Enzymatic Synthesis of Galactosylated 1d-Chiro-Inositol and 1d-Pinitol Derivatives Using the β-Galactosidase from Bacillus circulans

Abstract

The b-D-galactosidase from Bacillus circulans regioselectively galactosylates 1D-chiro-inositol and 1D-pinitol (1D-3-Omethyl-chiro-inositol) to give mono-and digalactosylated inositols in yields of up to 45%. The enzyme does not galactosylate 1L-chiroinositol or 1L-quebrachitol (1L-2-O-methyl-chiro-inositol). This is the first report of enzymatic glycosylation of chiro-inositol and its derivatives.Oligosaccharides containing inositol sugars such as 1D-chiro-inositol (DCI) (1) and 1D-pinitol (2) are the focus of current interest for their putative role as secondary messengers of insulin and their associated implications in the etiology of Type 2 diabetes. 1-5 Although the detailed structures of these secondary messengers remain elusive, they are believed to contain an amino sugar such as galactosamine linked to a phosphorylated inositol sugar such as 1, 2 or myo-inositol. 6 In addition, DCI and pinitol-based oligosaccharides have been isolated in high levels from the seeds of leguminous plants and are thought to provide protection for the organelles, enzymes, proteins etc. during dessiccation. 7,8 Glycoside hydrolases (glycosidases) are responsible for the cleavage of oligosaccharides into constituent monosaccharide units in vivo. In vitro, it is possible to drive the reaction in the reverse direction, i.e. to form interglycosidic linkages from monomeric sugar units. By employing glycosidases in this manner it is possible to circumvent classical organic synthesis methods and traditional labour-intensive extraction procedures in the production of oligosaccharides.The b-D-galactosidase from Bacillus circulans (EC 3.2.1.23) has been reported as being an excellent, high yielding catalyst for the formation of di-and trisaccharides with Galb1®4 linkages. 9-13 This enzyme catalyzes the transfer of Gal from p-nitrophenyl b-D-galactopyranoside (GalbOpNP) or lactose to Glc, GlcNAc, Gal and GalNAc, predominantly to the OH-4 position. 13Herein we describe the enzymatic synthesis and characterisation of the products from the enzyme catalyzed reactions of GalbOpNP with 1 and 2 employing the b-Dgalactosidase from B. circulans (Figures 1 and 2). 14 To our knowledge, this is the first report describing an enzymatic synthesis to prepare inositol containing oligosaccharides. β-Galactosidase from Bacillus circulans 5 OH OH HO OH OH OH OH HO HO OH OH

Enzyme-catalysed synthesis of galactosylated 1d- and 1l-chiro-inositol, 1d-pinitol, myo-inositol and selected derivatives using the β-galactosidase from the thermophile Thermoanaerobacter sp. strain TP6-B1

“…Previously, we reported that the galactosylation of 1D D -pinitol 2 by the b-galactosidase from B. circulans gave only one disaccharide product, 10, whereby galactosylation occurred on the axial position remote from the methyl group. 19 NMR spectroscopic analysis of the product mixture indicated that one of the disaccharide products was that observed previously, namely 1D D -1-O-(b-D Dgalactopyranosyl)-4-O-methyl-chiro-inositol 10. 19 A detailed analysis of the unassigned NMR correlations in the 2D-NMR spectra of the product mixture revealed that the second product was 1D D -1-O-(b-D D -galactopyranosyl)-3-O-methyl-chiro-inositol 11.…”

“…19 NMR spectroscopic analysis of the product mixture indicated that one of the disaccharide products was that observed previously, namely 1D D -1-O-(b-D Dgalactopyranosyl)-4-O-methyl-chiro-inositol 10. 19 A detailed analysis of the unassigned NMR correlations in the 2D-NMR spectra of the product mixture revealed that the second product was 1D D -1-O-(b-D D -galactopyranosyl)-3-O-methyl-chiro-inositol 11. It is apparent that disaccharide 10 is the result of galactosylation of 2 at the axial hydroxyl position remote from that of the methyl group of 2, that is OH-6, whereas 11 is the result of galactosylation at the other axial hydroxyl of 2, OH-1, two positions removed from that of the methyl group.…”

“…Instead, the approach can be thought of as being directed and biomimetic, because the regioselectivity encountered is more likely to be of biological significance than a corresponding random chemical approach. We have previously reported the galactosylation of 1D D -chiro-inositol 1 and 1D D -pinitol 2, using the b-galactosidase from Bacillus circulans, 19 the first report of the enzymatic glycosylation of inositols. An analysis of the products obtained from these glycosylations allowed us to gain some insight into the hydroxyl group configurations key to the recognition of the acceptor as a substrate in the active site of the enzyme.…”

“…An analysis of the product mixture by 1 H and 13 C NMR spectroscopy revealed that the resonances of the major component were identical to those of the disaccharide product observed from the enzymatic galactosylation of 1 using the b-galactosidase from B. circulans, namely 1D D -1-O-(b-D D -galactopyranosyl)-chiro-inositol 8. 19 The Table 1. Summary of galactosylation products obtained using TP6-B1 b-galactosidase with p-nitrophenyl b-D D -galactopyranoside as the donor second, minor, component was shown by 1 H and 13 C NMR analysis to be a disaccharide with a b-linkage to 1D D -chiro-inositol at a different position to that observed for compound 8.…”

“…12;16-18 Recently, considerable interest has focussed on the use of glycoside hydrolase enzymes (glycosidases) to synthesise di-and oligo-saccharides in only one step. [19][20][21][22][23][24] The particular characteristics of the glycosidase employed determines, which transglycosylation products, if any, are formed and the products formed are a function of the enzyme's acceptor and donor preferences as well as its glycosylation regio-and stereoselectivity. However, these selectivities need not be regarded as limitations to the use of glycosidases in oligosaccharide synthesis.…”