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.

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.

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.

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.

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.

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.

In vitro Effect of Harmine Alkaloid and Its N-Methyl Derivatives Against Toxoplasma gondii.

Toxoplasmosis is one of the most prevalent and neglected zoonotic global diseases caused by Toxoplasma gondii. The current pharmacological treatments show clinical limitations, and therefore, the search for new drugs is an urgent need in order to eradicate this infection. Due to their intrinsic biological activities, ß-carboline (ßC) alkaloids might represent a good alternative that deserves further investigations. In this context, the in vitro anti-T. gondii activity of three ßCs, harmine (1), 2-methyl-harminium (2), and 9-methyl-harmine (3), was evaluated herein. Briefly, the three alkaloids exerted direct effects on the parasite invasion and/or replication capability. Replication rates of intracellular treated tachyzoites were also affected in a dose-dependent manner, at noncytotoxic concentrations for host cells. Additionally, cell cycle analysis revealed that both methyl-derivatives 2 and 3 induce parasite arrest in S/M phases. Compound 3 showed the highest irreversible parasite growth inhibition, with a half maximal inhibitory concentration (IC50) value of 1.8 ± 0.2 µM and a selectivity index (SI) of 17.2 at 4 days post infection. Due to high replication rates, tachyzoites are frequently subjected to DNA double-strand breaks (DSBs). This highly toxic lesion triggers a series of DNA damage response reactions, starting with a kinase cascade that phosphorylates a large number of substrates, including the histone H2A.X to lead the early DSB marker γH2A.X. Western blot studies showed that basal expression of γH2A.X was reduced in the presence of 3. Interestingly, the typical increase in γH2A.X levels produced by camptothecin (CPT), a drug that generates DSB, was not observed when CPT was co-administered with 3. These findings suggest that 3 might disrupt Toxoplasma DNA damage response.

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.

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.

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.

ß-Carboline derivatives as novel antivirals for herpes simplex virus.

Several commercial and novel synthetic ß-carbolines (ßCs) were evaluated for their antiviral activity against herpes simplex virus type 1 (HSV-1) using an adapted MTS assay. Of 21 drugs tested, although 11 exerted antiviral activity at non-cytotoxic concentrations, only 3 of them [9-methyl-norharmane (9-Me-nHo), 9-methyl-harmane (9-Me-Ho) and 6-methoxy-harmane (6-MeO-Ho)] completely avoided virus-driven cytopathic effects. Half-maximal effective concentrations (EC50 values) (4.9 ± 0.4, 5.9 ± 0.8 and 19.5 ± 0.3 µM, respectively) and selectivity indexes (88.8, 40.2 and 7.0, respectively) of the latter three ßCs against HSV-1 were determined by MTS, flow cytometry and plaque reduction assays. The mode of action of these drugs was also evaluated. According to time-of-addition assays, the selected compounds were not virucidal and did not interfere with attachment or penetration of HSV-1, but interfered with later events of virus infection. Western blot studies showed that early and late protein expression was significantly delayed or even suppressed. Herpes simplex virus type 2 (HSV-2) was also inhibited by the selected substances in a similar manner. Interestingly, 6-MeO-Ho, 9-Me-Ho and 9-Me-nHo restricted HSV-1 ICP0 localisation to the nucleus during later stages of infection, possibly affecting its functionality in the cytoplasm where ICP0 normally inhibits antiviral signalling and promotes viral replication. In silico prediction of ADME (Absorption, Distribution, Metabolism and Excretion) properties showed that all compounds fulfilled Lipinski's rule and their calculated absorptions were >95%.

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.

Photophysical and Photochemical Properties of Naturally Occurring normelinonine F and Melinonine F Alkaloids and Structurally Related N(2)- and/or N(9)-methyl-ß-carboline Derivatives.

In the present work, we have synthesized and fully characterized the photophysical and photochemical properties of a selected group of N-methyl-ß-carboline derivatives (9-methyl-ß-carbolines and iodine salts of 2-methyl- and 2,9-dimethyl-ß-carbolinium) in aqueous solutions, in the pH range 4.0-14.5. Moreover, despite the quite extensive studies reported in the literature regarding the overall photophysical behavior of N-unsubstituted ßCs, this work constitutes the first full and unambiguous characterization of anionic species of N-unsubstituted ßCs (norharmane, harmane and harmine), present in aqueous solution under highly alkaline conditions (pH > 13.0). Acid dissociation constants (Ka ), thermal stabilities, room temperature UV-visible absorption and fluorescence emission and excitation spectra, fluorescence quantum yields (ФF ) and fluorescence lifetimes (τF ), as well as quantum yields of singlet oxygen production (Ð¤Δ ) have been measured for all the studied compounds. Furthermore, for the first time to our knowledge, chemometric techniques (MCR-ALS and PARAFAC) were applied on these systems, providing relevant information about the equilibria and species involved. The impact of all the foregoing observations on the biological role, as well as the potential biotechnological applications of these compounds, is discussed.

A comprehensive analysis of direct and photosensitized attenuation of Toxoplasma gondii tachyzoites.

In the present work, we have evaluated the effect of three different types of radiation: UVC (254±5nm), UVA (365±20nm) and visible (420±20nm) on different morphological and biological functions of Toxoplasma gondii tachyzoites. Briefly, UVC and UVA showed an inhibitory effect on parasite invasion in a dose-dependent manner. UVC showed the strongest effect inducing both structural damage (antigens) and functional inhibition (i.e., invasion and replication). On its own, visible light induces a quite distinctive and selective pattern of parasite-attenuation. This type of incident radiation inhibits the replication of the parasite affecting neither the capability of invasion/attachment nor the native structure of proteins (antigens) on parasites. Such effects are a consequence of photosensitized processes where phenol red might act as the active photosensitizer. The potential uses of the methodologies investigated herein are discussed.

UVA Photoactivation of Harmol Enhances Its Antifungal Activity against the Phytopathogens Penicillium digitatum and Botrytis cinerea.

Phytopathogenic fungi responsible for post-harvest diseases on fruit and vegetables cause important economic losses. We have previously reported that harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol) is active against the causal agents of green and gray molds Penicillium digitatum and Botrytis cinerea, respectively. Here, antifungal activity of harmol was characterized in terms of pH dependency and conidial targets; also photodynamic effects of UVA irradiation on the antimicrobial action were evaluated. Harmol was able to inhibit the growth of both post-harvest fungal disease agents only in acidic conditions (pH 5), when it was found in its protonated form. Conidia treated with harmol exhibited membrane integrity loss, cell wall disruption, and cytoplasm disorganization. All these deleterious effects were more evident for B. cinerea in comparison to P. digitatum. When conidial suspensions were irradiated with UVA in the presence of harmol, antimicrobial activity against both pathogens was enhanced, compared to non-irradiated conditions. B. cinerea exhibited a high intracellular production of reactive oxygen species (ROS) when was incubated with harmol in irradiated and non-irradiated treatments. P. digitatum showed a significant increase in ROS accumulation only when treated with photoexcited harmol. The present work contributes to unravel the antifungal activity of harmol and its photoexcited counterpart against phytopathogenic conidia, focusing on ROS accumulation which could account for damage on different cellular targets.