Zinc Phthalocyanine Photochemistry by Raman Imaging, Fluorescence Spectroscopy and Femtosecond Spectroscopy in Normal and Cancerous Human Colon Tissues and Single Cells.

Photodynamic therapy is a clinically approved alternative method for cancer treatment in which a combination of nontoxic drugs known as photosensitizers and oxygen is used. Despite intensive investigations and encouraging results, zinc phthalocyanines (ZnPcs) have not yet been approved as photosensitizers for clinical use. Label-free Raman imaging of nonfixed and unstained normal and cancerous colon human tissues and normal human CCD18-Co and cancerous CaCo-2 cell lines, without and after adding ZnPcS4 photosensitizer, was analyzed. The biochemical composition of normal and cancerous colon tissues and colon cells without and after adding ZnPcS4 at the subcellular level was determined. Analyzing the fluorescence/Raman signals of ZnPcS4, we found that in normal human colon tissue samples, in contrast to cancerous ones, there is a lower affinity to ZnPcS4 phthalocyanine. Moreover, a higher concentration in cancerous tissue was concomitant with a blue shift of the maximum peak position specific for the photosensitizer from 691-695 nm to 689 nm. Simultaneously for both types of samples, the signal was observed in the monomer region, confirming the excellent properties of ZnPcS4 for photo therapy (PDT). For colon cell experiments with a lower concentration of ZnPcS4 photosensitizer, c = 1 × 10-6 M, the phthalocyanine was localized in mitochondria/lipid structures; for a higher concentration, c = 9 × 10-6 M, localization inside the nucleus was predominant. Based on time-resolved experiments, we found that ZnPcS4 in the presence of biological interfaces features longer excited-state lifetime photosensitizers compared to the aqueous solution and bare ZnPcS4 film on CaF2 substrate, which is beneficial for application in PDT.

Exploring the ultrafast dynamics of a diarylethene derivative using sub-10 fs laser pulses.

A diarylethene derivative, 1,2-bis(2,4-dimethyl-5-phenyl-3-thienyl)perfluorocyclopentene (DMP), is a photoswitch molecule utilizing a reversible aromatic ring-opening reaction. The quantum yield of the ring-opening reaction is however remarkably low. We investigate the origin of this behaviour by means of ultrafast transient absorption spectroscopy utilizing sub-10 fs pulses, which is an invaluable tool for simultaneously studying both the electronic and the vibrational molecular dynamics. Namely, a noncollinear optical parametric amplifier (NOPA) generating sub-10 fs pulses in the spectral range 605-750 nm is employed. The transient absorption signal is modulated by several vibrational modes, which are compared with experimental and computational Raman spectra and then assigned to the ground or excited electronic state. We observe that the most pronounced vibrational mode - the ethylenic stretching mode at a frequency of 1501 cm-1 - exhibits instantaneous frequency and amplitude modulation. The observed modulations occur due to weak coupling with another 1431 cm-1 stretching mode mediated by a vibrational mode of low frequency, i.e. around 60 cm-1. Fast internal conversion S1 → S0 originates in a relaxation through a conical intersection (found by density-functional theory computations), facilitated by the two aforementioned stretching modes.

Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy.

Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity.

Spectroscopic properties of benzene at the air-ice interface: a combined experimental-computational approach.

A combined experimental and computational approach was used to study the spectroscopic properties of benzene at the ice-air interface at 253 and 77 K in comparison with its spectroscopic behavior in aqueous solutions. Benzene-contaminated ice samples were prepared either by shock-freezing of benzene aqueous solutions or by benzene vapor-deposition on pure ice grains and examined using UV diffuse reflectance and emission spectroscopies. Neither the absorption nor excitation nor emission spectra provided unambiguous evidence of benzene associates on the ice surface even at a higher surface coverage. Only a small increase in the fluorescence intensity in the region above 290 nm found experimentally might be associated with formation of benzene excimers perturbed by the interaction with the ice surface as shown by ADC(2) excited-state calculations. The benzene associates were found by MD simulations and ground-state DFT calculations, although not in the arrangement that corresponds to the excimer structures. Our experimental results clearly demonstrated that the energy of the S0 → S1 electronic transition of benzene is not markedly affected by the phase change or the microenvironment at the ice-air interface and its absorption is limited to the wavelengths below 268 nm. Neither benzene interactions with the water molecules of ice nor the formation of dimers and microcrystals at the air-ice interface thus causes any substantial bathochromic shift in its absorption spectrum. Such a critical evaluation of the photophysical properties of organic contaminants of snow and ice is essential for predictions and modeling of chemical processes occurring in polar regions.

The effect of point mutation on the equilibrium structural fluctuations of ferric Myoglobin.

The ultrafast equilibrium fluctuations of the Fe(III)-NO complex of a single point mutation of Myoglobin (H64Q) have been studied using Fourier Transform 2D-IR spectroscopy. Comparison with data from wild type Myoglobin (wt-Mb) shows the presence of two conformational substates of the mutant haem pocket where only one exists in the wild type form. One of the substates of the mutant exhibits an almost identical NO stretching frequency and spectral diffusion dynamics to wt-Mb while the other is distinctly different in both respects. The remarkably contrasting dynamics are largely attributable to interactions between the NO ligand and a nearby distal side chain which provides a basis for understanding the roles of these side chains in other ferric haem proteins.

Solution-phase photochemistry of a [FeFe]hydrogenase model compound: evidence of photoinduced isomerisation.

