Solid Phase Synthesis and TAR RNA-Binding Activity of Nucleopeptides Containing Nucleobases Linked to the Side Chains via 1,4-Linked-1,2,3-triazole.

Nucleopeptides (NPs) represent synthetic polymers created by attaching nucleobases to the side chains of amino acid residues within peptides. These compounds amalgamate the characteristics of peptides and nucleic acids, showcasing a unique ability to recognize RNA structures. In this study, we present the design and synthesis of Fmoc-protected nucleobase amino acids (1,4-TzlNBAs) and a new class of NPs, where canonical nucleobases are affixed to the side chain of L-homoalanine (Hal) through a 1,4-linked-1,2,3-triazole (HalTzl). Fmoc-protected 1,4-TzlNBAs suitable for HalTzl synthesis were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation of Fmoc-L-azidohomoalanine (Fmoc-Aha) and N1- or N9-propargylated nucleobases or their derivatives. Following this, two trinucleopeptides, HalTzlAAA and HalTzlAGA, and the hexanucleopeptide HalTzlTCCCAG, designed to complement bulge and outer loop structures of TAR (trans-activation response element) RNA HIV-1, were synthesized using the classical solid-phase peptide synthesis (SPPS) protocol. The binding between HalTzls and fluorescently labeled 5'-(FAM(6))-TAR UCU and UUU mutant was characterized using circular dichroism (CD) and fluorescence spectroscopy. CD results confirmed the binding of HalTzls to TAR RNA, which was evident by a decrease in ellipticity band intensity around 265 nm during complexation. CD thermal denaturation studies indicated a relatively modest effect of complexation on the stability of TAR RNA structure. The binding of HalTzls at an equimolar ratio only marginally increased the melting temperature (Tm) of the TAR RNA structure, with an increment of less than 2 °C in most cases. Fluorescence spectroscopy revealed that HalTzlAAA and HalTzlAGA, complementary to UUU or UCU bulges, respectively, exhibited disparate affinities for the TAR RNA structure (with Kd ≈ 30 and 256 µM, respectively). Hexamer HalTzlTCCCAG, binding to the outer loop of TARUCU, demonstrated a moderate affinity with Kd ≈ 38 µM. This study demonstrates that newly designed HalTzls effectively bind the TAR RNA structure, presenting a potential new class of RNA binders and may be a promising scaffold for the development of a new class of antiviral drugs.

Influence of Solvent Properties on Two Individuals Presented in the Excited State - Reverse Fluorosolvatochromism of CNAacDMA.

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.

Biological activity of 3-(2-benzoxazol-5-yl)alanine derivatives.

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.

Fluorescence Quenching Studies on the Interactions between Chosen Fluoroquinolones and Selected Stable TEMPO and PROXYL Nitroxides.

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-that is, Stern-Volmer quenching constants and bimolecular quenching rate constants-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'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 Product of Matrix Metalloproteinase Cleavage of Doxorubicin Conjugate for Anticancer Drug Delivery: Calorimetric, Spectroscopic, and Molecular Dynamics Studies on Peptide-Doxorubicin Binding to DNA.

Matrix metalloproteinases (MMPs) are extracellular matrix degradation factors, promoting cancer progression. Hence, they could provide an enzyme-assisted delivery of doxorubicin (DOX) in cancer treatment. In the current study, the intercalation process of DOX and tetrapeptide-DOX, the product of the MMPs' cleavage of carrier-linked DOX, into dsDNA was investigated using stationary and time-resolved fluorescence spectroscopy, UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). The molecular dynamics (MD) simulations on the same tetrapeptide-DOX…DNA and DOX…DNA systems were also performed. The undertaken studies indicate that DOX and tetrapeptide-DOX can effectively bond with dsDNA through the intercalation mode; however, tetrapeptide-DOX forms less stable complexes than free DOX. Moreover, the obtained results demonstrate that the differences in DNA affinity of both forms of DOX can be attributed to different intercalation modes. Tetrapeptide-DOX shows a preference to intercalate into DNA through the major groove, whereas DOX does it through the minor one. In summary, we can conclude that the tetrapeptide-DOX intercalation to DNA is significant and that even the lack of non-specific proteases releasing DOX from the tetrapeptide conjugate, the presence of which is suggested by the literature for the efficient release of DOX, should not prevent the cytostatic action of the anthracycline.

