Reactivity of -acyl hydrazone probes with the mammalian proteome.

Small molecule probes with distinct reactivities are useful tools for the identification and characterization of protein modifications and function. Herein, we show that hydrazone probes with an -carbamate structural motif react differently from -carbamates within the human proteome. Mass spectrometry analysis of probe-treated mammalian cell lysates identified several proteins that were covalently modified by the hydrazone probes, including the cytidine deaminase APOBEC3A.

Optimized aqueous Kinugasa reactions for bioorthogonal chemistry applications.

Kinugasa reactions hold potential for bioorthogonal chemistry in that the reagents can be biocompatible. Unlike other bioorthogonal reaction products, β-lactams are potentially reactive, which can be useful for synthesizing new biomaterials. A limiting factor for applications consists of slow reaction rates.

Rapid Screening and Identification of Living Pathogenic Organisms via Optimized Bioorthogonal Non-canonical Amino Acid Tagging.

Pathogenic bacteria can be a major cause of illness from environmental sources as well as the consumption of contaminated products, giving rise to public health concerns globally. The surveillance of such living organisms in food and water supplies remains an important challenge in mitigating their deleterious societal effects. Here, we have developed an optimized bioorthogonal non-canonical amino acid tagging approach to the imaging, capture, and interrogation of shigatoxigenic/verotoxigenic Escherichia coli (VTEC) and Listeria that enables the distinction between living wild-type pathogenic bacteria.

Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe.

To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells.

Dual Strain-Promoted Alkyne-Nitrone Cycloadditions for Simultaneous Labeling of Bacterial Peptidoglycans.

Bioorthogonal chemistry has been applied to study a multitude of biological processes in complex environments through incorporation and detection of small functional groups. However, few reactions are known to be compatible with each other to allow for studies of more than one biomolecule simultaneously. Here we describe a dual labeling method wherein two stereoelectronically contrasting nitrone tags are incorporated into bacteria peptidoglycan and detected via strain-promoted alkyne-nitrone cycloaddition (SPANC) simultaneously.

Bioorthogonal labelling of living bacteria using unnatural amino acids containing nitrones and a nitrone derivative of vancomycin.

Unnatural D-amino acids bearing endocyclic nitrones were developed for live-cell labelling of the bacterial peptidoglycan layer. Metabolic incorporation of D-Lys and D-Ala derivatives bearing different endocyclic nitrones was observed in E. coli, L.

Correction: Chigrinova, M., et al. Kinugasa reactions in water: from green chemistry to bioorthogonal labelling. Molecules 2015, 20, 6959-6969.

The authors wish to make the following correction to this paper [1]: The author name "Paul Pezacki" should be "John Paul Pezacki". [..

Kinugasa reactions in water: from green chemistry to bioorthogonal labelling.

The Kinugasa reaction has become an efficient method for the direct synthesis of β-lactams from substituted nitrones and copper(I) acetylides. In recent years, the reaction scope has been expanded to include the use of water as the solvent, and with micelle-promoted [3+2] cycloadditions followed by rearrangement furnishing high yields of β-lactams. The high yields of stable products under aqueous conditions render the modified Kinugasa reaction amenable to metabolic labelling and bioorthogonal applications.

Micelle-catalyzed domain swapping in the GlpG rhomboid protease cytoplasmic domain.

Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the Escherichia coli GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer-dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states.

Strain-promoted cycloadditions involving nitrones and alkynes--rapid tunable reactions for bioorthogonal labeling.

The development and applications of strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions have brought about new tools for rapid and specific functionalization of biomolecules in different settings. While a number of strain-promoted reactions have been successfully developed, SPANC reactions offer high reactivity with bimolecular rate constants of k2 that are as fast as 60M(-1)s(-1). SPANC reactions also offer stability of starting materials, particularly in the case of endocyclic nitrones, as well as stereoelectronic tunability of the nitrone moiety to optimize reactivity towards different alkyne reaction partners.

A new chemical probe for phosphatidylinositol kinase activity.

Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive "clickable" probe, PIK-BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK-BPyne to a wortmannin activity-based probe also known to target PIKs.

Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV core DII protein.

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics.

Activity-based protein profiling of the Escherichia coli GlpG rhomboid protein delineates the catalytic core.

Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site.

Insights into the effect of detergents on the full-length rhomboid protease from Pseudomonas aeruginosa and its cytosolic domain.

Rhomboids comprise a family of intramembrane serine proteases that catalyze the cleavage of transmembrane segments within the lipid membrane to achieve a wide range of biological functions. A subset of bacterial rhomboids possesses an N-terminal cytosolic domain that appears to enhance proteolytic activity via an unknown mechanism. Structural analysis of a full-length rhomboid would provide new insights into this mechanism, an objective that solution NMR has the potential to realize.