The leaving group strongly affects H2O2-induced dna cross-linking by arylboronates

Article abstract of DOI:10.1021/jo401901x

We evaluated the effects of the benzylic leaving group and core structure of arylboronates on H2O2-induced formation of bisquinone methides for DNA interstrand crosslinking. The mechanism of DNA cross-linking induced by these arylboronates involves genera

Full text of DOI:10.1021/jo401901x

Article
pubs.acs.org/joc
The Leaving Group Strongly Affects H2O2‑Induced DNA Cross-Linking
by Arylboronates
Sheng Cao,‡ Yibin Wang,‡ and Xiaohua Peng*
Department of Chemistry and Biochemistry, University of WisconsinMilwaukee, 3210 North Cramer Street, Milwaukee, Wisconsin
53211, United States
S
* Supporting Information
ABSTRACT: We evaluated the effects of the benzylic leaving
group and core structure of arylboronates on H2O2-induced
formation of bisquinone methides for DNA interstrand cross-
linking. The mechanism of DNA cross-linking induced by
these arylboronates involves generation of phenol intermedi-
ates followed by departure of benzylic leaving groups leading
to QMs which directly cross-link DNA via alkylation. The QM formation is the rate-determining step for DNA cross-linking. A
better leaving group (Br) and stepwise bisquinone methide formation increased interstrand cross-linking efficiency. These
findings provide essential guidelines for designing novel anticancer prodrugs.
INTRODUCTION
■
Interest in the development of cancer therapies with improved
selectivity and reduced host toxicity has been growing.1−3 One
effective approach is to design prodrugs that can be activated
under tumor-specific conditions.4 Tumor cells produce high
levels of reactive oxygen species (ROS), which makes them
distinctly different from normal cells.5−7 Taking advantage of
this difference, our group recently developed a novel prodrug
strategy involving H2O2-induced DNA cross-linking by a
nitrogen mustard cytotoxin for the selective destruction of
tumor cells.8 Quinone methides (QMs) are the ultimate
cytotoxins responsible for the activities of many anticancer
drugs.9,10 Therefore, the development of prodrugs that can be
activated under tumor-specific conditions to form QMs is a
promising approach for the targeted destruction of tumor cells.
Various methods have been developed for QM formation, such
as photoirradiation, oxidation, thermal extrusion reactions, acid-
or base-facilitated reactions, and fluoride- or H2O2-induced
reactions.10−19 However, most of these methods have
disadvantages, including the inaccessibility of precursors,
undesirably high reaction temperatures, long reaction times,
the requirement for additional reagents and acidic or basic
conditions, and the occurrence of various side reactions.20
Compared with these other methods, H2O2-induced QM
generation from arylboronates is more attractive for in vivo
applications because H2O2-induced cleavage of boronate esters
is a bioorthogonal reaction and H2O2 is bioavailable.
these findings, we synthesized three new arylboronic esters (1a,
1b, and 4c) in this study and used 1−4 to conduct a more-
detailed investigation of the effects of the leaving group and the
aromatic core structure on the DNA cross-linking ability of
these arylboronic esters.
RESULTS AND DISCUSSION
■
Compound 1a was synthesized starting from 2-bromo-m-xylene
(5) via palladium-catalyzed borylation followed by bromination
(Scheme 1). Quaternization of 1a with trimethylamine
provided 1b in nearly quantitative yield. Compounds 2−4
were synthesized as previously described.13
We investigated the DNA cross-linking abilities of 1−4 by
allowing them to react with 49-mer DNA duplex 7 in a
phosphate buffer at 37 °C for 24 h for 1a−4a or 48 h for 1b−
4b (Scheme 2). ICL formation and yields were analyzed via
denaturing polyacrylamide gel electrophoresis with phosphor
image analysis. Efficient ICL formation was observed with
Previously, we reported that arylboronic ester quaternary
ammonium salt 4b can be activated by H2O2 to generate a
bisQM that forms DNA interstrand cross-links (ICLs).13 In
contrast, QMs are not generated from arylboronic esters 2b and
3b, which have similar structures. Preliminary mechanistic
studies showed that the structure of the aromatic core, the
chemical properties of the benzylic leaving group, and the
aromatic substituents affect QM formation.13,21 Inspired by
Received: August 27, 2013
© XXXX American Chemical Society
A
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Scheme 1. Synthesis of 1a and 1b
Figure 1. H2O2-induced DNA ICL formation by compounds 1−4.
Lane 1: DNA only (cross-linking yield 0%). Lane 2: DNA with 100
μM H2O2 (0%). Lane 3: 2 mM 1a (25%). Lane 4: 2 mM 2a (24%).
