QSAR models for predicting enzymatic hydrolysis of new chemical entities in 'soft-drug' design.

The work described here is aimed at developing QSAR models capable of predicting in vitro human plasma lability/stability. They were built based on a dataset comprising about 200 known compounds. 3D structures of the molecules were drawn, optimized and submitted to the calculation of molecular descriptors that enabled selecting different TR/TS set pairs, subsequently exploited to develop QSAR models. Several 'machine learning' algorithms were explored in order to obtain suitable classification models, which were then validated on the relevant TS sets. Moreover the predictive ability of the best performing models was assessed on a Prediction set (PS) comprising about 40 molecules, not strictly related, from a structural point of view, to the initial dataset, but (obviously) comprised within the validity domain of the QSAR models obtained. The study allowed selecting predictive models enabling the classification of New Chemical Entities with regard to hydrolysis rate, that may be exploited for soft-drug design.

Metabolically labile cannabinoid esters: a 'soft drug' approach for the development of cannabinoid-based therapeutic drugs.

Biphenylic ester derivatives, designed by using a 'soft-drug' approach, proved to possess good binding properties toward cannabinoid CB(1) and CB(2) receptors and, at the same time, their metabolically labile ester portion would promote a rapid systemic inactivation. This may constitute a possible solution to the psychotropic side effects encountered when cannabinoids are therapeutically employed as local analgesic or antiglaucoma agents.

Development of an integrated method of skin phenotype measurement using the melanins.

Different types of melanin pigments have recently been identified and recognized as critical determinants of the photosensitivity of individuals. Eumelanin, the black to brown melanin pigments, are believed to protect against ultraviolet-induced cell damage, while phaeomelanin, the reddish brown variant, is thought to be photosensitizing. The relative, qualitative and absolute amount of melanin production under stimulation of solar radiation is likely to be genetically determined. The hypothesis of this study is that determination of these values can help in identifying those people who are less protected. However, these techniques must be evaluated at a population level and against traditional epidemiological measures. We assessed the amount and type of melanin in 195 subjects in four centres across Europe, relating the results to epidemiological measures such as skin characteristics, history of sunburns and number of naevi. The most important finding was that eumelanin and phaeomelanin have very different distributions in the population, being associated with other phenotype characteristics with different patterns. The relationship between phaeomelanin and eumelanin is linearly inverse in the range from black to dark blonde hair colour, while it is weakly directly proportional in the range from dark blonde to light blonde, with people with red hair showing a peculiar pattern. Phaeomelanin rather than eumelanin seemed to be independent of other skin characteristics. The results show the feasibility of a further study with an appropriate case-control design and accurate determination of melanin.

Zinc-catalyzed oxidation of 5-S-cysteinyldopa to 2,2'-bi(2H-1,4-benzothiazine): tracking the biosynthetic pathway of trichochromes, the characteristic pigments of red hair.

Trichochromes, the peculiar pigments of red human hair, featuring the Delta(2,2)(')-bi(2H-1,4-benzothiazine) skeleton, are known to arise from cysteinyldopas, mainly the 5-S-isomer (5). However, the mode of formation and the direct precursors have remained largely undefined. To fill this gap, we investigated the oxidation of 5 in air or with chemical and enzymatic agents under biomimetic conditions. In the presence of zinc ions, which occur in epidermal tissues at significant concentrations, the reaction course is diverted toward the formation of a labile 3-carboxy-2H-1,4-benzothiazine intermediate (11), which was identified by direct NMR analysis. Structural formulation was supported by characterization of the analogous compound 13 isolated from oxidation of the model 5-methyl-3-S-cysteinylcatechol (12) after methylation. In the further stages of the oxidation, diastereomeric 2,2'-bi(2H-1,4-benzothiazine) 15 and 14 were obtained from 5 and 12, respectively, the reaction proceeding at a higher rate and to a greater extent in the presence of acids. The dimers were shown to readily convert to each other in the presence of acids. In the case of the methylated dimers 14, a 2,2'-bi(4H-1,4-benzothiazine) intermediate (16) was isolated and characterized. In acidic media, trichochrome C (1a), the most abundant in red human hair, was smoothly formed from aerial oxidation of 15, and under similar conditions, trichochrome-related products (17 and 18) were obtained from 14 prior to or after methylation. The presence of 1a and precursors 5 and 15 was investigated by HPLC analysis of red hair samples following mild proteolytic digestion. On the basis of these data, a likely biosynthetic route to trichochrome pigments of red human hair is depicted.

