β(1→4) Galactosyltransferase from bovine milk (GalT, EC 2.4.1.22) is one of the most extensively studied mammalian glycosyltransferases with regard to synthesis and substrate specificity.1-5, 9-15 β(1→4) GalT catalyses the transfer of galactose from UDP-galactose (UDP-Gal) to the OH at the 4-position of Nacetyl glucosamine (GlcNAc) and also β-linked GlcNAc subunits to yield β-lactosamine (β-LacNAc) and β-Gal(1→4)-β-GlcNAc structures.16 When the enzyme forms a complex with α- lactalbumin, the specificity is altered and D-glucose becomes the preferred acceptor. Thus, addition of α-lactalbumin promotes the formation of lactose (β-Gal(1→4)-Glc). Both α- and β- glycosides of glucose were utilized as acceptors in enzymatic galactosidation as well. The α-glucosides required the presence of α-lactalbumin.4 Numerous other acceptor substrates for β(1→4)GalT catalyzed transfer of galactose have been described in the literature , e.g. 2-deoxyglucose, D-xylose, 5- thioglucose, N-acetylmuramic acid, and myoinositol. Moreover, 6-O-fucosylated and sialylated modifications served as acceptors17 as well as 3-O-methyl-GlcNAc,8, 18 3-deoxy-GlcNAc, 3-O-allyl-GlcNAcβOBu and 3-oxo-GlcNAc.19 Several modifications of GlcNAc that were employed as acceptor substrates are illustrated in Scheme 11 β(1→4)GalT has been employed in solid-phase oligosaccharide synthesis on polymer supports like polyacrylamide or watersoluble poly(vinyl alcohol). The resulting galactosylated oligosaccharides are cleaved from the polymers photochemically or by using chymotrypsin.20,21 N-Acetylglucosaminyl amino acids and peptides were successfully galactosylated to afford glycopeptides with a disaccharide moiety.22-24 Further extension of the carbohydrate chain was accomplished afterwards by employing α(2→6)Sialyltransferase.22-24 The preparation of an asparagine-bound trisaccharide was accomplished by combined chemo-enzymatic synthesis.22 Galactosidation of a N-acetylglucosaminyl oligopeptide followed by sialylation with α(2→3)Sialyltransferase and fucosylation with α(2→3)Fucosyltransferase yielded a glycopeptide containing a tetrasaccharide moiety.25 As different glycosides of N-acetylglucosamine and glucose can be used as acceptors in β(1→4)GalT catalyzed galactosidations, this enzymatic method was recently exploited in the modification of pharmacologically interesting glycosides.6, 7, 26, 27 Several currently published syntheses of new drug-sugar conjugates derived from the broad range of naturally occurring glycosides have accentuated the high potential of glycosylations in drug delivery, for example by increasing the solubility and bioavailability of large hydrophobic molecules under mild conditions. β(1→4)GalT catalyzed galactosidations of glycosides was successfully accomplished for elymoclavine-17-O-β-Dglucopyranoside,6 stevioside and steviolbioside,28 colchicoside,29 coumarinic glycoside fraxin,29 and different ginsenosides.30, 31

Modifications of GlcNAc employed as acceptors in β(1→4)GalT catalyzed transfer of galactose.

Scheme 1. Modifications of GlcNAc employed as acceptors in β(1→4)GalT catalyzed transfer of galactose.

Galactosylation of glycosides bearing a hydrophobic aglycone may suffer from poor solubility of the acceptor substrate. Recent systematic investigations of the stability of β(1→4)GalT in different aqueous reaction mixtures and the effect of organic cosolvents are very instructive for choosing an appropriate solvent mixture.29 Solvents like dimethyl sulfoxide, acetone, dioxane and ethanol seemed to be beneficial, increasing the stability of this enzyme, while other solvents such as N,Ndimethylformamide, acetonitrile and tetrahydrofuran enhanced the inactivation process. Transfer of galactose onto cyclodextrin was performed, because the recognition of the Gal-cyclodextrin conjugates by galectins was expected to enhance the drug delivery capabilities of the system.32 Employing C-glycoside analogues of the naturally occurring glycopeptide linkages (N-acetylglucosamine β-linked to either asparagine or serine) the corresponding C-lactosides were isolated in excellent yields.33 Neither D-mannose, D-allose, D-galactose, nor D-ribose are substrates.3, 4 Monosaccharides displaying a negative charge, such as glucuronic acid and α-glucose-1-phosphate are also not accepted as substrates. Azasugars and glucals are considered to be very weak acceptors.18 With regard to the nucleotide sugar donors, several modified substrates were utilized, but the rate of enzyme-catalyzed transfer turned out to be rather slow.3, 4

