Franco M. Cabrerizo

One- and Two-Photon Excitation of β-Carbolines in Aqueous Solution: pH-Dependent Spectroscopy, Photochemistry, and Photophysics

Beta-carboline (betaC) alkaloids are present in a wide range of biological systems and play a variety of significant photodependent roles. In this work, a study of the aqueous solution-phase photochemistry, photophysics, and spectroscopy of three important betaCs [norharmane (nHo), harmane (Ho), and harmine (Ha)] and two betaC derivatives [N-methylnorharmane (N-Me-nHo) and N-methylharmane (N-Me-Ho)] upon one- and two-photon excitation is presented. The results obtained depend significantly on pH, the ambient oxygen concentration, and the betaC substituent and provide unique insight into a variety of fundamental photophysical phenomena. The data reported herein should not only help to understand the roles played by betaC alkaloids in biological systems but should also help in the development of methods by which the photoinduced behavior of these important compounds can be controlled.

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).

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.

Photochemistry of norharmane in aqueous solution

The photochemistry of norharmane (9H-pyrido[3,4-b]indole) in acidic (pH 5.0+/-0.1) and alkaline (pH 10.0+/-0.1) aqueous solutions was studied. The photochemical reactions were monitored by TLC, UV/VIS absorption spectroscopy, high-performance liquid chromatography (HPLC), electronic ionization-mass spectrometry (EI-MS), UV-laser desorption/ionization-time of flight-mass spectrometry (UV-LDI-TOF-MS) and an enzymatic method for H2O2 determination. The neutral (nHoN) and the protonated (nHoH+) forms of norharmane irradiated under Ar atmosphere were photostable, but they suffered a photochemical transformation in the presence of O2, yielding as photoproducts norharmane dimers, trimers and tetramers. nHoN shown to be more photostable than nHoH+. The nHoH+ and nHoN consumption quantum yields were 1.82x10(-3) and 0.51x10(-3), respectively, and the mechanisms involved in its photochemistry are discussed. H2O2 and singlet oxygen (1O2) were also detected and quantified in irradiated solutions of norharmane, and their role in the photochemistry of norharmane is discussed.

Fluorescence Quenching by Oxygen: “Debunking” a Classic Rule

It has long been accepted that oxygen-induced deactivation of an organic molecule’s fluorescent state (i.e. the lowest excited singlet state, S1) proceeds exclusively via intersystem crossing (ISC) to produce the triplet excited state, T1. [1–4] Oxygeninduced S1!S0 internal conversion (IC) to produce the singlet ground state, S0, is thought not to occur. In a light-filled world where S1 generation is commonplace and where molecular oxygen can be found at appreciable concentrations, this phenomenon can have significant implications. Among other things, it means that much O2-dependent photochemistry derives from the T1 state and, as such, can yield unique and characteristic products. To our knowledge, only 9,10-diphenylanthracene has been presented as an exception to the rule that O2 always induces S1!T1 ISC. Even in this case, however, the data are not as convincing as one might otherwise desire. We recently suggested that b-carbolines, bCs, may provide substantive evidence that an O2-induced S1!S0 transition can actually occur. We now demonstrate that the O2-dependent photophysics of selected bCs indeed provide an “exception to the rule” and that O2-induced S1!S0 IC can efficiently compete with O2induced S1!T1 ISC. The present data, point to the possibility that other molecules will respond to O2 like the bCs, limiting T1-derived chemistry or behavior which, in turn, opens new options for the design and application of unique photochemical, photophysical, and photobiological systems. The bCs used, norharmane (nHo, R1 = R2 = H), harmane (Ho, R1 = H, R2 = CH3) and N-methylharmane (NMeHo, R1 = R2 = CH3), Scheme 1, are water soluble compounds of significant biological importance whose behavior depends on pH. Although the S1!S0 phenomenon presented herein is observed in both alkaline and acidic media, it is more pronounced in the latter and, as such, we limit our discussion to data recorded at pH 5. We also limit our discussion to Ho ; data for nHo and NMeHo can be found in the Supporting Information. For Ho, the fluorescence quantum yield, FF, decreases as the concentration of dissolved oxygen increases (Figure 1 a). This is expected with the bimolecular quenching of S1 by O2. Indeed,

Γ-Al2O3-Supported XMo6 Anderson Heteropolyoxomolybdates: Adsorption Studies for X = TeVI, AlIII, CoIII, CrIII and NiII by DR Spectroscopy and TPR Analysis

The support–overlayer adsorptive interactions, generated when solutions of Anderson heteropolyoxomolybdates were supported as a monolayer on γ-Al2O3 by means of the equilibrium adsorption method, were studied by XRD analysis, DR spectroscopy, electron microscopy (SEM–EDAX) and TPR methods. The adsorption isotherms and corresponding adsorption parameters (number of active sites and adsorption constants) of the γ-Al2O3-supported ammonium heptamolybdate were obtained and discussed. The following sequence of adsorption strength was suggested: CoMo6 > CrMo6 > TeMo6 ~ AlMo6 > NiMo6. The effectiveness of the TPR technique as a tool for elucidating interaction effects was clearly demonstrated. The formation of a γ-Al2O3-supported Te–Mo mixed valence oxide appears to provide new and interesting catalytic possibilities.

