Tandem one-pot biocatalytic oxidation and Wittig reaction in water

We explore biocatalytic aldehyde generation under aqueous conditions, concomitantly delivering access to a one-pot Wittig reaction using stabilized phosphoranes and granting diverse alkene products. Using a recombinant choline oxidase mutant, we first undertake biocatalytic alcohol oxidation across a range of functional aliphatic primary alcohols, demonstrating a remarkable substrate tolerance for this enzyme, including chloride, bromide, azide, S-methyl, and alkynyl groups. Following this, we extend capability and deliver a practicable milligram-scale one-pot Wittig reaction in water

Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors †

Upon undergoing mucoid conversion within the lungs of cystic fibrosis patients, the pathogenic bacterium Pseudomonas aeruginosa synthesises copious quantities of the virulence factor and exopolysaccharide alginate. The enzyme guanosine diphosphate mannose dehydrogenase (GMD) catalyses the rate-limiting step and irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Since there is no corresponding enzyme in humans, strategies that could prevent its mechanism of action could open a pathway for new and selective inhibitors to disrupt bacterial alginate production. Using virtual screening, a library of 1447 compounds within the Known Drug Space parameters were evaluated against the GMD active site using the Glide, FRED and GOLD algorithms. Compound hit evaluation with recombinant GMD refined the panel of 40 potential hits to 6 compounds which reduced NADH production in a time-dependent manner; of which, an usnic acid derivative demonstrated inhibition six-fold stronger than a previously established sugar nucleotide inhibitor, with an IC50 value of 17 μM. Further analysis by covalent docking and mass spectrometry confirm a single site of GMD alkylation

Bioprocess development for aliphatic alcohol oxidation and alkyl glycoside purification using biocatalysis

The detergents and skin care products are composed of surfactants that decrease interfacial tension between two phases (i.e. oil and water).1 Before the ecological dangers issued from nonrenewable sources surfactants (pollution of aqueous environments and their aqueous lives), biosurfactants conceived from renewable feedstocks are favoured : glycolipids, lipopeptides, fatty acids, sucrose esters, sorbitan esters, alkyl glucamides, methyl glucoside esters, alkyl polyglycosides (APGs).2 In a world where the market of APG biosurfactants reached US$ 1,024 million in 2019,3 the concerns to enhance production of APG biosurfactants and chemical reactions in accord to green chemistry principles are evolving daily.4 In the interest of furthering sustainable approaches, this work will focused on the bioprocess development of aliphatic chain compounds with oxidases, firstly, for an alternative to distillation of biosurfactant APGs and, secondly, for the formation of unsaturated carbon-carbon double bond in mild conditions via Wittig olefination in aqueous media. To this purpose, a process purification for APGs using oxidases-mediated bio-oxidation followed by sequestration solid-supported was investigated pre- and post-distillation from analytical scale to gram scale. Many challenges were encountered to cover the design of the process and the application to gram scale on manufacture APG biosurfactant. Finally, the work of Bergdahl and coworkers5 inspired a novel cascade reaction performed in deionised water, which combined enzymatic oxidation and Wittig olefination on functionalised alkyl alcohols. From fourteen alkyl alcohols, ten vinyl ester were successfully afforded using the novel cascade with a stabilised ylide

Tandem One-Pot Biocatalytic Oxidation and Wittig Reaction in Water

We explore biocatalytic aldehyde generation under aqueous conditions, concomitantly delivering access to a one-pot Wittig reaction using stabilized phosphoranes and granting diverse alkene products. Using a recombinant choline oxidase mutant, we first undertake biocatalytic alcohol oxidation across a range of functional aliphatic primary alcohols, demonstrating a remarkable substrate tolerance for this enzyme, including chloride, bromide, azide, S-methyl, and alkynyl groups. Following this, we extend capability and deliver a practicable milligram-scale one-pot Wittig reaction in water

Chemoenzymatic synthesis of an unnatural Manb1,4GlcNAc library using a glycoside phosphorylase with "reverse thiophosphorylase" activity

b-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manb1,4GlcNAc a constituent of the core N-glycan in eukaryotes, was recently identified as a STING immune pathway activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of b-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manb1,4GlcNAc analogues using the b-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer -mannan-like glycans. We also pioneer a reverse thiophosphorylase enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorylysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases

Harnessing a Biocatalyst to Bioremediate the Purification of Alkylglycosides

Wahart et al. demonstrate the use of an immobilised oxidase enzyme coupled to an extractive amino resin for the bioremediation of a common industrial surfactant. This allows the facile purification of alkyl monoglucosides without the need for high energy distillations. More information can be found in the Research Article by G. J. Miller, S. C. Cosgrove et al

Harnessing a Biocatalyst to Bioremediate the Purification of Alkylglycosides.

As the world moves towards net-zero carbon emissions, the development of sustainable chemical manufacturing processes is essential. Within manufacturing, purification by distillation is often used, however this process is energy intensive and methods that could obviate or reduce its use are desirable. Developed herein is an alternative, oxidative biocatalytic approach that enables purification of alkyl monoglucosides (essential bio-based surfactant components). Implementing an immobilised engineered alcohol oxidase, a long-chain alcohol by-product derived from alkyl monoglucoside synthesis (normally removed by distillation) is selectively oxidised to an aldehyde, conjugated to an amine resin and then removed by simple filtration. This affords recovery of the purified alkyl monoglucoside. The approach lays a blueprint for further development of sustainable alkylglycoside purification using biocatalysis and, importantly, for refining other important chemical feedstocks that currently rely on distillation. [Abstract copyright: © 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.

Correction: Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors

Correction for ‘Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors’ by Jonathan P. Dolan et al., RSC Chem. Biol., 2023, 4, 865–870, https://doi.org/10.1039/D3CB00126A

Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manβ1,4GlcNAc library

β-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manβ1,4GlcNAc a constituent of the core N-glycan in eukaryotes was recently identified as an immune activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of β-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manβ1,4GlcNAc analogues using the β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer β-mannan-like glycans. We also pioneer a “reverse thiophosphorylase” enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorolysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases

Reverse thiophosphorylase activity of a glycoside phosphorylase in the synthesis of an unnatural Manβ1,4GlcNAc library †

β-Mannosides are ubiquitous in nature, with diverse roles in many biological processes. Notably, Manβ1,4GlcNAc a constituent of the core N-glycan in eukaryotes was recently identified as an immune activator, highlighting its potential for use in immunotherapy. Despite their biological significance, the synthesis of β-mannosidic linkages remains one of the major challenges in glycoscience. Here we present a chemoenzymatic strategy that affords a series of novel unnatural Manβ1,4GlcNAc analogues using the β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase, BT1033. We show that the presence of fluorine in the GlcNAc acceptor facilitates the formation of longer β-mannan-like glycans. We also pioneer a “reverse thiophosphorylase” enzymatic activity, favouring the synthesis of longer glycans by catalysing the formation of a phosphorolysis-stable thioglycoside linkage, an approach that may be generally applicable to other phosphorylases