Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy


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 ten 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.

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Fig. 1: Structure of N-glycans and a bio-inspired strategy for their preparation.
Fig. 2: Two strategies for desymmetrizing N-glycans using the branch selectivity of the sialyltransferase ST6Gal1 and the galactosidase from E. coli, and subsequent preparation of asymmetric branched bi-antennary glycans such as 13.
Fig. 3: Synthesis of asymmetric branched tri-antennary glycosyl asparagines using MGAT5 and UDP-GlcNTFA.
Fig. 4: Synthesis of asymmetric branched tetra-antennary N-glycans using MGAT4 and MGAT5 in combination with UDP-GlcNTFA and subsequent conversion of the transferred GlcNTFA into GlcN3 or GlcNH2.

Data availability

All data related with this study are included in this article and the Supplementary Information, and also available from the authors upon request.


  1. 1.

    Moremen, K. W., Tiemeyer, M. & Nairn, A. V. Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 13, 448–462 (2012).

    CAS  Article  Google Scholar 

  2. 2.

    Ohtsubo, K. & Marth, J. D. Glycosylation in cellular mechanisms of health and disease. Cell 126, 855–867 (2006).

    CAS  Article  Google Scholar 

  3. 3.

    Lauc, G., Pezer, M., Rudan, I. & Campbell, H. Mechanisms of disease: the human N-glycome. Biochim. Biophys. Acta 1860, 1574–1582 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Hart, G. W. & Copeland, R. J. Glycomics hits the big time. Cell 143, 672–676 (2010).

    CAS  Article  Google Scholar 

  5. 5.

    Kiessling, L. L. & Splain, R. A. Chemical approaches to glycobiology. Annu. Rev. Biochem. 79, 619–653 (2010).

    CAS  Article  Google Scholar 

  6. 6.

    Cummings, R. D. & Pierce, J. M. The challenge and promise of glycomics. Chem. Biol. 21, 1–15 (2014).

    CAS  Article  Google Scholar 

  7. 7.

    Pilobello, K. T. & Mahal, L. K. Deciphering the glycocode: the complexity and analytical challenge of glycomics. Curr. Opin. Chem. Biol. 11, 300–305 (2007).

    CAS  Article  Google Scholar 

  8. 8.

    Wang, L. X. & Lomino, J. V. Emerging technologies for making glycan-defined glycoproteins. ACS Chem. Biol. 7, 110–122 (2012).

    CAS  Article  Google Scholar 

  9. 9.

    Hofmann, J. & Pagel, K. Glycan analysis by ion mobility-mass spectrometry. Angew. Chem. Int. Ed. 56, 8342–8349 (2017).

    CAS  Article  Google Scholar 

  10. 10.

    Hyun, J. Y., Pai, J. & Shin, I. The glycan microarray story from construction to applications. Acc. Chem. Res. 50, 1069–1078 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Wang, Z. et al. A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans. Science 341, 379–383 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    Li, L. et al. Efficient chemoenzymatic synthesis of an N-glycan isomer library. Chem. Sci. 6, 5652–5661 (2015).

  13. 13.

    Li, T. et al. Divergent chemoenzymatic synthesis of asymmetrical-core-fucosylated and core-unmodified N-glycans. Chem. Eur. J. 22, 18742–18746 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    Shivatare, S. S. et al. Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies. Nat. Chem. 8, 338–346 (2016).

    CAS  Article  Google Scholar 

  15. 15.

    Gagarinov, I. A. et al. Chemoenzymatic approach for the preparation of asymmetric bi-, tri- and tetra-antennary N-glycans from a common precursor. J. Am. Chem. Soc. 139, 1011–1018 (2017).

    CAS  Article  Google Scholar 

  16. 16.

    Schachter, H. & Freeze, H. H. Glycosylation diseases: quo vadis? Biochim. Biophys. Acta 1792, 925–930 (2009).

    CAS  Article  Google Scholar 

  17. 17.

    Kizuka, Y. & Taniguchi, N. Enzymes for N-glycan branching and their genetic and nongenetic regulation in cancer. Biomolecules 6, 25 (2016).

    Article  Google Scholar 

  18. 18.

    Cummings, R. D. The repertoire of glycan determinants in the human glycome. Mol. Biosyst. 5, 1087–1104 (2009).

    CAS  Article  Google Scholar 

  19. 19.

    Seko, A. et al. Occurence of a sialylglycopeptide and free sialylglycans in hen’s egg yolk. Biochim. Biophys. Acta 1335, 23–32 (1997).

    CAS  Article  Google Scholar 

  20. 20.

