Bifunctional rhodium intercalator conjugates as mismatch-directing DNA alkylating agents

Article abstract of DOI:10.1021/ja048543m

A conjugate of a DNA mismatch-specific rhodium intercalator, containing the bulky chrysenediimine ligand, and an aniline mustard has been prepared, and targeting of mismatches in DNA by this conjugate has been examined. The preferential alkylation of mism

Full text of DOI:10.1021/ja048543m

Published on Web 06/24/2004
Bifunctional Rhodium Intercalator Conjugates as Mismatch-Directing DNA
Alkylating Agents
Ulrich Schatzschneider and Jacqueline K. Barton*
DiVision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
Received March 12, 2004; E-mail: jkbarton@caltech.edu
To maintain the integrity of the genome, a complex cellular repair
machinery has evolved.1-3 Defects in this machinery, notably defi-
ciencies in mismatch repair, are associated with an increased cancer
susceptibility.4,5 In an effort to probe these mispairs, we have
designed metallointercalators that target base pair mismatches with
high selectivity.6-10 Metal complexes containing a bulky, intercalat-
ing ligand that is too expansive to insert within well-matched B-DNA
bind preferentially to mismatched sites. Thus, while rhodium com-
plexes containing the phenanthrenequinone diimine (phi) ligand bind
by intercalation without major perturbation to the B-form duplex,11
complexes containing the more expansive chrysenequinone diimine
(chrysi) ligand bind poorly to well-matched DNA and instead selec-
tively target mismatches. [Rh(bpy)2(chrysi)]3+ binds and, upon pho-
toactivation, cleaves the DNA backbone neighboring the destabi-
lized mismatch site;6,7 the site selectivity correlates with the thermo-
dynamic instability of the mismatch.8 Specific DNA cleavage is
observed with over 80% of mismatched sites in all sequence con-
texts. Moreover, the Rh complex has been shown to target a single
base mismatch in a 2725 base pair-linearized plasmid heteroduplex.7
Recently, [Rh(bpy)2(phzi)]3+ (phzi ) benzo[a]phenazinequinone
diimine), a mismatch-specific intercalator of higher affinity, was
applied in the differential cleavage of DNA obtained from cell lines
deficient versus proficient in mismatch repair.9
of the phenanthroline, chrysi, and aminoalkyl-substituted bipyridine
to the Rh center as described for the parent compound (Supporting
Information).21 The key step involved a mild coupling reaction of
the amino-functionalized Rh complex with the carboxylate-bearing
aniline mustard.
Reaction of 1 with 17mer oligonucleotides with two 5′-GNC-3′
binding sites12-14 either containing (AB) or lacking (AC) a central
CC mismatch was examined by an electrophoretic mobility shift
assay (Figure 1). Duplexes AB and AC (5 µM), 5′-32P-end-labeled
To explore alkylation of mismatch-containing DNA, we have
synthesized a conjugate 1 of [Rh(phen)(chrysi)(bpy)]3+ tethered to
an aniline mustard known to form covalent adducts at 5′-GNC-3′
sites.12-14 Bifunctional conjugates containing DNA alkylating agents
tethered to nonselective15,16 or site-specific17-20 DNA binding
moieties have been reported, but conjugates of metallointercalators
containing pendant alkylators have not been prepared. The Rh
conjugate 1 (Scheme 1) was constructed by sequential introduction
Figure 1. Autoradiogram of a denaturing gel showing the concentration
dependence of alkylation of AB (lanes 1-4) and AC (lanes 5-8) by
conjugate 1 (2, 5, 12.5, and 25 µM). * indicates 5′-32P-end-label of A.
Conditions: incubation for 1 h at 37 °C in the dark, 5 µM duplex DNA in
buffer (0.7 mM phosphate, 20 mM NaCl) at pH ) 7. Lane 9: DNA in
buffer alone. The arrow indicates the primary alkylation product relative
to the higher intensity parent band.
Scheme 1
on the A strand, were incubated with increasing concentrations of
conjugate 1 (2-25 µM) at 37 °C for 1 h in the dark, and the reaction
was then quenched by freezing. Autoradiography after denaturing
PAGE shows a band of retarded mobility that we assign to a
covalent DNA adduct containing conjugate 1; the retarded mobility
is expected with covalent attachment of the Rh complex. A weaker
band of still lower intensity is visible above. Importantly, the amount
formed is up to seven times higher with mismatched AB compared
to the fully matched duplex AC, with the difference most
pronounced at lower concentrations. Incubations as a function of
