Tandem biocatalytic continuous-flow synthesis of hydroxytyrosol and other tyrosol metabolites

Tyrosol and hydroxytyrosol are valuable dietary phenolic compounds mainly present in olive oil and wine, which have shown to possess a range of biological effects like anti-inflammatory, antioxidant, anticancer, antiviral, antibacterial and antifungal properties.1 The possibility to have access to gram amounts of pure tyrosol and hydroxytyrosol, their metabolites and derivatives, is highly appealing for their deep biological evaluation.2 In this context, the aim of our research was the design of a sustainable, scalable, and efficient protocol for the biocatalysed transformation of tyrosol into hydroxytyrosol and for the obtainment of their acetyl metabolites, characterised by increased lipophilicity and stability. To achieve our goal, two different biocatalysts were employed in a continuous flow system (Scheme 1). The first step was the oxidation of tyrosol into hydroxytyrosol by a free tyrosinase from Agaricus bisporus in presence of oxygen and ascorbic acid, necessary to avoid over-oxidation of the product. The aqueous flow stream was then extracted in-line with ethyl acetate in order to recirculate the aqueous layer containing the biocatalyst and the excess of ascorbic acid to improve the efficiency and the economy of the protocol in a self-sufficient closed-loop system. The organic phase was purified in-line thanks to a catch and release procedure using supported boronic acid to trap hydroxytyrosol, leaving unreacted tyrosol in solution. To obtain the acetyl metabolites, a bioreactor packed with an immobilised acyltransferase from Mycobacterium smegmatis (MsAcT) was used.3 Thanks to this modular set up, hydroxytyrosol was obtained with an isolated yield up to 75%, whereas the acetyl metabolites showed yields up to 80% in 10 minutes of residence time.