    Liu, L., Prudden, A. R., Bosman, G. P. & Boons, G. J. Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder. Carbohydr. Res. 452, 122–128 (2017).

    CAS  Article  Google Scholar 

  21. 21.

    Umekawa, M. et al. Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline. Biochim. Biophys. Acta 1800, 1203–1209 (2010).

    CAS  Article  Google Scholar 

  22. 22.

    Huang, W., Giddens, J., Fan, S. Q., Toonstra, C. & Wang, L. X. Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 134, 12308–12318 (2012).

    CAS  Article  Google Scholar 

  23. 23.

    Maki, Y., Okamoto, R., Izumi, M., Murase, T. & Kajihara, Y. Semisynthesis of intact complex-type triantennary oligosaccharides from a biantennary oligosaccharide isolated from a natural source by selective chemical and enzymatic glycosylation. J. Am. Chem. Soc. 138, 3461–3468 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Alexander, S. R., Lim, D., Amso, Z., Brimble, M. A. & Fairbanks, A. J. Protecting group free synthesis of glycosyl thiols from reducing sugars in water; application to the production of N-glycan glycoconjugates. Org. Biomol. Chem. 15, 2152–2156 (2017).

    CAS  Article  Google Scholar 

  25. 25.

    Peng, W. et al. Recent H3N2 viruses have evolved specificity for extended, branched human-type receptors, conferring potential for increased avidity. Cell Host Microbe 21, 23–34 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Paschinger, K., Staudacher, E., Stemmer, U., Fabini, G. & Wilson, I. B. Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates. Glycobiology 15, 463–474 (2005).

    CAS  Article  Google Scholar 

  27. 27.

    Voynow, J. A., Kaiser, R. S., Scanlin, T. F. & Glick, M. C. Purification and characterization of GDP-l-fucose-N-acetyl β-d-glucosaminide ɑ1→6fucosyltransferase from cultured human skin fibroblasts: equirement of a specific biantennary oligosaccharide as substrate. J. Biol. Chem. 266, 21572–21577 (1991).

  28. 28.

    Meng, L. et al. Enzymatic basis for N-glycan sialylation: structure of rat ɑ2,6-sialyltransferase (ST6GAL1) reveals conserved and unique features for glycan sialylation. J. Biol. Chem. 288, 34680–34698 (2013).

    CAS  Article  Google Scholar 

  29. 29.

    van den Eijnden, D. H., Blanken, W. M. & van Vliet, A. Branch specificity of β-d-galactosidase from Eschericha coli. Carbohydr. Res. 151, 329–335 (1986).

    Article  Google Scholar 

  30. 30.

    Choo, M. et al. Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4. J. Biol. Chem. 292, 6163–6176 (2017).

    CAS  Article  Google Scholar 

  31. 31.

    Zauner, G., Deelder, A. M. & Wuhrer, M. Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics. Electrophoresis 32, 3456–3466 (2011).

    CAS  Article  Google Scholar 

  32. 32.

    Lauber, M. A., Koza, S. M. & Fountain, K. J. Optimization of GlycoWorks HILIC SPE for the Quantitative and Robust Recovery of N-linked Glycans from mAb-type Samples Waters Application Note 720004717EN (Waters Corporation, Milford, 2013).

  33. 33.

    Xu, Y. et al. Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins. Science 334, 498–501 (2011).

    CAS  Article  Google Scholar 

  34. 34.

    Antonopoulos, A. et al. Loss of effector function of human cytolytic T lymphocytes is accompanied by major alterations in N- and O-glycosylation. J. Biol. Chem. 287, 11240–11251 (2012).

    CAS  Article  Google Scholar 

  35. 35.

    Goddard-Borger, E. D. & Stick, R. V. An efficient, inexpensive and shelf-stable diazotransfer reagent: imidazole-1-sulfonyl azide hydrochloride. Org. Lett. 9, 3797–3800 (2007).

    CAS  Article  Google Scholar 

  36. 36.

    Bayley, H., Standring, D. N. & Knowles, J. R. Propane-1,3-dithiol—selective reagent for efficient reduction of alkyl and aryl azides to amines. Tetrahedron Lett. 19, 3633–3634 (1978).

    Article  Google Scholar 

  37. 37.

    Aoki, D., Appert, H. E., Johnson, D., Wong, S. S. & Fukuda, M. N. Analysis of the substrate binding sites of human galactosyltransferase by protein engineering. EMBO J. 9, 3171–3178 (1990).

    CAS  Article  Google Scholar 

  38. 38.

    Unverzagt, C. Chemoenzymatic synthesis of a sialylated undecasaccharide–asparagine conjugate. Angew. Chem. Int. Ed. Engl. 35, 2350–2353 (1996).

