Jacob W. Petrich

Excited-State Intramolecular Hydrogen Atom Transfer and Solvation Dynamics of the Medicinal Pigment Curcumin

The potential use of the naturally occurring yellow-orange pigment curcumin as a photodynamic therapy agent is one of the most exciting applications of this medicinal compound. Although subnanosecond spectroscopy has been used to investigate the photophysical processes of curcumin, the time resolution is insufficient to detect important and faster photoinduced processes, including solvation and excited-state intramolecular hydrogen atom transfer (ESIHT). In this study, the excited-state photophysics of curcumin is studied by means of ultrafast fluorescence upconversion spectroscopy. The results show two decay components in the excited-state kinetics with time scales of 12-20 ps and approximately 100 ps in methanol and ethylene glycol. The resulting prominent isotope effect in the long component upon deuteration indicates that curcumin undergoes ESIHT in these solvents. The short component (12-20 ps) is insensitive to deuteration, and multiwavelength fluorescence upconversion results show that this decay component is due to solvation of excited-state curcumin.

Excited-State Intramolecular Hydrogen Atom Transfer of Curcumin in Surfactant Micelles

Femtosecond fluorescence upconversion experiments were performed on the naturally occurring medicinal pigment, curcumin, in anionic, cationic, and neutral micelles. In our studies, the micelles are composed of sodium dodecyl sulfate (SDS), dodecyl trimethyl ammonium bromide (DTAB), and triton X-100 (TX-100). We demonstrate that the excited-state kinetics of curcumin in micelles have a fast (3-8 ps) and slow (50-80 ps) component. While deuteration of curcumin has a negligible effect on the fast component, the slow component exhibits a pronounced isotope effect of approximately 1.6, indicating that micelle-captured curcumin undergoes excited-state intramolecular hydrogen atom transfer. Studies of solvation dynamics of curcumin in a 10 ps time window reveal a fast component (< or = 300 fs) followed by a 8, 6, and 3 ps component in the solvation correlation function for the TX-100, DTAB, and SDS micelles, respectively.

Excited-state intramolecular H-atom transfer in nearly symmetrical perylene quinones: Hypericin, hypocrellin, and their analogues

H-atom transfer and proton transfer reactions, like electron transfer reactions, are of fundamental importance in both the physical and biological sciences. Hatom transfer and proton transfer reactions lie at the heart of acid-base chemistry and of a wide range of catalytic reactions in biological systems. Although much progress has been made in understanding electron transfer reactions through the combination of experimental and theoretical work, many aspects of excited-state H-atom and proton transfer reactions are poorly understood, in particular, the way in which the solvent or the intramolecular modes of the solute couple to the reaction. We argue that hypericin and the hypocrellins undergo excited-state intramolecular H-atom transfer reactions. The hypericin and hypocrellin reactions are, relatively speaking, very slow, occurring in about 10 ps for hypericin and from 10-250 ps for hypocrellin A and may be explained in terms of a reaction coordinate that is dominated by intramolecular motions of the aromatic skeleton and the side chains. The observation of a 10 ps transient in hypocrellin A (which, like its analogue in hypericin, lacks a deuterium isotope effect) is essential in attaining a unified understanding of the hypericin and hypocrellin photophysics. Without this common 10 ps component, the photophysics of these two systems bear no similarities and are seemingly unrelated. Our assignment of intramolecular H-atom transfer to the excited-state kinetics has at times been controversial, owing largely to the mirror image symmetry between the absorption and emission spectra and to the absence of deuterium isotope effects. These topics are discussed in detail and we conclude that neither the absence of mirror image symmetry nor the presence of an isotope effect is a conditio sine qua non for a H-atom transfer reaction.

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F‐helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F‐helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen‐bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function. Proteins 2002;46:268–277.

Effect of pH on the Fluorescence and Absorption Spectra of Hypericin in Reverse Micelles

Abstract The well-characterized, monodispersed nature of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane and their usefulness in approximating a membrane-like environment have been exploited to investigate the effect of pH and water pool size on the photophysical properties of hypericin (Hyp). Our measurements reveal two titratable groups of pKa ∼1.5 and ∼12.5. These are assigned to the HypH+/Hyp equilibrium (the deprotonation of a carbonyl group) and the Hyp−/Hyp2− equilibrium (the deprotonation of a peri hydroxyl group). The low-energy absorbance maxima of HypH+, of Hyp and Hyp− and of Hyp2− are 583, 594 and 613 nm, respectively. Neither at pH 13 nor at 1 M HCl is the system entirely in the Hyp2− or the HypH+ forms. Ours is the first study of Hyp in reverse micelles as well as the first time-resolved study of Hyp as a function of pH.

Considerations for the Construction of the Solvation Correlation Function and Implications for the Interpretation of Dielectric Relaxation in Proteins

The dielectric response of proteins is conveniently measured by monitoring the time-dependent Stokes shift of an associated chromophore. The interpretation of these experiments depends critically upon the construction of the solvation correlation function, C(t), which describes the time-dependence of the Stokes shift and hence the dielectric response of the medium to a change in charge distribution. We provide an analysis of various methods of constructing this function and review selected examples from the literature. The naturally occurring amino acid, tryptophan, has been frequently used as a probe of solvation dynamics in proteins. Its nonexponential fluorescence decay has stimulated the generation of an alternative method of constructing C(t). In order to evaluate this method, we have studied a system mimicking tryptophan. The system is comprised of two coumarins (C153 and C152) having different fluorescence lifetimes but similar solvation times. The coumarins are combined in different proportions in methanol to make binary probe mixtures. We use fluorescence upconversion spectroscopy to obtain wavelength-resolved kinetics of the individual coumarins in methanol as well as the binary mixtures of 75:25, 50:50, and 25:75 of C153:C152. The solvation correlation functions are constructed for these systems using different methods and are compared.