time with 2 µM conjugate 1 also show a more rapid increase in
formation of the covalent adduct with mismatched AB versus the
matched duplex AC, as well as a corresponding decrease in the
intensity of the parent band. The presence of the Rh-chrysi unit
thus facilitates alkylation of the DNA by the tethered aniline mustard
preferentially on the mismatch-containing duplex.
To determine the binding site of the Rh intercalator as well as
the position of covalent modification by the aniline mustard, both
DNA photocleavage22 by the Rh complex and the enhanced
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10.1021/ja048543m CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Direct photocleavage neighboring the mismatch is evident with
higher intensity than on the A strand. Piperidine treatment to reveal
alkylation also shows significant reaction at the G 3 bases to the 5′
side of the mismatch.
These results demonstrate that the bifunctional rhodium complex
1 yields site-selective alkylation of mismatch-containing DNA. This
preferential targeting of mismatched DNA by 1 at low concentra-
tions, where untethered organic mustards show little reaction,
renders these compounds useful tools for the covalent tagging of
mismatched DNA and, potentially, for new chemotherapeutic
design.
Acknowledgment. We are grateful to the NIH (GM33309) for
their financial support. We also thank the Deutsche Forschungs-
gemeinschaft for a postdoctoral fellowship (U.S.).
Supporting Information Available: Schemes outlining the syn-
thesis of conjugate 1 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
References
Figure 2. Autoradiogram of a denaturing gel to determine the site
specificity of DNA alkylation. Conditions: 5 µM duplex DNA AB and 2
µM metal complex in buffer (0.7 mM phosphate, 20 mM NaCl) at pH )
7, incubated for 1 h at 37 °C in the dark prior to subsequent irradiation
(HeCd laser, 442 nm, 1 h, 12.5 mW) and/or piperidine treatment (30 min
at 90 °C). A+G and C+T, Maxam-Gilbert sequencing reactions. Lanes 1
and 2, buffer alone. Lanes 3-6, conjugate 1. Lanes 7 and 8, [Rh(bpy)2-
(chrysi)]3+. Lanes 9 and 10: melphalan (5 and 50 µM). Arrow: preferential
site of alkylation. *: position of the mismatch.
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The formation of a slow-moving covalent adduct above the parent
band is clearly visible after incubation with conjugate 1 (lanes 3-6).
Interestingly, incubation with melphalan alone at about the same
concentration (5 µM, lane 9) does not yield a resolved adduct, and
higher concentrations (50 µM, lane 10) lead to nonspecific
alkylation of the guanines. Subsequent treatment with piperidine
then reveals the site of alkylation.12 The primary alkylation site on
the labeled strand is at the G four bases away from the central CC
mismatch (lanes 4 and 6). Note that some damage, although of
lower intensity, is visible also at the G directly adjacent to the
mismatch. The preferential alkylation at the distal G is not surprising
given the length of the tether and the likely shielding of the proximal
site by the ancillary ligands of 1.23 Direct photocleavage, marking
the site of Rh-chrysi binding, occurs with still lower intensity at
the 5′-G neighboring the CC mismatch. It is noteworthy that
quantitation of these bands shows that the combined effects of
photocleavage and alkylation by 1 are similar to the sum of reactions
of the component parts.24 Thus, the tethered alkylator does not
inhibit binding of the intercalator at the mismatched site. 3′-end-
32P-labeling of the complementary B strand gives consistent results.
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(23) Given the lack of complete reaction with piperidine, the relative intensities
at the two sites may not reflect the relative amounts of alkylation.
(24) The amount of cleavage due to alkylation at the 5′-G of the distal
5′-GNC-3′ site is 1.0%, and at the 5′-G of the proximal site it is 0.4%
(lane 4). The damage due to direct photocleavage alone is 0.3% (lane 5).
The sum of these two values is similar to the combined effects of alkylation
and photocleavage, 0.7% in lane 6. Also, the amount of photocleavage at
the 5′-G of the proximal site in the presence of conjugate 1 is similar to
that observed with untethered intercalator 2 (0.3% in lane 7).
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