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Bioorganic & Medicinal Chemistry Letters 9 (1999) 1703-1708



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Taj Mohammad, a Chaoping Chen,b Peixuan Guo,b and Harry Morrisona Department ofaChemistry and bPathobiology, Purdue University, West Lafayette, IN 47907, U.S.A.

Received 4 March 1999; accepted 7 May 1999 Abstract: The metal complexes, eis-Rh(phen)2Cl2 + and its more hydrophobic analog eis-Rh(phen)(phi)Clz +, have been shown to photocross-link the 120-base d~29-encoded pRNA. Primer extension on the cis-Rh(phen)(phi)C12 +photocross-linked RNA revealed that guanines are responsible for the interstrand cross-links. © 1999 ElsevierScience Ltd. All rights reserved.

The development of reagents that can form interstrand cross-links in DNA or RNA continues to be a subject of considerable interest in the areas of molecular biology and rational drug design.2 The modification of the genome through the creation of such cross-links leads to the inhibition of nucleic acid replication or extension, so that in many cases such agents are potential antitumor drugs. 2a'3 Photochemical cross-linking reagents are of particular interest, since such chemicals can be selectively activated both in time and space, thus providing a "switch-like" mechanism that permits the chemical to be turned on precisely when and where a desired biological effect is intended.4 For example, psoralens are widely employed photoactivated cross-linking agents that are clinically used in the photochemotherapy of various skin diseases 5 and, more recently, in the photodetoxification of blood. 6 Psoralens predominantly target Us and Ts in RNA and DNA, respectively] and we are unaware of lightactivatable reagents that can create such cross-links between purines] '8 We thus report here two rhodium(III) octahedral complexes that, upon UVA (320-400 nm) excitation, form cross-links in nucleic acid through the involvement ofpurines which are proximal within the context of the tertiary structure of the pRNA.

The metal complexes are cis-Rh(phen)2Cl2÷ ("BISPHEN", phen = 1,10-phenanthroline)9 and cisRh(phen)(phi)C12 + ("PHENPHI", phi = 9,10-phenanthrenequinonediimine)l'l° (see structures below). We have 1+ +

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0960-894X/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.

PII: S0960-894X(99)00265-6 1704 T. Mohammad et al. / Bioorg. & Med. Chem. Lett. 9 (1999) 1703-1708 shown that BISPHEN is stable when mixed with DNA in the dark, but undergoes aquation and forms covalent dG adducts with calf thymus DNA upon irradiation with UVA light. 11 The complex preferentially targets purines, and of the purines does more so with guanine than with adenine analogs. 11a'llb There is evidence that metalation occurs at N1 and 0 6 o f deoxyguanosine, and at N3 of deoxyadenosine, ltb'12 via an electron transfer mechanism] la BISPHEN photosensitizes the inactivation of infectious bacteriophage dsDNA, 11c apparently by sensitizing pyrimidine dimerization. Ilc PHENPHI covalently binds to DNA with comparable quantum efficiency, and also preferentially targets guanine. 1° The major advantage of PHENPHI is its increased hydrophobicity by virtue of which it is taken up by tumor KB cells. It is phototoxic to both KB cells ~° and bacteriophage d06 virus] 3 We now report that both metal complexes effectively form interstrand cross-links in 120-base d029-encoded pRNA (see structure below) via covalent bond formation between two guanosine bases. This pRNA forms a hexameric complex to gear the DNA translocation process. 14 This is only the second class of compounds (in addition to psoralens) known to cross-link nucleic acid upon irradiation, and represents the first example of the photoinitiated cross-linking ofpurines. 2*'s Mixtures of BISPHEN (0.4 mM) and pRNA tS'16(ca. 1.0 ~tg) were irradiated with long wavelength UV light (365 nm) 17 at 0 °C for different intervals and analyzed on an 8% polyacrylamide/8 M urea denaturing gel. A representative gel from several experiments is shown in Figure 1 (panel A). It is clear that controls o f p R N A with BISPHEN/no light (lane 4) and with light/no BISPHEN (lane 2) produced no new bands. However, pRNA irradiated in the presence of BISPHEN (400 and 1000 ktM) produced a new band with slower mobility than the untreated pRNA (lanes 1, 3 and 5-8). As demonstrated below, this new band was assigned to cross-linked pRNA.