Lane 5: 2 mM 3a (9%). Lane 6: 2 mM 4a (3.5%). Lane 7: 2 mM 1b
(0%). Lane 8: 2 mM 2b (0%). Lane 9: 2 mM 3b (0%). Lane 10: 2 mM
4b (23%). Lane 11: 2 mM 4c (33.7%). Lane 12: DNA marker; [H2O2]
= 1 mM for 1, 2, and 4, and 2 mM for 3. (Reaction mixture was
incubated at 37 °C for 48 h.)
bromide 1a (cross-linking yield 25%) but not with the
corresponding quaternary ammonia salt 1b (0%; Figure 1,
compare lanes 3 and 7). To determine the generality of this
leaving-group effect, we investigated the cross-linking abilities
of 2a,b and 3a,b and found that, like 1a, bromides 2a (24%)
and 3a (9%) efficiently induced DNA cross-linking, whereas
quaternary ammonia salts 2b and 3b did not (Figure 1,
compare lanes 4 and 5 to lanes 8 and 9, respectively). Bromine,
which is a better leaving group than trimethylamine, greatly
improved the cross-linking yield. The cross-linking efficiencies
of compounds 1a−3a were affected by their concentrations, the
compound/H2O2 ratios, and the pH of the buffer solution
(Supporting Information, Figures S1 and S2). The best
compound/H2O2 ratios were 2:1 for 1a and 2a and 1:1 for
3a. Cross-linking yields for 1a−3a were lower under acidic
conditions than under basic conditions (Supporting Informa-
tion, Figure S2). These results are consistent with previously
reported results.13
Scheme 3. Synthesis of 4c That Contains Mixed Leaving
Groups
the ICLs formed from 1a−4a were stable to heating in
phosphate buffer. Obvious cleavage bands were observed with
dGs upon heating in 1.0 M piperidine (Figure 2 and Supporting
Information, Figures S3−S5), which is known to induce
cleavage of N-7-alkylated purines upon heating.22,23 Clearly, the
cross-linking reactions occurred mainly with dGs. To confirm
this, we synthesized two DNA duplexes with different
sequences (8 and 9, Scheme 2). Duplex 9 contains self-
complementary dAT sequences, whereas 8 contains dCs/dTs
in one strand and dGs/dAs in the other strand. ICLs were not
observed when 9 was treated with 1a−4a, which suggests that
cross-linking reactions did not take place with dT and dA
(Supporting Information, Figure S6). However, 1a−4a did
induce ICL formation with duplex 8 (2−12%, Supporting
Information, Figure S7). In addition, DNA cleavage bands were
observed with dCs when the purified cross-linked products
were heated in 1 M piperidine (Supporting Information, Figure
S8). Collectively, these results showed that 1a−4a alkylated
dGs and dCs. Thus, the ICL could occur between dG and dC,
two dGs, or two dCs in double-stranded DNA. This behavior is
different from that of the quaternary ammonia salt 4b, which
Surprisingly, the quaternary ammonia salt 4b, the core
structure of which bears two boronate groups, showed a higher
cross-linking yield (23%) than the corresponding bromide 4a
(3.5%; Figure 1, compare lanes 10 and 6), perhaps because 4b
is more water-soluble than 4a. In fact, 4a precipitated from the
reaction mixture. In order to test our hypothesis, we
synthesized compound 4c containing a mixed leaving group,
Br and NMe3 (Scheme 3), and investigated its cross-linking
ability. Compound 4c is completely dissolved in water or a
mixture of H2O/CH3CN. Precipitation was not observed
during the cross-linking reaction. As we expected, compound
4c displayed a much higher cross-linking yield (33.7%; Figure 1,
lane 11) than the bromide 4a (3.5%) and the quaternary
ammonia salt 4b (23.0%) under the same incubation
conditions. The result suggested that in addition to the leaving
groups the water solubility also affects the cross-linking
efficiency of 4a−c.
We further explored the reactivities of bromides 1a−4a by
determining the heat stability of purified cross-linked products
and monoalkylated single-stranded DNA. About 55−62% of
Scheme 2. DNA Duplexes Used in This Study
B
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Figure 2. Determination of the reaction sites of 1a. Phosphorimage
autoradiogram of 20% denaturing PAGE analysis of the isolated ICL
products and monoalkylated single-stranded DNA (7a′) upon heating
in piperidine or phosphate buffer. The ICL product and 7a′ were
produced by incubation of duplex 7 with 1 mM 1a and 1 mM H2O2.
7a was radiolabeled at the 5′-terminus. Lane 1: isolated monoalkylated
single stranded DNA (7a′). Lane 2: 7a′ was heated in phosphate
buffer at 90 °C for 30 min. Lane 3: 7a′ was heated in 1.0 M piperidine
at 90 °C for 30 min. Lane 4: the ICL was heated in phosphate buffer at
90 °C for 30 min. Lane 5: the ICL product was heated in 1.0 M
piperidine at 90 °C for 30 min. Lane 6: Fe·EDTA treatment of ICL.
Lane 7: G+A sequencing. Lane 8: Fe·EDTA treatment of 7.
