16 ACCOUNT Glycosylation Based on Glycosyl Phosphates as Glycosyl Donors GlycosylationBasedonGlycosylPhosphatesasGlycosylDonors Hariprasad Vankayalapati, Shende Jiang, Gurdial Singh* Department of Chemistry, University of Sunderland, Sunderland SR1 3SD, UK Fax +44(191)5153148; E-mail: gurdial.singh@sunderland.ac.uk Abstract: Glycosyl phosphates have emerged as useful glycosyl donors for stereoselective glycosidic bond formation in the synthesis of oligosaccharides. In this account, we describe some of our work in developing and further expanding this methodology, particularly with the use of propane-1,3-diyl phosphate as the anomeric leaving group. We have studied the reactions of several glycosyl propane-1,3-diyl phosphates with or without C-2 participating groups with a range of glycosyl acceptors in the presence of trimethylsilyl triflate in either stoichiometric or catalytic amount, and demonstrated their application in the synthesis of several disaccharides and also the synthesis of some trisaccharides. Key words: carbohydrates, glycosides, glycosyl phosphates, glycosylation, oligosaccharides Carbohydrate research has gone through a rapid phase of expansion during the last thirty years, not only because carbohydrates are being considered as extremely useful stereochemical building blocks for complex organic synthesis.1,2 More importantly, apart from being an energy source in living systems, carbohydrates increasingly are being recognised as playing important roles in a variety of biological processes, such as signaling, cell-cell communication, molecular and cellular targeting.3 It is the enormous advances in the latter that forms the basis of a new area of multidisciplinary research named glycobiology, which deals mainly with the nature and role of carbohydrates in biological events. In biological systems, although carbohydrates can exist as free monosaccharides and oligosaccharides, the majority are covalently attached to other non-carbohydrate biomolecules, such as proteins or lipids, to form what are called glycoconjugates that generally include glycoproteins, glycolipids and proteoglycans. The advancement of research in glycobiology has to a large extent depended on the technological progress in structural analysis of oligosaccharides in glycoconjugates. However, in order to study the biological functions of carbohydrates and to develop carbohydratebased therapeutic agents, sufficient quantities of pure oligosaccharides and glycoconjugates are often required, and some of these can only be accessed by chemical synthesis. Compared with proteins and nucleic acids, which have linear structures, the construction of oligosaccharides from the monomers is more complicated due to the number of hydroxyl groups available for linkage and also Synlett 2002, No. 1, 28 12 2001. Article Identifier: 1437-2096,E;2002,0,01,0016,0025,ftx,en;A27201ST.pdf. © Georg Thieme Verlag Stuttgart · New York ISSN 0936-5214 the creation of a- or b-glycosidic bond at the anomeric centre. Since the early glycosylation method reported by Koenigs and Knorr in 1901,4 stereoselective formation of the glycoside linkage in oligosaccharide synthesis has remained an active area of research. Over the last century, research in glycosylation chemistry has resulted in a better understanding of the glycoside reaction and produced an array of glycosylation methods that generally catered for almost every type of glycosidic bond formation.5 However, to date, there is still no generally applicable method for glycoside formation as in the case of peptide synthesis. Considering the complex stereoelectronic nature of carbohydrates, it is perhaps not surprising that a general glycosidation method is so elusive. There are numerous factors, such as anomeric leaving groups, activators, solvents, reaction temperature, and protecting groups, that all affect the outcome of glycosylation. It is perhaps this diversity in glycosylation chemistry that still challenges synthetic chemists to seek to modify current glycosylation methods and to search for alternative