The combination of sequential biocatalytic reactions, via non-natural synthetic cascades, is a rapidly developing field and leads to the generation of complex valuable chemicals from simple precursors. As the toolbox of available biocatalysts continues to expand, so do the options for biocatalytic retrosynthesis of a target molecule, leading to alternative routes employing enzymatic transformations. The implementation of such cascade reactions requires careful consideration, particularly with respect to whether the pathway is constructed in vitro or in vivo. In this Perspective, we describe the relative merits of in vitro, in vivo, and hybrid approaches to building biocatalytic cascades and showcase recent developments in the area. We also highlight the factors that influence the design and implementation of purely enzymatic or chemoenzymatic, one-pot, multistep pathways.Keywords: biocatalysis; biocatalytic retrosynthesis; cofactors; enzymatic cascades; enzymes; in vitro biotransformations; in vivo biotransformations; whole-cell biocatalysis;

The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric α/β glycan linkage within oligosaccharides remains a particular challenge. Here, we show that “memory” of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS2). These findings allow for integration of MS2 with ion mobility spectrometry (IM-MS2) and lead to a strategy to distinguish α- and β-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.

The identification of carbohydrate–protein interactions is central to our understanding of the roles of cell–surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate–protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

Screening of bacterial colonies to identify new biocatalytic activities is a widely adopted tool in biotechnology, but is constrained by the requirements for colorimetric or tag-based detection methods. Herein we report a label-free screening platform for biotransformations in live colonies using desorption electrospray ionization coupled with ion mobility mass spectrometry imaging (DiBT-IMMS). The screening method is demonstrated for both ammonia lyases and P450 monooxygenases expressed within live bacterial colonies and is shown to enable multiplexing of enzyme variants and substrate libraries simultaneously.

AbstractCarboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles.

N-Acetyl glucosamine (GlcNAc) is one of the most abundant biomolecules on Earth and is cheaply available from chitin, a major component of crustaceans. The key step in the conversion of GlcNAc to high-value products is the de-N-acetylation to glucosamine, in itself a valuable dietary supplement that is produced at over 29 000 tons scale per annum by chemical hydrolysis, a process that requires harsh reaction conditions and leads to side products requiring separation. Here, we report for the first time the isolation and characterisation of an enzyme, a deacetylase from Cyclobacterium marinum that is able to catalyse the highly selective quantitative hydrolysis of GlcNAc to glucosamine under mild reaction conditions. This enzyme is small (38 kDa), is easily obtainable by heterologous expression in E. coli, has high turnover rates (kcat = 61 s−1), tolerates high substrate concentrations (over 100 g L−1) and can be repeatedly re-used as an immobilised catalyst. When coupled with chitinase, the high selectivity of the enzyme for GlcNAc over other biomolecules allowed one-pot extraction of glucosamine from crude solid mushroom fractions containing chitin, thus allowing for alternative production of glucosamine from non-animal sources, of benefit to consumers with crustacean allergies and vegan diets. We suggest that the deacetylase fills an important gap in the sustainable exploitation of GlcNAc and chitin.

The combination of stable isotope labelling with direct infusion ion mobility mass spectrometry (IM-MS) enabled qualitative and quantitative monitoring of biocatalytic reactions with reduced analysis times, enhanced sensitivity and μL-level assay volumes. The new approach was demonstrated by applying to both lipase and monooxygenase enzymes, including multi-substrate screening.

•Bacteria were detected using field-effect transistors.•The devices enable faster and more sensitive bacteria detection than other techniques such as electrochemical impedance spectroscopy and MALDI-ToF.•The devices can be fabricated at low cost for the fast screening of pathogenic bacteria in point of care.While pathogenic bacteria contribute to a large number of globally important diseases and infections, current clinical diagnosis is based on processes that often involve culturing which can be time-consuming. Therefore, innovative, simple, rapid and low-cost solutions to effectively reduce the burden of bacterial infections are urgently needed. Here we demonstrate a label-free sensor for fast bacterial detection based on metal–oxide–semiconductor field-effect transistors (MOSFETs). The electric charge of bacteria binding to the glycosylated gates of a MOSFET enables quantification in a straightforward manner. We show that the limit of quantitation is 1.9×105 CFU/mL with this simple device, which is more than 10,000-times lower than is achieved with electrochemical impedance spectroscopy (EIS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF) on the same modified surfaces. Moreover, the measurements are extremely fast and the sensor can be mass produced at trivial cost as a tool for initial screening of pathogens.

