Sequence-specific DNA interstrand cross-linking by imidazole-pyrrole CPI conjugate

Full text of DOI:10.1021/ja003660c

5158
J. Am. Chem. Soc. 2001, 123, 5158-5159
Scheme 1
Sequence-Specific DNA Interstrand Cross-Linking by
Imidazole-Pyrrole CPI Conjugate
Toshikazu Bando,†,‡ Hirokazu Iida,†,‡ Isao Saito,†,§ and
Hiroshi Sugiyama*,‡
CREST
Japan Science and Technology Corporation (JST), Japan
DiVision of Biofunctional Molecules
Institute of Biomaterials and Bioengineering
Tokyo Medical and Dental UniVersity
2-3-10 Surugadai, Kanda, Chiyoda, Tokyo 101-0062, Japan
Department of Synthetic Chemistry and
Biological Chemistry, Faculty of Engineering
Kyoto UniVersity, Yoshida, Sakyo, Kyoto 606-8501, Japan
ReceiVed October 11, 2000
DNA interstrand cross-linking inhibits both DNA replication
and gene expression and therefore has considerable potential for
molecular biology and human medicine.1 Cross-linking agents,
including antitumor antibiotics such as mitomycin C and carzino-
philin A as well as synthetic antitumor agents such as Bizelesin
and nitrogen mustard derivatives, show intrinsic sequence-
selectivity in the formation of interstrand cross-links.2 However,
an interstrand cross-linking agent that targets a predetermined
base-pair sequence has not been achieved. Minor-groove binding
polyamides that contain N-methylimidazole (Im)-N-methylpyrrole
(Py)-hydroxylpyrrole (Hp),3 which uniquely recognize each of
the four Watson-Crick base pairs, can be used as novel
recognition parts of sequence-specific DNA alkylating agents.
Indeed, we demonstrated that hybrid molecules between segment
A of duocarmycin A4 and Im/Py diamides and hairpin polyamides
specifically alkylate at predetermined base-pair sequences.5 Der-
van and collegues have recently achieved similar sequence-
specific DNA alkylations6 by the conjugation of hairpin polyamide
and seco-CBI.7 We also demonstrated that Im/Py diamide-CPI
conjugate with a vinyl linker, ImPyLDu86, alkylates double-
stranded DNA at predetermined sequences through highly co-
operative homodimer formation.8 Herein we describe the synthesis
of a covalent dimer of ImPyLDu86 connected with various linkers
and their DNA interstrand cross-linking abilities.
We have designed an interstrand cross-linking system by
forming a 1:2 complex of an alkylating dimer component with
partner Im/Py triamides to achieve efficient interstrand cross-
linking by synthesizing dimers of ImPyLDu86 possessing various
linkers (7a-f). Scheme 1 shows that the dimeric units (4a-f)
were synthesized by the reduction of 3 followed by coupling with
six different linkers. After subsequent deprotection, carboxyl
groups were activated by CDI to form 6a-f, which were coupled
with segment A of Du86,9 to give 7a-f.10
The interstrand cross-linking abilities of 7a-f were examined
by denaturing polyacrylamide gel electrophoresis using a 5′-
TexasRed-labeled DNA oligomer and its complementary strand
as shown in Figure 1. Compounds 7c and 7d generated slow
migrating bands only in the presence of ImImPy (lane 6 and 7),
while other compounds such as 7a,b,e, and f did not generate
such slow migrating bands under the same conditions. When a
longer complementary 21 mer was employed in the same reaction,
a slower migrating band appeared, suggesting that these slow
migrating bands are due to cross-linked products (Figure 1S).
These results suggest that the combination of 7d and ImImPy
efficiently produces DNA interstrand cross-links. Interestingly,
rigid linker compounds did not produce slow migrating bands,
and only tri- and tetramethylene linker compounds gave cross-
† CREST, Japan Science and Technology Corporation (JST).
‡ Tokyo Medical and Dental University.
§ Kyoto University.
(1) Rajski, S. R.; Williams, R. M. Chem. ReV. 1998, 98, 2723.