ones with regard to the stability of the glycosyl donors, regioselectivity, stereoselectivity, and chemical yield. For the past few years, we have been interested in using phosphates as anomeric leaving groups in glycoside synthesis. It is well known that glycosyl phosphates are key intermediates in the biosynthesis of oligosaccharides and glycocojugates. In biosynthesis along the Leloir pathway, the monosaccharide is converted to a nucleoside diphosphate monosaccharide, this then acts under the catalysis of the glycosyltransferase as a glycosyl donor with the nucleoside diphosphate as the anomeric leaving group to couple with another monosaccharide as a glycosyl acceptor to form stereospecifically the glycosidic linkage.6 In spite of the significant importance of glycosyl phosphates in the biosynthesis of oligosaccharides, until recently, there were only a few reports on the use of phosphate-based anomeric leaving groups in glycoside synthesis. This account deals with the application of glycosyl phosphates as glycosyl donors in glycoside synthesis with different substrates and under various conditions. In 1985, Inazu and co-workers7 reported the preparation of 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl dimethylphosphothioate 2 as a glycosylating agent (Scheme 1). This was based on the observation that dimethylphosphinothioyl chloride had been used in peptide chemistry for the protection of hydroxyl groups and also for the activation of carboxyl group as stable mixed anhydride,8 and therefore the dimethylphosphiothionate was expected to ACCOUNT 17 Glycosylation Based on Glycosyl Phosphates as Glycosyl Donors be a good anomeric leaving group. Dimethylphosphinothioate 2 was a stable, crystalline solid which coupled with methanol, cyclohexanol, 3b-cholestanol, Z-L-SerOme, methyl 2,3,4-tri-O-benzyl-a-D-glucopyranoside, and mehtyl 2,3,6-tri-O-benzyl-a-D-glucopyranoside in the presence of silver perchlorate as an activator to give predominantly 1,2-cis-a-linked glycoside (a:b ratio 2:1– 9:1) with good chemical yields (69% to 91%). They also prepared dimethylphosphinothionates 3 and 4 (Figure 1).9,10 Dimethylphosphinothionates 3 had participating substituent on C-2, it coupled with 3b-cholestanol to give 1,2-trans-b-linked product, while 4 gave predominantly 1,2-cis-a-linked glycoside. OBn OBn O BnO BnO OBn 1. n-BuLi OH 2. Me2P(S)Cl O BnO BnO BnO 2 1 S O P Me Me Scheme 1 Biographical Sketches Hariprasad Vankayalapati received his MPharm degree in pharmaceutical chemistry from Karnatak University (India) in 1991. He was a lecturer at the University of Mysore from 1991 to 1993. He studied for his PhD degree in medicinal chemistry at the Department of Chemical Technology (UDCT) University of Bombay, working with Prof. V. M. Kulkarni from 1993 to 1996. He was also the recipient of a UGC (University Grants Commission) fellowship. Later he worked for Ranbaxy Research Laboratories (New Delhi, India) as a senior research associate in drug discovery. From 1998 to 2000 he was a postdoctoral researcher with Prof. Gurdial Singh at the University of Sunderland. He is currently a senior postdoctoral fellow in medicinal chemistry working with Prof. Laurence H. Hur- ley at the Arizona Cancer Center/College of Pharmacy, University of Arizona, Tucson. His research interests include design and synthesis of new anticancer agent quinobenzoxazines, application of computation chemistry methods for the rational design, new stereoselective glycosylation methods, assembly of oligosaccharides and synthesis of glycomimetics. Shende Jiang was born and raised in the rural area of Shandong peninsula (P. R. China). He graduated from Liaocheng Teachers College (BSc in chemistry in 1983) and studied medicinal chemistry with Prof. Wang Huicai at Shandong Medical University (MSc, 1986). He worked with the late Prof. Shi Mingli at Shandong Teachers University from 1986 to 1988 as a lecturer of organic chemistry. He came to the University of Nottingham as a Leverhulme Trust scholar (1988–1991), there he spent two years in the School