A synthetic perfluoroalkyl-tagged lactosyl glycolipid has been shown to form lipid microdomains in fluid phospholipid bilayers. When embedded in the membranes of phospholipid vesicles, this glycolipid was trans-sialylated by soluble T. cruzi trans-sialidase (TcTS) to give a perfluoroalkyl-tagged glycolipid that displayed the ganglioside GM3 epitope, with up to 35% trans-sialylation from fetuin after 18 h. Following sialylation, vesicles bearing this Neu5Ac(α2-3)Gal(β1-4)Glc sequence in their “glycocalyx” were recognised and agglomerated by the lectin M. amurensis leukoagglutinin. Monitoring TcTS-mediated trans-sialylation by HPLC over the first 6 h revealed that enzymatic transformation of bilayer-embedded substrate was much slower than that of a soluble lactosyl substrate. Furthermore, clustering of the lactose-capped glycolipid into “acceptor” microdomains did not increase the rate of sialic acid transfer from fetuin by soluble TcTS, instead producing slight inhibition.

This review gives an overview of enzymatic reactions that have been conducted on substrates attached to solid surfaces. Such biochemical reactions have become more important with the drive to miniaturisation and automation in chemistry, biology and medicine. Technical aspects such as choice of solid surface and analytical methods are discussed and examples of enzyme reactions that have been successful on these surfaces are provided.

Glycoconjugates containing either terminal α-2,3- or α-2,6-Neu5Ac-Gal disaccharides are found on cell surfaces of many animal glycans. Each linkage can be specifically recognized by lectins and enzymes such as neuraminidases. Here we describe a one-step enzymatic synthesis of two colorimetric substrates that allow for fast distinction of specific neuraminidase activity.

O-Mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galβ1-4GlcNAcβ1-2Manα, which is reported to be the most abundant O-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a “one-pot” enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described.

We report a highly efficient and selective method for the coupling of peptides and glycoconjugates bearing N-terminal cysteines to activated surfaces. This chemoselective method generates stable amide linkages without using any thiol additives.

There is a wide range of immobilisation reactions to tether substrates to a variety of surfaces for array-based analysis. Most of these immobilisation strategies are specific for a particular surface and require an additional linker to be attached to the substrate or the surface. Furthermore, the analysis of functionalised surfaces is often restricted to certain analytical techniques and therefore, different immobilisation strategies for different surfaces are desirable. Here we have tested an S-tritylated linker for non-covalent or covalent immobilisation of mannosides to polystyrene or gold surfaces. S-Tritylated mannosides with varying linkers were readily synthesised and used to add to biorepulsive maleimide-terminated preformed SAMs after in situ deprotection of the S-trityl group. In addition, S-tritylated mannosides themselves formed stable glycoarrays on polystyrene microtiter plates. The glycoarrays were successfully analysed by MALDI-ToF mass spectrometry, SPR spectroscopy, and interrogated with GFP-transfected Escherichia coli cells. This work has shown that a dual purpose linker can be used on multiple surfaces to form arrays allowing for different testing as well as analytical approaches.

Sialooligosaccharides were generated by direct enzymatic glycosylation on arrays and the resulting surfaces were suitable for the study of carbohydrate-specific cell adhesion.

A chimeric oxygenase, in which the P450cam domain was fused to the reductase host domains of a P450RhF from Rhodococcus sp. strain NCIMB 9784 was optimised to allow for a biotransformation at 30 mM substrate in 80% overall yield, with the linker region between P450 and FMN domain proving to be important for the effective biotransformation of (+)-camphor to 5-exo-hydroxycamphor.

We have previously shown that dipeptides can be synthesised in high yields from amino acids using protease catalysis in aqueous media, if the amino component is immobilised on porous PEGA resin (a copolymer of polyethylene glycol and polyacrylamide). Here we explore the scope of this methodology for using protected and glycosylated amino acids as well as the synthesis of longer peptides on resin and show that such a method can also be applied on non-porous surfaces, in particular on gold.

