Synthesis of Glycosides by Glycosynthases

Abstract

The many advances in glycoscience have more and more brought to light the crucial role of glycosides and glycoconjugates in biological processes. Their major influence on the functionality and stability of peptides, cell recognition, health and immunity and many other processes throughout biology has increased the demand for simple synthetic methods allowing the defined syntheses of target glycosides. Additional interest in glycoside synthesis has arisen with the prospect of producing sustainable materials from these abundant polymers. Enzymatic synthesis has proven itself to be a promising alternative to the laborious chemical synthesis of glycosides by avoiding the necessity of numerous protecting group strategies. Among the biocatalytic strategies, glycosynthases, genetically engineered glycosidases void of hydrolytic activity, have gained much interest in recent years, enabling not only the selective synthesis of small glycosides and glycoconjugates, but also the production of highly functionalized polysaccharides. This review provides a detailed overview over the glycosylation possibilities of the variety of glycosynthases produced until now, focusing on the transfer of the most common glucosyl-, galactosyl-, xylosyl-, mannosyl-, fucosyl-residues and of whole glycan blocks by the different glycosynthase enzyme variants.

Keywords: biocatalysis; glycosidase; glycoside; glycosylation; glycosynthase; polysaccharide; synthesis.

Scheme 1

Mechanisms of glycosyl hydrolases and synthases. (A) Retaining hydrolases follow a double displacement mechanism in which the enzyme forms a glycosyl-enzyme-intermediate by attacking the anomeric centre with a nucleophilic amino acid residue. The anomeric configuration of the substrate is retained in the product; (B) Inverting hydrolases follow a single displacement mechanism, which is supported by acid/base residues. The anomeric configuration of the product is inverted compared to the substrate; (C) ‘Classic’ glycosynthase mechanism involving an activated glycosyl donor in opposite configuration to the natural substrate. The product cannot be hydrolyzed due to the lack of a nucleophilic residue; (D) Alternative glycosynthase mechanism producing the activated donor in situ with an external nucleophile (azide, formate or acetate ion); (E) Glycosynthases derived from endo-β-N-acetylglucosaminidases transfer oxazoline glycoside donors to variable acceptors.

Scheme 2

Glucosylation of natural and unnatural glycosides. (a) Synthesis of glucosylated methyl β-acarviosin (16) by Fairweather et al. [24]. The mono- and diglycosylated products 17, 18 were isolated in yields 42% and 6%; (b) Glucosylation of erythromycin A (19) by the glucosynthase EryBI D257G [26]. The enzyme showed high tolerance towards the bulky dimethylated amino group in the C3 position.

Scheme 3

Direct glycosylation of flavonoids 21 and 22 utilizing the glycosynthase HiCel7B E197S [30]. The enzyme exhibited high specificity for glycosylation of the hydroxyl function in the Y position (as for flavonoid 22) only deviating in the case of absence of this group as observed for the flavonoid 21. The yields of the flavonoid glycosylation ranged from 72–95%.

Scheme 4

Synthesis of the methyl umbelliferone derivative of the type 2 blood group A oligosaccharide 26 performed by Kwan et al. [33]. The synthesis combined the engineered glycosynthase Abg 2F6 (A19T, E358G, Q248R, M407V) with two glycosyltransferases WbgL and BgtA. The glycosyltranferases could also be employed in a one-pot reaction giving a higher yield of 62% compared to the total yield of 36% for the sequential reaction.

Scheme 5

Synthesis of the glycolipid lyso-G_M3 (31) by Rich et al. [36]. The production of the lactosyl sphingosine acceptor 32 for the Cst-I α-2,3-sialyltransferase was catalyzed by the EGCase II glycosynthase in an overall yield of 61%. ^a Yield encompassing the chemical synthesis of LacF (23) and the glycosynthase reaction.

Scheme 6

In situ formation of the glycosyl donors βGalN₃ (35) or βGal formate (36) by incubation of αGalF (2) with glycosynthase BtGH97b D415G the additional external nucleophiles sodium azide of formate [41]. The in situ produced donor can then be subsequently transferred to a suitable acceptor (R²OH).

Scheme 7

Synthesis of various xylosides 40, 42–48 by the glycosynthase Abg 2F6 derived from the β-glucosidase Abg of A. tumefaciens [44]. The enzyme exhibited variable selectivity depending on the acceptor substrate, producing predominantly β-1,4 linkages. ^a Yields determined by HPLC analysis of the reaction mixture; ^b Yield of isolated, acetylated product.

Scheme 8

Production of xylooligomers comprising 6–100 monomers by exploitation of the self-condensation of αXylF (14) and Xyl₂F (49) catalyzed by the enzyme combination of XynB2 E335G and XT6 E265G [48,49].

Scheme 9

Combinatorial glycosynthase/glycosyltransferase approach for the production of defined, homogenous xyloglucans [53]. Polysaccharide synthesis using compound 50 or 51 was catalyzed by PpXG5 E323G resulting in polymers with a maximal molecular weight of 30,000 (n = 29) and 60,000 (n = 44), respectively. Subsequent fucosylation by AtFUT1 reached a fucosylation of 75% of the oligosaccharide repeats. Nomenclature: X = Xyl-α1,6-Glc; L = Gal-β1,2-Xyl-α1,6-Glc; F = Fuc-α1,2- Gal-β1,2-Xyl-α1,6-Glc.

Scheme 10

In situ production of the glycosylation donor αManF (15) by exploiting the chemical rescue of hydrolytic activity of Man2a E519S in the presence of sodium fluoride as an external nucleophile [54].

Scheme 11

α-Fucosylation of LNB 66 or LacNAc 67 for the production of the Lewis antigens Le^a (64) and Le^x (65), respectively, catalyzed by the α-1,3-1,4-l-fucosynthase BbAfcB D703S [59].

Scheme 12

Selective introduction of an α-fucosyl residue to the glycoside of the GM1 ganglioside (68) demonstrated by Sugiyama et al. [61]. The enzyme BbAfcA N423H showed a broad acceptor range exhibiting fucosylation activity also towards N- and O-glycans and a nonasaccharide xyloglucan.

Scheme 13

Synthesis of two glycoforms of the sperm antigen CD52 (70a/b) by the endo-β-N-acetylglucosaminidase derived glycosynthase Endo-M N175Q [65]. The high-mannose and complex type glycoforms were produced under utilization of oxazoline donors 72, 73 and the GlcNAc-CD52 peptide (71).