixture of NaI/CuI and trans-N,N′-dimethyl-
1,2-cyclohexanediamine (11) in a pressure bottle in a manner
similar to that previously described by Kool.30,31 The reaction
1
must be followed closely by H NMR to avoid forming the
reduction product (12). The iodide (6) was also carried on to
10b via standard methods.
The phosphoramidites of the halogenated nucleotide
analogues were incorporated into oligonucleotides 13−15 via
automated solid-phase synthesis using standard procedures and
reagents, with the exception that an extended (15 min) time
was used for coupling the modified phosphoramidites.
Oligonucleotides containing 4−6 were deprotected using
“AMA” conditions (1:1 aqueous methylamine and concentrated
NH4OH) at 65 °C.32 The oligonucleotides were purified by
20% denaturing polyacrylamide gel electrophoresis and
characterized by MALDI-TOF-MS following desalting.33
RESULTS AND DISCUSSION
■
The molecules described in this study were based on a
combination of our own cross-linking results using aryl halide
C-nucleotides (e.g., 2, 3) and investigations that revealed the
importance of a hydrogen bond acceptor in the minor groove
for polymerase interactions.27,28 Consequently, oligonucleo-
tides containing 4−6 were synthesized and evaluated for ICL
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position equivalent to the bromine or iodine in BrdU and IdU,
respectively. Cross-link formation would require rotation about
the glycosidic bond into the syn-equivalent conformation. C-
Nucleotides 5 and 6 contain the halide at the position
analogous to C4 in a native pyrimidine, and the orientation of
the halide (and subsequent radical center) with respect to the
opposing strand will be relatively insensitive to the con-
formation about the pseudoglycosidic bond. However, if
responsible for the observed selectivity these factors should
also affect cross-linking induced by γ-radiolysis, which does not
exhibit the same preference (see below). Alternatively, the
differences in UV-induced cross-linking yields may be due to
the involvement of a mechanism other than direct excited state
homolysis of the aryl halide bond. For instance, the 5-
halopyrimidines are converted into 1 (Scheme 1) via
photoinduced electron transfer.34,35 Such a mechanism cannot
be discounted for these molecules, nor is it certain whether 4−
6 would behave differently from each other in this type of
process. However, it would be consistent with differences in
cross-linking yields between UV and γ irradiation, provided that
σ-radical yields from a photoinduced electron transfer process
are different for 4-6.
Interstrand Cross-Link Formation upon UV Irradiation
of Halogenated C-Nucleotides. Cross-link formation upon
UV irradiation (30 min) under aerobic conditions in a Rayonet
photoreactor (λmax = 300 nm) of duplexes containing modified
nucleotides 4−6 was determined using 5′-32P-16a−d to 5′-32P-
18a−d (Table 1) in which the strand containing the C-
Table 1. Interstrand Cross-Link Yields Following UV
a
Irradiation of 5′-32P-16a−d to 5′-32P-18a−d
ICL yield (%)
X
Y = A
Y = C
Y = G
Y = T
Despite the significant difference in ICL yields between 4
and the C4-halogenated nucleotides (5 and 6), their preferred
cross-linking site(s), as determined by reaction with hydroxyl
radical, were quite similar.36 The major site of cross-linking was
T14 in all three duplexes containing dC opposite the C-
nucleotide (16b−18b).33 However, the cross-linking prefer-
ences for 4−6 were different than those of 2 and 3. The latter
formed the majority of cross-links with an opposing dC.26
Interstrand Cross-Link formation upon 137Cs Irradi-
ation of Halogenated C-Nucleotides. 137Cs irradiation (315
Gy) of duplexes containing C-nucleotides 4−6 under anaerobic
conditions also produced interstrand cross-links (Table 2). In
4
5
6
7.7 2.3
17.7 3.1
24.5 4.5
11.0 1.6
27.2 4.1
34.8 3.4
10.4 1.5
65.1 12.4
24.4 1.2
12.9 2.6
25.0 6.1
27.4 2.2
a
Yields are the average standard deviation of three samples.
nucleotide was radiolabeled. ICL yields from 4 were
consistently lower, by at least 50%, than the respective duplexes
containing either 5 or 6. UV-induced cross-linking yields
obtained from 5 (5′-32P-17a−d) and 6 (5′-32P-18a−d) are
more similar to one another, with the exception that when aryl
bromide 5 was opposite dG (5′-32P-17c) the yield reached 65%.
Although average ICL yields are (with the exception of when
dG is opposite the modification) slightly higher for the aryl
iodide (6, 5′-32P-18) than 5, the variations are such that they
are within experimental error of one another. It is not known
why the ICL yield for 5 opposite dG is so much greater than in
all other duplexes examined. At this time we can only speculate
regarding the variable photochemical efficiencies from substrate
to substrate to explain the lower ICL yields from duplexes
containing 4.
