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.

In Vitro Effect of 9,9'-Norharmane Dimer against Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the ß-carboline (ßC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.

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.

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.

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.

Photophysics and photochemistry of carminic acid and related natural pigments.

Carminic acid (CA) and other related compounds have been widely used as dyes in cultural heritage, cosmetics and the food industry. Therefore, the study of their properties upon photoexcitation is particularly important. In this work, the photophysical and photochemical properties of CA, carminic lake and other related pigments in aqueous solutions are revisited. Novel quantitative information regarding the fate of the photoexcited states is provided including the efficiency of reactive oxygen species (ROS) photosensitized production (i.e., singlet oxygen and hydrogen peroxide) as well as the efficiency of nonradiative deactivation pathways. Laser-induced optoacoustic spectroscopy (LIOAS) data revealed that for all the investigated compounds, almost all the absorbed energy is released as prompt heat to the media. This is in agreement with the fact that other deactivation pathways, including fluorescence (ΦF ∼ 10-3-10-5), photochemical degradation (ΦR ∼ 10-4) and/or photosensitized ROS formation (ΦH2O2 < 10-5 and ΦΔ âˆ¼ 0), are negligible or null. In addition, a comprehensive investigation of the photodegradation of CA and lake is herein reported. The influence of different experimental parameters such as irradiation wavelength and oxygen partial pressure was evaluated. UV-vis absorption and fluorescence emission spectroscopy in combination with chemometric data analysis were used to elucidate the relevant aspects of the photodegradation mechanism involved and the spectroscopic features of the photoproducts generated. In aqueous media, CA follows an O2-dependent photochemical degradation when subject to elapsed photoexcitation in the UVB, UVA and visible regions. The photoproduct profile depends on the excitation wavelength giving rise to quite distinctive spectroscopic profiles. With respect to lake, our data suggest that upon photoexcitation, this pigment releases a CA-like chromophore that follows a similar fate to CA.

DNA Oxidation Photoinduced by Norharmane Rhenium(I) Polypyridyl Complexes: Effect of the Bidentate N,N'-Ligands on the Damage Profile.

ReI -polypyridyl complexes have interesting and distinctive photochemical and photosensitizing properties. This work describes the capability to induce (or photoinduce) DNA damage of three ReI -complexes with a naturally occurring alkaloid called norharmane (nHo) as ligand: [Re(CO)3 (nHo)(L)]CF3 SO3 where L=2,2'-bipyridine (ReBpy), phenanthroline (RePhen) or dipyrido[3,2-a:2',3'-c]phenazine (ReDppz). The interaction of the complexes with DNA was investigated by steady-state and time-resolved spectroscopy. Data show that the mode and strength of interaction depend on the chemical structure of the bidentate ligand. The complexes show a major static contribution to the overall interaction, giving rise to the formation of noncovalent adducts with DNA, and the particular trend observed was RePhen>ReDppz>ReBpy. Photo-oxidation at the purine bases represents the major DNA damaging mechanism. RePhen also induces single-strand breaks in a yield similar to that of base damage, suggesting an additional photosensitizing pathway. We also performed the Ames test to evaluate the cytotoxic and mutagenic properties of both non-irradiated and photoexcited complexes. RePhen, but not the other complexes, turned out to be both toxic and phototoxic for the bacteria.

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.

Antifungal activity of ß-carbolines on Penicillium digitatum and Botrytis cinerea.

ß-carbolines (ßCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six ßCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic ßCs (harmine, harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-ßCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases.

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.

DNA damage induced by bare and loaded microporous coordination polymers from their ground and electronic excited states.

We report on interactions of cell free double-stranded DNA (dsDNA) with a selected subgroup of Microporous Coordination Polymers (MCPs). In particular, we have studied the influence of different metal ion constituents and chemically modified linkers using a set of five benzene carboxylate-based MCPs. Our results suggest that the DNA moiety can be structurally modified in two different ways: by direct MCPs-dsDNA interaction and/or through photosensitized processes. The extent of the observed damage was found to be strongly dependent on the charge density of the material. The potential use of the MCPs tested as inert carriers of photosensitizers was demonstrated by analyzing the interaction between dsDNA and harmine-loaded Cr-based materials, both in the absence of light and upon UVA irradiation.

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.

