Efficient chemoenzymatic synthesis of an N-glycan isomer library

Li, Lei; Liu, Yunpeng; Ma, Cheng; Qu, Jingyao; Calderon, Angie D.; Wu, Baolin; Wei, Na; Wang, Xuan; Guo, Yuxi; Xiao, Zhongying; Song, Jing; Sugiarto, Go; Li, Yanhong; Yu, Hai; Chen, Xi; Wang, Peng George

doi:10.1039/c5sc02025e

Cited by 125 publications

(108 citation statements)

References 47 publications

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“…With this convergent synthesis strategy in place, a chemo-enzymatic approach to the synthesis of D1 and D2/D3 arm donors was explored to allow a rapid assembly of diverse N -glycans 55,56 . To this end, various glycosyl transferases were used, including β-1,4-galactosyltransferases 51,57 , α-2,3/2,6-sialyltransferases 52,53 and α-1,3/1,2-fucosyltransferases 49,51,58 , for the preparation of linear and branched modules by enzymatic extension of chemically synthesized acceptors 16–20 (Fig. 3 and Supplementary Scheme 22).…”

Section: Resultsmentioning

confidence: 99%

“…Access to gp120-related N -glycans is a formidable task, because such structures are species- and cell-specific 33,37 and difficult to obtain due to their structural diversity and micro-heterogeneity, as well as synthetic challenges. However, a major advance in N -glycan synthesis was achieved recently 49–51 with the stepwise enzymatic extension of a chemically synthesized tri-antennary acceptor by Boons 49 and a similar strategy by Wang 51 . Despite these advances, the development of a more efficient strategy for the synthesis of diverse N -glycans of high-mannose, hybrid- and complex-type structures (estimated to be around 20,000) remains a major challenge.…”

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confidence: 99%

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Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

et al. 2016

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A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120, thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

et al. 2016

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“…In animals and humans, core-fucosylation is catalyzed solely by the mammalian α1,6-fucosyltransferase, FUT8 27,28 . However, FUT8 has a very strict substrate specificity, requires the presence of a free GlcNAc at the α1,3-linked mannose arm in the N-glycan as the substrate and usually is unable to fucosylate full-size mature N-glycans 29–32 . Only until recently we have provided the first examples showing that FUT8 could catalyze in vitro fucosylation of some high-mannose N-glycans lacking a free GlcNAc at the α1,3-linked mannose arm when the glycan is present in an appropriate protein or other context 33 .…”

Section: Introductionmentioning

confidence: 99%

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Zhu

Christopher

et al. 2017

J. Am. Chem. Soc.

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Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multi-step chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core-fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide, but also to high-mannose and complex type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

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“…Recently, we have developed an efficient core synthesis/enzymatic extension (CSEE) strategy for the rapid preparation of N -glycan libraries. 10 In this study, a similar strategy was successfully applied for HMO synthesis. Briefly, 3 core oligosaccharides with 1 or 2 GlcNAc residue(s) at the nonreducing end were first synthesized by the convergent assembly of 3 simple building blocks followed by extension of the cores by 4 robust glycosyltransferases to produce a library of 31 HMOs (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Chemoenzymatic Synthesis of a Library of Human Milk Oligosaccharides

et al. 2016

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Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist in human milk as one of the major components. Characterization, quantification, and biofunctional studies of HMOs remain a great challenge due to their diversity and complexity. The accessibility of a homogeneous HMO library is essential to solve these issues which have beset academia for several decades. In this study, an efficient chemoenzymatic strategy, namely core synthesis/enzymatic extension (CSEE), for rapid production of diverse HMOs was reported. On the basis of 3 versatile building blocks, 3 core structures were chemically synthesized via consistent use of oligosaccharyl thioether and oligosaccharyl bromide as glycosylation donors in a convergent fragment coupling strategy. Each of these core structures was then extended to up to 11 HMOs by 4 robust glycosyltransferases. A library of 31 HMOs were chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a practical approach to harvest structurally defined HMOs for various applications.

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Efficient chemoenzymatic synthesis of an N-glycan isomer library

Abstract: An efficient chemoenzymatic synthesis strategy and a HILIC-based purification approach enabled rapid access to an N-glycan isomer library.

Cited by 125 publications

References 47 publications

Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Chemoenzymatic Synthesis of a Library of Human Milk Oligosaccharides

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