Mannosyl glycolipids with perfluoroalkyl membrane anchors have been synthesised. When inserted into vesicles, these mannosyl lipids either dispersed evenly over the surface or, in the presence of cholesterol, phase-separated into artificial lipid rafts. At 1% mol/mol, the affinity of dispersed mannosyl lipids for Con A was 3-fold weaker than in solution, perhaps reflecting steric blocking by the surface. However increasing membrane loading 5-fold increased Con A affinity by up to 75% and indicated weak intramembrane chelation of Con A. Despite this observation, concentrating the mannosyl lipids into artificial lipid rafts did not significantly improve affinity for Con A. This lack of a cluster glycoside effect was ascribed to lipid congestion inhibiting intra-raft chelation of Con A, and implies that glycolipids located in lipid rafts may not necessarily be preorganised for multivalent binding.

Carbohydrate arrays (glycoarrays) have recently emerged as a high-throughput tool for studying carbohydrate-binding proteins and carbohydrate-processing enzymes. A number of sophisticated array platforms that allow for qualitative and quantitative analysis of carbohydrate binding and modification on the array surface have been developed, including analysis by fluorescence spectroscopy, mass spectrometry and surface plasmon resonance spectroscopy. These platforms, together with examples of biologically-relevant applications are reviewed in this Feature Article.

Hydroxymethylphenoxy linkers that are commonly used in solid phase peptide synthesis are surprisingly susceptible to efficient cleavage by the protease chymotrypsin with a broad range of amino acid residues being tolerated at the scissile bond; this enzyme-cleavable linker system has been applied to peptide and glycopeptide synthesis.

•Carbohydrates are involved in a diverse variety of biological processes.•Characterization of isomeric carbohydrates is challenging using current techniques.•Mass spectrometry is routinely used to ascertain primary connectivity information.•Coupling ion mobility spectrometry improves separation of isomeric carbohydrates.•Structural information can be ascertained through computational calculations.BackgroundDiverse varieties of often heterogeneous glycans are ubiquitous in nature. They play critical roles in recognition events, act as energy stores and provide structural stability at both molecular and cellular levels. Technologies capable of fully elucidating the structures of glycans are far behind the other ‘-omic’ fields. Liquid chromatography (LC) and mass spectrometry (MS) are currently the most useful techniques for high-throughput analysis of glycans. However, these techniques do not provide full unambiguous structural information and instead the gap in full sequence assignment is frequently filled by a priori knowledge of the biosynthetic pathways and the assumption that these pathways are highly conserved.Scope of the reviewThis comprehensive review details the rise of the emerging analytical technique ion mobility spectrometry (IMS) (coupled to MS) to facilitate the determination of three-dimensional shape: the separation and characterization of isobaric glycans, glyco(peptides/proteins), glycolipids, glycosaminoglycans and other polysaccharides; localization of sites of glycosylation; or interpretation of the conformational change to proteins upon glycan binding.Major conclusionsIMS is a highly promising new analytical route, able to provide rapid isomeric separation (ms timescale) of either precursor or product ions facilitating MS characterization. This additional separation also enables the deconvolution of carbohydrate MS(/MS) information from contaminating ions, improving sensitivity and reducing chemical noise. Derivation of collision cross sections (CCS) from IM-MS(/MS) data and subsequent calculations validate putative structures of carbohydrates from ab initio derived candidates. IM-MS has demonstrated that amounts of specific glycan isomers vary between disease states, which would be challenging to detect using standard analytical approaches.General significanceIM-MS is a promising technique that fills an important gap within the Glycomics toolbox, namely identifying and differentiating the three-dimensional structure of chemically similar carbohydrates and glycoconjugates. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.Download high-res image (356KB)Download full-size image

Mannosyl glycolipids with perfluoroalkyl membrane anchors have been synthesised. When inserted into vesicles, these mannosyl lipids either dispersed evenly over the surface or, in the presence of cholesterol, phase-separated into artificial lipid rafts. At 1% mol/mol, the affinity of dispersed mannosyl lipids for Con A was 3-fold weaker than in solution, perhaps reflecting steric blocking by the surface. However increasing membrane loading 5-fold increased Con A affinity by up to 75% and indicated weak intramembrane chelation of Con A. Despite this observation, concentrating the mannosyl lipids into artificial lipid rafts did not significantly improve affinity for Con A. This lack of a cluster glycoside effect was ascribed to lipid congestion inhibiting intra-raft chelation of Con A, and implies that glycolipids located in lipid rafts may not necessarily be preorganised for multivalent binding.