An unusual decarboxylative Maillard reaction between L-DOPA and D-glucose under biomimetic conditions: factors governing competition with Pictet-Spengler condensation.

In 0.1 M phosphate buffer at pH 7.4 and 37 degrees C, the tyrosine metabolite L-3,4-dihydroxyphenylalanine (L-DOPA) reacts smoothly with D-glucose to afford, besides diastereoisomeric tetrahydroisoquinolines 1 and 2 by Pictet-Spengler condensation, a main product shown to be the unexpected decarboxylated Amadori compound N-(1-deoxy-D-fructos-1-yl)-dopamine (3). Under similar conditions, dopamine gave only tetrahydroisoquinoline products 4 and 5, whereas L-tyrosine gave exclusively the typical Amadori compound 6. Fe(3+) and Cu(2+) ions, which accumulate in relatively high levels in parkinsonian substantia nigra, both inhibited the formation of 3. Cu(2+) ions also inhibited the formation of 1 and 2 to a similar degree, whereas Fe(3+) ions increased the yields of 1 and 2. Apparently, the formation of 3 would not be compatible with a simple decarboxylation of the initial Schiff base adduct, but would rather involve the decarboxylative decomposition of a putative oxazolidine-5-one intermediate assisted by the catechol ring. These results report the first decarboxylative Maillard reaction between an amino acid and a carbohydrate under biomimetic conditions and highlight the critical role of transition metal ions in the competition with Pictet-Spengler condensation.

Pictet-Spengler condensation of the antiparkinsonian drug L-DOPA with D-glyceraldehyde. Opposite kinetic effects of Fe3+ and Cu2+ ions and possible implications for the origin of therapeutic side effects.

In 0.05 M phosphate buffer, pH 7.4, and at 37 degrees C. L-DOPA, a widely used antiparkinsonian drug, reacted smoothly with D-glyceraldehyde to afford diastereoisomeric (1R, 1'S,3S)-3-carboxy-1-(1',2'-dihydroxyethyl)-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline (1) and (1S,1'5S,3S)-3-carboxy-1-(1',2'-dihydroxyethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (2) in an approx. 3:2 ratio. The prevalent formation of 1 over 2 reflects stereoselective cyclisation of a transient Schiff base in accord with the Felkin-Anh model. Fe3+ ions, present at relatively high levels in parkinsonian brains, markedly accelerated formation of 1 and 2, whereas Cu2+ decreased the reaction rate, due apparently to different sites of chelate formation between L-DOPA and the metal ions. Both metal ions markedly decreased the stereoselectivity of the reaction. Product 1 exhibited chelating properties toward metal ions comparable or stronger than those of L-DOPA. These results throw new light on the effects of transition metal ions on the Pictet-Spengler reaction and suggest a possible role of tetrahydroisoquinoline products from L-DOPA and carbohydrate metabolites in the severe side effects of the drug.

Melanin density and melanin type predict melanocytic naevi in 19-20 year olds of northern European ancestry.

Recent advances in estimating the density of cutaneous melanin by spectrophotometry and the concentration of the two types of melanin (eumelanin and phaeomelanin) in hair offer the potential to define the risk of skin cancer in individuals more accurately. The presence of common melanocytic naevi on the arm is associated with an increased risk of melanoma, and in this study the associations of arm naevi with melanin density at the upper inner arm and with melanin type in hair samples were examined in a representative sample (n = 267) of 19-20 year olds of northern European ancestry. Particularly in men, the association with naevus count was stronger for cutaneous melanin density than for follicular melanin type. Adjusted for recreational sun exposure, the rank correlation coefficients were r = -0.25, 0.12 and 0.01 for men, and r = -0.17, -0.12 and 0.14 for women, for cutaneous melanin, hair eumelanin and hair phaeomelanin, respectively. The associations with less objective markers of phenotype (hair colour, eye colour, nurse-assessed skin colour, and self-reported skin reaction to unaccustomed sun) were weaker. These findings provide important new information that human susceptibility to mutations of melanocytes can be estimated by objective biological measures. The next step is to determine whether these measures also predict the risk of melanoma.

