Thermal and solvatochromic effects on the emission properties of a thienyl-based dansyl derivative.

Environmental conditions have a profound effect on the photophysical behavior of highly conjugated compounds, which can be exploited in a large variety of applications. In this context, we use a combination of experimental and computational methods to investigate thermal and solvatochromic effects on the fluorescence properties of a dansyl derivative bearing a thienyl substituent, namely 2-(3-thienyl)ethyl dansylglycinate (TEDG). In particular, we analyze how the solvent polarity and temperature affect the ground and excited state energies of TEDG by using time-resolved and steady-state fluorescence techniques. We determine the changes in dipole moment of the TEDG molecule upon photoexcitation, as well as the solvent polarity effects on the excited state lifetime. Besides, we provide theoretical modeling of the HOMO-LUMO orbitals and the vertical absorption and emission energies using time-dependent density functional theory (TDDFT) as well as the polarizable continuum model (PCM) to include the solvent contribution to the absorption and emission energies. Our results show that the emission mechanism of TEDG involves locally excited states derived from hybrid molecular orbitals, accompanied by a moderate variation of the molecular dipole moment upon light excitation. Our findings demonstrate that TEDG exhibits desirable fluorescence properties that make it a promising candidate for use as a photoactive material in electrochromic, optical thermometry, and thermography applications.

Interface-driven Sr-morin complexation at Langmuir monolayers for bioactive coating design.

Flavonoid-metal complexes are widely studied because of their interesting luminescent behavior and biological activity. Despite the extensive exploration of flavonoid-metal coordination processes in solution, the formation of complexes using the flavonoid molecule inserted in a lipid membrane has been little investigated. This effect could provide important insight into the biological activity of flavonoids at lipid membranes and could represent an attractive strategy to design supramolecular structures. Here, we studied the complexation between Sr2+ and morin inserted in an octadecylphosphonic acid (OPA) Langmuir monolayer. This is a relevant system due to the synergism imposed by the association of the Sr2+ ability to control bone formation/resorption with the morin antioxidative effect. Morin incorporation into the OPA monolayers and further Sr2+ complexation were monitored by surface pressure isotherms. Electronic absorption spectroscopy and fluorescence techniques showed Sr-morin complexation both in solution and at the air-liquid interface. Although morin complexation has been described to occur only at basic pH, the specific thermodynamic properties at the air-liquid interface drove metal complexation. LB films were deposited on Ti surfaces, and the resulting OPA/Sr-morin coatings exhibited high surface free energy and increase on its polar component. This optimized surface feature supported further serum protein adsorption and osteoblast growth and differentiation, indicating that these lipid-based coatings are promising for bioactive coating design. This study paves the way for the use of this lipid-based coating in the design of implants for faster osteointegration. Moreover, flavonoid-metal complexation at membranes could also help to shed light on the biological role played by flavonoids.

Evolution of electronic and vibronic transitions in metal(II) meso-tetra(4-pyridyl)porphyrins.

The changing of the electronic and vibronic states due to the insertion of Zn(II), Cu(II), Ni(II) or Co(II) ions in the meso-tetrakis(4-pyridyl)porphyrin ring center is investigated. The combination of absorption, photoluminescence, Raman and infrared spectroscopies with second-derivative-based spectral deconvolution analysis reveals that the structuration of both B- and Q-bands is very sensitive to the decorating ion. Similar to free base porphyrins, metal(II) meso-tetra(4-pyridyl)porphyrins also present their Q-band constituted of multiple electronic transitions, where the central ion plays an important role in the selection of vibration modes that mediate the vibronic transitions. Our novel results will expand and reinterpret current assignments for metal(II) meso-tetra(4-pyridyl)porphyrins vibrational modes available in the literature.

Effects of artepillin C on model membranes displaying liquid immiscibility.

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Novel insights on the vibronic transitions in free base meso-tetrapyridyl porphyrin.

