**Angling for Uniqueness in Enzymatic Preparation of Glycosides**

#### **Antonio Trincone**

**Abstract:** In the early days of biocatalysis, limitations of an enzyme modeled the enzymatic applications; nowadays the enzyme can be engineered to be suitable for the process requirements. This is a general bird's-eye view and as such cannot be specific for articulated situations found in different classes of enzymes or for selected enzymatic processes. As far as the enzymatic preparation of glycosides is concerned, recent scientific literature is awash with examples of uniqueness related to the features of the biocatalyst (yield, substrate specificity, regioselectivity, and resistance to a particular reaction condition). The invention of glycosynthases is just one of the aspects that has thrust forward the research in this field. Protein engineering, metagenomics and reaction engineering have led to the discovery of an expanding number of novel enzymes and to the setting up of new bio-based processes for the preparation of glycosides. In this review, new examples from the last decade are compiled with attention both to cases in which naturally present, as well as genetically inserted, characteristics of the catalysts make them attractive for biocatalysis.

Reprinted from *Biomolecules*. Cite as: Trincone, A. Angling for Uniqueness in Enzymatic Preparation of Glycosides. *Biomolecules* **2013**, *3*, 334-350.

#### **1. Introduction**

In a brilliant, recently published analysis of the research-guided development in the field of biocatalysis during the last century, different authors recognized three historical waves of innovations that totally changed the field of biocatalysis to the present industrially accomplished level [1]. In a nutshell, while in the past limitations of an enzyme modeled the enzymatic process, today the enzyme can be engineered to be suitable for the process requirements. However this general bird's-eye view cannot be specific for articulated contexts in which each single class of enzyme or selected enzymatic process is at the present state. In another similar general bird's-eye analysis, Riva identified a long wave of successes still far from reaching the end in biocatalysis [2], due to the difficulties encountered in the shift from "classical" processes to biobased ones. It is clear that exploiting natural catalysts to obtain selective transformations of non-natural substrates is far from being fully explored; among many others, the cases represented by the new concept of "third generation biorefineries" [2] (producing chemicals from biomasses), or by the new glycoside hydrolases and other enzymes found in marine environments [3] have re-fostered new research trends in the field. As a matter of example, although investigation into hemicellulases as biorefining enzymes has been slow, as reported in a recent analysis [4], xylan-related biocatalysis has continued to make steady progress in many areas, including the discovery and characterization of a wide range of hemicellulases. Talking more specifically about biobased glycosynthesis, these studies are

opening new prospects for the use of pentose sugars as building blocks for engineered pentosides as non-ionic surfactants or prebiotic food/feed ingredients.

Carbohydrates are involved in a broad range of functions in cell living systems. Structural roles and energy storage as functions were recognized during the first half of the last century while attainments in glycobiology and glycochemistry, during the last twenty years, have further revealed that carbohydrate parts of biomolecules (glycoproteins, glycolipids, *etc.*) are involved in important biological functions mainly related to cell recognition events [5–7]. It should not be neglected that carbohydrates are important molecules also in the technological domain. Synthetic carbohydrate-containing polymers have a wide range of applications in medical biotechnology [8]. A number of novel dietary carbohydrates produced by enzymatic syntheses have been introduced into food technology during the last decade [9]. In innovative fine chemical manufacturing solutions, straightforward synthesis of products is of interest (e.g., chromophoric oligosaccharides of strictly defined structure as valuable tools for the kinetic analysis of hydrolytic activities and for characterization of new exo- or endo-glycosidases). Finally, in cosmetics, prodrug action of enzymatically glycosylated natural lipophilic antioxidants is currently under consideration.

In general glycosylation is considered to be an important and quite special method for the structural modification of a compound. It allows the conversion of a lipophilic compound into a hydrophilic one changing pharmacokinetic properties or creating drug delivery systems. It could also be generalized that in a glycoside, the type of aglycon determines the application: long alkyl chains allow glycosides to possess useful properties as surfactants and emulsifiers; aglycon based on unsaturated alkyl chains are said to be valuable, as glycosides, for fungal infections or as antimicrobial agents; glycosides of peptides and steroids are used in antitumor formulations and cardiac-related drugs, respectively; and glycosides of flavors and fragrances are used as "controlled release" compounds [10,11].

