Synthesis of macrocyclic thiolactone peptides via photochemical intramolecular radical acyl thiol-ene ligation.

A photochemical acyl thiol-ene reaction can be used to rapidly cyclise fully unprotected peptides bearing both a thioacid and alkene to form peptide thiolactones. This strategy represents the first reported synthesis of peptide thiolactones under radical-mediated conditions.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

Fluorescent Probes for Disease Diagnosis.

The identification and detection of disease-related biomarkers is essential for early clinical diagnosis, evaluating disease progression, and for the development of therapeutics. Possessing the advantages of high sensitivity and selectivity, fluorescent probes have become effective tools for monitoring disease-related active molecules at the cellular level and in vivo. In this review, we describe current fluorescent probes designed for the detection and quantification of key bioactive molecules associated with common diseases, such as organ damage, inflammation, cancers, cardiovascular diseases, and brain disorders. We emphasize the strategies behind the design of fluorescent probes capable of disease biomarker detection and diagnosis and cover some aspects of combined diagnostic/therapeutic strategies based on regulating disease-related molecules. This review concludes with a discussion of the challenges and outlook for fluorescent probes, highlighting future avenues of research that should enable these probes to achieve accurate detection and identification of disease-related biomarkers for biomedical research and clinical applications.

Radical Mediated Decarboxylation of Amino Acids via Photochemical Carbonyl Sulfide (COS) Elimination.

Herein, we present the first examples of amino acid decarboxylation via photochemically activated carbonyl sulfide (COS) elimination of the corresponding thioacids. This method offers a mild approach for the decarboxylation of amino acids, furnishing N-alkyl amino derivatives. The methodology was compatible with amino acids displaying both polar and hydrophobic sidechains and was tolerant towards widely used amino acid-protecting groups. The compatibility of the reaction with continuous-flow conditions demonstrates the scalability of the process.

A thiol-ene mediated approach for peptide bioconjugation using 'green' solvents under continuous flow.

Flow chemistry has emerged as an integral process within the chemical sector permitting energy efficient synthetic scale-up while improving safety and minimising solvent usage. Herein, we report the first applications of the photoactivated, radical-mediated thiol-ene reaction for peptide bioconjugation under continuous flow. Bioconjugation reactions employing deep eutectic solvents, bio-based solvents and fully aqueous systems are reported here for a range of biologically relevant peptide substrates. The use of a water soluble photoinitiator, Irgacure 2959, permitted synthesis of glycosylated peptides in fully aqueous conditions, obviating the need for addition of organic solvents and enhancing the green credentials of these rapid, photoactivated, bioconjugation reactions.

Traceless Thioacid-Mediated Radical Cyclization of 1,6-Dienes.

Five-membered ring systems are ubiquitous throughout natural products and synthetic therapeutics, and thus, efficient methods to access this essential scaffold are required. Herein, we report the thioacid-mediated, 5-exo-trig cyclization of various 1,6-dienes, with high yields of up to 98%. The labile thioester functionality can be exploited to generate a free thiol residue which can be used as a functional handle or removed entirely to provide the traceless cyclized product.

Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein N-acyltransferase.

Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein N-acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.

Glycosylated quantum dots as fluorometric nanoprobes for trehalase.

Trehalase is an important enzyme in the metabolic cascades of many organisms, catalysing the hydrolysis of the disaccharide trehalose. Herein we describe the first examples of fluorometric nanoprobes for detection of trehalase, based on trehalose-functionalised quantum dots (QDs). QDs cross-linked with trehalose form aggregates, which are released upon enzymatic cleavage of the trehalose glycosidic bond proportionally to the enzyme concentration, offering a unique and efficient approach for specific sensing of this biologically important enzyme.

Glycosidase activated prodrugs for targeted cancer therapy.

In this review glycosidase activated prodrugs that target cancer cells are discussed. Glycosylated prodrugs undergo enzymatic bioconversion, cleaving the prodrug to release the anticancer drug at the desired site of action, hence minimising the toxic side effects associated with many current anticancer drugs. In addition, the presence of the carbohydrate moiety increases the aqueous solubility of the drugs, allowing for a more effective treatment. In the past decade, significant advancements have been made in this field that have led to the development of many novel carbohydrate-based prodrugs - ranging from simple glycoconjugates to complex self-assemblies and materials, which are discussed in detail herein.

