Henry C. Brenner

THE TRIPLET STATE AS A PROBE OF DYNAMICS AND STRUCTURE IN BIOLOGICAL MACROMOLECULES

Because of their long lifetimes, triplet excited states offer important advantages over singlet excited state probes. In this review, we discuss the characteristics of triplet excited states, with special reference to tryptophan in proteins, and aromatic molecules which have been employed as probes in studying the dynamics and other properties of proteins and DNA

Optically detected magnetic resonance study of benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide covalently bound to DNA.

Recent investigations of the potent carcinogenic activity of benzo [a] pyrene have indicated that the major metabolite of this molecule responsible for in vivo binding to nucleic acids is a specific 7,8-dihydrodiol9,10-oxide derivative [l-7] denoted by BPDE and shown in fig.1. The in vitro adduct, prepared by the reaction of BPDE and DNA [8], exhibits an extent of binding -loo-times greater than the in vivo product, making it more convenient for physico-chemical studies of the binding process. Evidence was recently obtained by fluorescence quenching [8,9] and electric field induced linear dichroism techniques [lo] that in this in vitro adduct, the BPDE moiety, and in particular the pyrene-like chromophore, is not intercalated between the nucleic acid base pairs, but is instead located in a region external to the DNA helix. Other workers have asserted, however, that the pyrene-like chromophore is intercalated, based on the observations that BPDE

Energy exchange in a coherently coupled ensemble

Abstract The effect of energy transfer between translationally inequivalent molecular sites on spin coherence in photoexcited triplet states is investigated using density matrix techniques. Starting with the theoretical description of triplet—triplet transfer given by Dexter, in which the spatial alignment of the donor spin is conserved in the transfer, it is found that the triplet density matrix produced at the acceptor site depends not only on the angles relating the spin axes at the two sites, but also on the point within the Larmor period of oscillation at which the transfer occurs. This is interpreted in terms of the relationships between the off-diagonal elements of the density matrix and the components of triplet magnetization along molecular spin axes. When it is assumed that the spins transfer randomly with respect to the Larmor oscillation, it is found that (a) the populations of the donor sublevels are transferred into the acceptor sublevels with relative probabilities given by the squared cosines of the angles relating the spin axes at donor and acceptor sites (in agreement with previous studies), and (b) the coherent terms are multiplied by a geometrical factor F which depends explicitly on the Euler angles relating the two sites. Unless the sites are equivalent, F is less than one, so that even when exchange occurs between molecules with identical zero-field splittings, the spin coherence decays. The contribution of inequivalent site exchange to coherence decay in a variety of experimental techniques, e.g. spin echoes and spin locking, is evaluated.

Fluorescence spectroscopy of benzo[a]pyrene diol epoxide-DNA adducts. Conformation-specific emission spectra.

The fluorescence characteristics of adducts derived from the covalent binding of the highly tumorigenic (+) and the non‐tumorigenic (‐) enantiomers of trans‐7,8‐dihydroxy‐anti‐9,10‐epoxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene (BPDE) to native calf thymus DNA are significantly different from one another both at room temperature and at 77 K. The ratio R of fluorescence intensities of the (0,0) band I (situated near 380 nm) and vibronic band V (near 400 nm) of the pyrene ring system in the BPDE‐DNA adducts and of the tetraol (BPT) hydrolysis product of BPDE is very sensitive to the polarity of the solvent, thus mimicking the well known behavior of pyrene itself (A. Nakajima, 1971, Bull. Chem. Soc. Jpn. 44, 3272). The fluorescence excitation and emission spectra of the(+)‐BPDE‐DNA adducts are relatively sharp and only slightly red‐shifted (2–3 nm) with respect to those of BPT in aqueous buffer solution, and R= 1.07 when the fluorescence is excited at the maximum of the absorption spectrum; this compares with R= 1.17 for BPT in water, R= 0.75 in ether, and R= 0.84 for noncovalently intercalated BPT. These results suggest that the pyrene ring system in the covalent (+)‐BPDE‐DNA adducts is located in an environment which is relatively exposed to the aqueous environment, while physically intercalated BPT molecules are located at hydrophobic binding sites. The fluorescence characteristics of the (‐)‐BPDE‐DNA adducts are more heterogeneous and thus more complex than those of the (+)‐adducts. The R ratio depends rather strongly on the wavelength of excitation; a minor, more highly fluorescent and relatively solvent‐accessible form of adducts exhibits an R ratio of 1.01. The major, less solvent accessible form is characterized by a larger red shift in the absorption spectrum (10 nm) and emission spectrum (6 nm for the (0,0) band) relative to BPT, and an R ratio of 1.07. These characteristics suggest that the local environments of the pyrenyl residues in the (‐)‐BPDE‐DNA adducts are significantly different from those of BPT bound noncovalently to DNA by the intercalation mechanism. Fluorescence methods, particularly at low temperatures where the bands are better resolved and the fluorescence yields are significantly greater than at room temperature, can also be used to distinguish covalent DNA adducts derived from the binding of (+)‐BPDE and (‐)‐BPDE to native double‐stranded DNA.

