Highly cooperative DNA dialkylation by the homodimer of imidazole - pyrrole diamide - CPI conjugate with vinyl linker

Article abstract of DOI:10.1021/ja9926212

We synthesized new type of diamide - CPI conjugate possessing a vinyl linker, 7. Sequence-selective alkylation of double-stranded DNA by 7 was investigated by high-resolution denaturing gel electrophoresis using ～400 bp DNA fragments. Highly efficient alkylation predominantly occurs simultaneously at the purines of 5′-PyG(A/T)CPu-3′ site on both strands at a nanomolar concentration of 7. These results suggest that the homodimer of conjugate 7 dialkylates both strands according to Dervan's pairing rule together with a new mode of recognition in which the Im-vinyl linker (L) pair targets G/C base pairs. In addition to the major dialkylation sites, a minor alkylation site was also observed at 5′-GT(A/T)GC-3′. This alkylation can be explained by an analogous slipped homodimer recognition mode in which the L-L pair recognizes the A/T base pair. Efficient dialkylation by the homodimer of 7 was further confirmed using oligonucleotides (ODNs). HPLC analysis revealed that the conjugate 7 simultaneously alkylates GN3/AN3 of the target sequences on both strands of ODNs.

Full text of DOI:10.1021/ja9926212

1602
J. Am. Chem. Soc. 2000, 122, 1602-1608
Highly Cooperative DNA Dialkylation by the Homodimer of
Imidazole-Pyrrole Diamide-CPI Conjugate with Vinyl Linker
Zhi-Fu Tao,†,‡ Isao Saito,†,§ and Hiroshi Sugiyama*,‡
Contribution from the 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, and Department of
Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto UniVersity,
Yoshida, Sakyo, and Kyoto 606-8501, Japan
ReceiVed July 26, 1999
Abstract: We synthesized new type of diamide-CPI conjugate possessing a vinyl linker, 7. Sequence-selective
alkylation of double-stranded DNA by 7 was investigated by high-resolution denaturing gel electrophoresis
using ∼400 bp DNA fragments. Highly efficient alkylation predominantly occurs simultaneously at the purines
of 5′-PyG(A/T)CPu-3′ site on both strands at a nanomolar concentration of 7. These results suggest that the
homodimer of conjugate 7 dialkylates both strands according to Dervan’s pairing rule together with a new
mode of recognition in which the Im-vinyl linker (L) pair targets G/C base pairs. In addition to the major
dialkylation sites, a minor alkylation site was also observed at 5′-GT(A/T)GC-3′. This alkylation can be explained
by an analogous slipped homodimer recognition mode in which the L-L pair recognizes the A/T base pair.
Efficient dialkylation by the homodimer of 7 was further confirmed using oligonucleotides (ODNs). HPLC
analysis revealed that the conjugate 7 simultaneously alkylates GN3/AN3 of the target sequences on both
strands of ODNs.
Introduction
helix approach is associated with poor cellular uptake and is
limited to purine tracks, a fact which severely restricts the
availability of target sites. Minor-groove binding polyamides
that contain N-methylimidazole (Im)-N-methylpyrrole (Py)-
hydroxylpyrrole (Hp),5 which uniquely recognize each of the
four Watson-Crick base pairs, have potential as novel recogni-
tion parts of sequence-specific DNA alkylating agents. Indeed,
we recently demonstrated that hybrid molecules between
segment A of duocarmycin A and Py-Im diamides alkylate a
predetermined sequence in the presence of distamycin A.6 More
recently, the alkylation of predetermined seven-base pair
sequences has been achieved by duocarmycin A derivatives
bearing six-ring Py-Im hairpin polyamides.7 However, these
molecules only alkylate one strand of the DNA helix, and the
double-strand alkylation at predetermined mixture DNA se-
quences still remains a challenge.8 Molecular modeling sug-
gested that the insertion of a vinyl linker (L) between polyamide
and CPI adjusts the location of the cyclopropane ring of the
conjugate and in this way improves the alkylation efficacy of
Sequence-specific DNA alkylation has significant potential
for use in molecular biology and human medicine.1,2 Although,
many antitumor agents alkylate DNA, most have only limited
sequence specificity and often exhibit severe toxicity to normal
tissues. Rational design of alkylating agents targeting specific
sequences in the human genome may provide useful molecules
for various applications, including gene-targeted drugs.3 The
sequence specificities induced by DNA alkylators are usually
the result of a combination of their inherent reactivities toward
purine bases and the binding selectivities to DNA. While
alkylation of predetermined sequences has reportedly been
achieved by the triple helix formation through tethering the
alkylating group to polypyrimidine oligonucleotides,4 the triple
† CREST, Japan Science and Technology Corporation (JST).