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T.; Saito, I.; Sugiyama, H. Tetrahedron Lett. 1999, 40, 315. (c) Seaman, F.
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J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Nature 1998, 391, 468. (d)
Kielkopf, C. L.; Baird, E. E.; Dervan, P. B.; Rees, D. C. Nat. Struct. Biol.
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Wang, A. H.-J.; Lee, M.; Sugiyama, H. J. Am. Chem. Soc. 1999, 121, 7706.
(g) Mapp, A. K.; Ansari, A. Z.; Ptashne, M.; Dervan, P. B. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 3930.
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(9) Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E.; Gomi, K.; Saito,
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(10) The purity of 7d was more than 85% as determined by HPLC analysis
(Wakopak 5C18, 0.05 M ammonium formate containing 0-100% acetonitrile,
linear gradient, 20 min, at a flow rate of 1.0 mL/min; retention time 7d: 14.4
min). 1H NMR (DMSO-d6) δ 1.29 (m, 2H), 1.58 (s, 4H), 2.09 (m, 2H), 2.33
(s, 4H), 2.47 (s, 6H), 3.45 (m, 2H), 3.72 (s, 6H), 3.73 (s, 6H), 3.95 (s, 6H),
4.18 (m, 2H), 4.28 (m, 2H), 6.57 (d, J ) 14.5 Hz, 2H), 6.83 (brs, 2H), 6.99
(s, 2H), 7.41 (s, 2H), 7.44 (s, 2H), 7.58 (d, J ) 14.5 Hz, 2H), 9.98 (s, 2H),
10.23 (s, 2H), 12.36 (brs, 2H); ESIMS m/e calcd for C60H61N14O12 (M + H)
1169.5, found 1169.6. The synthesis of other compounds 7a-c and 7e-f were
performed in a manner similar to that for 7d.
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10.1021/ja003660c CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/08/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5159
Figure 1. Denaturing polyacrylamide gel electrophoresis of 5′-TexasRed-
labeled 5′-[TR]-TTACAGTGGCTGCCAGCA-3′ and 5′-TGCTGGCAGC-
CACTG-3′ cross-linked by 7a-f in the presence and absence of ImImPy.
(a) Lane 1, DNA control; lane 2, 30 µM ImImPy; lane 3, 24 µM 7d;
lanes 4-9, 30 µM ImImPy and 24 µM 7a-f, respectively. DNA fragment
(3 µM) labeled with 5′-TexasRed was incubated in 10 µL of 5 µM Na
cacodylate buffer (pH 7.0) at 37 °C for 15 h. The reaction was quenched
by adding 1 µL of 1 µM calf thymus DNA. To this solution was added
89 µL of H2O. A 1 µL of aliquot was concentrated, and the resulting
residue was redissolved in 8 µL of loading dye (formamide with New
Fuchsine). A 2 µL aliquot was electrophoresed on 15% denaturing
polyacrylamide gel using a Hitachi 5500-S DNA sequencer.
Figure 2. Denaturing polyacrylamide gel electrophoresis of 5′-TexasRed-
labeled oligonucleotides. Lanes 1-5 and lanes 6-10 use 5′-[TR]-TTAC-
AGTGGCTGCCAGCA-3′ and 5′-[TR]-TTATGCTGGCAGCCACTG-3′,
respectively. Lanes 1 and 6, DNA controls; lanes 2 and 7, reaction
mixture; lanes 3 and 8, reaction mixture after exposure to heat and hot
piperidine; lanes 4 and 9, samples of lane 3 and 8 after exposure to alkaline
phosphatase. Lanes 5 and 10, 5′-[TR]-TTACAGTGGCTGCC-3′ and 5′-
[TR]-TTATGCTGGCAGCC-3′. DNA cross-linking was carried out as
described in the legend to Figure 1. Heat degradation of cross-linking
product was performed at 90 °C for 20 min and followed by heating in
0.1 M piperidine at 90 °C for 20 min. Dephosphorylation was carried
out with calf intestine alkaline phosphatase at 37 °C for 1 h. The samples
were analyzed as described in the legend to Figure 1.
linking products. The compound with a pentamethylene linker
gave less cross-linked product, indicating that tetramethylene
linker is an optimal spacer for the formation of interstrand cross-
linking (Figure 3S). The reason for an efficient cross-linking by
7d is not known; however, flexibility of the linker region would
be important for the reaction. The band which appeared just below
the cross-linking product observed in the case of 7c was assumed
to be a mono-alkylated product, since this band was predominantly
formed when the same reaction by 7c was carried out with 5′-
[TR]-TTACAGTGGCTGCCAGCA-3′/5′-TGCTGGCAGCCTCTG-
3′ in which target A in the lower strand was substituted with T.