Spin relaxation in photo-excited triplet states via vibronic excitation: rotation of spin axes

Abstract We present a model for the effect of promotion to an excited vibronic level on triplet spin coherence and spin-lattice relaxation. A general case is considered in which the zero field splittings and the fine structure axes orientations differ in the vibrationless triplet and the vibronic level. The expressions obtained are found to be functions of both the zero field splittings of the states involved and of the angles relating the spin axes. We compare the results to those of recent experimental investigations.

Phosphorescence and ODMR studies of isotopically mixed monoclinic and triclinic 1,4-dichlorobenzene

Abstract Phosphorescence and optically detected magnetic resonance (ODMR) studies were performed on the perprotonated triplet traps in isotopically mixed ( h 4 in d 4 ) 1,4-dichlorobenzene at 1.75 K. We find evidence for two distinct crystalline phases of the d 4 host crystal which are stable in temperature ranges near room temperature, each of which induces a distinct set of ODMR parameters on the trap species being monitored. Both cw and pulsed ODMR are used to probe for evidence of trap-to-trap triplet energy transfer at several trap concentrations. We find that at low concentrations ODMR signals are strong, and the excitations are definitely localized. At concentrations above 9%, both coherent and cw ODMR signals become hard to detect. The results are discussed in terms of likely mechanisms for energy exchange among the triplet traps.

Spin coherence studies of a quasi-one-dimensional crystal: Trap-to-trap energy migration and stimulated echo decay

Abstract Optically detected spin coherence experiments including spin locking, two-pulse Hahn echoes and three-pulse stimulated echoes were performed on h 2 traps in isotopically mixed 1,2,4,5-tetrachlorobenzene. Values for the rate constants for trap-to-trap triplet energy migration are obtained for crystals of several different trap concentrations, and the relative importance of various energy transfer pathways is estimated. The results suggest direct superexchange as an important mechanism for triplet energy migration in these crystals. We also find that some out-of-chain energy transfer must occur to account for our results.

ODMR in ethylene glycol-aqueous buffer mixtures: Enhancement of triplet spin lattice relaxation at high aqueous concentrations

Abstract Phosphorescence and optical detection of magnetic resonance (ODMR) intensities of the pyrene triplet chromophore in benzo(a)pyrene tetraol are studied as a function of the fraction of aqueous buffer present in the solvent. It is found that the ODMR signal intensity is abruptly attenuated to zero between 95% and 100% buffer, even though appreciable phosphorescence can still be detected. This loss of signal is found to be due to the onset of rapid spin lattice relaxation, induced most probably by a change in the phonon structure of the solvent medium and its coupling with the triplet spin system at high buffer concentrations.

ODMR and fluorescence studies of pyrene solubilized in anionic and cationic micelles

Abstract The luminescence of pyrene solubilized in anionic sodium n-alkyl sulfate (NaC n S, n =10, 12) and cationic trimethyl-n-alkyl-ammonium bromide (C n TABr, n =10, 12, 14, 16) micelles was studied using zero field optically detected magnetic resonance (ODMR) at cryogenic temperatures and fluorescence quenching techniques at room temperature. The values of triplet state phosphorescence wavelengths and zero-field splitting parameters D in the micelles are consistent with a pyrene microenvironment which is intermediate in polarity and polarizability between an alkyl chain-like environment and an aqueous solvent. The triplet state properties vary reproducibly with surfactant chain length, indicating slight differences in the pyrene environment in the different micelles, but there are no monotonic trends. Pyrene phosphorescence and fluorescence lifetimes in the C n TABr micelles are shortened by the external heavy atom effect of the bromide counterions; the C 10 TABr micelle shows the shortest lifetimes, indicating enhanced counterion proximity to pyrene. Quenching of pyrene fluorescence by added acrylamide was measured for the C n TABr micelles by both steady state and fluorescence decay methods. The apparent dynamic quenching constant in the micelles is nearly three orders of magnitude smaller than its value in aqueous solution, and shows a small but monotonic decrease with increasing surfactant chain length.

Methods for optical detection of electron spin coherence in nonphosphorescent triplet states

A method is described whereby the time development of the coherent components within the spin sublevels of an excited triplet state can be monitored, using an observable which can measure only the total population of the sublevel manifold, such as absorbance to higher triplet states or singlet fluorescence. The approach is very similar to the ’’probe’’ pulse method recently developed for the optical detection of electron spin coherence in phosphorescent triplet states, but the procedure for extracting the value of the coherent component is different. In the present approach it is shown that the recovery of the total population of the sublevel manifold to steady state, after the pulse sequence has been applied, is sensitive to the coherence. By measuring the amplitude of the recovery curve as a function of pulse timing the lifetime of the coherent component can be obtained. Application of the method to some common coherence experiments is discussed.