SpringerLink
Log in

Chemo-enzymatic synthesis of a glycosylated peptide containing a complex N-glycan based on unprotected oligosaccharides by using DMT-MM and Endo-M

  • Short Communication
  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

For chemo-enzymatic synthesis of a glycosylated peptide, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used for the synthesis of a N-acetylglucosaminyl peptide and a pseudoglycopeptide by solid-phase peptide synthesis without the requirement of protecting groups on the carbohydrate. We also performed transglycosylation of an N-glycan to the N-acetylglucosaminyl peptide using endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) to synthesize a glycopeptide containing a complex N-glycan.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Germany)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Notes

  1. IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by McNaught, A. D. and Wilkinson, A. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by Nic, M., Jirat, J., Kosata, B.: updates compiled by Jenkins, A. ISBN 0–9678550–9-8.

  2. A large variance in the methionine residue was observed. Antioxidant was not added to the reaction mixture during the hydrolysis of the peptides, therefore leading to partial oxidation of methionine to methionine sulfoxide.

  3. The biological assay was entrusted to Anti Viral Screening System Co. ltd. (AVSS).

Abbreviations

Boc:

Tert-butoxycarbonyl

tBu:

Tert-butyl

DIEA:

N,N′-diisopropylethylamine

DMT-MM:

4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride

EDT:

Ethanedithiol

Endo-M:

Endo-β-N-acetylglucosaminidase from Mucor hiemalis

ENGase:

Endo-β-N-acetylglucosaminidase

ESI:

Electrospray ionization

Fmoc:

9-fluorenylmethoxycarbonyl

Gal:

Galactose

GlcNAc:

N-acetylglucosamine

HIV:

Human immunodeficiency virus

HRMS:

High-resolution mass spectrometry

IC50 :

Half maximal inhibitory concentration

LCMS–IT–TOF:

Liquid chromatography/mass spectrometry-ion trap-time-of-flight

Man:

Mannose

NeuAc:

N-acetylneuraminic acid

NMP:

1-methyl-2-pyrrolidinone

RP-HPLC:

Reversed-phase high-performance liquid chromatography

SGP:

Sialylglycopeptide

SG-oxazoline:

Sialylglyco-oxazoline

SPPS:

Solid phase peptide synthesis

TMSOTf:

Trifluoromethanesulfonic acid trimethylsilyl ester

TFA:

Trifluoroacetic acid

TfOH:

Trifluoromethanesulfonic acid

Trt:

Trityl

TIS:

Triisopropylsilane

VIRIP:

Virus-inhibitory peptide

WST:

Water soluble tetrazolium salts

References

  1. Varki, A.: Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 3, 97–130 (1993)

    Article  CAS  PubMed  Google Scholar 

  2. Jefferis, R.: Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov. 8, 226–234 (2009)

    Article  CAS  PubMed  Google Scholar 

  3. Dalziel, M., Crispin, M., Scanlan, C.N., Zitzmann, N., Dwek, R.A.: Emerging principles for the therapeutic exploitation of glycosylation. Science. 343, 1235681 (2014)

    Article  PubMed  Google Scholar 

  4. Kadowaki, S., Yamamoto, K., Fujisaki, M., Izumi, K., Tochikura, T., Yokoyama, T.: Purification and characterization of a novel fungal endo-β-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins. Agric Biol Chem. 54, 97–106 (1990)

    CAS  PubMed  Google Scholar 

  5. Yamamoto, K., Kadowaki, S., Watanabe, J.: Kumagai, H.:Transglycosylation activity of Mucor hiemalis endo-β-N-acetylglucosaminidase which transfers complex oligosaccharides to the N-acetylglucosamine moieties of peptides. Biochem Biophys Res Commun. 203, 244–252 (1994)

    Article  CAS  PubMed  Google Scholar 

  6. Li, B., Zeng, Y., Hauser, S., Song, H., Wang, L.X.: Highly efficient endoglycosidase-catalyzed synthesis of glycopeptides using oligosaccharide oxazolines as donor substrates. J Am Chem Soc. 127, 9692–9693 (2005)

    Article  CAS  PubMed  Google Scholar 

  7. Umekawa, M., Huang, W., Li, B., Fujita, K., Ashida, H., Wang, L.-X., Yamamoto, K.: Mutants of Mucor hiemalis endo-beta-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J Biol Chem. 283, 4469–4479 (2008)

    Article  CAS  PubMed  Google Scholar 

  8. Umekawa, M., Li, C., Higashiyama, T., Huang, W., Ashida, H., Yamamoto, K., Wang, L.-X.: Efficient glycosynthase mutant derived from Mucor hiemalis endo-β-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan. J Biol Chem. 285, 511–521 (2010)