The solution-phase photochemistry of the [FeFe] hydrogenase subsite model (µ-S(CH(2))(3)S)Fe(2)(CO)(4)(PMe(3))(2) has been studied using ultrafast time-resolved infrared spectroscopy supported by density functional theory calculations. In three different solvents, n-heptane, methanol, and acetonitrile, relaxation of the tricarbonyl intermediate formed by UV photolysis of a carbonyl ligand leads to geminate recombination with a bias towards a thermodynamically less stable isomeric form, suggesting that facile interconversion of the ligand groups at the Fe center is possible in the unsaturated species. In a polar or hydrogen bonding solvent, this process competes with solvent substitution leading to the formation of stable solvent adduct species. The data provide further insight into the effect of incorporating non-carbonyl ligands on the dynamics and photochemistry of hydrogenase-derived biomimetic compounds.

The role of CN and CO ligands in the vibrational relaxation dynamics of model compounds of the [FeFe]-hydrogenase enzyme.

The vibrational dynamics of (µ-propanedithiolate)Fe(2)(CO)(4)(CN)(2)(2-), a model compound of the active site of the [FeFe]-hydrogenase enzyme, have been examined via ultrafast 2D-IR spectroscopy. The results indicate that the vibrational coupling between the stretching modes of the CO and CN ligands is small and restricted to certain modes but the slow growth of off-diagonal peaks is assigned to population transfer processes occurring between these modes on timescales of 30-40 ps. Analysis of the dynamics in concert with anharmonic density functional theory simulations shows that the presence of CN ligands alters the vibrational relaxation dynamics of the CO modes in comparison to all-carbonyl model systems and suggests that the presence of these ligands in the enzyme may be an important feature in terms of directing the vibrational relaxation mechanism.

Series of M(I)[Co(bpy)3][Mo(CN)8] x nH2O (M(I) = Li (1), K (2), Rb (3), Cs (4); n = 7-8) exhibiting reversible diamagnetic to paramagnetic transition coupled with dehydration-rehydration process.

In this paper we report the synthesis and the structural and magnetic properties of the series of ionic compounds with general formula: M(I)[Co(bpy)(3)][Mo(CN)(8)] x nH(2)O (M(I) = Li, n = 8 (1), M(I) = K, n = 8 (2), M(I) = Rb, n = 8 (3), M(I) = Cs, n = 7.5 (4)). Solids 1-4 are characterized by the optical outer-sphere metal-to-metal charge transfer (MMCT) transition from Mo(IV) center to Co(III) center in the visible region and the Co(III)Mo(IV) <==> Co(II)Mo(V) spin equilibrium strongly dominated by the Co(III)Mo(IV) form. We show a gentle thermal treatment of diamagnetic compounds 1-4 leading to the dehydrated forms 1a-4a, which reveal a significant increase of paramagnetic contribution (from 0.5 to 2% to 30-40%). The rehydration allows to recover the diamagnetic phases 1b-4b of compositions and properties similar to those of 1-4. The irradiation of the dehydrated form 2a within the MMCT band in the Superconducting Quantum Interference Device (SQUID) cavity at T = 10 K causes further increase of the Co(II)Mo(V) contribution giving the metastable phase annealed back to the 2a phase after heating above T = 290 K. The IR, electron paramagnetic resonance (EPR), and X-ray photoelectron spectroscopy (XPS) spectroscopic data along with the magnetic data are interpreted in terms of strong modification of the Co(III)Mo(IV) <==> Co(II)Mo(V) equilibrium occurring in these systems.

Investigating the vibrational dynamics of a 17e(-) metallocarbonyl intermediate using ultrafast two dimensional infrared spectroscopy.

Ultrafast two dimensional infrared (2D-IR) spectroscopic methods have been used to study the vibrational dynamics of a transient 17e(-) metallocarbonyl species formed following photolysis of the n-propyl-cyclopentadienyl tungsten tricarbonyl dimer [(nPr-Cp)W(CO)(3)](2) in solution. A combination of ground-state 2D-IR and transient 2D-IR techniques has revealed interesting differences in the vibrational relaxation processes of the parent dimer and the monomeric photoproduct that are ascribed to changes in solvent interactions arising from the unsaturated coordination sphere of the intermediate. Additionally, the infrared spectrum of the lesser-populated gauche isomeric form of the dimer in the carbonyl-stretching region has been determined using the presence of off-diagonal peaks in the 2D-IR spectra.

Encapsulating [FeFe]-hydrogenase model compounds in peptide hydrogels dramatically modifies stability and photochemistry.

A [FeFe]-hydrogenase model compound (µ-S(CH(2))(3)S)Fe(2)(CO)(4)(PMe(3))(2) [1] has been encapsulated in a low molecular weight (LMW) hydrogelator (Fmoc-Leu-Leu). Linear infrared absorption spectroscopy, gel melting and ultrafast time-resolved infrared spectroscopy experiments reveal significant contrasts in chemical environment and photochemistry between the encapsulated molecules and solution phase systems. Specifically, the gel provides a more rigid hydrogen bonding environment, which restricts isomerisation following photolysis while imparting significant increases in stability relative to a similarly aqueous solution. Since understanding and ultimately controlling the mechanistic role of ligands near Fe centres is likely to be crucial in exploiting artificial hydrogenases, these gels may offer a new option for future materials design involving catalysts.