We present novel results on the free base 5,10,15,20-meso-tetra(pyridyl)-21H,23H-porphyrin (H2TPyP). This molecule presents complex electronic and vibrational properties and despite the vast literature reporting the transitions observed in its absorption and fluorescence spectra, a more accurate interpretation has been kept elusive. In particular, we show that the molecule's Q-band develops into many electronic and vibronic transitions, whose the well-known "four orbital model" finds it difficult to reconcile. Using distinct spectroscopy techniques, we conclude that both Qx- and Qy-bands comprise, in fact, two quasi-degenerated electronic states together with their respective vibronic progressions each. The analysis of the Huang-Rhys factors and complementary time- and polarization-resolved measurements reinforce the need for the proposed Q-band multi features remodeling.

Effects of artepillin C on model membranes displaying liquid immiscibility

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Optical Characterization of Parathyroid Tissues.

The parathyroid glands are small and often similar to lymph nodes, fat, and thyroid tissue. These glands are difficult to identify during surgery and a biopsy of the parathyroid for identification can lead to damage of the gland. The use of static and time-resolved fluorescence techniques to detect biochemical composition and tissue structure alterations could help to develop a portable, minimally invasive, and nondestructive method to assist medical evaluation of parathyroid tissues. In this study, we investigated 10 human parathyroid samples by absorbance, fluorescence, excitation, and time-resolved fluorescence measurements. Moreover, we compared the results of time-resolved fluorescence measurements with 59 samples of thyroid tissues. The fluorescence lifetimes with emission at 340 nm were 1.09 ± 0.10 and 4.46 ± 0.06 ns for healthy tissue, 1.01 ± 0.25 and 4.39 ± 0.36 ns for benign lesions, and 0.67 ± 0.36 and 3.92 ± 0.72 ns for malignant lesions. The lifetimes for benign and malignant lesions were significantly different, as attested by the analysis of variance with confidence levels higher than 87%. For each class of samples (healthy, benign, and malignant) we perceived statistical differences between the thyroid and parathyroid tissue, independently. After further investigations, fluorescence methods could become a tool to identify normal and pathological parathyroid tissues and distinguish thyroid from parathyroid tissues.

Optical characterization of normal, benign, and malignant thyroid tissue: a pilot study.

Fine-needle aspiration cytology is the standard technique to diagnose thyroid pathologies. However, this method has a high percentage of inconclusive and false-negative results for benign and malignant lesions. Hence, it is important to search for a new method to assist medical evaluation during these surgical procedures. The use of time-resolved fluorescence techniques to detect biochemical composition and tissue structure alterations could help to develop a portable, minimally invasive, and non-destructive method to assist medical evaluation. In this study, we investigated 17 human thyroid samples by absorbance, fluorescence, excitation, and time-resolved fluorescence measurements. This initial investigation has demonstrated that thyroid fluorescence originates from many endogenous fluorophores and culminates in several bands. The fluorescence lifetimes of benign and malignant lesions were significantly different, as attested by analysis of variance using Tukey test with individual confidence level of 98.06%. Our results suggest that fluorescence lifetimes of benign and malignant lesions can potentially assist diagnosis. After further investigations, fluorescence methods could become a tool for the surgeon to identify differences between normal and pathological thyroid tissues.

End-to-end distance distribution in fluorescent derivatives of bradykinin in interaction with lipid vesicles.

