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Oxidative cross-coupling reactions of substituted o-aminophenols were catalyzed by a commercial laccase to produce non-symmetrically substituted phenoxazinones for the first time. Identification by 1H-, 13C- and 31P-NMR, and by HPLC-PDA and HPLC-MS/MS of exclusively two kinds of substituted phenoxazinones out of four potential heterocyclic frameworks was confirmed by a DFT study. The redox-properties of the substrates, their relative rates of conversion and the rigid docking of selected substrates led to a revisited mechanistic pathway for phenoxazinones biosynthesis. Our suggestions concern both the first formal two-electron oxidation by laccase and the first intermolecular 1,4-conjugated addition which secures the observed regioselectivity.

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize

o-Aminophenols have been long recognised as tyrosinase substrates. However their exact mode of interaction with the enzyme's active site is unclear. Properly vic-substituted o-aminophenols could help gain some insight into tyrosinase catalytic mechanism. Methods: Eight vic-substituted o-aminophenols belonging to two isomeric series were systematically evaluated as tyrosinase substrates and/or activators and/or inhibitors, by means of spectrophotometric techniques and HPLC-MS analysis. Some relevant kinetic parameters have also been obtained. Results: Four o-aminophenolic compounds derived from 3-hydroxyorthanilic acid (2-amino-3-hydroxybenzenesulfonic acid) and their four counterparts derived from the isomeric 2-hydroxymetanilic acid (3-amino-2- hydroxybenzenesulfonic acid) were synthesised and tested as putative substrates for mushroom tyrosinase. While the hydroxyorthanilic derivatives were quite inactive as both substrates and inhibitors, the hydroxymetanilic compounds on the contrary all acted as substrates for the enzyme, which oxidised them to the corresponding phenoxazinone derivatives. General significance: Based on the available structures of the active sites of tyrosinases, the different affinities of the fourmetanilic derivatives for the enzyme, and their oxidation rates,we propose a new hypothesis regarding the interaction between o-aminophenols and the active site of tyrosinase that is in agreement with the obtained experimental results.

Laccases are members of the multicopper oxidase family which are involved in numerous biosynthetic pathways in fungi. Among these, one laccase has been shown to produce in vitro a natural pigment, namely the cinnabarinic acid, which is related to the actinomycin chromophore. The core structure of this chromophore, i.e 2-amino-3H-phenoxazin-3-one, was found to possess a marked potential for the fluorescent labelling in the field of molecular biology. As water-solubility is one of the critical features for biological application, efficient and original synthetic routes are needed to provide new hydrophilic fluorophores inspired from natural pigments. In the frame of the European research project SOPHIED, we have investigated the biomimetic synthesis of novel aminophenoxazinone dyes featuring tuneable water-solubility. Our synthetic route involves a key step of biocatalysed oxidative cascade with a total of 6e- oxidation mediated by a laccase. Sulfonylated and phosphorylated aminophenol building-blocks were synthesised with a total regioselectivity control and further processed as water-soluble substrates by our biocatalyst to afford novel fluorophores. The investigation of the scope and limitations of our biocatalytic process led to a new mechanistic proposal for the key step of oxidative cyclisation initiated by the laccase. Fluorescent properties of these heterocyclic scaffolds were evaluated and further assayed in a live-cell imaging application. This results in the discovery of a promising fluorophore, presumably a bulk endocytic tracer with targeting by default to lysosomes. Lastly the serendipitous discovery of an intramolecular cyclisation due to a neighbouring substituent effect of the acidic group (SO3H and PO(OH)2) in our aminophenol derivatives led to the development of an alternative synthetic route towards novel benzoxazole derivatives of potential interest in medicinal chemistry., Les Laccases font partie de la famille des oxydases à cuivre, enzymes principalement impliquées dans de nombreuses voies de biosynthèse chez les champignons. Parmi les isozymes répertoriés, une laccase a permis de réaliser avec succès la formation in vitro d’un pigment d’origine naturelle dénommé « acide cinnabarinique ». Ce composé comprend un motif hétérocyclique identique à celui du groupement chromophore de l’Actinomycin D qui s’avère être un puissant agent antitumoral d’origine fongique. Le motif hétérocyclique commun dénommé 2-amino-3H-phenoxazin-3-one, possède un potentiel avéré pour une application telle que le marquage par fluorescence dans le domaine de la biologie cellulaire et/ou moléculaire. Une excellente solubilité en milieux aqueux représente une caractéristique incontournable pour de telles applications biologiques. Dés lors, de nouvelles voies de synthèses présentant simultanément originalité et efficacité doivent être continuellement élaborées afin de produire de nouveaux marqueurs fluorescents hydrophiles inspirés de pigments naturels. Dans le cadre d’un projet de recherche européen nommé SOPHIED, nous avons élaboré la synthèse biomimétique de nouveaux colorants de type aminophenoxazinone possédant une solubilité en milieux aqueux modulable grâce aux groupements fonctionnels présents sur ceux-ci. Notre voie de synthèse implique une étape clé de cascade oxydante pour un total de six électrons initiée par un bio-catalyseur, une laccase. Nos blocs de constructions, des molécules de type aminophénols présentant des groupements fonctionnels dérivés d’acides sulfoniques et phosphoniques, ont été synthétisés avec un contrôle total de la régio-sélectivité. Ces substrats hydrophiles ont été transformés par une laccase en nouveaux colorants fluorescents avec une complète sélectivité et d’excellents rendements. Une investigation des potentialités et limitations de notre synthèse bio-catalytique a permis de suggérer une proposition mécanistique pour la casca, (CHIM 3) -- UCL, 2010