‡ Tokyo Medical and Dental University.
§ Kyoto University.
(1) For example and applications, see: (a) Hartley, J. A. Molecular Basis
of Specificity in Nucleic Acid-Drug Interaction; Pullman, B., Jortner, J.,
Eds.; Kluwer Academic Publishers: Netherlands, 1990; pp 513-530. (b)
Nucleic Acid Targeted Drug Design; Prospt, C. L., Perun, T. J., Eds.;
Dekker: New York, 1992. (c) Rajski, S. R.; Williams, R. M. Chem. ReV.
1998, 98, 2723.
(2) (a) AdVance in DNA Sequence Specific Agent; Hurley, L. H., Ed.;
JAI Press: London, 1992; Vol. 1. (b) AdVances in DNA Sequence Specific
Agents; Hurley, L. H.; Chaires, J. B., Ed.; JAI Press: London, 1996; Vol.
2. (c) AdVances in DNA Sequence Specific Agents; Jones, G. B., Palumbo,
M., Ed.; JAI Press: London, 1998; Vol. 3.
(5) (a) For recent reviews, see: Wemmer, D. E.; Dervan, P. B. Curr.
Opin. Str. Biol. 1997, 7, 355. Nielsen, P. E. Chem. Eur. J. 1997, 3, 505. (b)
Lamamie de Clairac, R. P.; Geierstanger, B. H.; Mrksich, M.; Dervan, P.
B. Wemmer, D. E. J. Am. Chem. Soc. 1997, 119, 7909. (c) White, S.;
Szewczyk, 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. 1998, 5, 104. (e) Yang, X.-L.; Kaenzig, C.; Lee, M.; Wang,
A. H.-J. Eur. J. Biochem. 1999, 263, 646. (f) Fujiwara, T.; Tao, Z.-F.; Ozeki,
Y.; Saito, I.; Wang, A. H.-J.; Lee, M.; Sugiyama, H. J. Am. Chem. Soc.
1999, 121, 7706.
(3) (a) Wittung-Stafshede, P. Science 1998, 281, 657. (b) Denison, C.;
Kodadek, T. Chem. Biol. 1998, 5, R129.
(6) Tao, Z.-F.; Fujiwara, T.; Saito, I.; Sugiyama, H. Angew. Chem.,
Int. Ed. 1999, 38, 650.
(4) (a) Moser, H. E.; Dervan, P. B. Science, 1987, 238, 645. (b) Thoung,
N. T.; Helene, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 666. (c) Lukhtanov,
E. A.; Mills, A. G.; Kutyavin, I. V.; Gorn, V. V.; Reed, M. W.; Meyer, R.
B. Nucleic Acids Res. 1997, 25, 5077. (d) Belousov, E. S.; Afonina, I. A.;
Podyminogin, M A.; Gamper, H. B.; Reed, M. W.; Wydro, R. M.; Meyer,
R. B. Nucleic Acids Res. 1997, 25, 3440. (e) Taylor, M. J.; Dervan, P. B.
Bioconjugate Chem. 1997, 8, 354.
(7) Tao, Z.-F.; Fujiwara, T.; Saito, I.; Sugiyama, H. J. Am. Chem. Soc.
1999, 121, 4961.
(8) Double-strand alkylation at homopurine sequences of DNA through
triplex formation were attempted: (a) Povsic, T. J.; Strobel, S. A.; Dervan,
P. B. J. Am. Chem. Soc. 1992, 114, 5934 and references therein. (b) Reed,
M. W.; Wald, A.; Meyer, R. B. J. Am. Chem. Soc. 1998, 120, 9729.
10.1021/ja9926212 CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/11/2000

Dialkylation by Im-Py Conjugate with Vinyl Linker
J. Am. Chem. Soc., Vol. 122, No. 8, 2000 1603
Scheme 1a
a (a) Benzotriazole-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate (BOP), THF, then NaBH4; (b) MnO2, THF; (c) triethyl
phosphonoacetate, NaH, THF; (d) NaOH/H2O/MeOH; (e) 1,1′-carbonyldiimidazole, DMF; (f) NaH, DMF, segment A of DU86.
the conjugates. In fact, Lown et al. demonstrated that incorpora-
tion of a vinyl linker dramatically enhances the efficiency of
DNA alkylation as well as cytotoxicity. However, their mol-
ecules are limited to the recognition of A-T rich sequences.9
Thus, diamide-CPI conjugate 7 was designed, synthesized, and
evaluated as a novel sequence-specific DNA alkylating agent.
