Contents lists available at ScienceDirect Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig Enzymatic synthesis of novel water-soluble indigoid compounds Mikas Sadauskas ∗ , Roberta Statkevičiūtė, Justas Vaitekūnas, Vytautas Petkevičius, Vida Časaitė, Renata Gasparavičiūtė, Rolandas Meškys Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Sauletekio al. 7, Vilnius, LT-10257, Lithuania ARTICLE INFO Keywords: Indigo Indirubin Bacterial flavin-dependent monooxygenases ABSTRACT This work was aimed at expanding the diversity of indigoid compounds with novel characteristics by employing biological catalysts. A total of 16 novel indigoid compounds were synthesized using indole derivatives as sub- strates containing either aminomethyl-, hydroxymethyl-, carboxaldehyde or carboxyl groups at positions 4, 5, 6 or 7 of indole ring. Two different monooxygenase systems – a flavin dependent monooxygenase Hind8 and the mutant G109Q of multicomponent soluble diiron monooxygenase PML were employed to achieve the conversion of all those substrates. Characterization of purified indigo dicarboxylic acids revealed that the produced in- digoids were soluble in water (solubility increased more than 1000-fold compared to indigo), also in methanol and DMSO. A bioconversion of indole-7-carboxylic acid resulted in the mixture of indigo-7,7′-dicarboxylic acid and indirubin-7,7′-dicarboxylic acid. Both of those products were separated from each another and purified thus providing the basis for the purification of water-soluble indigoids of similar structure. These findings show that (1) biological catalysts can be an easy approach for synthesizing novel indigoids with important characteristics and (2) introduction of carboxyl group can increase the water-solubility of indigo and indirubin significantly, thus providing a new tool in search for bioactive indigoids. 1. Introduction Indigo is one of the oldest examples of green biotechnology, when leaf extracts of Indigofera spp. were used for cotton dyeing more than 6000 years ago [1]. In the 20th century, the rate of synthetic indigo production increased and reached approx. 17000 tons a year in 2012 [2]. These amounts are used for cotton and jeans dyeing mainly. Composed of two aromatic indolinone ring systems, indigo is highly insoluble in water; however, two carbonyl groups are also present, which can be reduced to form leuco-indigo, a water-soluble form of indigo. These features are exploited during a vat dyeing. Another ancient indigoid compound is indirubin, a structural isomer of indigo. Indirubin is not being used in textile industry due to low stability, albeit having a clear purple color. Instead, indirubin has been known to be a constituent of the Chinese herbal medicine qing-dai, also known as Indigo naturalis. This medicine has long been used as an anti- inflammatory drug [3]. Indirubin can act as an inhibitor of cyclin-de- pendent kinase 1 [4] and glycogen synthase kinase [5], inhibit the proliferation of cancer cells and also the proliferation of tumor-derived endothelial cells [6,7]. In addition, isoindigo, another structural isomer of indigo, has become an excellent tool for the production of organic electronics, particularly organic photovoltaics and organic field effect transistors [8]. Substituent chemical groups can be introduced into the indigo backbone and the resulting compounds are regarded as indigoids. Those compounds receive increasing attention due to exceptional character- istics. Owing to their reversible two-electron reduction and oxidation, indigoids can act as both electron donors and acceptors, thus providing the basis for production of indigoids-based materials for green organic electronics [9]. Due to low toxicity and chemical stability in aerated conditions, halogenated indigoids, such as 5-bromoindigo, 6,6′-di- chloroindigo and Tyrian purple (6,6′-dibromoindigo), regarded as one of the most precious ancient compounds, are potential compounds for semiconductor synthesis [10]. Indigo N,N′-bis(arylimine)s (Nindigo), modified variants of indigo containing two β-diketiminatetype motifs, were shown to form coordination complexes with different redox active metals [11–13] giving these indigoids novel absorbance and redox properties. Different methods exist for the synthesis of indigoids. Chemical synthesis has been the preferred method until recently, when enzymatic https://doi.org/10.1016/j.dyepig.2019.107882 Received 31 July 2019; Received in revised form 12 September 2019; Accepted 12 September 2019 ∗ Corresponding author. E-mail addresses: mikas.sadauskas@bchi.vu.lt (M. Sadauskas), statkeviciuteroberta@gmail.com (R. Statkevičiūtė), justas.vaitekunas@bchi.vu.lt (J. Vaitekūnas), vytautas.petkevicius@bchi.vu.lt (V. Petkevičius), vida.casaite@bchi.vu.lt (V. Časaitė), renata.gasparaviciute@bchi.vu.lt (R. Gasparavičiūtė), rolandas.meskys@bchi.vu.lt (R. Meškys). Dyes and Pigments 173 (2020) 107882 Available online 13 September 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved. T