Antony J. Fairbanks

Enhanced glycosylation with mutants of endohexosaminidase A (endo A).

Glycosylation of proteins is the most diverse form of posttranslational modification, and can play a key role in protein folding, and can also crucially affect important protein properties. However, since the biosynthesis of glycans is not under direct genetic control, glycoproteins are produced intracellularly as heterogeneous mixtures of glycoforms, in which different oligosaccharide structures are linked to the same peptide chain. Access to pure single glycoforms of glycoproteins has now become a major scientific objective since it is not only a prerequisite for more precise biological investigations into the different effects glycans have on protein properties, but also an important commercial goal in the field of glycoprotein therapeutics, which are currently marketed as heterogeneous mixtures of glycoforms. Access to single glycoforms of glycoproteins can be achieved by total synthesis of both glycan and polypeptide components, and some outstanding achievements in this area have recently been published. However, such ACHTUNGTRENNUNGsynthesis approaches are particularly arduous and do not realistically represent a practical approach that could be applied to widespread and large-scale glycoprotein production. Alternative approaches based on bioengineering of cell lines in order to optimise production of glycoproteins that bear particular oligosaccharide structures have also been reported and are being exploited commercially, though such approaches have no guarantee of complete glycan homogeneity. An alternative method for achieving homogeneous protein glycosylation involves the use of enzymatic catalysis, and one particular class of enzyme that displays considerable synthesis potential in this respect comprises the endohexosaminidases. Endohexosaminidases are a class of enzyme that specifically cleave the chitobiose core [GlcNAcb ACHTUNGTRENNUNG(1-4)GlcNAc] of Nlinked glycans between the two N-acetyl glucosamine residues, and since they cleave this linkage they can also be used to selectively synthesise it. Two members of this class that have been demonstrated to display useful synthesis glycosylation activity are Endo M from Mucor hiemalis and Endo A from Arthrobacter protophormiae. However, since these enzymecatalysed reactions are reversible, competitive product hydrolysis can greatly reduce synthesis efficiency, particularly when transglycosylations are undertaken with unactivated donors. Seminal work in the field by Shoda and co-workers demonstrated that carbohydrate oxazolines are useful activated glycosyl donors for these enzymes, presumably because they mimic the putative oxazolinium ions, which are proposed intermediates in the enzyme-catalysed hydrolysis reaction. Subsequently, extensive work from the group of Wang has detailed the efficient synthesis of a series of glycopeptides by transglycosylation with Endo A; they have also recently reported the synthesis of single glycoforms of ribonuclease B by using this approach. In order to circumvent the problem of competitive product hydrolysis previous work has focussed on the attempted development of irreversible glycosylation reactions with structurally modified oxazolines as glycosyl donors; the synthesis products of these reactions are generally not hydrolysed by the endohexosaminidase used to promote their synthesis ; the enzymes therefore act as glycoligases. However, another potential way to circumvent this problem is to either use specifically mutated enzymes or glycosynthases—as developed by Withers and Planas—which are not capable of product hydrolysis. The term “glycosynthase” was first applied by Withers to retaining glycosidases in which the nucleophilic of the two catalytic acid residues in the enzyme active site had been replaced by site-directed mutagenesis with a nonparticipating residue, for example, by alanine. The use of an activated glycosyl donor, such as a glycosyl fluoride, allows this mutant enzyme to promote a synthesis reaction, but the mutant enzyme is not capable of hydrolysing the product glycosidic linkage as the key nucleophilic residue was absent. Endo A is a member of family 85 of the glycohydrolases (GH85). These enzymes, though they are retaining glycosidases, are thought to catalyse hydrolysis by a neighbouringgroup-participation mechanism in which the carbonyl oxygen of the 2-acetamide of the second GlcNAc residue is the actual nucleophile, rather than an enzyme-bound aspartate or glutamate. These enzymes, therefore, do not possess a nucleophilic residue at the active site, and as such it is not possible to envisage the production of a glycosynthase along the lines of the accepted Withers and Planas precedents. However, Wang et al. have very recently reported the production of a series of mutants of Endo M—another family 85 endohexosaminidase— which they screened for hydrolytic and transglycosylation activity. In particular they identified an N175A mutant of Endo M in which Asn175—a conserved residue in the GH85 family— was replaced by alanine; this mutant displayed glycosynthase activity by using oxazolines as glycosyl donors. Since this N175A mutant displayed only marginal hydrolysis activity it [a] Dr. C. D. Heidecke, T. B. Parsons, Dr. A. J. Fairbanks Department of Chemistry, Chemistry Research Laboratory University of Oxford, Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+44)1865-275674 E-mail : antony.fairbanks@chem.ox.ac.uk [b] Dr. Z. Ling, Prof. N. C. Bruce, Dr. J. W. B. Moir Department of Biology, University of York YO10 5YW (UK) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Synthesis of 1-epihydantocidin from d-ribose

Abstract A key step in a short synthesis of 1- epi hydantocidin (2) is the unanticipated transformation of azidolactones (8) and (10) to a bicyclic amine (12) induced by tetra- n -propylammonium perruthenate in the presence of morpholine-N-oxide. The structure of (12) was established by X-ray crystallographic analysis.