Table 2. Interstrand Cross-Link Yields Following 137Cs
a
Irradiation of 5′-32P-16a−d to 5′-32P-18a−d
ICL yield (%)
X
Y = A
Y = C
Y = G
Y = T
4
5
6
15.1 1.0
9.6 1.2
15.2 2.0
13.3 0.5
9.3 0.5
17.5 1.0
11.1 0.8
6.5 0.6
9.4 1.5
13.2 0.8
9.6 1.3
13.0 1.1
It is tempting to ascribe the large difference in ICL yields
between 4 and the other C-nucleotides to differences in the
molecules’ conformations. If 4−6 adopt conformations (as
drawn) equivalent to that of a native nucleotide in its anti form,
the halide in 4 lies in the major groove (Scheme 4) in a
a
Yields are the average standard deviation of three samples.
comparison to UV irradiation, γ-radiolysis produced much
more similar yields of ICLs among the 12 duplexes examined.
Cross-link formation was slightly less efficient in duplexes
containing the aryl bromide (5). This was true regardless of the
identity of the nucleotide opposite the C-nucleotide. Although
the difference is less than 2-fold within any one family of
duplexes containing the same C-nucleotide, ICL formation was
least efficient when dG was opposite the modified nucleotide.
Exposing 5′-32P-16a−d to 5′-32P-18a−d to the same dose of
radiation under aerobic conditions produced less than 2% ICLs,
and adding t-BuOH (10 mM) prior to irradiation had no effect
on cross-link yield (data not shown). These effects are
consistent with generation of the respective σ-radicals by loss
of halide ion from the radical anions following reaction of the
aryl halides with a solvated electron. Cross-link formation by
Scheme 4
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4−6 was more efficient than the previous aryl iodides (e.g., 2,
3), which also produce ICLs via σ-radicals, despite being
exposed to less than one-half the dose.26
same as when dC was in the template. In contrast, Deep Vent
(exo−) incorporated 6 very weakly opposite dA and not at all
when dG was in the template under these conditions. A direct
comparison to data in the literature is not available. However,
Romesberg found that of the four native nucleotides, dA
incorporation opposite 12 was most efficient.27 Incorporation
of the other three native nucleotides was too slow to measure.
In contrast, Klenow exhibited the same order of nucleotide
incorporation opposite 4 (dC > T > dA > dG) as observed here
for incorporation of 6 opposite native nucleotides by Deep
Vent (exo)−.29
Extension of the nascent strand is typically even more
challenging than nonnative nucleotide incorporation.27,29 The
effect of 6 in the growing strand on polymerization was
qualitatively examined in two ways. Full-length extension of 20
was examined in the presence of all four native dNTPs (200
μM each) and compared to extension in which 19 (200 μM)
was substituted for dGTP. While Deep Vent (exo−) produced
full-length product within 5 min when all four native nucleotide
triphosphates were present, the reaction containing 19 gave no
full-length material after 1 h (Figure 1). Multiple extension
DNA Polymerase Incorporation of 6 via its C-
Nucleotide Triphosphate (19). The above experiments
indicate that 4−6 will function as radiosensitizing agents
when present in DNA. To be a complete radiosensitizing agent,
the triphosphate of such molecules must be incorporated into
cellular DNA by a polymerase(s) and the same molecule or an
appropriate precursor must pass through the cell membrane.
Romesberg reported on the incorporation of 4 into DNA, as
well as its effect on polymerase activity when present in a DNA
template.29 The Klenow fragment of E. coli DNA polymerase I
(Klenow), a model polymerase, incorporated three of the four
native 2′-deoxynucleotides opposite 4, although only dA was
introduced with moderate efficiency. This was desirable for the
authors’ goals but averse to our own. As a proof of principle, we
chose to examine the incorporation of 6 by DNA polymerase
instead of 5 because it provided higher ICL yields when
exposed to 137Cs. Choosing a model polymerase was difficult, as
there are more than a dozen DNA polymerases in human cells,
several of which have evolved to be promiscuous and error
prone. Any one of these might achieve our goal and incorporate
low levels of 6 opposite native nucleotides in a DNA template.
Deep Vent (exo−) was selected as a model polymerase because
it tolerates other non-native nucleotide triphosphates and
backbone modifications.37,38
The nucleotide triphosphate of 6 (19) was synthesized by
standard methods and purified by ion-exchange and C18-
reversed-phase HPLC. The kinetics of its incorporation
opposite dC in 20 were examined quantitatively and compared
to that of dGTP because a duplex containing this nucleotide
opposite 6 yielded the highest yield of radiolytically induced
ICLs (Table 2). Under steady-state conditions, dG was
incorporated ∼1300 times more efficiently than 6 (Table
3).39 The predominant source of this selectivity was an ∼650-
fold lower apparent Km (Km(app)) for dGTP. The ability of Deep
Vent (exo−) to accept 19 and incorporate 6 opposite the other
three native nucleotides was examined qualitatively at 70 μM
(the Km(app) opposite dC). At this single concentration of 19,
the rate of incorporation opposite T was approximately the
Figure 1. Full-length extension of 20 in the absence or presence of 19.
