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Searching for new drugs is still a challenge for science, mainly because of civilization development and globalization which promote the rapid spread of diseases, which is particularly dangerous in the case of infectious ones. Moreover, readily available already known antibiotics are often overused or misused, possibly contributing to the increase in the number of multidrug-resistant microorganisms. A consequence of this is the need for new structures of potential drugs. One of them is a benzoxazole moiety, a basic skeleton of a group of fluorescent heterocyclic compounds already widely used in chemistry, industry, and medicine, which is also present in naturally occurring biologically active compounds. Moreover, synthetic benzoxazoles are also biologically active. Considering all of that, a large group of non-proteinogenic amino acids based on 3-(2-benzoxazol-5-yl)alanine skeleton was studied in search for new antimicrobial and anticancer agents. Screening tests revealed that antibacterial potential of 41 compounds studied is not very high; however, they are selective acting only against Gram-positive bacteria (B. subtilis). Moreover, almost half of the studied compounds have antifungal properties, also against pathogens (C. albicans). Most of studied compounds are toxic to both normal and cancer cells. However, in a few cases, toxicity to normal cells is much lower than for cancer cells indicating these compounds as future anticancer agents. The research carried out on such a large group of compounds allowed to establish a structure–activity relationship which enables to select candidates for further modifications, necessary to improve their biological activity and obtain a new lead structure with potential for therapeutic use.

The photophysical behavior of 9-(2-(4-(N,N-dimethylamino)phenyl)ethynyl)anthracene-10-carbonitrile (CNAacDMA) was investigated with absorption and steady-state fluorometry. Studies have shown extremely interesting properties of the analyzed derivative. The studied molecule is characterized by dual fluorescence and bidirectional fluorescence. These two observed phenomena are interrelated. Dual fluorescence analysis (part 1) explains bidirectional fluorescence. The compound in polar solvents shows only one emission band, however in moderate polar solvents shows dual emission. The appearance of the long-wavelength CT band in moderate polar solvents and its lack in polar solvents leads to bidirectional solvatofluorochromism. The solvatochrome analysis was performed in a wide range of 24 solvents. To determine the contribution of specific and nonspecific interaction of this compound with solvents linear and multi-parametric correlation of the solvent polarity were analyzed. The novelty and innovation in this article is to carry out such an analysis separately for two different individuals occurring in the excited state. Single-parameter scale with ET(30) as a solvent polarity parameter and multi-parameter scales such as the Catalán four-parameter solvent scale were used. The excited state dipole moment was determined based on solvatochromic method. The hydrogen bond energy change after excitation in the Franck-Condon and relaxed excited state were estimated.

The influence of the stable 2,2,6,6-tetramethylpiperidinyl-N-oxyl (TEMPO) nitroxide and its six C4-substituted derivatives, as well as two C3-substituted analogues of 2,2,5,5-tetramethylpyrrolidynyl-N-oxyl (PROXYL) nitroxide on the chosen fluoroquinolone antibiotics (marbofloxacin, ciprofloxacin, danofloxacin, norfloxacin, enrofloxacin, levofloxacin and ofloxacin), has been examined in aqueous solutions by UV absorption as well as steady-state and time-resolved fluorescence spectroscopies. The mechanism of fluorescence quenching has been specified and proved to be purely dynamic (collisional) for all the studied systems, which was additionally confirmed by temperature dependence experiments. Moreover, the selected quenching parameters&mdash, that is, Stern&ndash, Volmer quenching constants and bimolecular quenching rate constants&mdash, have been determined and explained. The possibility of electron transfer was ruled out, and the quenching was found to be diffusion-limited, being a result of the increase in non-radiative processes. Furthermore, as the chosen nitroxides affected the fluorescence of fluoroquinolone antibiotics in different ways, an influence of the structure and the type of substituents in the molecules of both fluoroquinolones and stable radicals on the quenching efficiency has been determined and discussed. Finally, the impact of the solvent&rsquo, s polarity on the values of bimolecular quenching rate constants has been explained. The significance of the project comes from many applications of nitroxides in chemistry, biology and industry.

