Chemical Biology Discussion Group Year-End Meeting

Agenda

* Presentation times are subject to change

Abstracts

Stereoselective Reactions of Oxocarbenium Ions: Conformational Analysis, Stereoelectronic Effects, and Reactivity

Keith Woerpel, PhD, New York University

Because oxocarbenium ions are important intermediates in both synthetic chemistry and glycobiology, a deeper understanding of their structures and reactivities would benefit both fields. Our research demonstrates that the structures of oxocarbenium ions related to carbohydrate systems cannot be explained by consideration of steric effects. Both stereoselectivities and spectroscopic analysis show that electronic effects exert powerful influences on the conformational preferences of these cations. The reactions of oxocarbenium ions, which generally conform to stereoelectronic models, can be more difficult to analyze than might be expected. Nucleophilic additions to these cations can occur through the lowest energy conformer, but in many cases nucleophiles add to higher-energy forms of the cations, consistent with the Curtin–Hammett principle.

Transition State of ADP-ribosylation of Acetyllysine Catalyzed by Archeaglobus fulgidus Sir2 Determined by Kinetic Isotope Effects and Computational Approaches

Yana Cen, PhD, Weill Cornell Medical College

Chemically stable transition state analogues are powerful inhibitors by trapping energy of catalysis as binding energy. Accumulating evidence has established that transition state structures of enzyme isoforms may differ from one another, which makes the development of isozyme specific inhibitors highly possible. Sirtuins are protein modifying enzymes distributed throughout all forms of life. These enzymes bind NAD+, a universal metabolite, and react it with acetyllysine residues, to effect deacetylation of protein side chains. The first chemical step of deacetylation is the reaction of NAD+ with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. We solved the transition state for the ADP-ribosylation of acetyllysine for an Archaeaglobus fulgidus sirtuin (Af2Sir2). Multiple kinetic isotope effects (KIEs) were obtained by the competitive substrate method, and they provided a boundary condition for the quantum chemistry calculations. Transition state geometry was varied until the calculated KIEs matched the experimentally determined KIEs. Our results indicate that the enzyme stabilizes a highly dissociated oxacarbenium ion-like transition state with very low bond orders to leaving group nicotinamide and nucleophile acetyllysine. These findings reveal that the enzyme proceeds via a concerted yet highly asynchronous substitution mechanism in formation of the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction. This transition state structure provides the blueprint to design molecules of similar shape and charge but with increased chemical stability.

Design and Synthesis of Immuno–potentiating Saponins to Probe the Mechanism of Action of the Vaccine Adjuvant QS-21

Eric Chea, Memorial Sloan Kettering Cancer Center

QS-21 is an immuno-stimulatory saponin adjuvant used in vaccines to improve efficacy, and has found use in over 100 clinical trials that span various pathogenic, oncogenic, and cognitive diseases. Even with many therapeutic indications, little is known about its mechanism of action – partly due to the lack of tools to better understand its immuno-potentiating effects. We designed/synthesized the first set of chemically defined QS-21 analogues to serve as probes, which have allowed us to demonstrate for the first time that immuno-active saponins accumulate in the draining lymph nodes. Contrary to conventional wisdom, preliminary evidence suggests that these saponins do not simply associate with the plasma membrane, but rather they are internalized in a cell-specific fashion. Implications of this internalization on the mechanism of action of immuno-potentiating saponins are currently being investigated..

Synthesis of Complex Natural Products: Discoveries and Challenges in the Application of the Rauhut-Currier Reaction 

Alpay Dermenci, Yale University

Work in the Miller lab focuses on asymmetric catalysis and natural product synthesis and modification. In particular, this work involves a cysteine-catalyzed Rauhut-Currier reaction for the synthesis of natural products, namely Sch-642305 and Glabramycin C. This work has furthered our understanding of catalyst-substrate recognition in complex systems and substantiated the effectiveness of employing amino acids as catalysts. We have showcased the multifunctional nature of single amino acids highlighting both the chirality and the functional groups that constitute natural amino acids. In addition to our synthetic discoveries, we have also tested Sch-642305 and related analogs in HIV-infected cells and various cancer cell lines. Ultimately, as we make progress toward understanding nature’s manner of preparing Sch-642305 and its congeners through application of single amino acid catalysts in total synthesis, we expect that our work may inspire future discoveries of nature’s biosynthetic pathways for constructing biologically active molecules..

Chemical Reporter for Protein AMPylation in Bacterial Pathogenesis

Markus Grammel, The Rockefeller University

Bacterial pathogens establish intricate interactions with the host during the course of an infection. Protein AMPylation is an emerging posttranslational modification (PTM), used by bacterial pathogens to interfere with host signaling processes. AMPylation refers to the addition of an adenosine 5’-monophosphate onto hydroxyl groups of amino acid side-chains. Several secreted bacterial AMPylation enzymes have been identified that modify host proteins and additional putative AMPylation enzymes are present in numerous bacteria. Since analytical tools for AMPylation are currently very limited, we developed a general alkynyl chemical reporter for protein AMPylation to further the understanding of this understudied PTM. Covalent functionalization of AMPylation substrates with the alkynyl chemical reporter in lieu of adenylyl 5’-monophosphate allows its subsequent detection by Cu(I)-catalyzed alkyne-azide cycloaddition. We show that this chemical reporter is transferred by two recently identified AMPylation enzymes, VopS and Fic2, onto its cognate substrate Cdc42 and allows rapid detection of AMPylation.