A Pentapeptide with Tyrosine Moiety as Fluorescent Chemosensor for Selective Nanomolar-Level Detection of Copper(II) Ions.

Herein, we have investigated principally with the use of UV and fluorescence (steady-state and time-resolved) spectroscopy the interactions between selected pentapeptides with tyrosine residue (EYHHQ, EHYHQ, EHHQY, and KYHHE) and various metal ions (Cu2+, Mn2+, Co2+, Ni2+, Zn2+, Cr3+, Cd2+, Ag+, Pb2+, Sr2+, Ba2+, Ca2+, Mg2+, Al3+, Fe2+, and Ga3+) in order to establish the relationship between the position of a tyrosine residue in the peptide sequence and the metal ion-binding properties. Among the peptides studied, EHYHQ was evaluated as an efficient and selective ligand for developing a chemosensor for the detection of copper(II) ions. While significant fluorescence emission quenching was observed for that peptide in the presence of Cu2+ cations, other metal cations used at the same and at considerably higher concentrations caused a negligible change of the fluorescence emission spectrum, indicating a high selectivity of EHYHQ for Cu2+ ions. Under optimum conditions, fluorescence intensity was inversely proportional to the concentration of Cu2+ ions. The limit of detection of Cu2+ ions with the use of EHYHQ was determined at the level of 26.6 nM. The binding stoichiometry of the complexes of the studied peptides with Cu2+ ions was evaluated spectrophotometrically and fluorimetrically (as in the case of EHYHQ confirmed by mass spectrometry) and found to be 1:2 (Cu2+-peptide) for all the investigated systems. Furthermore, the stability constant (K) values of these complexes were determined. The reversibility of the proposed Cu2+ ions sensor was confirmed, the pH range where the sensor acts was determined, while its analytical performance was compared with some other reported recently fluorescent sensors. The mechanism of the interactions between EHYHQ and Cu2+ was proposed on the basis of NMR spectroscopy investigations.

Dihydroxy-Substituted Coumarins as Fluorescent Probes for Nanomolar-Level Detection of the 4-Amino-TEMPO Spin Label.

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-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-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).

Copper(II) complexation by fragment of central part of FBP28 protein from Mus musculus.

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.

Probing the binding of Cu(2+) ions to a fragment of the Aß(1-42) polypeptide using fluorescence spectroscopy, isothermal titration calorimetry and molecular dynamics simulations.

Steady-state and time-resolved fluorescence quenching measurements supported by isothermal titration calorimetry (ITC) and molecular dynamics simulations (MD), with the NMR-derived restraints, were used to investigate the interactions of Cu(2+) ions with a fragment of the Aß(1-42) polypeptide, Aß(5-16) with the following sequence: Ac-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-NH2, denoted as HZ1. The studies presented in this paper, when compared with our previous results (Makowska et al., Spectrochim. Acta A 153: 451-456), show that the affinity of the peptide to metal ions is conformation-dependent. All the measurements were carried out in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, pH6.0. The Stern-Volmer equations, along with spectroscopic observations, were used to determine the quenching and binding parameters. The obtained results unequivocally suggest that Cu(2+) ions quench the fluorescence of HZ1 only through a static quenching mechanism, in contrast to the fragment from the N-terminal part of the FPB28 protein, with sequence Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr- NH2 (D9) and its derivative with a single point mutation: Ac-Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr- NH2 (D9_M), where dynamic quenching occurred. The thermodynamic parameters (ΔITCH, ΔITCS) for the interactions between Cu(2+) ions and the HZ1 peptide were determined from the calorimetric data. The conditional thermodynamic parameters suggest that, under the experimental conditions, the formation of the Cu(2+)-HZ1 complex is both an enthalpy and entropy driven process.

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.

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.

Photophysical properties of symmetrically substituted diarylacetylenes and diarylbuta-1,3-diynes.

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.

Experimental and theoretical studies of spectroscopic properties of simple symmetrically substituted diphenylbuta-1,3-diyne 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.

Fluorescence quenching of 7-amino-4-methylcoumarin by different TEMPO derivatives.

