Alberto L. Capparelli

Fluorescence of pterin, 6-formylpterin, 6-carboxypterin and folic acid in aqueous solution: pH effects

Steady-state and time-resolved studies have been performed on four compounds of the pterin family (pterin, 6-carboxypterin, 6-formylpterin and folic acid) in aqueous solution, using the single photon counting technique. The fluorescence characteristics (spectra, quantum yields, lifetimes) of these compounds and their dependence on the pH have been investigated. Most pterins can exist in two acid-base forms over the pH range between 3 and 13. Emission spectra and excitation spectra were obtained for both forms of each compound studied. Fluorescence quantum yields (phi(F)) in acidic and basic media were measured. The phi(F) of folic acid (< 0.005 in both media) is very low compared to those of pterin (0.27 in basic media and 0.33 in acidic media), 6-carboxypterin (0.18 in basic media and 0.28 in acidic media) and 6-formylpterin (0.07 in basic media and 0.12 in acidic media). The variation in integrated fluorescence intensity and fluorescence lifetimes (tau(F)) was analysed as a function of pH. Dynamic quenching by OH- was observed and the corresponding bimolecular rate constants for quenching of fluorescence (k(q)) were calculated. The reported values for k(q) (M(-1) s(-1)) are 3.6 x 10(9), 1.9 x 10(9) and 1.1 x 10(10) M(-1) s(-1) for pterin, 6-carboxypterin and 6-formylpterin, respectively.

Singlet oxygen (1Δg) production by pterin derivatives in aqueous solutions

Six compounds of the pterin family (pterin, 6-carboxypterin, 6-formylpterin, folic acid, biopterin and neopterin) have been investigated for their efficiencies of singlet oxygen (O2(1Δg)) production and quenching in aqueous solutions. The quantum yields of 1O2 production (ΦΔ) have been determined by measurements of the 1O2 luminescence in the near-infrared (1270 nm) upon continuous excitation of the sensitizer. Under our experimental conditions, all studied compounds (except folic acid) are relatively efficient 1O2 sensitizers with ΦΔ values of up to 0.47. Results show that the nature of the substituent at position 6 on the pterin moiety, as well as the pH, affect considerably the capacity of pterins to produce 1O2. All compounds investigated are efficient 1O2 quenchers: the rate constant of 1O2 total quenching (kt) by folic acid (3.0(± 0.3) × 107 M−1 s−1) is one order of magnitude larger than those for the other pterin derivatives investigated (1.4(± 0.1) × 106 M−1 s−1to 2.9(± 0.3) × 106 M−1 s−1).

Competition kinetics using the UV/H2O2 process: a structure reactivity correlation for the rate constants of hydroxyl radicals toward nitroaromatic compounds.

The rate constants for hydroxyl radical reaction toward a set of nitroaromatic substrates kS, have been measured at 25 degrees C using competition experiments in the UV/H2O2 process. For a given pair of substrates S1 and S2, the relative reactivity beta (defined as kS1/kS2) was calculated from the slope of the corresponding double logarithmic plot, i.e., of ln[S1] vs. ln[S2]. This method is more accurate and remained linear for larger conversions in comparison with the plots of ln[S1] and ln[S2] against time. The rate constants measured ranged from 0.33 to 8.6 x 10(9) M(-1)s(-1). A quantitative structure-reactivity relationship was found using the Hammett equation. Assuming sigma values to be additive, a value of -0.60 was obtained for the reaction constant rho. This value agrees with the high reactivity and the electrophilic nature of HO* radical.