Figure 3. HPLC profiles of the enzymatic analysis: (A) DNA duplex 7
only as control; (B) 1a-treated single-stranded 7a and 7b; (C) ICL
products induced by 1a, obtained by digestion with snake venom
phosphodiesterase followed by alkaline phosphatase (analyzed by
reversed-phase HPLC (RP-18, at 260 nm) using gradient: 0−30 min
2−20% MeOH in water, 30−35 min 20−50% MeOH in water, 35−42
min 50−100% MeOH in water, 42−50 min 100% MeOH in water, at
a flow rate 1.0 mL/min).
indicated that intrastrand cross-linking took place between dGs
and dCs too. Although we have tried to synthesize an authentic
sample of 11 by reacting 1a with dG and dC, we could not
succeed in any of our efforts in isolation of these adducts from
the monomer reactions. However, we observed that other
alkylating agents formed N7-alkylated dG which decomposed
to N7-alkylated guanine (unpublished data).
reacted mainly with dG, dA and dCs.13 That is, the leaving
group influenced both the cross-linking efficiency and the cross-
linking site.
Around 50% of the ICLs were stable to piperidine treatment,
which suggests that alkylations may also have occurred at the
exocyclic amines of dG and dC to form heat-stable adducts. In
addition, similar cleavage patterns were observed with single-
stranded DNA, indicating monoalkylation or intrastrand cross-
link formation (Supporting Information, Figures S3−S5).
Furthermore, enzymatic digestion assay of the isolated ICL
product and drug-treated single stranded DNA with snake-
venom phosphodiesterase and alkaline phosphatase has been
performed to acquire more detailed information about the
cross-linking sites.
Enzyme-digested nucleotide mixtures were purified by HPLC
and analyzed by mass spectroscopy (Figure 3). An obvious new
peak with a retention time of ∼7.9 min was observed and
accumulated in measurable quantities. It was characterized by
LC−MS, which showed the exact mass of guanine-dC adduct
11 [11 + H]+ calcd 497.2, found 497.3) (Supporting
Information, Figure S9). We propose that compound 11
might result from deglycosylation of the corresponding N7
adduct of dG (10). Rokita and co-workers showed that
deglycosylation easily occurred with N7-alkylated dG leading to
an N7 adduct of guanine.12 Therefore, the cross-linking sites
induced by 1a most likely occurred at dGs and dCs. Similarly,
an adduct with a retention time of 7.8 min and a mass of 497.3
was observed with drug-treated single stranded DNA, which
Next, we determined the rate constants for ICL formation
induced by 1a−3a, 4b, and 4c (Table 1). For all compounds,
ICL growth followed first-order kinetics (Supporting Informa-
Table 1. Rate of ICL Growth from 7 upon Treatment with
Bromides and Salts
compd
kobs, 10−5 s−1
t1/2, min
1a
2a
3a
4b
4c
8.8 1.3
14.1 1.5
13.8 1.2
4.9 0.5
3.7 0.3
130 13
82
84
8
9
9
234
312 10
C
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tion, Figure S10). The rate constants for bromides 1a−3a were
2−3 times the rate constant for quaternary ammonium salt
4b.13 This indicated that better leaving groups facilitate the QM
formation.21 Surprisingly, the cross-link yields (4c > 4b ≈ 1a ≈
2a > 3a) do not correlate well with the kinetics (2a ≈ 3a > 1a >
4b ≈ 4c). Bromides 1a−3a showed a larger k but a lower ICL
yield than the salt 4b or 4c. One of the possible reasons is that
the better leaving properties of −Br lead to nucelophilic
substitution of bromides 1a−3a by water under the reaction
conditions. The NMR analysis of 1a−3a in a mixture of DMSO
and deuterated phosphate buffer (10:1) without addition of
H2O2 showed formation of the hydrolysis intermediates
(Supporting Information, Figures S11B, 12B, and 13B). The
degree of the bromide hydrolysis increased with an increase in
the percentage of water. To ensure all compounds were soluble
in a mixture of DMSO and phosphate buffer, we could use a
maximum of 9% buffer. Finally, a solvent mixture of 10:1
DMSO/buffer was used for NMR measurements. The
hydrolysis of compounds 1a−3a can be seen from the
appearance of a variety of new peaks in the region between
4.0 and 6.0 ppm. However, this was not observed with 4b
(Supporting Information, Figure S14B). Our recent studies
about the substituent effects on QM formation also indicated
that both boronate ester and the benzylic bromo group were
easily hydrolyzed to generate (2-(hydroxymethyl)phenyl)-
boronic acid derivatives, which underwent intramolecular
esterification to form benzo[c][1,2]oxaborol-1(3H)-ol deriva-
tives (A).21 In addition, bromides could be directly oxidized by
excess H2O2, leading to 1,2-dihydroxybenzene derivatives (B).
However, this phenomenon was not observed with the
corresponding quaternary ammonium salts.21 In summary,
both hydrolysis and oxidization reaction of bromide analogues
might lead to the lower DNA ICL yields.
of 1b with H2O2 produced stable phenol product 1e, which was
isolated in quantitative yield (Scheme 5C and Supporting
Information, Figure S16). Furthermore, no QM−EVE adduct
was detected from the reaction of 1e with EVE. This result is
consistent with the results of the cross-linking study.