The interactions between a group of different coumarins {7-hydroxycoumarin, 7-methoxycoumarin, 7-hydroxy-6-methoxycoumarin, 7-amino-4-methylcoumarin, 7-hydroxy-4-methylcoumarin, 7-hydroxy-4-(trifluoromethyl)coumarin} and 2,2,6,6-tetramethylpiperidinyl-N-oxyl (TEMPO) and its C4-substituted derivatives (4-hydroxy-TEMPO, 4-methoxy-TEMPO, 4-carboxy-TEMPO, 4-acetamido-TEMPO, 4-oxo-TEMPO) have been studied mainly in aqueous solutions with the use of spectroscopic techniques – namely UV-Vis absorption as well as steady-state and time-resolved fluorescence spectroscopy – and supported by theoretical calculations of molecular radii and ionization potentials. Since it has been found that the fluorescence intensity of the chosen coumarins is efficiently decreased by the nitroxides, the mechanism of fluorescence quenching has been determined. The work constitutes a continuation of our previous experiments on the interactions between different coumarin derivatives and 4-hydroxy-TEMPO (J Fluoresc 24, 713–718, 2014). The biological significance of this work is proven by the fact that TEMPO nitroxides have already many applications and their detection and quantitative determination under physiological conditions might help to understand the mechanism of an oxidative stress.

New derivatives of non-proteinogenic amino acids benzoxazol-5-yl-alanine containing substituted acetylene derivative were synthesized according to Sonogashira coupling reaction. All of the obtained compounds are fluorescent. They are characterized by high or moderate molar absorption coefficients, large Stokes shifts, high fluorescence quantum yields and very high brightness. All of these parameters as well as the positions of absorption and emission bands depend on the type and size of substituent and the solvent polarity. Their high brightness enables working with low concentrations, simple and easy detection of spectral absorption and fluorescence analyzes. Moreover, amino acid part of studied compounds allow to use them as covalently attached to a peptide or protein fluorescent probes in biological system studies.

Variable-temperature absorption and emission data over the range 80–300 K have been collected for 1,2-diphenylacetylene (DPA) and its derivatives, as well as 1,4-diphenylbuta-1,3-diyne (DPB) derivatives in 2-methyltetrahydrofuran (2-MeTHF). Moreover, the influence of temperature on fluorescence lifetime and quantum yield has also been investigated. A strong coupling between solvation dynamics and solute structure is observed in the low-temperature regime as well as at intermediate temperature where the structure of the compound is evolving in the course of excited-state relaxation. Moreover, the dependency of fluorescence quantum yield on temperature indicates that the energy of activation of radiationless transition for 1,2-diphenylacetylene and its derivatives is larger than the activation energy of viscous flow of 2-MeTHF, while for 1,4-diphenylbuta-1,3-diyne derivatives the opposite is observed. This finding implies that the conformational change of DPA and its derivatives in the excited state in 2-MeTHF shows an activation energy, contrary to DPB and its derivatives for which conformational changes are barrierless.

Diphenylacetylene derivatives containing electron-donor (amino) and electron-acceptor (ester) groups in 2,2′ or 3,2′ positions in phenyl rings were synthesized to study the effects of intramolecular charge transfer and stiffening of the conformation by intramolecular hydrogen bond on the photophysical properties. Additionally, the derivatives with and without a steric hindrance were studied to determine the effect of conformational freedom on photophysical properties. The persistence of the intramolecular hydrogen bond and the corresponding twisted conformations as well as the conformational flexibility were also established by DFT calculations.

