Chemoenzymatic Labeling, Detection and Profiling of Core Fucosylation in Live Cells.

Core fucosylation, the attachment of an α-1,6-linked-fucose to the N-glycan core pentasaccharide, is an abundant protein modification that plays critical roles in various biological processes such as cell signaling, B cell development, antibody-dependent cellular cytotoxicity, and oncogenesis. However, the tools currently used to detect core fucosylation suffer from poor specificity, exhibiting cross-reactivity against all types of fucosylation. Herein we report the development of a new chemoenzymatic strategy for the rapid and selective detection of core fucosylated glycans. This approach employs a galactosyltransferase enzyme identified fromCaenorhabditis elegansthat specifically transfers an azido-appended galactose residue onto core fucose via a ß-1,4 glycosidic linkage. We demonstrate that the approach exhibits superior specificity toward core fucose on a variety of complex N-glycans. The method enables detection of core fucosylated glycoproteins from complex cell lysates, as well as on live cell surfaces, and it can be integrated into a diagnostic platform to profile protein-specific core fucosylation levels. This chemoenzymatic labeling approach offers a new strategy for the identification of disease biomarkers and will allow researchers to further characterize the fundamental role of this important glycan in normal and disease physiology.

Bioorthogonal Hydroamination of Push-Pull-Activated Linear Alkynes.

A bioorthogonal reaction between N,N-dialkylhydroxylamines and push-pull-activated halogenated alkynes is described. We explore the use of rehybridization effects in activating alkynes, and we show that electronic effects, when competing stereoelectronic and inductive factors are properly balanced, sufficiently activate a linear alkyne in the uncatalyzed conjugative retro-Cope elimination reaction while adequately protecting it against cellular nucleophiles. This design preserves the low steric profile of an alkyne and pairs it with a comparably unobtrusive hydroxylamine. The kinetics are on par with those of the fastest strain-promoted azide-alkyne cycloaddition reactions, the products regioselectively formed, the components sufficiently stable and easily installed, and the reaction suitable for cellular labeling.

Enamine N-Oxides: Synthesis and Application to Hypoxia-Responsive Prodrugs and Imaging Agents.

Tumor hypoxia induces the large-scale adaptive reprogramming of cancer cells, promoting their transformation into highly invasive and metastatic species that lead to highly negative prognoses for cancer patients. We describe the synthesis and application of a hypoxia-responsive trigger derived from previously inaccessible enamine N-oxide structures. Hypoxia-dependent reduction of this motif by hemeproteins results in the concomitant activation of a caged molecule and a latent electrophile. We exploit the former in a hypoxia-activated prodrug application using a caged staurosporine molecule as a proof-of-principle. We demonstrate the latter in in vivo tumor labeling applications with enamine-N-oxide-modified near-infrared probes. Hypoxia-activated prodrug development has long been complicated by the heterogeneity of tumor hypoxia in patients. The dual drug release and imaging modalities of the highly versatile enamine N-oxide motif present an attractive opportunity for theranostic development that can address the need not only for new therapeutics but paired methods for patient stratification.

Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sulfation.

Glycosaminoglycans (GAGs) play critical roles in diverse processes ranging from viral infection to neuroregeneration. Their regiospecific sulfation patterns, which are generated by sulfotransferases, are key structural determinants that underlie their biological activity. Small-molecule modulators of these sulfotransferases could serve as powerful tools for understanding the physiological functions of GAGs, as well as potential therapeutic leads for human diseases. Here, we report the development of the first cell-permeable, small-molecule inhibitor selective for GAG sulfotransferases, which was obtained using a high-throughput screen targeted against Chst15, the sulfotransferase responsible for biosynthesis of chondroitin sulfate-E (CS-E). We demonstrate that the molecule specifically inhibits GAG sulfotransferases in vitro, decreases CS-E and overall sulfation levels on cell-surface and secreted chondroitin sulfate proteoglycans (CSPGs), and reverses CSPG-mediated inhibition of axonal growth. These studies pave the way toward a new set of pharmacological tools for interrogating GAG sulfation-dependent processes and may represent a novel therapeutic approach for neuroregeneration.