products were formed, some of which based upon their length
could contain as many as three molecules of 6 if it were the
only nucleotide inserted opposite dC. Extension products alone
do not distinguish between incorporation of 6 or any of the
native nucleotides. Consequently, the ability of Deep Vent
a
Table 3. Steady-State Incorporation Kinetics of Nucleotides Opposite dC in 20
X
dNTP
Vmax(%/min)
Km(app) (nM)
Vmax/Km (%/(min·nM))
G
6
dGTP
12.4 + 0.5
6.9 0.3
94.9 8.0
(63.0 6.4) × 103
130.5 12.2
0.1 0.01
19
a
Kinetic constants are the average standard deviation of three experiments, each containing three replicates.
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(exo−) to extend a primer following incorporation of 6 was
examined by extending 5′-32P-20 in the presence of 19 only,
isolating the extension product by denaturing polyacrylamide
gel electrophoresis (PAGE), and rehybridizing with the
complement to form 5′-32P-21. Subsequent denaturing PAGE
analysis of freshly isolated 5′-32P-21 incubated with Deep Vent
(exo−) and native dNTPs (1 mM) for 2 h showed no extension
of the material containing 6 at its 3′-terminus (data not shown),
indicating that the C-nucleotide is an absolute block for the
polymerase under these conditions.
pressure of argon atmosphere and monitored by TLC on silica gel G-
25 UV254 (0.25 mm) unless stated otherwise. Spots were detected
under UV light and/or by charring with a solution of ammonium
molybdate or ceric ammonium sulfate in water and H2SO4. Column
chromatography was performed on silica gel 60 (40−60 μm). The
ratio between silica gel and crude product ranged from 100:1 to 20:1
(w/w).
Oligonucleotides were synthesized via standard automated DNA
synthesis on an Applied Biosystems model 394 instrument. The
coupling time for the phosphoramidites of modified nucleotides 15
min. The phosphoramidite for 4 and nucleoside analogue 4 were
synthesized as previously described.29 The UV spectrum of 4 was not
reported previously (MeOH, λmax = 246 nm, ε = 1700 M−1 cm−1).
Oligonucleotides were deprotected using 1:1 methylamine (40% in
water)−concentrated NH4OH at 65 °C for 75 min (oligonucleotides
containing 4−6) or concentrated NH4OH at 25 °C for 9 h
(oligonucleotides containing native nucleotides only). Oligonucleo-
tides were purified by 20% denaturing polyacrylamide gel electro-
phoresis (PAGE). All oligonucleotides containing modified nucleo-
tides were characterized by MALDI-TOF MS. Oligonucleotides were
5′-32P-labeled by polynucleotide T4 kinase (New England Biolabs)
and γ-32P-ATP (Perkin-Elmer) using standard protocols.44 Radio-
labeled oligonucleotides were hybridized with 1.5 equiv of
complementary oligonucleotides in 10 mM potassium phosphate
(pH 7.2) and 100 mM NaCl at 90 °C for 5 min and cooled to room
temperature. All anaerobic reactions were carried out in sealed Pyrex
tubes, which were degassed and sealed using freeze−pump−thaw
(three cycles, 3 min each) degassing techniques. Experiments involving
radiolabeled oligonucleotides were analyzed following PAGE using a
Storm 840 phosphorimager.
Since Deep Vent (exo−) was developed for use in PCR, we
explored the possibility that its acceptance of 19 would be
enhanced under conditions in which such experiments are
typically carried out. Consequently, a 287 bp PCR product was
prepared from single-stranded M13mp7 plasmid using 24 nt
primers (one of which was labeled with 32P at its 5′-terminus),
as previously described.40 A longer substrate than 20 also
increased the statistical probability for incorporating a single
molecule of 6, which would be sufficient for producing an ICL.
The four native nucleotide triphosphates (200 μM) and 19 (2
mM) were present in the reaction mixture. Control reactions
contained only native dNTPs (200 μM). Following 25 PCR
cycles, the reaction was phenol extracted and the full-length
product purified by gel electrophoresis using DNA standards as
markers. The presence of 6 was probed for by exposing the
5′-32P-PCR products to 137Cs (21−105 Gy) under anaerobic
conditions. However, no ICL formation above that formed in
the control that was produced only from native dNTPs was
detected (data not shown). Finally, to further increase the
statistical probability of incorporating 6, linearized single-
stranded M13mp7 plasmid (7200 nt) was copied in the
presence of native dNTPs (1 mM) and 19 (10 mM) using a
5′-32P-primer and Sequenase as previously described.41
However, γ-radiolysis up to 210 Gy also failed to produce
any ICLs above the background established by a control
produced in the absence of 19 (data not shown).
Summary. Unlike the 5-halopyrimidines (BrdU and IdU),
C-nucleotide aryl halides 4−6 produce interstrand cross-links
when duplex DNA containing them is exposed to ionizing or
UV irradiation. γ-Radiolysis of DNA containing 6 in the
presence of a hydroxyl radical quencher indicates that this
species is not responsible for interstrand cross-linking.