The video image digitized data 18 of the cross-linked bands indicated that ca. 5% of pRNA had been cross-linked in

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Figure 1: Denaturing 8% polyacrylamide/8 M urea gel of phototreated mixtures of pRNA with BISPHEN (panel A) and PHENPHI (panel B). Panel A: lane 1, 10 h irradiation; lane 2, pRNA irradiated for 31 h in the absence of BISPHEN; lane 3, 10 h irradiation with 1000 gM BISPHEN; lane 4, 15 h dark of pRNA/BISPHEN; lane 5, 5 h irradiation; lane 6, 15 h irradiation; lane 7, 25 h irradiation; lane 8, 31 h irradiation. All irradiations were in air at 0 °C with 400 IJM BISPHEN except for lane 2 and 3 as noted above. Panel B: lane 1, pRNA irradiated in absence of PHENPHI; lane 2-6, pRNA irradiated in presence of 15, 30, 60, 120 and 250 ~tM PHENPHI, respectively. All irradiations were under air at 0 °C for 16 h; pRNA degradation is evident in the last lane.

7". Mohammad et al. /Bioorg. & Med. Chem. Lett. 9 (1999) 1703-1708 in lanes 6-8. At higher concentrations of BISPHEN (ca.1.0 mM) there was also some degradation of the nucleic acid as is evident from the reduction in the density of the intact pRNA band (lane 3). The degradation was found to be a function o f the absorbed UV dose. 19It should be noted that the band labeled as "uncross-linked" is likely to be a mixture o f unmodified RNA and RNA containing monoadduct(s); these are indistinguishable under the electrophoretic conditions.

pRNA (ca. 1.0 lag) was irradiated under similar conditions for 16 h with increasing amounts of PHENPHI (0-250 laM). The resultant gel is also presented in Figure 1 (panel B). One major cross-linked band was produced with slower mobility than the uncross-linked pRNA ((lanes 2-5). This band was absent when the pRNA was irradiated without PHENPHI (lane 1). Visual examination indicates that 15-120 laM PHENPHI cross-linked ca.

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5-10% o f the pRNA, with 100 ktM giving optimal results. In a separate experiment, the mobility of the crosslinked band was found to be comparable to that observed for the crosszlinked pRNA produced by irradiation of the RNA with 4'-aminomethyl-4, 5',8-trimethylpsoralen2° (data not shown). As was the case with BISPHEN (vide supra), degradation of the pRNA was observed at concentrations 120 ~M (lane 6).

To identify the cross-linking sites, the cross-linked and uncross-linked bands were separately excised, and extracted overnight with 100 ~L of elution buffer at 37 °C. The extracted pRNAs were precipitated with ethanol and the pellets dissolved in nuclease-free water (2.4 ~L). The isolated RNA samples served as templates for primer extension. The cross-linked pRNA was annealed with 5'[y-32p]-labeled P11 or P71 primers at 75 °C for 10 min, followed by cooling to ca. 30 °C for 20 min. These two primers target the 3' end and the middle region of the pRNA sequence, respectively. Avian myeloblastosis virus (AMV) reverse transcriptase was used for chain elongation, and the eDNA was run on an 8% polyacrylamide/8 M urea sequencing gel. DNA synthesis on the photocross-linked pRNA template generated a population of DNA fragments (cf. lane 3 and 5 in Figure 2), suggesting that duplex synthesis had been terminated. To aid in the identification of the termination sites, the DNA fragments containing a U ladder were generated (lane 7) by reverse transcription in the presence ofddATP, 2I and were electrophoresed in parallel with the samples in lanes 5 and 6. Primer extension on the untreated pRNA (lane 1) served as a background control for non-specific stops. The uncross-linked pRNA in lanes 4 and 6 gave more specific chain termination points than the untreated pRNA (lane 1), with monoadducts presumably accounting for most of the stops. Analysis of the termination sites from the cross-linked pRNA template produced strong stops prior to G28 and G30 (see lane 5 in Figure 2). This is consistent with our earlier observations that the