Similarly, QM−EVE adducts 13−15 were obtained from
QM-trapping reactions of bromides 2a−4a (Scheme 5B). This
result indicated that the phenol intermediates generated by the
reactions of 2a−4a with H2O2 efficiently formed QMs under
physiological conditions. Unlike the reactions of 2a and 3a, the
reactions of 2b and 3b with H2O2 yielded stable phenol
products that did not undergo QM formation.13 In addition,
the QM−EVE adduct 15 was formed from 4a, 4b, and 4c. The
reaction of 4b and 4c containing trimethyl amine leaving group
with EVE took more than 48 h, whereas the QM-trapping
reaction of 4a was complete within 3 h. All these results
indicated that the bromo leaving group greatly facilitated H2O2-
induced QM formation from the arylboronates. Different from
1b−3b, compounds 4b and 4c produced QMs even though
they contain the poor leaving group (NMe3). As previously
described, the reaction of bisboronates 4b and 4c with H2O2
induced two electron-donating groups (OH) to the benzene
ring (see 4e) (Scheme 4c).13 The addition of the second
donating group facilitated QM formation from 4e.13
In order to determine whether formation of the phenol
intermediates or QM generation is the rate-determining step
for DNA ICL formation, the reaction of these compounds with
H2O2 was monitored by NMR analysis (Supporting Informa-
tion, Figure S11−14, S17, and S18). Due to the poor solubility
of boronate esters in D2O, all compounds were hydrolyzed to
the corresponding boronic acids in DMSO/phosphate buffer
(10:1) prior to the addition of H2O2. Then, more D2O was
added to reach a condition which was similar to the DNA cross-
linking condition. However, if the ratio of phosphate buffer to
DMSO was more than 2:3, the boronic acids also precipitated
out. Finally, we used a mixture of phosphate buffer/DMSO
(2:3) for the kinetic study. From the disappearance of the peaks
at about 5.0 ppm (peak d), we were able to figure out the
relative rate for the phenol intermediate formation. The relative
reaction rates of these compounds with H2O2 are in the order
of 4a ≈ 4b ≈ 4c ≥ 3a > 1a > 2a (Table 2, Supporting
Information, Figure S19). The reactions of diboronates 4a−c
were too fast to determine the rate constant by NMR under
these conditions (Supporting Information, Figures S14D−E,
S17D−E, and S18C−D). The relative rates of QM formation
were estimated by the formation of the final products (peak f,
its hydrolyzed compounds or peak f′, the formed free NMe3),
which showed the following trend: 4a > 3a > 1a > 2a ≥ 4b ≈
4c (Table 2, Supporting Information, Figure S19). The kinetic
data for DNA ICL formation (2a ≈ 3a > 1a > 4b ≈ 4c) (Table
1) showed a trend similar to those for QM formation but
different from those for formation of the phenol intermediates
(Figure 4), which suggested that QM formation is the rate-
limiting step for DNA cross-linking. For 4c with a mixed
leaving group −Br and NMe3, departure of NMe3 is the rate-
determining step which took about 40 h while departure of −Br
occurred within 3 min (Supporting Information, Figure S17).
Among the four bromide analogues, 1a and 2a showed ICL
yields that were 3 times the ICL yield from 3a and 9 times that
from 4a. Compound 4c with a mixed leaving group −Br and
NMe3 proved to be the most efficient for inducing ICL
formation. We propose another two possible explanations for
the greater cross-linking efficiency of 1a, 2a, and 4c: (1) better
Considering that compound 4c also contains one bromo
group but led to the highest ICL yield, we fully investigated the
effect of other factors, such as the mechanism and the kinetics
of the QM generation, on the ICL formation. We propose that
the mechanism of ICL formation induced by 1a−4a and 4c is
similar to that for 4b:13 that is, 1a−4a or 4c react with H2O2 to
form phenol intermediates, which directly cross-link DNA via
QMs (Scheme 4 and Supporting Information, Scheme S1).
However, owing to the instability and high reactivity of the
phenol intermediates, they were not isolated from the reaction
mixtures. NMR analysis of the reaction of 1a with H2O2
showed a variety of new peaks in the region between 4.0 and
6.0 ppm (Supporting Information, Figure S15). As described
previously, the phenol products generated from the arylboro-
nates having −Br as a leaving group quickly form QMs which
react with various nucleophiles or the phenol analogues which
could be oxidized by excess H2O2 leading to 1,2-dihydrox-
ybenzene derivatives (B).21 To confirm fast QM formation
from 1a, we performed a QM-trapping experiment with a large
excess of ethyl vinyl ether (EVE). When 1a was incubated at 37
°C for 24 h in the presence of H2O2 and EVE, QM−EVE
adduct 12 was generated (Scheme 5A). In contrast, the reaction
D
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Scheme 4. Mechanism of H2O2-Induced QM Generation and ICL Formation by 1a, 2a, and 4c
a
Scheme 5. Effect of the Leaving Group on QM Formation
Table 2. Rate of Starting Material Disappearance and QM
Formation
disappearance of starting
materials
QM formation
time of
time of
completion
(min)
kobs
completion
(min)
kobs
compd
(10−5 s−1
)
(10−5 s−1
)
1a
2a
3a
4a
4b
4c
60
90
30
<3
<3
<3
39.0 1.5
36.7 3.8
77.0 2.1
nd
60
90
9.5 0.2
12.2 1.1
20.0 4.0
nd
30
<3
nd
∼3000
∼2400
4.6 0.3
4.8 0.5
nd
a
nd: not determined.