Latanoprost stimulates eumelanogenesis in iridial melanocytes of cynomolgus monkeys.

Latanoprost, the active principle of Xalatan eye drops, is a prostaglandin F2alpha analogue in widespread use for the treatment of glaucoma. During chronic treatment with the drug, an increased pigmentation of the iris was observed in both primates and man. To gain an insight into the nature of this effect, we analyzed the stroma of the irides of cynomolgus monkeys subjected to 25-38 weeks of treatment. A highly sensitive procedure, based on chemical degradation by alkaline hydrogen peroxide oxidation, or hydriodic acid hydrolysis, was developed, which allowed eumelanin and pheomelanin analysis of a single iris at a time. Untreated monkey irides were found to be essentially pheomelanic, providing further support to the recently reported occurrence of these pigments in human irides. In the Latanoprost-treated eyes, the amount of eumelanin increased from three to sevenfold, while the variation of pheomelanin did not exceed 25%. The increase in eumelanin/pheomelanin ratio in the treated eyes, as compared with the contralateral control eyes, varied from three to fivefold, and the change was statistically significant (P < 0.01; t-test). Based on the results of parallel studies, showing that Latanoprost does not induce proliferation of iridial melanocytes, and that the other pigmented layers of the iris which do not contain melanocytes are not affected by the drug, it can be concluded that the observed effect is a result of a direct interaction with the melanogenic mechanism. This probably involves activation of tyrosinase, as suggested, to account for the stimulation of melanin synthesis by related compounds, including natural prostaglandins.

Human melanocytes and melanomas express novel mRNA isoforms of the tyrosinase-related protein-2/DOPAchrome tautomerase gene: molecular and functional characterization.

We previously reported that a melanoma antigen, recognized by tumor-specific cytotoxic T lymphocytes, was encoded by intron sequences retained in a partially spliced transcript of the tyrosinase-related protein-2/DOPAchrome tautomerase gene. At difference with the mRNA encoding tyrosinase-related protein-2, this anomalous transcript was not expressed in melanocytes. This study examined whether neoplastic and/or normal cells of the melanocytic lineage could express additional forms of tyrosinase-related protein-2 mRNA. Screening of a melanoma-derived cDNA library with a tyrosinase-related protein-2 probe allowed identification of two novel isoforms. The first, tyrosinase-related protein-2-long tail, corresponds to the dominant transcript detected on melanomas and melanocytes by northern blot analysis. Tyrosinase-related protein-2-long tail is identical to the tyrosinase-related protein-2-encoding published cDNA sequence except for an extended 3'-untranslated region and is originated by alternative polyadenylation. This novel 3'-untranslated region contains an alternatively spliced, tyrosinase-related protein-2 last exon in the second isoform (tyrosinase-related protein-2-8b). The protein encoded by tyrosinase-related protein-2-8b is identical to tyrosinase-related protein-2 in its first 460 amino acids but possesses a different carboxyl-terminus devoid of transmembrane domain. Tyrosinase-related protein-2-long tail exhibited DOPA-chrome tautomerase activity, when transiently transfected into COS-7 cells. On the contrary, no detectable activity was exhibited by tyrosinase-related protein-2-8b. Reverse transcription-polymerase chain reaction analysis indicated that tyrosinase-related protein-2-long tail and tyrosinase-related protein-2-8b are expressed by tyrosinase-related protein-2-positive melanomas and normal melanocytes. Moreover all cell lines positive for tyrosinase-related protein-2 isoforms expressed tyrosinase and, all but one, tyrosinase-related protein-1. These data show that the human tyrosinase-related protein-2/DOPAchrome tautomerase gene can yield different isoforms by alternative poly(A) site usage or by alternative splicing. The pattern of expression of these isoforms suggest that they might play a part in the normal pathway of melanin biosynthesis.