Thioether analogues of the pituitary neuropeptide oxytocin via thiol-ene macrocyclisation of unprotected peptides.

Disulfide bonds are an essential feature of many bioactive peptides, however, they are labile to reducing conditions which can limit therapeutic application. Herein, we report an efficient methodology for peptide macrocyclisation, furnishing thioether mimetics of disulfide linkages via thiol-ene click chemistry. Furthermore, this methodology is applied to the efficient synthesis of analogues of the neuropeptide oxytocin and in a highly efficient route to the clinical therapeutic carbetocin.

Macrophage innate training induced by IL-4 and IL-13 activation enhances OXPHOS driven anti-mycobacterial responses.

Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating bacteria such as Mycobacterium tuberculosis. By contrast, 'alternatively activated' M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here, we demonstrate that activation with IL-4 and IL-13 counterintuitively induces protective innate memory against mycobacterial challenge. In human and murine models, prior activation with IL-4/13 enhances pro-inflammatory cytokine secretion in response to a secondary stimulation with mycobacterial ligands. In our murine model, enhanced killing capacity is also demonstrated. Despite this switch in phenotype, IL-4/13 trained murine macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4/13 trained macrophages. Lastly, this work identifies that IL-10 attenuates protective IL-4/13 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.

Real-Time Multi-Photon Tracking and Bioimaging of Glycosylated Theranostic Prodrugs upon Specific Enzyme Triggered Release.

Real-time tracking of prodrug uptake, delivery and activation in vivo represents a major challenge for prodrug development. Herein, we demonstrate the use of novel glycosylated theranostics of the cancer pharmacophore Amonafide in highly-selective, enzymatic triggered release. We show that the use of endogenous enzymes for activated release of the therapeutic component can be observed, in real time, and monitored using one and two-photon bioimaging, offering unique insight into the prodrug pharmacokinetic profile. Furthermore, we demonstrate that the potent cytotoxicity of Amonafide is preserved using this targeted approach.

Synthetic Strategies for FRET-Enabled Carbohydrate Active Enzyme Probes.

Carbohydrates are an essential class of biomolecule and carbohydrate active enzymes (CAZys) catalyze their synthesis, refinement, and degradation, hence contributing an overall regulatory capacity to their underpinning physiological roles. Here we survey recent accomplishments for accessing defined carbohydrate structures, suitably equipped with FRET probe capability, followed by their utilization in studying particular classes of CAZy.

Cysteinyl radicals in chemical synthesis and in nature.

Nature harnesses the unique properties of cysteinyl radical intermediates for a diverse range of essential biological transformations including DNA biosynthesis and repair, metabolism, and biological photochemistry. In parallel, the synthetic accessibility and redox chemistry of cysteinyl radicals renders them versatile reactive intermediates for use in a vast array of synthetic applications such as lipidation, glycosylation and fluorescent labelling of proteins, peptide macrocyclization and stapling, desulfurisation of peptides and proteins, and development of novel therapeutics. This review provides the reader with an overview of the role of cysteinyl radical intermediates in both chemical synthesis and biological systems, with a critical focus on mechanistic details. Direct insights from biological systems, where applied to chemical synthesis, are highlighted and potential avenues from nature which are yet to be explored synthetically are presented.

Controlling the Carbon-Bio Interface via Glycan Functional Adlayers for Applications in Microbial Fuel Cell Bioanodes.

Surface modification of electrodes with glycans was investigated as a strategy for modulating the development of electrocatalytic biofilms for microbial fuel cell applications. Covalent attachment of phenyl-mannoside and phenyl-lactoside adlayers on graphite rod electrodes was achieved via electrochemically assisted grafting of aryldiazonium cations from solution. To test the effects of the specific bio-functionalities, modified and unmodified graphite rods were used as anodes in two-chamber microbial fuel cell devices. Devices were set up with wastewater as inoculum and acetate as nutrient and their performance, in terms of output potential (open circuit and 1 kΩ load) and peak power output, was monitored over two months. The presence of glycans was found to lead to significant differences in startup times and peak power outputs. Lactosides were found to inhibit the development of biofilms when compared to bare graphite. Mannosides were found, instead, to promote exoelectrogenic biofilm adhesion and anode colonization, a finding that is supported by quartz crystal microbalance experiments in inoculum media. These differences were observed despite both adlayers possessing thickness in the nm range and similar hydrophilic character. This suggests that specific glycan-mediated bioaffinity interactions can be leveraged to direct the development of biotic electrocatalysts in bioelectrochemical systems and microbial fuel cell devices.