The study is devoted to a luminescent glass and its devitrified counterpart (nano-glass ceramics) both triply co-doped with Eu 3+ , Tb 3+ and Tm 3+ ions. Besides for comparison glasses singly doped with Eu 3+ , Tb 3+ or Tm 3+ and a glass doubly co-doped with Eu 3+ and Tb 3+ were studied. The singly Tb 3+ doped glass did not exhibit cross-relaxation effect. In the case of the di-doped glass energy transfers Tb 3+ → Eu 3+ was certified. Then, for the tri-doped glass energy transfers Tb 3+ → Eu 3+ and in reverse Eu 3+ → Tb 3+ as well as Tm 3+ → Tb 3+ were observed. In the emission spectrum of the triply co-doped devitrified glass, similarly as in the other Eu 3+ doped glasses, band peaked at 612 nm ascribed to 5 D 0 → 7 F 2 transition in Eu 3+ was dominated above the 5 D 0 → 7 F 2 band. It suggests that the majority of Eu 3+ (and very probably also Tb 3+ and Tm 3+ ) ions were incorporated into the residual vitreous phase of the glass-ceramics. Reduced luminescence lifetimes τ 1 of the Tb 3+ and Tm 3+ dopants in the triply co-doped glass in comparison to τ values of these dopants singly embedded in the glasses additionally confirms presence of the energy transfer effects in the tri-doped material. Devitrification of the tri-doped glassy phosphor did not improve its white color emission.

The GeO 2 -PbO-Bi 2 O 3 -SrF 2 (GePbBiSr) glass systems doped with Eu 3 + , Tb 3 + , Tm 3 + and triply-doped with Eu 3 + /Tb 3 + /Tm 3 + ions were successfully synthesized. Their structural and luminescent properties have been investigated with various techniques. Obtained results indicate that these glass systems are suitable materials for the RE 3 + ions hosts. RE 3 + ions can be excited by single UV light wavelength (355/378/395 nm) and simultaneously emit red, green and blue light originating from Eu 3 + , Tb 3 + and Tm 3 + transitions. Emission color changes from bluish-purple for 355 nm excitation to white when irradiated with 378 nm and 395 nm. The GePbBiSr glass triply-doped by Eu 3 + /Tb 3 + /Tm 3 + ions could be a potential candidate for white LED applications.

Unsymmetrically substituted diphenylacetylene derivatives containing a different type of an electron-acceptor substituent were synthetized and studied using spectroscopic methods in order to establish the influence of electron affinity of substituent on the photo physics of donor-acceptor pairs. Spectral and photophysical properties were studied in 14 solvents (non-polar, polar aprotic and protic). Influence of solvent properties on spectral and photophysical properties of studied compounds was analyzed using Catalan multi-parameter solvent scale. The change of the excited state dipole moment was estimated based on Bilot-Kawski theory.

Steady-state and time-resolved fluorescence spectroscopy, UV spectrophotometry and isothermal titration calorimetry techniques were used to study the coordinating properties of the 17aa peptide fragment (D17) derived from the central part of the mouse formin binding protein (FBP28 with the PDB code: 1E0L ) towards Cu2+ ions. All the measurements were run in the 2-(N-morpholino)ethanesulfonic acid buffer (20 mM, pH 6.0). Under experimental conditions the formation of the 1:1 complex of Cu2+ ions with D17 is an entropy-driven process. Cu2+ ions cause the static fluorescence quenching of the peptide studied through the formation of a non-fluorescent complex. Furthermore, the thermal stability of D17 was discussed based on the results obtained from differential scanning fluorimetry (nanoDSF) data.

A series of symmetrically substituted diphenylacetylene (DPA) derivatives possessing electron-donating (N,N-dimethylamino or methoxy) or electron-accepting (nitrile, ester or aldehyde) character were prepared and studied with respect to their spectral and photophysical properties. The photophysical characteristics of these compounds were studied in relation to their structures and influence of temperature. The observed spectral and photophysical properties are explained with the help of potential energy maps obtained from density functional theory (DFT, B3LYP, def2TZVP basis set) calculations. The structure–property relationships of all of the compounds are discussed.

Due to the biological significance of 4-hydroxy-TEMPO, its detection in biological systems such as blood serum is of great importance in both research and clinical applications. The following paper reports a novel fluorimetric method for the detection and quantitative determination of 4-hydroxy-TEMPO radicals in an aqueous solution using dihydroxycoumarins as fluorescent probes. Among the 17 coumarin derivatives studied, only some dihydroxycoumarins show high sensitivity, specificity and selectivity for 4-hydroxy-TEMPO. Among them, 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement under the action of 4-hydroxy-TEMPO. In this assay, esculetin reacts with 4-hydroxy-TEMPO to exclusively yield a dimer. 6,7-Dihydroxycoumarin responds to 4-hydroxy-TEMPO quickly and shows a 2.5-fold fluorescence enhancement with an estimated detection limit of 285 μM.