There is a wide range of immobilisation reactions to tether substrates to a variety of surfaces for array-based analysis. Most of these immobilisation strategies are specific for a particular surface and require an additional linker to be attached to the substrate or the surface. Furthermore, the analysis of functionalised surfaces is often restricted to certain analytical techniques and therefore, different immobilisation strategies for different surfaces are desirable. Here we have tested an S-tritylated linker for non-covalent or covalent immobilisation of mannosides to polystyrene or gold surfaces. S-Tritylated mannosides with varying linkers were readily synthesised and used to add to biorepulsive maleimide-terminated preformed SAMs after in situ deprotection of the S-trityl group. In addition, S-tritylated mannosides themselves formed stable glycoarrays on polystyrene microtiter plates. The glycoarrays were successfully analysed by MALDI-ToF mass spectrometry, SPR spectroscopy, and interrogated with GFP-transfected Escherichia coli cells. This work has shown that a dual purpose linker can be used on multiple surfaces to form arrays allowing for different testing as well as analytical approaches.

A synthetic perfluoroalkyl-tagged lactosyl glycolipid has been shown to form lipid microdomains in fluid phospholipid bilayers. When embedded in the membranes of phospholipid vesicles, this glycolipid was trans-sialylated by soluble T. cruzi trans-sialidase (TcTS) to give a perfluoroalkyl-tagged glycolipid that displayed the ganglioside GM3 epitope, with up to 35% trans-sialylation from fetuin after 18 h. Following sialylation, vesicles bearing this Neu5Ac(α2-3)Gal(β1-4)Glc sequence in their “glycocalyx” were recognised and agglomerated by the lectin M. amurensis leukoagglutinin. Monitoring TcTS-mediated trans-sialylation by HPLC over the first 6 h revealed that enzymatic transformation of bilayer-embedded substrate was much slower than that of a soluble lactosyl substrate. Furthermore, clustering of the lactose-capped glycolipid into “acceptor” microdomains did not increase the rate of sialic acid transfer from fetuin by soluble TcTS, instead producing slight inhibition.

Carbohydrate arrays (glycoarrays) have recently emerged as a high-throughput tool for studying carbohydrate-binding proteins and carbohydrate-processing enzymes. A number of sophisticated array platforms that allow for qualitative and quantitative analysis of carbohydrate binding and modification on the array surface have been developed, including analysis by fluorescence spectroscopy, mass spectrometry and surface plasmon resonance spectroscopy. These platforms, together with examples of biologically-relevant applications are reviewed in this Feature Article.

We have previously shown that dipeptides can be synthesised in high yields from amino acids using protease catalysis in aqueous media, if the amino component is immobilised on porous PEGA resin (a copolymer of polyethylene glycol and polyacrylamide). Here we explore the scope of this methodology for using protected and glycosylated amino acids as well as the synthesis of longer peptides on resin and show that such a method can also be applied on non-porous surfaces, in particular on gold.

A chimeric oxygenase, in which the P450cam domain was fused to the reductase host domains of a P450RhF from Rhodococcus sp. strain NCIMB 9784 was optimised to allow for a biotransformation at 30 mM substrate in 80% overall yield, with the linker region between P450 and FMN domain proving to be important for the effective biotransformation of (+)-camphor to 5-exo-hydroxycamphor.

We report a highly efficient and selective method for the coupling of peptides and glycoconjugates bearing N-terminal cysteines to activated surfaces. This chemoselective method generates stable amide linkages without using any thiol additives.

This review gives an overview of enzymatic reactions that have been conducted on substrates attached to solid surfaces. Such biochemical reactions have become more important with the drive to miniaturisation and automation in chemistry, biology and medicine. Technical aspects such as choice of solid surface and analytical methods are discussed and examples of enzyme reactions that have been successful on these surfaces are provided.

Sialooligosaccharides were generated by direct enzymatic glycosylation on arrays and the resulting surfaces were suitable for the study of carbohydrate-specific cell adhesion.