Aggregation of protein therapeutics enhances their immunogenicity: causes and mitigation strategies.

Protein aggregation in biotherapeutics has been identified to increase immunogenicity, leading to immune-mediated adverse effects, such as severe allergic responses including anaphylaxis. The induction of anti-drug antibodies (ADAs) moreover enhances drug clearance rates, and can directly block therapeutic function. In this review, identified immune activation mechanisms triggered by protein aggregates are discussed, as well as physicochemical properties of aggregates, such as size and shape, which contribute to immunogenicity. Furthermore, factors which contribute to protein stability and aggregation are considered. Lastly, with these factors in mind, we encourage an innovative and multidisciplinary approach with regard to further research in the field, with the overall aim to avoid immunogenic aggregation in future drug development.

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis.

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.

Applications of Thiol-Ene Chemistry for Peptide Science.

Radical thiol-ene chemistry has been demonstrated for a range of applications in peptide science, including macrocyclization, glycosylation and lipidation amongst a myriad of others. The thiol-ene reaction offers a number of advantages in this area, primarily those characteristic of "click" reactions. This provides a chemical approach to peptide modification that is compatible with aqueous conditions with high orthogonality and functional group tolerance. Additionally, the use of a chemical approach for peptide modification affords homogeneous peptides, compared to heterogeneous mixtures often obtained through biological methods. In addition to peptide modification, thiol-ene chemistry has been applied in novel approaches to biological studies through synthesis of mimetics and use in development of probes. This review will cover the range of applications of the radical-mediated thiol-ene reaction in peptide and protein science.

Mycobacterial para-Hydroxybenzoic Acid-Derivatives (pHBADs) and Related Structures Induce Macrophage Innate Memory.

Macrophages are key immune cells for combatting Mycobacterium tuberculosis. However, M. tuberculosis possesses means to evade macrophage bactericidal responses by, for instance, secretion of the immunomodulatory para-hydroxybenzoic acid derivatives (pHBADs). While these molecules have been implicated in inhibiting macrophage responses in an acute context, little is known about their ability to reprogram macrophages via induction of long-term innate memory. Since innate memory has been highlighted as a promising strategy to augment bactericidal immune responses against M. tuberculosis, investigating corresponding immune evasion mechanisms is highly relevant. Our results reveal for the first time that pHBAD I and related molecules (unmethylated pHBAD I and the hexose l-rhamnose) reduce macrophage bactericidal mechanisms in both the short- and the long-term. Moreover, we demonstrate how methyl-p-anisate hinders bactericidal responses soon after exposure yet results in enhanced pro-inflammatory responses in the long-term. This work highlights new roles for these compounds in M. tuberculosis pathogenesis.

Tailored glycosylated anode surfaces: Addressing the exoelectrogen bacterial community via functional layers for microbial fuel cell applications.

Grafting of aryldiazonium cations bearing a p-mannoside functionality over microbial fuel cell (MFC) anode materials was performed to investigate the ability of aryl-glycoside layers to regulate colonisation by biocatalytic biofilms. Covalent attachment was achieved via spontaneous reactions and via electrochemically-assisted grafting using potential step experiments. The effect of different functionalisation protocols on MFC performance is discussed in terms of changes in wettability, roughness and electrochemical response of modified electrodes. Water contact angle measurements (WCA) show that aryl-mannoside grafting yields a significant increase in hydrophilic character. Surface roughness determinations via atomic force microscopy (AFM) suggest a more disordered glycan adlayer when electrografting is used to facilitate chemisorption. MFCs were used as living sensors to successfully test the coated electrodes: the response of the MFCs in terms of start-up time was accelerated when compared to that of MFC equipped with non-modified electrodes, this suggests a faster development of a mature biofilm community resulting from aryldiazonium modifications, as confirmed by cyclic voltammetry of MFC anodes. These results therefore indicate that modification with glycans offers a bioinspired route to accelerating biofilm colonisation without any adverse effects on final MFC outputs.