1.
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Wong CH, Whitesides GM. 1994. in Enzymes in Synthetic Organic Chemistry, Tetrahedron Organic Chemistry Series, Vol. 12, Elsevier Science Ltd, Oxford. . 252
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Koeller KM, Wong C. 2000. Synthesis of Complex Carbohydrates and Glycoconjugates:  Enzyme-Based and Programmable One-Pot Strategies. Chem. Rev.. 100(12):4465-4494. http://dx.doi.org/10.1021/cr990297n
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K?en V, Augé C, Sedmera P, Havlí?ek V. ?-Glucosyl and ?-galactosyl transfer catalysed by ?-1,4-galactosyltransferase in preparation of glycosylated alkaloids. J. Chem. Soc., Perkin Trans. 1.(17):2481-2484. http://dx.doi.org/10.1039/p19940002481
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Riva S. 2002. Enzymatic modification of the sugar moieties of natural glycosides. Journal of Molecular Catalysis B: Enzymatic. 19-2043-54. http://dx.doi.org/10.1016/s1381-1177(02)00150-9
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Augé C, David S, Mathieu C, Gautheron C. 1984. Synthesis with immobilized enzymes of two trisaccharides, one of them active as the determinant of a stage antigen.. Tetrahedron Letters. 25(14):1467-1470. http://dx.doi.org/10.1016/s0040-4039(01)80188-x
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Schanbacher FL, Ebner KE. 1970. J. Biol. Chem.. 245, 5057
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1980. Organization section. Diabetologia. 19(5):489-489. http://dx.doi.org/10.1007/bf00281837
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Trayer IP, Hill RL. 1970. J. Biol. Chem.. 245, 5057.
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Andrews P. 1970. Purification of lactose synthetase a protein from human milk and demonstration of its interaction with ?-lactalbumin. 9(5):297-300. http://dx.doi.org/10.1016/0014-5793(70)80382-9
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Barker R. 1972. et al., J. Biol. Chem.. 247, 7135.
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Rao AK, Garver F, Mendicino J. 1976. Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes. Biochemistry. 15(23):5001-5009. http://dx.doi.org/10.1021/bi00668a009

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Baisch G, Öhrlein R, Ernst B. 1996. Enzymatic galactosylation of non-natural glucosamide-acceptors. Bioorganic & Medicinal Chemistry Letters. 6(7):749-754. http://dx.doi.org/10.1016/0960-894x(96)00117-5
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Palcic MM, Srivastava OP, Hindsgaul O. 1987. Transfer of d-galactosyl groups to 6-O-substituted 2-acetamido-2-deoxy-d-glucose residues by use of bovine d-galactosyltransferase. Carbohydrate Research. 159(2):315-324. http://dx.doi.org/10.1016/s0008-6215(00)90224-6
18.
David S, Auge C. 1987. Immobilized enzymes in preparative carbohydrate chemistry. 59(11):1501-1508. http://dx.doi.org/10.1351/pac198759111501
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Wong CH, Ichikawa Y, Krach T, Gautheron-Le Narvor C, Dumas DP, Look GC. 1991. Probing the acceptor specificity of .beta.-1,4-galactosyltransferase for the development of enzymatic synthesis of novel oligosaccharides. J. Am. Chem. Soc.. 113(21):8137-8145. http://dx.doi.org/10.1021/ja00021a045
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David S, Auge C. 1987. Immobilized enzymes in preparative carbohydrate chemistry. 59(11):1501-1508. http://dx.doi.org/10.1351/pac198759111501
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Wong CH, Ichikawa Y, Krach T, Gautheron-Le Narvor C, Dumas DP, Look GC. 1991. Probing the acceptor specificity of .beta.-1,4-galactosyltransferase for the development of enzymatic synthesis of novel oligosaccharides. J. Am. Chem. Soc.. 113(21):8137-8145. http://dx.doi.org/10.1021/ja00021a045
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Zehavi U, Herchman M. 1984. Enzymic synthesis of oligosaccharides on an ?-chymotrypsin-sensitive polymer. O-(?-d-Galactopyranosyl)-(1?4)-O-(?-d-glucopyranosyl)-(1?4)-d-glucopyranose. Carbohydrate Research. 133(2):339-342. http://dx.doi.org/10.1016/0008-6215(84)85212-x
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Unverzagt C, Kunz H, Paulson JC. 1990. High-efficiency synthesis of sialyloligosaccharides and sialoglycopeptides. J. Am. Chem. Soc.. 112(25):9308-9309. http://dx.doi.org/10.1021/ja00181a037
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Panza L, Chiappini PL, Russo G, Monti D, Riva S. 1997. Stereoselective enzymatic galactosylation of C-glucosides. J. Chem. Soc., Perkin Trans. 1.(9):1255-1256. http://dx.doi.org/10.1039/a701747b
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Danieli B, Luisetti M, Schubert-Zsilavecz M, Likussar W, Steurer S, Riva S, Monti D, Reiner J. 1997. Regioselective Enzyme-Mediated Glycosylation of Natural Polyhydroxy Compounds. Part 1. Galactosylation of stevioside and steviolbioside. Helv. Chim. Acta. 80(4):1153-1160. http://dx.doi.org/10.1002/hlca.19970800412
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Riva S, Sennino B, Zambianchi F, Danieli B, Panza L. 1997. Effect of organic cosolvents on the stability and activity of the ?-1,4-galactosyltransferase from bovine colostrum. Carbohydrate Research. 305(3-4):525-531. http://dx.doi.org/10.1016/s0008-6215(97)00273-5
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Danieli B, Falcone L, Monti D, Riva S, Gebhardt S, Schubert-Zsilavecz M. 2001. Regioselective Enzymatic Glycosylation of Natural Polyhydroxylated Compounds:  Galactosylation and Glucosylation of Protopanaxatriol Ginsenosides1. J. Org. Chem.. 66(1):262-269. http://dx.doi.org/10.1021/jo001424e
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Leray E, Parrot-Lopez H, Augé C, Coleman AW, Finance C, Bonaly R. Chemical?enzymatic synthesis and bioactivity of mono-6-[Gal-?-1,4-GlcNAc-?-(1,6?)-hexyl]amido-6-deoxy-cycloheptaamylose. J. Chem. Soc., Chem. Commun..(10):1019-1020. http://dx.doi.org/10.1039/c39950001019
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Tarantini L, Monti D, Panza L, Prosperi D, Riva S. 2001. Enzymatic galactosylation of C-glycosides analogues en route to C-glycopeptides. Journal of Molecular Catalysis B: Enzymatic. 11(4-6):343-348. http://dx.doi.org/10.1016/s1381-1177(00)00020-5

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