A potent, water-soluble and photoinducible DNA cross-linking agent

Article abstract of DOI:10.1021/ja029040o

Water-soluble DNA cross-linking phenol and biphenol derivatives 3 and 6 have been synthesized by a Mannich reaction. Both of them can cross-link DNA by photoactivation using visible light (wavelength > 400 nm). Compound 6 can cross-link DNA at pH 5.0 and

Full text of DOI:10.1021/ja029040o

Published on Web 01/09/2003
A Potent, Water-Soluble and Photoinducible DNA Cross-Linking Agent
†
†
†
†
§
|
Ping Wang, Renpeng Liu, Xiaojun Wu, Hongjuan Ma, Xiaoping Cao, Ping Zhou,
Jiangye Zhang, Xiaocheng Weng, Xiao-Lian Zhang, Jun Qi, Xiang Zhou,* and Linhong Weng⊥
†
†
‡
†
,†
College of Chemistry and Molecular Sciences, Wuhan UniVersity, Hubei Wuhan 430072, P. R. of China,
School of Medicine, Wuhan UniVersity, Hubei Wuhan 430072, P. R. of China, National Laboratory of Applied
Organic Chemistry, Lanzhou UniVersity, Gansu Lanzhou 730000, P. R. of China, Department of Macromolecular
Science, Fudan UniVersity, Shanghai, 200433, P. R. of China, and Department of Chemistry, Fudan UniVersity,
Shanghai 200433, P. R. of China
Received October 22, 2002 ; E-mail: zhoux@chem.whu.edu.cn or zhouxiang65@hotmail.com
Induced DNA interstrand cross-links (ISC) by chemical agents
or photoactivation play very important roles for cancer therapy.1
Several important clinical drugs (e.g., cisplatin, psoralens, and
mitomycin C) are known to induce DNA ISC formation which can
2
disrupt cell maintenance and replication. The design of novel
dimeric agents targeting DNA has been investigated by several
groups.3 Among these antitumor agents, one mechanism was
involved in o-quinone methide (o-QM, Figure 1a) intermediate.
o-QM is a quinone methide derivative which has played important
roles in organic syntheses as well as in chemical and biological
processes.4 o-QM has been found to have significance for their
,5
6
nucleic acid bases and DNA alkylation. More recently, the Wan,
Freccero, and Kresge groups7 have significantly reported the
-9
generation of o-QMs upon photochemical and thermal activation
Figure 1. o-Quinone methide formation by photoactivation.
in aqueous solution. According to Freccero’s report, phenol
possessing a quaternary ammonium group can form o-quinone
methide by photoactivation in aqueous solution (Figure 1a).8
Meanwhile, photo-cross-links of DNA by irradiation with light
under mild conditions and without any additives such as metals,
oxidative agents, and reducing agents will offer considerable
potential in medicine.1 These encouraged us to design and
synthesize the water-soluble phenol biquaternary ammonium 3 and
biphenol biquaternary ammonium 6 derivatives which may have
potential applications for DNA ISC under photochemical activation.
To our surprise, compound 6 demonstrated very potent ISC
properties upon photoactivation using visible light. Furthermore,
we found that the structural conformation might be the key factor
for this mode of action. Herein, we report our preliminary results.
Two phenol and biphenol quaternary ammonium derivatives have
been synthesized according to the procedure shown in Figure 2.
Compound 2 was obtained by a Mannich reaction. Quaternarization
was carried out by reaction with methyl iodide. Compound 5 was
obtained by a reported procedure,10 and its quaternarization was
performed as above. Purification of the two derivatives was
performed by crystallization with methanol and ethyl ether. Both
compounds were fully characterized by NMR, HRMS, and elemen-
tal analysis (see Supporting Information).
Figure 2. Synthesis of compounds 3 and 6. Reagents and conditions: (a)
33% aqueous (CH3)2NH, 37% formaldehyde aqueous solution, 30 °C for
30 min, then 90-95 °C 2 h, 39.0%. (b) CH3I, CH3CN, 61.0%. (c) (CH2O)n,
3
3% aqueous (CH3)2NH, C2H5OH, overnight, 13.2%. (d) CH3I, CH3CN,
2.4%.
6
were carried out in phosphate buffer (pH ) 7.7). Samples were
exposed to a 50-W high mercury lamp (wavelength > 400 nm)
placed 20 cm away at 25 °C. The crude reaction mixtures were
loaded onto a denaturing 0.9% alkaline agarose gel. Lambda HindIII
DNA was used as a molecular weight marker.
The DNA-DNA cross-linking ability of compounds 3 and 6
was investigated using linearized plasmid DNA by denaturing
11
alkaline agarose gel electrophoresis as reported by Cech and
Williams.12 The duplex DNA was linearized using the EcoRI
Results of concentration dependences for compounds 3 and 6
are illustrated in Figures 3 and 4. DNA cross-linking by compound
restriction endonuclease digestion. DNA cross-linking experiments
6
was observed at a concentration as low as 1.0 µM. At 10 µM
*
To whom correspondence should be addressed. E-mail: zhoux@chem.whu.
concentration, compound 6 afforded predominantly cross-linked
DNA. Upon comparison with compound 3, compound 6 was found
to be around 100-fold more potent as a DNA cross-linking agent.
