Developing deprotectase biocatalysts for synthesis.

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.

Development and Application of Desorption Electrospray Ionization Mass Spectrometry for Historical Dye Analysis.

A desorption electrospray ionization (DESI) source was built and attached to a Bruker 7T SolariX FT-ICR-MS for the in situ analysis of 14 early synthetic dyestuffs. Optimization using silk and wool cloths dyed with rhodamine B concluded that when using a commercial electrospray emitter (part number: 0601815, Bruker Daltonik), a nebulizing gas (N2) pressure of 3.9 bar and a sprayer voltage of 4.5 kV (positive ionization mode) or 4.2 kV (negative ionization mode), a solvent system of 3:1 v/v ACN:H2O, and a sprayer incident angle, α, of 35° gave the highest signal-to-noise ratios on both silk and wool for the samples investigated. The system was applied to modern early synthetic dye references on silk and wool as well as historical samples from the 1893 edition of Adolf Lehne's Tabellarische Übersicht über die künstliche organischen Farbstoffe und ihre Anwendung in Färberei und Zeugdruck [Tabular overview of the synthetic organic dyestuffs and their use in dyeing and printing]. The successful analysis of six chemically different dye families in both negative and positive modes showed the presence of known degradation products and byproducts arising from the original synthetic processes in the historical samples. This study demonstrates the applicability and potential of DESI-MS to the field of historical dye analysis.

Stretching the Bisalkyne Raman Spectral Palette Reveals a New Electrophilic Covalent Motif.

Small heteroaryl-diyne (Het-DY) tags with distinct vibrational frequencies, and physiologically relevant cLog P were designed for multiplexed bioorthogonal Raman imaging. Pd-Cu catalyzed coupling, combined with the use of Lei ligand, was shown to improve overall yields of the desired heterocoupled Het-DY tags, minimizing the production of homocoupled side-products. Spectral data were in agreement with the trends predicted by DFT calculations and systematic introduction of electron- rich/poor rings stretched the frequency limit of aryl-capped diynes (2209-2243 cm-1 ). The improved Log P of these Het-DY tags was evident from their diffuse distribution in cellular uptake studies and functionalizing tags with organelle markers allowed the acquisition of location-specific biological images. LC-MS- and NMR-based assays showed that some heteroaryl-capped internal alkynes are potential nucleophile traps with structure-dependent reactivity. These biocompatible Het-DY tags, equipped with covalent reactivity, open up new avenues for Raman bioorthogonal imaging.

Targeting glioblastoma through nano- and micro-particle-mediated immune modulation.

Glioblastoma Multiforme (GBM) is a multifaceted and complex disease, which has experienced no changes in treatment for nearly two decades and has a 5-year survival rate of only 5.4%. Alongside challenges in delivering chemotherapeutic agents across the blood brain barrier (BBB) to the tumour, the immune microenvironment is also heavily influenced by tumour signalling. Immunosuppression is a major aspect of GBM; however, evidence remains conflicted as to whether pro-inflammatory or anti-inflammatory therapies are the key to improving GBM treatment. To address both of these issues, particle delivery systems can be designed to overcome BBB transport while delivering a wide variety of immune-stimulatory molecules to investigate their effect on GBM. This review explores literature from the past 3 years that combines particle delivery systems alongside immunotherapy for the effective treatment of GBM.

Recent advances in the use of stimulated Raman scattering in histopathology.

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.

Halomethyl-Triazoles for Rapid, Site-Selective Protein Modification.

Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.

New Methods for the Synthesis of Spirocyclic Cephalosporin Analogues.

Spiro compounds provide attractive targets in drug discovery due to their inherent three-dimensional structures, which enhance protein interactions, aid solubility and facilitate molecular modelling. However, synthetic methodology for the spiro-functionalisation of important classes of penicillin and cephalosporin ß-lactam antibiotics is comparatively limited. We report a novel method for the generation of spiro-cephalosporin compounds through a Michael-type addition to the dihydrothiazine ring. Coupling of a range of catechols is achieved under mildly basic conditions (K2CO3, DMF), giving the stereoselective formation of spiro-cephalosporins (d.r. 14:1 to 8:1) in moderate to good yields (28-65%).

An Alkyne-Metathesis-Based Approach to the Synthesis of the Anti-Malarial Macrodiolide Samroiyotmycin†A.

We report the first total synthesis of samroiyotmycin A (1), a C2 -symmetric 20-membered anti-malarial macrodiolide isolated from Streptomyces sp. The convergent synthetic strategy orchestrates bisalkyne fragment-assembly using an unprecedented Schöllkopf-type condensation on a substituted ß-lactone and an ambitious late-stage one-pot alkyne cross metathesis-ring-closing metathesis (ACM-RCAM) reaction. The demanding alkyne metathesis sequence is achieved using the latest generation of molybdenum alkylidynes endowed with a tripodal silanolate ligand framework. Subsequent conversion to the required E-alkenes uses contemporary hydrometallation chemistry catalysed by tetrameric cluster [{Cp*RuCl}4 ].

