Simon J. Charnock

Prozomix Limited · Enzyme Discovery

ATAs engineered for having an enlarged small binding pocket were applied for the synthesis of enantiomerically pure (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine, a chiral component of human leukocyte elastase inhibitor DMP 777 (L‐694,458). Kinetic resolution of the racemic amine was performed by using the L59A variant of the (S)‐selective ATA from Chromoba...

Chiral β‐hydroxysulfides are an important class of organic compounds which find broad application in organic and pharmaceutical chemistry. Herein we describe the development of novel biocatalytic and chemoenzymatic methods for the enantioselective synthesis of β‐hydroxysulfides by exploiting ketoreductase (KRED) enzymes. Four KREDs were discovered...

Chiral β‐hydroxysulfides are an important class of organic compounds which find broad application in organic and pharmaceutical chemistry. Herein we describe the development of novel biocatalytic and chemoenzymatic methods for the enantioselective synthesis of β‐hydroxysulfides by exploiting ketoreductase (KRED) enzymes. Four KREDs were discovered...

The catalytic asymmetric construction of Csp3–Csp3 bonds remains one of the foremost challenges in organic synthesis. Metal-catalyzed cross-electrophile couplings have emerged as a powerful tool for C–C bond formation. However, these catalysts struggle to differentiate Csp3-electrophiles leading to dimerization of the starting material, and there a...

Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals¹, yet their preparation often relies on low-efficiency multi-step synthesis². These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocata...

In the last two decades, several PET‐degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high‐throughput screening protocols for PET‐hydrolyzing activity that avoid the use of...

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient...

Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreductase was identified using metagenomic mining to catalyze a highly enantio- and diastereoselective dynamic kinetic resolution of a 𝛽-ketolactone. Subsequent lactone reduction with diisobutylaluminium hydride and phase transfer cyclization affords the bic...

Herein, we describe a chemoenzymatic synthesis of the bicyclic fragment of Darunavir. A ketoreductase was identified using metagenomic mining to catalyze a highly enantio- and diastereoselective dynamic kinetic resolution of a 𝛽-ketolactone. Subsequent lactone reduction with diisobutylaluminium hydride and phase transfer cyclization affords the bic...

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the developm...

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the developm...

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this...

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this...

Aldolase and transaminase coexpressed in Escherichia coli cells and lyophilized (i.e., lyophilized whole-cell biocatalyst (LWCB)) were used as biocatalysts for the one-pot cascade synthesis of l-homoserine with substrate cycling. The kinetic analysis of enzymes within lyophilized cells was performed to evaluate the behavior of the system. The best...

2‐Aminotetralin and 3‐aminochroman derivatives are key structural motifs present in a wide range of pharmaceutically important molecules. Herein, we report an effective biocatalytic approach towards these molecules through the enantioselective reductive coupling of 2‐tetralones and 3‐chromanones with a diverse range of primary amine partners. Metag...

2‐Aminotetralin and 3‐aminochroman derivatives are key structural motifs present in a wide range of pharmaceutically important molecules. Herein, we report an effective biocatalytic approach towards these molecules through the enantioselective reductive coupling of 2‐tetralones and 3‐chromanones with a diverse range of primary amine partners. Metag...

The COVID-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue currently in phase 3 clinical trials as an orally available treatment for SARS-CoV-2. Key to the success of this process was the developm...

A major challenge in chemical synthesis is to develop catalytic systems that convert simple molecules to complex high-value products. Often these valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Of great interest are enantioenriched amine diastereomers, which...

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of bio...

N ‐Substituted α‐amino esters are widely used as chiral intermediates in a range of pharmaceuticals. Here we report the enantioselective biocatalyic synthesis of N ‐substituted α‐amino esters through the direct reductive coupling of α‐ketoesters and amines employing sequence diverse metagenomic imine reductases (IREDs). Both enantiomers of N ‐subst...

N ‐Substituted α‐amino esters are widely used as chiral intermediates in a range of pharmaceuticals. Here we report the enantioselective biocatalyic synthesis of N ‐substituted α‐amino esters through the direct reductive coupling of α‐ketoesters and amines employing sequence diverse metagenomic imine reductases (IREDs). Both enantiomers of N ‐subst...