To elucidate the site of interstrand cross-linking, we chemically
degraded the products using two set of oligomers in which the
upper or lower strand was labeled. Both systems provided slow
migrating bands (Figure 2, lanes 2 and 7). Heating with piperidine
(90 °C, 20 min) converted these bands to a faster migrating
product (lanes 3, and 8). The faster bands were converted into
slightly slower migrating bands by dephosphorylation with
alkaline phosphatase (lanes 4 and 9), the mobilities of which were
identical to that of the authentic fragments (lanes 5 and 10). These
results demonstrated that interstrand cross-linking selectively
occurred at target A’s colored in red.11 For efficient cross-linking,
one A‚T base pair between two recognition moieties was required
to accommodate the linker region. Deletion of the A‚T base pair
and insertion of two A‚T base pairs significantly and slightly
reduced cross-linking, respectively. Substitution of the A‚T linker
with a G‚C base pair almost completely inhibited the cross-linking.
These results suggest that the linker region possesses A‚T base-
pair preference.12
In conclusion, we developed a novel DNA interstrand cross-
linking agent 7d that cross-linked double strands only in the
presence of ImImPy at a nine-base-pair sequence, 5′-PyGGC(T/
A)GCCPu-3′. The present system will provide a promising
approach for the design of novel sequence-specific DNA inter-
strand cross-linking agents. Targeting specific sequences in the
human genome by such sequence-specific cross-linking agent
would constitute a powerful gene-regulating tool.3g,13 Further
studies on the applicability of this novel class of cross-linking
agents are currently in progress.
Acknowledgment. This study was partly supported by a Grant-in-
Aid for Priority Research from the Ministry of Education, Science, Sports,
and Culture, Japan, The Sagawa Foundation for Promotion of Cancer
Research, and from the Atsuko Ohuchi Memorial Fund (Tokyo Medical
and Dental University).
Supporting Information Available: Figures 1S-3S show denaturing
polyacrylamide gel electrophoresis of 5′-[TR]-TTACAGTGGCTGCCA-
GCA-3′/5′-TTTTTATGCTGGCAGCCACTG-3′ and 5′-[TR]-TTACA-
GCGGCTGCCGGCA-3′/5′-TGCCGGCAGCCGCTG-3′ cross-linked by
7c, 7d, and pentamethylene linker compound in the presence of ImImPy
(PDF). This material is available via the Internet at http://pubs.acs.org.
We examined the sequence specificity of the interstrand cross-
linking using 7d in the presence of various triamides. In contrast
to ImImPy, significant cross-linked bands were not observed for
ImImIm, ImPyPy, and PyImPy. Densitometric analysis of gel
electrophoresis indicated that the efficiency of cross-linking by
these compounds was less than 10%. These results indicate that
interstrand cross-linking proceeds sequence-specifically according
to Dervan’s base-pair recognition rule.3
JA003660C
(12) Analogous A‚T base-pair preference of γ-turn and â-tail of hairpin
polyamides was reported. Swalley, S. E.; Baird, E. E.; Dervan, P. B. J. Am.
Chem. Soc. 1999, 121, 1113.
(11) It was found that 7d also cross-linked to the corresponding oligomers
possessing G’s at cross-link sites in the presence of ImImPy with a slightly
low efficiency (Figure 2S).
(13) (a) Gottesfeld, J. M.; Neely, L.; Trauger, J. W.; Baird, E. E.; Dervan,
P. B. Nature 1997, 387, 202. (b) Weisz, K. Angew. Chem., Int. Ed. Engl.
1997, 36, 2592.