However, O2 prevents cross-linking by γ-radiolysis but not
UV irradiation, suggesting that solvated electrons produced by
the former react with the aryl halides to initiate product
formation via σ-radicals. Selective cross-link formation under
anaerobic conditions suggests that 4−6 could be useful as
radiosensitizing agents in hypoxic cells, provided the nucleotide
analogues could be incorporated enzymatically in DNA. The
nucleotide triphosphate of 6 is incorporated preferentially
opposite pyrimidines, but the product formed is not extended
further. These nucleotide analogues provide motivation for
designing next-generation molecules that could serve as
radiosensitizing agents in cells. In addition, the utility of such
nonnative nucleotide analogues in cells may be increased by the
evolution of DNA polymerases containing expanded substrate
tolerance.42,43
Synthesis of 5. A mixture of palladium acetate (60 mg, 0.27 mol)
and triphenylarsine (159 mg, 0.52 mmol) in DMF (10 mL) was stirred
under argon atmosphere at room temperature for 30 min. Then 8
(820 mg, 2.62 mmol), 1,4-anhydro-3,5-bis-O-(tert-butyldimethylsilyl)-
2-deoxy-D-erythro-pent-1-enitol (4, 810 mg, 2.35 mmol), and tributyl-
amine (1.02 mL, 4.24 mmol) in DMF (10 mL) were added, and the
resulting reaction mixture was stirred under argon at 70 °C for 15 h.
The mixture was cooled to 0 °C, 1 M tetrabutylammonium fluoride in
THF (6 mL, 6 mmol) was added, and the mixture was stirred for 1.5 h.
The reaction mixture was quenched with H2O (30 mL) and extracted
with EtOAc (50 mL × 2). The combined EtOAc layers were washed
with saturated NaHCO3 aq (50 mL) and then dried over anhydrous
MgSO4. After filtration and evaporation to dryness under reduced
pressure, the residue was purified by silica gel column chromatography
(EtOAc−hexanes, 1:2) to afford 9 (301 mg, 43%). Without further
purification or characterization, 9 was dissolved in acetic acid (5 mL)
and acetonitrile (5 mL), the solution was cooled to 0 °C, sodium
triacetoxyborohydride (318 mg, 1.5 mmol) was added, and the mixture
was stirred for 1 h. The reaction mixture was extracted with EtOAc (50
mL × 2) and saturated NaHCO3 aq (50 mL). The combined organic
layers were dried over anhydrous MgSO4. After filtration and
evaporation, the residue was purified by silica gel column
chromatography (CH2Cl2−CH3OH, 20:1) to afford 5 (260 mg,
1
86%) as a white foam: H NMR (CD3OD) δ 7.42−7.40 (m, 1H),
7.07−7.05 (m, 2H), 5.32 (dd, 1H, J = 10.0, 5.6 Hz), 4.27−4.24 (m,
1H), 3.92−3.89 (m, 1H), 3.80 (s, 3H), 3.66−3.63 (m, 2H), 2.33−2.27
(m, 1H), 1.76−1.69 (m, 1H); 13C NMR (CD3OD) δ 158.3, 131.4,
128.4, 124.5, 122.2, 114.7, 88.6, 76.0, 74.3, 64.0, 56.2, 43.1; IR (NaCl
plate) 3418, 3056, 2987, 1489, 1266, 1031 cm−1; UV (MeOH) λmax
=
280 nm (ε = 2285 M−1 cm−1); MALDI-TOF HRMS C12H15O4BrNa
+
(M + Na ) calcd 325.0045, obsd 325.0050.
Synthesis of 10a. Diol 5 (100 mg, 0.33 mmol) was azeotroped
with pyridine (2 mL), after which a 2 mL solution of 4,4′-
dimethoxytrityl chloride (168 mg, 0.50 mmol) in pyridine was
added. The reaction mixture was stirred at room temperature for 6 h,
at which time methanol (3 mL) was added to quench the reaction.
The organic solution was removed in vacuo, and the residue was
purified by flash chromatography (EtOAc−hexanes, 4:1 to 2:1) to
afford compound the dimethoxytritylated C-nucleoside (133 mg, 67%)
EXPERIMENTAL SECTION
■
General Methods. Solvents used in reactions were purified by
distillation before use. All reagents used in reactions were purchased
from commercial sources and were used without further purification
unless noted otherwise. All reactions were carried out under a positive
1
as a white foam: H NMR (CDCl3) δ 7.52−7.49 (m, 2H), 7.42−7.25
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(m, 8H), 7.10−7.07 (m, 1H), 7.00 (s, 1H), 6.89−6.85 (m, 4H), 5.39
(dd, 1H, J = 6.0, 9.6 Hz), 4.41−4.39 (m, 1H), 4.08−4.07 (m, 1H),
3.83 (s, 9H), 3.42 (dd, 1H, J = 4.8, 9.8 Hz), 3.30 (dd, 1H, J = 5.6, 9.8
Hz), 2.38−2.36 (m, 1H), 1.92−1.86 (m, 1H); 13C NMR (CDCl3) δ
158.6, 156.8, 145.0, 136.2, 136.1, 130.24, 130.22, 130.19, 128.3, 128.0,
127.4, 126.9, 123.6, 121.3, 113.8, 113.3, 86.4, 85.7, 74.76, 74.72, 64.5,
55.7, 55.4, 42.1; IR (NaCl plate) 3055, 2938, 1509, 1463, 1285, 1033
cm−1; MALDI-TOF HRMS C33H33O6BrNa (M + Na +) calcd
627.1353, obsd 627.1357.
