Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues.

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.

Influence of local microenvironment on the double hydrogen transfer in porphycene.

We performed time-resolved transient absorption and fluorescence anisotropy measurements in order to study tautomerization of porphycene in rigid polymer matrices at cryogenic temperatures. Studies were carried out in poly(methyl methacrylate) (PMMA), poly(vinyl butyral) (PVB), and poly(vinyl alcohol) (PVA). The results prove that in all studied media hydrogen tunnelling plays a significant role in the double hydrogen transfer which becomes very sensitive to properties of the environment below approx. 150 K. We also demonstrate that there exist two populations of porphycene molecules in rigid media: "hydrogen-transferring" molecules, in which tautomerization occurs on time scales below 1 ns and "frozen" molecules in which double hydrogen transfer is too slow to be monitored with nanosecond techniques. The number of "frozen" molecules increases when the sample is cooled. We explain this effect by interactions of guest molecules with a rigid host matrix which disturbs symmetry of porphycene and hinders tunnelling. Temperature dependence of the number of hydrogen-transferring molecules suggests that the factor which restores the symmetry of the double-minimum potential well in porphycene are intermolecular vibrations localized in separated regions of the amorphous polymer.

Interfacial Generation of a Carbanion: The Key Step of PTC Reaction Directly Observed by Second Harmonic Generation.

We present the first unambiguous evidence of the interfacial mechanism of phase-transfer catalysis (PTC) by direct observation of the formation of carbanions in the interfacial region between the aqueous and the organic phase by using a surface-sensitive spectroscopic method known as second harmonic generation (SHG). Ion exchange of carbanions adsorbed at the surface after addition of lipophilic tetraalkylammonium salts (TAA) to organic phase and transport of the lipophilic ion-pairs to the organic phase is observed. Results allow for the formulation of a more detailed mechanism of PTC.

Deciphering Photoluminescence in an Aryl Iodides-Gold Nanoparticles System: Au-Mediated Homocoupling Reaction at a Low Temperature.

The study of photoactive materials often unveils intriguing findings, showcasing the value of an interdisciplinary approach. We examined the purported metal-enhanced luminescence thought to result from the chemisorption of aryl iodides on poly(N-vinylpyrrolidone)-stabilized gold nanoparticles. Our discovery deviates from previous assumptions: the fluorescence observed does not originate from excimers of iodophenols chemisorbed on Au:PVP. Instead, it arises from biphenol products, resulting from a gold-mediated Ullmann homocoupling reaction that occurs within the system. Notably, this reaction, known for its demanding nature, proceeds in methanol under purely ambient conditions: room temperature and air atmosphere, without the need for a base. Therefore, these findings not only offer a complete understanding of the observed luminescence but also provide a substantial contribution to the field of carbon-carbon coupling reactions.

Excited-state dynamics of charged dyes at alkane/water interfaces in the presence of salts and ionic surfactants.

The excited-state dynamics of the cationic dye malachite green (MG) and of the dianionic dye eosin B at the dodecane/water interface has been investigated using femtosecond time-resolved surface second harmonic generation (TR-SSHG). By using different probe wavelengths, the contributions of monomeric and aggregated MG to the signal could be spectroscopically distinguished. The effect of the addition of a small amount of surfactants was found to strongly depend on the relative charges of surfactant and dye. For surfactant/dye pairs with opposite charges, the TR-SSHG signal is dominated by the contribution from aggregates, whereas for pairs with the same charges, the signal intensity becomes vanishingly small. These effects are explained in terms of electrostatic interactions between surfactants and dyes that favor either attraction of the dye toward the interface or its repulsion toward the bulk. As a very similar behavior is observed with MG upon addition of NaSCN, we conclude that, in this case, this effect reflects the affinity of SCN¯ for the interface. On the other hand, the guanidinium cation was found to have a different effect than that of a positively charged surfactant on the SSHG signal of MG, indicating this cation does not accumulate in the interfacial region.

Molecular dynamics simulations of liquid phase interfaces: understanding the structure of the glycerol/water-dodecane system.