Cellular membranes have relevant roles in processes related to proteases like human kallikreins and cathepsins. As enzyme and substrate may interact with cell membranes and associated co-factors, it is important to take into account the behavior of peptide substrates in the lipid environment. In this paper we report an study based on energy transfer in two bradykinin derived peptides labeled with the donor-acceptor pair Abz/Eddnp (ortho-aminobenzoic acid/N-[2,4-dinitrophenyl]-ethylenediamine). Time-resolved fluorescence experiments were performed in phosphate buffer and in the presence of large unilamelar vesicles of phospholipids, and of micelles of sodium dodecyl sulphate (SDS). The decay kinetics were analyzed using the program CONTIN to obtain end-to-end distance distribution functions f(r). Despite of the large difference in the number of residues the end-to-end distance of the longer peptide (9 amino acid residues) is only 20 % larger than the values obtained for the shorter peptide (5 amino acid residues). The proline residue, in position 4 of the bradykinin sequence promotes a turn in the longer peptide chain, shortening its end-to-end distance. The surfactant SDS has a strong disorganizing effect, substantially broadening the distance distributions, while temperature increase has mild effects in the flexibility of the chains, causing small increase in the distribution width. The interaction with phospholipid vesicles stabilizes more compact conformations, decreasing end-to-end distances in the peptides. Anisotropy experiments showed that rotational diffusion was not severely affected by the interaction with the vesicles, suggesting a location for the peptides in the surface region of the bilayer, a result consistent with small effect of lipid phase transition on the peptides conformations.

Investigation of ground- and excited-state photophysical properties of 5,10,15,20-tetra(4-pyridyl)-21H,23H-porphyrin with ruthenium outlying complexes.

The present work employs a set of complementary techniques to investigate the influence of outlying Ru(II) groups on the ground- and excited-state photophysical properties of free-base tetrapyridyl porphyrin (H(2)TPyP). Single pulse and pulse train Z-scan techniques used in association with laser flash photolysis, absorbance and fluorescence spectroscopy, and fluorescence decay measurements, allowed us to conclude that the presence of outlying Ru(II) groups causes significant changes on both electronic structure and vibrational properties of porphyrin. Such modifications take place mainly due to the activation of nonradiative decay channels responsible for the emission quenching, as well as by favoring some vibrational modes in the light absorption process. It is also observed that, differently from what happens when the Ru(II) is placed at the center of the macrocycle, the peripheral groups cause an increase of the intersystem crossing processes, probably due to the structural distortion of the ring that implies a worse spin-orbit coupling, responsible for the intersystem crossing mechanism.

Effects of environment on the photophysical characteristics of mesotetrakis methylpyridiniumyl porphyrin (TMPyP).

Porphyrins are an important class of organic molecules, with interesting linear and nonlinear optical properties given mainly by their extended π-conjugation structure. Their photophysical properties can be greatly affected by the surrounding environment, which can be used to tune its final properties. Here we report on an experimental study of the photophysical properties of meso-tetrakis (methylpyridiniumyl) porphyrin (TMPyP) in aqueous and in several organic solvents and its interaction with micelles formed from negatively charged sodium dodecylsulphate (SDS), positively charged cetyl trimethyl ammonium bromide (CTAB) and neutral TRITON X-100. By using the Z-scan technique, flash-photolysis and time-resolved fluorescence techniques, we were able to evaluate the excited state dynamics of the TMPyP, and observed that the tetrapyrrole ring plays important role due to hydrogen bonds formation between nitrogen atom and water, while the side groups determine the porphyrin localization in non-aqueous micelle part.

Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: fluorescence study.

The peptide hormone bradykinin (BK) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) and its shorter homolog BK(1-5) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)) were labeled with the extrinsic fluorescent probe ortho-aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH(2) and Abz-BK(1-5)-NH(2)). The fragment des-Arg(9)-BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg(9)-BK-DAP(Abz)-NH(2). The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide-lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy-Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK(1-5)-NH(2) presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer.

Fluorescence study of conformational properties of melanotropins labeled with aminobenzoic acid.

