Synthesis of N-glycan oxazolines: donors for endohexosaminidase catalysed glycosylation

Rising, Thomas W. D. F.; Claridge, Timothy D. W.; Davies, Norman; Gamblin, David P.; Moir, James; Fairbanks, Antony J.

doi:10.1016/j.carres.2006.03.007

Cited by 76 publications

(42 citation statements)

References 35 publications

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“…A similar series of reactions produced the manno tetrasaccharide oxazoline 4 [34] from the previously described trisaccharide diol 20 [33] which underwent regioselective glycosylation at the primary hydroxyl with donor 14. An identical sequence of protecting group manipulations and oxazoline formation furnished manno tetrasaccharide oxazoline 4 (Scheme 2).…”

Section: Synthesis Of Oxazolinesmentioning

confidence: 97%

“…[33] The (1!6)-linked trisaccharide 3 and tetrasaccharide 4 were synthesised as shown in Schemes 1 and 2, respectively. The previously described disaccharide 12 [33] underwent regioselective reductive ring opening after treatment with Et 3 SiH and PhBCl 2 to yield the alcohol 13, which was then glycosylated with the trichloroacetimidate donor 14 to give (1!6)-linked trisaccharide 15. A series of protecting group manipulations that was common to most reaction sequences then followed.…”

Section: Synthesis Of Oxazolinesmentioning

confidence: 99%

“…Endohexosaminidase-catalysed glycosylations were carried out using model glycosyl amino acid 11 [33] as the acceptor, which possessed Cbz protection to facilitate time course studies of the extent of reaction by UV/HPLC analysis.…”

Section: Enzyme Catalysed Glycosylation Reactionsmentioning

confidence: 99%

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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: Synthesis Of Oxazolinesmentioning

confidence: 97%

Section: Synthesis Of Oxazolinesmentioning

confidence: 99%

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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.…”

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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

220

229

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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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“…1c). [23][24][25][26][27][28] The efficiency of any transglycosylation process with a wild-type enzyme is inevitably limited by enzyme-catalyzed hydrolysis of the product. In order to address this issue, we have recently performed mutagenesis studies on Endo-A in order to produce enzymes that retain transglycosylation activity 29 while showing reduced hydrolysis of the products.…”

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confidence: 99%

The X-ray Crystal Structure of an Arthrobacter protophormiae Endo-β-N-Acetylglucosaminidase Reveals a (β/α)8 Catalytic Domain, Two Ancillary Domains and Active Site Residues Key for Transglycosylation Activity

Ling

Suits

Bingham

et al. 2009

Journal of Molecular Biology

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Synthesis of N-glycan oxazolines: donors for endohexosaminidase catalysed glycosylation

Cited by 76 publications

References 35 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

The X-ray Crystal Structure of an Arthrobacter protophormiae Endo-β-N-Acetylglucosaminidase Reveals a (β/α)8 Catalytic Domain, Two Ancillary Domains and Active Site Residues Key for Transglycosylation Activity

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