Modern spectroscopic techniques such as time-resolved second-harmonic-generation spectroscopy allow molecules to be examined selectively directly at phase interfaces. Two-phase systems formed by glycerol/water and alkane layers have previously been studied by time-resolved second-harmonic-generation spectroscopic measurements. In this molecular dynamics study, a triphenylmethane dye was inserted at the glycerol/water-alkane interface and was used as a probe for local properties such as viscosity. We now show how extensive simulations over a wide range of concentrations can be used to obtain a detailed view of the molecular structure at the glycerol/water-alkane interface. Glycerol is accumulated in a double layer adjacent to the alkane interface, which results in increased viscosity of the glycerol/water phase in the direct vicinity of the interface. We also show that conformational ensembles created by classical molecular-dynamics simulations can serve as input for QM/MM calculations, yielding further information such as transition dipoles, which can be compared with spectroscopic measurements.

Tautomerization in 2,7,12,17-tetraphenylporphycene and 9-amino-2,7,12,17-tetraphenylporphycene: influence of asymmetry on the direction of the transition moment.

Femtosecond transient absorption anisotropy studies have been performed for two porphycenes of different symmetry. In 2,7,12,17-tetraphenylporphycene, the chemical identity of two trans forms implies a change in the S(0)-S(1) transition-moment direction upon tautomerization. Exploiting this phenomenon, the rates of double hydrogen transfer in both the S(0) and S(1) states (1.4×10(12)  s(-1) and 2.7×10(11)  s(-1) , respectively) have been determined by performing time-resolved anisotropy studies. In the asymmetric 9-amino-2,7,12,17-tetraphenylporphycene, tautomerization occurs with a similar rate in the ground state. In the S(1) state, the reaction is hindered in its vibrationally relaxed form, but the excitation spectra suggest that it may occur for an unrelaxed molecule. Unlike all porphycenes that have been studied so far, 9-amino-2,7,12,17-tetraphenylporphycene does not reveal significant changes in anisotropy owing to intramolecular double hydrogen transfer; rather, the transition-moment directions are similar in the two tautomeric forms. Analysis of the molecular orbitals allows for an explanation of the "locking" of the transition moments: it is due to a large splitting of the two HOMO orbitals, which retain the order of their energies in the two tautomers.

Two-photon excited lasing for detection of amyloids in brain tissue.

Two-photon excitation of emissive markers with near-infrared (NIR) light is of a particular interest for imaging in biology and medicine because NIR light is relatively weakly absorbed and scattered by tissues. At the same time the mechanism of two-photon absorption allows excitation of molecules located deep inside a scattering medium. In this work we demonstrate that the two-photon excitation combined with the effect of light amplification in the stimulated emission process provides a sensitive method for detecting amyloids of different forms. We investigate the two-photon excited amplified spontaneous emission (ASE) of a fluorescent dye, coumarin 307, in the brain tissue infiltrated with various amyloid phantoms i.e. oligomers, protofibrils and mature fibrils. All these forms of amyloids can be detected by observation of ASE and determination of thresholds for light amplification. On this basis we suggest that a relatively simple extension of currently used emission-based optical spectroscopy techniques can provide key information on pathogenic amyloid structures in tissue.

Ground and excited state double hydrogen transfer in symmetric and asymmetric potentials: comparison of 2,7,12,17-tetra-n-propylporphycene with 9-acetoxy-2,7,12,17-tetra-n-propylporphycene.

Analysis of time-resolved anisotropy of transient absorption enabled determination of room temperature ground and excited state rate constants for intramolecular double hydrogen transfer in two similar porphycenes, one of them with symmetric and the other, with asymmetric character of a double minimum potential for hydrogen motion. The perturbation preserves a quasi-symmetric minimum in S(0), but the rate decreases approximately two times. In S(1), the perturbed potential becomes strongly asymmetric, and the downhill hydrogen transfer occurs with a rate higher than that observed for a symmetrical compound.

Hydrogen-bond-assisted excited-state deactivation at liquid/water interfaces.

