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The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.

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1H NMR has been used to study the interactions of over 70 clinical and experimental antitumor drugs with DNA. Spectra of the low-field (H-bonded imino proton) resonances of DNA were studied as a function of drug per base pair ratio. From the spectral changes observed, it was possible to distinguish three modes of drug binding (intercalation, groove binding, and nonspecific outside binding), to determine the kinetics of drug binding (approximate lifetime of the bound drug), and, in favorable cases, to determine the specificity of the drugs for A X T or G X C base pairs. This method is a useful assay for general drug-binding characteristics. For the intercalating compounds there appears to be a correlation between drug-binding kinetics and useful antitumor activity.

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)

A variety of experimental tests have been applied to the methylene-blue-sensitized photooxidation of amino acids to distinguish between singlet oxygen and non-singlet oxidation mechanisms. Conventional flash photolysis and laser photolysis were used to measure the rate constants for the quenching of excited triplet sensitizer and singlet oxygen by the amino acids histidine. tryptophan and methionine and the nucleotide guanosine-5'-monophosphate. In the case of histidine, the rate constants alone rule out an oxidation mechanism involving direct reaction with excited dye. With the other amino acids, and with guanosine monophosphate, the oxidation rates might be accounted for by either mechanism. The inhibition of the photooxidation of both tryptophan and methionine as well as histidine by the singlet-oxygen quenchers N3-and tetramethylethylene suggests that these reactions occur via a singlet-oxygen mechanism. A newly developed test of singlet oxygen reactions involving a comparison of photooxidation rates in normal and perdeuterated solvents has been used to establish that the photooxidation of tryptophan proceeds primarily by a singlet-oxygen mechanism. These experiments appear to constitute the first proof that singlet oxygen is involved in the photooxidation of the three amino acids tryptophan, methionine and histidine. I N T R O D U C T I O NINSIGHT into the mechanism of photodynamic action has been seriously hampered by the lack of knowledge concerning the primary events in the dye-sensitized photooxidations of biologically important compounds in the presence of oxygen[ 1,2].Although it has been amply demonstrated that oxygen is involved[ 1,2], the nature of the primary oxidizing species still remains to be identified. In trying to deduce the mechanism of any dye-sensitized photooxidation, there are at least two major pathways to be considered, including: (a) direct reaction between sensitizer triplets and the oxidizable substrate, followed eventually by reaction with oxygen, and (b) reaction between oxidizable substrates and singlet oxygen which has been generated by energy transfer from triplet-state sensitizers.In pathway (a) there are many possible mechanisms whereby oxygen can participate in the processes following the initial reaction between dye triplet, 3 S , and substrate. For example, molecular oxygen may reoxidize semireduced dye intermediates. Alternatively, it may act as an electron acceptor and cause irreversible formation of oxidized products by electron transfer from half-oxidized substrate molecules (X') as follows:3s+x-+ s -+ xx*+o, + x+ + 0,-2 0 , -+ 2 H + + 02+H20,

The single -, triplet absorption spectra of 16 photochemically active aromatic ketones and aldehydes have been investigated by the phosphorescence excitation method. Both n,r and r,r triplet states have been located in a number of molecules. The relation between our experimental findings and a recent theoretical interpretation of intersystem-crossing mechanisms in carbonyl compounds is discussed. The intensity of singlet + triplet transitions in carbonyl compounds is examined theoretically, and it is predicted that the S-T,,, transitions in aromatic compounds should be enhanced by the presence of the > G O substituent. This prediction is experimentally verified with several examples. Within the accuracy of our experiments, there was no external heavy atom effect on the intensity of the %Tu,, transitions, although the addition of ethyl iodide to the solvent did enhance the somewhat weaker S-rT,,, transitions by about a factor of 2. This difference in sensitivity to external heavy atoms provides a useful criterion for distinguishing ST,,, and S+T,,, transitions. The relation of the spectroscopic results to the interpretation of the photochemical properties of these molecules is discussed. nterest in the triplet-state properties of organic (15) S. K. Lower and M. A.

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