Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Apr 16;147(15):12937-12948.
doi: 10.1021/jacs.5c02356. Epub 2025 Apr 7.

Expedient Assembly of Multiantennary N-Glycans from Common N-Glycan Cores with Orthogonal Protection for the Profiling of Glycan-Binding Proteins

Ruofan Li  1 , Pengxi Chen  1 , Yi-Fang Zeng  1 , Tzu-Hao Tseng  1 , Veeranjaneyulu Gannedi  1 , Larissa Krasnova  1 , Chi-Huey Wong  1   2
Affiliations

Expedient Assembly of Multiantennary N-Glycans from Common N-Glycan Cores with Orthogonal Protection for the Profiling of Glycan-Binding Proteins

Ruofan Li et al. J Am Chem Soc. .

Abstract

Complex-type N-glycans are structurally diverse molecules, responsible for many biological processes, yet the specific sequences of N-glycans involved in biological recognition remain largely unknown. Despite the recent development of many efficient chemoenzymatic approaches, it is still lacking a general approach to produce structurally diverse complex-type N-glycans. Here, we designed two common precursors equipped with orthogonal protecting groups for antennary differentiation and selective glycan elongation. The N-acetyllactosamine (LacNAc) repeat modules were synthesized separately based on iterative Au(I) promoted glycosylation and programmable one-pot strategy and were incorporated into the N-glycan core structure in a site-specific manner. The final removal of benzyl groups was cleanly achieved using pressurized flow chemistry. A total of 51 N-glycans were assembled and presented as an array to study the binding specificity toward a panel of influenza hemagglutinins and other lectins. The established method allows a rapid and previously infeasible synthesis of asymmetric bi- and triantennary N-glycans, especially with the LacNAc repeats residing at a specific arm, bringing in new opportunities to study carbohydrate-receptor interactions.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
General approach to asymmetric bi- and triantennary N-glycans: synthetic precursors 1 and 2 with orthogonally protected GlcNAc units and modules are coupled to give a diverse array of N-glycan intermediates that are further elaborated by enzymatic synthesis.
Scheme 1
Scheme 1. Rapid Assembly of Common Precursors 1 and 2 Using Mannosyl and GlcNPhth BBLs
Reagents and conditions: (a) 3 (1.6 equiv), BSP (1.6 equiv), TTBP (3.2 equiv), Tf2O (1.6 equiv), CH2Cl2, 4 Å MS, −60 °C, 5 min; then 11 (1.0 equiv), −60 → −20 °C, 1 h, 57%; (b) cyclopropyl acetylene (5.0 equiv), (Ph3P)2PdCl2 (0.1 equiv), CuI (0.2 equiv), DMF/Et3N = 1:3 (v/v), r.t., 24 h, 90%; (c) 12 (1.1 equiv), Ph3PAuOTf (0.2 equiv), CH2Cl2, 4 Å MS, 0 °C, 30 min, 78%; (d) DDQ (1.2 equiv), CH2Cl2:H2O = 10:1 (v/v), 0 °C, 6 h, 82%; (e) 5 (1.0 equiv), 7 (1.05 equiv) or 8 (1.1 equiv), NIS (1.2 equiv), TfOH (0.1 equiv), 4 Å MS, CH2Cl2, −50 → 0 °C, 30 min, 64% for Lev-disaccharide, 83% for TBS-disaccharide; (f) CAN (3.0 equiv), PhMe:ACN:H2O (1:4:1, v/v/v), 0 °C, 1 h; (g) DAST (2.0 equiv), 4 Å MS, CH2Cl2, −40 °C, 30 min, 72% for 15 over the two steps, 75% for 16 over the two steps, 69% for 18 over the two steps; (h) 4 (1.0 equiv), 9 (1.1 equiv), NIS (1.2 equiv), TMSOTf (0.1 equiv), 4 Å MS, CH2Cl2, −40 → 0 °C, 30 min; (i) PhBCl2 (2.0 equiv), Et3SiH (5.0 equiv), 4 Å MS, CH2Cl2, −78 °C, 2 h, 70% over the two steps; (j) 17 (1.0 equiv), 10 (1.25 equiv), NIS (1.25 equiv), TfOH (0.15 equiv), 4 Å MS, CH2Cl2, −40 → 0 °C, 30 min, 82%; (k) 14 (1.0 equiv), 16 (1.2 equiv), Cp2HfCl2 (3.5 equiv), AgOTf (5.0 equiv), 4 Å MS, PhMe, −60 → −30 °C, 1 h, 70%; (l) PhBCl2 (2.0 equiv), Et3SiH (5.0 equiv), 4 Å MS, CH2Cl2, −78 → −60 °C, 2 h, 78%; (m) 19 (1.0 equiv), 6 (3.0 equiv), Ph3PAuOTf (0.5 equiv), 4 Å MS, CH2Cl2, 0 °C, 80%; (n) Et3N:CH2Cl2 = 1:10 (v/v), r.t., 12 h; (o) 20 (1.0 equiv), 7 (3.0 equiv), NIS (3.0 equiv), TMSOTf (0.6 equiv), 4 Å MS, CH2Cl2, −40 → 0 °C, 54% for the two steps; (p) 14 (1.0 equiv), 15 (1.1 equiv), Cp2HfCl2 (3.5 equiv), AgOTf (5.0 equiv), 4 Å MS, PhMe, −60 → −20 °C, 1 h, 53%; (q) CSA (5.0 equiv), EtSH (10.0 equiv), CH2Cl2:MeOH (20:1, v/v), r.t., 12 h, 59% (26% recovered S.M.); and (r) 18 (1.5 equiv), Cp2HfCl2 (3.5 equiv), AgOTf (5.0 equiv), 4 Å MS, PhMe, −20 °C, 1 h, 72% combined yield (α:β ca. 1.5:1). BSP = 1-(phenylsulfinyl)piperidine, TTBP = 2,4,6-tritert-butylpyrimidine, DMF = N,N-dimethylformamide, DDQ = 2,3-dichloro-5,6-dicyano-p-benzoquinone, NIS = N-iodosuccinimide, CAN = ammonium cerium(IV)nitrate, DAST = (diethylamino)sulfur trifluoride, MS = molecular sieves, TMSOTf = trimethylsilyl trifluoromethanesulfonate, and CSA = camphor-10-sulfonic acid.
Scheme 2
Scheme 2. An Iterative Strategy for the Synthesis of LacNAc Repeats
Reagents and conditions: (a) 7 (1.1 equiv), 22 (1.0 equiv), NIS (1.2 equiv), TMSOTf (0.1 equiv), 4 Å MS, CH2Cl2, −50 °C, 1 h, 72%; (b) cyclopropyl acetylene (5.0 equiv), (Ph3P)2PdCl2 (0.1 equiv), CuI (0.2 equiv), DMF:Et3N (1:4, v/v), r.t., 24 h, 90% for 24, 82% for 27, 94% for 30; (c) N2H4·AcOH (2.0 equiv), CH2Cl2/MeOH (20:1, v/v), r.t., 12 h, 88% for 25, 90% for 28; and (d) Ph3PAuOTf (0.25 equiv), 4 Å MS, CH2Cl2, 0 °C, 82% for 26, 82% for 29.
Scheme 3
Scheme 3. Programmable One-Pot Synthesis of (Fucosylated) LacNAc Modules 33, 36, 39, 42, and 44
Reagents and conditions: (a) 31 (1.1 equiv), 32 (1.0 equiv), NIS (1.08 equiv), TfOH (0.15 equiv), 4 Å MS, CH2Cl2, −50 → −40 °C, 1 h; then 22 (0.9 equiv), NIS (1.1 equiv), TfOH (0.15 equiv), −40 → 0 °C, 30 min, 41% overall. (b) (i) 34 (1.1 equiv), 35 (1.0 equiv), NIS (1.08 equiv), TfOH (0.15 equiv), 4 Å MS, CH2Cl2, −50 → −40 °C, 1 h; then 22 (0.9 equiv), NIS (1.1 equiv), TfOH (0.15 equiv), −40 → 0 °C, 30 min, 84% overall; (ii) DDQ (2.0 equiv), CH2Cl2/H2O (10:1, v/v), 0 °C, 4 h, 68%. (c) (i) 31 (1.2 equiv), 38 (1.0 equiv), NIS (1.18 equiv), TfOH (0.15 equiv), 4 Å MS, CH2Cl2, −50 → −40 °C, 1 h; then 22 (1.0 equiv), NIS (1.2 equiv), TfOH (0.15 equiv), −40 → 0 °C, 30 min, 67% overall; (ii) DDQ (1.2 equiv), CH2Cl2/H2O (10:1, v/v), 0 °C, 6 h, 69%. (d) 31 (1.1 equiv), 41 (1.0 equiv), NIS (1.1 equiv), TfOH (0.15 equiv), 4 Å MS, CH2Cl2, −50 → −40 °C, 1 h; then 37 (0.9 equiv), NIS (1.1 equiv), TfOH (0.15 equiv), −40 → 0 °C, 30 min, 46% overall. (e) 43 (1.1 equiv), 35 (1.0 equiv), NIS (1.1 equiv), TfOH (0.15 equiv), 4 Å MS, CH2Cl2, −50 → −40 °C, 1 h; then 40 (0.9 equiv), NIS (1.1 equiv), TfOH (0.15 equiv), −40 → 0 °C, 30 min, 55% overall.
Figure 2
Figure 2
(a) Chemical elongation of common precursors 1 and 2 using galactosyl donors A-I. (b) Representative enzymatic derivatizations of synthetic bi- and triantennary N-glycans.
Figure 3
Figure 3
(a) Representative microarray binding results from a plant lectin, influenza Has, and human Siglec-10. (b) N-glycans used in the array.
See this image and copyright information in PMC