a
(A) QM-trapping product 12 was generated by reaction of 1a, H2O2,
and EVE. (B) QM-trapping products 13−15 were generated from 2a−
4a. (C) no QM-EVE adduct was generated from quaternary ammonia
salt 1b. Phenol product 1e was obtained instead.
water solubility and (2) stepwise generation of bisQMs from
the corresponding phenol derivatives (Scheme 4). Compounds
1a, 2a, and 4c were much more soluble than 3a and 4a under
the reaction conditions tested. Furthermore, the two quinone
methides (C, D or E, F) were generated from 1a or 2a in a
stepwise process (Scheme 4). Once the first QM (C or E)
reacted with one DNA strand, the stepwise formed second QM
(D or F) would have better interaction with the nucleophilic
centers in the complementary DNA strand, which resulted in
more efficient DNA ICL formation. For 4c, the fast formation
of the first QM (G) plays an important role for its higher ICL
yield than compound 4b. In contrast, the reaction of 3a likely
generated two methide groups simultaneously (Scheme S1A,
Figure 4. Relationship between the rate constant (k) of ICL formation
(y-axis) and that of QM formation (x-axis).
Supporting Information), in which case only molecules that
were already well-positioned between two nucleophilic centers
of two DNA strands would generate DNA ICLs, and the ICL
yield would drop if either of the two methide groups reacted
E
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with H2O prior to addition of a nucleophile from the DNA
duplex.24 However, we cannot exclude other explanations for
the greater cross-linking efficiency of 1a, 2a, and 4c, such as
DNA sequence, molecular structure, or distance between cross-
linking sites.
The crystal structures of bromides 1a−4a suggest that they
could easily enter the minor or major groove of DNA (Figure 5
useful for designing novel arylboronic esters that can serve as
ROS biosensors and other applications in medicine and
chemical biology.
EXPERIMENTAL SECTION
■
General Methods. All chemicals were commercially purchased
and used without further purification. Thin-layer chromatography
(TLC) was carried out on precoated silica gel plates and visualized
under UV light. Oligonucleotides were synthesized via standard
automated DNA synthesis techniques. Deprotection of the synthesized
DNA was carried out under mild deprotection conditions (28% aq
NH3, room temperature, overnight). Oligonucleotides were purified by
20% denaturing polyacrylamide gel electrophoresis. Radiolabeling was
carried out according to the standard protocols. Quantification of
radiolabeled oligonucleotides was carried out using a Molecular
Dynamics Phosphorimager equipped with ImageQuant Version 5.1
software. Enzymatic digestion products were purified with a HPLC,
and mass spectra were available on an electron spray injection mass
Spectrometer (ESI). 1H, 13C NMR spectra were collected on 300 and
500 MHz FT-NMR spectrometers. High-resolution mass spectrometry
was carried out on an atmospheric-pressure chemical ionization
(APCI) TOF mass spectrometer. X-ray crystallography was performed
on a diffractometer and crystal structures were solved with the Olex2
software.
2-(2,6-Bismethylphenyl)-4,4,5,5-tetramethyl[1,3,2]-
dioxaborolane (6). 2-Bromo-1,3-dimethyl-benzene (5: 0.74 g, 4
mmol), bis(pinacolato)diboron (1.53 g, 6 mmol), KOAc (1.18 g, 12
mmol), and PdCl2(dppf) (98 mg, 0.12 mmol) were dissolved in DMF
(40 mL) under argon atmosphere. The mixture was heated at 85 °C
for 48 h and cooled to room temperature. Then, water (100 mL) was
added, and the mixture was extracted with CH2Cl2 (3 × 50 mL). The
combined organic layer was washed with water and brine, dried over
anhydrous Na2SO4, and filtrated, and the solvent was evaporated. The
crude product was purified through column chromatography with 0−
Figure 5. X-ray crystal structures of 1a and 2a.
and Supporting Information, and Figures S38 and S39). For
example, the distance between B1A and B2A for the largest
molecule (3a) is 10.2 Å; comparatively, the widths of the DNA
minor groove and the major groove are 12 Å and 22 Å
respectively. Thus, these molecules can interact with DNA
either through minor groove or major groove. The bromo-
methylene groups extended almost perpendicularly from the
central benzene ring like two arms and were in a syn orientation
for 2a and 3a and an anti orientation for 1a and 4a. This result
indicates that the C−Br bonds could freely rotate. The
distances between the two methide groups were 5.1 Å for 1a,
5.0 Å for 2a, 8.6 Å for 3a, and 5.8 Å for 4a. In addition, the two
central benzene rings in 3a were rotated relative to each other
at a torsion angle C2−C1−C14−C19 of 44.9(7)°. This angle
results in a more flexible spatial conformation. Overall, the
medium size, the structural flexibility, and the medium distance
between the two formed methide groups make all these
molecules suitable for interaction with DNA duplexes to form
ICLs.