Endohexosaminidase M : Exploring and exploiting enzyme substrate specificity

Post-translational modification of proteins by glycosylation can play a key role in protein folding and can also crucially affect important protein properties such as conformation and stability, susceptibility to proteases and circulatory lifetime. In addition, protein glycosylation plays a role in many other key biological processes such as cell–cell signalling, development and immune response. The synthesis of the carbohydrate portion of glycoproteins is not under direct genetic control, and, as a result, glycoproteins are typically biosynthesised as complex heterogeneous mixtures, known as glycoforms, in which different oligosaccharide structures are linked to the same peptide chain. These mixtures are to all intents and purposes inseparable, since their physical properties are so similar. However, access to pure single glycoforms of glycoproteins is not only a prerequisite for precise biological investigation but is also becoming an increasingly important goal in relation to glycoprotein therapeutics, which are currently marketed as heterogeneous glycoform mixtures. Whilst several chemoselective methods have been developed in order to synthetically access proteins glycosylated with defined oligosaccharides, 10] these methods suffer from the disadvantage that the carbohydrates are connected to the peptide backbone by non-native linkages. As an alternative method for achieving homogenous protein glycosylation, several groups have recently promoted the use of enzyme catalysis. One particular class of enzymes that display considerable synthetic potential in this respect is the endohexosaminidases, which specifically cleave the chitobiose core [GlcNAcbACHTUNGTRENNUNG(1– 4) lcNAc] of N-linked glycans between the two N-acetyl glucosamine residues. Two members of this class that have been shown to display useful synthetic glycosylation activity are Endo M from Mucor hiemalis and Endo A from Arthrobacter protophormiae. Previous work, particularly on the use of Endo A, has demonstrated that carbohydrate oxazolines are useful activated glycosyl donors for these enzymes, presumably since they mimic the putative oxazolonium ions, which are intermediates in the enzyme-catalysed hydrolysis reaction. Indeed, the efficient synthesis of a series of glycopeptides has been achieved by transglycosylation with Endo A; moreover a recent paper also details investigations of a correlation between the efficiency of glycosylation and substrate structure. 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, or so called glycosynthases, developed by Withers and Planas, which are not capable of product hydrolysis. Another potential solution to this problem is the use of highly activated donors that are structurally slightly modified. In this case, the activated donor could well be processed by the enzyme at a reasonably efficient rate, particularly if the donor itself can be considered as a transition-state mimic, and yet the product might not be hydrolysed due to the minor structural modification. As part of a long-term program aimed at developing synthetic routes to pure, single glycoforms of glycoproteins, we recently became interested in the use of Endo M as a catalyst for the conjugation of synthetic oligosaccharides to glycopeptides and proteins bearing single GlcNAc residues. Herein we report investigations into the substrate tolerance and efficiency of glycosylation for Endo M-mediated reactions of a model glycosyl amino acid acceptor with a range of natural N-glycan oxazoline donors, and also several donors that contain a minor structural modification. Oxazoline donors 1–4 (Scheme 1), which correspond to fragments of natural high-mannose N-glycans, were synthetically accessed as previously described. In addition, in order to probe the substrate tolerance of Endo M and to be able to later investigate the importance and role of the central mannose unit of the core N-glycan pentasaccharide, the gluco-containing diand trisaccharide oxazolines 5 and 6 were also ACHTUNGTRENNUNGaccessed as follows. Methyl triflate-mediated glycosylation of the gluco thioglycoside donor 7 with protected glucosamine acceptor 8 gave the bACHTUNGTRENNUNG(1–4)-linked disaccharide 9, which was then converted into the deprotected oxazoline 5 by following a series of standard protecting-group manipulations and finally Zemplen deacetylation (Scheme 2). Likewise, the previously described ACHTUNGTRENNUNGglucose-containing trisaccharide 13 was converted into the ACHTUNGTRENNUNGdeprotected trisaccharide oxazoline 6 by following a similar series of protecting-group manipulations, and a final global ACHTUNGTRENNUNGdeprotection step, again by Zemplen deacetylation. Endo M-catalysed glycosylations were carried out by using model glycosyl amino acid 17 as the acceptor, since it possessed Z protection of nitrogen as a chromophore to facilitate HPLC analysis (Scheme 3). Endo M-catalysed reaction of monosaccharide donor 1 yielded no product; this indicated that at least a disaccharide is required as the minimum donor structure. That only a minimum of disaccharide is required for efficient glycosylation was confirmed by observing that the ManGlcNAc disaccharide oxazoline donor 2 glycosylated acceptor 17 to give trisaccharide 19 in a yield of 98% after 3 h [a] T. W. D. F. Rising, Dr. T. D. W. Claridge, Dr. A. J. Fairbanks Chemistry Research Laboratory, Oxford University Mansfield Road, Oxford, OX1 3TA (UK) Fax: (+44)1865-275674 E-mail : antony.fairbanks@chem.ox.ac.uk [b] Dr. J. W. B. Moir Department of Biology, University of York York, YO10 5YW (UK) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Synthesis of Arabino glycosyl triazoles as potential inhibitors of mycobacterial cell wall biosynthesis