The native hormone alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analog [Nle(4),D-Phe(7)]alpha-MSH (NDP-alpha MSH), labeled at the amino terminal with the fluorescent aminobenzoic acid (Abz) isomers, were examined by fluorescence methods. We observed energy transfer between the tryptophan(9) residue acting as donor and Abz as acceptor, the transfer being more pronounced to the ortho-form of the acceptor. Within the hypothesis that different peptide conformations coexist in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function f(r). From the time-resolved fluorescence experimental data, we recovered the distance distribution between Abz and Trp(9), using the CONTIN program, within the framework of the Förster resonance energy transfer model. The methodology proved to be useful to provide quantitative information about conformational dynamics of melanotropins and its dependency on the solvent. In aqueous medium, alpha-MSH has a broad Abz-Trp(9) distance distribution, reflecting the structural flexibility of the peptide. Three different distance populations could be identified in the labeled analog NDP-alpha MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abz-Trp(9) distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides.

A pH-induced dissociation of the dimeric form of a lysine 49-phospholipase A2 abolishes Ca2+-independent membrane damaging activity.

The hydrolysis of phospholipids by class II phospholipase A2 (PLA2) involves a Ca2+ ion cofactor bound to the Asp49 residue in the active site region. In the lysine 49 phospholipase A2 homologues (Lys49-PLA2), the Asp49 residue is substituted by Lys, and consequently the Lys49-PLA2s show no Ca2+ binding and no detectable phospholipid hydrolysis. Nevertheless, the Lys49-PLA2s demonstrate membrane damaging activity by an incompletely understood Ca2+-independent mechanism of action. Using a combination of steady-state and time-resolved fluorescence techniques, we have examined the effect of pH on the monomer-dimer equilibrium of bothropstoxin I (BthTX-I), a Lys49-PLA2 from the venom of Bothrops jararacussu which contains a single Trp77 residue located at the dimer interface. At pH 5.0, we observe a decreased quantum yield, a decreased rotational correlation time, and an increased bimolecular quenching rate constant with iodide. These results are consistent with a pH-induced dissociation of the BthTX-I dimer, with the consequent exposure of the Trp77 residue to aqueous solvent. In the presence of liposomes, membrane damaging activity is observed only under conditions in which the dimeric form of the BthTX-I is favored. These results demonstrate that the dimeric form of the protein is essential for the initiation of the Ca2+-independent membrane damaging activity.

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides.

Similar to melanocyte stimulating hormone (alpha-MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha-MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac-Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.

How bradykinin alters the lipid membrane structure: a spin label comparative study with bradykinin fragments and other cations.

Electron spin resonance spectroscopy of several different spin labels was used to comparatively study the interaction of the cationic peptide hormone bradykinin (BK; Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), and some BK fragments (des-Arg(9)-BK, des-Arg(1)-BK, and Arg-Pro-Pro-Gly-Phe or BK(1-5)), with anionic vesicles of dimyristoyl phosphatidylglycerol (DMPG). For temperatures above the lipid gel-liquid crystal thermal transition (T(m) approximately 20 degrees C), membrane-incorporated spin labels indicated that all peptides (total concentration of 10 mol % relative to lipid) interact with the bilayer, turning the membrane less fluid, both at its surface and center, suggesting a partial penetration of the peptides into the membrane core. However, in the lipid gel phase (t < T(m)), BK was found to display a much stronger interaction with the membrane, decreasing the bilayer fluidity. At temperatures around 15 degrees C the BK-DMPG system was found to present a hysteresis, evinced by the different electron spin resonance spectra yielded upon cooling and heating the sample. System reversibility was found at all other temperatures (0-45 degrees C). That effect could not be assigned to the BK higher concentration at the membrane surface, due to its higher net charge (2(+)) compared to the fragments (1(+)), because ten times more des-Arg(9)-BK (100 mol %) yielded opposite result. Further, that was found to be a result rather different from those elicited by the other cations tested: the monovalent Na(+), the divalent Zn(2+), and the peptide pentalysine. The data presented here are discussed in the light of the different BK and BK fragments biological activities.

Tryptophan fluorescence studies of melanotropins in the amphiphile-water interface of reversed micelles.