We found that 7 alkylates double-stranded DNA at predeter-
mined DNA sequences through highly cooperative homodimer
formation.
Highly Cooperative Double-Strand Alkylation within over
400-Base Pair DNA Fragments. Sequence-selective alkylation
by 7 was investigated on both strands of 5′-end Texas Red-
labeled DNA of three sets of ∼400 bp DNA fragments using a
DNA sequencer as previously described.5f-7 Surprisingly, in all
three sets of DNA fragments most of the alkylation occurred in
close proximity of both strands. A typical sequencing analysis
of the alkylated fragment after heat treatment is shown in Figure
1. Under the present experimental conditions, alkylation by 7
occurred predominantly at the purines of sequence 5′-PyG(A/
T)CPu-3′ of both strands (sites 1-3) with reasonable efficiency
at a nanomolar concentration within a 450 bp DNA fragment.
Dialkylation at sites 1-3 can be explained by the mode of
recognition shown in Figure 2, in which the Im-L pair targets
the G/C base pair. In addition to the major dialkylation sites,
minor alkylation was also observed at sequence 5′-GT(A/T)-
GC-3′ of site 4. This alkylation can be explained by an
analogous slipped homodimer recognition mode in which the
L-L pair recognizes the A/T base pair (Figure 2). It is important
to note that almost all alkylation on the two strand occurred
simultaneously. These results clearly indicate that conjugate 7
dialkylates both strands through cooperative homodimer forma-
tion. Densitometric analysis of the cleavage bands indicated that
the efficiency of alkylation by 7, which is the amount of DNA
cleavage divided by that of the agent approached 69%, confirm-
ing the unusually high efficiency of dialkylation.
Results and Discussion
Synthesis. Although Lown et al. reported on the importance
of the vinyl linker for biological activity, their molecules are
limited to pyrrole triamide, and no detailed synthetic procedures
have yet been reported.9 We developed an alternative synthetic
methodology, as shown in Scheme 1. The imidazole moiety
was incorporated into the novel conjugate for the first time.
Hydroxybenzotriazolyl ester, formed in situ from carboxylic acid
1 and a BOP reagent, reacts with sodium borohydride in THF
to give alcohol 2 at a moderate yield.10 Oxidation of 2
quantitatively produces aldehyde 3. The key intermediate 4 was
obtained through Wadsworth-Emmons reaction11 at a good
yield. Hydrolysis and activation were performed as previously
described6,7 to give activated amide 6. As the alkylating moiety,
segment A of DU86 was employed due to its superior chemical
stability compared to that of duocarmycin A, which was
synthesized through five steps according to reported proce-
dures.12 Finally, the coupling reaction between 6 and segment
A of DU86 accomplished the synthesis of 7 at an excellent yield.
Highly Cooperative Double-Strand Alkylation Confirmed
at the Oligonucleotide Level Using HPLC Product Analysis.
To clarify the mechanism of DNA alkylation by 7, we
investigated the alkylation of two oligonucleotides, ODN 1 and
2, both of which were designed according to the gel experiments
described above. To obtain better separation of the two strands
of ODNs when using our HPLC separation conditions, we added
two T residues to the 3′-end of the lower strand (ODN 2). HPLC
(9) (a)Wang, Y.; Gupta, R.; Huang, L.; Luo, W.; Lown, J. W. Anti-
cancer Drug Des. 1996, 11, 15. (b) Fregeau, N. L.; Wang, Y.; Pon, R. T.;
Wylie, W. A.; Lown, J. W. J. Am. Chem. Soc. 1995, 117, 8917. (c) Iida,
H.; Lown, J. W. Recent Res. DeVel. Synth. Org. Chem. 1998, 1, 17.
(10) McGeary, R. P. Tetrahedron Lett. 1998, 39, 3319.
(11) Wadsworth, W. S., Jr.; Emmons, W. D. J. Am. Chem. Soc. 1961,
83, 1733.
(12) Nagamura, S.; Asai, A.; Kanda, Y.; Kobayashi, E.; Gomi, K.; Saito,
H. Chem. Pharm. Bull. 1996, 44, 1723.

1604 J. Am. Chem. Soc., Vol. 122, No. 8, 2000
Tao et al.
