Carolina Lorente

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).

Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways†

Here, 10 guidelines are presented for a standardized definition of type I and type II photosensitized oxidation reactions. Because of varied notions of reactions mediated by photosensitizers, a checklist of recommendations is provided for their definitions. Type I and type II photoreactions are oxygen‐dependent and involve unstable species such as the initial formation of radical cation or neutral radicals from the substrates and/or singlet oxygen (1O2 1∆g) by energy transfer to molecular oxygen. In addition, superoxide anion radical ( O2·− ) can be generated by a charge‐transfer reaction involving O2 or more likely indirectly as the result of O2‐mediated oxidation of the radical anion of type I photosensitizers. In subsequent reactions, O2·− may add and/or reduce a few highly oxidizing radicals that arise from the deprotonation of the radical cations of key biological targets. O2·− can also undergo dismutation into H2O2, the precursor of the highly reactive hydroxyl radical ( ·OH ) that may induce delayed oxidation reactions in cells. In the second part, several examples of type I and type II photosensitized oxidation reactions are provided to illustrate the complexity and the diversity of the degradation pathways of mostly relevant biomolecules upon one‐electron oxidation and singlet oxygen reactions.

Tryptophan oxidation photosensitized by pterin

Pterins are normal components of cells and they have been previously identified as good photosensitizers under UV-A irradiation, inducing DNA damage and oxidation of nucleotides. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the oxidation of another class of biomolecules, amino acids, using tryptophan (Trp) as a model compound. Irradiation of Ptr in the UV-A spectral range (350 nm) in aerated aqueous solutions containing Trp led to the consumption of the latter, whereas the Ptr concentration remained unchanged. Concomitantly, hydrogen peroxide (H₂O₂) was produced. Although Ptr is a singlet oxygen ((1)O₂) sensitizer, the degradation of Trp was inhibited in O₂-saturated solutions, indicating that a (1)O₂-mediated process (type II oxidation) was not an important pathway leading to Trp oxidation. By combining different analytical techniques, we could establish that a type I photooxidation was the prevailing mechanism, initiated by an electron transfer from the Trp molecule to the Ptr triplet excited state, yielding the corresponding radical ions (Trp(·+)/Trp(-H)· and Ptr(·-)). The Trp reaction products that could be identified by UPLC-mass spectrometry are in agreement with this conclusion.

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).

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.

Healing of bony defects in the irradiated and unirradiated rat mandible

A model system of the irradiated rat mandible has been developed and used in conjunction with a non-spontaneously healing mandibular defect. The contribution of the tissue components in the healing of bony defects was studied using demineralized bone powder (DBP) prepared from unirradiated or in vivo irradiated rat long bones. Better bony fill of the defects occurred in the irradiated beds filled with unirradiated DBP than in the unirradiated beds containing irradiated DBP. This suggests that, at least in the early postirradiation period, the bed is not the limiting factor in healing of bony defects and the osteogenic components of bone in the DBP may be most affected by irradiation. In the irradiated bed, the defects grafted 2 weeks after irradiation healed better than those grafted at 4 weeks. Thus, the timing of surgery after irradiation also plays a role in the healing process, with early surgery producing better results.

Production and quenching of reactive oxygen species by pterin derivatives, an intriguing class of biomolecules

Pterins, a family of heterocyclic compounds derived from 2-aminopteridin-4(1H)-one, are widespread in living systems and participate in important biological functions, such as metabolic redox processes. Under UV-A excitation (320–400 nm), aromatic pterins (Pt) can generate reactive oxygen species (ROS), as a consequence of both energy- and electron-transfer processes from their triplet excited state. Quantum yields of singlet oxygen (1O2) production depend largely on the nature of the substituents on the pterin moiety and on the pH. Formation of the superoxide anion by electron transfer between the pterin radical anion and molecular oxygen leads to the production of significant amounts of hydrogen peroxide (H2O2) by disproportionation. Dihydropterins (H2Pt) do not produce 1O2 but are oxidized by this species with high rate constants yielding pterins as well as H2O2. In contrast to aromatic derivatives, H2Pt are oxidized by H2O2, and rates and products strongly depend on the nature of the substituents on the H2Pt moiety. Aromatic pterins have been found in vivo under pathological conditions, e.g., biopterin or 6-carboxypterin are present in the skin of patients affected by vitiligo, a depigmentation disorder. The biomedical implications of the production of ROS by pterin derivatives and their reactivity with these species are discussed.

Electron-transfer processes induced by the triplet state of pterins in aqueous solutions

Pterins (Pt) are heterocyclic compounds widespread in living systems. They participate in relevant biological processes, such as metabolic redox reactions, and can photoinduce the oxidation of biomolecules through electron-transfer mechanisms. We have investigated the electron-transfer pathways initiated by excited states of pterin (Ptr) and 6-methylpterin (Mep), selected as model compounds. The experiments were carried out in aqueous solutions under continuous UV-A irradiation, in the presence and in the absence of ethylenediaminetetraacetic acid (EDTA), used as an electron donor. The reactions were followed by UV/Vis spectrophotometry, HPLC, and an enzymatic method for H(2)O(2) determination. The formation of the superoxide anion (O(2)(*-)) was investigated by electron paramagnetic resonance-spin trapping. The triplet excited states of Ptr and Mep are efficient electron acceptors, able to oxidize a Pt molecule in its ground state. The resulting radical anion (Pt(*-)) reacts with dissolved O(2) to yield O(2)(*-), regenerating the pterin. In the presence of EDTA, this reaction competes efficiently with the anaerobic reaction between Pt(*-) and EDTA(*+), yielding the corresponding stable dihydroderivatives H(2)Pt. The effects of EDTA and dissolved O(2) concentrations on the efficiencies of the different competing pathways were analyzed.