Reactivity of Conjugated and Unconjugated Pterins with Singlet Oxygen (O2(1Δg)): Physical Quenching and Chemical Reaction†

Pterins (PTs) belong to a class of heterocyclic compounds present in a wide range of living systems. They participate in relevant biological functions and are involved in different photobiological processes. We have investigated the reactivity of conjugated PTs (folic acid [FA], 10‐methylfolic acid [MFA], pteroic acid [PA]) and unconjugated PTs (PT, 6‐hydroxymethylpterin [HPT], 6‐methylpterin [MPT], 6,7‐dimethylpterin [DPT], rhamnopterin [RPT]) with singlet oxygen (1O2) in aqueous solutions, and compared the efficiencies of chemical reaction and physical quenching. The chemical reactions between 1O2, produced by photosensitization, and PT derivatives were followed by UV‐visible spectrophotometry and high‐performance liquid chromatography, and corresponding rate constants (kr) were evaluated. Whenever possible, products were identified and quantified. Rate constants of 1O2 total quenching by the PT derivatives investigated were obtained from steady‐state 1O2 luminescence measurements. Results show that the behavior of conjugated PTs differs considerably from that of unconjugated derivatives, and the mechanisms of 1O2 physical quenching by these compounds and of their chemical reaction with 1O2 are discussed in relation to their structural features.

Substituent Effects on the Photophysical Properties of Pterin Derivatives in Acidic and Alkaline Aqueous Solutions

Abstract Pterins are heterocyclic compounds with important biological functions, and most of them may exist in two acid-base forms in the pH range between 3 and 13 in aqueous solution. In this work, the photophysical properties of acid and basic forms of six compounds of the pterin family (6-hydroxymethylpterin [HPT], 6-methylpterin [MPT], 6,7-dimethylpterin [DPT], rhamnopterin [RPT], N-methylfolic acid [MFA], and pteroic acid [PA]) have been studied. The effects of the chemical nature of the substituents at position 6 of the pterin moiety and the effects of the pH on the absorption and emission properties are analyzed. The fluorescence characteristics (spectra, quantum yields, lifetimes) of these compounds have been investigated using the single-photon-counting technique. Results obtained for pterin derivatives containing small substituents with 1 carbon atom (HPT, MPT, DPT) and short hydrocarbon chain (4 carbon atoms) (RPT) are different from those found for pterin derivatives containing a p-aminobenzoic acid (PABA) moiety in the substituent (MFA and PA). Fluorescence quantum yields (ΦF) of the first group of compounds are relatively high (≥0.4), whereas MFA and PA exhibit very small ΦF values (≤0.01).

Degradation of nitroaromatic compounds by the UV–H2O2 process using polychromatic radiation sources

The UV-H2O2 process, a standard advanced oxidation process (AOP) for water treatment, has been applied to the degradation of a series of nitroaromatic compounds (nitrobenzene, 1-chloro-2,4-dinitrobenzene, 2,4-dinitrophenol, 3-nitrophenol, 4-nitrophenol and 4-chloro-3,5-dinitrobenzoic acid) using polychromatic radiation sources. The optimal concentration of hydrogen peroxide ([H2O2]OPT) leading to the fastest degradation rate of a given substrate (S) was determined experimentally and estimated using a simplified kinetic model based on the main reactions involved in the first stages of the oxidation. We have shown that, under conditions of monochromatic irradiation, the ratio ROPT (= [H2O2]OPT/[S]0) is given by a simple mathematical expression containing only a few parameters, whereas, under conditions of polychromatic irradiation, ROPT is expressed by a complex mathematical equation (involving the spectral distribution of the lamp emission and the absorption spectra of H2O2 and the substrate). Two numerical analysis procedures are proposed for obtaining the bimolecular rate constants for the reaction of hydroxyl radicals with a substrate (kS) from this equation. The rate constants, kS, determined for the substrates investigated in this work are in agreement with the expected reactivity trend, taking into account the effects of substituents on the distribution of electron density in the aromatic ring. The methods proposed in this work offer a double advantage: i) a standard AOP may be used to evaluate the rate constants of reaction of substrates with hydroxyl radicals under polychromatic as well as under monochromatic irradiation, ii) optimal amounts of additive may be obtained using only a few parameters as predictive tools.

