by in situ regenerating and recycling of sugar nucleotides.
Source: Modified from Wong et al. [21].
Some natural products of nonsugar substances such as complex glycosides of ergot alkaloids required for immunological studies can also be prepared by glycosylation using GalTs. For example, bovine β‐1,4‐GalT was used for catalyzing UDP‐Gal and elymoclavine 17‐O‐β‐D‐glucopyranoside (R = OH) or elymoclavine 17‐O‐(2‐acetamido‐2‐deoxy‐β‐D‐glucopyranoside) (R = NHAc) to produce the corresponding lactose and lactosamine derivatives (Scheme 3.9) [15, 22, 23]. In this reaction scheme, UDP‐Gal was generated in situ from UDP‐Glc by the use of UDP‐Gal 4′‐epimerase originated from yeast. It is interesting to note that glucose in the form of UDP‐Glc can be concomitantly transferred to give in parallel β‐1,4‐D‐glucopyranosyl elymoclavine 17‐O‐(2‐acetamido‐2‐deoxy‐β‐D‐glucopyranoside) by β‐1,4‐GalT [15, 22]. Following this protocol, other natural glycosides such as the sweetener stevioside and its congener steviolbioside can be transformed with good degree of conversion to their monogalactosyl derivatives with absolute regioselectivity and site‐selectivity [23].
There are a group of non‐Leloir glycosyltransferases such as cyclodextrin glucosyltransferase (CGTase). CGTases are cultivated and produced from microorganisms [15, 24]. They catalyze four different kinds of reactions: cyclization, disproportionation, coupling, and hydrolysis so that cyclodextrins (CDs) can be formed from amylose or starch through the intramolecular circularization reaction; a linear malto‐oligosaccharide is cleaved and one part is transferred to an acceptor sugar molecule in the disproportionation reaction; CD ring is opened and the resulting linear malto‐oligosaccharide is transferred to a sugar molecule; and the glycosidic linkages in starch can be hydrolyzed to form oligosaccharides [25]. Since CDs of CD6, CD7, and CD8 (α‐CD, β‐CD, and γ‐CD) have been extensively used in the food, cosmetic, and pharmaceutical industries, the synthesis of CD by CGTases from starch is important. However, the formation of CD by native CGTases has the major advantage of lacking product specificity that they produce a mixture of CD6, CD7, CD8, and larger‐ring CD (≥CD9) and make the separation difficult. To solve the product specificity problem, CGTases from Paenibacillus sp. A11 and Bacillus macerans was crosslinked imprinted, thus the size of the CD products formed was shifted toward CD8 and ≥CD9, and the overall CD yield was increased. The crosslinked imprinted cyclodextrin glycosyltransferases also showed better stability in organic solvents and can be recycled several times [24].
Scheme 3.9 β‐1,4‐GalT catalyzed galactosylation of natural glycosides and concomitant transfer of glucose.
Source: Based on Křen and Thiem [15]; Křen [22]; Riva [23].
Glycogen is a branched polysaccharide using glucose as monomer that contains a series of α‐1,4‐glycosidic linkages with branch points about every 10–13 glucose residues through the formation of α‐1,6‐glycosidic linkages. The synthesis of glycogen is as of other biological polymers a two‐step process: the initiation stage and the elongation stage. The initiation stage of glycogen synthesis is catalyzed by glycogenin in an autocatalytic manner. The elongation stage is catalyzed by glycogen synthase in synergy with the branching enzyme. Glycogenin is a member of the glycosyltransferase family 8 (GT‐8) that is a metal (Mn2+) dependent retaining enzyme responsible for the synthesizing 6‐10 α‐1,4‐linked glucose residues using UDP‐Glc as the substrate donor during glycogen synthesis [26, 27]. The two‐step reaction scheme is illustrated by Scheme 3.10 [27, 28]. In the first reaction step, the role for the aspartate residue at position 159 (Asp‐159) of glycogenin serves in binding and activating the acceptor oligosaccharide chain that is covalently attached to the tyrosine residue at position 194 (Tyr‐194), and the aspartate residue at position 162 (Asp‐162) serves the role of chemical reaction of glucosyltransferase [27–29].
Protein glycosylation is one of the most common posttranslational modifications of proteins in eukaryotes that affect a wide range of protein functions, from folding and secretion to biomolecular recognition (targeting) and serum half‐life (stability), and many other intercellular communication processes [30, 31]. However, owing to glycoprotein microheterogeneity [32], the specific covalently bound oligosaccharides to protein is difficult because protein glycosylation is not under direct genetic control. Therefore, glycoproteins are often produced as a mixture of glycoforms to make the isolation of individual glycoforms difficult and drive the development of new synthetic methods for producing well‐defined oligosaccharides bound glycoproteins. The advancement in the field has provided some strategies to resolve this problem by synthesizing the glycoproteins in vitro that include the remodeling of recombinant glycoproteins with glycosidases and glycosyltransferases, the ligation of synthetic glycopeptides by enzymatic and chemical methods, the intein‐mediated coupling of glycopeptides to larger proteins expressed as intein‐fusion protein, the ligation of glycopeptides to larger proteins containing N‐terminal cysteine expressed as TEV protease cleavable fusion proteins, in vitro translation, and the pathway reengineering in yeast systems to produce human‐type N‐linked glycoforms [18, 31,33–35].
Scheme 3.10 Two‐step synthesis of glycogen with glycogenin functioning as an autocatalytic initiator.
Source: Based on Hurley et al. [27]; Smythe and Cohen [28].
Besides in vitro synthesis of glycoproteins, in vivo synthesis method using suppressor tRNA has been described for the recombinant production of neoglycoproteins and glycoproteins [35]. The strategy to produce unique glycoforms in E. coli has been reported by evolving an orthogonal synthetase‐tRNA pair that genetically encodes a glycosylated amino acid in responds to the amber stop codon (TAG). Further, a naturally occurring homogeneous glycoprotein can be produced in E. coli via the direct incorporation of the core glycosyl amino acids N‐acetylglucosamine‐β‐serine and N‐acetylgalactosamine‐α‐threonine [30, 31, 36, 37]. The sugar chains of these glycoproteins can be further elongated in vitro using glycosyltransferase.
Glycolipids are responsible for the organism’s toxic and immunological properties. Like glycoproteins, glycolipids reside in cell membranes with their carbohydrate segments extending into the fluid surrounding the cells. In this location they function as receptors that are essential for recognizing chemical messengers, other cells, pathogens, and drugs [38]. The synthesis of glycolipid oligosaccharides is performed in the Golgi apparatus by a complex membrane‐bound glycosyltransferases together with sugar nucleotide transporters and ceramide‐bound accepter [39, 40]. The details are not discussed here for their fewer industrial applications.
3.3 Phosphorylation with Kinases
The fundamental roles played by kinases in cells are in different signal transduction pathways and in many cellular processes such as cell metabolism, division, differentiation, survival and apoptosis, as well as development and oncogenesis. They effectively catalyzed the transfer of the γ‐phosphoryl group (PO3 –) of ATP to a broad number of hydroxyl group containing substrates with the release of a proton (Scheme 3.11) [41]. Nowadays, the structural study of many different kinase classes indicates