of Continuing Education with Prof. Teddy Thomas and the late Prof. Michael Stephens, and one year with Prof. Stephen Clark in the School of Chemistry. In 1991 he moved to Nottingham Trent University working with Dr. Ian Coutts to work on heterocycles. He joined Prof. Singh's group at the University of Teesside in 1992 and moved with him to Sunderland in 1996. He has been with Prof. Singh for nine years, first as a PhD student and then as a postdoctoral worker. His academic interests are in the area of synthetic organic chemistry. Gurdial Singh is currently Research Professor of Organic Chemistry at the University of Sunderland. He received his BSc. (Hons) in chemistry from the University of Liverpool in 1977 and PhD in organic chemistry from UMIST with Prof. Bob Ramage in 1980 working on b-lactam chemistry. He did two years postdoctoral work with Prof. Mal- colm Campbell at the University of Bath and was appointed lecturer at Heriot–Watt University in 1982. He moved to the University of Teesside in 1984 as a senior lecturer and was promoted to reader in 1991. He has been at Sunderland since 1996. His research interests include natural product synthesis using carbohydrates as chiral tem- plates, development of Oand C-glycosylation methods for assembling complex molecules with interesting biological properties, chemical syntheses of the shikimate pathway intermediates and their analogues, development of novel b-lactams, terpene and perfume chemistry, polymers, and the development of new dental materials. Synlett 2002, No. 1, 16–25 ISSN 0936-5214 © Thieme Stuttgart · New York 18 ACCOUNT H. Vankayalapati et al. OBn O BnO BnO TBDPSO OBn RHN S BnO BnO O OH S Ph2POCl O P Me O N-methylimidazole O Me O P Me 3 R = Ac, Cbz O Me 4 5 O TBDPSO Figure 1 O O P O TBDPSO Ph O P O Ph + In order to extend the scope of this methodology we investigated using the diphenylphosphinate as the anomeric leaving group for the coupling with sugars and peptides/ amino acids.11 The attraction for this approach was also based on the premise that diphenylhposphinyl chloride had been used successfully for the activation of the carboxyl function of amino acids for the synthesis of peptides.12 In the peptide synthesis, with diphenylphosphinate as the leaving group an amide bond was formed, while in the case of glycosylation a glycosidic bond was formed. At the outset we chose to study the glycosylation reaction with protected D-ribofuranosyl diphenylphosphinates as glycosyl donors. The attraction to this was multi-faceted, with the main reason being that this process had received scant attention in the literature and in addition we wished to extend this method to the synthesis of a wide range of furanosides. Furthermore as the anomeric effect in furanoses results in equal stabilisation of both a- and b-anomers, we would be able to determine the significance of the steric interactions in the subsequent transition states of these systems. The phosphinates 6, 7 and 9, 10 were prepared by the reaction of diphenylphosphinyl chloride and the corresponding protected D-ribofuranoses 5 and 8 in yields of 80% and 60% for the pair of anomers, respectively. In both of cases, the a-anomer was the major product and the anomeric mixture was separated by flash chromatography. Separate reactions of the phosphinates 6 and 7 in the presence of TMSOTf as activator with butanol as a simple glycosyl acceptor invariably led to an almost equal amount of a- and b-glycosides in excellent chemical yields (Scheme 2). Similar results were obtained with 9 and 10. The low stereoselectivities in these reactions suggest that glycosylation is proceeding via a planar oxonium ion or a loosely bound ion pair. Following these observations and being undeterred by the results, we went on to prepare the six-membered D-glucopyranosyl phosphinates 12 and 13 with an anomeric mixture of 10:1 from 2,3,4,6-tetra-O-benzyl-D-glucopyranose 11 in 95% yield (Scheme 3). Glycosylation of pure 12 with butanol as glycosyl acceptor in the presence of trimethylsilyl triflate (TMSOTf) gave a 1:2 