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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. doi: 10.1101/glycobiology.3e.054
Essentials of Glycobiology [Internet]. 3rd edition.
Show detailsGlycosyltransferases are the biosynthetic enzymes responsible for the construction of interglycosidic linkages, and glycosidases catalyze the opposite reaction, hydrolysis of interglycosidic linkages (Chapter 6). The diversity of natural glycans is reflected by the numerous glycosyltransferases and glycosidases encountered in nature, each showing a defined substrate specificity. Natural glycans are often encountered in heterogeneous form and often produced in minute amounts, making their isolation and characterization from natural sources often cumbersome. Therefore, glycobiology relies heavily on synthetic glycans, and synthetic methodology to produce glycans has witnessed tremendous progress in the past two decades (Chapter 53). Glycosyltransferases and glycosidases offer several advantages in the construction of glycans. Especially under controlled conditions, these biocatalysts are very powerful. Here, recent developments in the use of (mutant) glycosidases and glycosyltransferases for the synthesis of tailored glycans, including combinations of both enzyme classes and in conjunction with synthetic intermediates, are presented.
MECHANISM OF GLYCOSYLTRANSFERASES AND GLYCOSIDASES
Biological evolution has spent millions of years optimizing enzymatic processing for the biosynthesis and cleavage of specific glycans with highly specific glycosidic linkages. Thus, it is logical to turn to naturally occurring glycosyltransferases and glycosidases to address some difficult problems in synthesis of glycans in vitro. The mechanism of action of glycosyltransferases even resembles the way an interglycosidic linkage is installed by chemical synthesis (Chapter 53). An activated donor saccharide, represented by UDP-glucose (Figure 54.1), is condensed with an acceptor moiety (here, ceramide) to give, after expulsion of the leaving group (here, UDP), the glycoconjugate (glucosylceramide). In contrast to chemical synthesis, the regioselectivity is regulated by the enzyme active site that also selects for donor- and acceptor-glycosides. Glucosylceramide synthase (GCS) catalyzed synthesis of glucosylceramide (Figure 54.1) proceeds with inversion of the anomeric configuration, with the α-glucosidic linkage in UDP-glucose transformed into the β-glucosidic linkage in glucosylceramide. Nature uses a limited set of donor glycosides—most prominently the Leloir donor glycosides, next to UDP-glucose (e.g., CMP-sialic acid, GDP-mannose)—and the nature of the glycosyltransferase determines whether glycosylation proceeds with retention or inversion of configuration at the anomeric center (Chapter 6). The mechanisms used by glycosyltransferases are not resolved yet and are the subject of intense research.
Glucosylceramide is hydrolyzed by the lysosomal exo-glucosidase, acid glucosylceramidase (GBA). Hydrolysis takes place with retention of configuration and is the result of a double displacement mechanism. Upon protonation of the aglycon, ceramide is displaced in a formal SN2 substitution to yield a covalent enzyme-glycoside adduct. On entry of water in the enzyme active site, the formed acylal linkage is hydrolyzed in another formal SN2 event to release glucose from the enzyme active site. Next to retention of configuration, the hydrolysis of interglycosidic bonds can also take place with inversion of configuration and is normally the result of direct SN2 displacement of a protonated aglycon by water. Although not necessarily relevant for the product formation in nature (sugar hemiacetals being prone to anomerize at physiological pH), the different mechanisms used by retaining glycosidases (involvement of a covalent intermediate) and inverting glycosidases (no covalent intermediate involved) bear consequences for their use in glycan synthesis.
GLYCOSYLTRANSFERASE-MEDIATED SYNTHESIS OF GLYCANS
The use of glycosyltransferases in glycan synthesis requires access to the natural donor glycosides, most often Leloir-type nucleotide sugars. Thus, the intrinsic advantage of glycosyltransferase-mediated synthesis (excellent regio- and stereoselectivity) can be offset by the relative scarcity of the required donor glycosides. However, this disadvantage may be counteracted by adding the biosynthetic enzymes that regenerate the consumed sugar nucleotides. The power of glycosyltransferase-mediated glycan synthesis was shown in the 1992 synthesis of sialyl Lewis x derivative 5 (Figure 54.2). Allyl lactoside 2, derived by chemical synthesis (Chapter 53), is reacted with CMP-sialic acid 1 using recombinant α2-3-sialyltransferase (α2-3-SiaT) as the catalyst. The resulting trisaccharide 3 is further extended with GDP-fucose 4 as the donor and recombinant fucosyltransferase (FucT) as the catalyst to deliver allyl-sialyl Lewis x.
