Enzymatic Synthesis of Hydroxycinnamic Acid Amides in Water Using the Promiscuous Hydrolase/Acyltransferase PestE from Pyrobaculum calidifontis VA1

Abstract

Hydroxycinnamic acid amides are believed to have antioxidant, antidiabetic, cytotoxic, anticancer, neuroprotective, and antiinflammatory properties, making them interesting target molecules for potential applications in the food, cosmetics, and pharmaceutical industries. Here, we describe the synthesis of hydroxycinnamic acid amides starting from hydroxycinnamic acid esters and the corresponding amines by using variants of the promiscuous hydrolase/acyltransferase from Pyrobaculum calidifontis VA1 (PestE) in water as a solvent. Up to 97% conversion within two hours at 60 °C was achieved with methyl ferulate and tyramine as substrates. This is a promising, environmentally friendly alternative strategy to established chemical synthesis routes or enzymatic methods using lipases in nonaqueous organic solvents.

Key words

acyltransferase - amides - biocatalysis - carboxylesterase - hydroxycinnamic amides

Publication History

Received: 12 October 2023

Accepted after revision: 25 January 2024

Accepted Manuscript online:
14 February 2024

Article published online:
01 March 2024

• References and Notes

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• 23 Initial Screening of Starting Enzyme CandidatesA 2 M solution of vinyl ferulate in acetonitrile was prepared. Tyramine was dissolved in 200 mM potassium phosphate buffer (pH 8). Due to the addition of tyramine, the pH shifted to pH 10. Final reaction concentrations: tyramine (44.5 mM), potassium phosphate buffer (178 mM), vinyl ferulate (100 mM), enzyme (0.3 mg/mL), 5% (v/v) MeCN. Potassium phosphate buffer served as a control. Incubation was performed for 16 h at 25 °C and 1000 rpm. Samples were analyzed by using TLC, and N-trans-feruloyltyramine formation was quantified on a UHPLC system by using a calibration curve.
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• 25 Enzyme Assays Amines were dissolved in 200 mM potassium phosphate buffer, CHES buffer, or CAPS buffer and, by using HCl or NaOH, the pH was adjusted to pH 7, 7.5, and 8 for the phosphate buffer; pH 8.5, 9, 9.5, and 10 for the CHES buffer; and pH 10, 10.5, and 11 for the CAPS buffer. If ascorbic acid was used, this was dissolved in the respective buffer together with the amine, and the pH was adjusted accordingly. Ascorbic acid was used in a fivefold molar excess compared with the combined concentration of the amine and hydroxycinnamic acid ester. The hydroxycinnamic acid esters were dissolved in MeCN at concentrations such that 5% (v/v) of the cosolvent was present in the reactions. In all reactions, final concentrations of 5 mM amine, 178 mM buffer, 5% (v/v) MeCN, and 0.21 mg/mL of PestE variant were used. Samples were incubated at 1000 rpm. The reactions were stopped by the addition of MeCN to a final concentration of 50% (v/v) after full consumption of the hydroxycinnamic acid ester. Samples were analyzed by TLC, and N-trans-feruloyltyramine and N-trans-caffeoyltyramine formation was quantified by using a UHPLC system with a calibration curve. For temperature profiles, conversions after 2 h of incubation were measured.
• 26 Expression of PestE variants was performed according to a protocol previously described.²¹
• 27 Preparative-Scale Synthesis of N-trans-Feruloyltyramine with PestE Ethyl ferulate (567.3 mg), tyramine (88 mg; molar ratio 4:1), ascorbic acid (2.81 g), CHES (5.29 g), MeCN (6.383 mL) and H₂O (127 mL) were mixed, and the pH was adjusted to pH 10 by using NaOH. The reaction was started by using PestE_I208A_L209F_N288A (26.8 mg) and the mixture was incubated at 70 °C. When the ethyl ferulate was completely consumed (TLC), the product was extracted with EtOAc (×3). The organic phase was washed with 2 M HCl and brine, dried (MgSO₄), filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography [silica gel, hexane–EtOAc (2:3)] to give a white, slightly pale-brown solid; yield: 168 mg (84%). ¹H NMR (300 MHz, DMSO) δ = 9.40 (s, 1 H), 9.16 (s, 1 H), 7.30 (d, J = 15.7 Hz, 1 H), 7.11 (d, J = 1.7 Hz, 1 H), 7.04–6.93 (m, 3 H), 6.79 (dd, J = 8.1, 1.6 Hz, 1 H), 6.68 (d, J = 8.5 Hz, 2 H), 6.43 (d, J = 15.7 Hz, 1 H), 3.80 (s, 3 H), 3.33–3.26 (m, 2 H), 2.64 (t, J = 7.3 Hz, 2 H). The ¹H NMR was in accordance with the reported NMR spectrum (see the Supporting Information).²⁸
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