Figure 1. Thermally induced strand cleavage of 5′-end Texas Red-labeled 450-base pair (bp) DNA fragments by conjugate 7. Results using 5′-end
labeled upper strand (pUC 18 F 780-1229) (a) and 5′-end labeled lower strand (pUC 18 R 1459-1908) (b) DNA fragments are shown. These two
DNA fragments are complementary. Lanes 1 and 6, DNA control; G, C, T, A are Saenger sequencing standard. Drug concentrations are indicated
in lanes 2-5. Sequences containing dialkylation sites 1-4 are represented (c). The arrows indicate the site of alkylation.
analysis of the reaction mixture of ODN 1 and 2 with 7 revealed
that conjugate 7 simultaneously alkylated GN3 of both ODN 1
and 2 at the target sequences and thus produced a dialkylated
product (Figure 3). It is noteworthy that the complex of ODN
1-ODN 2-7 in HPLC profile consistently showed one peak,
indicating the strong stability of the complex. Direct observation
of molecular ions (-5, -6, -7, -8, and -9) for the ODN
1-ODN 2-7 complex by electron-spray MS further confirmed
that double alkylation by 7 greatly stabilizes duplex DNA
(Figure 4a). Similarly, ODNs 3 and 4 were used to investigate
the alkylation of slipped homodimer recognition mode at site 4
of the gel results. The alkylation of ODNs 3 and 4 by 7 exhibited
a moderate efficiency and produced different kinetics compared
with that of ODNs 1 and 2, which is consistent with the DNA
alkylation efficiency demonstrated by the high-resolution gel
experiments (Figure 4b inset). As before, the complex ODN 3,
4 -7 showed high stability and this was confirmed by electron
spray MS (Figure 4b), suggesting cooperative dialkylation.
High Stability of Oligonucleotide-Drug Dialkylation
Complex Confirmed by Melting Temperature Experiment.
To determine how much stability the drug gives to the DNA
duplex, a UV-monitoring Tm experiment was performed. Under
the present experimental conditions, duplex 5′-GATCGACGCTC-
3′/5′-GAGCGTCGATC-3′ has Tm ) 38 °C, and 5′-GATC-
GACGCTC-3′/5′-GAGCGTCGATC-3′-7 dialkylation complex
does not start to decompose until it reaches a temperature of
90 °C, which we refer to as the decomposing temperature (Td)
for the complex, suggesting that 7 stabilizes the DNA duplex

Dialkylation by Im-Py Conjugate with Vinyl Linker
J. Am. Chem. Soc., Vol. 122, No. 8, 2000 1605
Figure 2. Schematic representation of conjugate 7-DNA interacting modes. The arrows represent the site of alkylation. The imidazole and pyrrole
rings are represented by red and blue circles, respectively; the red triangle represents segment A of DU86.
Figure 3. HPLCprofiles of alkylation of ODN1, 2 by conjugate 7 after the indicated incubation periods. The arrows represent the site of alkylation.
by at least 52 °C (Td - Tm). Similarly, 7 stabilizes the ODN 3
and 4 duplex at least by 42 °C [Td (80 °C) - Tm (38 °C)] (data
not shown).
Characterization of the Dialkylation Complex by Thermal
Degradation. Although gel and oligonucleotide experiments
demonstrated that 7 shows a high efficiency in alkylating both
strands of DNA, one may argue that alkylation comes from one
of the two monomers in the dimer and possibly never occurs
on both strands at the same time. Therefore, unambigous
characterization of the dialkylation complex by thermal degra-
dation was performed. Despite of the fact that complex ODN-7
is too stable to be denatured, it is well-known that covalently
alkylated duocarmycin-oligonucleotide adducts are easily con-
verted to abasic site-containing oligonucleotides, and ultimately

1606 J. Am. Chem. Soc., Vol. 122, No. 8, 2000
Tao et al.
Figure 4. Electron spray mass spectra of oligonucleotide-7 dialkylation complex in solution. The calculated m/z value is shown in the parenthesis.
y-axis indicates relative peak intensity. (a) ODN1,2-7 dialkylation complex. (b) ODN 3,4-7 dialkylation complex; inset, interaction mode of ODN3,4-
conjugate 7 and the alkylation efficiency after various indicated incubation time at 0 °C is also included.