The fluorescence quenching of norfloxacin, danofloxacin, enrofloxacin and levofloxacin, belonging to a group of fluoroquinolone antibiotics, by 4-hydroxy-TEMPO was studied in aqueous solutions with the use of steady-state, time-resolved fluorescence spectroscopy as well as UV-VIS absorption spectroscopy methods. In order to understand the mechanism of quenching the absorption and fluorescence emission spectra of all fluoroquinolone antibiotics studied as well as decreases of their fluorescence were registered as a function of the 4-hydroxy-TEMPO concentration. No deviations from a linearity in the Stern-Volmer plots (determined from both, steady-state and time-resolved measurements) were observed. The fluorescence quenching mechanism was proved to be totally dynamic, what was additionally confirmed by the registration of Stern-Volmer plots at 5 temperatures ranging from 15 to 55°C. On the basis of theoretical calculations of fluoroquinolones' molecular radii and ionization potentials the mechanism of electron transfer was rejected. It seems that the fluorescence quenching is diffusion-limited and is caused by the increase of nonradiative processes, such as internal conversion or intersystem crossing. The Stern-Volmer quenching constants and bimolecular quenching constants were determined at the room temperature for all fluoroquinolone antibiotics studied.

The spectral and photophysical properties of newly synthesized 3,6-di-tert-butyl-8H-indolo[3,2,1-de]acridin-8-one were studied. To determine the contribution of specific and nonspecific interactions of this compound with solvents, the solvatochromic methods were used. It was found that bathochromic shifts of absorption and fluorescence spectra were mostly caused by polarizability/dipolarity interactions of the solute with solvent. The excited state dipole moment, determined based on a solvatochromic method, is two times higher than that in the ground state. Also, the spectral and photophysical properties of the compound studied correlate with the ENT solvent polarity scale. The radiative rate constant does not depend on the solvent properties, while the non-radiative rate constant decreases with increasing solvent polarity. Such a decrease of the non-radiative rate constant with the lowering of the energy gap between the S0 and S1 states indicates the participation of the triplet state in the deactivation of the excited state. After photoexcitation hydrogen bond energy increases in both the locally excited Franck–Condon and the relaxed excited state, however, the change of hydrogen bond energy is three times higher in the relaxed excited state. The hydrogen bond energy change measured in five alcohols correlates with the Kamlet–Taft or Catalan solvent acidity parameter.

This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM&mdash, a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH&mdash, the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

The fluorescence quenching of different coumarin derivatives (7-hydroxy-4-methylcoumarin, 5,7-dimethoxycoumarin, 7-amino-4-methyl-3-coumarinylacetic acid, 7-ethoxy-4-methylcoumarin, 7-methoxycoumarin, 7-hydroxycoumarin, 7-hydroxy-4-methyl-3-coumarinylacetic acid and 7-amino-4-methylcoumarin) by 4-hydroxy-TEMPO in aqueous solutions at the room temperature was studied with the use of UV–Vis absorption spectroscopy as well as a steady-state and time-resolved fluorescence spectroscopy. In order to understand the mechanism of quenching the absorption and fluorescence emission spectra of all coumarins along with fluorescence decays were recorded under the action of 4-hydroxy-TEMPO. The Stern-Volmer plots (both from time-averaged and time-resolved measurements) displayed no positive (upward) deviation from a linearity. The fluorescence quenching mechanism was found to be entirely dynamic, what was additionally confirmed by the registration of Stern-Volmer plots at different temperatures. The Stern-Volmer quenching constants and bimolecular quenching rate constants were obtained for all coumarins studied at the room temperature. The findings demonstrate the possibility of developing an analytical method for the quantitative determination of the free radicals’ scavenger, 4-hydroxy-TEMPO.