mixture of aand b-glycosides. The same result was also obtained when the mixture of 12 and 13 was used instead of 12. When the mixture of 12 and 13 was coupled with methanol, isopro- Synlett 2002, No. 1, 16–25 ISSN 0936-5214 Ph Ph O O O O 7 6 BnO O OH Ph2POCl BnO N-methylimidazole OBn 8 O BnO O O P O BnO Ph O P O Ph Ph Ph + BnO OBn BnO OBn 10 9 Scheme 2 panol, and other acceptors 14–18 (Figure 2), only moderate selectivity was observed in favour of b-glycosides (a:b ratio from 1:2 to 1:4).13 OBn O BnO BnO Ph2POCl OH N-methylimidazole OBn 11 OBn OBn O BnO BnO BnO + O O P Ph Ph 12 BnO BnO O O O P OBn Ph Ph 13 Scheme 3 Ikegami and co-workers14 have demonstrated that glycopyranosyl phosphates 19, 20, and 21 with non-participating substituents on C-2, and phosphates 22, 23, and 24 (Figure 3) with C-2 participating substituents coupled with a range of glycosyl acceptors (mostly protected monosaccharides) upon activation with TMSOTf give 1,2-trans-b-linked glycosides with high stereoselectivities. © Thieme Stuttgart · New York ACCOUNT OH NHZ OH CO2Me H 19 Glycosylation Based on Glycosyl Phosphates as Glycosyl Donors NHZ H BnO BnO CO2Me P O N-methylimidazole OH OBn 11 OBn Me Me H O O BnO BnO Me H Cl O 15 14 OO OBn O P O O OBn H 25 16 HO Scheme 4 HO OMe O O OH O AcO AcO O AcO OBn OBn O BnO BnO OMe O OBn OBn 17 O BnO BnO O OMe O 18 O AcO AcO Figure 2 AcO O O 27 26 OBn OBn OBn O BnO BnO BnO BnO O 19 BnO OPh 20 O conduct these glycosylation reactions employing a catalytic amount of TMSOTf as activator.15 O P OPh OPh Seeberger and co-workers16 have prepared some differentially protected glycosyl phosphates using glycal as precursors. The glycal 28 was oxidised with dimethyldioxirane (DMDO) and the epoxide was opened with a phosphoric acid (Scheme 5). The ratio of the resulting a- and b-glycosyl phosphates 29 and 30 depended on the solvents, where use of toluene and dichloromethane gave preferentially the b-phosphates and the use of THF produced almost exclusively the a-phosphates. This procedure allowed the selective introduction of the participating pivaloyl ester group on C-2 as well as the nonparticipating triethylsilyl ether group. They reported that the C-2 protected (with participating or nonparticipating groups) a- and b-glycosyl phosphates were treated respectively with acceptors 31 and 32 (Figure 5) in the presence of TMSOTf to form b-glycosidic linkage with OBz MeO2C O BnO BnO OBn O O P OPh BzO BzO O OBz OPh O O P OPh OPh 21 OBz Figure 4 O O P OPh 22 OBz O BzO OBz O O P OPh BzO BzO O OBz 23 O O P OPh OPh OPh OMe 24 Figure 3 As a result we proceeded to prepare the propane-1,3-diyl phosphate 25 (Scheme 4) (a and b mixture, a:b, 10:1) on the basis that an anomeric phosphate function would be a better leaving group compared to the diphenylphosphinate function, and also the cyclic phosphate group would have steric influence on the outcome the glycosyation.13 In this instance, the two anomers of phosphate 25 could not be separated by chromatography. The reactions of phosphate 25 with glycosyl acceptors methanol, isopropanol and 14–18 proceeded readily in the presence of one equiv of TMSOTf in good chemical yields and good stereoselectivities. In the case of glycosyl acceptors 17 and 18 we were able to prepare exclusively the b-glycosides 26 and 27 (Figure 4). More recently we have been able to OBn O BnO BnO 1. dimethyldioxirane 2. HOP(O)(OBu)2 28 OBn OBn O BnO BnO OH + O O P BnO BnO OBu O O O P OBu OH OBu OBu 30 29 Scheme 5 Synlett 2002, No. 1, 16–25 ISSN 0936-5214 © Thieme Stuttgart · New York 20 ACCOUNT H. Vankayalapati et al. O Yamanoi and co-workers20 reported the stereoselective aand b-mannopyranoside formation from dimethylphosphinothioate 37 with acceptors 31, 38, 39, and 40 (Figure 6). Reaction of 37 with acceptors 31, 38, and 39 with 1 equiv of AgClO4 as activator gave only a-mannopyranosides, while reaction of 37 with acceptors 31 and 40 with iodine (1 equiv) and TiClO4 (0.05 equiv) as activator gave b-mannopyranoside as the major product. OH OBn O O O O BnO BnO O OMe OH 32 31 Figure 5 complete