Sugar units have more than one site through which the chains are extended. Each of these sites frequently shows very similar reactivity, thus the masking of reactive centers by protecting groups is essential in order to direct coupling through the right position. For this reason protection and deprotection steps of functional groups are in use extensively in the arsenal of the synthetic carbohydrate chemist; moreover ensuring the correct stereochemistry of the glycosidic linkage formed entails additional difficulty. Carbohydrate related synthetic chemistry can still be considered one of the well-explored branches of organic chemistry and very rich in significant and spectacular successes, although important alternative biomethodologies for assembling glycosidic linkages are presently known and acknowledged. It is worth noting that in comparison with chemical methods, enzymatic glycosylation is particularly useful for the modification of complex biologically active substances, when generally harsh conditions or use of toxic (heavy metals) catalysts are undesirable. Enzymes may represent an imperative choice in fields such as agriculture and food or cosmetics where chemical strategies are not acceptable [12]. In a very recent report detailing different examples of enzymatic glycosylation of small molecules, the authors concluded that challenging substrates require tailored catalysts, and the progress in the field of enzyme engineering and screening of new catalytic activities are both expected to result in new applications of biocatalytic glycosylations in various industrial sectors [13].

The enzymes responsible for the synthesis of glycosidic linkage have been recognized as transglycosylases and named glycosyltransferases, specifying the glycosyl donor and the reaction product. These enzymes transfer sugar moieties from activated donors to specific acceptors, forming stereochemically specific glycosidic bonds, and are responsible *in vivo* for the synthesis of most cell-surface glycoconjugates, using eight common sugar nucleotides as activated donors (Leloir pathway). Sugar phosphates act as donors for other glycosyltransferases (non-Leloir pathway). Another widespread group of enzymes, named glycoside hydrolases (glycosidases), exists; they are involved in the carbohydrate metabolism being responsible for the hydrolysis of glycosidic linkages; they can act as exo- or endo-glycosidases and are involved in a series of important biological events such as energy uptake, in processes inherent cell wall metabolism, in glycan processing during *in vivo* glycoprotein synthesis, *etc.* Based on historical grounds, glycoside hydrolases were implicated in most experimental observations during the early studies into the biological synthesis of glycosidic linkages at the beginning of the last century. Hence, the concepts of enzymatically promoted synthesis by both hydrolysis-reversal and glycosyl transfer soon appeared [14]. By the end of the 1980s, several research projects [15] testified the importance of different and interesting glycoside hydrolases, especially from the marine environment; their main application was centered on the structural identification efforts that faced the complexity of oligosaccharide structures before the instrumental exploit of 2D NMR and MS spectroscopy.

Different wild-type glycosidases and their modified versions are enzymes deserving new expectations in research and development today. Significant progress has been made in recent years for the application of these enzymes: even while the major breakthrough was the invention of glycosynthases, protein engineering, metagenomics and reaction engineering led to the discovery of an expanding number of novel enzymes and to the setting up of new bio-based processes for the preparation of important glycosides. This review will compile different examples where glycoside hydrolases are the key enzymes in the process.

#### **2. Natural Enzymes for the Synthesis of Glycosidic Linkages**

In chemical terms, considering both hydrolytic or synthetic aspects of esterases, glycosidases, phosphatases, transglycosidases and peptidases, the enzymatic mechanisms are based on displacement reactions and could be grouped together. This line of thought proved to be highly productive in historical terms, allowing the collection and rationalization of the amount of mechanistic data especially for glycosidases and transglycosylases. The stereochemistry of the mechanisms of glycoside hydrolases was analyzed by Koshland [16] more than 60 years ago and allowed the classification of inverting and retaining enzymes according to the anomeric configuration found in the product with respect to that in the starting substrate. Very recently, it has become clear that other mechanisms have evolved, such as the one based on elimination [17]. In 2010, in an interesting review on diversity of catalytic base nucleophile of glycoside hydrolases, it was reported that a variety of systems are used to replace this function, including substrate-assisted catalysis, a network of several residues, and the use of non-carboxylate residues or exogenous nucleophiles [18].

Glycosyltransferases-mediated reactions are thought to proceed via an oxocarbenium-ion-like transition state as proposed for glycosidase reactions on the basis of solid structural, mechanistic and *ab initio* molecular orbital calculations data [19]. Glycosyltransferases are catalysts for natural glycosylation reactions, known as "Leloir" glycosyl transferases (GT). Glycoside phosphorylases (GP), requiring glycosyl phosphates and transglycosidases (TG), employing non-activated carbohydrates (e.g., sucrose), are additional examples of synthetic enzymes. However glycoside hydrolases (GH) can also be used for synthetic purposes under either kinetic (transglycosylation) or thermodynamic (reverse hydrolysis) control.

In this paragraph new examples related to the transfer of glycosyl residues between two oxygen nucleophiles are compiled with attention to those cases in which the natural characteristics of the catalyst make it attractive for biocatalysis (importance of molecular skeleton of substrates, yield, regioselectivity, resistance to particular reaction condition, *etc.*).