Herein we report the conversion of cytidine 2 to N-hydroxycytidine 7 catalysed by cytidine deaminase (CD). The wild-type enzyme operates efficiently at high sustrate loadings and hydroxylamine concentrations to favor N-hydroxy-cytidine formation over uridine. Although the wild-type enzyme demonstrated good activity, we were able to further enhance...

Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination,...

Statins are a group of drugs used for lowering cholesterol levels in the human bloodstream to prevent cardiovascular diseases and its synthesis is a challenge because of chemical and stereochemical requirements. In this work, a multi-enzyme one-pot sequential strategy was employed to obtain lactonized statin side-chain precursor and was compared to...

p>The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical timescale. Here we show the application of direct infusion mass s...

A multi-enzyme route for the production of an atorvastatin side-chain was proposed. The approach includes the consecutive double aldol addition of acetaldehyde to the phenylacetamide amino-protected propanal by 2-deoxyribose-5-phosphate aldolase (DERA), the oxidation by ketoreductase, the lipase-catalysed acylation and amino group deprotection by p...

In 2012, David Rozzell published an excellent and concise survey of commercial enzyme suppliers. This chapter includes details of companies that provide one or more of the many services focused towards bioprocess development. This survey is primarily aimed at the industrial and academic synthetic chemist who needs to source enzymes or services for...

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorina...

In biological science, metagenomics has revolutionised the process of drug discovery. This is because metagenomics serves as a tool for in depth characterisation by examining, sequencing, replicating and identifying the whole DNA/genome collected from any mixed population (Mahapatra et al. , 2019). Metagenomics is used to study Carbohydrate Active...

The enantioselective synthesis of α‐thiocarboxylic acids via biocatalytic dynamic kinetic resolution (DKR) of nitrile precursors exploiting nitrilase enzymes is described. A panel of 35 nitrilase biocatalysts were screened and enzymes Nit27 and Nit34 were found to catalyze the DKR of racemic α‐thionitriles under mild conditions, affording the corre...

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. H...

Aromatic β-hydroxyaldehydes, 1,3-diols, and α,β-unsaturated aldehydes are valuable precursors to biologically active natural products and drug molecules. Herein we report the biocatalytic aldol condensation of acetaldehyde with various aromatic aldehydes to give a number of aromatic α,β-unsaturated aldehydes using a previously engineered variant of...

L-Homoserine is an important compound and a building block in chemical and pharmaceutical industry. The one-pot cascade synthesis of L-homoserine with substrate recycling catalysed by an aldolase and a transaminase was investigated in detail, with special interest in the reaction’s kinetics and process development. Complex relationships between the...

Cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases. P450s are found in all Kingdoms of life and exhibit incredible diversity, both at sequence level and also on a biochemical basis. In the majority of cases, P450s can be assigned into one of ten classes based on their associated redox partners, domain architecture an...

3-Hydroxyisobutyric acid is an important intermediate in the biosynthesis of methacrylic acid. Its biocatalytic synthesis can be performed by aldolase-catalysed aldol addition of propanal to formaldehyde followed by an enzymatic oxidation of the resulting 3-hydroxy- 2- methylpropanal to 3-hydroxyisobutyric acid. In this work, D-fructose-6-phosphate...

Motivation Residue-residue coevolution has been used to elucidate structural information of enzymes. Networks of coevolution patterns have also been analyzed to discover residues important for the function of individual enzymes. In this work, we take advantage of the functional importance of coevolving residues to perform network-based clustering o...

BACKGROUND The statin side‐chain synthesis by the sequential aldol condensation catalyzed by DERA (EC 4.1.2.4) is the most known and promising route. The advantage of this process is the formation of two stereocenters in a single step starting from inexpensive achiral components, acetaldehyde and chloroacetaldehyde. The limitation for the industria...

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, antidepressants and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from sim...

Enzymes are attractive tools for synthetic applications. To be viable for industrial use enzymes need sufficient stability towards the desired reaction conditions such as high substrate and cosolvent concentration, non‐neutral pH and elevated temperatures. Thermal stability is an attractive feature not only because it allows for protein purificatio...

