Glycosyltransferase Mechanisms:  Impact of a 5-Fluoro Substituent in Acceptor and Donor Substrates on Catalysis

Abstract

In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electronwithdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogs on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and β-Nacetylglucosaminyl-β-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc β-octyl glycoside acceptor; however, is an excellent substrate for GalT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration.Glycosyltransferases are involved in a wide variety of biological processes ranging from the processing of N-linked glycan structures (1) to signal transduction (2) to bacterial cell wall (3) and natural product synthesis (4). A thorough understanding of these enzyme-catalyzed reactions will be important for the design of molecular therapeutics targeting these enzymes. Based on sequence homology, glycosyltransferases have been classified into an expanding list of families (5) (89 as of April 2007; available at http://afmb.cnrs-mrs.fr/CAZY. Despite this sequence diversity, glycosyltransferases can be classified into three superfamilies based on current crystal structures (6). Although there are likely to be some subtle differences between the superfamilies, the mechanisms of glycosyltransferase-catalyzed reactions fall into two categories: retaining, where the product glycoside has the same stereochemistry as the activated † This research was supported in part by the Vahlteich Research Fund administered by the College of Pharmacy, University of Michigan (JKC) and by NIH grant GM36065 (CJW).*To whom correspondence should be addressed: Phone: 734-936-2843. FAX: 734-647-4865. E-mail: jkcoward@umich.edu NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript leaving group, and inverting, where the reaction proceeds with inversion of stereochemistry at C-1.A number of studies using substrate analogs (7-9) have suggested that inverting glycosyltransferase-catalyzed reactions, like those of g...

UDP-(5F)-GlcNAc Acts as a Slow-Binding Inhibitor of MshA, a Retaining Glycosyltransferase

“…5 The 5-fluoro compound did not act as a viable donor substrate for the MshA-catalyzed reaction at concentrations up to 200 µM under conditions similar to those in the inhibition assays. These results are similar to previous studies demonstrating that the 5-fluoro compound will not act as a donor substrate, but acts as a functional acceptor substrate in glycosyltransferase reactions 8. The results shown here provide further support for the formation of substantial charge development on the donor sugar in glycosyltransferase reactions and demonstrate the power of 5-F sugars as mechanistic probes.…”

Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions

“…A second rational strategy for development of A4GALT inhibitors could be the synthesis of UDP-Gal mimics. Following the success of GH inhibitors, fluorinated donors with a fluorine at their C2 or C5 position functionalized with a UDP group at the anomeric position have been developed as slow inhibitors of retaining glycosyltransferases [ 147 , 148 , 149 ]. UDP-carba-Gal analogues, in which the pyranose oxygen atom is replaced by a carbon atom, have also been developed as GT inhibitors and, in general, are very stable competitive inhibitors [ 150 ].…”

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides

“…24 Although improved yields have been reported using 1H-tetrazole as a catalyst 25 or through extensive co-evaporations with pyridine, 26 many coupling reactions of this type take several days to reach completion and result in only low to moderate yields (<50%) following purification. [27][28][29][30][31] Herein, we report the synthesis and purification of eight sugar nucleotides via coupling of sugar-1-phosphates with nucleoside 5 0 -monophosphates activated using trifluoroacetic anhydride and N-methylimidazole. This activation method was first reported by Bogachev for use in the synthesis of nucleoside 5 0 -triphosphates, 32 and has since been used in the synthesis of UDP-a-D Dgalactofuranose by Marlow and Kiessling, 33 for the preparation of GDP-hexanolamine by Vincent and Gastinel 34 and in the synthesis of electron-deficient nucleoside 5 0 -b,c-methylenetriphosphate analogues by Mohamady and Jakeman.…”