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An Enxyme that Adds Whole Sugar Chains without Breaking Down Products: Glycosynthase

 *1 unit will convert 1 μmol of pNP-GlcNAc to SG-GlcNAc-pNP per min. at pH7.0 at 30°C

 Glycosynthase (Endo-M-N175Q) is an enzyme developed by Yamamoto, Umekawa, et al. through site-directed mutation of areas around the active center of Endo-M1) which is already marketed. Since the feature of Glycosynthase is efficient transglycosylation activity by using oxazoline derivatives as glycosyl donors while suppressing sugar hydrolysis activity, the resulting glycosylated products are obtained in high yield with less digestion of the products by the enzyme. Due to this feature Glycosynthase is expected to be applied as useful tool in glycotechnology.

Fig. 1 Scheme of transglycosylation reaction

 Umekawa and her colleagues caused transglycosylation reactions at the GlcNAc site of sperm antigen CD52 using oxazoline derivatives of the high-mannose type sugar chains or the complex type sugar chains as glycosyl donors2). They succeeded in obtaining glycosylated products in high yield of 84% and 76%, respectively. Moreover, they also achieved transglycosylation reactions using two biologically active blood-pressure-lowing peptides, PAMP12 and Substance P, as glycosyl acceptors and the oxazoline derivative of a complex type sugar chain containing sialic acids as a glycosyl donor in 95% and 98% yield, respectively3). The articles in 2009 describe the advantages of this glycosylation method using sugar-oxazoline derivatives4-5).

Fig. 2 Experiment example of the transglycosylation

 Practical realization of efficient transglycosylation reactions would also be useful for expansion into glycoprotein synthesis, such as the area of biosimilars, and creation of new functional sugar complexes can be expected.

 A set of the transglycosylation data using a glycosynthase (Endo-M-N175Q) is shown below (Fig. 3). The oxazoline derivative of a complex type sugar as a glycosyl donor was transglycosylated with GlcNAc-β-pNP as a glycosyl acceptor, and the glycosylated product was afforded in a high yield of 95% after 24 hours. The sustained and effective production of the glycosylated compound was identified from HPLC profiles and MALDI-TOF MS as shown below (Fig. 4 and 5).

Fig. 3 Transglycosylation of oxazoline derivative and GlcNAc-β-pNP

HPLC: ODS, CH3CN / H2O, UV 260nm
⇒(space):GlcNAc-b-pNP       ⇒(fill):glycosylated product

Fig. 4 HPLC profiles of transglycosylation reaction

Fig. 5 MALDI-TOF MS spectrum of transglycosylated product

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