A multifunctional anomeric linker for the chemoenzymatic synthesis of complex oligosaccharides (original) (raw)
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Journal of the American Chemical Society, 2017
Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manβ(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the β-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and β-Man t...
Efficient Chemoenzymatic Synthesis of an N-glycan Isomer Library
Chemical Science, 2015
Quantification, characterization and biofunctional studies of N-glycans on proteins remain challenging tasks due to the complexity, diversity and low abundance of these glycans. The availability of structurally defined N-glycan (especially isomer) libraries is essential to help solve these tasks. We report herein an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse N-glycans. Starting with 5 chemically prepared building blocks, 8 N-glycan core structures containing one or two terminal N-acetyl-D-glucosamine (GlcNAc) residue(s) were chemically synthesized via consistent use of oligosaccharyl thioethers as glycosylation donors in a convergent fragment coupling strategy. Each of these core structures was then extended to 5 to 15 N-glycan sequences by enzymatic reactions catalyzed by 4 robust glycosyltransferases. Success in synthesizing N-glycans with Neu5Gc and core-fucosylation further expanded the ability of the enzymatic extension. Meanwhile, high performance liquid chromatography with an amide column enabled rapid and efficient purification (>98% purity) of N-glycans in milligram scales. A total of 73 N-glycans (63 isomers) were successfully prepared and characterized by MS 2 and NMR. In summary, the CSEE strategy provides a practical approach for "mass production" of structurally defined N-glycans, which are important standards and probes for glycoscience.
Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy
Nature Chemistry, 2018
Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch selective manner to give asymmetrical structures commonly found in Nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in 10 or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart allowing each arm to be elaborated into a unique appendage.
A short synthesis of the trisaccharide building block of the N-linked glycans
Tetrahedron Letters, 2003
An efficient preparation of the core trisaccharide of N-linked glycoproteins containing b-azido functionality at the reducing terminus is described. In the synthesis, triflate-mediated direct b-mannosylation was employed for the formation of the b-D-Man-(14)-GlcNAc linkage; the anomeric azide installation was achieved through oxazoline ring opening.
Nature Chemistry, 2010
Solid phase oligosaccharide synthesis (SPOS) offers the promise to provide libraries of oligosaccharides for glycomics research. A major stumbling block to SPOS has been a lack of general methods to stereoselectively install 1,2-cis-glycosides, and intractable mixtures of compounds will be obtained if several of such glycosides need to be installed. We have prepared on-resin a biologically important glucoside containing multiple 1,2-cis-glycosidic linkages with complete anomeric control by using glycosyl donors having a participating (S)-(phenylthiomethyl)benzyl chiral auxiliary at C-2. A branching point could be installed by employing 9-fluorenylmethyloxycarbonyl (Fmoc) and allyloxycarbonyl (Alloc) as a versatile set of orthogonal protecting groups. The synthetic strategy made it possible for partial on-resin deprotection of the completed oligosaccharide thereby increasing the overall efficiency of the synthesis. The combination of classical and auxiliary mediated neighboring group participation for controlling anomeric selectivity is bringing the promise of routine automated solid supported oligosaccharides synthesis closer. As many as 50% of human proteins are O-or N-glycosylated and the carbohydrate moieties of these glycoproteins have been implicated as essential mediators of cellular processes such as protein folding, regulation of cell signaling, fertilization, embryogenesis, neuronal development and hormone activities.1 However, carbohydrates are also important for pathogen recognition, modulation of innate immune responses, control of immune cell homeostasis, inflammation, and the development of autoimmune diseases and cancer.2-4 The ability of cells to generate information rich glycans has created a new field of research termed "glycomics", which seeks to identify and understand the processes involved in the formation of cell type and developmental stage specific oligosaccharide patterns.5-8 In this respect, collections of well-defined oligosaccharides are needed for the development of algorithms for the assignment of oligosaccharide MS spectra, for fabricating microarrays, Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Automated assembly of oligosaccharides containing multiple cis-glycosidic linkages
Nature Communications, 2016
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.
Journal of the American Chemical Society, 2017
Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), Nacetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manβ(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the β-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and β-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal β-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric biand triantennary glycans could be obtained by removal of a terminal β-GlcNAc moiety by treatment with β-N-acetylglucosaminidase and selective extension of the other arms. The power of
Chemoselective Reagents for Covalent Capture and Display of Glycans in Microarrays
European Journal of Organic Chemistry, 2010
Glycobiology has made very significant progress in the past decades. However, further progress will significantly depend on the establishment of novel methods for miniaturized, high-throughput analysis of glycan-protein interactions. Robust solid-phase chemical tools and new, chemoselective reagents for biologically meaningful display of surface-immobilized glycans are likely to play a key role. Here we present four new bifunctional linkers that allow highly chemoselective capture of unprotected glycans in solution to form glycan-linker conjugates for direct construction of glycan microarrays (glycochips). The bifunctional linkers carry O-linked aminooxy moieties, some with N-substituents at one end and an amino group at the other. In addition, they contain a substituted benzene ring for UV traceability and improved puri-[a] IGM,