References

    1. Reily C.; Stewart T. J.; Renfrow M. B.; Novak J. Glycosylation on health and disease. Nat. Rev. Nephrol. 2019, 15, 346.10.1038/s41581-019-0129-4. - DOI - PMC - PubMed
    1. Shriver Z.; Raguram S.; Sasisekharan R. Glycomics: a pathway to a class of new and improved therapeutics. Nat. Rev. Drug Discovery 2004, 3, 863.10.1038/nrd1521. - DOI - PubMed
    2. Gong Y.; Qin S.; Dai L.; Tian Z. The glycosylation in SARS-CoV-2 and its receptor ACE2. Signal Transduct. Targeted Ther. 2021, 6, 396.10.1038/s41392-021-00809-8. - DOI - PMC - PubMed
    3. Cao L.; Diedrich J. K.; Kulp D. W.; Pauthner M.; He L.; Park S. K. R.; Sok D.; Su C. Y.; Delahunty C. M.; Menis S.; Andrabi R.; Guenaga J.; Georgeson E.; Kubitz M.; Adachi Y.; Burton D. R.; Schief W. R.; Yates III J. R.; Paulson J. C. Global site-specific N-glycosylation analysis of HIV envelope glycoprotein. Nat. Commun. 2017, 8, 14954.10.1038/ncomms14954. - DOI - PMC - PubMed
    1. Wallace E. N.; West C. A.; McDowell C. T.; Lu X.; Bruner E.; Mehta A. S.; Aoki-Kinoshita K. F.; Angel P. M.; Drake R. R. An N-glycome tissue atlas of 15 human normal and cancer tissue types determined by MALDI-imaging mass spectrometry. Sci. Rep. 2024, 14, 489.10.1038/s41598-023-50957-w. - DOI - PMC - PubMed
    2. Turiák L.; Shao C.; Meng L.; Khatri K.; Leymarie N.; Wang Q.; Pantazopoulos H.; Leon D. R.; Zaia J. Workflow for combined proteomics and glycomics profiling from histological tissues. Anal. Chem. 2014, 86, 9670.10.1021/ac5022216. - DOI - PMC - PubMed
    1. Wang Z.; Chinoy Z. S.; Ambre S. G.; Peng W.; Mcbride R.; de Vries R. P.; Glushka J.; Paulson J. C.; Boons G.-J. A general Strategy for the Chemoenzymatic Synthesis of Asymmetrically Branched N-glycans. Science 2013, 341, 379.10.1126/science.1236231. - DOI - PMC - PubMed
    2. Gagarinov I. A.; Li T.; Toraño J. S.; Caval T.; Srivastava A. D.; Kruijtzer J. A. W.; Heck A. J. R.; Boons G.-J. Chemoenzymatic Approach for the Preparation of Asymmetric Bi-, Tri-, and Tetra-Antennary N-Glycans from a Common Precursor. J. Am. Chem. Soc. 2017, 139, 1011.10.1021/jacs.6b12080. - DOI - PMC - PubMed
    1. Liu L.; Prudden A. R.; Capicciotti C. J.; Bosman G. P.; Yang J.-Y.; Chapla D. G.; Moremen K. W.; Boons G.-J. Streamlining the Chemoenzymatic Synthesis of Complex N-glycans by a Stop and Go Strategy. Nat. Chem. 2019, 11, 161.10.1038/s41557-018-0188-3. - DOI - PMC - PubMed
    2. Ma S.; Liu L.; Eggink D.; Herfst S.; Fouchier R. A. M.; de Vries R. P.; Boons G.-J. Asymmetrical Biantennary Glycans Prepared by a Stop-and-Go Strategy Reveal Receptor Binding Evolution of Human Influenza A Viruses. JACS Au 2024, 4, 607.10.1021/jacsau.3c00695. - DOI - PMC - PubMed

MeSH terms

LinkOut - more resources