1
50% EtOAc in hexane to provide 6 as colorless oil (0.74 g, 80%): H
NMR (300 MHz, CDCl3) δ 7.13 (t, J = 7.0 Hz, 1H), 6.95 (d, J = 7.0
Hz, 2H), 2.42 (s, 6H), 1.41 (s, 12H); 13C NMR (500 MHz, CDCl3) δ
141.6, 129.1, 126.4, 83.4, 24.9, 22.2. The NMR spectra were consistent
with literature values.25
2-(2,6-Bisbromomethylphenyl)-4,4,5,5-tetramethyl[1,3,2]-
dioxaborolane (1a). Compound 6 (0.83 g, 3.6 mmol) was dissolved
in CH3CN (55 mL), and NBS (1.6 g, 9 mmol) and AIBN (62.9 mg)
were added. The mixture was refluxed at 90 °C for 3 h. Then the
mixture was concentrated and dissolved in DCM (100 mL). The
organic phase was washed with H2O (3 × 50 mL) and dried with
anhydrous Na2SO4. The solution was evaporated, and the residue was
subjected to column chromatography on silica gel with 0−50% DCM
in hexane to give 1a as a white solid (0.7 g, 50%): mp 159−163 °C; 1H
NMR (300 MHz, CDCl3) δ 7.31 (m, 3H), 4.84 (s, 4H), 1.49 (s, 12H);
13C NMR (500 MHz, CDCl3) δ 144.4, 130.8, 130.0, 84.4, 34.0, 25.2;
CONCLUSIONS
HRMS (EI) m/z calcd for C14H19BBr2O2 [M]+ 387.9845, found
387.9829. The NMR spectra were consistent with literature value.25
1,1′-(2-(4,4,5,5-Tetramethyl[1,3,2]dioxaborolan-2-yl)-1,3-
henylene)bis(N,N,N-trimethylmethanaminium) Bromide (1b).
Compound 1a (0.182 g, 0.47 mmol) was suspended in CH3CN (10
mL), and 4.2 M trimethylamine (0.34 mL, 1.41 mmol) in ethanol was
added dropwise with stirring. The reaction mixture was stirred at rt for
12 h and concentrated, resulting in 1b as a white solid (0.22 g, 95%):
■
In summary, our investigation of H2O2-induced reactions of
nine arylboronate esters with DNA revealed that the benzylic
leaving group and the aromatic core structure significantly
affected the DNA cross-linking ability of the arylboronates. The
mechanism of ICL formation induced by these arylboronates
involves generation of phenol intermediates which directly
produce QMs capable of cross-linking DNA. The QM
formation is the rate-determining step for DNA cross-linking.
Bromine, the better of the two leaving groups, facilitated the
efficient generation of QMs. Meanwhile, the core structure
determined whether bisQM formation was stepwise or
simultaneous, which greatly altered the cross-linking ability of
the bisQMs. More importantly, incorporation of mixed leaving
groups such as Br and NMe3 greatly enhances the DNA cross-
linking efficiency. Overall, our results suggest an effective
strategy for designing promising anticancer drugs that release
QMs upon activation by ROS in vivo. Our findings may also be
1
mp 250−256 °C; H NMR (300 MHz, D2O) δ 7.73−7.70 (m, 3H),
4.79 (s, 4H), 3.05 (s, 18H), 1.41 (s, 12H); 13C NMR (500 MHz,
DMSO) δ 136.3, 135.0, 131.3, 85.9, 68.1, 52.9, 25.3; HRMS (ESI) m/z
calcd for C20H37BBr2N2O2 [(M − 2Br)/2]+ 174.1474, found
174.1460.
1-(4-(Bromomethyl)-2,5-bis(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenyl)-N,N,N-trimethylmethanaminium Bro-
mide (4c). Bromide 4a (50 mg, 0.1 mmol) was dissolved in
CH3CN (2 mL), and 4.2 M trimethylamine (24 μL, 0.1 mmol) in
ethanol was added dropwise with stirring. The reaction mixture was
concentrated after 24 h at room temperature. The residue was purified
by column chromatography with 0−15% methanol in DCM to afford
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The Journal of Organic Chemistry
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1
compound 4c as a white solid (7 mg, 12%): mp 216−220 °C; H
NMR (300 MHz, CDCl3) δ 8.02 (s, 1H), 7.87 (s, 1H), 4.99 (s, 2H),
4.91 (s, 2H), 3.46 (s, 9H), 1.39 (s, 24H); 13C NMR (500 MHz,
CDCl3) δ 146.3, 140.6, 139.2, 131.8, 85.1, 84.7,68.3, 53.1, 32.3, 25.0,
24.9; HRMS (ESI) m/z calcd for C23H39B2Br2NO4 [M − Br]+
494.2243, found 494.2247.
volume of 90% formamide loading buffer, and then subjected to 20%
denaturing polyacrylamide gel analysis. For kinetics study, aliquots
(final concentration: 50 nM 32P-labeled oligonucleotide duplex, 100
mM NaCl, 10 mM potassium phosphate, 1 mM H2O2, 2 mM of 1−4)
were taken at the prescribed times, immediately quenched by 90%
formamide loading buffer, and stored at −20 °C until being subjected
to 20% denaturing PAGE analysis.