The excited-state dynamics of eosin B (EB) at dodecane/water and decanol/water interfaces has been investigated with polarization-dependent and time-resolved surface second harmonic generation. The results of the polarization-dependent measurements vary substantially with (1) the EB concentration, (2) the age of the sample, and (3) the nature of the organic phase. All of these effects are ascribed to the formation of EB aggregates at the interface. Aggregation also manifests itself in the time-resolved measurements as a substantial shortening of the excited-state lifetime of EB. However, independently of the dye concentration used, the excited-state lifetime of EB at both dodecane/water and decanol/water interfaces is much longer than in bulk water, where the excited-state population undergoes hydrogen-bond-assisted non-radiative deactivation in a few picoseconds. These results indicate that hydrogen bonding between EB and water molecules at liquid/water interfaces is either much less efficient than in bulk water or does not enhance non-radiative deactivation. This strong increase of the excited-state lifetime of EB at liquid/water interfaces opens promising avenues of applying this molecule as a fluorescent interfacial probe.

Ultrafast excited-state dynamics of eosin B: a potential probe of the hydrogen-bonding properties of the environment.

The photophysics of two dyes from the xanthene family, eosin B (EB), and eosin Y (EY) has been investigated in various solvents by femtosecond transient absorption spectroscopy, first, to clarify the huge disparity of the EB fluorescence lifetimes reported in literature, and, second, to understand the mechanism responsible for the ultrafast excited-state deactivation of EB in water. The excited-state lifetime of EB was found to be much shorter in water and in other protic solvents, due to the occurrence of hydrogen-bond assisted nonradiative deactivation. This mechanism is associated with the hydrogen bonds between the solvent molecules and the nitro groups of EB, which become stronger upon optical excitation due to the charge-transfer character of the excited-state. This process is not operative with EY, where the nitro groups are replaced by bromine atoms. Therefore, the excited-state lifetime of EB in solution is directly related to the strength of the solvent as a hydrogen-bond donor, offering the possibility to build a corresponding scale based on the fluorescence quantum yield or lifetime of EB. This scale of hydrogen-bonding strength could be especially useful for studies of liquid interfaces by time-resolved surface second harmonic generation.

Laser Emission of Thioflavin T Uncovers Protein Aggregation in Amyloid Nucleation Phase.

There is currently no definitive test for early detection of neurodegeneration which is linked with protein aggregation. Finding methods capable of detecting intermediate states of protein aggregates, named oligomers, is critical for the early stage diagnosis of over 30 neurodegenerative diseases including Alzheimer's or Parkinson's. Currently, fluorescence-based imaging using Thioflavin T (ThT) dye is the gold standard for detecting protein aggregation. It is used to detect aggregation in vitro and in various tissues, including the cerebrospinal fluid (CSF), whereby the disease-related protein recombinant is seeded with the patient's fluid. The major drawback of ThT is its lack of sensitivity to oligomeric forms of protein aggregates. Here, we overcome this limitation by transferring a ThT-oligomer mixture into solid state thin films and detecting fluorescence of ThT amplified in the process of stimulated emission. By monitoring the amplified spontaneous emission (ASE) we achieved a remarkable recognition sensitivity to prefibrillar oligomeric forms of insulin and lysozyme aggregates in vitro, to Aß42 oligomers in the human protein recombinants seeded with CSF and to Aß42 oligomers doped into brain tissue. Seeding with Alzheimer patient's CSF containing Aß42 and Tau aggregates revealed that only Aß42 oligomers allowed generating ASE. Thus, we demonstrated that, in contrast to the current state-of-the-art, ASE of ThT, a commonly used histological dye, can be used to detect and differentiate amyloid oligomers and evaluate the risk levels of neurodegenerative diseases to potential patients before the clinical symptoms occur.

Unusual, solvent viscosity-controlled tautomerism and photophysics: meso-alkylated porphycenes.