Scheme 2
give rise to the cleavage of the DNA strand upon heating. The
characterization of the latter two processes has been well
documented.13 The ODN 1- and 2-7 complex in Figure 3 was
thus collected and heated at 90 °C for 5 min, and two abasic
site-containing oligonucleotides 8 and 9 were immediately
produced at a high yield without producing any intact oligo-
nucleotide (Scheme 2 and Figure 5a). Cleavage of the abasic
site in 8 and 9 with hot alkali (0.1 N NaOH, 90 °C, 5 min)
produced the corresponding cleavage oligonucleotides (Scheme
2 and Figure 5b), whose compositions were unambiguously
confirmed by enzymatic digestion with snake venom phos-
phodiesterase and alkaline phosphatase (Scheme 2). It is worth
mentioning that even when the concentration ratio of the ODN
1 and 2 duplex and 7 is 1:1, the drug was almost quantitatively
utilized to alkylate both strands at the same time without
producing any monoalkylation (data not shown). The ODN 3-
and 4-7 dialkylation complex was similarly characterized (data
purine alkylation sites in the DNA target give dsDNA fragments
not shown). The unambiguous characterization of oligonucle-
with residual phosphates that can be used as substrates for
otides-7 dialkylation complex provides further evidence that
ligase.8a In this sense, conjugate 7 is the first example of an
conjugate 7 highly cooperatively, efficiently, and simultaneously
artificial “restriction enzyme”. Recently, synthetic Py-Im
alkylates both strands of DNA in a predictable manner.
polyamides have been shown to disrupt specific transcription
factor-DNA interaction and to inhibit basal and activated
transcription from various RNA polymerase II and III promo-
Conclusions
The present study is the first report of sequence-specific
(13) (a) Sugiyama, H.; Hosoda, M.; Saito, I.; Asai, A.; Saito, H.
cooperative double-strand alkylation of DNA. The development
Tetrahedron Lett. 1990, 31, 7197. (b) Sugiyama, H.; Ohmori, K.; Chan, K.
L.; Hosoda, M.; Asai, A.; Saito, H.; Saito, I. Tetrahedron Lett. 1993, 34,
2179. (c) Sugiyama, H.; Fujiwara, T.; Ura, A.; Tashiro, T.; Yamamoto, K.;
Kawanishi, S.; Saito, I. Chem. Res. Toxicol. 1994, 7, 673.
of sequence-specific DNA cleaving molecules that go beyond
the specificities of the natural enzymes is a long-term dream at
the interface of chemistry and biology. It has been reported that

Dialkylation by Im-Py Conjugate with Vinyl Linker
J. Am. Chem. Soc., Vol. 122, No. 8, 2000 1607
tube (Suprec-02) were purchased from Takara Co., thermo sequenase
core sequencing kit and loading dye (dimethylformamide with fushin
red) from Amersham Co., Ltd, 5′-end Texas Red-modified DNA
oligomer (18mer) from Kurabo Co., Ltd, and 50% Long Ranger gel
solution from FMC Bioproducts. Calf intestine alkaline phosphatase
(AP, 1000 units/mL) and snake venom phosphodiesterase (s.v. PDE, 3
units/mL) were purchased from Boehringer Mannheim.
4-[[4-(Acetylamino)-1-methylimidazol-2-yl]carbonylamino]-2-hy-
droxylmethyl-1-methylpyrrole (2). To a mixture of 205 mg (0.67
mmol) of 1, 326 mg (0.74 mmol) of BOP, and 170 µL of DIEA in 30
mL of THF was added 98 mg (2.59 mmol) of NaBH4. The mixture
was stirred for 3 h at room temperature, and the solvent was evaporated
to give a residue, to which was added 20 mL of CH3OH and 5 mL
H2O. The resulting mixture was stirred for 1 h to produce a clear
solution. Removal of the solvents under reduced pressure gave a light
yellow residue, which was subjected to flash chromatography using a
mixture of CH3OH and CH2Cl2 as eluent to afford 92.6 mg of 2 as a
white solid in 47.4% yield. 1H NMR (DMSO-d6) δ 10.24 (s, 1H), 9.62
(s, 1H), 7.38 (s, 1H), 7.10 (d, J ) 2.0 Hz, 1H), 6.09 (d, J ) 2.0 Hz,
1H), 4.86 (t, J ) 5.5 Hz, 1H), 4.34 (d, J ) 5.5 Hz, 2H), 3.93 (s, 3H),
3.54 (s, 3H), 2.01 (s, 3H). HREIMS m/e calcd for C13H17N5O3 291.1331,
found 291.0987.
Figure 5. HPLC profiles of characterization of the dialkylation
complex. (a) abasic site-containing oligonucleotides; (b) cleavage
products at the abasic site; (c) enzymatic digestion analysis of the
cleavage products.