We report studies on the interaction of alpha-melanocyte stimulating hormone (alpha-MSH) and a synthetic analogue (MSH-I) with reverse micelles prepared from the amphiphilic sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. The tripeptide lysyl-tryptophyl-lysine and the isolated amino acid tryptophan were also investigated as simpler compounds interacting with the micelles. Tryptophan fluorescence parameters (spectral position of emission band, anisotropy, and lifetime decay) demonstrated that in the presence of reverse micelles the environment around the fluorophore is less polar and more rigid than bulk water. Those parameters are sensitive to the changes induced in the micelles by the presence of water. In large micelles having a water/ amphiphile molar ratio above 10, the modifications detected by fluorescence are small and the location of the fluorophore is not affected by a further increase in the concentration of the bulk water. The results, with additional support from quenching experiments, indicated that the different compounds occupy different positions in the large reverse micelles, but in any case they are in the interface region, without dispersing into the bulk water. From decay associated spectra, conformations were identified showing different degrees of tryptophan exposition to polar and nonpolar local environments. The conformation related to the long lifetime has its tryptophan more exposed to water while that associated to the intermediate lifetime has that residue stabilized in nonpolar media. The native hormone alpha-MSH and the analogue MSH-I present similar conformations in dry micelles. However, in buffer and in the large hydrated micelles, differences in conformations are evident, and could be related to the different physiological activity of the peptides.

End-to-end distance distribution in bradykinin observed by Förster resonance energy transfer.

Förster resonance energy transfer (FRET) was used to study the conformational dynamics of bradykinin related peptides. The fluorescent probe aminobenzoic acid (Abz) bound to the amino terminal of bradykinin maintained its fluorescence characteristics, like high quantum yield and excited state decay dominated by a lifetime of 8.3 ns. The binding of the acceptor group N-[2, 4-dinitrophenyl]-ethylenediamine (EDDnp) to the carboxy terminal of Abz labeled bradykinin resulted in a drastic decrease of the fluorescence intensity and in a fastening of the excited state decay. The change of the decay kinetics to an heterogeneous process, precludes the use of energy transfer models based on a single fixed distance between donor and acceptor. The computational package CONTIN was employed to the analysis of time-resolved fluorescence data, allowing the recovery of a distance distribution between donor and acceptor corresponding to the end-to-end distance of the labeled peptide. The distance distribution reflects the occurrence of distinct conformations for the peptide, that coexist in equilibrium during the fluorescence lifetime. We observed three distance populations for bradykinin in water, that merged to two populations when the solvent was trifluoroethanol (TFE). The results were consistent with those obtained from circular dichroism spectroscopy, that showed structural flexibility in water and the presence of more defined secondary structure in TFE. We also studied several peptides related to bradykinin, and the results emphasized the formation of turns involving the proline residues and the decrease of conformational flexibility induced by using TFE as the solvent.

Molecular dynamics simulation of alpha-melanocyte stimulating hormone in a water-membrane model interface.

The conformation of the tridecapeptide alpha-melanocyte stimulating hormone in the presence of a double water-membrane interface was studied by molecular dynamics simulation, using the computational package THOR. In this program the solvent is represented by a continuous medium with dielectric constant epsilon, and the interface between different media is simulated by a surface of discontinuity of the dielectric constant. The electrostatic image method was used to write down the terms, added to the force field, that describe the polarisation effects induced in the interface by the atomic charges. The program was further improved by the introduction of a second surface, parallel to the first one, to mimic the membrane. A conformational search using the software Prelude was employed to find an initial geometry for the peptide in water. The molecular dynamics simulation performed during 10 ns showed that the peptide structure is flexible in water, without stabilisation of any preferential conformation. In the presence of the model membrane, the peptide moved to the medium representing the interior of the membrane. Inside the low dielectric constant medium, the structure of the peptide showed a turn in the central sequence of amino acids and a packed conformation remained stabilised during more than 7.0 ns of simulation.