Stationary and time-resolved studies of 9,10,19,20-tetramethylporphycene and 9,10,19,20-tetra-n-propylporphycene in condensed phases reveal the coexistence of trans and cis tautomeric forms. Two cis configurations, cis-1 and cis-2, play a crucial role in understanding the excited-state deactivation and tautomer conversion dynamics. The trans-trans tautomerization, involving intramolecular transfer of two hydrogen atoms, is extremely rapid (k ≥ 10(13) s(-1)), both in the ground and lowest electronically excited states. The cis-1-trans conversion rate, even though the process is thermodynamically more favorable, is much slower and solvent-dependent. This is explained by the coupling of alkyl group rotation with the hydrogen motion. Excited-state deactivation is controlled by solvent viscosity: the S(1) depopulation rate decreases by more than 2 orders of magnitude when the chromophore is transferred from a low-viscosity solution to a polymer film. Such behavior confirms a model for excited state deactivation in porphycene, which postulates that a conical intersection exists along the single hydrogen transfer path leading from the trans to a high energy cis-2 tautomeric form. For this process, the tautomerization coordinate includes not only hydrogen translocation but also large-amplitude twisting of the two protonated pyrrole moieties attached to the opposite sides of the ethylene bridge.

Reaction kinetics of sonochemical oxidation of potassium hexacyanoferrate(II) in aqueous solutions.

We studied sonochemical reactions resulting from ultrasonic treatment of potassium hexacyanoferrate(II) in aqueous solutions using a custom-built apparatus working at 536 kHz. We concluded that primary reactions are completely dominated by oxidation of Fe(II) to Fe(III) and did not find any evidences for degradation of cyanide. At the highest concentration used in the present study (0.1 M) we detected formation of pentacyanoaquaferrate(II) complex, which is most probably formed in reactions between hexacyanoferrate(III) anions and hydrogen atoms or hydrated electrons formed in sonochemical processes. We also determined that hydroxyl radicals formation rate in our system, (8.7 ± 1.5)∙10-8 M∙s-1, is relatively high compared to other reported experiments. We attribute this to focusing of the ultrasonic wave in the sample vessel. Finally, we suggest that oxidation rate of hexacyanoferrate(II) anions can be a convenient benchmark of efficiency of sonochemical reactors.

Fluorinated Porphycenes: Synthesis, Spectroscopy, Photophysics, and Tautomerism.

Six porphycenes have been synthesized, bearing one, two, or three fluorine atoms attached directly to the 18-π-electron system at the meso positions. These novel compounds have been characterized by structural, electrochemical, and spectral techniques, combined with quantum chemical calculations. In three fluoroporphycenes, the unsymmetric substitution pattern leads to the presence of two nonequivalent trans tautomeric forms. They have been identified using electronic absorption, emission, and magnetic circular dichroism spectroscopies. Their relative energies have been estimated for the ground and lowest excited electronic states. Tautomerization potential is quasi-symmetric in S0 , but becomes strongly nonsymmetric in S1 . Femtosecond transient absorption studies allowed determination of tautomerization rates, larger and similar for both directions of the double hydrogen transfer in S0 , lower and disparate in S1 . Fluoroporphycenes emerge as good candidates for detailed studies of mechanisms of double hydrogen transfer, as well as processes responsible for rapid radiationless excited state depopulation.

Two-photon excited lasing of Coumarin 307 for lysozyme amyloid fibrils detection.

Amyloid fibrils are a well-recognized hallmark of neurodegeneration. A common approach to detect amyloid fibrils is staining with organic molecules and monitoring optical properties using fluorescence spectroscopy. However, the structural diversity of amyloids necessitates new sensitive methods and probes that can be reliably used to characterize them. Here, Coumarin 307 is applied for lysozyme fibrils detection by observation of laser action in the process of two-photon excited stimulated emission. It is shown that the lasing threshold and spectrum significantly depend on the adopted structure (α-helix or ß-sheet) of the lysozyme protein, whereas fluorescence spectrum is insensitive to the protein structure. The applications of coherent stimulated emission light that can be emitted deep inside a scattering medium can be particularly promising for imaging and therapeutic purposes in the neurodegeneration field. Two-photon excitation with the near-infrared light, which allows the deepest penetration of tissues, is an important advantage of the method.

