Mariusz Szabelski

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.

Instrument response standard in time-resolved fluorescence

The fluorescence of LDS 798 dye in aqueous solution has a very short lifetime of 24 ps, independent of excitation wavelength. The time response of common photon counting detectors depends on the wavelength of the registered photon. In lifetime measurements, the instrument response function (IRF) is usually approximated by the temporal profile of the scattered excitation light. Because lambda(Exc) is typically much shorter than lambda(Em), a systematic error may be present in these measurements. We demonstrate that the fluorescence decay of LDS 798 is a better approximation of IRF, in particular, for avalanche photodiodes used in the near infrared spectral region.

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.

Collisional Quenching of Erythrosine B as a Potential Reference Dye for Impulse Response Function Evaluation

We studied the collisional quenching of the erythrosine B fluorophore by potassium iodide. The quenching follows a Stern–Volmer dependence up to the highest quencher concentration. The lifetime of erythrosine B decreases to 24 ps in 5.02 M of potassium iodide. The quantum yield of erythrosine B in the presence of 5.02 M KI is 0.0035. The relatively high brightness makes this compound attractive as an ultrashort reference in time-resolved measurements. In both frequency- and time-domain fluorescence techniques, there is a need for lifetime standards with extremely short decay times. Mimicking the instantaneous scattering at longer wavelengths allows color-effect-free measurements in the emission region. Another motivation is the problem of obtaining the impulse response function in the case of two-photon excitation. Time-resolved microscopy also benefits from fast-decaying dyes because the impulse response function can be evaluated at the emission wavelength of the investigated specimen without changing filters. We demonstrated that impulse response functions for commonly used detectors are practically the same for scattering as for quenched erythrosine B emission. We also analyzed a complex fluorescence decay using both elastic scattering and quenched erythrosine B emission as a response function.

Fluorescence Instrument Response Standards in Two-Photon Time-Resolved Spectroscopy

We studied the fluorescence properties of several potential picosecond lifetime standards suitable for two-photon excitation from a Ti:sapphire femtosecond laser. The fluorescence emission of the selected fluorophores (rose bengal, pyridine 1, and LDS 798) covered the visible to near-infrared wavelength range from 550 to 850 nm. We suggest that these compounds can be used to measure the appropriate instrument response functions needed for accurate deconvolution of fluorescence lifetime data. Lifetime measurements with multiphoton excitation that use scatterers as a reference may fail to properly resolve fluorescence intensity decays. This is because of the different sensitivities of photodetectors in different spectral regions. Also, detectors often lose sensitivity in the near-infrared region. We demonstrate that the proposed references allow a proper reconvolution of measured lifetimes. We believe that picosecond lifetime standards for two-photon excitation will find broad applications in multiphoton spectroscopy and in fluorescence lifetime imaging microscopy (FLIM).

Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

Forster resonance energy transfer (FRET) can be utilized to achieve ultrashort fluorescence responses in time-domain fluorometry. In a poly(vinyl) alcohol matrix, the presence of 60 mM Rhodamine 800 acceptor shortens the fluorescence lifetime of a pyridine 1 donor to about 20 ps. Such a fast fluorescence response is very similar to the instrument response function (IRF) obtained using scattered excitation light. A solid fluorescent sample (e.g a film) with picosecond lifetime is ideal for IRF measurements and particularly useful for time-resolved microscopy. Avalanche photodiode detectors, commonly used in this field, feature color- dependent-timing responses. We demonstrate that recording the fluorescence decay of the proposed FRET-based reference sample yields a better IRF approximation than the conventional light-scattering method and therefore avoids systematic errors in decay curve analysis.

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.