0022-202X/ 8 1/ 770 1-00J9$02.00/ 0 THE JOURNAL OF I NV r, STIGATIVE DEIlMATOLOGY, 77:39-44, IH8 1 Vol. 77, No. I Copy righ t © 198 1 by The Williams & Wilkins Co. Printed ill U.S.A. Psoralen Photochemistry and Nucleic Acid Structure JOHN E. H EARST, PH.D. Departm.ent of Chemistry, University of California, B erkeley, California, U.S.A . Many new psorale n derivatives have b een synthesized nated in the production of many new psoralens, several of which in an effort to enhance their water solubility and their have superior photoreactivity with both DNA and RNA as binding to nucleic acids. Availability of the very soluble compared to TMP and 8-MOP. The 5 new derivatives shown strongly binding compounds has improved our abilities here are 4' adducts of TMP and their structW"es ru-e shown (3 - to follow the optical changes associated with the photo­ 7). T he complete characterization of methoxymethyltrioxsalen chemistry of psoralens with DNA. Changes in both ab­ (MMT, 4) , hydroxymethyltrioxsalen (HMT, 5) and aminome­ sorbance and fluoresce nce are prese nted in this r eview. thyltrioxsalen (AMT, 7) wi th respect to their reactivity with A kinetic model for the photochemistry concludes that nucleic acids, including a theoretical treatment, is described in the detailed kinetics is dominated by the equilibrium a recent paper by Isaacs et al [6]. A related set of soluble 8- constant for intercalation of the psoralen in the DNA, methoxypsoralen derivatives have been synthesized by Isaacs, the quantum yield for photoaddition to DNA once inter­ Chun, a nd Hearst [7] a nd ru'e listed as compounds 8-11. Ad­ calated and the quantum yield for photodestruction of ditional advances in psora len synthetic chemistry have been the drug in water. With these 3 parameter s the kinetics presented by Bender, Hearst, and Rapoport [8] a nd in 3 asso­ of photochemistry is predictable. The values of these ciated patents [9-11]. parameters for numerous derivatives of 8-me thoxypso­ Briefly, th e advantages of the new compounds MMT (4), ralen and 4,5',8 trimethylpsoralen are presented. Appli­ HMT (5), AMT (7), MMX (9), and HMX (10) are higher cation of this photoche mistry to a study of nucleic acid aqueous solubility and, in the case of the aminomethyl com­ secondary structure in chromatin, fd bacteriophage, and pound (7), a positively charged side chain. T hese factors greatly in ribosomes is reviewed. enhance the binding of the compound to nucleic acids and thus allow more dl'U g to be covalently bound to DNA or RNA in the photoaddition reaction for a given dose of drug and light. One At present there exist over 100 psoralen derivatives in the example of the advantage gained here is found in viral inacti­ chemical literature of which approximately half are natW"ally vation, as discussed below. occurring and the remainder synthetically prepared. Of this The current position on the therapeutic potential of these large number, however, only 2 derivatives al'e cUJTently in new derivatives falls mainly into 2 areas which should not be widespread scientific and clinical use in the United States. interpreted as limiting. A method enabling selective control of These 2 compounds ar e known by their trade names as Tri­ nucleic acid replication, hence viral and cellulru' reproduction, oxsalen (4, 5',8-trimethylpsoralen or TMP, 1) and Methoxsalen obviously lends itself to a broad range of application. (8-methoxypsoralen or 8-MOP, 2). ANTIVIRAL VACCINE PRODUCTION Current methods for inactivating viJ"U ses for the purpose of ~3 vaccine production consist primarily of fo rmaldehyde or heat ~ denatW"ation of the intact virus. The disadvantage of such c~o~o "O~~O proceduTes is the concomitant denaturation or ch emical modi­ CH 3 OCH 3 fi cation of vU'al protein, ch anging the antigenic structures to 4,5',B-trimethylpsoralen (TMP) 8- methoxypsoralen (B-MOP) which a ntibodies are formed. With psoralens, which have very weak reactivity with protein, I 2 it has been possible to completely inactivate both DNA and ~ RNA viruses with an efficiency several orders of magnitude My laboratory has become involved in many fundamental greater than the denaturation methods. In the case of the RNA aspects of psoralen chemistry, including probing the secondary vU'us, VesiculaT Stomatitis vU'us (Hearst and Thu'y [12], Tri­ structW"es of Escherichia coli 16 S ribosomal RNA in vitro and oxsalen was found capable of reducing the number of survivors in th e 30 S ribosomal subunit [1-3], of Drosophila melanogas­ to 10% of the original vU'al titer for a given dose; an equivalent ter 5 S ribosomal RNA in vitro [4], and of the circular single­ dose of the new derivative AMT was found to leave a fraction stranded