The fluorescence quenching of 7-amino-4-methylcoumarin by different TEMPO derivatives 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 distinguish each TEMPO derivative from the others and to understand the mechanism of quenching, the absorption and fluorescence emission spectra as well as decays of the fluorescence of 7-amino-4-methylcoumarin were registered as a function of each TEMPO derivative concentration. There were no deviations from a linearity in the Stern-Volmer plots (determined from both, steady-state and time-resolved measurements). The fluorescence quenching mechanism was found to be entirely collisional, what was additionally confirmed by the registration of Stern-Volmer plots at 5 temperatures ranging from 15 to 55°C. Based on theoretical calculations of molecular radii and ionization potentials of all TEMPO derivatives the mechanism of electron transfer was rejected. The fluorescence quenching which was being studied seems to be diffusion-limited and caused by the increase of non-radiative processes, such as an internal conversion and an intersystem crossing. The Stern-Volmer quenching constants and bimolecular quenching constants were determined at the room temperature for all TEMPO derivatives studied. Among all TEMPO derivatives studied TEMPO-4-amino-4-carboxylic acid (TOAC) was found to be the most effective quencher of 7-amino-4-methylcoumarin fluorescence (kq for TOAC was approximately 1.5 higher than kq for other TEMPO compounds studied). The findings demonstrate the possibility of developing an analytical method for the quantitative determination of TOAC, which incorporation into membrane proteins may provide a direct detection of peptide backbone dynamics.

Fluorescence quenching of fluoroquinolone antibiotics by 4-hydroxy-TEMPO in aqueous solution.

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.

Fluorescent analogs of trypsin inhibitor SFTI-1 isolated from sunflower seeds--synthesis and applications.

This article describes the synthesis and enzymatic study of newly synthesized analogs of trypsin inhibitors SFTI-1 that were fluorescent labeled on their N-terminal amino groups. Two fluorescent derivatives of benzoxazole (3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-L-alanine-[(4NPh2 )Ph]Box-Ala and 3-[2-(2',4',5'-trimethoxyphenyl)benzoxazol-5-yl]-L-alanine-[2,4,5-(OMe)3Ph]Box-Ala) were used as efficient fluorescent labels. The compounds obtained preserved their inhibitory activity and were efficient inhibitors of bovine trypsin or chymotrypsin. Nevertheless, their association inhibition constants were one or two orders of magnitude lower than those determined for unlabeled monocyclic SFTI-1 or [Phe(5)]SFTI-1, respectively. The conjugates obtained were found to be proteolytically stable in the presence of cognate enzymes. Applying such fluorescent peptides, we were able to investigate enzyme-inhibitor complex formation using fluorescent techniques. We found that such compounds were rapidly internalized by the fibroblast or cancer cells with no cytotoxic effects.

Analysis of fluorescence quenching of coumarin derivatives by 4-hydroxy-TEMPO in aqueous solution.

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.

Photophysical properties of 3-[2-(N-phenylcarbazolyl)benzoxazol-5-yl]alanine derivatives--experimental and theoretical studies.

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.

Influence of substituents on the nitrogen atom of 3-[2-(4-aminophenyl)benzoxazol-5-yl]alanine derivatives on their photophysical properties--solvatochromic studies.

Spectral and photophysical properties of three derivatives of 3-[2-(4-aminophenyl)benzoxazol-5-yl]alanine were studied in 36 solvents. The amino group was substituted by methyl and/or phenyl in various combinations (N,N-dimethyl, N-phenyl, N-methyl-N-phenyl). It has been found that the type of substituents on the nitrogen atom determines the spectral and photophysical properties of the compounds studied. The change of the dipole moment in going from the ground to the excited state as well as the dependence of the spectral and photophysical properties of compounds on the solvent polarity parameter E(N)(T) were analyzed. Additionally, spectral and photophysical properties of the compounds studied were analyzed applying multi-linear correlation using the three-parameter solvents scales of Kamlet-Taft and Catalán. Moreover, the new four-parameter Catalán solvent scale, which takes into account polarizability, dipolarity, acidity, and basicity of the solvents, was also applied giving a better fit than the three-parameter solvent scales. The correlation analysis reveals that the position of the UV/Vis absorption band depends primarily on the change of polarizability of the environment of the dye, although the solvent dipolarity cannot be neglected. However, the position of the fluorescence band and photophysical properties such as the fluorescence rate constant depend mostly on the solvent dipolarity.

Unexpected photoproduct generated via the acetone-sensitized photolysis of 5-bromo-2'-deoxyuridine in a water/isopropanol solution: experimental and computational studies.

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.

Highly specific substrates of proteinase 3 containing 3-(2-benzoxazol-5-yl)-l-alanine and their application for detection of this enzyme in human serum.

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.