4-[[4-(Acetylamino)-1-methylimidazol-2-yl]carbonylamino]-1-me-
thylpyrrole-2-aldehyde (3). A mixture of 85 mg (0.29 mmol) of 2
and 550 mg of activated MnO2 (85%) in 30 mL of THF was stirred
for 1.5 h at room temperature, and the solid was removed by filtration.
ters.14 More recently, Py-Im polyamides have also been used
to activate transcription by blocking the DNA-binding activity
of a repressor protein.15 The stability of the polyamide-DNA
complex is one of the key factors necessary for efficient
regulation of specific genes. Anchoring the polyamides at both
strands of predetermined sequences dramatically stabilizes the
drug-DNA complex, which would in turn be expected to
enhance the gene-regulating capacity. In addition, conjugate 7
would also benefit from the inherent DNA damage by alkylation
compared with general polyamides in terms of biological
activity. The new DNA dialkylating agent developed in the
present investigation may provide a promising approach for
developing new types of sequence-specific DNA dialkylating
and cross-linking agents. Further studies on the specificity,
optimization, and applicability of this new class of DNA
dialkylating agents are currently in progress.
1
After removal of the solvent, crude product was obtained. H NMR
analysis indicated that the purity of the product was >95%, and
therefore it was directly used for the next reaction without further
purification. 1H NMR (DMSO-d6) δ 10.21 (s, 1H), 10.18 (s, 1H), 9.50
(s, 1H), 7.63 (s, 1H), 7.43 (s, 1H), 7.10 (d, J ) 2.0 Hz, 1H), 3.94 (s,
3H), 2.84 (s, 3H), 2.02 (s, 3H). HREIMS m/e calcd for C13H15N5O3
289.1175, found 289.1204.
Ethyl 4-[[4-(Acetylamino)-1-methylimidazol-2-yl]carbonylamino]-
1-methylpyrrole-2-acrylate (4). To a suspension of 23 mg (0.58 mmol)
of NaH (60%) in 6 mL of THF in an ice bath was added 116 mL of
triethyl phosphonoacetate. The mixture was stirred for 5 min and 3
(all) in 25 mL of THF was added. The resulting mixture was stirred
overnight. Evaporation of THF gave a residue, which was subjected to
flash chromatography using ethyl acetate as eluent to afford 88.5 mg
1
of 4 as a yellow solid in 84% yield (two steps based on 2). H NMR
(DMSO-d6) δ 10.25 (s, 1H), 9.87 (s, 1H), 7.51 (d, J ) 15.9 Hz, 1H),
7.44 (d, J ) 1.8 Hz, 1H), 7.42 (s, 1H), 6.84 (d, J ) 1.8 Hz, 1H), 6.11
(d, J ) 15.9 Hz, 1H), 4.16 (q, J ) 7.0 Hz, 2H), 4.13 (s, 3H), 3.70 (s,
3H), 2.02 (s, 3H), 1.24 (t, J ) 7.0 Hz, 3H). HREIMS m/e calcd for
C17H21N5O4 359.1593, found 359.1602.
Experimental Section
General Methods. Reagents and solvents were purchased from
standard suppliers without further purification. Abbreviations of some
reagents are the following: DIEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide; BOP, benzotriazole-1-yloxytris (dimethyl-
amino)-phosphonium hexafluorophosphate. Reactions were monitored
by thin-layer chromatography (TLC) using 0.25 mm silica gel 60 plates
impregnated with 254 nm fluorescent indicator (purchased from Merck).
Plates were visualized by UV light. NMR spectra were recorded on a
JEOL JNM-A 500 magnetic resonance spectrometer, and tetrameth-
ylsilane was used as the internal standard. Proton NMR spectra were
recorded in parts per million (ppm) downfield relative to a tertra-
methylsilane. The following abbreviations apply to spin multiplicity:
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), b (broad).
Electron impact (EI) mass spectra were recorded on a JNM-AX 505
mass spectrometer, electrospray ionization mass spectra (ESIMS) on a
PE SCIEX API 165 mass spectrometer and high-resolution fast atom
bombardment mass spectra (FABMS) on a JEOL-JMS-SX102A mass
spectrometer. Polyacrylamide gel electrophoresis was performed on a
HITACHI 5500-S DNA sequencer. Ex Taq DNA polymerase and filter
4-[[4-(Acetylamino)-1-methylimidazol-2-yl]carbonylamino]-1-me-
thylpyrrole-2-acrylic acid (5). To a solution of 70 mg (0.2 mmol) of
4 in 5 mL of CH3OH was added 1.5 mL of 2N aqueous NaOH and 3
mL of H2O. The mixture was stirred for 4.5 h at room temperature.