DNA genome of fd bacteriophage virus in the virus of 10- 4 sW"viving plaques after treatment. In both cases the [5]. In the coW"se of our investigations it became clear that required time of uTadiation for inactivation was on the order of modification of the psoralen nucleus in various ways would minutes. Even more effective AMT inactivation has been dem­ enable different types of experiments to be carried out which onstrated by H anson, Riggs, and Lennete [13] using Western are not possible to do with either TMP or 8-MOP. With this in Equine Encephalitis vU·us. mind, an organic synthesis project was initiated which culm i- In theory, a psoralen inactivated vU'us should produce a superior vaccine, as the protein a ntigenic component of the This work was supported in part by Grants GM 11180 and GM 25 151 virus is, in aUprobability, unmodified by the inactivation pro­ fro m the United States Public Health Service, and Grant NP 185 fro m cedure. the America n Ca ncer Society. A final point on vu"a l inactivation deserves comment. Recent Reprint requests to: John E. Hearst, Ph.D., Department of Chemis­ a ttention has been given to the oncogene theory. If this theory try, University of California, Berkeley, Cali fornia 94720. is correct, an inactivated vU'us may contain a n oncogene which Abbreviations: could be excised and reinserted into the host genome, giving AMT: aminomethyltrioxsalen HMT: hydroxmethyltriosalen rise to transformation. With psoralen inactivation, however, it MMT: methoxymethyltrioxsalen is possible to chemically alter lout of every 4 base pau's, cleru'ly 8-MOP: 8-methoxypsoralen eliminating the possibility of the repair or recombination of TMP: 4,5',8-trimethylpsoralen viral information. 39 40 HEARST Vol. 77, No.1 CH 3 H CH3 ' CH3 CH3 " , I' , 1 I" )!. 1 /. 1 /. CH 0 /. 0 0 CH 0 0 CH 0 0 M3 CH lm3 CH lm3 CH 3 3 3 4'-ChloromethYltrioxsolen 4'-Methoxymethyltrioxsolen 4'- Hydroxymethyltrioxsolen 3 4 o CH3 1/ N2:¢"",: ~ 1 t /. o CH 0 0 0 o , 3 CH 3 4' -N - phtha limidomethyltrioxsolen 4' - Ami nomethy Itrioxsolen hydrochloride 1 5 -Chloromethyl- 8 -methoxypsorolen 5- Methoxymethyl-8 -methoxypsorolen 5-Hydrox ymethyl-8-met hoxypsorolen 8 9 10 Pj1 N~ QO¥O~O~ OCH 3 N-(5-Me thylene-8-methoxypsorolen) -pyridinium chloride TUMOR CHEMOTHERAPY oadduct and that only this monoadduct will be fluorescent One possible approach to tumor chemotherapy employing when excited at 365 nm [14-16]. These conclusions have re­ psoralens and light combines oral or locai administration of the cently been verified in our laboratory. drug followed by local irradiation of solid tumor mass via needle Figure 2 shows the excitation (left) and emission spectra guided light pipes. Such an approach is essentially the same in (right) of AMT alone, where the excitation spectrum was re­ principle as the photochemotherapy of psoriasis, but it does not corded at the fluorescence wavelength of 450 nm while the limit itself to an external surface. If found to be workable, the emission spectrum was recorded utilizing exciting radiation at procedure would provide a means. to affect the controlled killing 330 nm [17]. The fluorescence peaks at 457 nm independent of of neoplastic cells without the trauma of surgery or high energy the wavelength of the exciting light between 240 nm and 380 ionizing radiation. nm. There are 3 obvious bands in the excitation spectrum corresponding to the 3 absorption bands seen in Fig 1. These bands lead to fluoresence with an efficiency which is the reverse OPTicAL PROPERTIES OF THE PSORALENS AND of their absorption efficiencies, the shortest wavelength band THEIR REACTION PRODUCTS WITH DNA leading to the least fluorescence. The absorption spectrum of AMT is shown in Fig 1. Typi­ In the presence of poly d(A-T) (Fig 3), there is a 23% cally, the photochemistry is activated at wavelengths between depression of fluorescence intensity of AMT at 457 nm. Under 340 and 380 nm, well away from the absorption bands of nucleic the conditions of this experim'ent, essentially all the AMT was acids alone, which absorb below 300 nm. The absorption spec­ intercalated into the alternating poly d(A-T) before photoreac­ trum of the psoralens is similar to that of the corresponding tion because of its strong binding constant to DNA. coumarins, suggesting that conjugation in the furan ring is As this reaction mixture is irradiated at 365 nm, Fig 3 shows rather weak. In fact, benzofuran does not absorb significantly that the 457 nm fluorescent peak is depressed and a new peak above 320 nm. For this reason one would predict that the furan at 380 nm appears, corresponding to the buildup of furan ring monoadduct to pyrimidine would still absorb between 320 monoadduct.