In the first step of the assembly line, the expensive nucleotide sugar, CMP-sialic acid, is consumed, and upon transfer of the sialic acid, saccharide cytosine monophosphate (CMP) is produced. With the aid of two consecutive kinases (nucleoside monophosphate kinase and pyruvate kinase), CMP can be transformed in situ into the corresponding triphosphate (CTP), which is then condensed by the enzyme CMP-Neu5Ac with sialic acid to regenerate CMP-sialic acid 2.
The synthesis of GalNAc-GD1a heptasaccharide 7 equipped with a biotin at the reducing end (replacing the sphingolipids present in the natural product) was accomplished by submitting synthetic lactoside 6 to the consecutive action of four glycosyltransferases, one of which (α2-3-SiaT) was used twice (Figure 54.3). By using this method with various donor nucleotide sugars and glycosyltransferases, a comprehensive series of glycosphingolipids and their analogs have been obtained. The methodology, especially with respect to enzymatic sialic acid introduction, is competitive when compared with chemical gangliosides synthesis.
The synthesis of a comprehensive set of complex, asymmetrically branched mammalian N-glycans was accomplished using the combined power of chemical and glycosyltransferase-mediated enzymatic synthesis. As an example (Figure 54.4), decasaccharide 8 was prepared via contemporary solution phase chemical oligosaccharide synthesis (Chapter 53). The asymmetrically branched decasaccharide features two nonreducing galactopyranose moieties, one of which is introduced as the tetra-acetate (in bold) whereas the other is unprotected. Decasaccharide 8 is thus designed to allow for specific sialylation of the unprotected galactose residue. After saponification, the intermediate undecasaccharide is expanded to well-defined oligosaccharide 9 making use of the substrate specificity of α1-3-fucosyltransferase (α1-3-FucT, introduction of two fucopyranoses), β1-4-galactosyltransferase (β1-2-GalT, twice), β1-3-N-acetylglucosaminetransferase (β1-3-GlcNAcT), and finally the sialic acid transferase, ST6Gal-I. The chemoenzymatic strategy proved flexible and allowed for the generation of a diverse panel of N-glycans where the reducing end is available for bioconjugation and for the preparation of glycan microarrays for protein binding studies.
FROM TRANSGLYCOSYLATION TO GLYCOSYNTHASES
In contrast to glycosyltransferase-mediated reactions, in which the equilibrium is shifted predominantly to (natural) product formation because of the intrinsic reactivity of donor glycosides, the equilibrium in a glycosidase-mediated reaction can be influenced such that the reaction proceeds in the opposite direction.
Under physiological conditions, with high water concentrations, glycosidases hydrolyze glycosidic linkages to produce the corresponding hemiacetal, either with retention of configuration (Figure 54.5A) or with inversion of configuration at the anomeric center. Performing a glycosidase reaction in partly nonaqueous conditions, by addition of large excess of aglycon, by inducing kinetic conditions or by a combination of these, allows for partial reversal of the reaction equilibrium. By this means and in a transglycosylation event, glycans have been constructed. Disadvantages of this method are that reaction conditions may be adverse to enzyme reactivity and/or stability and, moreover, that the formed product is in essence a substrate for glycosidase-catalyzed hydrolysis. This caveat can be circumvented by making use of mutant glycosidases (Figure 54.5B), in which the catalytic nucleophile in case of retaining glycosidases is mutated to an innocent bystander (depicted is an Asp to Ala substitution). Such a glycosynthase can be used to react a synthetic donor glycoside that bears the anomeric configuration corresponding to the intermediate enzyme glycosyl covalent adduct (Figure 54.1) with an appropriate nucleophile to construct an interglycosidic linkage. The main advantage of this strategy is that, in principle, the mutant glycosidase has largely lost the ability to hydrolyze the formed product, because of the absence of the catalytic nucleophile. Many glycosidases and, in recent years, inverting glycosidases have been adapted to glycosynthases, and have produced an array of glycans.
