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Organic synthesis often requires multiple steps where a functional group (FG) is concealed from reaction by a protecting group (PG). Common PGs include N-carbobenzyloxy (Cbz or Z) of amines and tert-butyloxycarbonyl (OtBu) of acids. An essential step is the removal of the PG, but this often requires excess reagents, extensive time and can have low % yield. An overarching goal of biocatalysis is to use "green" or "enzymatic" methods to catalyse chemical transformations. One under-utilised approach is the use of "deprotectase" biocatalysts to selectively remove PGs from various organic substrates. The advantage of this methodology is the exquisite selectivity of the biocatalyst to only act on its target, leaving other FGs and PGs untouched. A number of deprotectase biocatalysts have been reported but they are not commonly used in mainstream synthetic routes. This study describes the construction of a cascade to deprotect doubly-protected amino acids. The well known Bacillus BS2 esterase was used to remove the OtBu PG from various amino acid substrates. The more obscure Sphingomonas Cbz-ase (amidohydrolase) was screened with a range of N-Cbz-modified amino acid substrates. We then combined both the BS2 and Cbz-ase together for a 1 pot, 2 step deprotection of the model substrate CBz- L -Phe OtBu to produce the free L -Phe. We also provide some insight into the residues involved in substrate recognition and catalysis using docked ligands in the crystal structure of BS2. Similarly, a structural model of the Cbz-ase identifies a potential di-metal binding site and reveals conserved active site residues. This new biocatalytic cascade should be further explored for its application in chemical synthesis. [ABSTRACT FROM AUTHOR]

Stimulated Raman histopathology (SRH) utilises the intrinsic vibrational properties of lipids, proteins and nucleic acids to generate contrast providing rapid image acquisition that allows visualisation of histopathological features. It is currently being trialled in the intraoperative setting, where the ability to image unprocessed samples rapidly and with high resolution offers several potential advantages over the use of conventional haematoxylin and eosin stained images. Here we review recent advances in the field including new updates in instrumentation and computer aided diagnosis. We also discuss how other non-linear modalities can be used to provide additional diagnostic contrast which together pave the way for enhanced histopathology and open up possibilities for in vivo pathology. [ABSTRACT FROM AUTHOR]

Raman spectroscopy is well-suited to the study of bioorthogonal reaction processes because it is a non-destructive technique, which employs relatively low energy laser irradiation, and water is only very weakly scattered in the Raman spectrum enabling live cell imaging. In addition, Raman spectroscopy allows species-specific label-free visualisation; chemical contrast may be achieved when imaging a cell in its native environment without fixatives or stains. Combined with the rapid advances in the field of Raman imaging over the last decade, particularly in stimulated Raman spectroscopy (SRS), this technique has the potential to revolutionise our mechanistic understanding of the biochemical and medicinal chemistry applications of bioorthogonal reactions. Current approaches to the kinetic analysis of bioorthogonal reactions (including heat flow calorimetry, UV-vis spectroscopy, fluorescence, IR, NMR and MS) have a number of practical shortcomings for intracellular applications. We highlight the advantages offered by Raman microscopy for reaction analysis in the context of both established and emerging bioorthogonal reactions, including the copper(I) catalysed azide–alkyne cycloaddition (CuAAC) click reaction and Glaser–Hay coupling. [ABSTRACT FROM AUTHOR]