The solvent was removed by evaporation under reduced pressure, and
20 mL of H2O was added. The resulting mixture was filtered, and the
filtrate was acidified with 2N HCl to pH 2-3. The gel-like precipitate
was collected by filtration and dried to give 43 mg of 5 in 67% yield.
1H NMR (DMSO-d6) δ 10.24 (s, 1H), 9.84 (s, 1H), 7.43 (d, J ) 15.0
Hz, 1H), 7.41 (s, 1H), 7.40 (s, 1H), 6.78 (s, 1H), 6.03 (d, J ) 15.0 Hz,
1H), 3.94 (s, 3H), 3.67 (s, 3H), 3.86 (s, 3H); ESIMS m/e calcd for
C15H16N5O4 (M - H) 330.3, found 330.2.
AcImPyLCOIm (6). To a solution of 26.4 mg (0.08 mmol) of 5 in
2 mL of DMF was added 49.9 mg (0.31 mmol) of 1,1′-carboxyldiimi-
dazole. The mixture was stirred overnight at room temperature, and
20 mL of H2O was added. The yellow precipitate was collected by
filtration to afford 20.5 mg of 6 in 68% yield. 1H NMR (DMSO-d6) δ
10.23 (s, 1H), 10.04 (s, 1H), 8.67 (s, 1H), 7.90 (d, J ) 1.0 Hz, 1H),
7.88 (d, J ) 15.5 Hz, 1H), 7.50 (d, J ) 2.0 Hz, 1H), 7.44 (s, 1H), 7.32
(d, J ) 2.0 Hz, 1H), 7.16 (d, J ) 15.5 Hz, 1H), 7.10 (S, 1H), 3.96 (s,
3H), 3.79 (s, 3H), 2.03 (s, 3H); C18H18N7O3 (M - H) 380.4, found
380.4.
(14) (a) Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382,
559. (b) Gottesfeld, J. M.; Neely, L.; Trauger, J. W.; Baird, E. E.; Dervan,
P. B. Nature 1997, 387, 202. (c) Dickinson, L. A.; Gulizia, R. J.; Trauger,
J. W.; Baird, E. E.; Mosier, D. E.; Gottesfeld, J. M.; Dervan, P. B. Proc.
Natl. Acd. Sci. U.S.A. 1998, 95, 1298.
(15) Dickinson, L. A.; Trauger, J. W.; Baird, E. E.; Ghazal, P.; Dervan,
P. B.; Gottesfeld, J. M. Biochemistry 1999, 38, 10801.
AcImPyLCOCPI (7). To a suspension of 3.2 mg (0.08 mmol) of
sodium hydride (60%) in 0.3 mL of DMF at -50 °C was injected 6.1
mg (0.024 mmol) of segment A of DU86 in 0.3 mL of DMF. The

1608 J. Am. Chem. Soc., Vol. 122, No. 8, 2000
Tao et al.
mixture was stirred for 3 h at -50 to -40 °C. After 10.8 mg (0.028
mmol) of 6 in 1 mL of DMF was injected at -50 °C, the reaction
mixture was further stirred for 5 h at -40 °C and kept at -30 °C in a
refrigerator for 2 days. Next, 3 mL of 0.01 M of sodium phosphate
buffer was added, and the mixture was stirred for 5 min at room
temperature. The evaporation of the solvent gave a yellow residue,
which was subjected to flash chromatography using a mixture of
chloroform and methanol as eluent to afford 12.3 mg of 7 in 91% yield.
1H NMR (DMSO-d6) δ 12.36 (s, 1H), 10.24 (s, 1H), 9.97 (s, 1H), 7.58
(d, J ) 15.0 Hz, 1H), 7.43 (s, 1H), 7.41 (d, J ) 2.0 Hz, 1H), 6.99 (d,
J ) 2.0 Hz, 1H), 6.85 (s, br, 1 H), 6.58 (d, J ) 15.0 Hz, 1H), 4.29 (d,
J ) 10.5 Hz, 1H), 4.19 (dd, J ) 5.0 Hz and 4.5 Hz, 1H), 3.95 (s, 3H),
3.73 (s, 3H), 3.72 (s, 3H), 3.46 (m, 1H), 2.47 (s, 3H), 2.08 (m, 1H),
2.02 (s, 3H), 1.29 (t, J ) 4.5 and 3.5 Hz, 1H); ESIMS m/e calcd for
C29H28N7O6 (M - H) 570.6, found 570.4. HRFABMS m/e calcd for
C29H30N7O6 (M + H) 572.2257, found 572.2262.