1,3-Mercaptoalkanols constitute an important class of volatile sulfur compounds and contribute to both agreeable and disagreeable flavours and fragrances of many foods and beverages. Most mercaptoalkanols exist as chiral isomers and their olfactory perception may depend on their diastereomeric and enantiomeric configuration. A number of mercaptoalk...

1,3-Mercaptoalkanols constitute an important class of volatile sulfur compounds and contribute to both agreeable and disagreeable flavours and fragrances of many foods and beverages. Most mercaptoalkanols exist as chiral isomers and their olfactory perception may depend on their diastereomeric and enantiomeric configuration. A number of mercaptoalk...

The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annota...

Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its...

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a Systems Biocatalysis approach comprising a biocatalytic one...

Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450. nm, ie, "Pigment-450. nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example,...

Oxidoreductases have been employed in the synthesis of chiral alcohols from achiral ketones, primary alcohols from aldehydes, and in the oxidative direction towards carbonyl formation increasingly over approximately the last 30 years. The class of enzymes involved, frequently referred to as ketoreductases (KREDs), comprises multiple protein familie...

Biocatalytic oxidation reactions have the potential to substitute many chemically catalyzed oxidations in the pharmaceutical and fine chemical industry due to their superior regio- and stereoselectivity and low environmental impact. Galactose oxidase (GOase) has been shown to be a promising biocatalyst for the oxidation of primary and secondary alc...

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of β-sheets, the β-domain. Simulation flexibility analys...

Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore...

Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called 'flesh eating disease') and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside hydrolases, has been proposed to be a virulence factor. In the present...

Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successful...

Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyo...

To allow continued innovation in the development of test kits for the cereals, foods and beverage industries, Megazyme has established a Molecular Biology division within the company. The sole function of this division is to produce a range of novel enzymes allowing the development of new and improved test kits. In this presentation, we will descri...

In an attempt to understand better the organisation of genes encoding enzymes of the ribulose monophosphate pathway (RuMP), the 3-hexulose 6-phosphate synthase gene (hps) and flanking sequences were cloned from the obligate methylotroph Aminomonas aminovorus C2A1. To date only three hps containing gene clusters from methylotrophs have been characte...

Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere. The hydrolysis of xylan, by endo-beta-1,4-xylanases (xylanases), is one of the key reactions in this process. Although amino acid sequence variations are evident in the substrate binding cleft of "family GH10" xylanases (see afmb.cnrs...

Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the mult...

Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9 Å structure of the mul...

Trehalose is a nonreducing disaccharide that plays a major role in many organisms, most notably in survival and stress responses. In Mycobacterium tuberculosis, it plays a central role as the carbohydrate core of numerous immunogenic glycolipids including "cord factor" (trehalose 6,6'-dimycolate). The classical pathway for trehalose synthesis invol...

Carbohydrate-protein recognition is central to many biological processes. Enzymes that act on polysaccharide substrates frequently contain noncatalytic domains, "carbohydrate-binding modules" (CBMs), that target the enzyme to the appropriate substrate. CBMs that recognize specific plant structural polysaccharides are often able to accommodate both...

Cellvibrio japonicus arabinanase Arb43A hydrolyzes the alpha-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans. The three-dimensional structure of Arb43A, determined at 1.9 A resolution, reveals a five-bladed beta-propeller fold. Arb43A is the first enzyme known to display this topology. A long V-shaped surface groove, partially...

Enzyme-catalyzed -elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a "family PL-10" polysacchari...

The Ots gene cluster of Escherichia coli encodes the synthetic apparatus for the formation of alpha,alpha-1,1-trehalose, a non-reducing glucose disaccharide. The otsA gene encodes a trehalose-6-phosphate synthase, a glycosyltransferase which catalyses the synthesis of alpha,alpha-1,1-trehalose-6-phosphate from glucose-6-phosphate using a UDP-glucos...

Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degr...

The vast majority of glycosidic-bond synthesis in nature is performed by glycosyltransferases, which use activated glycosides as the sugar donor. Typically, the activated leaving group is a nucleoside phosphate, lipid phosphate or phosphate. The nucleotide-sugar-dependent glycosyltransferases fall into over 50 sequence-based families, with the larg...