Combining SPR with atomic-force microscopy enables single-molecule insights into activation and suppression of the complement cascade.

Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·µm-2 AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.

Alkyne-Tagged PLGA Allows Direct Visualization of Nanoparticles In Vitro and Ex Vivo by Stimulated Raman Scattering Microscopy.

Polymeric nanoparticles (NPs) are attractive candidates for the controlled and targeted delivery of therapeutics in vitro and in vivo. However, detailed understanding of the uptake, location, and ultimate cellular fate of the NPs is necessary to satisfy safety concerns, which is difficult because of the nanoscale size of these carriers. In this work, we show how small chemical labels can be appended to poly(lactic acid-co-glycolic acid) (PLGA) to synthesize NPs that can then be imaged by stimulated Raman scattering microscopy, a vibrational imaging technique that can elucidate bond-specific information in biological environments, such as the identification of alkyne signatures in modified PLGA terpolymers. We show that both deuterium and alkyne labeled NPs can be imaged within primary rat microglia, and the alkyne NPs can also be imaged in ex vivo cortical mouse brain tissue. Immunohistochemical analysis confirms that the NPs localize in microglia in the mouse brain tissue, demonstrating that these NPs have the potential to deliver therapeutics selectively to microglia.

Kinetic analysis of bioorthogonal reaction mechanisms using Raman microscopy.

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.

A Catch-and-Release Approach to Selective Modification of Accessible Tyrosine Residues.

The tyrosine side chain is amphiphilic leading to significant variations in the surface exposure of tyrosine residues in the folded structure of a native sequence protein. This variability can be exploited to give residue-selective functionalization of a protein substrate by using a highly reactive diazonium group tethered to an agarose-based resin. This novel catch-and-release approach to protein modification has been demonstrated for proteins with accessible tyrosine residues, which are compared with a control group of proteins in which there are no accessible tyrosine residues. MS analysis of the modified proteins showed that functionalization was highly selective, but reactivity was further attenuated by the electrostatic environment of any individual residue. Automated screening of PDB structures allows identification of potential candidates for selective modification by comparison with the accessibility of the tyrosine residue in a benchmark peptide (GYG).

Mono-Substituted Hydrocarbon Diastereomer Combinations Reveal Stapled Peptides with High Structural Fidelity.

Modified peptides, such as stapled peptides, which replicate the structure of α-helical protein segments, represent a potential therapeutic advance. However, the 3D solution structure of these stapled peptides is rarely explored beyond the acquisition of circular dichroism (CD) data to quantify bulk peptide helicity; the detailed backbone structure, which underlies this, is typically undefined. Diastereomeric stapled peptides based on helical sections of three proteins (αSyn, Cks1 and CK1α) were generated; their overall helicity was quantified by CD; and the most helical peptide from each series was selected for structural analysis. Solution-phase models for the optimised peptides were generated using NMR-derived restraints and a modified CHARMM22 force field. Comparing these models with PDB structures allowed deviation between the stapled peptides and critical helical regions to be evaluated. These studies demonstrate that CD alone is not sufficient to assess the structural fidelity of a stapled peptide.

Optimizing the Readout of Lanthanide-DOTA Complexes for the Detection of Ligand-Bound Copper(I).

The CuAAC 'click' reaction was used to couple alkyne-functionalized lanthanide-DOTA complexes to a range of fluorescent antennae. Screening of the antenna components was aided by comparison of the luminescent output of the resultant sensors using data normalized to account for reaction conversion as assessed by IR. A maximum 82-fold enhanced signal:background luminescence output was achieved using a Eu(III)-DOTA complex coupled to a coumarin-azide, in a reaction which is specific to the presence of copper(I). This optimized complex provides a new lead design for lanthanide-DOTA complexes which can act as irreversible 'turn-on' catalytic sensors for the detection of ligand-bound copper(I).

Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-Targeting Thiazole Analogue of Bisebromoamide.

Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid-phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose-dependent response in IRS-1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed.

Selective and Efficient Generation of ortho-Brominated para-Substituted Phenols in ACS-Grade Methanol.

The mono ortho-bromination of phenolic building blocks by NBS has been achieved in short reaction times (15-20 min) using ACS-grade methanol as a solvent. The reactions can be conducted on phenol, naphthol and biphenol substrates, giving yields of >86% on gram scale. Excellent selectivity for the desired mono ortho-brominated products is achieved in the presence of 10 mol % para-TsOH, and the reaction is shown to be tolerant of a range of substituents, including CH3, F, and NHBoc.

Self-Assembly of Disorazole†C1 through a One-Pot Alkyne Metathesis Homodimerization Strategy.

Alkyne metathesis is increasingly explored as a reliable method to close macrocyclic rings, but there are no prior examples of an alkyne-metathesis-based homodimerization approach to natural products. In this approach to the cytotoxic C2-symmetric marine-derived bis(lactone) disorazole C1, a highly convergent, modular strategy is employed featuring cyclization through an ambitious one-pot alkyne cross-metathesis/ring-closing metathesis self-assembly process.

Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues.

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.