A Highly Efficient Chemoenzymatic Approach toward Glycoprotein Synthesis

Li, Bing; Song, Haijing; Hauser, Steven; Wang, Lai-Xi

doi:10.1021/ol061056m

Cited by 111 publications

(111 citation statements)

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“…[28] More recent work has also reported the application of the glycoprotein remodelling strategy using Endo A to access single glycoforms of ribonuclease B. [29] However, as with many enzyme-catalysed transglycosylations one particular problem that can greatly reduce synthetic efficiency and utility is product hydrolysis, since in general the product is itself an enzyme substrate. One particularly elegant way of circumventing this problem is the use of specifically mutated enzymes called glycosynthases, as developed by Withers [30] and Planas, [31] which are not capable of product hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

Endohexosaminidase‐Catalysed Glycosylation with Oxazoline Donors: Fine Tuning of Catalytic Efficiency and Reversibility

Rising

Heidecke

Moir

et al. 2008

Chemistry A European J

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A complete series of oxazoline di-, tri-, tetra-, and hexasaccharides, corresponding to the core sections of N-linked glycoprotein high mannose glycans, together with the corresponding oligosaccharides containing a central glucose unit, were synthesised and tested as glycosyl donors for glycosylation of a GlcNAcAsn glycosyl amino acid catalysed by the endohexosaminidases M (Endo M), A (Endo A) and H (Endo H). Whilst Endo H did not catalyse any glycosylation reactions, both Endo M and Endo A efficiently catalysed glycosylations that were not limited to donors containing the Manbeta(1-->4)GlcNAc linkage. Precise structure activity relationships and time course studies have revealed fine-tuning of the efficiency of the synthetic processes which correlated both with the enzyme used and the precise oxazoline structure. Efficient irreversible glycosylation was achievable with both Endo M and Endo A, further demonstrating the use of structurally modified oxazoline donors as transition state mimics in order to promote enzyme-catalysed synthesis, whilst precluding product hydrolysis; enzymes in these cases display "glycoligase" activity.

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Section: Introductionmentioning

confidence: 99%

Endohexosaminidase‐Catalysed Glycosylation with Oxazoline Donors: Fine Tuning of Catalytic Efficiency and Reversibility

Rising

Heidecke

Moir

et al. 2008

Chemistry A European J

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“…As an effort to address these problems, we and others have recently explored synthetic sugar oxazolines, a highly active species mimicking the presumed transition state, as donor substrates for the enzymatic transglycosylation (16 -22). It was found that this strategy was particularly useful for the synthesis of glycopeptides carrying truncated and/or modified N-glycans, since the highly activated sugar oxazolines could tolerate certain modifications, but the resulting (ground state) glycopeptide product would become resistant to enzymatic hydrolysis due to the slight modification, allowing accumulation of the product (17,19,20,22). However, when glycopeptides and glycoproteins carrying natural, full-size N-glycans are concerned, the rapid hydrolysis of the product by the wild-type endoglycosidases is difficult to avoid, thus limiting its wide application.…”

mentioning

confidence: 99%

Mutants of Mucor hiemalis Endo-β-N-acetylglucosaminidase Show Enhanced Transglycosylation and Glycosynthase-like Activities

Umekawa

Huang

et al. 2008

Journal of Biological Chemistry

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Endo-␤-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the ␤1,4 linkage of N,N-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glycoengineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the sitedirected mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the K m value of Y217F for an acceptor substrate 4-methylumbelliferyl-␤-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn 175 "knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-␤-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.Endo-␤-N-acetylglucosaminidase (EC 3.2.1.96) (ENGase) 3 catalyzes hydrolysis of the ␤1,4-glycosidic linkage of the N,NЈ-diacetylchitobiose moiety in the core of asparagine-linked glycan of various glycoproteins and glycopeptides. This type of enzyme is widely distributed in animals, plants, fungi, and bacteria. Several bacterial enzymes, such as Endo-H from Streptomyces plicatus (1) and Endo-F 1 from Flavobacterium meningosepticum (2), were cloned and classified into glycoside hydrolase (GH) family 18 in the CAZy data base (available on the World Wide Web), which may share a common evolutional origin with GH18 chitinases. The other ENGases are distinct from the enzymes of the GH18 chitinase family and are classified into the GH family 85. We and others have previously reported that several ENGases of the GH85 family showed significant transglycosylation activity (i.e. the ability to transfer the releasing glycan to an acceptor other than water to form a new glycosidic linkage) (3-6). These ENGases include Endo-M from Mucor hiemalis (3), Endo-A from Arthrobactor protophormiae (4), Endo-CE from Caenorhabditi...

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“…The essential role of this residue for hydrolysis was confirmed by the fact that mutation of this residue abolished the hydrolytic activity of ENGases (11)(12)(13)(14)(15)(16). These mechanistic and mutagenesis studies laid the basis for exploring synthetic sugar oxazolines as donor substrates for transglycosylation, which resulted in significant enhancement of the transglycosylation efficiency for glycopeptide and glycoprotein synthesis (17)(18)(19)(20)(21)(22). Moreover, it was further demonstrated that novel glycosynthases could be generated by site-directed mutation at the critical Asn residue that promotes oxazolinium intermediate formation in hydrolysis (Asn-175 in Endo-M and Asn-171 in Endo-A).…”

mentioning

confidence: 89%

Remarkable Transglycosylation Activity of Glycosynthase Mutants of Endo-D, an Endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae

Fan¹,

Huang

Wang

2012

Journal of Biological Chemistry

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Background: Endo-D is an endoglycosidase that can deglycosylate N-glycan core from IgG antibodies. Results: Endo-D showed distinct activities toward core-fucosylated and nonfucosylated substrates, and novel mutants were generated that demonstrated remarkable transglycosylation activity. Conclusion:The new discovery expands the repertoire of endoenzymes for glycoprotein research. Significance: This study reveals interesting substrate specificity of Endo-D and provides new enzymatic tools for glycosylation engineering of glycoproteins such as IgG-Fc.

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A Highly Efficient Chemoenzymatic Approach toward Glycoprotein Synthesis

Cited by 111 publications

References 40 publications

Endohexosaminidase‐Catalysed Glycosylation with Oxazoline Donors: Fine Tuning of Catalytic Efficiency and Reversibility

Endohexosaminidase‐Catalysed Glycosylation with Oxazoline Donors: Fine Tuning of Catalytic Efficiency and Reversibility

Mutants of Mucor hiemalis Endo-β-N-acetylglucosaminidase Show Enhanced Transglycosylation and Glycosynthase-like Activities

Remarkable Transglycosylation Activity of Glycosynthase Mutants of Endo-D, an Endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae

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