Acid–base properties of 3-[2-(2-pyridyl)benzoxazol-5-yl]alanine and 3-[2-(4-pyridyl)benzoxazol-5-yl]alanine as well as a model compound without heterocyclic substituent (3-[2-(4-methylphenyl)benzoxazol-5-yl]alanine) were studied by means of absorption and fluorescence spectroscopy as well as theoretical calculations. It was found that basicity of heterocyclic derivatives in the ground and excited state depends on the position of the heteroatom in the substituent. Contrary to 2-(pyridyl)-benzimidazole, dual fluorescence and formation of dication were not detected for both compounds studied. Moreover, experimental as well as theoretical calculations revealed that pyridyl nitrogen atom is the first protonation center.

Solvatochromic probes are often used in biophysical studies to obtain information about polarity of the microenvironment. As there is not much natural fluorophores with such properties, there is still need for new synthetic compounds such as 3-(2-benzoxazol-5-yl)alanine derivatives. Among this group of non-proteinogenic fluorescent amino acids especially interesting are 3-[2-(4-aminophenyl)benzoxazol-5-yl]alanine derivatives whose solvatochromism depends on the substituents on the nitrogen atom, as revealed by our recent studies. To expand them we synthesized two new derivatives with an N-phenylcarbazole moiety in position 2 of the benzoxazole ring and studied their photophysical properties in solvents of different polarity and ability to form hydrogen bonds using absorption and steady-state and time-resolved fluorescence spectroscopy. Applying single parameter and multi-linear correlations with different solvent parameters, the excited state dipole moments were determined as well as the influence of solvent parameters on each photophysical property was estimated. Moreover, the geometry of compounds and vertical absorption transition were theoretically calculated (DFT and TD DFT methods). It was found that the place of substitution of the N-phenylcarbazole part by the benzoxazole unit determines the character of the electron transition (π-π* or ICT) and thereby the spectral and photophysical properties of the compounds studied.

Two 3-[2-(boronophenyl)benzoxazol-5-yl]alanine derivatives were synthesized and their potential application as fluorescent monosaccharide sensors was studied. It was found that both non-proteinogenic amino acids bound glucose and fructose at physiological pH, however, the latter much stronger. As a result they are selective sensors for fructose. Moreover, one of them (3-[2-(3-boronophenyl)benzoxazol-5-yl]alanine methyl ester) can be used to quickly distinguish, which monosaccharide is present in the solution because of the different character of fluorescence intensity changes (increase in the presence of fructose and decrease in the presence of glucose).

A series of symmetrically substituted diarylacetylenes and diaryl-1,3-butadiynes were prepared and studied with an emphasis on their spectral and photophysical properties. The photophysical characteristics of these compounds were studied in relation to their structures and the influence of solvent or temperature. The observed spectral and photophysical properties are explained using potential energy maps of the ground and excited states obtained from density functional theory calculations. The structure–property relationships of all of the compounds are discussed and compared with the appropriate diphenylacetylene and diphenyl-1,3-butadiyne derivatives.

A series of symmetrically substituted diphenylbuta-1,3-diyne (DPB) derivatives possessing electron-donating (N,N-dimethylamino or methoxy) or electron-accepting (nitrile, ester or aldehyde) groups have been prepared and studied with emphasis on their spectral and photophysical properties. The photophysical characteristics of these compounds have been studied in relation to their structures and influence of solvents or temperature. The observed spectral and photophysical properties are explained with the help of potential energy maps in the ground and excited states obtained from density functional theory (DFT, B3LYP, def2TZVP basis set) calculations. The structure-property relationship of all of the compounds is discussed and compared with appropriate diphenylacetylene derivatives.

The influence of peptide sequence and Leu chirality in linear and cyclic peptides containing 3-[2-(9-anthryl)benzoxazol-5-yl]alanine on interaction with β-cyclodextrin were studied using fluorescence and NMR spectroscopy. The analysis of enthalpy-entropy compensation effect (α=1.05±0.02 and TΔS00=15.1±0.5 kJ mol−1) indicates that the entropic contribution connected with the solvent reorganization is the major factor governing the peptides-β-cyclodextrin complexation. Moreover, spatial orientation of guest-host molecule depends more than association constant on Leu residue configuration. However, the cyclization of the peptide chain substantially decrease the association constant with β-CD. An analysis of 2D NMR spectra reveals that inclusion complex is formed by penetration of cyclodextrin cavity from wider and narrow rims by anthryl group in the case of Box(Ant)-SPKL or anthryl and Leu residues for Box(Ant)-SPK(D)L analogue.