(m, 12H), 6.84−6.80 (m, 4H), 5.36−5.34 (m, 1H), 4.50−4.46 (m,
1H), 4.20 (s, 1H), 3.84−3.54 (m, 12H), 3.30−3.23 (m, 2H), 2.65−
2.40 (m, 3H), 1.84−1.79 (m, 1H), 1.28−1.05 (m, 13H); 31P NMR
(CDCl3) δ 148.2, 147.7; MALDI-TOF HRMS C42H50N2O7INaP (M
+
+ Na ) calcd 875.2293, obsd 875.2294.
Synthesis 19. To a solution of 6 (53 mg, 0.15 mmol) and 1,8-
bis(dimethylamino)naphthalene (Proton Sponge, 48 mg, 0.22 mmol)
in trimethyl phosphate (2 mL) at 0 °C was added phosphorus
oxytrichloride (17 μL, 0.18 mmol). After the mixture was stirred at 0
°C for 3 h, a solution of tributylammonium pyrophosphate (178 mg,
0.32 mmol) in anhydrous DMF (1 mL) and tributylamine (220 μL,
0.92 mmol) was added dropwise. The reaction was stirred at room
temperature for 10 min, followed by quenching with 1 M
triethylammonium bicarbonate buffer (30 mL, pH 8.5). The quenched
reaction was stirred for an additional 10 min. Lyophilization gave the
crude product. The crude product was subjected to ion-exchange
column (DEAE) and eluted using a 0−1 M TEAB gradient. Fractions
was monitored by UV and checked by ESI-mass. Fractions was
collected, lyophilized, and purified by reversed-phase (C18) HPLC
(0−50% CH3CN in 0.1 M TEAB, pH 7.5) followed by lyophilization
to afford the triphosphate as its triethylammonium salt (yield 3.5%) as
a fluffy, white solid. The concentration of the triphosphate is
determined by using the extinction coefficient at 260 nm (1650 M−1
To a solution of dimethoxytritylated C-nucleoside (80 mg, 0.13
mmol) and diisopropylethylamine (46 μL, 0.26 mmol) in dichloro-
methane (3 mL) was added 2-cyanoethyl N,N-diisopropylphosphor-
amidic chloride (39 μL, 0.17 mmol) at 0 °C. After being warmed to
room temperature and stirred for 3 h, the reaction mixture was diluted
with dichloromethane (20 mL) and washed with saturated aq
NaHCO3 (20 mL). The organic layer was dried over anhydrous
Na2SO4, filtered, and evaporated to dryness in vacuo. The crude
product was purified by silica gel column chromatography (EtOAc−
1
hexanes, 2:1) to afford 10a (83 mg, 78%) as a white foam: H NMR
(CDCl3) δ 7.51−7.21 (m, 10H), 7.06−7.04 (m, 1H), 6.98−6.96 (m,
1H), 6.84−6.79 (m, 4H), 5.40−5.30 (m, 1H), 4.52−4.41 (m, 1H),
4.20 (s, 1H), 3.82−3.51 (m, 12H), 3.39−3.20 (m, 2H), 2.68−2.40 (m,
3H), 1.89−1.75 (m, 1H), 1.30−1.05 (m, 13H); 31P NMR (CDCl3) δ
+
1
cm−1) for the nucleoside: H NMR (D2O) δ 7.44−7.35 (m, 2H),
148.3, 147.8. MALDI-TOF HRMS C42H50N2O7BrNaP (M + Na )
calcd 827.2431, obsd 827.2444.
7.28−7.25 (m, 1H), 5.41 (br s, 1H), 4.53 (br s, 1H), 4.23−4.08 (m,
3H), 3.75 (s, 3H), 2.30−2.23 (m, 1H), 2.06−1.93 (m, 1H); 31P NMR
(D2O) δ −6.37 (br s), −11.11 (br s), −22.53 (br s); MS (ESI) m/z
588.9 [M + 3H], calcd m/z 588.9.