A novel octahydropyridobenzothiazepine metabolite in human urine: biomimetic formation from the melanogen 5-S-cysteinyldopa and formaldehyde via a peculiar sulfur-controlled double Pictet-Spengler condensation.

HPLC evidence is reported demonstrating the occurrence in some human urine samples of a novel catecholic metabolite, (3R,7S)-3, 7-dicarboxy-10,11-dihydroxy-2,3,4,5,6,7,8,9-octahydropyrido[ 4,3-g][1, 4]benzothiazepine (2). The compound was shown to arise by a double Pictet-Spengler condensation of the urinary melanogen 5-S-cysteinyldopa (1) with formaldehyde, in which regioselective formation of the six-membered ring ortho to the activating hydroxyl group lends assistance to the subsequent closure of the seven-membered 1,4-thiazepine moiety. Under physiologically relevant conditions, i.e., in 0.1 M phosphate buffer pH 7.4 and at 37 degrees C, the 7,8-tetrahydroisoquinoline 5 was the sole detectable intermediate in the formation of 2. N-Acetylcysteinyldopa (4) reacted likewise with formaldehyde to give the 7, 8-dihydroxytetrahydroisoquinoline 6. The anomalous regiochemistry underlying formation of 5 and 6 was rationalized with the aid of AM1/PM3 calculations on the model alkylthiocatechol 10, predicting a higher HOMO-controlled reactivity on the position ortho rather than para to the activating hydroxyl group. The potential of the reported chemistry as a convenient synthetic access to the 2,3,4, 5-tetrahydro[1,4]benzothiazepine ring system is suggested by the efficient conversion of the cysteinylcatechol 3 to 8 in the presence of formaldehyde.

Isolation and characterization of mammalian eumelanins from hair and irides.

A new enzymatic procedure was developed for isolation of eumelanin from black human hair which might provide a substantially intact pigment for structural characterization. Sequential digestion with protease, proteinase K and papaine in the presence of dithiothreitol afforded a pigment with a 6% w/w protein content. HPLC analysis of pyrrole acids resulting from alkaline H(2)O(2) degradation, carboxyl content determination, and ferricyanide titration showed that the isolated pigment is made up of 5,6-dihydroxyindole (DHI)- and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units at a 6:1 ratio, exhibiting a significant degree of oxidative degradation. For comparison, a different eumelanin isolated from black bovine irides by a similar enzymatic procedure was analyzed. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the final pigment provided evidence for homologous series of DHICA oligomers, while chemical analysis allowed an estimate of 2:1 DHICA/DHI-derived units in the polymer, with a substantial proportion of intact o-diphenolic functions. Iris melanin proved able to promote the Fenton oxidation of deoxyribose while hair melanin was ineffective. Overall, these results provide, for the first time, unambiguous evidence for marked structural differences of mammalian eumelanins which may be directly related to the diversity of the sites of biosynthesis and storage, as well as to functional role of these pigments.

Microanalysis of melanins in mammalian hair by alkaline hydrogen peroxide degradation: identification of a new structural marker of pheomelanins.

A highly sensitive, easy-to-perform method for melanin analysis in pigmented tissues based on alkaline hydrogen peroxide degradation has been developed and accomplishes simultaneous determination of eumelanins and pheomelanins. Pyrrole-2,3,5- tricarboxylic acid, the typical eumelanin marker, was obtained in higher yields than in previous procedures. A benzothiazole acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, characterized in our previous studies as a specific marker of pheomelanins, and the newly identified 1,3-thiazole-2,4, 5-tricarboxylic acid were also used for pigment analysis. Optimal yields of the pigment markers were obtained at 24 h reaction time. Pyrrole-2,3,5-tricarboxylic acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, and 1, 3-thiazole-2,4,5- tricarboxylic acid were quantified in a single chromatographic analysis without fractionation or work up of the degradation mixture. The linearity (linearity coefficient from 0.997 to 0.999) was excellent and the inter-assay (percentage coefficient of variation values in the range 0.2-2, n = 6) and intra-assay (percentage coefficient of variation values

New regulatory mechanisms in the biosynthesis of pheomelanins: rearrangement vs. redox exchange reaction routes of a transient 2H-1, 4-benzothiazine-o-quinonimine intermediate.