The acetone-sensitized photolysis of 5-bromo-2'-deoxyuridine (5-BrdU) in a water/isopropanol solution with 300 nm photons leads to the formation of 2'-deoxyuridine (dU) and a comparable amount of another photoproduct that has not been reported in the literature so far. The negative and positive mass spectra recorded for this species indicate that they originate from the molecular mass of 286 Da, which corresponds to an adduct of 2'-deoxyuridine and 2-propanol. Quantum chemical calculations carried out at the DFT and TDDFT levels reveal both the structure and the UV spectrum of that adduct. The latter computational characteristic matches well the experimental UV spectrum of the new photoproduct. Our findings indicate that the acetone-sensitized photolysis of 5-BrdU is more complicated than has hitherto been assumed. Nevertheless, since electron transfer is one of the pathways responsible for 5-BrdU decay, acetone-sensitized photolysis of the halogen derivatives of nucleobases could be a convenient tool for studying their radiosensitivity in aqueous solutions.

The ability of new chelate ligands, benzoxazol-5-yl-alanine derivatives substituted in position 2 by heteroaromatic substituent, to form complexes with selected metal ions in acetonitrile are studied by means of absorption and steady-state and time-resolved fluorescence spectroscopy. Among the ligands studied, only azaaromatic derivatives form stable complexes with transition metal ions in the ground state. Their absorption bands are bathochromically shifted enabling to use those ligands as ratiometric sensors. The fluorescence of each ligand is quenched by metal ions, however, in the presence of Cd(II) and Zn(II) ions a new red shifted emission band is observed.

A set of benzoxazolyl-l-alanine derivates along with the MCA moiety (donors of fluorescence) were introduced into a proteinase 3 (PR3) substrate with a C-terminal ANB-NH(2) that serves as a fluorescence acceptor. Five substrates with general formula X-Tyr-Tyr-Abu-ANB-NH(2) were synthesized, and their kinetic parameters against proteinase 3 were determined. The highest k(cat)/K(M) value, 1.5 x 10(6) M(-1) s(-1), was obtained for (Pyr)Box-Ala-Tyr-Tyr-Abu-NH(2) where (Pyr)Box-Ala stands for N-methylpyrrole benzoxazole-l-alanine. Titration of this peptide with proteinase 3 resulted in measurable fluorescence at an enzyme amount equal to 29 pmol. This substrate was selected used to detect quantifiable levels of proteinase 3 in serum samples, including those of normal subjects. For all c-ANCA-positive samples (diagnosed Wegener granulomatosis), a significant increase of PR3 concentration was observed. Wegener granulomatosis is a severe autoimmune disease causing inflammation of the blood vessels. Our results clearly show that this substrate can be used for the construction of a very reliable, inexpensive, and easy to use diagnostic test for PR3 determination.

Natural enkephalins and their analogues are very important as potential therapeutic agents (analgetics). Herein we describe the influence of Dab and Pro chirality of cyclic [Leu]enkephalins (X1-c[Dab2-Pro3-βNal(2)4-Leu5], where X = Tyr or Phe) on the binding constant with β-cyclodextrin and spatial and mutual orientation of guest and host molecules. The formation of complexes is enthalpy driven for all cyclic [Leu]enkephalins studied as well as for Nal and AcNalNH2. Moreover, change of Dab residue configuration has a greater influence on changes of the binding constant of cyclic enkephalin with β-CD than change of Pro chirality has. Also, the replacement of Tyr1 residue by Phe1 substantially changes the peptide chain conformation. An analysis of 2D NMR spectra reveals that, apart from inclusion complex formed by penetration of cyclodextrin cavity from wider and narrow rims by Nal, Tyr or Phe or Leu residue, a side and/or bottom association complexes are formed.

