Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: overview and feasibility: properties of diazirine as a precursor.

Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C-H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra :CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.

4'-Aminomethyl-4,5',8-trimethylpsoralen photochemistry: the effect of concentration and UVA fluence on photoadduct formation in poly(dA-dT) and calf thymus DNA.

The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4- and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10(5) M-1) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320-400 nm radiation) fluence.

Site-specific targeting of psoralen photoadducts with a triple helix-forming oligonucleotide: characterization of psoralen monoadduct and crosslink formation.

A polypurine tract in the supF gene of bacteriophage lambda (base pairs 167-176) was selected as the target for triple helix formation and targeted mutagenesis by an oligopurine (5'-AGGAAGGGGG-3') containing a chemically linked psoralen derivative (4'-hydroxymethyl-4,5',8-trimethylpsoralen) at its 5' terminus (psoAG10). The thymines at base pairs 166 and 167, a 5'ApT site, were targeted for photomodification. Exposure of the triple helical complex to long wavelength ultraviolet radiation led to the covalent binding of psoAG10 to the targeted region in the supF gene and to the induction of site-specific mutations. We report here experiments to characterize the photomodification of the targeted region of the supF gene in the context of triple helix formation. An electrophoretic mobility-shift assay showed that, at low radiation doses, monoadducts at base pair 166 were the major photoadducts. At higher doses the monoadducts were converted to crosslinks between base pairs 166 and 167. HPLC analysis of enzymatically hydrolyzed photoreaction mixtures was used to confirm the electrophoresis results. A strong strand preference for specific photoadduct formation was also detected.

The specific effects of 8-methoxypsoralen photoadducts on cell growth: HPLC analysis of monoadduct and crosslink formation in cells exposed to split-dose treatment.

The effects of 8-methoxypsoralen (8-MOP) monoadducts and crosslinks on growth and viability of mastocytoma cells were investigated. To induce monoadduct formation (4',5'-monoadducts and 3,4-monoadducts), the cells were incubated with 8-MOP (1 microgram ml-1) and exposed to 419 nm radiation, resulting in the formation of more than 96% monoadducts. After washing and resuspension, the cells were exposed to a small dose of long-wavelength UV radiation (UVA, 2 J cm-2) to convert monoadducts into crosslinks. Similar adduct levels were obtained after either 8-MOP plus visible light treatment or 8-MOP plus split-dose protocol. Cells treated with 419 nm light resumed normal growth rates more rapidly than cells which also received the UVA dose. High performance liquid chromatography (HPLC) analysis of DNA obtained from each group of cells showed that the UVA step resulted in an increase in crosslinks from 3.2% after 419 nm radiation to 56.5% after UVA irradiation.

Phototoxicity of lumidoxycycline.

Doxycycline (DOTC) is a photosensitizing drug whose mechanism of phototoxicity is complicated by the large variety of stable photoproducts formed. To assess the role of a DOTC photoproduct, lumidoxycycline (LuDOTC), in the photosensitization mechanism of DOTC, MGH-U1 human bladder carcinoma cells were treated in vitro with either DOTC or LuDOTC, and irradiated with the 351-nm emission of an argon-ion laser. Both DOTC and LuDOTC were phototoxic and caused radiant-exposure-dependent inhibition of cellular incorporation of tritiated thymidine. On an absorbed-photon basis, DOTC was about five times as phototoxic as LuDOTC. Cellular uptake of DOTC was about five times as great as that of LuDOTC. Epifluorescence microscopy showed localization of LuDOTC predominantly within cellular membranes, particularly of mitochondria, as well as a low level of LuDOTC fluorescence diffusely within the cytoplasm. Epifluorescence microscopy of cells labeled with the mitochondrial probe, rhodamine 123, showed mitochondrial fragmentation and altered mitochondrial membrane integrity after LuDOTC photosensitization; these effects depended on radiant exposure and were partially reversible by 24 h after irradiation. For both DOTC and LuDOTC, phototoxicity was increased by irradiation in the presence of deuterium oxide and decreased in the presence of sodium azide, effects consistent with an important mechanistic role for singlet oxygen, O2(1 delta g), in the injury. In solution, LuDOTC and DOTC had similar quantum yields for generation of O2(1 delta g) as measured by time-resolved spectroscopy and by O2(1 delta g) trapping. LuDOTC was photostable in solution, but DOTC underwent significant photodegradation. These data demonstrate that DOTC photo-products such as LuDOTC have significant photobiologic activity and may play an important role in the phototoxicity mechanism of DOTC.

The excitation of 8-methoxypsoralen with visible light: reversed phase HPLC quantitation of monoadducts and cross-links.

The formation of 8-methoxypsoralen-DNA monoadducts and cross-links is presumed to be responsible for the efficacy of photochemotherapies that employ 8-methoxypsoralen activated with long-wavelength ultraviolet radiation (UVA, 320-400 nm). In this report it is shown that 8-methoxypsoralen can also be activated with visible light (419 nm). Bovine aorta smooth muscle cells were treated with 8-methoxypsoralen (1,000 ng/mL) and 419 nm light (up to 12 J/cm2). Cellular DNA was isolated, hydrolyzed using nucleolytic enzymes and then analyzed by reversed-phase high-performance liquid chromatography. The primary effect of using visible light instead of long-wavelength ultraviolet radiation is a more than 10-fold reduction in the extent of cross-link formation. Because the extent of monoadduct and cross-link formation has not been routinely measured in experiments in which cellular assays have been performed, it is difficult to correlate cell response to the presence of a particular type of 8-methoxypsoralen photoadduct (monoadduct or cross-link). Thus, the use of visible light allows the study of cells containing nearly 100% monoadducts. In addition, the reduction in cross-link formation when visible light is used to activate the compound may also reduce the mutagenicity of 8-methoxypsoralen and hence enhance its therapeutic efficacy.

Improved high-performance liquid chromatographic analysis of 8-methoxypsoralen monoadducts and cross-links in polynucleotide, DNA, and cellular systems: analysis of split-dose protocols.

The distribution of 8-methoxypsoralen-thymidine photoadducts from polynucleotides, calf thymus DNA and mammalian cells treated with [3H]8-methoxypsoralen under a variety of irradiation conditions was determined using high-performance liquid chromatography and scintillation analysis. The split-dose protocol, with samples treated with 8-methoxypsoralen and low doses of long-wavelength UV radiation to generate monoadducts, washed to remove unreacted 8-methoxypsoralen, then irradiated further to convert the monoadducts to cross-links, was examined. The photoadduct distribution in the first step is dependent upon the UVA dose and the wavelength of the radiation, but it is relatively independent of 8-methoxypsoralen concentration. Low fluence and longer wavelengths generate mainly 4',5'-monoadducts, whereas higher fluences and shorter wavelengths yield more cross-links. The second irradiation step converts the 4',5'-monoadducts to cross-links as well as to 3,4-monoadducts. The overall yield of cross-links after the second irradiation step is not dependent upon the wavelength used in the first step. Cellular studies demonstrated that the split-dose protocol is applicable to mammalian systems. These results may affect the interpretation of mutagenesis studies based on the split-dose protocol, because the second step can convert 4',5'-monoadducts to both 3,4-monoadducts, the expected cross-links. Therefore, interpretations that link increases in mutagenicity after the second step in a split-dose study solely to cross-link formation may need re-examination.