1,1′-(2-Hydroxy-1,3-phenylene)bis(N,N,N-trimethylmetha-
naminium) Bromide (1e). A solution of 1b (50 mg) in a mixture of
H2O (3 mL), 1 M potassium phosphate buffer (52 μL, pH 8), and
H2O2 (1.9 equiv of 1b) was incubated at 37 °C for 3 h and then rinsed
with ethyl acetate (3 × 5 mL) and DCM (3 × 5 mL). The aqueous
phase was dried under vacuum yielding 1e as a white solid
Enzyme Digestion of Cross-Linked Oligonucleotides. Inter-
strand cross-linked oligonucleotide (38 nmol) was dissolved in 0.1 M
Tris−HCl buffer, pH 8.0 (300 μL) and snake-venom phosphodiester-
ase (8.0 μL, 0.34 U) in a buffer of 110 mM Tris−HCl, pH 8.9, 110
mM NaCl, 15 mM MgCl2, and 50% glycerol was added. The mixture
was incubated at 37 °C for 1 h. Then alkaline phosphatase (8.0 μL, 80
U) in 16 μL of alkaline phosphatase buffer (100 mM NaCl, 50 mM
Tris−HCl, 10 mM MgCl2 and 1 mM dithiothreitol) was added. The
reaction mixture was incubated at 37 °C for another 1 h. The digested
products were passed through a Microcon cellulose filter (10000
molecular cutoff, Amicon, Inc.) by centrifugation at 15000 rpm. The
filtrate was collected, lyophilized, redissolved in H2O (500 μL), and
analyzed by reversed-phase HPLC (RP-18, at 260 nm) using the
following gradient: 0−30 min 2−20% MeOH in water, 30−35 min
20−50% MeOH in water, 35−42 min 50−0% MeOH in water, 42−50
min 0% MeOH in water, at a flow rate 1.0 mL/min.
Stability Study of ICL Product Formed with DNA Duplex.
After the cross-linking reaction, the reaction mixtures (0.35 μM DNA
duplex, 20 μL) were coprecipitated with calf thymus DNA (2.5 mg/
mL, 5 μL) and NaOAc (3 M, 5 μL) in the presence of EtOH (90 μL)
at −80 °C for 30 min, followed by centrifuging for 5 min at 15000
rmp. The supernatant was removed, and the pellet was washed with
cold 75% EtOH and lyophilized for 30 min in a Centrivap
Concentrator of LABCONCO at 37 °C. The dried DNA fragments
were dissolved in H2O (30 μL) and divided into three portions. One
portion (10 μL) was incubated with piperidine (2 M, 10 μL) at 90 °C
for 30 min, and the second portion (10 μL) was incubated with 0.1 M
NaCl and 10 mM potassium phosphate buffer (pH 8, 10 μL) under
the same conditions, and the third portion was used as control sample.
The samples were subjected to electrophoresis on a 20% denaturing
polyacrylamide gel.
1
quantitatively: mp 214−218 °C; H NMR (500 MHz, D2O) δ 7.32
(d, J = 7.5 Hz, 2H), 6.56 (t, J = 7.5 Hz, 1H), 4.36 (s, 4H), 2.96 (s,
18H). 13C NMR (500 MHz, DMSO) δ 137.3, 118.2, 112.8, 65.6, 52.2;
HRMS (ESI) m/z calcd for C14H26Br2N2O [(M − 2Br)/2]+ 119.1017,
found 119.1022.
QM Trapping Assay. General Procedure. A solution of
bromides 1a−4a (50 mg) in a mixture of CH3CN (3 mL) and 1 M
potassium phosphate buffer (52 μL, pH 8) was incubated at 37 °C for
30 min with excess ethyl vinyl ether (EVE). Then H2O2 (1.9
equivalent of bromides) was added to initiate the reaction. The
reaction mixture was stirred at 37 °C for 24 h and then evaporated.
Water (2 mL) was added to the residue, and the resulting mixture was
extracted with ethyl acetate (3 × 5 mL). The organic phase was
combined, dried over anhydrous Na2SO4, and evaporated. The crude
product was purified through column chromatography with 0−50%
EtOAc in hexane to provide QM−EVE adducts 12−15.
(2-Ethoxychroman-8-yl)methanol (12): colorless oil, 16% yield
(4.9 mg); 1H NMR (300 MHz, CDCl3) δ 7.20−6.82 (m, 3H), 5.35 (s,
1H), 4.78−4.58 (m, 2H), 3.98−3.84 (m, 1H), 3.72−3.58 (m, 1H),
3.05−2.88 (m, 1H), 2.68−2.56 (m, 1H), 2.28 (s, 1H), 2.12−1.92 (m,
2H), 1.21 (t, J = 7.2 Hz, 3H); 13C NMR (500 MHz, CDCl3) δ 150.1,
128.9, 128.5, 126.5, 122.6, 120.4, 97.2, 63.9, 62.0, 26.5, 20.5, 15.1;
HRMS (APCI) m/z Calcd for C12H16O3 [M − H]+ 207.1021, found
207.1025.