Peptoids as Antimicrobial Agents

Mia Huang, New York University

We investigate the antimicrobial activities of peptoid oligomers comprising cationic and hydrophobic sequences. We demonstrate that they are potent and selective agents against gram-negative and gram-positive bacteria. These oligomers display low (g/mL) minimum inhibitory concentrations (MICs) against E. coli, S. aureus, and B. subtilis and are non-toxic to mammalian cells. Structure-activity relationships establish the dependence of activities on conformational rigidity and a balance of cationic and hydrophobic surface area. We demonstrate the potency of a model cationic peptoid macrocycle against clinical and community-acquired pathogenic strains of Staphylococcus aureus while deterring the emergence of antimicrobial resistance. These results demonstrate the potential of peptoid oligomers as advanced therapeutics that can address the growing need for new agents to combat antimicrobial resistance.

The Molecular Basis of Nitric Oxide Regulated C-Di-GMP Synthesis and Biofilm Formation in Shewanella Woodyi

Niu Liu, Stony Brook University

Diguanylate cyclases (DGCs) synthesize cyclic-di-GMP (c-di-GMP) in bacteria. C-di-GMP is important for many biological processes, including virulence, luminescence, and biofilm formation. However, regulation of DGCs as well as the downstream targets of c-di-GMP, are still under investigation. In this work, we have characterized a DGC (Swoo_2750; SwDGC) protein from Shewanella woodyi. We have demonstrated its role in c-di-GMP production and biofilm formation. We hypothesize that the cyclase activity of SwDGC is regulated by a neighboring gene product, Swoo_2751. Studies in our laboratory have shown that Swoo_2751 is a member of the H-NOX family (heme-nitric oxide/oxygen binding domain), a family of proteins found from bacteria to man that are demonstrated nitric oxide (NO) sensors. Indeed, enzymatic and cell-based assays have shown that NO-bound SwH-NOX regulates the activity of SwDGC, leading to a change in c-di-GMP concentration in bacteria. Moreover, immuno-precipitation results have demonstrated direct evidence of a binding interaction between SwH-NOX and SwDGC. Knock-out studies also show that SwH-NOX/SwDGC is involved in biofilm formation. We will present data demonstrating a new role for NO and H-NOX in bacterial biofilm formation via regulation of intracellular c-di-GMP concentration.

Reproducible enrichment of extracellular heat shock proteins from blood serum

Doina Mihai, Hunter College, CUNY

Heat shock proteins (Hsps) are well-known cytosolic proteins, playing important roles in the maintenance of correct protein folding in the cell. Recently, Hsps have also been found in the extracellular milieus, such as tumor surface and blood circulation, suggesting broader but as-yet uncharacterized functions of these proteins. Characterization of extracellular Hsps (eHsps), however, has been hampered by the lack of method to enrich minuscule quantities of extracellular Hsps (eHsps) from serum samples. Here we present a chemical approach using monomeric avidin to reproducibly enrich eHsps from serum. Biochemical mechanism of this eHsps enrichment and its implications in biomarker discovery is discussed.

Tracking N-acetyllactosamine on Cell Surface Glycans in vivo

Tianqing Zheng, Albert Einstein College of Medicine

Many mammalian glycans associated with signaling receptors contain terminal or penultimate N-acetyllactosamine (LacNAc; Galb1,4GlcNAc), which functions in the regulation of growth factor signaling. We develop a rapid, highly specific and practical method for labeling this disaccharide on cell surface glycoproteins of cultured cells and zebrafish embryos. The method utilizes a recombinant H. pylori α(1,3)fucosyltransferase to transfer a C-6 azide- or alkyne-tagged fucose residue to the 3-OH of N-acetylglucosamine (GlcNAc) of the LacNAc disaccharide. The tag may then be selectively derivatized with probes via bioorthogonal click chemistry. We used this strategy to detect LacNAc-bearing glycans on the surface of live mammalian cells, and characterized the specificity of labeling using Chinese hamster ovary (CHO) cell glycosylation mutants. Ex vivo we discovered that spleen lymphocytes with an activated/memory phenotype exhibited higher LacNAc levels compared to their unactivated counterparts. This method also allowed, for the first time, noninvasive imaging of LacNAc in glycans of zebrafish embryos at late gastrula and tissue segmentation stages. Therefore, this chemoenzymatic method serves as a general approach for the detection of LacNAc-containing glycans in living systems, and may be used to reveal changes in LacNAc status that occur during cellular differentiation.

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