    Article  CAS  PubMed  Google Scholar 

  9. Sakaguchi, K., Katoh, T., Yamamoto, K.: Transglycosidase-like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor Biotechnol. Appl Biochem. 63, 812–819 (2016)

    Article  CAS  Google Scholar 

  10. Katoh, T., Katayama, T., Tomabechi, Y., Nishikawa, Y., Kumada, J., Matsuzaki, Y., Yamamoto, K.: Generation of a mutant Mucor hiemalis endoglycosidase that acts on core-fucosylated N-glycans. J Biol Chem. 291, 23305–23317 (2016)

    Article  CAS  PubMed  Google Scholar 

  11. Mizuno, M., Haneda, K., Iguchi, R., Muramoto, I., Kawakami, T., Aimoto, S., Yamamoto, K., Inazu, T.: Synthesis of a glycopeptide containing oligosaccharides: chemoenzymic synthesis of eel calcitonin analogs having natural N-linked oligosaccharides. J Am Chem Soc. 121, 284–290 (1999)

    Article  CAS  Google Scholar 

  12. Yamamoto, K., Fujimori, K., Haneda, K., Mizuno, M., Inazu, T., Kumagai, H.: Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase. Carbohydr Res. 305, 415–422 (1997)

    Article  CAS  PubMed  Google Scholar 

  13. Li, H., Singh, S., Zeng, Y., Song, H., Wang, L.-X.: Chemoenzymatic synthesis of CD52 glycoproteins carrying native N-glycans. Bioorg Med Chem Lett. 15, 895–898 (2005)

    Article  CAS  PubMed  Google Scholar 

  14. Hojo, H., Tanaka, H., Hagiwara, M., Asahina, Y., Ueki, A., Katayama, H., Nakahara, Y., Yoneshige, A., Matsuda, J., Ito, Y., Nakahara, Y.: Chemoenzymatic synthesis of hydrophobic glycoprotein: synthesis of Saposin C carrying complex-type carbohydrate. J Organomet Chem. 77, 9437–9446 (2012)

    Article  CAS  Google Scholar 

  15. Tomabechi, Y., Krippner, G., Rendle, P.M., Squire, M.A., Fairbanks, A.J.: Glycosylation of pramlintide: synthetic glycopeptides that display in vitro and in vivo activities as amylin receptor agonists. Chem Eur J. 19, 15084–15088 (2013)

    Article  CAS  PubMed  Google Scholar 

  16. Kowalczyk, R., Brimble, M.A., Tomabechi, Y., Fairbanks, A.J., Fletcher, M., Hay, D.L.: Convergent chemoenzymatic synthesis of a library of glycosylated analogues of pramlintide: structure–activity relationships for amylin receptor agonism. Org Biomol Chem. 12, 8142–8151 (2014)

    Article  CAS  PubMed  Google Scholar 

  17. McIntosh, J.D., Brimble, M.A., Brooks, A.E.S., Dunbar, P.R., Kowalczyk, R., Tomabechi, Y., Fairbanks, A.J.: Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells. Chem Sci. 6, 4636–4642 (2015)

    Article  CAS  Google Scholar 

  18. Toonstra, C., Amin, M.N., Wang, L.-X.: Site-selective chemoenzymatic glycosylation of an HIV-1 polypeptide antigen with two distinct N-glycans via an orthogonal protecting group strategy. J Organomet Chem. 81, 6176–6185 (2016)

    Article  CAS  Google Scholar 

  19. Tomabechi, Y., Odate, Y., Izumi, R., Haneda, K., Inazu, T.: Acceptor specificity in the transglycosylation reaction using Endo-M. Carbohydr Res. 345, 2458–2463 (2010)

    Article  CAS  PubMed  Google Scholar 

  20. Tomabechi, Y., Squire, M.A., Fairbanks, A.J.: Endo-β-N-acetylglucosaminidase catalyzed glycosylation: tolerance of enzymes to structural variation of the glycosyl amino acid acceptor. Org Biomol Chem. 12, 942–955 (2014)

    Article  CAS  PubMed  Google Scholar 

  21. Tomabechi, Y., Inazu, T.: Preparation of pseudo glycoamino acid and its application to glycopeptide synthesis. Tetrahedron Lett. 52, 6504–6507 (2011)

    Article  CAS  Google Scholar 

  22. Asahina, Y., Fujimoto, R., Suzuki, A., Hojo, H.: Synthesis of Fmoc-Thr unit carrying core 1 O-linked sugar with acid-sensitive O-protecting group and its application to the synthesis of glycosylated peptide thioester. J Carbohydr Chem. 34, 12–27 (2015)