Supercontinuum Stimulated Emission Depletion Fluorescence Lifetime Imaging

Supercontinuum (SC) stimulated emission depletion (STED) fluorescence lifetime imaging is demonstrated by using time-correlated single-photon counting (TCSPC) detection. The spatial resolution of the developed STED instrument was measured by imaging monodispersed 40-nm fluorescent beads and then determining their fwhm, and was 36 ± 9 and 40 ± 10 nm in the X and Y coordinates, respectively. The same beads measured by confocal microscopy were 450 ± 50 and 430 ± 30 nm, which is larger than the diffraction limit of light due to underfilling the microscope objective. Underfilling the objective and time gating the signal were necessary to achieve the stated STED spatial resolution. The same fluorescence lifetime (2.0 ± 0.1 ns) was measured for the fluorescent beads by using confocal or STED lifetime imaging. The instrument has been applied to study Alexa Fluor 594-phalloidin labeled F-actin-rich projections with dimensions smaller than the diffraction limit of light in cultured cells. Fluorescence lifetimes of the actin-rich projections range from 2.2 to 2.9 ns as measured by STED lifetime imaging.

Accumulation and Interaction of Hypericin in Low‐density Lipoprotein— A Photophysical Study

The accumulation and interaction of hypericin with the biologically important macromolecule, low‐density lipoprotein (LDL), is investigated using various steady‐state and time‐resolved fluorescence measurements. It is concluded that multiple hypericins can penetrate considerably deeply into the LDL molecule. Up to ∼20 nonaggregated hypericin molecules can enter LDL; but upon increasing the hypericin concentration, the fluorescence lifetime of hypericin decreases drastically, suggesting most likely the self‐quenching of aggregated hypericin. There is also evidence of energy transfer from tryptophans of the constituent protein, apoB‐100, to hypericin in LDL. The results demonstrate the ability of LDL to solubilize hypericin (a known photosensitizer) in nonaggregated form, which has implications for the construction of drug delivery systems.

Photophysics and Multifunctionality of Hypericin‐Like Pigments in Heterotrich Ciliates: A Phylogenetic Perspective

In this paper, we review the literature and present some new data to examine the occurrence and photophysics of the diverse hypericin‐like chromophores in heterotrichs, the photoresponses of the cells, the various roles of the pigments and the taxa that might be studied to advance our understanding of these pigments. Hypericin‐like chromophores are known chemically and spectrally so far only from the stentorids and Fabrea, the latter now seen to be sister to stentorids in the phylogenetic tree. For three hypericin‐like pigments, the structures are known but these probably do not account for all the colors seen in stentorids. At least eight physiological groups of Stentor exist depending on pigment color and presence/absence of zoochlorellae, and some species can be bleached, leading to many opportunities for comparison of pigment chemistry and cell behavior. Several different responses to light are exhibited among heterotrichs, sometimes by the same cell; in particular, cells with algal symbionts are photophilic in contrast to the well‐studied sciaphilous (shade‐loving) species. Hypericin‐like pigments are involved in some well‐known photophobic reactions but other pigments (rhodopsin and flavins) are also involved in photoresponses in heterotrichs and other protists. The best characterized role of hypericin‐like pigments in heterotrichs is in photoresponses and they have at least twice evolved a role as photoreceptors. However, hypericin and hypericin‐like pigments in diverse organisms more commonly serve as predator defense and the pigments are multifunctional in heterotrichs. A direct role for the pigments in UV protection is possible but evidence is equivocal. New observations are presented on a folliculinid from deep water, including physical characterization of its hypericin‐like pigment and its phylogenetic position based on SSU rRNA sequences. The photophysics of hypericin and hypericin‐like pigments is reviewed. Particular attention is given to how their excited‐state properties are modified by the environment. Dramatic changes in excited‐state behavior are observed as hypericin is moved from the homogeneous environment of organic solvents to the much more structured surroundings provided by the complexes it forms with proteins. Among these complexes, it is useful to consider the differences between environments where hypericin is not found naturally and those where it is, notably, for example, in heterotrichs. It is clear that interaction with a protein modifies the photophysics of hypericin and understanding the molecular basis of this interaction is one of the outstanding problems in elucidating the function of hypericin and hypericin‐like chromophores.

Influence of Chiral Ionic Liquids on Stereoselective Fluorescence Quenching by Photoinduced Electron Transfer in a Naproxen Dyad

In a previous study of a naproxen dyad in a pair of N-methylimidazoliummethyl menthylether-NTf(2) chiral ionic liquids (J. Phys. Chem. B 2008, 112, 7555), we observed that though intramolecular electron transfer was impeded, a consistent small stereodifferentiation in the fluorescence lifetime of the dyad was obtained. We proposed that this discrimination was purely electronic in nature and did not arise from geometrical effects, which can influence nonradiative rate processes, such as intramolecular electron transfer. In our present work, we have studied the interaction of the same chiral naproxen dyad molecule in both the previously studied menthyl-based NTf(2) ionic liquids and also in bis(tertrabutylphosphonium) (TBP) d-,l-tartrate ionic liquids. Unlike in the menthyl-based IL pair, the amount of quenching is different in the bis(TBP) tartrate enantiomeric liquids and the tartrate enantiomers have a different temperature dependence on the nonradiative rate of the dyad. This chiral discrimination most likely arises from the steric effects of the different conformations of the chiral molecules. We have shown that the viscosity and polarity of the solvents can influence the rate of electron transfer. On the other hand, no such electron transfer quenching is observed in the menthyl-based NTf(2) IL solvents. To our knowledge, this is the first example of chiral ionic liquids inducing a stereoselective fluorescence quenching by photoinduced, intramolecular electron transfer.