Exploring potooxidative degradation pathways of harmol and harmalol alkaloids in water: effects of pH, excitation sources and atmospheric conditions.

This work explores the photochemical degradation of cationic species of 7-hydroxy-1-methyl-2H-pyrido[3,4-b]indole or harmol (1C) and the corresponding partially hydrogenated derivative 7-hydroxy-1-methyl-3,4-dihydro-2H-pyrido[3,4-b]indole or harmalol (2C) in aqueous solution. UV-visible absorption and fluorescence emission spectroscopy coupled with multivariate data analysis (MCR-ALS and PARAFAC), HPLC and HRESI-MS techniques were used for both quantitative and qualitative analysis. The formation of hydrogen peroxide reactive oxygen species (ROS) was quantified, and the influence of pH, oxygen partial pressure and photoexcitation source on the photochemical degradation of both compounds was assessed. The potential implications on the biosynthesis of ßCs and their biological role in living systems are discussed.

Photosensitizing properties and subcellular localisation of 3,4-dihydro-ß-carbolines harmaline and harmalol.

Harmaline (1) and harmalol (2) represent two 3,4-dihydro-ß-carboline (DHßCs) most frequently reported in a vast number of living systems. Fundamental aspects including the photosensitizing properties, cellular uptake, as well as the cyto- and phototoxicity of 1 and 2 were investigated herein. The molecular basis underlying the investigated processes are elucidated. Data reveal that both alkaloids show a distinctive pattern of extracellular DNA photodamage. Compound 1 induces a DNA photodamage profile dominated by oxidised purines and sites of base loss (AP sites), whereas 2 mostly induces single-strand breaks (SSBs) in addition to a small extent of purine oxidative damage. In both cases, DNA oxidative damage would occur through type I mechanism. In addition, a concerted hydrolytic attack is suggested as an extra mechanism accounting for the SSBs formation photoinduced by 2. Subcellular internalisation, cyto- and phototoxicity of 1 and 2 and the corresponding full-aromatic derivatives harmine (3) and harmol (4) also showed quite distinctive patterns in a structure-dependent manner. These results are discussed in the framework of the potential biological, biomedical and/or pharmacological roles reported for these alkaloids. The subtle structural difference (i.e., the exchange of a methoxy group for a hydroxyl substituent at C(7)) between harmaline and harmalol, gives rise to distinctive photosensitizing and subcellular localisation patterns.

Spectroscopic and quantum chemical characterization of the ground and lowest electronically excited singlet and triplet states of halo- and nitro-harmines in aqueous media.

Halogenated and nitro ß-carboline (ßCs) alkaloids have garnered increasing interest for their role in a broad range of biological, pharmacological and biotechnological processes. Addressing their spectroscopic and photophysical properties provide tools to further explore the presence of these alkaloids in complex biological matrices. In addition, these studies help to elucidate processes where these alkaloids are involved. The UV-visible and steady-state room temperature fluorescence of bromo- and nitro-harmines in an aqueous environment at different pHs, low-temperature phosphorescence (at 77 K) and quantum yields of singlet oxygen production are reported herein. Singlet (S0 and S1) and triplet (T1) electronic states are further analyzed using density functional theory (DFT) and the results compared with experimental data. Data are discussed in the framework of potential biotechnological applications of these ßC alkaloids.

Photophysical and spectroscopic features of 3,4-dihydro-ß-carbolines: a combined experimental and theoretical approach.

3,4-Dihydro-ß-carbolines (DHßCs) are a set of endogenously synthesized alkaloids spread over a great variety of living species (e.g., plants, animals and microorganisms), playing a broad spectrum of biological, biochemical and/or pharmacological roles, in a structure-dependent manner. Addressing unresolved fundamental aspects related to the photophysical properties of DHßCs might help to gain further insights into the molecular basis of the mechanisms of the biological processes where these alkaloids are involved. In this work, the UV-visible spectroscopic features of DHßCs are revisited and they are further analyzed by calculations at the Density Functional Theory (DFT) level. In addition, steady-state and time-resolved fluorescence spectroscopy, as well as quantitative singlet oxygen production analysis is reported. Data obtained herein are discussed in the framework of the potential biological role of these alkaloids.

N-Methyl-ß-carboline alkaloids: structure-dependent photosensitizing properties and localization in subcellular domains.