It suggests that compound 3 might not be involved in spontaneous
reaction and that its mechanism of action is transient and consists
edu.cn or zhouxiang65@hotmail.com.
†
Wuhan University, College of Chemistry and Molecular Sciences.
‡
Wuhan University, School of Medicine.
§
Lanzhou University, National Laboratory of Applied Organic Chemistry.
|
Fudan University, Department of Macromolecular Science.
⊥
Fudan University, Department of Chemistry.
1116
9
J. AM. CHEM. SOC. 2003, 125, 1116-1117
10.1021/ja029040o CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
The significant difference between compounds 3 and 6 concern-
ing cross-linking properties probably involves two factors. First,
the formation of cross-link by 3 requires two steps, whereas cross-
link by 6 is probably by formation of a bisquinone methide
intermediate18 or by nonspontaneous quinone methide formation.
Second, on comparison with planar 3, compound 6, like many
DNA-specific binders,16 apparently has good shape orientation to
adopt a helical conformation (twist ≈ 35°). Therefore, compound
6 might naturally exhibit the strongest DNA binding and cross-
linking abilities upon photoactivation.
In summary, we prepared a potent, water-soluble and photoin-
ducible DNA cross-linking agent. This finding will encourage us
to modify its structure for multiple applications in biological and
medicinal chemistry. Further tethering to DNA binding agents for
potential drug applications is currently under investigation.
Figure 3. Concentration dependence of 6 for DNA cross-linking. Lane 1,
.5 µg lambda DNA/HindIII (molecular weight standard); lane 2, 0.7 µg
pBR322 (control); lane 3, 0.7 µg pBR322 + 50 µM 6 ; lane 4, 0.7 µg
pBR322 + hν (30 min); lane 5, 0.7 µg pBR322 + 0.1 µM 6 + hν (30
min); lane 6, 0.7 µg pBR322 + 1 µM 6 + hν (30min); lane 7, 0.7 µg
pBR322 + 10 µM 6 + hν (30 min).
1
17
Acknowledgment. This research was supported in part by the
National Science of Foundation of China (NSFC, No.20142001,
20272046). We thank Professors Dr. Dehua Pei in the Department
of Chemistry of The Ohio State University and Dr. Kin Shing Chan
in the Department of Chemistry of The Chinese University of Hong
Kong for their critical reading of our manuscript and we thank three
reviewers for their critical comments.
Figure 4. Concentration dependence of 3 for DNA cross-linking. Lane 1,
1
.5 µg lambda DNA/HindIII (molecular weight standard); lane 2, 0.7 µg
pBR322 (control); lane 3, 0.7 µg pBR322 + 1000 µM 3; lane 4, 0.7 µg
pBR322 + hν (30 min); lane 5, 0.7 µg pBR322 + 10 µM 3 + hν (30 min);
lane 6, 0.7 µg pBR322 + 100 µM 3 + hν (30 min); lane 7, 0.7 µg pBR322
+
500 µM 3 + hν (30 min); lane 8, 0.7 µg pBR322 + 1000 µM 3 + hν
(
30 min).
Supporting Information Available: Synthesis and characterization
of 3 and 6, DNA experimental data and detailed calculation for 6 (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(
1) (a) Kohn, K. W. In Topics in Molecular and Structural Biology 3.
Molecular Aspects of Anti-cancer Drug Action; Neidle, S., Waring, M.,
Eds.; Verlag Chemie Gmbh: D-6940, Weinheim, 1994; p 315. (b) Rajski,
S. R.; Williams, R. M. Chem. ReV. 1998, 98, 2723. (c) Wolkenberg, S.
E.; Boger, D. L. Chem. ReV. 2002, 102, 2477.
Figure 5. pH dependence of DNA cross-link by compound 6. Lane 1, 1.5
µg lambda DNA/HindIII (molecular weight standard); lane 2, 0.7 µg
pBR322 (control); lane 3, 0.7 µg pBR322 + 10 µM 6 (pH ) 5.0); lane 4,
0
6
7
8
.7 µg pBR322 + hν (30 min, pH ) 5.0); lane 5, 0.7 µg pBR322 + 10 µM
(pH ) 5.0) + hν (30 min); lane 6, 0.7 µg pBR322 + 10 µM 6 (pH )
.7) + hν (30 min); lane 7, 0.7 µg pBR322 + 10 µM 6 (pH ) 10.0); lane
, 0.7 µg pBR322 + hν (30 min, pH ) 10.0); lane 9, 0.7 µg pBR322 + 10
(
2) (a) Cimino, G. D.; Gamper, H. B.; Isaacs, S. T.; Hearst, T. E. Annu. ReV.
Biochem. 1985, 54, 1151. (b) Sherman, S. E.; Lippard, S. J. Chem. ReV.