The strategy is exemplified by the synthesis of flavonoid glucoside 13 (Figure 54.6). Synthetic α-fluorolactoside 10 is treated with phenol 11 in the presence of the E197S mutant of the Humicola insolens Cel7B endoglucosidase. This glycosynthase proved highly flexible with respect to the acceptor phenol, allowing for the construction of a small library of flavonoid glycosides represented by phenolic disaccharide 12. Subsequent enzymatic removal of the nonreducing galactospyranoside provided flavonoid glucoside 13.
By combining of the strengths of glycosyltransferases, glycosynthases, and chemical synthesis, the total synthesis of ganglioside LLG-3 from the neurogenic starfish was accomplished (Figure 54.7). N-Carboxybenzyl(Cbz)-protected CMP-sialic acid 17 was prepared by first performing a Neu5Ac aldolase catalyzed aldol reaction of mannosamine derivative 14 and pyruvate 15. The resulting sialic-acid derivative 16 was reacted with CTP under the agency of CMP-Neu5Ac synthetase to give donor sialoside 17. In a sialyl transferase catalyzed reaction, compound 17 was condensed with α-fluorolactoside 10 to give trisaccharide 18, where the amine was unmasked by palladium-catalyzed hydrogenolysis to give trisaccharide, equipped with an anomeric fluoride for glycosynthesis and an amine for chemical amide bond formation. The sequence commenced by condensation of synthetic sialic acid derivative 20 with the free amine in 19 under amide bond-forming conditions, to give 21. Condensation of tetrasaccharidyl fluoride 21 with the double (E351S/D341Y) mutant of the bacterial endoglycosidases, EGCase II, gave lysolipids 23 in good yield. The free amine in 23 can be condensed with a fatty acid or alternatively with a fluorescent reporter group.
Glycosynthases derived from endoglycosidases have finally been subject of numerous studies aimed at the construction of structurally well-defined N-glycoproteins (Figure 54.8). Hexosaminidases, both exo- and endo-, hydrolyze N-acetylglucosamine containing glycosidic linkages with retention of configuration. In contrast to most other retaining glycosidases, hexosaminidases do not use an enzyme active site nucleophile in the nucleophilic displacement of the aglycon, but rather use the N-acetyl group for this purpose (Figure 54.8 insert). As a result, an intermediate oxazolinium ion intermediate is produced that after nucleophilic attack of water yields the hemi-acetal with retention of configuration. Endo-hexosaminidases can be used in transglycosylation reactions and, in mutant form, as glycosynthases.
A demonstration of an endo-hexosaminidase derived glycosynthase in action is given by the synthesis of the Man9GlcNAc2-glycopeptide 28 from the glycoprotein HIV-1. The sequence of steps commences with homogeneous Man9GlcNAc2Asn 24 that can be prepared by exhaustive (pronase) proteolytic digestion of soybean glycoproteins. Wild-type endohexosaminidase, Endo-A, recognizes this high-mannose N-glycan and hydrolyses the GlcNAc-GlcNAc interglycosidic linkage, to yield Man9GlcNAc 25. After peracetylation, treatment with trimethylsilyl bromide and borontrifluoride etherate and global deprotection provided oxazolidinium ion 27. This compound is a good substrate for mutant (N175A) endohexosaminidase, endo-M, which was identified by site-directed mutagenesis as a putative glycosynthase activity. In the final step, the efficacy of the endo-M glycosynthase was shown by the construction, from 26 and 27, of high-mannose N-glycan 28.
