Revisiting alcohol dehydrogenases: Self-sufficient regio- and enantio- selective formation of bi- and tri-cyclic lactones

The field of biocatalysis has witnessed over the past few years a renewed interest in the design of synthetic routes with high atom-economy, particularly of those employing redox enzymes. For long, the implementation of efficient recycling systems for cofactors (typically nicotinamide) essential to these enzymes was considered attractive for economical reasons, since the use of co-substrate and co-enzyme approaches [1] usually implied co-conversion of a cheap auxiliary substrate (e.g., glucose, ethanol, acetone, formate). In line with growing awareness for sustainable technologies and the desire to reduce the environmental footprint of synthetic processes, strategies that bypass the need for stoichiometric amounts of co-subtrate or reagents are now being considered. For redox enzymes, this translates for instance into the enlargement of the scope of intramolecular hydride transfer, which allows biotransformations to run in closed loop. Exemplary are redox isomerization reactions applied to allylic alcohols or recently cyclic hydroxy-ketones [2]. In both cases, no new bond is created, and the hydride shift is responsible for a switch of functionality. Owing to the dual redox reactivity of aldehydes and complementary activity of alcohol dehydrogenases (ADHs) on the aldehyde functionality (oxidation/reduction), a formal intramolecular biocatalytic hydride shift can be considered with dialdehyde molecules. Following our work on the disproporationation of aldehydes and the establishment of a biocatalytic Cannizzaro-type reaction using ADHs [3], we are now disclosing a broadly applicable enzymatic platform for the synthesis of bi- and tri-cyclic lactones starting from dialdehydes (Scheme 1). An intramolecular bio-Tishchenko reaction was developed with particular attention to redox economy. High turn-over numbers for the nicotinamide cofactor (up to 1.6x103 half-reactions) along with efficient 1,4-, 1,5- and 1,6-hydride shift on dialdehydes (1:1 ratio enzyme/cofactor) could be demonstrated, following reduction-oxidation sequence through lactol intermediate. Noteworthy, regio- and enantioselectivity were observed with a range of wild-type and engineered ADHs and preparative scale synthesis allowed isolation of several lactone products, with no concomitant waste generation [4]. Application of these lactones in (cross-)polymerization reactions is currently being investigated. Please click Additional Files below to see the full abstract

Discovery of novel Fe(II)/α-ketoglutarate-dependent dioxygenases for oxidation of L-proline

Genome-mining for novel Fe(II)/α-ketoglutarate-dependent dioxygenases (αKGDs) to expand the enzymatic repertoire in the oxidation of L-proline is reported. Through clustering of genes, we predicted regio- and stereoselectivity in hydroxylation reaction and validated this hypothesis experimentally. Two novel by-products in reactions with BcePH and Ssp5PH were observed, isolated and structure was determined as an epoxide and a 3,4-diol, respectively. Mechanism for formation of epoxide is suggested and validated by using 18O-labelling experiment, that proceeds via cis-3-hydroxylation step first, followed by ring closure. A Biocatalytic step was performed on sub-gram quantities of starting material without any significant condition optimization. The substrate concentration, however, is already up to 40-fold higher than the usually reported titers for P450-mediated hydroxylations, showing the synthetic potential of αKGDs on preparative scal

Conjugation of Hydroxytyrosol with Other Natural Phenolic Fragments: From Waste to Antioxidants and Antitumour Compounds

Hydroxytyrosol, a natural polyphenol that can be extracted from olive mill waste waters, was converted into a series of more complex and lipophilic analogues by conjugation with other naturally derived (poly)phenolic fragments. Ether and triazole linkers were employed. A small library of compounds was prepared, stressing step economy and operational simplicity. Some of these substances were proven to have activity equal or superior to that of the parent hydroxytyrosol in radical scavenging assays as well as in cytotoxicity tests against tumour cells

Dataset to "An Enzymatic Route to α‐Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3"

Aromatic hydroxylation of pseudocumene and mesitylene with P450 BM3 yields key phenolic building blocks for a-tocopherol synthesis. Site-saturation mutagenesis generated a new P450 BM3 mutant, named “variant M3” (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3- to 8-fold) and activity (75- to 230-fold) for the conversion of pseudocumene and mesitylene. Here the dataset to “An Enzymatic Route to α‐Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3” (Alexander Dennig, Alexandra Maria Weingartner, Tsvetan Kardashliev, Christina Andrea Müller, Erika Tassano, Martin Schürmann, Anna Joëlle Ruff, Ulrich Schwaneberg, Chemistry. 2017 Dec 19;23(71):17981-17991., doi: 10.1002/chem.201703647) is given. This study provides an enzymatic route to key phenolic synthons for a-tocopherols and the first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2.Source data of the performed substrate docking, and mechanistic considerations are given. Docking of mesitylene into the active site of P450 BM3 WT (source data to Fig1) and the homology model of P450 BM3 variant M3, as well as Docking of pseudocumene into the active site of P450 BM3 WT (1BU7) (source data to Fig2) and P450 BM3 variant M3 (R47S, Y51W, A330F, I401M) (source data to Fig3) are submitted. In the description of dataset a summary of the docking source data is given