Pheomelanins, the typical epidermal pigments of red haired, Celtic-type Caucasians, arise from oxidative cyclization of cysteinyldopas, mainly the 5-S-isomer CD, via 1,4-benzothiazines. However, the mechanism and the relative yields of formation of these intermediates have remained poorly defined. We have now examined the course of the oxidation of CD at physiological pHs, under different reaction conditions. Surprisingly, a consumption of CD far exceeding the stoichiometry of the oxidant was observed at low oxidant-to-substrate ratios, low temperatures and high substrate concentrations. The yields of the 3,4-dihydro-1,4-benzothiazine-3-carboxylic acid DHBCA vs. the non-carboxylated analogue DHB in the oxidation mixture, after NaBH4 reduction, were also found to depend markedly on the reaction conditions. Based on these and other results, a reaction scheme is proposed involving a transient o-quinonimine generated by oxidative cyclization of CD to which three different paths are offered, namely redox exchange with CD to give DHBCA (path A) or intramolecular rearrangement with (path B) or without (path C) decarboxylation, leading to the benzothiazine BTZ and the 3-carboxy analogue BTZCA, respectively. The relative operation of path A vs. path C was assessed by deuterium labeling experiments. These findings point to new mechanisms of regulation of the initial steps of pheomelanogenesis, bearing significant implications on the structure of the final pigment.

New reaction pathways of dopamine under oxidative stress conditions: nonenzymatic iron-assisted conversion to norepinephrine and the neurotoxins 6-hydroxydopamine and 6, 7-dihydroxytetrahydroisoquinoline.

Aerial oxidation of dopamine at concentrations as low as 50 microM in the presence of ferrous ions in phosphate buffer (pH 7.4) led in the early stages (6-8 h) to the formation of the quinone of the neurotoxin 6-hydroxydopamine, 2, followed (24 h) by a complex product pattern comprising main components norepinephrine (5), 3, 4-dihydroxybenzaldehyde (4), and the neurotoxic alkaloid 6, 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (3). Product formation required the assistance of metal ions such as Mn(II), Zn(II), and iron, in either the ferrous or ferric form. Product yields were shown to vary linearly with iron and dopamine concentration in the early phases of the reaction (2 h). Biologically relevant antioxidants, like glutathione and ascorbate, and metal chelators, e. g., 2,2'-bipyridyl, inhibited dopamine conversion to products 2-5, but not substrate consumption, while hydroxyl radical scavengers such as DMSO and mannitol did not alter the course of the reaction. On the contrary, mannitol increased product yields, an effect seen for other monosaccharides. Catalase exhibited a significant inhibitory effect particularly on the formation of 3 and 4. By using (18)O(2), evidence was obtained for incorporation of the label into the carbonyl oxygen of 4, but not into the hydroxyl group of 5. On the basis of these and other results, a complete mechanistic picture of the oxidation is drawn involving conversion of dopamine to the corresponding o-quinone and its quinonemethide tautomer with concomitant reduction of O(2) to H(2)O(2). Nucleophilic attack by H(2)O to the quinonemethide gives rise to 5, while H(2)O(2) addition leads to benzaldehyde 4 via a beta-aminohydroperoxide intermediate. This latter reaction path also gives formaldehyde which yields the isoquinoline 3 by Pictet-Spengler condensation with dopamine. The quinone 2 results from H(2)O(2) attack at the 6-position of dopamine o-quinone in agreement with previous studies. These results provide an insight into new routes of nonenzymatic conversion of dopamine to its metabolite norepinephrine and neurotoxic species which may become operative under conditions relevant to neurodegeneration.

5,6-Dihydroxyindoles in the fenton reaction: a model study of the role of melanin precursors in oxidative stress and hyperpigmentary processes.