OUTLOOK
Enzymatic synthesis has proven to be a powerful methodology for the construction of complex glycans. Chemoenzymatic synthesis is thus emerging as a suitable alternative for chemical synthesis, as shown by the many applications of sialyltransferases for the construction of complicated or intractable sialic acid–containing glycans. The marriage of enzymatic synthesis and chemical synthesis is very promising. The elaboration by means of glycosyltransferases and/or glycosynthases has proven to be a highly effective strategy to prepare large and complex glycoconjugates. To date, no all-encompassing solution for the preparation of each and every glycan exists—not by means of enzymatic synthesis, not through chemical synthesis nor by a combination of these methods. Advances in chemical and enzymatic syntheses provides, in principle, the means to construct most natural or designed glycans.
ACKNOWLEDGMENTS
The authors acknowledge contributions to previous versions of this chapter by Nathaniel Finney and David Rabuka and appreciate helpful comments and suggestions from Abubakar Jalloh, Chelsea Painter, Eillen Tecle, and Paeton L. Wantuch.
FURTHER READING
- Ichikawa Y, Lin YC, Dumas DP, Shen GJ, Garcia-Junceda E, Williams MA, Bayer R, Ketcgam C, Walker LE, Paulson JC, Wong CH. 1992. Chemical-enzymatic synthesis and conformational analysis of sialyl Lewis x and derivatives. J Am Chem Soc 114: 9283–9298.
- Lu Y, Ye J, Wold F. 1993. Isolation of oligomannose-type glycans from bean glycoproteins. Anal Biochem 209: 79–84. [PubMed: 8465965]
- Henrissat B, Davies G. 1997. Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7: 637–644. [PubMed: 9345621]
- Williams SJ, Withers SG. 2002. Glycosynthases: Mutant glycosidases for glycoside synthesis. Aust J Chem 55: 3–12.
- Coutinho PM, Deleury E, Davies GJ, Henrissat B. 2003. An evolving hierarchical family classification for glycosyltransferases. J Mol Biol 328: 307–317. [PubMed: 12691742]
- Blixt O, Vasiliu D, Allin K, Jacobsen N, Warnock D, Razi N, Paulson JC, Bernatchez, Gilbert M, Wakarchuk W. 2005. Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a. Carbohydr Res 340: 1963–1972. [PubMed: 16005859]
- Rising TWDF, Claridge TDW, Moir JWB, Fairbanks AJ. 2006. Endohexosaminidase M: exploring and exploiting enzyme substrate specificity. ChemBioChem 7: 1177–1180. [PubMed: 16800015]
- Bennet CS, Wong CH. 2007. Chemoenzymatic approaches to glycoprotein synthesis. Chem Soc Rev 36: 1227–1238. [PubMed: 17619683]
- Yang M, Davies GJ, Davis BG. 2007. A glycosynthase catalyst for the synthesis of flavonoid glycosides. Angew Chem Int Ed 46: 3885–3888. [PubMed: 17304599]
- Umekawa M, Huang W, Li B, Fujita K, Ashida H, Wang LX, Yamamoto K. 2008. Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J Biol Chem 283: 4469–4479. [PubMed: 18096701]
- Pukin AV, Florack DEA, Brochu D, van Lagen B, Visser GM, Wennekes T, Gilbert M, Zuilhof H. 2011. Chemoenzymatic synthesis of biotin-appended analogues of gangliosides GM2, GM1, GD1a and GalNAc-GD1a for solid-phase applications and improved ELISA tests. Org Biomol Chem 9: 5809–5815. [PubMed: 21727969]
- Schmaltz RM, Hanson SR, Wong CH. 2011. Enzymes in the synthesis of glycoconjugates. Chem Rev 111: 4259–4307. [PubMed: 21749134]
- Rich JR, Withers SG. 2012. A chemoenzymatic total synthesis of the neurogenic starfish ganglioside LLG-3 using an engineered and evolved synthase. Angew Chem Int Ed 51: 8640–8643. [PubMed: 22821741]
- Armstrong A, Withers SG. 2013. Synthesis of glycans and glycopolymers through engineered enzymes. Biopolymers 99: 666–674. [PubMed: 23821499]
- Wang Z, Chinoy ZS, Ambre SG, Peng W, McBride R, de Vries RP, Glushka J, Paulson JC, Boons GJ. 2013. A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans. Science 341: 379–383. [PMC free article: PMC3826785] [PubMed: 23888036]
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