Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides Shannon C. Timmons a and David L. Jakeman a,b, * a Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J3 b College of Pharmacy, Dalhousie University, 5968 College Street, Halifax, Nova Scotia, Canada B3H 3J5 Received 13 November 2007; received in revised form 23 January 2008; accepted 31 January 2008 Available online 9 February 2008 Abstract—As Leloir glycosyltransferases are increasingly being used to prepare oligosaccharides, glycoconjugates, and glycosylated natural products, efficient access to stereopure sugar nucleotide donor substrates is required. Herein, the rapid synthesis and purifi- cation of eight sugar nucleotides is described by a facile 30 min activation of nucleoside 5 0 -monophosphates bearing purine and pyrimidine bases with trifluoroacetic anhydride and N-methylimidazole, followed by a 2 h coupling with stereospecifically prepared sugar-1-phosphates. Tributylammonium bicarbonate and tributylammonium acetate were the ion-pair reagents of choice for the C18 reversed-phase purification of 6-deoxysugar nucleotides, and hexose or pentose-derived sugar nucleotides, respectively. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Sugar nucleotides; Glycosyltransferases; Sugar-1-phosphates 1. Introduction The glycosylation of an array of structurally diverse bio- molecules including proteins, lipids, and many classes of natural products mediates a wide variety of important biological processes. 1–3 In contrast to protein and nucleic acid research, the development of tools to study glycosylation has been limited by the structural and functional diversity of carbohydrates, resulting in a chasm of understanding between these groups of bio- molecules. 4 Recent advances in chemical glycobiology 5 have begun to bridge this gap in knowledge and innova- tive strategies for engineering glycosylation have been described. 6–8 The ubiquitous enzymes responsible for the transfer of carbohydrates from sugar nucleoside diphosphate donors to various acceptors are Leloir glycosyltransfer- ases. 9 Until recently, the membrane-associated nature of these anabolic enzymes largely limited their access for use in glycosylation studies. 10 Advances in molecular biology have facilitated the overexpression, purification, and crystallization of an increasing number of glycosyl- transferases, revealing a higher degree of structural diversity than originally anticipated. 11 A second major obstacle to investigating the functional role of glycosyl- ation in numerous biological systems using glycosyl- transferases is a lack of efficient access to stereopure sugar nucleotides. 12 The recent observation that the glycosyltransferase VinC is capable of processing both a- and b-anomer of a sugar nucleotide to give glycosylated products with corresponding inversion of stereochemistry at the anomeric linkage 13 underlies the importance of stereospecific synthetic methods for the preparation of these substrates. Although the enzymatic preparation of these compounds has emerged, 14–18 the versatility available via chemical synthesis is unparalleled. One synthetic strategy used to prepare sugar nucleo- tides involves the direct coupling of electrophilic sugar donors with nucleoside 5 0 -diphosphates. 19–21 In addition to low yields, these routes typically suffer from low anomeric diastereoselectivites, although an improve- ment involving neighboring group participation has recently been described. 22 A second synthetic approach 0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2008.01.046 * Corresponding author. Tel.: +1 902 494 7159; fax: +1 902 494 1396; e-mail: david.jakeman@dal.ca Available online at www.sciencedirect.com Carbohydrate Research 343 (2008) 865–874