Aminophenoxazinone dyes with variable water solubilities were assayed for the first time in a live-cell imaging application. Among a library of ten sulfonylated chromophores, one compound gave excellent results as an endocytic marker, showing a precise subcellular distribution. The compound was compared to four commercial vital tracers, including Lucifer Yellow. The first laccase-mediated regioselective synthesis of a diphosphorylated 2-aminophenoxazinone dye was also described. This compound, water-soluble at 10(-2) M, displayed modest fluorescence properties and the ability to complex Mg(2+) and Ca(2+) cations, therefore giving fluorescence quenching.

The efficient synthesis of sulfonylated benzoxazoles at positions C4 or C7 is reported. The condensation reactions involve original anilide acetal reagents which, upon acid catalysis, allow an easy cyclization reaction with the sulfonylated o-aminophenol partners. This method circumvents the classical use of orthoesters which drawback is the limited access to aromatic reagents.

The enzymatic synthesis of an azoanthraquinone by Perenniporia ochroleuca MUCL 41114 laccase was undertaken. The major product was purified and its structure identified by NMR, MS, IR analyses. In order to scale Lip the production of this azoanthraquinone named Laccase Acid Red 1, a commercial laccase from Trametes versicolor immobilised on perlite as an inexpensive carrier was used. Laccase Acid Red 1 showed lower toxicity than other commercial red dyes, was not mutagenic and displayed low ecotoxicity. In addition, its dyeing properties were assayed on polyamide and the industrial potential of the dye was demonstrated. To our knowledge, this is the first example of sulfonic azoanthraquinone production through enzymatic coupling of aromatic amine monomers. These results show promise for new, safer and environmental friendly routes to azo dye biosynthesis. (C) 2009 Elsevier ltd. All rights reserved.

Laccases are members of the blue copper oxidases family found in nature. They commonly oxidise a wide range of phenol and aniline derivatives, which in turn are involved in oxidative coupling reactions. Yet, laccases remain rarely described as biocatalysts in organic synthesis. This paper describes the chemical preparation of original sulfonated aminophenol substrates and their enzyme-mediated dimerisation into phenoxazine chromophores that feature tuneable water solubility as a function of the sulfonyl substituent. The scope and limitations of the biocatalysed synthetic process are outlined. Kinetic data were collected to evaluate the influence of physicochemical parameters. The structure of the novel phenoxazine dyes ("head-to-head" or "head-to-tail" dimer) was assessed by NMR spectroscopic analysis. Two crystalline compounds were analysed by X-ray diffraction. Such laccase-mediated synthesis (a green chemistry process) was proven to be more efficient than the chemical oxidation of o-aminophenols with silver oxide.

A natural phenoxazinone derivative, cinnabarinic acid (2, CA, R = CO2H), could be obtained in vitro with the aid of purified laccase from the fungi Pycnoporus cinnabarinus by oxidative dimerization of 3-hydroxyanthranilic acid (1, 3-HAA, R = CO2H). With the aim of gaining access to a new class of water-soluble chromophores and potential bioactive molecules, the regioselective sulfonation of 2-hydroxyanihne was investigated. Straightforward insertion of a sulfonate group into a carbon-metal bond provided an efficient and selective process for the synthesis of 3-hydroxyorthanilic acid (3, 3-HOA, R = SO3H). This sulfonated compound was then subjected to laccase oxidation in order to mimic the cinnabarinic acid synthesis. A practical HPLC method to study the oxidized products was developed, and the major product of biotransformation - namely 2-aniino-3-oxo-3H-phenoxazin-1,9-disulfonic acid (4, LAO, R = SO3H) - was isolated and identified as the sulfonate analogue of cinnabarinic acid. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008).

2-Amino-5-(p-hydroxyphenyl)-1,4-pyrazine and 2-amino-3,5-bis(p-hydroxyphenyl)- 1,4-pyrazine are endowed with excellent antioxidative properties. For a possible development in medicinal chemistry, the lipophilicity of these lead compounds has to be increased. Therefore various methods of N-alkylation were systematically explored. The best results were obtained by quenching aminopyrazinyl anion with alkyl iodides, and by coupling aldehydes (reductive amination) in the presence of phenylsilane and tin catalyst. Aminopyrazines equipped with linear alkyl side-chains of at least six carbons showed improved radical-scavenging properties in lipidic media.