Electronic and vibrational relaxation of porphycene in solution.

The relaxation of electronically excited porphycene in acetonitrile solution has been studied by transient absorption spectroscopy supported by global analysis techniques. Three processes following the femtosecond pulse excitation to the S 2 state have been identified: the intramolecular vibrational redistribution on the time scale of tens of femtoseconds, the internal conversion S 2 right arrow-wavy S 1 (750 fs) and thermal equilibration of the molecule by energy exchange with the solvent (16 ps). The recorded transient absorption kinetics exhibit oscillations which have been assigned to the evolution of wavepackets in both S 1 and S 0 states.

In vitro photodynamic activity of lipid vesicles with zinc phthalocyanine derivative against Enterococcus faecalis.

Zinc(II) phthalocyanine bearing eight non-peripheral 2-propoxy substituents was subjected to physicochemical study and, after incorporation in lipid vesicles, assessed as a potential photosensitizer for antibacterial photodynamic therapy. The phthalocyanine derivative obtained in the macrocyclization reaction was characterized by MS and NMR techniques. Moreover, its chemical purity was confirmed by HPLC analysis. X-ray structural analysis revealed that overcrowding of the phthalocyanine derivative leads to a strong out-of-plane distortion of the π-system of the macrocycle core. In the UV-Vis absorption spectra of zinc(II) phthalocyanine two characteristic bands were found: the Soret (300-450 nm) and the Q band (600-800 nm). Photophysical properties of mono- and diprotonated forms of phthalocyanine derivative were studied with time-resolved fluorescence spectroscopy. Its tri- and tetraprotonated forms could not be obtained, because compound decomposes in higher acid concentrations. The presented zinc(II) phthalocyanine showed values of singlet oxygen generation ΦΔ = 0.18 and 0.16, the quantum yield of the photodecomposition ΦP = 3.06∙10-4 and 1.23∙10-5 and the quantum yield of fluorescence ΦFL = 0.005 and 0.004, designated in DMF and DMSO, respectively. For biological studies, phthalocyanine has been incorporated into modified liposome vesicles containing ethanol. In vitro bacteria photoinactivation study revealed no activity against Escherichia coli and 5.7 log reduction of the Enterococcus faecalis growth.

Evidence for Dominant Role of Tunneling in Condensed Phases and at High Temperatures: Double Hydrogen Transfer in Porphycenes.

Investigation of the double hydrogen transfer in porphycene, its 2,7,12,17-tetra-tert-butyl derivative, and their N-deuterated isotopologues revealed the dominant role of tunneling, even at room temperature in condensed phase. Ultrafast optical spectroscopy with polarized light employed in a wide range of temperatures allowed the identification and evaluation of contributions of two tunneling modes: vibrational ground-state tunneling, occurring from the zero vibrational level, and vibrationally activated, via a large amplitude, low-frequency mode. Good correspondence was found between the rates of incoherent tunneling occurring in condensed phase and the values estimated on the basis of tunneling splittings observed in molecules isolated in supersonic jets or helium nanodroplets. The results provide solid experimental insight into widely proposed quantum facets of ubiquitous hydrogen-transfer phenomena.

Tautomerism in porphycenes: analysis of rate-affecting factors.

Double hydrogen transfer occurring in both ground and the lowest electronically excited singlet states was studied for a series of 19 differently substituted porphycenes. The rates of tautomerization have been determined using femtosecond pump-probe spectroscopy with polarized light. The values vary by over 3 orders of magnitude, suggesting the importance of tunneling. Good correlation exists between the values of the rates and the parameters characterizing the strength of two intramolecular hydrogen bonds: proton NMR shift, distance between the hydrogen-bonded nitrogen atoms, and the NH stretching frequency. While hydrogen-bond strength is the main factor determining the rate of double hydrogen transfer, other factors, such as static and dynamic symmetry breaking and the population of low-frequency vibrations also have to be taken into account.