N-Methyl-ß-carboline (ßC) alkaloids, including normelinonine F (1b) and melinonine F (2b), have been found in a vast range of living species playing different biological, biomedical and/or pharmacological roles. Despite this, molecular bases of the mechanisms through which these alkaloids would exert their effect still remain unknown. Fundamental aspects including the photosensitizing properties and intracellular internalization of a selected group of N-methyl-ßC alkaloids were investigated herein. Data reveal that methylation of the ßC main ring enhances its photosensitizing properties either by increasing its binding affinity with DNA as a biomolecular target and/or by increasing its oxidation potential, in a structure-dependent manner. As a general rule, N(9)-substituted ßCs showed the highest photosensitizing efficiency. With the exception of 2-methyl-harminium, all the N-methyl-ßCs investigated herein induce a similar DNA photodamage profile, dominated largely by oxidized purines. This fact represents a distinctive behavior when comparing with N-unsubstituted-ßCs. On the other hand, although all the investigated compounds might accumulate mainly into the mitochondria of HeLa cells, methylation provides a distinctive dynamic pattern for mitochondrial uptake. While rapid (passive) diffusion is most probably reponsible for the prompt uptake/release of neutral ßCs, an active transport appears to mediate the (reatively slow) uptake of the quaternary cationic ßCs. This might be a consequence of a distinctive subcellular localization (mitochondrial membrane and/or matrix) or interaction with intracellular components. Biomedical and biotechnological implications are also discussed herein.

Light-induced full aromatization and hydroxylation of 7-methoxy-1-methyl-3,4-dihydro-2H-pyrido[3,4-b]indole alkaloid: Oxygen partial pressure as a key modulator of the photoproducts distribution.

Full-aromatic and partially hydrogenated ß-carboline (ßC) derivatives constitute a group of alkaloids widely distributed in a great variety of living systems. In plants and bacteria, tetrahydro-ßCs are the primary product of the Pictet-Spengler enzymatically catalyzed condensation. Tetrahydro-ßC skeleton is further modified giving rise to the formation of a vast set of derivatives including dihydro- and full-aromatic ßCs. However, in most of the cases, the later processes still remain unclear and other sources, such as photo-triggered reactions, deserve to be explored. In this context, the photophysic and photochemistry of 7-methoxy-1-methyl-3,4-dihydro-2H-pyrido[3,4-b]indole or harmaline (Hlina) in aqueous solution is reported herein. UV-visible absorption and fluorescence emission spectroscopy coupled with multivariate data analysis (PARAFAC), HPLC and HRESI-MS techniques were used for both quantitative and qualitative analysis. The formation singlet oxygen and hydrogen peroxide reactive oxygen species (ROS) was quantified and their role together with the influence of pH and oxygen partial pressure on the photochemical degradation of HlinaH+ was assessed. We report herein the first study on photochemical full-aromatization of a dihydro-ßC derivative. These results can shed some light on the ßCs biosynthesis and role in living systems.

DNA damage photo-induced by chloroharmine isomers: hydrolysis versus oxidation of nucleobases.

Photodynamic therapy (PDT) is an emerging clinical treatment currently being used against a wide range of both cancerous and noncancerous diseases. The search for new active photosensitizers as well as the development of novel selective delivery systems are the major challenges faced in the application of PDT. We investigated herein three chloroharmine derivatives (6-, 8- and 6,8-dichloroharmines) with quite promising intrinsic photochemical tunable properties and their ability to photoinduce DNA damage in order to elucidate the underlying photochemical mechanisms. Data revealed that the three compounds are quite efficient photosensitizers. The overall extent of photo-oxidative DNA damage induced by both 8-chloro-substituted ß-carbolines is higher than that induced by 6-chloro-harmine. The predominant type of lesion generated also depends on the position of the chlorine atom in the ß-carboline ring. Both 8-chloro-substituted ß-carbolines mostly oxidize purines via type I mechanism, whereas 6-chloro-harmine mainly behaves as a "clean" artificial photonuclease inducing single-strand breaks and site of base loss via proton transfer and concerted (HO--mediated) hydrolytic attack. The latter finding represents an exception to the general photosensitizing reactions and, to the best of our knowledge, this is the first time that this process is well documented. The controlled and selective production of different oxygen-independent lesions could be fine-tuned by simply changing the substituent groups in the ß-carboline ring. This could be a promising tool for the design and development of novel photo-therapeutic agents aimed to tackle hypoxic conditions shown in certain types of tumours.

Chemical and photochemical properties of chloroharmine derivatives in aqueous solutions.

