Indigo dye production by enzymatic mimicking based on an iron(III)porphyrin Susana L.H. Rebelo a,⇑ , Margarida Linhares a , Mário M.Q. Simões b , Artur M.S. Silva b , M. Graça P.M.S. Neves b,⇑ , José A.S. Cavaleiro b , Cristina Freire a a REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal b QOPNA, Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal article info Article history: Received 10 February 2014 Revised 15 April 2014 Accepted 18 April 2014 Keywords: Indigo Indole Iron(III)porphyrin Oxidation Catalysis Hydrogen peroxide Mechanism Ethanol abstract A novel indigo synthesis is based on a simple and cost-effective model system of the enzymes involved in the natural and biocatalytic productions. The method considers the oxidation of indole by hydrogen peroxide, being catalyzed by an iron(III)porphyrin in ethanol, as solvent, and no other additives. The yields of indigo and of the other oxidized indolinoid derivatives were found to be dependent on the metalloporphyrin system used and on the control of the oxidation conditions. Significant reductions of the environmental impact relatively to the present industrial production and of the costs relatively to the biocatalytic methodologies were obtained. The enhanced indigo production in the presence of the iron(III)porphyrin-ethanol catalytic system relatively to the manganese(III)porphyrin-acetonitrile system can be rationalized by the formation of different active species in the two systems. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Indigo is a historical dye used by almost all the ancient civiliza- tions and with relevance until our days, mainly due to its incidence as the primary color of blue jeans [1]. Other indigoid compounds are also prominent targets in textile industries and for alimentary, medicinal and cosmetic purposes [2–4]. Until the late 19th century, indigo was obtained from natural sources and, with the advent of the modern chemical industry, became one of the first natural molecules to be synthesized [5]. Today, almost all the traded indigo is produced synthetically and, in general, the branded industrial production is based on variations of the Pflegers’s method. In the largely used process, the synthesis involves the reaction of aniline, formaldehyde and hydrogen cyanide, affording phenylglycinonitrile that is then hydrolyzed to N-phenylglycine. Subsequently, the N-phenylglycine is treated with molten mixtures of caustic soda and sodamide at 200 °C to afford indoxyl, which undergoes further oxidative dimerization to form indigo [6]. The processes are highly energy demanding, result in the production of high amounts of toxic waste products, and require sophisticated purification processes [7]. The high production rates and the amount of hazardous wastes have been raising significant environmental concern that have stimulated the reediting of the natural production [8], and since the work of Ensley et al. in 1983, the microbial productions have been considered as an important eco-sustainable alternative [9]. These authors reported an Escherichia coli mutant encoding the enzyme naphthalene dioxygenase (NDO) that was able to synthe- size indigo from indole. Several other mutant strains have been tested in order to improve the methodology [10,11], which is still expensive and has significant drawbacks in obtaining pure indigo from the culture media (fermentation broth). Enzymatic systems were also tested, such as cytochrome P450 monooxygenases [12], non-heme oxygenases such as NDO [13], and engineered mutants [14,15]. Recently, myoglobin mutants were developed and succeed to directly activate hydrogen peroxide; the system allowed to avoid the use of expensive co-factors, NADPH or NADH, required for the activation of molecular oxygen, thus affording a more prac- tical and green indigo production [16]. The best indigo yield obtained was 12%, based on the consumed indole [16]. However, the cost and purification drawbacks in enzymatic synthesis also need to be considered and, furthermore, significant quantities of http://dx.doi.org/10.1016/j.jcat.2014.04.012 0021-9517/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding authors. Fax: +351 220402659. E-mail addresses: susana.rebelo@fc.up.pt (S.L.H. Rebelo), gneves@ua.pt (M. Graça P.M.S. Neves). Journal of Catalysis 315 (2014) 33–40 Contents lists available at ScienceDirect Journal of Catalysis journal homepage: www.elsevier.com/locate/jcat