Automated glycan assembly (AGA) has advanced from a concept to a commercial technology that rapidly provides access to diverse oligosaccharide chains as long as 30-mers. To date, AGA was mainly ...employed to incorporate trans-glycosidic linkages, where C2 participating protecting groups ensure stereoselective couplings. Stereocontrol during the installation of cis-glycosidic linkages cannot rely on C2-participation and anomeric mixtures are typically formed. Here, we demonstrate that oligosaccharides containing multiple cis-glycosidic linkages can be prepared efficiently by AGA using monosaccharide building blocks equipped with remote participating protecting groups. The concept is illustrated by the automated syntheses of biologically relevant oligosaccharides bearing various cis-galactosidic and cis-glucosidic linkages. This work provides further proof that AGA facilitates the synthesis of complex oligosaccharides with multiple cis-linkages and other biologically important oligosaccharides.
Glycosaminoglycans (GAGs) are important sulfated carbohydrates prevalent in the extracellular matrix. The synthesis of structurally defined GAGs requires laborious procedures, and incorporating ...defined sulfation patterns is challenging. The automated synthesis of defined sulfated chondroitin hexasaccharides on solid support has been achieved using a photolabile linker that is efficiently cleaved in a continuous‐flow photoreactor.
Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics ...(e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.
Using a high-throughput chemical screen, we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action, we discovered ...an essential role of E-cadherin signaling for ESC survival. Specifically, we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling, which then leads to a fatal perturbation of integrin signaling. Furthermore, we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally, we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology, growth factor requirement, and sensitivity to enzymatic cell dissociation.
Reliable and rapid access to defined biopolymers by automated DNA and peptide synthesis has fundamentally altered biological research and medical practice. Similarly, the procurement of defined ...glycans is key to establishing structure–activity relationships and thereby progress in the glycosciences. Here, we describe the rapid assembly of oligosaccharides using the commercially available Glyconeer 2.1 automated glycan synthesizer, monosaccharide building blocks, and a linker-functionalized polystyrene solid support. Purification and quality-control protocols for the oligosaccharide products have been standardized. Synthetic glycans prepared in this way are useful reagents as the basis for glycan arrays, diagnostics, and carbohydratebased vaccines.
The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we ...describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.
Recent breakthroughs in using viral transduction of a few genes for the reprogramming of both mouse and human somatic cells into induced pluripotent stem (iPS) cells have opened up tremendous ...opportunities for the stem cell field (Maherali et al., 2007, Meissner et al., 2007, Nakagawa et al., 2008, Okita et al., 2007, Takahashi et al., 2007, Takahashi and Yamanaka, 2006, Wernig et al., 2007, Wernig et al., 2008 and Yu et al., 2007). For the iPS cell approach to be clinically relevant, several challenges remain to be addressed, including the elimination of the risks and drawbacks associated with the current iPS cell method, such as the use of genetic manipulation and the low efficiency and slow kinetics of induction. Recent studies have shown that c-Myc, one of the four genes originally thought to be required for iPS cell generation, is dispensable, but in its absence the reprogramming efficiency is reduced (Nakagawa et al., 2008 and Wernig et al., 2008). Here, we have explored two approaches toward identifying conditions that can replace viral transduction of oncogenic transcription factors (TFs) and enhance reprogramming efficiency. In one, we have found that neural progenitor cells can be reprogrammed with fewer genetic manipulations than previously reported somatic cells, and in the other we have found that small molecules may be able to replace viral integration of certain transcription factors and promote the reprogramming process.
Arabinogalactan proteins are heavily glycosylated proteoglycans in plants. Their glycan portion consists of type-II arabinogalactan polysaccharides whose heterogeneity hampers the assignment of the ...arabinogalactan protein function. Synthetic chemistry is key to the procurement of molecular probes for plant biologists. Described is the automated glycan assembly of 14 oligosaccharides from four monosaccharide building blocks. These linear and branched glycans represent key structural features of natural type-II arabinogalactans and will serve as tools for arabinogalactan biology.
Sulfonyl‐triazoles have emerged as a new reactive group for covalent modification of tyrosine sites on proteins through sulfur‐triazole exchange (SuTEx) chemistry. The extent to which this sulfur ...electrophile can be tuned for developing ligands with cellular activity remains largely underexplored. Here, we performed fragment‐based ligand discovery in live cells to identify SuTEx compounds capable of liganding tyrosine sites on diverse protein targets. We verified our quantitative chemical proteomic findings by demonstrating concentration‐dependent activity of SuTEx ligands, but not inactive counterparts, against recombinant protein targets directly in live cells. Our structure‐activity relationship studies identified the SuTEx ligand HHS‐0701 as a cell‐active inhibitor capable of blocking prostaglandin reductase 2 (PTGR2) biochemical activity.
A tyrosine‐reactive PTGR2 inhibitor: A cell‐active sulfonyl‐triazole compound (HHS‐0701) that ligands Y100 on prostaglandin reductase 2 (PTGR2) was discovered by quantitative chemical proteomics. Biochemical assays support HHS‐0701 blockade of PTGR2 metabolism of the lipid substrate 15‐Keto‐PGE2. These findings bolster the potential for developing SuTEx compounds as pharmacological modulators of proteins in live cells.
Dermatan sulfates are glycosaminoglycan polysaccharides that serve a multitude of biological roles as part of the extracellular matrix. Orthogonally protected D-galactosamine and L-iduronic acid ...building blocks and a photo-cleavable linker are instrumental for the automated synthesis of dermatan sulfate oligosaccharides. Conjugation-ready oligosaccharides were obtained in good yield.
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