The influence of substituent on amide nitrogen atom on the interactions of N-acetyl tyrosine amides with β-cyclodextrin was studied by means of steady-state and time-resolved fluorescence spectroscopy, 2D 1H NMR, and microcalorimetry. In comparison with AcTyr-OH a primary amide group only in a small degree modified the binding constant with β-CD, regardless of the structure (linear or branched) and the length of n-alkyl substituent which for primary amides (methyl, ethyl, n-propyl, iso-propyl, n-butyl, and sec-butyl), as determined from the microcalorimetric titrations, is in the range from 122 M−1 to 190 M−1, except for t-butyl substituent for which the highest binding constant (over 500 M−1) was determined. Moreover, for a branched substituent binding constants are a little higher in comparison with n-alkyl ones. For secondary amides (di-methyl, di-ethyl, di-n-propyl, di-iso-propyl, and di-iso-butyl) the binding constants are higher (in the range from 270 M−1 to 410 M−1).

Natural enkephalins and their analogues are very important as potential therapeutic agents (analgetics). In this paper we describe the influence of Leu chirality of cyclic [Leu]enkephalins on the binding constant with β-cyclodextrin and spatial and mutual orientation of guest and host molecules. The formation of complexes is enthalpy driven for both cyclic [Leu]enkephalins. Moreover, d-configuration of Leu residue causes an increase of the binding constant of cyclic enkephalin compared to l-analogue. An analysis of 2D NMR spectra reveals that, apart from inclusion complex formed by penetration of cyclodextrin cavity from wider and narrow rims by Trp or Leu residue, a side and/or bottom association complexes are formed.

In the current work, a synthesis of new sensitive fluorescence substrates of cathepsin G is reported. The substrate sequence was selected using combinatorial chemistry methods. The starting structure of chromogenic cathepsin G substrate Ac-Phe-Val-Thr-Gnf-ANB-NH(2), where Gnf stands for 4-guanidine-l-phenylalanine, was modified by replacing the acetyl moiety with a residue of 7-methoxycoumarin-4-yl acetic acid (Mca) that served as a fluorescence donor. An amide of amino benzoic acid (ANB-NH(2)) was used as an acceptor. This peptide, exhibiting effective fluorescence resonance energy transfer (FRET) phenomena, was used as a starting structure to construct the library Mca-Phe-Val-Thr-Gnf-X(1)-X(2)-ANB-NH(2), where in both variable X positions all proteinogenic amino acid residues except Cys were introduced. Deconvolution of such a library, performed by the iterative method in solution, revealed prime site preferences of cathepsin G. Finally, the most susceptible sequence, Mca-Phe-Val-Thr-Gnf-Ser-Trp-ANB-NH(2), was selected. The determined value of the specificity constant (k(cat)/K(M) = 252 x 10(3)M(-1)xs(-1)) was two orders of magnitude higher than that obtained for the parent compound. By the use of this substrate, we were able to detect as little as 70 pM of the enzyme studied.

Excitation energy transfer between donor (Box(benzfur)) and acceptor (Tyr(NO 2 )) is studied for uniaxially stretched and unstretched poly(vinyl) alcohol films. Free D–A pair and short linear peptides are examined. D–A distance for the longest peptide in unstretched poly(vinyl alcohol) film is very similar to that determined in aqueous solution which indicates the immobilization of the peptide native conformation chain in PVA. The results for stretched PVA matrix indicate partial stretching of the peptide chain. However, for free non-linked D–A pair for stretched PVA matrix substantial increase of D–A distance is observed. Emission anisotropy increases with the degree of stretching but for the longest peptide the saturation effect is not reached. Moreover, the donor emission anisotropy spectra remain practically constant for a given stretching factor R S of the film.

N-(tert-butoxycarbonyl)-3-(benzoxazol-5-yl)alanine methyl ester derivatives substituted in position 2 by heterocyclic group were synthesized and their photophysical properties in methanol, acetonitrile and methylcyclohexane were studied by means of absorption and fluorescence spectroscopies. The positions of their emission bands depend on the solvent polarity and are shifted to longer wavelengths in more polar solvents as a result of a charge transfer from a substituent to the benzoxazole moiety. High molar absorption coefficient values and fluorescence quantum yields are characteristic for most of the compounds studied making them efficient fluorescent probes. Moreover, the presence of additional heteroatom in the substituent enables those compounds to act as chemosensors for metal ions or protons.