In vitro evaluation of β-carboline alkaloids as potential anti-Toxoplasma agents

BackgroundToxoplasmosis is a worldwide infection caused by the protozoan parasite Toxoplasma gondii, which causes chorioretinitis and neurological defects in congenitally infected newborns or immunodeficient patients. The efficacy of the current treatment is limited, primarily by serious host toxicity. In recent years, research has focused on the development of new drugs against T. gondii. β-Carbolines (βCs), such as harmane, norharmane and harmine, are a group of naturally occurring alkaloids that show microbicidal activity. In this work, harmane, norharmane and harmine were tested against T. gondii.FindingsThe treatment of extracellular tachyzoites with harmane, norharmane and harmine showed a 2.5 to 3.5-fold decrease in the invasion rates at doses of 40 μM (harmane and harmine) and 2.5 μM (norharmane) compared with the untreated parasites. Furthermore, an effect on the replication rate could also be observed with a decrease of 1 (harmane) and 2 (norharmane and harmine) division rounds at doses of 5 to 12.5 μM. In addition, the treated parasites presented either delayed or no monolayer lysis compared with the untreated parasites.ConclusionsThe three βC alkaloids studied (norharmane, harmane and harmine) exhibit anti-T. gondii effects as evidenced by the partial inhibition of parasite invasion and replication. A dose–response effect was observed at a relatively low drug concentration (< 40 μM), at which no cytotoxic effect was observed on the host cell line (Vero).

Photochemical Behavior of 6-Methylpterin in Aqueous Solutions: Generation of Reactive Oxygen Species¶

Abstract Pterins are a family of heterocyclic compounds present in a wide range of living systems that participate in relevant biological functions and are involved in different photobiological processes. 6-Methylpterin (MPT) was investigated for its efficiency of singlet-oxygen (1O2) production and quenching in aqueous solution. The quantum yields of 1O2 production (ΦΔ) was determined by measurements of the 1O2 luminescence in the near-infrared upon continuous excitation of the sensitizer. Values of ΦΔ were found to be 0.10 ± 0.02 and 0.14 ± 0.02 in acidic and alkaline media, respectively. Studies of the photooxidation of MPT in acidic (pH = 5.0–6.0) and alkaline (pH = 10.2–10.8) aqueous solutions at 350 nm and room temperature have been performed. The photochemical reactions were followed by UV-visible spectrophotometry, high-performance liquid chromatography and an enzymatic method for H2O2 determination. MPT is not light sensitive in the absence of oxygen, but it undergoes a photooxidation reaction in the presence of oxygen, yielding several nonpteridinic products. The quantum yields of MPT disappearance were determined and values of 2.4 (±0.5) × 10−4 and 8.1 (±0.8) × 10−4 were obtained in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of MPT. The rate constant of the chemical reaction between 1O2 and MPT (kr) was determined to be 4.9 × 106 M−1 s−1 in alkaline medium and the role of 1O2 in the photooxidation of MPT is discussed.

New Results on the Photochemistry of Biopterin and Neopterin in Aqueous Solution

New photochemical studies of the reactivity of biopterin (BPT) and neopterin (NPT) in acidic (pH = 5.5) and alkaline (pH = 10.5) aqueous solutions at 350 nm and room temperature were performed. The photochemical properties of BPT are of particular interest because the photolysis of this compound takes place in the white skin patches of patients affected by vitiligo. The photochemical reactions were followed by UV/VIS spectrophotometry, HPLC, electrochemical measurement of dissolved O2 and enzymatic methods for hydrogen peroxide (H2O2) and superoxide anion (O2•−) determinations. When BPT or NPT are exposed to UVA radiation, a red intermediate, very likely 6‐formyl‐5,8‐dihydropterin, is generated in an O2‐independent process. That product is rapidly oxidized on admission of O2 to yield 6‐formylpterin and H2O2. When the photolysis takes place in aerobic conditions, no additional pathways exist. On the other hand, in the absence of O2, the intermediate generated is not stable and leads to the formation of many products. O2•− is also generated during photo‐oxidation of BPT and NPT. The quantum yields of reactant consumption depends on the O2 concentration: the higher the O2 concentration, the lower the quantum yields. This behavior is discussed in connection with the excited state of the pterins.