stereoselectivity, indicating that the participation of the C-2 protecting group was not required for the b-selectivity. They have also exploited the differences in the reactivities of thioethyl glycosides and glycosyl phosphates to assemble the trisaccharide 36 (Scheme 6). OBn BnO BnO O P OBu OPiv + O O P Me O O 31 37 Me Me H Me SEt H 34 33 O Me OPiv O BnO BnO OBu OH S OH O O BnO BnO O OBn BnO O H 38 HO OBn O BnO BnO TMSOTf -78 °C OPiv O OPiv BnO BnO BnO SEt BnO BnO O OPiv O BnO BnO OBn O BnO Figure 6 O HO BnO O OPiv O In their synthesis of Bleomycin A2, Boger and Honda21 coupled the 3-O-carbamoyl-2,4,6-tri-O-acetyl-a-D-mannopyranosyl diphenylphosphate 41 with protected gulopyranoside 42 using 1.8 equiv of TMSOTf as activator to give exclusively the a-mannoside 43 (Scheme 7). 36 Scheme 6 AcO H2NOCO OAc AcO O ISSN 0936-5214 OBn O BnO O O P OPh + OBn OH OBn OPh The “Holy Grail” in carbohydrate chemistry has been the goal of synthesising b-mannopyranosides in a stereoselective and efficient manner. The b-mannose linkage exists widely in glycoproteins, which often exhibit interesting biological properties.17 The challenging problem for the formation of b-mannopyranosides derives from two factors. The first is the anomeric effect, which favours the axial glycosidic bond formation to give the amannopyranosides. The second factor is the existence of an axial C–O bond adjacent to the anomeric centre in mannose, which not only disfavours the equatorial approach of the acceptor, but also in the case of acyl protecting groups participates to promote the axial glycoside formation through anchimeric assistance. There are several methodologies developed for the synthesis of b-mannopyranosides,18 particularly the intramolecular aglycon delivery which tethers the glycosyl donor and the acceptor together on the same molecule.19 Synlett 2002, No. 1, 16–25 OMe 40 39 OBn OBn O BnO BnO 35 MeOTf, DTBP 0 °C OH OH O 41 42 TMSOTf CH2Cl2, 0 °C 93% AcO H2NOCO OAc AcO O OBn O BnO OBn O OBn 43 Scheme 7 Using our previous methodology, we prepared the phosphate 45 from 2,3,4,6-tetra-O-benzyl-D-mannopyranose © Thieme Stuttgart · New York ACCOUNT Glycosylation Based on Glycosyl Phosphates as Glycosyl Donors 44 and propane-1,3-diylphosphoryl chloride in 68% yield as a mixture of a and b anomers in a ratio of 19:1 (Scheme 8). We have reacted the phosphate 45 with a range of acceptors.22 HO HO HO 46 47 48 HO BnO BnO OH OO OBn BnO O Cl NHZ 44 BnO BnO CO2Me H N-methylimidazole NHZ O O O P O O O O CO2Me H 14 OBn BnO O OMe O OH P O OH 21 15 17 Me Me O H O HO H HO Scheme 8 49 Glycosylation experiments with Bu3SnOTf and TESOTf as the activators resulted in only small amounts of glycosides being isolated, 10–20%. However with 0.2 equiv of TMSOTf as activator, the glycosidation reaction with a number of acceptors led to the formation of mannosides that had approximate a:b ratio of 1:1. The use of a 1.5 equiv of TMSOTf as activator resulted in the formation of only the a-mannosides in good yields. The stereochemical integrity of the anomeric centre was assigned on the basis of the 13C-1H coupling constant for the anomeric proton, which in the case of the a-mannoside was in range of 165– 175 Hz whilst the b-mannoside had values ranging from 150–162 Hz (Figure 7).23 The interest in the preparation of S-glycosides has mainly arisen due to their increased stability towards hydrolysis by glycosidases with the premise that these may produce effective biological agents for the treatment of carcinomas and other disease states.24 Due to the availability of the mannosylphosphate 45 we proceeded to investigate the displacement of the phosphate group by thiol acceptors. As the nucleophilicity of thiols is greater than their oxygen counterparts reaction of mannosyl phosphate (predominantly a-anomer) with thiols should favour an SN2like process over an SN1-type reaction leading to the formation of b-linked mannosides. In the case of thiols that are non-planar and good nucleophiles, this is indeed the outcome of the reactions of 45 with thiols 55–58, where we have isolated