Preparation of 5′-Texas Red-End-Modified 450-Base Pair DNA
Fragments. The 5′-end Texas Red-modified 450-base pair DNA
fragments pUC18 F780*-1229 and pUC18 R1459*-1908 (they are
complementary) were prepared by the PCR method using 5′-end Texas
Red-modified 18mers 5′-AGAATCAGGGGATAACGCAG-3′ (pUC 18
forward 780-799), and 5′-TTACCAGTGGCTGCTGCCAG-3′ (pUC
18 reverse 1459-1478) as primers and purified by filtration using
Suprec-02. Their concentration was determined by ethidium bromide
staining. The asterisk indicates Texas Red modification, and the
nucleotide numbering starts with the replication site.
was performed using 0.05 M tetraethylammonium acetate (TEAA) and
a 9-11% acetonitrile linear gradient (0-50 min) at a flow rate of 1.0
mL/min. Detection was performed at 254 nm. ESIMS detection of
ODN-7 dialkylation complexes. A reaction mixture (50 µL) containing
conjugate 7 (75 µM) and the duplex oligonucleotides (25 µM duplex
concentration) in 50 mM sodium cacodylate buffer (pH 7.0) was
incubated at 0 °C for 2 days and then subjected to HPLC (Elution was
performed with 0.05 M ammonium formate and a 0-50% acetonitrile
linear gradient (0-40 min) at a flow rate of 1.0 mL/min.); the complex
(main peak at retention time)10 min) was collected and subjected to
electrospray mass spectrometer. ESMS spectra are shown in Figure 4.
Melting Temperature Experiments. Thermal denaturation profiles
were obtained on a Beckman DU650 spectrophotometer equipped with
a high performance temperature controller. Measurements were con-
ducted in 50 mM sodium cacodylate buffer (pH 7.0) and the samples
had a 13 µM (duplex) concentration. The absorbance of the samples
was monitored at 260 nm from 4 °C to 100 °C with a heating rate of
1 °C per minute.
Characterization of ODN-7 Dialkylation Complex. The dialky-
lation complex with retention time at 26 min in Figure 3 was collected
and then lyophilized. After TEAA was removed by another HPLC using
ammonium formate-acetonitrile as eluent, the resulting complex was
heated at 90 °C for 5 min and subjected to HPLC (Figure 5a). Products
A and B in Figure 5a were collected and heated in the presence of 0.1
N NaOH at 90 °C for 5 min, respectively, and their cleavage products
were separated by HPLC [Elution was performed with 0.05 M
ammonium formate and a 0-15% acetonitrile linear gradient (0-20
min) at a flow rate of 1.0 mL/min] (Figure 5b). The composition of
the cleavage oligonucleotides were confirmed by enzymatic digestion
according to a previously reported procedure.15
High-Resolution Gel Eletrophoresis. The 5′-end Texas Red-labeled
DNA fragment (75 nM) was alkylated by various concentrations of 7
in 10 µL of 10 mM Na phosphate buffer (pH 7.0) containing 10%
DMF at room-temperature overnight. The reaction was quenched by
the addition of calf thymus DNA (5 mM, 1 µL) and by heating for 5
min at 90 °C. DNA was collected by ethanol precipitation. The pellet
was dissolved in 8 µL of loading dye (formamide with fushin red),
heated at 94 °C for 30 min, and then immediately cooled to 0 °C. A 2
µL of aliquot was electrophoresed on 6% denaturing polyacrylamide
gel using a Hitachi 5500-S DNA sequencer.
Acknowledgment. We thank Dr. J. W. Lown for providing
a detailed experimental procedure for the synthesis of poly-
amide-CPI conjugate with vinyl linker. Duocarmycin B2 was
obtained from Kyowa Hakko through the assistance of Dr. C.
Murakata.
Alkylation of Oligonucleotides by Conjugate 7 as Monitored by
HPLC. Oligonucleotides were synthesized on an automated DNA
synthesizer. A reaction mixture (50 µL) containing conjugate 7 (75
µM) and the duplex oligonucleotides (25 µM duplex concentration) in
50 mM sodium cacodylate buffer (pH 7.0) was incubated at 0 °C for
the indicated periods. The progress of the reaction was monitored by
HPLC using a Chemcobond 5-ODS-H column (4.6 × 150 mm). Elution
Supporting Information Available: The full sequences of
pUC 18 F 780-1229 and pUC 18 R 1459-1908 DNA fragments
with specific alkylation sites (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA9926212