The excitation-emission spectra of flavin mononucleotide (FMN) were measured in rigid PVA films for concentrations ranging from 6.92 x 10(-4)M to 1.03 M. The theoretical three-linear decomposition of the excitation-emission spectra indicated the presence of two absorption and emission centers corresponding to FMN monomer and dimer, respectively. The component of the fluorescence profile corresponding to the FMN monomer has a large negative part which is the mirror image of the emission band profile of the dimer. The elimination of this part by taking a linear combination of the emission components of the monomer and of the dimer resulted in emission spectrum, which is in a very good agreement with the monomer spectrum measured directly. The appearance of a negative part of the monomer emission profile obtained by trilinear decomposition of the emission-absorption spectra of FMN can be explained in terms of the non-radiative reverse energy transfer from the FMN dimers to the FMN monomers. The presented results confirm that the FMN molecules in rigid PVA form dimers but not higher order aggregates. Moreover, they enable to obtain fluorescence spectra of dimers and suggest that FMN dimers may take part in the process of non-radiative energy transfer occurring in photoreception phenomena.

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu(2+) ions are discussed.

A series of N-(tert-butoxycarbonyl)-3-[2-(phenyl)benzoxazol-5-yl]alanine methyl ester derivatives possessing electron-donor and electron-acceptor substitutents in the phenyl ring were synthesized and their photophysical properties in methanol, acetonitrile and methylcyclohexane were studied by means of absorption and fluorescence spectroscopies as well as theoretical calculations. The structures of compounds studied in the ground and excited state were calculated using the MOPAC 2002 package with COSMO model of the solvent whereas the positions of vertical absorption and emission transitions were calculated using a INDO/S or CNDO/Param method within a SCRF approximation for the solvent but they are not in good agreement with the experimental values. High molar absorption coefficient values and fluorescence quantum yields are characteristic for all compounds studied. Moreover, in the case of derivatives possessing electron-donor substituents in position 2 of the phenyl ring the fluorescence quantum yield increases with the solvent polarity whereas the opposite effect is observed for derivatives substituted by electron-acceptor groups. Also, the position of the absorption and emission band depends on the solvent polarity and is shifted to longer wavelengths in more polar solvents as a result of a charge transfer from a substituent to the benzoxazole moiety. In the case of derivatives substituted by 2-hydroxyphenyl, the intramolecular proton transfer process is observed. However, the character of the group in position 4 of the phenyl ring influences on it. An electron-acceptor substituent facilitates the proton transfer whereas the opposite is true for an electron-donor group.

The photophysical properties of N -Boc-3-[2-(9-anthryl)benzoxazol-5-yl]- l -alanine methyl ester (BoxAnt) and N -Boc-3-[2-[4-(9′-(10′-butyl)anthryl)phenyl]benzoxazol-5-yl]- l -alanine methyl ester (BoxPhAnt) were studied in a series of solvents. Their absorption spectra are less sensitive to the solvent polarity than the corresponding fluorescence spectra which show a pronounced solvatochromic effect leading to large Stokes shifts. Using an efficient solvatochromic method, based on the empirical solvent polarity parameter E T N , a large change of the dipole moment on excitation for BoxPhAnt has been found. From an analysis of the solvatochromic behaviour of the absorption and fluorescence spectra in terms of bulk solvent polarity functions, f ( ɛ r , n ) and g ( n ), a larger excited-state dipole moment (about 8 D, ψ = 56) was obtained for BoxPhAnt than for BoxAnt (about 3 D, ψ = 0). Both applied methods gave similar values of the excited-state dipole moments for both compounds studied.

A novel aromatic amino acid, 3-[2-(8-quinolinyl)benzoxazol-5-yl]alanine derivative was synthesized as a potential Zn(II) and rare-earth metal, Eu(III) and Tb(III), ion chemosensor. The fluorophore was obtained using lead tetraacetate in DMSO to oxidize the Schiff base obtained from N -Boc-3-amino-tyrosine methyl ester and quinoline-8-carboxaldehyde. Preliminary photophysical properties of this ligand show that it possesses the properties necessary to be an effective chemosensor for Zn 2+ , Tb 3+ and Eu 3+ ions.