A series of arabino glycosyl triazoles with varying hydrophobic groups were synthesised as putative mimics of decaprenolphosphoarabinose (DPA) as potential inhibitors of mycobacterial cell wall biosynthesis. Biological testing against Mycobacterium bovis BCG revealed low to moderate anti-mycobacterial activity, with strong dependence on the identity of the hydrophobic side chain.

Glycosylation Catalyzed by a Chiral Brønsted Acid

The use of a chiral Brønsted acid catalyst for the activation of trichloroacetimidate glycosyl donors has been demonstrated for the first time. In toluene the chirality of the acid catalyst is seen to influence the stereochemical outcome of the glycosylation processes, hinting that perhaps diastereocontrol of glycosylation processes may become achievable through the judicious use of chiral organic catalysts.

Glycosyl phenylthiosulfonates (Glyco-PTS): novel reagents for glycoprotein synthesis

Controlled site-selective glycosylation can be achieved by combining site-directed cysteine mutagenesis with chemical modification of the introduced thiol; a new class of more efficient chemoselective reagents, glycosyl phenylthiosulfonates, allow rapid glycosylations of representative simple thiols, peptides and proteins.

Stereoselective synthesis of C-glycosides via Tebbe methylenation and Claisen rearrangement

Abstract A variety of β- C -glycosides may be readily accessed in a stereoselective fashion from 3-OH glycal esters, via the use of Tebbe methylenation and subsequent thermal Claisen rearrangement. The use of carbohydrate ester substrates allows the formation of β(1→6) linked C -disaccharides.

Total synthesis of the Glc3Man N-glycan tetrasaccharide

Abstract The total synthesis of the tetrasaccharide Glcα(1→2)Glcα(1→3)Glcα(1→3)ManαOMe, which corresponds to the terminal tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved by the use of differentially protected selenoglycosides and thioglycosides as glycosyl donors, all of which possessed non-participating protection of the 2-hydroxyl group. Favourable anomeric stereoselectivity was achieved for the glycosylation reactions by the use of ether as solvent, or co-solvent. Global deprotection by catalytic hydrogenation with palladium acetate in a mixture of ethanol and acetic acid yielded the target tetrasaccharide.

Stereoselective 1,2‐cis Glycosylation of 2‐O‐Allyl Protected Thioglycosides

The technique of intramolecular aglycon delivery (IAD), whereby a glycosyl acceptor is temporarily appended to a hydroxyl group of a glycosyl donor is an attractive method that can allow the synthesis of 1,2-cis glycosides in an entirely stereoselective fashion. 2-O-Allyl protected thioglycoside donors are excellent substrates for IAD, and may be glycosylated stereoselectively through a three-step reaction sequence. This sequence consists of quantitative yielding allyl bond isomerisation, to produce vinyl ethers that can then undergo N-iodosuccinimide mediated tethering of the desired glycosyl acceptor, and subsequent intramolecular glycosylation, to yield either alpha-glucosides or beta-mannosides accordingly. Although attempted one-pot tethering and glycosylation is hampered by competitive intermolecular reaction with excess glycosyl acceptor, this problem can be simply overcome by the use of excess glycosyl donor. Allyl mediated IAD is a widely applicable practical alternative to other IAD approaches for the synthesis of beta-mannosides, that is equally applicable for alpha-gluco linkages. It is advantageous in terms of both simplicity of application and yield, and in addition has no requirement for cyclic 4,6-protection of the glycosyl donor.

Anomeric spirohydantoins of mannofuranose : approaches to novel anomeric amino acids by an oxidative ring contraction

Bromine-induced oxidative ring contraction of an α-amino-δ-lactone gave a mixture of α-aminotetrahydrofurancarboxylic esters which may allow the preparation of stable amino acid derivatives at the anomeric position of mannofuranose. The synthesis of N-phenylhydantoins at the anomeric position of mannofuranose is reported.