Photochemical Behavior of Folic Acid in Alkaline Aqueous Solutions and Evolution of Its Photoproducts

The photolysis of folic acid (=N-(4-{[(2-amino-1,4-dihydro-4-oxopteridin-6-yl)methyl]amino}benzoyl)-L-glutamic acid) in alkaline aqueous solution (pH 10.0–11.0) was carried out at 350 nm at room temperature and monitored by UV/VIS spectrophotometry, anal. and prep. thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC, HPLC/MS). The folate species underwent at least two independent photo-oxidation pathways, which were not observed when the acid form was photolyzed at pH<7. The presence of O2 was essential in these oxidation pathways. Evidence for the role of singlet oxygen was established. In one of the pathways, the folate underwent cleavage, yielding 6-formylpterin (=2-amino-1,4-dihydro-4-oxopteridine-6-carboxaldehyde) and (4-aminobenzoyl)glutamic acid as photoproducts. The other pathway yielded a new photostable product A of molecular mass 455, which could be isolated and stored in acidic or neutral aqueous solution. However, A was rather unstable in alkaline media undergoing a thermal reaction to a product B of lower molecular mass (427). The kinetics of this thermal reaction was analyzed with a stopped-flow spectrophotometer. A linear dependence of the first-order rate constant with the OH− concentration was observed. The corresponding bimolecular rate constant was 1.1 M−1 s−1. The quantum yields of substrate consumption and of photoproduct formation were determined. The here-reported photochemical behavior of folate solutions departs from results in acid media, where phototransformation proceeded via the cleavage of the acid form into 6-formylpterin and (4-aminobenzoyl)glutamic acid as the first major photoproducts, and where no thermal reactions were observed.

Quenching of the fluorescence of pterin derivatives by anions

Steady-state and time-resolved studies of the fluorescence of pterins in aqueous solutions in the presence of different anions have been performed using the single-photon counting technique. In the pH range between 3 and 13, most pterins exist in a protonated and a deprotonated form. Results obtained for both acid and basic forms of five compounds belonging to the pterin family (pterin, 6-carboxypterin, 6-formylpterin, biopterin and neopterin) show that the fluorescence of the acid forms is dynamically quenched by phosphate and acetate, and the corresponding bimolecular rate constants of fluorescence quenching (k(q)) are reported. These results are of importance from the technical and analytical points of view because measurements of the fluorescence of pterin derivatives for a variety of purposes are often performed in the presence of salts, especially buffers, and significant quenching of the pterin fluorescence by the buffer might lead to errors in interpretation and erroneous conclusions. No quenching of the fluorescence of the acid forms by chloride, sulfate or nitrate was detected. The fluorescence of the basic forms was either not quenched by anions or any such quenching was negligible in comparison with that observed for the acid forms.

Photoinduced cleavage of plasmid DNA in the presence of pterin

Summary The photoinduced cleavage of plasmid DNA by UV -A light in the presence of pterin was investigated. Electrophoretic analysis of the irradiated plasmid pUCI8 in the presence of pterin showed that UV light of 350 nm induced the transformation of a significant proportion of the supercoiled plasmid to its relaxed form. A minor proportion of plasmid forms are also converted to the linear plasmid isomer at the longer irradiation times. All these transformations during irradiation can be observed in the absorption spectrum of DNA as function of time. Such spectral modifications correlated with the extent and the kinetics of plasmid relaxation, but not with the appearance of the linear plasmid. None of the exchanges were operative without the irradiation with UV-A light. Control experiments with pterin or plasmid DNA irradiated separately, showed no photochemical changes. Results taken together suggest that the observed changes in the supercoiled plasmid as well as the spectral modifications both derive from the generation of single-strand break in the DNA.

Effect of temperature on hydrogen peroxide photolysis in aqueous solutions

Abstract The photolysis of hydrogen peroxide in dilute aqueous solution (1 × 10−4 M) at various temperatures (15–85°C) and pH (ph 2.5–7) was studied by flash photolysis. The rate of oxygen production under continuous photolysis conditions was measured at room temperature. The rate constants and activation parameters are reported. Evidence for the formation of complexes between hydrogen peroxide and intermediate radicals is presented. The liquid phase data are discussed and compared with those available for the gas phase.