Rewiring Photosynthesis by Water-Soluble Fullerene Derivatives for Solar-Powered Electricity Generation.

Natural photosynthesis holds great potential to generate clean electricity from solar energy. In order to utilize this process for power generation, it is necessary to rewire photosynthetic electron transport chains (PETCs) of living photosynthetic organisms to redirect more electron flux toward an extracellular electrode. In this study, a semi-artificial rewiring strategy, which use a water-soluble fullerene derivative to capture electrons from PETCs and donate them for electrical current generation, is proposed. A positively charged fullerene derivative, functionalized with N,N-dimethyl pyrrolidinium iodide, is found to be efficiently taken up by the cyanobacterium Synechocystis sp. PCC 6803. The distribution of this fullerene derivative near the thylakoid membrane, as well as site-specific inhibitor assays and transient absorption spectroscopy, suggest that it can directly interact with the redox centers in the PETCs, particularly the acceptor side of photosystem I (PSI). The internalized fullerene derivatives facilitate the extraction of photosynthetic electrons and significantly enhance the photocurrent density of Synechocystis by approximately tenfold. This work opens up new possibility for the application of fullerenes as an excellent 3D electron carrier in living biophotovoltaics.

Distribution of photoactive ß-carboline alkaloids across Passiflora caerulea floral organs.

This study reports valuable information regarding the presence and concentration of a series of photoactive ß-carboline (ßCs) alkaloids (norharmane, harmane, harmine, harmol, harmaline, and harmalol) and their distribution across the floral age and organs of Passiflora caerulea. UHPLC-MS/MS data reported herein reveal that the ßCs' content ranged from 1 to 110 µg kg-1 , depending on the floral organ and age. In certain physiologically relevant organs, such as anthers, ßCs' content was one order of magnitude higher than in other organs, suggesting a special role for ßCs in this specific organ. ßCs' content also varied in a structure-dependent manner. Alkaloids bearing a hydroxyl group at position C(7) of the main ßC ring were present at concentrations one order of magnitude higher than other ßC derivatives investigated. UV-visible and fluorescence spectroscopy of the flower extracts provided complementary information regarding other biologically relevant groups of chromophores (phenolic/indolic derivatives, flavonoids/carotenes, and chlorophylls). Since flowers are constantly exposed to solar radiation, the presence of photoactive ßCs in floral organs may have several (photo)biological implications that are further discussed.

Mechanisms of DNA damage by photoexcited 9-methyl-ß-carbolines.

It has been well documented that ß-carboline alkaloids, particularly the 9-methyl derivatives, are efficient photosensitizers. However, structure-activity relationships are missing and the photochemical mechanisms involved in the DNA photodamage still remain unknown. In the present work, we examined the capability of three 9-methyl-ß-carbolines (9-methyl-norharmane, 9-methyl-harmane and 9-methyl-harmine) to induce DNA damage upon UVA excitation at physiological pH. The type and extent of the damage was analyzed together with the photophysical and binding properties of the ß-carboline derivatives investigated. The results indicate that even at neutral pH most of the DNA damage is generated from the protonated form of the excited ß-carbolines in a type-I reaction. Oxidized purine residues are produced in high excess over oxidized pyrimidines, single-strand breaks and sites of base loss. In addition, the excited neutral form of the ß-carbolines is responsible for significant generation of cyclobutane pyrimidine dimers (CPDs) by triplet-triplet-energy transfer. In the case of 9-methyl-norharmane, the yield of CPDs is increased in D2O, probably due to less rapid protonation in the deuterated solvent.

Glucosinolates, a natural chemical arsenal: More to tell than the myrosinase story.

Glucosinolates are a group of thioglucosides that belong to the class of plant nitrogen-containing natural products. So far, very little biological activity has been associated with intact glucosinolates. The hydrolysis of glucosinolates has, for long, attracted attention because of the potent biological activity of the hydrolysis products. From allelopathic to antiparasitic, antimicrobial and antineoplastic effects, the activity spectrum of the degradation products of typical glucosinolates has been the subject of much research. The present review seeks to address the various means of glucosinolate degradation (thermal, enzymatic, or chemical degradation) and the ensuing products. It also aims to draw a comparative profile of the various antimicrobial effects of these degradation products to provide a further understanding of the biological function of these important compounds.