Peptide stapling is an effective strategy to stabilise α-helical peptides, enhancing their bioactive conformation and improving physiochemical properties. In this study, we apply our novel diyne-girder stapling approach to the MDM2/MDMX α-helical binding region of the p53 transactivation domain. By incorporation of an unnatural amino acid to create an optimal i , i + 7 bridge length, we developed a highly α-helical stapled peptide, 4, confirmed via circular dichroism. This diyne-girder-stapled peptide demonstrated enhanced helicity and nanomolar binding affinity for MDM2, as assessed by fluorescence polarisation. Crucially, peptide 4 exhibited strong selectivity for MDM2, with approximately 100-fold reduced affinity for MDMX. Molecular modeling and docking studies suggested that this selectivity arose from diminished hydrophobic interactions at the MDMX binding site, driven by the diyne-girder's constrained geometry. The use of the diyne-girder, a unique feature amongst stapled peptide analogues, for cellular visualisation using Raman spectroscopy in the "cell-silent" region was explored. This capability potentially offers a novel method for tracking stapled peptides in biological systems without the need for large fluorophores. Overall, peptide 4 represents a promising tool for probing MDM2 activity and a valuable addition to the arsenal of peptide-based therapeutic strategies., Competing Interests: A patent application [(GB) patent application no: 2219576.2] has been submitted on this work. D. T. H. is a consultant for Triana Biomedicines. There are no other conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Cyclophilins (Cyps) are a major family of drug targets that are challenging to prosecute with small molecules because the shallow nature and high degree of conservation of the active site across human isoforms offers limited opportunities for potent and selective inhibition. Herein a computational approach based on molecular dynamics simulations and free energy calculations was combined with biophysical assays and X-ray crystallography to explore a flip in the binding mode of a reported urea-based Cyp inhibitor. This approach enabled access to a distal pocket that is poorly conserved among key Cyp isoforms, and led to the discovery of a new family of sub-micromolar cell-active inhibitors that offer unprecedented opportunities for the development of next-generation drug therapies based on Cyp inhibition. The computational approach is applicable to a broad range of organic functional groups and could prove widely enabling in molecular design.

A Heck cyclisation approach is described for the rapid synthesis of a library of natural product-like small molecules, based on the phenanthridine core. The synthesis of a range of substituted benzylamine building blocks and their incorporation into the library is reported, together with a highly selective cis-dihydroxylation protocol that enables access to the target compounds in an efficient manner. Biological evaluation of the library using zebrafish phenotyping has led to the discovery of compound 20c, a novel inhibitor of early-stage zebrafish embryo development.

The chemical functionalization of glycosaminoglycans is very challenging due to their structural heterogeneity and polyanionic character; but as an enabling technology it promises rich rewards in terms of the structural and biological data it will afford. This review surveys the known methods for the preparation of glycosaminoglycan oligosaccharides and conditions for the selective functionalization of both the reducing and non-reducing ends. The synthetic merits of each approach are discussed, together with the structural modification of the glycosaminoglycan oligosaccharide which they confer. Recent applications of this methodology are highlighted, including introduction of functional labels for gel mobility shift assays and NMR studies of glycosaminoglycan-protein complexes, and synthesis of immobilised glycosaminoglycan arrays.

Efficient functionalisation of the non-reducing end of uronic acid derivatives and glycosaminoglycan-derived disaccharides using peptide coupling has been achieved, mediated by the water-soluble agent DMT-MM.

A truncated approach to the design of molecular probes from small molecule libraries is outlined, based upon the incorporation of a bioorthogonal marker. The applicability of this strategy to small molecule chemical genetics screens has been demonstrated using analogues of the known stress activated protein kinase (SAPK) pathway activator, anisomycin. Compounds marked with a propargyl group have shown activation of the SAPK pathways comparable to that induced by their parent structures, as demonstrated by immunoblot assays against the downstream target JNK1/2. The considerable advantages of this new approach to molecular probe design have been illustrated through the rapid development of a functionally active fluorescent molecular probe, through coupling of the marked analogues to fluorescent azides using the copper(i)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. Active molecular probes generated in this study were used to investigate cellular uptake through FACS analysis and confocal microscopy.

The synthesis of C(4)H and C(4)Me analogues of the JNK/p38 pathway activator anisomycin, based upon an aldol or Claisen construction of the C(3)-C(4) bond, has been demonstrated. The relative activation of the JNK/SAPK1 and p38/SAPK2 pathways in RAW macrophages by these analogues, and their synthetic precursors, has been assessed using immunoblot assays against phosphorylated c-Jun and MAPKAP-K2. These studies demonstrate that some of the synthetic C(4) analogues are also potent activators of these stress kinase pathways.