the corresponding b-thiomannosides in b:a ratios greater than 9:1 (Figure 8). In the case of thiol 54 and the other two planar and less nucleophilic glycosyl acceptors 59 and 60, the stereoselectivity of the coupling reaction was invariably 1:1 for the resulting anomers.25 16 OH O AcO O O O HO O AcO AcO O OAc 50 31 OAc OAc OH O AcO AcO O AcO AcO OH OMe 18 OAc 51 OH OBn BnO O BnO HO O O O OMe 52 O 53 Figure 7 HS OMe HS HS O 54 55 56 HS SH 57 58 N SH SH S 26 Seeberger and co-workers examined the coupling reactions of mannosyl phosphates 61, 62, and 63 with three monosaccharide acceptors, 31, 32, and 64 under controlled reaction conditions (Figure 9). With phosphate 61, where a non-participating group was present in the C-2 position they found that the selectivity of the glycosylation reaction was strongly dependent on the nature of the glycosyl acceptor and the solvent used for the coupling re- H Me O 45 O 59 60 Figure 8 action. When phosphate 61 was coupled with the hindered acceptor 32 with 1.3 equiv of TMSOTf as activator, the bmannoside was preferentially formed (a:b, 1:3) in dichlo- Synlett 2002, No. 1, 16–25 ISSN 0936-5214 © Thieme Stuttgart · New York 22 ACCOUNT H. Vankayalapati et al. romethane, while in acetonitrile the a-mannoside was preferentially obtained (a:b, 5.5:1). In the case of the acceptor 31, its coupling with phosphate 61 showed a-selectivity in both dichloromethane and acetonitrile. Reactions of mannosyl phosphates 62 and 63, which contained the participating pivaloyl group in C-2 position with acceptors 31, 32, and 64 in the presence of 1.3 equiv of TMSOTf gave a-mannosides with complete selectivity. BnO BnO 25 (α:β 10:1) OAc O O BnO BnO O P O H3C AcO AcO OPh 62 OBn PivO O OBn O OAc OAc 66 (α:β 20:1) O P O O O OAc O O P OPh 67 (α:β 5:1) O BnO BnO OMe Figure 10 OH OPh O 32 63 O O P O OAc O O O P OPh 61 H3C O 65 (α:β 20:1) O OPh 45 (α:β 19:1) O O PivO O O P OPh O O P O O OAc O O TBSO BnO BnO O P O O OBn OAc BnO BnO O O AcO OBn BnO O OBn BnO O OBn H3C OH O O O O BnO BnO O OAc OAc OBn O O P O OAc O O CN 64 H3C O O OAc OAc Figure 9 68 To further investigate and explore the general scope of this type of chemistry and to further study the efficacy of the propane-1,3-diylphosphate as an anomeric leaving group, we prepared the acetyl protected glycosyl phosphates 65, 66, and 67 using the standard protocol (Figure 10). In this regard we investigated the reaction of phosphates 25, 45, 65, 66, and 67 with the acceptors allyltrimethylsilane26 and trimethylsilyl cyanide (TMSCN) for the preparation of C-glycosides (Scheme 9). The coupling reactions of phosphates 25 and 65 with allyltrimethylsilane proceeded smoothly in the presence of a catalytic amount of TMSOTf to afford the b-anomers as the major products, whereas in the case of phosphates 66 and 67, a-anomers were obained as the major products. Mannosyl phosphate 45 reacted with allyltrimethylsilane under the same conditions to give a 1:1 anomeric mixture. Glycosylation of phosphates 25 and 45 with trimethylsilyl cyanide using a catalytic amount of TMSOTf as activator showed no anomeric selectivity, giving a mixture of aand b-glycosyl cyanides. For phosphates 65, 66 and 67 with participating groups at C-2, their coupling with trimethylsilyl cyanide using a catalytic amount of TMSOTf produced stable intermediates, which resulted from the trapping of the acetoxonium ion by cyanide. However, exposure of these intermediate cyanoacetals to 1 equiv of TMSOTf gave preferentially a-glycosyl cyanides. Synlett 2002, No. 1, 16–25 ISSN 0936-5214 TMSOTf (cat.) -78 °C 66 (α:β 20:1) OEt OH 31 TMSCN Me TMSOTf (1 equiv) rt H3C O OAc OAc CN OAc 69 Scheme 9 To apply our methodology to the synthesis of some biologically important disaccharides, we prepared a-L-Fuc1,2-b-D-Galp and a-L-Fuc-1,3-b-D-Galp, each bearing a 4-methylumbellifery moiety attached at the anomeric centre of the D-galactose as a fluorescent label.27,28 The coupling of fucopyranosyl phosphate 66 and 1,3,4,6tetra-O-acetyl-a-D-galactopyranose 52 with 1.5 equiv of TMSOTf as activating agent gave