Issue 13, 2022

Chemoenzymatic deracemization of lisofylline catalyzed by a (laccase/TEMPO)-alcohol dehydrogenase system


Lisofylline (LSF) is a synthetic methylxanthine active agent exhibiting potent anti-inflammatory and immunomodulatory properties; therefore, it has been widely investigated as a promising drug candidate for treating various autoimmune disorders, including type 1 diabetes. In this study, we report on developing a sequential chemoenzymatic one-pot two-step deracemization protocol for racemic LSF. This task was accomplished in a stereo-complementary manner via a tandem bi-enzymatic oxidation–reduction reaction sequence composed of (i) non-selective chemoenzymatic aerobic oxidation of LSF to pentoxifylline (PTX) catalyzed by commercially available laccase from Trametes versicolor (LTv) and 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) as a redox mediator, and (ii) stereoselective bioreduction of in situ formed PTX to give enantiomeric LSF, which was catalyzed by home-made lyophilized E. coli cells harboring overexpressed alcohol dehydrogenases (ADHs) with complementary stereospecificity. Firstly, a multi-step optimization procedure of LTv/TEMPO-catalyzed oxidation of LSF allowed achieving dramatic improvement of the conversion rates from an initial 16% up to 95%, demonstrating the high synthetic potency of this method compared to traditional chemical reactions requiring toxic oxidants used in stoichiometric amounts. In turn, separate stereoselective bioreductions of PTX using recombinant ADHs from Rhodococcus ruber (E. coli/ADH-A) and Lactobacillus kefiri (E. coli/LK-ADH Prince) furnished both LSF enantiomers (>99% ee) with high 93–94% conversion and in 65–67% yield range, respectively. The coupling of the above-mentioned chemoenzymatic steps afforded both antipodes of LSF on a preparative scale (0.16 mmol of racemic LSF) in the range of 56–67% yield and 94% ee depending on the employed ADHs.

Graphical abstract: Chemoenzymatic deracemization of lisofylline catalyzed by a (laccase/TEMPO)-alcohol dehydrogenase system

Supplementary files

Article information

Article type
21 Jan 2022
16 May 2022
First published
16 May 2022
This article is Open Access
Creative Commons BY license

Catal. Sci. Technol., 2022,12, 4312-4324

Chemoenzymatic deracemization of lisofylline catalyzed by a (laccase/TEMPO)-alcohol dehydrogenase system

P. Borowiecki, A. Rudzka, T. Reiter and W. Kroutil, Catal. Sci. Technol., 2022, 12, 4312 DOI: 10.1039/D2CY00145D

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

Read more about how to correctly acknowledge RSC content.

Social activity