    CAS  Article  Google Scholar 

  39. 39.

    Hanashima, S., Manabe, S. & Ito, Y. Divergent synthesis of sialylated glycan chains: combined use of polymer support, resin capture-release and chemoenzymatic strategies. Angew. Chem. Int. Ed. 44, 4218–4224 (2005).

    CAS  Article  Google Scholar 

  40. 40.

    Jonke, S., Liu, K. G. & Schmidt, R. R. Solid-phase oligosaccharide synthesis of a small library of N-glycans. Chem. Eur. J. 12, 1274–1290 (2006).

    CAS  Article  Google Scholar 

  41. 41.

    Sun, B., Srinivasan, B. & Huang, X. F. Pre-activation-based one-pot synthesis of an ɑ-(2,3)-sialylated core-fucosylated complex type bi-antennary N-glycan dodecasaccharide. Chem. Eur. J. 14, 7072–7081 (2008).

    CAS  Article  Google Scholar 

  42. 42.

    Unverzagt, C. et al. Synthesis of multiantennary complex type N-glycans by use of modular building blocks. Chem. Eur. J. 15, 12292–12302 (2009).

    CAS  Article  Google Scholar 

  43. 43.

    Serna, S., Etxebarria, J., Ruiz, N., Martin-Lomas, M. & Reichardt, N. C. Construction of N-glycan microarrays by using modular synthesis and on-chip nanoscale enzymatic glycosylation. Chem. Eur. J. 16, 13163–13175 (2010).

    CAS  Article  Google Scholar 

  44. 44.

    Walczak, M. A. & Danishefsky, S. J. Solving the convergence problem in the synthesis of triantennary N-glycan relevant to prostate-specific membrane antigen (PSMA). J. Am. Chem. Soc. 134, 16430–16433 (2012).

    CAS  Article  Google Scholar 

  45. 45.

    Hamilton, B. S. et al. A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors. Sci. Rep. 7, 15907 (2017).

    Article  Google Scholar 

  46. 46.

    Calderon, A. D. et al. An enzymatic strategy to asymmetrically branched N-glycans. Org. Biomol. Chem. 15, 7258–7262 (2017).

    CAS  Article  Google Scholar 

  47. 47.

    Maki, Y., Mima, T., Okamoto, R., Izumi, M. & Kajihara, Y. Semisynthesis of complex-type biantennary oligosaccharides containing lactosamine repeating units from a biantennary oligosaccharide isolated from a natural source. J. Org. Chem. 83, 443–451 (2018).

    CAS  Article  Google Scholar 

  48. 48.

    Prudden, A. R. et al. Synthesis of asymmetrical multiantennary human milk oligosaccharides. Proc. Natl Acad. Sci. USA 114, 6954–6959 (2017).

    CAS  Article  Google Scholar 

  49. 49.

    Cai, L. Recent progress in enzymatic synthesis of sugar nucleotides. J. Carbohydr. Chem. 31, 535–552 (2012).

    CAS  Article  Google Scholar 

  50. 50.

    Moremen, K. W. et al. Expression system for structural and functional studies of human glycosylation enzymes. Nat. Chem. Biol. 14, 156–162 (2018).

    CAS  Article  Google Scholar 

  51. 51.

    Muthana, S., Cao, H. & Chen, X. Recent progress in chemical and chemoenzymatic synthesis of carbohydrates. Curr. Opin. Chem. Biol. 13, 573–581 (2009).

    CAS  Article  Google Scholar 

  52. 52.

    Schmaltz, R. M., Hanson, S. R. & Wong, C. H. Enzymes in the synthesis of glycoconjugates. Chem. Rev. 111, 4259–4307 (2011).

    CAS  Article  Google Scholar 

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This research was supported by the National Institute of General Medical Sciences (P01GM107012, P41GM103390 and U01GM120408 to G.-J.B. and K.W.M.) and the National Cancer Institute (F31CA180478 to A.R.P.) from the US National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The research benefitted from instrumentation provided by NIH grant S10 RR027097.

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L.L., A.R.P., C.J.C., K.W.M. and G.-J.B. designed research. L.L., A.R.P., C.J.C., G.P.B., D.G.C. and J.-Y.Y. performed research. D.G.C. and J.-Y.Y. contributed new reagents/analytic tools. L.L., A.R.P., C.J.C. and G.-J.B. wrote the paper.

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Correspondence to Geert-Jan Boons.

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Materials and Methods, Supplementary Figures 1-13, Supplementary Table 1 and NMR spectra

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Liu, L., Prudden, A.R., Capicciotti, C.J. et al. Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy. Nature Chem 11, 161–169 (2019).

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