Synthesis of 6. Diol 5 (100 mg, 0.33 mmol) was dissolved in
pentanol (1 mL). To this solution were added sodium iodide (989 mg,
6.6 mmol) and trans-N,N′-dimethyl-1,2-cyclohexanediamine (11, 50
mg, 0.35 mmol). The flask was evacuated and backfilled with argon
three times. The reaction mixture was stirred at 130 °C for 3 h. The
resulting suspension was cooled to room temperature and diluted with
Et2O (30 mL). After filtration, the organic layer was washed with
saturated aq NaHCO3 (20 mL) and brine (20 mL). The organic layer
was dried over Na2SO4. After filtration and evaporation, the residue
was purified by flash chromatography (CH2Cl2−MeOH, 20:1) to
afford 6 (75 mg, 65%) as a yellow foam: 1H NMR (CD3OD) δ 7.27−
7.22 (m, 3H), 5.34−5.30 (m, 1H), 4.26−4.25 (m, 1H), 3.91−3.89 (m,
1H), 3.79 (s, 3H), 3.65−3.63 (m, 2H), 2.32−2.27 (m, 1H), 1.76−1.68
(m, 1H); 13C NMR (CD3OD) δ 158.2, 132.2, 130.9, 128.7, 120.6,
93.2, 88.6, 76.1, 74.4, 64.1, 56.2, 43.2; IR (NaCl plate) 3600, 3054,
2987, 1488, 1264, 1081 cm−1; UV (MeOH) λmax = 260 nm (ε = 1650
M−1cm−1); MALDI-TOF HRMS C12H16O4I (M + H +) calcd
351.0088, obsd 351.0093.
Kinetic Study of Incorporation of 19 by Deep Vent (Exo−)
DNA Polymerase. The primer−template duplex was obtained by
hybridizing the 5′-32P-radiolabeled primer (1 μM) and the cold
template (1.5 μM) in 20 mM Tris-HCl pH 8.8, 10 mM ammonium
sulfate, 10 mM KCl, 2 mM MgCl2, and 0.1% Triton X-100. The DNA
was denatured at 90 °C (5 min) and slowly cooled to room
temperature. A DNA duplex−enzyme cocktail (2 × ) stock solution
(150 μL) was prepared by mixing Deep Vent (exo−) DNA polymerase
solution (10 μL, 2 nM) with the primer−template solution (30 μL,
200 nM), 100× BSA (3 μL), 10× thermopol buffer (30 μL), 1 mM
DTT (3 μL), and water (74 μL). The extension reactions were carried
out by adding 5 μL of the cocktail to the appropriate 2 × dNTP
solutions (5 μL, 50−175 nM for dGTP, 40−90 μM for 19), which
were freshly prepared. After 6 min (19 or dGTP) at 37 °C, the
reactions were quenched with 95% formamide loading buffer (5 μL)
containing 10 mM EDTA. The mixtures were heated at 90 °C for 2
min and cooled immediately in an ice bath. Aliquots of the mixtures
were subjected to 20% denaturing PAGE. Kinetic parameters were
obtained by nonlinear regression analysis of velocity versus [dNTP].
The dNTP concentrations used were as follows: 50, 75, 100, 125, 150,
175 nM for dGTP; 40, 50, 60, 70, 80, 9 μM for 19. Reaction
conditions were chosen such that the maximum amount of extension
was <30%.
Synthesis of 10b. Diol 6 (70 mg, 0.20 mmol) was azeotroped
with pyridine (2 mL), after which 2 mL of a solution of 4,4′-
dimethoxytrityl chloride (102 mg, 0.30 mmol) in pyridine was added.
The reaction mixture was stirred at room temperature for 16 h and
then quenched with methanol (3 mL). The organic solution was
removed in vacuo, and the residue was purified by flash
chromatography (EtOAc−hexanes, 5:1 to 2:1) to afford the
1
dimethoxytritylated C-nucleotide (79 mg, 61%) as a white foam: H
Full Length Extension Reactions. A DNA primer−template
enzyme solution (50 μL) was prepared by mixing 2 μL of enzyme
(500 nM) with the DNA solution (2 μL, 1 μM), 100 × BSA (5 μL), 1
mM DTT (1 μL), 10 × thermopol buffer (10 μL), and water (34 μL).
The extension reactions were initiated by adding 10 μL of a premixed
dNTP solution (200 μM dATP, 200 μM dCTP, 200 μM dTTP, 200
μM dTTP, or 200 μM 19) to 10 μL of DNA primer−template enzyme
solution. Aliquots (8 μL) were taken and quenched with 95%
formamide loading buffer (8 μL) containing 10 mM EDTA at 5, 15,
30, and 60 min. The mixture was heated to 90 °C for 2 min and chilled
on ice. An aliquot of the mixture was loaded on to a 20% denaturing
polyacrylamide gel.
Photoreactions. Photoreactions of the duplexes were carried out
in Pyrex tubes in a Rayonet photoreactor fitted with 16 lamps having a
maximum output at 300 nm. All photoreactions were carried out for
30 min in 10 mM potassium phosphate (pH 7.2) and 100 mM NaCl.
