Katarzyna Guzow

Photophysical properties of tyrosine at low pH range

Abstract The global analysis of the fluorescence intensity decays of tyrosine across the emission spectrum at pH=0 revealed that its fluorescence intensity decay is bi-exponential with fluorescence lifetimes equal to 63±7 ps and 1.030±0.009 ns, whereas, the global analysis of the fluorescence intensity decays of tyrosine at different pH revealed that the quenching of tyrosine fluorescence by H 3 O + ions is connected with the presence of protonated carboxyl group. The lack of acid–base equilibrium in the excited state or disturbance of this equilibrium by an additional quenching process caused the discrepancy between experimental points and Hendersson–Haselbalch relationship.

Synthesis and photophysical properties of 3-[2-(pyridyl)benzoxazol-5-yl]-l-alanine derivatives

Abstract The syntheses of N -( tert -butyloxycarbonyl)-3-[2-(4-pyridyl)benzoxazol-5-yl]- l -alanine methyl ester and N -( tert -butyloxycarbonyl)-3-[2-(2-pyridyl)benzoxazol-5-yl]- l -alanine methyl ester using different oxidizing agents (NBS, lead tetraacetate, Mitsunobu reaction) in oxidative cyclization of Schiff base are described. The compounds obtained are fluorescent. The position of the emission band and the fluorescence quantum yield depend on solvent polarity. The fluorescence decays of all the compounds studied are heterogeneous.

New highly fluorescent amino-acid derivatives: Substituted 3-[2-(phenyl)benzoxazol-5-yl]-alanines: synthesis and photophysical properties

Abstract New derivatives of 3-[2-(phenyl)benzoxazol-5-yl]alanine (Box-Ala) substituted at position 2′ and 4′ with OH, OMe, NMe 2 groups were prepared. Fluorescence properties (spectra, quantum yields) of new compounds were determined and compared with parent molecules without amino acid moiety. It was established that fluorescence properties of known benzoxazoles and new ones are similar. In the case of 3-[2-(2′-hydroxyphenyl)benzoxazol-5-yl]alanine (( o -OH)Box-Ala), presence of many different tautomeric forms in the ground state were found depending on solvent used.

Synthesis of a New, Highly Fluorescent Amino Acid Derivative: N-[(tert-Butoxy)carbonyl]-3-[2-(1H-indol-3-yl)-benzoxazol-5-yl]-L-alanine Methyl Ester

A simple method of synthesis of a new, highly fluorescent amino acid derivative from the simple and generally available substrates 3-nitro-L-tyrosine and 1H-indole-3-carbaldehyde is described. The obtained compound, N-[(tert-butoxy)carbonyl]-3-[2-(1H-indol-3-yl)benzoxazol-5-yl]-L-alanine methyl ester (4), possesses a high fluorescence quantum yield. The described method illustrates a new possibility of synthesis of amino acid derivatives possessing desirable photophysical properties.

Acidity of carboxyl group of tyrosine and its analogues and derivatives studied by steady-state fluorescence spectroscopy

Abstract The acidity of the carboxyl group of tyrosine and its derivatives and analogues was studied by means of fluorimetric titration using a steady-state fluorescence method. The pKa value of carboxyl group of tyrosine, its analogues and derivatives with blocked amino or hydroxyl group or both determined from the fluorimetric titration curve indicates that the methylation of hydroxyl group of phenolic ring, as well as the position of carboxyl group with respect to the phenol ring (Tyr, β-Tyr, β-Hty, Phg, Tic(OH)) have a minor influence on the value of pKa. The conversion of a protonated amino group of tyrosine or its analogues to the N-acetyl derivatives or its removal result in major lowering of the acidity of carboxyl group. The introduction of an additional hydroxyl group into a phenolic ring (Dopa) slightly increased acidity of the carboxyl group compared to tyrosine, whereas the replacement of α-hydrogen atom in Dopa by α-methyl group caused increase of pKa to the value observed for tyrosine. The pKa values of acetyl group of amino acid studied are in the range from 0.3 to 0.6.

The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH(2), where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k(cat)/K(M) = 275,000 M(-1) s(-1)), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.

Development of sensitive cathepsin G fluorogenic substrate using combinatorial chemistry methods.

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.

Determination of stoichiometry and equilibrium constants of complexes of tyrosine with cyclodextrins by time-resolved fluorescence spectroscopy and global analysis of fluorescence decays

Abstract Time-resolved fluorescence spectroscopy and global analysis of fluorescence decay times were applied to determine stoichiometry and equilibrium constants of complexes of tyrosine with α-, β- and γ-cyclodextrins (CDs). This study reveals from Global analysis of fluorescence decay curves of tyrosine with CDs in water the presence of only two fluorescence life-times of tyrosine: 3.35±0.10 ns for free tyrosine and 4.3±0.3 ns for the tyrosine–CD complex. The formation constants of tyrosine–CD complexes were determined from the pre-exponential factors as well as the fractional intensities and average fluorescence life-times. The cavity size of CD influenced the equilibrium constant but had no influence on the fluorescence life-time of the tyrosine–CD complex.

A DFT/TD DFT study of the structure and spectroscopic properties of 5-methyl-2-(8-quinolinyl)benzoxazole and its complexes with Zn(II) ion.

The structure and spectroscopic properties of 5-methyl-2-(8-quinolinyl)benzoxazole and its complexes with Zn(II) ion were studied using a DFT and TD DFT methods with def2-TZVP basis set. It was shown that the type of functional used (B3-LYP or pbe0) implemented in TURBOMOLE package does not have essential influence on the geometry (small differences in bond length, valence and dihedral angles) of studied compounds in both ground and excited states. However, significant differences were obtained for the position of vertical absorption and emission transition but not for the oscillator strength of transition. Application of pbe0 functional seems to reproduce better the experimental spectrum.

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.