    Article  CAS  Google Scholar 

  23. Buskas, T., Ingale, S., Boons, G.-J.: Glycopeptides as versatile tools for glycobiology. Glycobiology. 16, 113R–136R (2006)

    Article  CAS  PubMed  Google Scholar 

  24. Kunishima, M., Kawachi, C., Iwasaki, F., Terao, K., Tani, S.: Synthesis and characterization of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. Tetrahedron Lett. 40, 5327–5330 (1999)

    Article  CAS  Google Scholar 

  25. Kunishima, M., Kawachi, C., Morita, J., Terao, K., Iwasaki, F., Tani, S.: 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride: an efficient condensing agent leading to the formation of amides and esters. Tetrahedron. 55, 13159–13170 (1999)

    Article  CAS  Google Scholar 

  26. Kunishima, M., Kawachi, C., Hioki, K., Terao, K., Tani, S.: Formation of carboxamides by direct condensation of carboxylic acids and amines in alcohols using a new alcohol- and water-soluble condensing agent: DMT-MM. Tetrahedron. 57, 1551–1558 (2001)

    Article  CAS  Google Scholar 

  27. Münch, J., Ständker, L., Adermann, K., Schulz, A., Schindler, M., Chinnadurai, R., Pöhlmann, S., Chaipan, C., Biet, T., Peters, T., Meyer, B., Wilhelm, D., Lu, H., **g, W., Jiang, S., Forssmann, W.G., Kirchhoff, F.: Discovery and optimization of a natural HIV-1 entry inhibitor targeting the gp41 fusion peptide. Cell. 129, 263–275 (2007)

    Article  PubMed  Google Scholar 

  28. Strohalm, M., Kavan, D., Novák, P., Volný, M., Havlíček, V.: mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal Chem. 82, 4648–4651 (2010)

    Article  CAS  PubMed  Google Scholar 

  29. Sun, B., Bao, W., Tian, X., Li, M., Liu, H., Dong, J., Huang, W.: A simplified procedure for gram-scale production of sialylglycopeptide (SGP) from egg yolks and subsequent semi-synthesis of Man3GlcNAc oxazoline. Carbohydr Res. 396, 62–69 (2014)

    Article  CAS  PubMed  Google Scholar 

  30. Umekawa, M., Higashiyama, T., Koga, Y., Tanaka, T., Noguchi, M., Kobayashi, A., Shoda, S.-I., Huang, W., Wang, L.-X., Ashida, H., Yamamoto, K.: 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)

    Article  CAS  PubMed  Google Scholar 

  31. Akaike, E., Tsutsumida, M., Osumi, K., Fujita, M., Yamanoi, T., Yamamoto, K., Fujita, K.: High efficiency of transferring a native sugar chain from a glycopeptide by a microbial endoglycosidase in organic solvents. Carbohydr Res. 339, 719–722 (2004)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Drs. Yoshimi and Osamu Kanie at Tokai University for their assistance in high-resolution mass spectrometry. We thank associate professor Hidekazu Katayama (Tokai University) for his help with running the amino acid analysis and Tokyo Chemical Industry Co., Ltd., (TCI) for kindly providing the Endo-M N175Q enzyme and SG-oxazoline. We also thank Daiki Katou (Kanazawa University) and Rina Akashi (Tokai University) for their support. A part of this work was conducted in JAIST, supported by the Nanotechnology Platform Program (Molecule and Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Author information

Authors and Affiliations

  1. Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, 921-8836, Japan

    Yusuke Tomabechi, Toshihiko Katoh & Kenji Yamamoto

  2. Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan

    Toshihiko Katoh

  3. Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan

    Munetaka Kunishima

  4. Department of Applied Chemistry, School of Engineering, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan

    Toshiyuki Inazu

Authors
  1. Yusuke Tomabechi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toshihiko Katoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Munetaka Kunishima
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Toshiyuki Inazu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenji Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yusuke Tomabechi.

Ethics declarations

Conflict of interest

No potential conflict of interest was reported by the authors.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

ESM 1

(DOCX 1278 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tomabechi, Y., Katoh, T., Kunishima, M. et al. Chemo-enzymatic synthesis of a glycosylated peptide containing a complex N-glycan based on unprotected oligosaccharides by using DMT-MM and Endo-M. Glycoconj J 34, 481–487 (2017). https://doi.org/10.1007/s10719-017-9770-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-017-9770-y

Keywords

Search

Navigation