(2-Ethoxychroman-6-yl)methanol (13): colorless oil, 11% yield
(3.4 mg); 1H NMR (300 MHz, CDCl3) δ 7.16−7.08 (m, 2H), 6.82 (d,
J = 8.1 Hz, 1H), 5.27 (s, 1H), 4.61 (s, 2H), 3.98−3.84 (m, 1H), 3.72−
3.58 (m, 1H), 3.08−2.92 (m, 1H), 2.72−2.58 (m, 1H), 2.12−1.92 (m,
2H), 1.61 (br, 1H), 1.21 (t, J = 7.2 Hz, 3H). 13C NMR (500 MHz,
CDCl3): δ 151.9, 133.0, 128.5, 126.5, 122.7, 117.1, 97.0, 65.3, 63.7,
26.5, 20.5, 15.1; HRMS (APCI) m/z calcd for C12H16O3 [M − H]+
207.1021, found 207.1023.
2,7-Diethoxy-2,3,4,7,8,9-hexahydropyrano[2,3-g]chromene
(14): colorless oil, 21% yield (8.8 mg); 1H NMR (300 MHz, CDCl3) δ
6.54 (s, 2H) 5.21 (s, 2H), 3.96−3.86 (m, 2H), 3.72−3.58 (m, 2H),
3.04−2.90 (m, 2H), 2.64−2.54 (m, 2H), 2.08−1.90 (m, 4H), 1.21 (t, J
= 7.2 Hz, 6H); 13C NMR (500 MHz, CDCl3) δ 145.8, 121.5, 116.5,
96.8, 63.5, 26.7, 20.5, 15.1; HRMS (APCI) m/z calcd for C16H22O4
[M + NH4]+ 296.1862, found 296.1861.
Hydroxyl Radical Reaction (Fe·EDTA Reaction). Fe(II)·EDTA
cleavage reactions of 32P-labeled oligonucleotide (0.1 μM) were
performed in a buffer containing 50 μM (NH4)2Fe(SO4)2, 100 μM
EDTA, 5 mM sodium ascorbate, 0.5 M NaCl, 50 mM sodium
phosphate (pH 7.2), and 1 mM H2O2 for 3 min at room temperature
(total substrate volume 20 μL) and then quenched with 100 mM
thiourea (10 μL). Samples were lyophilized and incubated with 1 M
piperidine (20 μL) at 90 °C for 30 min. The mixture was lyophilized
again, dissolved in 20 μL H2O: 90% formamide loading buffer (1:1),
and subjected to 20% denaturing PAGE analysis.
ASSOCIATED CONTENT
■
S
* Supporting Information
2,2′-Diethoxy-6,6′-bichroman (15): colorless oil, 26% yield
1
Copies of H and 13C NMR spectra for compounds 1a,b, 4c,
1
(13.8 mg); H NMR (300 MHz, CDCl3) δ 7.34−7.24 (m, 4H), 6.87
10b, and 12−15, DNA experiments, NMR analysis, and X-ray
crystal structures and analysis data of 1a−4a. This material is
available free of charge via the Internet at http://pubs.acs.org.
(d, 2H, J = 8.1 Hz), 5.30 (s, 2H), 3.98−3.88 (m, 2H), 3.72−3.62 (m,
2H), 3.08−2.96 (m, 2H), 2.74−2.62 (m, 2H), 2.08−1.92 (m, 4H),
1.21 (t, J = 7.2 Hz, 6H); 13C NMR (500 MHz, CDCl3) δ 151.3, 133.7,
127.6, 125.7, 122.7, 117.2, 97.0, 63.7, 26.6, 20.7, 15.2; HRMS (APCI)
m/z calcd for C22H26O4 [M + NH4]+ 372.2175, found 372.2178.
Interstrand Cross-Link Formation and Kinetics Study with
Duplex DNA. The 32P-labeled oligonucleotide (0.5 μM) was annealed
with 1.5 equiv of the complementary strand by heating to 65 °C for 3
min in a buffer of 10 mM potassium phosphate (pH 7) and 100 mM
NaCl, followed by slow cooling to room temperature overnight. The
32P-labeled oligonucleotide duplex (0.5 μM, 2 μL) was mixed with 1 M
AUTHOR INFORMATION
Corresponding Author
*E-mail: pengx@uwm.edu.
■
Author Contributions
‡These authors contributed equally to this work.
Notes
NaCl (2 μL), 100 mM potassium phosphate (2 μL, pH 8.0), 10 mM
H2O2 (2 μL), compounds 1b−4b (concentration range: 10 μM to 7
mM), and an appropriate amount of autoclaved distilled water to give
a final volume of 20 μL. For Bromides 1a−4a, 6 μL CH3CN was
added in the reaction mixture to facilitate their dissolution. The
reaction was incubated at 37 °C for 24 h, quenched by an equal
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Cancer Institute
(1R15CA152914-01), Great Milwaukee Foundation (Shaw
■
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Article
Scientist Award), and UWM Research Growth Initiative
(RGI101X234).
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dx.doi.org/10.1021/jo401901x | J. Org. Chem. XXXX, XXX, XXX−XXX