Mechanistic and structural studies into the biosynthesis of the bacterial sugar pseudaminic acid (Pse5Ac7Ac)

The non-mammalian nonulosonic acid sugar pseudaminic acid (Pse) is present on the surface of a number of human pathogens including Campylobacter jejuni and Helicobacter pylori and other bacteria such as multidrug resistant Acinetobacter baumannii. It is likely important for evasion of the host immune sysyem, and also plays a role in bacterial motility through flagellin glycosylation. Herein we review the mechanistic and structural characterisation of the enzymes responsible for the biosynthesis of the Pse parent structure, Pse5Ac7Ac in bacteria

Synthetic Approaches for Accessing Pseudaminic Acid (Pse) Bacterial Glycans

Sugars to order: A summary of work in the field of pseudaminic acid (Pse) synthesis is provided. This non‐mammalian sugar is of increasing biological importance as an essential component in cell‐surface glycoconjugates of a number of pathogenic bacteria. Pioneering studies into biosynthesis of Pse5Ac7Ac have provided inspiration to carbohydrate chemists

Developments in mannose-based treatments for Uropathogenic Escherichia coli induced urinary tract infections

During their lifetime almost half of women will experience a symptomatic urinary tract infection (UTI) with a further half experiencing a relapse within six months. Currently UTIs are treated with antibiotics, but increasing antibiotic resistance rates highlight the need for new treatments. Uropathogenic Escherichia coli (UPEC) is responsible for the majority of symptomatic UTI cases and thus has become a key pathological target. Adhesion of type one pilus subunit FimH at the surface of UPEC strains to mannose-saturated oligosaccharides located on the urothelium is critical to pathogenesis. Since the identification of FimH as a therapeutic target in the late 1980s, a substantial body of research has been generated focusing on the development of FimH-targeting mannose-based anti-adhesion therapies. In this review we will discuss the design of different classes of these mannose-based compounds and their utility and potential as UPEC therapeutics

Palladium-unleashed proteins: gentle aldehyde decaging for site-selective protein modification

Protein bioconjugation frequently makes use of aldehydes as reactive handles, with methods for their installation being highly valued. Here a new, powerful strategy to unmask a reactive protein aldehyde is presented. A genetically encoded caged glyoxyl aldehyde, situated in solvent-accessible locations, can be rapidly decade through treatment with just one equivalent of allylpalladium(II) chloride dimer at physiological pH. The protein aldehyde can undergo subsequent oxime ligation for site-selective protein modification. Quick yet mild conditions, orthogonality and powerful exposed reactivity make this strategy of great potential in protein modification

Palladium-unleashed proteins: gentle aldehyde decaging for site-selective protein modification

Protein bioconjugation frequently makes use of aldehydes as reactive handles, with methods for their installation being highly valued. Here a new, powerful strategy to unmask a reactive protein aldehyde is presented. A genetically encoded caged glyoxyl aldehyde, situated in solvent-accessible locations, can be rapidly decade through treatment with just one equivalent of allylpalladium(II) chloride dimer at physiological pH. The protein aldehyde can undergo subsequent oxime ligation for site-selective protein modification. Quick yet mild conditions, orthogonality and powerful exposed reactivity make this strategy of great potential in protein modification

Chemical Bioconjugation of Proteins in an Undergraduate Lab: One-Pot Oxidation and Derivatization of the N-Terminus

A laboratory experiment introducing the concept of chemical bioconjugation of proteins to undergraduate students in a therapeutically relevant context was developed. Initially, students installed an aldehyde functionality into a protein via the oxidation of the N-terminal threonine residue of the cholera toxin subunit B (CTB) protein, which was followed by subsequent modification via hydrazone addition under mild conditions with a chromophore bearing a distinct UV–vis-absorption peak. Students determined the yield of the reaction to be ca. 11% by HPLC coupled to UV–vis spectroscopy and developed key skills such as preparation of stock solutions, chemical manipulation of proteins, and analysis via HPLC. The reported experiment can be readily adapted for use with other proteins and may contribute to enhancing constructive alignment in interdisciplinary degree programs at the chemistry–biology interface

Aldehyde-mediated protein-to-surface tethering via controlled diazonium electrode functionalization using protected hydroxylamines

We report a diazonium electro-grafting method for the covalent modification of conducting surfaces with aldehyde-reactive hydroxylamine functionalities that facilitate the wiring of redox-active (bio)molecules to electrode surfaces. Hydroxylamine monolayer formation is achieved via a phthalimide-protection and hydrazine-deprotection strategy that overcomes the multilayer formation that typically complicates diazonium surface modification. This surface modification strategy is characterized using electrochemistry (electrochemical impedance spectroscopy and cyclic voltammetry), X-ray photoelectron spectroscopy and quartz crystal microbalance with dissipation monitoring. Thus-modified glassy carbon, boron-doped diamond and gold surfaces are all shown to ligate to small molecule aldehydes, yielding surface coverages of 150-170, 40 and 100 pmol cm-2, respectively. Bio-conjugation is demonstrated via the coupling of a dilute (50 µM) solution of periodate-oxidized horseradish peroxidase enzyme to a functionalized gold surface under bio-compatible conditions (H2O solvent, pH 4.5, 25 °C)

Profiling substrate promiscuity of wild-type sugar kinases for multifluorinated monosaccharides

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 fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluoro galactose, giving insight into changes in the active site that may account for the specificity of the enzyme towards certain substrate analogues