Increasing evidence supports the view that diffusible melanin-related metabolites do not serve merely as pigment precursors, but may also act as modulators of the responses of the pigmentary cell melanocyte to external stimuli, especially to inflammation. In this study, the effect of melanin precursors 5, 6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) on the Fenton-induced oxidation of deoxyribose was investigated as a model of the oxidative stress processes triggered by the release of iron during inflammation. DHICA caused a powerful inhibition of the H(2)O(2)-Fe(II)/EDTA oxidation under both aerobic and anaerobic conditions, proving to be more efficient than typical hydroxyl radical (HO(*)) scavengers even at low concentrations with respect to deoxyribose. Conversely, DHI in air was a prooxidant at low indole:Fe(II) ratios, but shifted to an antioxidant at higher ratios (>6). The magnitude of the prooxidant effect increased by lowering the pH of the medium or by replacing Fe(II) with Fe(III), but was suppressed by exclusion of oxygen. Both the indoles retained their effects on the Fenton reaction in the absence of EDTA, as a result of their ability to chelate iron ions as evidenced by spectrophotometric experiments. Investigation of the reaction of DHI and DHICA with the Fenton reagent led to the conclusion that the indoles interact efficiently with HO(*), yielding indolesemiquinone species which are then converted to melanin pigments by self-coupling or disproportionation. At low DHI:iron molar ratios, the ability of semiquinones, generated by autoxidation of indoles, to recycle Fe(II) ions prevails, accounting for the observed prooxidant effect. Collectively, the results of this study provide new evidence for melanogenic 5,6-dihydroxyindoles as a novel class of biological antioxidants and point to these compounds as the key to interpreting the response of melanocytes to oxidative injuries. Moreover, the rapid formation of melanin following the exposure of 5, 6-dihydroxyindoles to the Fenton oxidation suggests new mechanisms of skin hyperpigmentation associated with inflammation.

Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.

Reaction of dopamine with D-glyceraldehyde under biomimetic conditions: stereoselective formation of tetrahydroisoquinolines and rate-accelerating effects of transition metal ions.

In 0.05 M phosphate buffer, pH 7.4, and at 37 degrees C, dopamine underwent a smooth Pictet-Spengler condensation with D-glyceraldehyde and D,L-glyceraldehyde-3-phosphate to afford diastereoisomeric tetrahydroisoquinolines. In the case of D-glyceraldehyde 1a/1b were formed in ca. 2:1 ratio. Treatment with carbonyldiimidazole converted 1a and 1b into the corresponding oxazinoisoquinolinones 2a and 2b which were separated and stereochemically characterised by NMR analysis. Transition metal ions commonly occurring in biological systems (e.g. Cu2+ and Fe3+) markedly accelerated the formation of 1a-1b without affecting the product ratio. Mechanistic evidence suggested the reversible generation of Schiff base intermediates, detected by 1H NMR, which undergo stereoselective cyclisation according to the Felkin-Anh model. Metal-chelation at the catechol group facilitates the rate-determining nucleophilic attack to the imine moiety by enhancing the electron density at the site of cyclisation. These results highlight an apparently overlooked effect of transition metal ions on the Pictet-Spengler reaction under biomimetic conditions and provide a chemical basis to postulate a role of carbohydrate metabolites as modulatory agents of dopaminergic neurotransmission via conversion to potentially bioactive tetrahydroisoquinoline derivatives.

Nitric oxide-induced oxidation of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid under aerobic conditions: non-enzymatic route to melanin pigments of potential relevance to skin (photo)protection.

Diffusible melanin-related metabolites have recently been suggested to subserve a variety of functions that are critical for protection of skin against inflammatory stimuli and oxidative tissue injury. We report here the results of in vitro studies showing that 5,6-dihydroxyindole (DHI) and its 2-carboxylic acid (DHICA) exhibit a marked reactivity toward potentially cytotoxic nitrogen oxides produced by autoxidation of nitric oxide (NO) under physiologically relevant conditions. Exposure of DHI or DHICA to NO in air-equilibrated 0.1 M phosphate buffer, pH 7.4, resulted in a fast, concentration-dependent consumption of the substrates and the concomitant deposition of dark melanin-like pigments. All NO-induced oxidations were completely inhibited in the absence of oxygen. Addition of 10 microM DHI and DHICA completely prevented the oxidation of 10 microM alpha-tocopherol in 0.1 M phosphate buffer, pH 7.4 in the presence of 300 microM NO. Overall, these results shed light on novel oxidative pathways of melanin-related metabolites of possible relevance to the mechanisms of skin hyperpigmentation under oxidative stress conditions.