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metal complexes preferentially target Gs (see above). 22-24 In addition, primer 11, which is complementary to the 3' end of pRNA, produced a strong band corresponding to a stop prior to G75 (lane 3). G75 is well positioned relative to G28 and G30 in the three-helix junction area of the pRNA. From these data we conclude that RNA cross-linking results from the covalent bond formation of PHENPHI to G75 and G28 or G30. However, the data in hand do not allow us to determine the relative contribution of stops at G28 and G30 to the overall cross-linking to G75. It may be added that the possibility for a cross-link between G28 and G30 themselves was excluded because such modification is not expected to result in a significant change in the pRNA mobility.

This site selectivity exhibited by these metal complexes is reminiscent of the covalent adducts cisplatin (cis-diamminedichloroplatinum(II), cis-Pt(NH3)2C12) forms with DNA in the dark, which involve predominantly intrastrand cross-links at N7 of neighboring guanines. These cross-links are believed to be responsible for the antitumor activity of cisplatin, 25 and we have noted above that both BISPHEN and PHENPHI are photocytotoxic.

Further studies of these and related complexes as potential photochemotherapeutic agents, and as photoactive probes for nucleic acid structure and function, are in progress.

Acknowledgement: Partial support of grants (to PG) from NIH (GM48159) and NSF (MCB9723923) is gratefully acknowledged.

References and Notes

1. Presented, in part, at the 26 th Meeting of the American Society for Photobiology, Snowbird, UT, July 11



2. (a) Rajski, S. R.; Williams, R. M. Chem. Rev. 1998, 98, 2723. (b) Antsypovich, S. I.; Oretskaya, T. S. Russ.

Chem. Rev. 1998, 67, 245.

3. (a) Hurley, L. H. J. Med. Chem. 1989, 32, 2027. (b) Shabarova, Z. A. Biochimie 1988, 70, 1323. (c) Kohn, K. W. In Molecular Aspects of Anti-Cancer Drug Action; Neidle, S.; Waring, M. J., Eds.; Macmillan, London, 1983, pp. 315-361.

4. Morrison, H., Ed.; Biological Applications of Photochemical Switches; John Wiley & Sons, Inc.; New York, NY, 1993.

5. (a) Parrish, J. A.; Fitzpatrick, T. B.; Tannenbaum, L.; Pathak, M. A. New Engl. J. Med. 1974, 291, 1207.

(b) Bethea, D.; Fullmer, B.; Syed, S.; Seltzer, G.; Tiano, J.; Rischko, C.; Gillespie, L.; Brown, D.;

Gasparro, F. P. J. Dermatol. Sci. 1999, 19, 78. (c) Knol31er, R. M.; Hrnigsmann, H.; Edelson, R. L. In Psoralen DNA Photobiology; Gasparro, F. P., Ed.; CRC Press, Inc., Boca Raton, FL, 1988; Vol. II, pp.


6. (a) Lin, L.; Cook, D. N.; Wiesehahn, G. P.; Alfonso, R.; Behrman, B.; Cimino, G. D.; Corten, L.; Damonte, P. B.; Dikeman, R.; Dupuis, K.; Fang, Y. M.; Hanson, C. V.; Hearst, J. E.; Lin, C. Y.; Londe, H. F.;

Metchette, K.; Nerio, A. T.; Pu, J. T.; Reames, A. A.; Rheinschmidt, M.; Tessman, J.; Isaacs, S. T.;

Wollowitz, S.; Corash, L. Transfusion 1997, 37, 423. (b) Lin, L.; Londe, H.; Hanson, C. V.; Wiesehahn, G.;

Wollowitz, S.; Cimino, G.; Corash, L. Blood 1993, 82, 292. (c) Lin, L.; Wiesehahn, G. P.; Morel, P. A.;

Corash, L. Blood 1989, 74, 517. (d) Wagner, S. J.; White, R.; Wolf, L.; Chapman, J.; Robinette, D.;

Lawlor, T. E.; Dodd, R. Y. Photochem. Photobiol. 1993, 57, 819. (e) Margolis-Nunno, H.; Robinson, R.;

Horowitz, B.; Geacintov, N. E.; Ben-Hur, E. Photochem. Photobiol. 1995, 62, 917.