After reaction, each sample (20 nM, 40 μL) was aliquoted into a 0.6-
mL Eppendorf tube and mixed with formamide loading buffer and
subjected to 20% denaturing PAGE analysis. ICL yields were
NMR (CDCl3) δ 7.50−7.47 (m, 2H), 7.38−7.21 (m, 9H), 7.15 (s,
1H), 6.86−6.83 (m, 4H), 5.39−5.36 (m, 1H), 4.39−4.38 (m, 1H),
4.07−4.06 (m, 1H), 3.80 (s, 9H), 3.41−3.37 (m, 1H), 3.30−3.26 (m,
1H), 2.40−2.35 (m, 1H), 1.89−1.82 (m, 1H); 13C NMR (CDCl3) δ
158.6, 156.7, 144.9, 136.1, 131.0, 130.2, 129.8, 128.3, 127.9, 127.6,
126.9, 119.4, 113.2, 92.5, 86.3, 85.7, 74.8, 74.6, 64.5, 55.6, 55.3, 42.1;
IR (NaCl plate) 3054, 2989, 1588, 1422, 1264, 1178 cm−1; MALDI-
TOF HRMS C33H33O6INa (M + Na +) calcd 675.1214, obsd
675.1214.
To a solution of the dimethoxytritylated C-nucleoside (69 mg, 0.11
mmol) and diisopropylethylamine (37 μL, 0.22 mmol) in dichloro-
methane (3 mL) was added 2-cyanoethyl N, N-diisopropylphosphor-
amidic chloride (31 μL, 0.14 mmol) at 0 °C. After being stirred for 3 h
at room temperature, the reaction mixture was diluted with
dichloromethane (20 mL) and washed with saturated aq NaHCO3
(20 mL). The organic layer was dried over anhydrous Na2SO4, filtered,
and evaporated to dryness in vacuo. The crude product was purified by
silica gel column chromatography (EtOAc−hexanes, 2:1) to afford
1
10b (63 mg, 70%) as a white foam: H NMR (CDCl3) δ 7.51−7.14
1882
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The Journal of Organic Chemistry
Featured Article
determined using the phosphor image by dividing the volume of the
cross-link product by the summation of all of the DNA in the lane
(cross-link product, intact DNA, cleaved DNA) and multiplying by
100.
γ-Radiolysis. γ-Radiolysis of the duplexes were carried out in Pyrex
tubes in a J. L. Shepherd Mark I 137Cs irradiator that has an output of
23 Gy/min. After reaction (315 Gy), each sample (20 nM, 40 μL) was
aliquoted into a 0.6-mL Eppendorf tube and mixed with formamide
loading buffer and subjected to 20% denaturing PAGE analysis. ICL
yields were determined as described above.
of Centricon leads to loss of most of the products. The concentration
of the plasmid was determined by UV absorbance (ε260 = 7.152 × 107
L/mol·cm). One liter of GW5100 cell growth produced 2.7 nmol of
plasmid. The quality of plasmid was confirmed by 1% agarose gel.
Plasmid was linearized by an EcoR I restriction cut. Restriction
digestion of M13mp7 (100 pmol) with EcoR I (100 units) was carried
out in 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 50 mM NaCl, and 1
mM dithiothreitol at 37 °C for 4 h. After incubation, the solution was
heated to 90 °C for 5 min and immediately cooled in ice−water to
inactivate the enzyme. The linearized plasmid was stored at −20 °C
until use. Complete digestion of M13mp7 was confirmed by
comparison of the mobility in 1% agarose gel electrophoresis between
native plasmid and digested linear plasmid. The gel was stained with
Syber-green. EcoR I cut plasmid migrated farther down the gel than
native plasmid.
Polymerization of Linearized Plasmid by Sequenase.
Linearized plasmid (30 pmol) was hybridized with 5′-32P labeled
primer (10 pmol, 5′-d(CAC TGA ATC ATG GTC ATA GCT GTT))
in 40 mM Tris·HCl (pH 7.5), 20 mM MgCl2, and 50 mM NaCl at 90
°C for 5 min, followed by slow cooling to room temperature.
Extension was carried out using Sequenase (26 units, Version 2.0 DNA
polymerase) in the presence of 19 (10 mM) and native dNTPs (1
mM) at 37 °C for 30 min. After reaction, plasmid duplex was purified
by Microcon filter (YM = 3) to remove excess reagents. Complete
polymerization of linearized M13mp7 was confirmed by comparison of
the mobility in 1% alkaline agarose gel electrophoresis between
enzymatic reaction product and 5′-32P-labeled linear plasmid.
Fe(II)−EDTA Digestion of Cross-Linked DNA. Fe(II)−EDTA
cleavage reactions of ICLs were carried out in 50 μM (NH4)2Fe-
(SO4)2, 100 μM EDTA, 1 mM sodium ascorbate, 5.0 mM H2O2, 100
mM NaCl, and 10 mM potassium phosphate (pH 7.2) for 1 min at 25
°C (total volume of 20 μL each). The reactions were quenched with
100 mM thiourea (10 μL). Samples were lyophilized, resuspended in
formamide loading buffer, and subjected to 20% PAGE analysis.