1
987, 87, 1153. (c) Li, V.-S.; Choi, D.; Tang, M.-S.; Kohn, H. J. Am.
Chem. Soc. 1996, 118, 3765.
µM 6 (pH ) 10.0) + hν (30 min).
(3) (a) Bose, D. S.; Thompson, A. S.; Ching, J.; Hartley, J. A.; Berardine, M.
D.; Jenkins, T. C.; Neidle, S.; Hurley, L. H.; Thurston, D. E. J. Am. Chem.
Soc. 1992, 114, 4939. (b) Hartley, J. A.; Hazrati, A.; Hodgkinson, T. J.;
Kelland, L. R.; Khanim, R.; Shipman, M.; Suzenet, F. Chem. Commun.
of sequential formation of two o-quinone methide intermediates13
2000, 2325. (c) Na, Y.; Li, V.-S.; Nakanishi, Y.; Bastow, K. F.; Kohn,
after photoactivation. When time-courses of cross-link formation
by compounds 3 and 6 at the same concentration (10 µM) were
performed (Figures 6 and 8, see Supporting Information), compound
H. J. Med. Chem. 2001, 44, 3453. (d) Nagatsugi, F.; Tokuda, N.; Maeda,
M.; Sasaki, S. Bioorg. Med. Chem. Lett. 2001, 11, 2577.
4) (a) Iyer, V. N.; Szybalski, W. Science 1964, 145, 55. (b) Moore, H. W.;
Czerniak, R. Med. Res. ReV. 1981, 1, 249.
(
6
was obviously found to be very sensitive to time increase, whereas
(5) (a) Peter, M. G. Angew. Chem., Int. Ed. Engl. 1989, 28, 555. (b) Gaikwad,
N. W.; Bodell, W. J. Chem.-Biol. Interact. 2001, 138, 217. (c) Amouri,
H.; Le Bras, A. J. Acc. Chem. Res. 2002, 35, 501.
compound 3 was inert. When the concentration of compound 3
was increased to 500 µM, no significant increase of DNA cross-
linking was observed (Figure 7, see Supporting Information).
Meanwhile, we found that DNA cross-linking by compound 6 was
affected by pH variation (Figure 5). These results show that the
most intense cross-linking occurred at pH 7.7 and the weaker cross-
linking was at pH 5.0. On the contrary, no DNA cross-linking
occurred at all at pH 10.0, implying that the decay of o-quinone
(6) (a) Pande, P.; Shearer, J.; Yang, J.; Greenberg, W. A.; Rokita, S. E. J.
Am. Chem. Soc. 1999, 121, 6773. (b) Zhou, Q.; Turnbull, K. D. J. Org.
Chem. 2000, 65, 2022.
(7) Brousmiche, D.; Wan, P. Chem. Commun. 1998, 491.
(8) Modica, E.; Zanaletti, R.; Freccero, M.; Mella, M. J. Org. Chem. 2001,
66, 41
(9) Chiang, Y.; Kresge, A. J.; Zhu, Y. J. Am. Chem. Soc. 2001, 123, 8089.
10) Mathai, K. P. J. Indian Chem. Soc. 1966, 43, 422.
(
(
(
11) Cech, T. R. Biochemistry 1981, 20, 1431.
12) Tepe, J. J.; Williams, R. M. J. Am. Chem. Soc. 1999, 121, 2951.
9
(13) Zeng, Q.-P.; Rokita, S. E. J. Org. Chem. 1996, 61, 9080.
methide intermediate was accelerated in basic buffer and that the
(
14) Zhang, R. Q.; Wong, N. B.; Lee, S. T.; Zhu, R. S.; Han, K. L. Chem.
elimination to form quinone methide occurred with difficulty
because of deprotonation of phenol in acid buffer.
Phys. Lett. 2000, 319, 213.
(15) de Dios, A. C.; Pearson, J. G.; Oldfield, E. Science 1993, 260, 1491.
16) (a) Barton, J. K. Science 1986, 233, 727. (b) Hurley, L. H. J. Med. Chem.
989, 32, 2027. (c) Qu, X.-G.; Trent, J. O.; Fokt, I.; Priebe, W.; Chaires,
(
We predicted the structure of 6 on the basis of DFT geometry
optimization and then from their 13C chemical shift calculations.
We found that the dihedral angle between the two phenyl rings
is about 35° and that the two quaternary ammonium groups were
on the same side like two anchors, thus forming a twist structure
1
14,15
J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12032. (d) Dervan, P. B.
Bioorg. Med. Chem. 2001, 9, 2215. (e) Satz, A. L.; Bruice, T. C. Acc.
Chem. Res. 2002, 35, 86.
(17) Boger, D. L.; Garbaccio, R. M. Acc. Chem. Res. 1999, 32, 1043.
18) Nakatani, K.; Higshida, N.; Saito, I. Tetrahedron Lett. 1997, 38, 5005.
(
(see Supporting Information for calculation details).
JA029040O
J. AM. CHEM. SOC.
9
VOL. 125, NO. 5, 2003 1117