7. (a) Takasugi, M.; Guendouz, A.; Chassignol, M.; Decout, J. L.; Lhomme, J.; Thuong, N. T.; Hrlrne, C.

Proc. Natl., Acad. Sci. U.S.A. 1991, 88, 5602. (b) Meyer, Jr., R. B.; Tabone, J. C.; Hurst, G. D.; Smith, T.

M.; Gamper, H. J. Am. Chem. Soc. 1989, 111, 8517. (c) Song, P. S.; Tapley, Jr., K. J. Photochem.

Photobiol. 1979, 29, 1177. (d) Shi, Y.; Hearst, J. E. Biochemistry 1987, 26, 3792. (e) Thompson, J. F.;

Hearst, J. E. Cell 1983, 32, 1355. (f) Cimino, G. D.; Gamper, H. B.; Isaacs, S. T.; Hearst, J. E. Ann. Rev.

Biochem. 1985, 54, 1151. (g) Van Houten, B.; Gamper, H.; Hearst, J. E.; Sancar, A. J. Biol. Chem. 1986, 1708 T. Mohammad et al. / Bioorg. & Med. Chem. Lett. 9 (1999) 1703-1708 261, 14135. (h) Lee, M.; Roldan, M. C.; Haskell, M. K.; McAdam, S. R.; Hartley, J. A. d. Med. Chem.

1994, 37, 1208. (i) Chen, C.; Guo, P.J. Virology 1997, 71,495.

8. For a recent example of the use of oligonucleotides conjugated to photoactivatable groups to form crosslinks to a G on a complementary strand, see: Nakatani, K.; Shirai, J.; Sando, S.; Saito, I. J. Am Chem. Soc.

1997, 119, 7626. While our manuscript was under review, a report has appeared describing DNA crosslinking by a light-activatable progenitor of dehydromonocrotaline which then reacts with the nucleic acid in the dark. This pyrrolizidine alkaloid also targets GC-rich regions of duplex DNA preceding (photo)chemical activation, see: Tepe, J. J.; Williams, R. M. J. Am Chem. Soc. 1999, 121, 2951.

9. Gidney, P. M.; Gillard, R. D.; Heaton, B. T. J. Chem. Soc., Dalton Trans. 1972, 2621.

10. Terrian, D. L. Ph.D. Dissertation 1996, Purdue University, West Lafayette, IN.

11. (a) Mahnken, R. E.; Bina, M; Deibel, R. M.; Luebke, K.; Morrison, H. Photochem. Photobiol. 1989, 49, 519. (b) Mahnken, R. E; Billadeau, M. A.; Nikonowicz, E. P.; Morrison, H. J. Am. Chem. Soc. 1992, 114, 9253. (c) Harmon, H. L.; Morrison, H. Inorg. Chem. 1995, 34, 4937. (d) Billadeau, M. A.; Wood, K. V.;

Morrison, H. lnorg. Chem. 1994, 33, 5780. (e) Mohammad, T.; Tessman, I.; Morrison, H.; Kennedy, M.

A.; Simmonds, S. W. Photochem. Photobiol. 1994, 59, 189.

12. Interestingly, when oxygen is excluded from the reaction medium, photolysis of BISPHEN and dG leads to metal coordination at the N7 of the guanine via a chain reaction.lie

13. Terrian, D. L.; Wagner, S. W.; Morrison, H. Photochem. Photobiol. 1997, 65, 68S.

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