Regioselective Synthesis of 3‐Hydroxyorthanilic Acid and Its Biotransformation into a Novel Phenoxazinone Dye by Use of Laccase

Abstract

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‐hydroxyaniline 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‐amino‐3‐oxo‐3H‐phenoxazin‐1,9‐disulfonic acid (4, LAO, R = SO3H) – was isolated and identified as the sulfonate analogue of cinnabarinic acid.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Heterologous laccase production and its role in industrial applications

“…As a fact, laccases can be adopted (1) to bleach textiles; 12 (2) to eliminate undesirable phenolics responsible for the browning, haze formation and turbidity development in clear fruit juice, beer and wine; 13 (3) to bleach wood pulp; 14 (4) to synthesize various functional organic compounds such as drug, dyes; 15,16 (5) to produce various polymers; 17 (6) to detect molecules in biosensor devices; 18 (7) to produce power in biofuel cells; 19 (8) and to dye hair. 20 As far as bioremediation is concerning, laccases may be applied to decolorize textile effluents; 21 to degrade plastic waste having olefin units; 20 to eliminate odor emitted from places such as garbage disposal sites, livestock farms, or pulp mills; 20 to remove phenolic compounds from olive concentration is required for the production of active laccase at a post-translational step in a heterologous host.…”

Laccase‐Mediated Synthesis of Novel Substituted Phenoxazine Chromophores Featuring Tuneable Water Solubility

“…Indeed, a directed ortho-metallation reaction was successfully used for the regioselective sulfonation of aniline and derivatives. [18,22] Optional selectivity in the metallation site has been reported before for N-(tert-butoxycarbonyl)anisidine. [23] In our hands, the use of Schlosser superbasic mixture gave unsatisfactory results (data not shown).…”

“…Synthesis of substrates 4 a-g from 2-tert-butylbenzoxazole-4-sulfonic acid. [18] Reagents: a) PCl 5 (5 equiv), OPCl 3 (5 equiv), CH 2 Cl 2 , 4 h at 40 8C, 95 %; b) RNH 2 (10 %), CH 2 Cl 2 , overnight at 25 8C, 95 %; c) 6 n aqueous HCl, overnight at 100 8C, 50 % for 4 a (R = H), 30 % for 4 d (R = phenyl), 85 % for 4 b (R = cyclohexyl), c (R = (CH 2 ) 3 N(Me) 2 ), e (R = (CH 2 ) 2 NH 2 ), f (R = CH 2 CO 2 Me), g (R = (CH 2 ) 2 CH 2 OH) (see Table 1). …”

“…The preparation of 3-HOA and the key intermediate 1 were previously published. [18] Compounds 4 a-g (Table 1) were obtained by using benzoxazole 1 as the starting material (Scheme 2). The sulfonic acid was converted into the corresponding sulfonyl chloride 2 in high yield, which was directly submitted to substitution by ammonia, alkyl or aryl amines in a biphasic medium.…”

“…[18] A phosphate-buffered medium was initially discarded because phosphate salts would impair the isolation of the desired water-soluble heterocycles; however, working in pure water led to slight variations of pH during the reactions. Furthermore, the previously isolated laccase acid orange (LAO) dye [18] (Table 2 and Scheme 4), when placed in water equilibrated between two protonated forms (NMR spectroscopy study not shown). Thus a reaction Scheme 3.…”