Preparation of a 287 nt PCR Fragment. A 287 nt PCR fragment
was prepared from M13mp7 plasmid (10 fmol), which was amplified
with primer 1 and primer 2 (250 pmol each), dNTP (0.5 mM each),
and Taq DNA polymerase (5 units) in 100 μL of Taq DNA
polymerase buffer (20 mM Tris, 10 mM (NH4)2SO4, 10 mM KCl, 2
mM MgSO4, 0.1% Triton X-100, pH 8.8). PCR was performed using
the following conditions: 94 °C, 30 s for melting and 58 °C, 1 min for
annealing, and then 72 °C, 1 min for polymerase reaction. After
repeating the cycle 60 times, the reaction solution was extracted by
phenol and further purified by Microcon (MY-30) using a standard
protocol. The concentration of the PCR fragment was determined by
UV (ε260 = 20 g−1·cm−1·L), and the quality of PCR fragment was
determined by agrose gel (3%). Sequences of three primers and PCR
fragment: 5′-CAC TGA ATC ATGGTC ATA GCT GTT-3′ (primer
1), and 5′-GGT GAA GGG CAA TCA GCT GTT-3′ (primer 2) used
for primers. The sequence of the PCR fragment is 5′-GGT GAA GGG
CAA TCA GCT GTT GCC CGT CTC ACT GGT GAA AAG AAA
AAC CAC CCT GGC GCC CAA TAC GCA AAC CGC CTC TCC
CCG CGC GTT GGC CGA TTC ATT AAT GCA GCT GGC ACG
ACA GGT TTC CCG ACT GGA AAG CGG GCA GTG AGC GCA
ACG CAA TTA ATG TGA GTT AGC TCA CTC ATT AGG CAC
CCC AGG CTT TAC ACT TTA TGC TTC CGG CTC GTA TGT
TGT GTG GAA TTG TGA GCG GAT AAC AAT TTC ACA CAG
GAA ACA GCT ATG ACC ATG ATT CAG TG-3′.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for PCR and Sequenase experiments.
Representative hydroxyl radical cleavage autoradiogram for
cross-link identification. Sample kinetic plots for incorporation
of dG and 6 opposite dC in 20. Spectral data for previously
unreported compounds. MALDI-TOF-MS for oligonucleotides
containing C-nucleotides and cross-linked products. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 410-516-8095. Fax: 410-516-7044. E-mail:
mgreenberg@jhu.edu.
■
PCR Experiments. PCR was performed in an overall volume of 50
μL containing 5 pM of the 287 nt template in thermopol buffer (20
mM Tris-HCl pH 8.8, 10 mM ammonium sulfate, 10 mM KCl, 2 mM
MgCl2, and 0.1% Triton X-100). The final mixtures contained dNTPs
(200 μM of each dATP, dGTP, dCTP, and dTTP) in the presence or
absence of 19 (2 mM), primers (25 pmol of each primer), and 30 nM
of Deep Vent (exo−) DNA polymerase. PCR amplifications were
performed employing the following program: initial denaturation at 95
°C for 2.5 min, followed by 25 cycles of denaturation at 94 °C for 30 s,
primer annealing at 45 °C for 1 min, and extension at 65 °C for 5 min.
The PCR solution was filtered with a Microcon YM-30 filter
(Millipore) to remove the excess unincorporated dNTPs. The quality
of PCR product was determined by 8% native PAGE analysis.
Preparation of the M13mp7 Plasmid. GW5100 cells were
grown overnight in 10 mL of LB at 37 °C. The stock solution (2 mL)
was diluted to 1 L in 2X-YT media and incubated at 37 °C for 2 h. To
this solution was added 10 mL of M13mp7 and the resulting solution
incubated for further 9 h. After that, the cells were store in ice for 10
min and centrifuged at 9500 rpm for 15 min at 4 °C. The supernatant
was decanted and precipitated at 4 °C overnight in NaCl (500 mM)
and PEG (4%) buffer. The solution was pelleted at 9500 rpm for 15
min. The resulting pellet was resuspended in 10 mL of TE buffer (10
mM Tris, 10 mM EDTA, pH 8.0). The plasmid was purified by
extraction with 3 mL of phenol/isoamyl alcohol/chloroform three
times until the aqueous layer was clear. The aqueous layer was
subjected to a hydroxyapatite column (2 g, 18 × 1.2 cm) and eluted
with 10 mL of potassium phosphate (79 mM, pH 7). The sample was
concentrated using a YM 100 Centricon filter (2 mL) and spun for 10
min at 1600g. Note: longer centrifuge time and use of the wrong size
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for generous support of this research from the
National Institute of General Medical Sciences (GM-054996).
We thank an anonymous reviewer for helpful suggestions.
REFERENCES
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