Thermal and photochemical stability (Φ(R)), room temperature UV-vis absorption and fluorescence spectra, fluorescence quantum yields (Φ(F)) and lifetimes (τ(F)), quantum yields of hydrogen peroxide (Φ(H2O2)) and singlet oxygen (Φ(Δ)) production, and triplet lifetimes (τ(T)) have been obtained for the neutral and protonated forms of 6-chloroharmine, 8-chloroharmine and 6,8-dichloroharmine, in aqueous media. When it was possible, the effect of pH and oxygen concentration was evaluated. The nature of electronic transitions of protonated and neutral species of the three investigated chloroharmines was established using Time-Dependent Density Functional Theory (TD-DFT) calculations. The impact of all the foregoing observations on the biological role of the studied compounds is discussed.

Experimental and computational study of solvent effects on one- and two-photon absorption spectra of chlorinated harmines.

A combined experimental and computational study of solvent effects on one- and two-photon absorption spectra of three chlorinated harmine derivatives is presented. The systems studied were protonated forms of 6-chloroharmine, 8-chloroharmine and 6,8-dichloroharmine in two solvents, acetonitrile and water. For the computations, polarizable embedding density functional and coupled cluster response theory methods were used. The computations were able to model the solvent-dependent experimental data well. These results demonstrate that reasonably sophisticated computational methods can be successfully applied to accurately study linear and nonlinear spectroscopic properties of comparatively large organic molecules in solution.

Determining the molecular basis for the pH-dependent interaction between 2'-deoxynucleotides and 9H-pyrido[3,4-b]indole in its ground and electronic excited states.

Interaction between norharmane and three different 2'-deoxynucleotides (dNMP) (2'-deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxyadenosine 5'-monophosphate (dAMP) and 2'-deoxycytidine 5'-monophosphate (dCMP)), in aqueous solution, was studied in the ground state by means of UV-vis and (1)H-NMR spectroscopy and in the first electronic excited state using steady-state and time-resolved fluorescence spectroscopy. In all cases, the norharmane-dNMP interaction dependence on the pH was examined. Possible mechanisms for the interaction of both ground and electronic excited states of norharmane with nucleotides are discussed. Spectroscopic, molecular modeling and chemometric analysis were performed to further characterize the chemical structure of the complexes formed and to get additional information concerning the interaction between dNMPs and norharmane.

Selective quenching of triplet excited states of pteridines.

Steady-state and time-resolved studies on quenching of excited states of pterin (Ptr) and lumazine (Lum) in the presence of iodide in aqueous solution have been performed. In contrast to the typical iodide enhancement in the triplet state population, iodide promotes a fast non-radiative T1→ S0 transition for both Ptr and Lum. In this work, we present evidence for the effective iodide-induced deactivation of singlet and triplet excited states, with rate constants close to the diffusion-controlled limit (between 3 × 10(9) M(-1) s(-1) and 1 × 10(10) M(-1) s(-1)). The longer lifetimes of the triplet excited states over the singlet excited states increase the probability of deactivation (k(T)(q)τ(0)(T)≫k(S)(q)τ(0)(S)). Therefore, at micromolar concentrations of iodide, where the deactivation of the singlet excited state is negligible, an efficient deactivation of the triplet excited states is observed. This selective deactivation of the excited triplet state is an analytical tool for the study of photosensitized reactions where pteridines are involved.

Mechanism of electron transfer processes photoinduced by lumazine.

UV-A (320-400 nm) and UV-B (280-320 nm) radiation causes damage to DNA and other biomolecules through reactions induced by different endogenous or exogenous photosensitizers. Lumazines are heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. The parent and unsubstituted compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) is able to act as photosensitizer through electron transfer-initiated oxidations. To get further insight into the mechanisms involved, we have studied in detail the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by Lum in aqueous solution. After UV-A or UV-B excitation of Lum and formation of its triplet excited state ((3)Lum*), three reaction pathways compete for the deactivation of the latter: intersystem crossing to singlet ground state, energy transfer to O(2), and electron transfer between dAMP and (3)Lum* yielding the corresponding pair of radical ions (Lum˙(-) and dAMP˙(+)). In the following step, the electron transfer from Lum˙(-) to O(2) regenerates Lum and forms the superoxide anion (O(2)˙(-)), which undergoes disproportionation into H(2)O(2) and O(2). Finally dAMP˙(+) participates in subsequent reactions to yield products.

Photodynamic effects of pterin on HeLa cells.

Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. In the present study, we ascertained that 2-aminopteridin-4(3H)-one, abbreviated as Ptr, is readily incorporated into and/or onto cervical cancer cells (HeLa) and that these cells die upon UV-A irradiation of Ptr. Cell death was assessed using two tests: (1) the Rhodamine 123 fluorescence assay for mitochondrial viability and (2) the Trypan Blue assay for membrane integrity. The data suggest that, for Ptr-dependent photoinitiated cell death, events related to mitochondrial failure precede those associated with the failure of the cell membrane.

Mechanism of photooxidation of folic acid sensitized by unconjugated pterins.

Folic acid, or pteroyl-l-glutamic acid (PteGlu), is a precursor of coenzymes involved in the metabolism of nucleotides and amino acids. PteGlu is composed of three moieties: a 6-methylpterin (Mep) residue, a p-aminobenzoic acid (PABA) residue, and a glutamic acid (Glu) residue. Accumulated evidence indicates that photolysis of PteGlu leads to increased risk of several pathologies. Thus, a study of PteGlu photodegradation can have significant ramifications. When an air-equilibrated aqueous solution of PteGlu is exposed to UV-A radiation, the rate of the degradation increases with irradiation time. The mechanism involved in this "auto-photo-catalytic" effect was investigated in aqueous solutions using a variety of tools. Whereas PteGlu is photostable under anaerobic conditions, it is converted into 6-formylpterin (Fop) and p-aminobenzoyl-l-glutamic acid (PABA-Glu) in the presence of oxygen. As the reaction proceeds and enough Fop accumulates in the solution, a photosensitized electron-transfer process starts, where Fop photoinduces the oxidation of PteGlu to Fop, and H(2)O(2) is formed. This process also takes place with other pterins as photosensitizers. The results are discussed with the context of previous mechanisms for processes photosensitized by pterins, and their biological implications are evaluated.

Oxidation of 2'-deoxyadenosine 5'-monophosphate photoinduced by lumazine.

UV radiation induces damages to the DNA molecule and its components through photosensitized reactions. Among these processes, photosensitized oxidations may occur through electron transfer or hydrogen abstraction (type I mechanism) and/or the production of singlet molecular oxygen ((1)O(2)) (type II mechanism). Lumazines are an important family of heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. To evaluate the capability of lumazines to act as photosensitizers through type I mechanism, we have investigated the oxidation of 2'-deoxyadenosine 5'-monophosphate (dAMP) photosensitized by the specific compound called lumazine (pteridine-2,4(1,3H)-dione; Lum) in aqueous solutions under UV irradiation. The photochemical reactions were followed by UV/vis spectrophotometry, HPLC, electrochemical measurement of dissolved O(2), and an enzymatic method for H(2)O(2) determination. The effect of pH was evaluated and the participation of oxygen was investigated. In aerated solutions, oxidation of dAMP photoinduced by the acid form of Lum (pH 5.5) takes place through a type I mechanism, in which the excitation of Lum is followed by an electron transfer from dAMP molecule to the Lum triplet excited state. During the process, O(2) is consumed and H(2)O(2) is generated, whereas the photosensitizer is not consumed. In contrast, no evidence of a photochemical reaction induced by the basic form of Lum (pH 10.5) was observed.

The photosensitizing activity of lumazine using 2'-deoxyguanosine 5'-monophosphate and HeLa cells as targets.

Lumazines are an important family of heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. Upon UV irradiation, the specific compound called lumazine (pteridine-2,4(1,3H)-dione) is able to generate singlet oxygen (1O2), which is one of the main chemical species responsible for photodynamic effects. To further assess the photosensitizing capability of lumazine (Lum) experiments were performed using the nucleotide 2'-deoxyguanosine 5'-monophosphate (dGMP) and, independently, cervical cancer cells (HeLa cell line) as targets. In the dGMP experiments, the data revealed that dGMP indeed undergoes oxidation/oxygenation photoinduced by Lum. Moreover, dGMP disappearance proceeds through two competing pathways: (1) electron transfer between dGMP and excited-state Lum (Type I process) and (2) reaction of dGMP with 1O2 produced by Lum (Type II process). The multistep processes involved are convoluted and susceptible to changes in experimental conditions. The independent studies with HeLa cells included fluorescence analysis of cell extracts and phototoxicity experiments performed at the single-cell level. Results showed that, upon Lum uptake and irradiation, photodynamic effects occur. In particular, the mitochondria and cell membrane were perturbed, both of which reflect key stages in cell death. The data reported herein illustrate how the irradiation of an endogenous biological compound can have various effects which, depending on the system, can be manifested in different ways.

One- and two-photon excitation of beta-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.