exclusively the alinked disaccharide 70 in 63% yield. However, if a catalytic amount of TMSOTf was used, a complete reversal of anomeric selectivity was observed for the coupling of phosphate 66 and acceptor 52 to give a b-linked disaccharide (46%) together with some recovered starting material (39%). These results suggested that the a-linked fucoside 70 was the thermodynamic product, which was probably formed via an oxonium ion intermediate while the blinked disaccharide was formed via a SN2-like process or participation of the C-2 acetoxy group. The anomeric acetate group in 70 was deprotected and further treated with propane-1,3-diylphosphoryl chloride to afford the phosphate 72 as an anomeric mixture (a:b, 20:1). 4-Methylumbelliferone had been used extensively in the fluorimetry assay of glycosides, and its coupling with phosphate 72 in the presence of 0.2 equiv of TMSOTf yielded exclusively © Thieme Stuttgart · New York ACCOUNT the b-linked disaccharide 73. Further deprotection then gave the final disaccharide 74 (Scheme 10). OAc HO OAc O OAc O P O OAc O O OAc OAc OAc O O P O AcO TMSOTf (cat.) -78 °C, 83% 65 (α:β 20:1) 63% OAc OAc OAc OAc O OH OAc MeCN, 0 °C H3C 69% OAc OH O OTBDPS H3C O O OAc OAc OAc 70 O HO O O OO O O P O AcO O OH P O O O O N-methylimidazole H3C OAc H 3C OAc OAc OAc OAc 72 77 OH OAc OH O O AcO O O H3C OH O O OH H3C O O OH OH OH OAc OAc OAc O O O -78 °C, 56% O 1. Bu4NF, THF 2. NaOMe, MeOH O O TMSOTf (cat.) O OH O OAc OAc O 66 OTBDPS O O O TMSOTf (1 equiv) -78 °C, 52% 76 OAc O P O OAc O OAc OAc OH 71 OAc O 1. NaOMe, MeOH 2. TBDPSCl, Im, DMF 75 O NH3 (g) O HO O OAc AcO OAc OAc O O AcO H3C O AcO OAc O O OAc 52 OAc O O OH 66 (α:β 20:1) Cl OAc TMSOTf (1.5 equiv) O + O O AcO H3C 23 Glycosylation Based on Glycosyl Phosphates as Glycosyl Donors 73 78 Scheme 11 OH O NaOMe, MeOH 79% O HO O O syl phosphate 65 reacted with 1 equiv of mannoside 79 and a catalytic amount of TMSOTf as activator, the trisaccharide 80 was obtained in 43% yield with both new glycoside bonds having the b stereochemistry (Scheme 12). In another example, we used galactopyranoside 81 as the glycosyl acceptor, which was sequentially treated with 1 equiv of galactopyranosyl phosphate 65 and 1 equiv of O H3C OH OH O OH 74 Scheme 10 For the preparation of disaccharide 78, we used galactopyranosyl phosphate 65 as the starting material, and its coupling with 4-methylumbelliferone in the presence of 0.2 equiv of TMSOTf as activator gave the b-galactopyranoside 75 in 63% yield. After further deprotection and selective protection, the partially protected 4-methylumbellifery-b-D-galactopyranoside 76 was used as an acceptor to couple with fucopyranosyl phosphate 66 using 1.0 equiv of TMSOTf as activator. This resulted in the formation of the a-linked disaccharide 77 (52%), and a trace amount of another a-linked disaccharide attached on the C-2 position of the galactose. Further deprotection of 77 produced the a-L-Fucp-1,3-b-D-Gal(1)-4¢-methylumbelliferone (Scheme 11). We have also used these phosphates as glycosyl donors to react with minimally protected monosaccharide acceptors to prepare trisaccharides. When 2 equiv of galactopyrano- OAc OAc AcO OAc OH OO O O O P O + O HO O 65 79 OAc OMe OAc O O AcO OAc TMSOTf (0.2 equiv) -78 °C, 43% OAc OAc OO O O AcO O OAc 80 OMe Scheme 12 Synlett 2002, No. 1, 16–25 ISSN 0936-5214 © Thieme Stuttgart · New York 24 ACCOUNT H. Vankayalapati et al. rhamanopyranosyl phosphate 67 in the presence of a catatlytic amount of TMSOTf to give the trisaccharide 82 in 51% yield (Scheme 13). Me Me HO OAc HO H OAc O O O P O AcO OAc OAc OMe TMSOTf (0.2 equiv) BnO OH OH OMe 86 OMe 18 OH O O O H3C AcO AcO O P O O O O AcO AcO O OMe + AcO OBn 85 O -78 °C, 51% OAc O AcO AcO OAc OH H3C AcO AcO O O AcO 81 16 OH HO OAc O O H O + O HO 48 46 65 O H Me CO2Me O O O HO O OAc OAc O Me O 82 67 O OMe MeO Me OMe MeO OMe OH Scheme 13 87 Recently we have come back almost full circle to reinvestigate the glycosylation using furanosyl phosphates as glycosyl donors. Treatment of 2,3,5-tri-O-benzyl-D-arabinofuranose 83 with propane-1,3-diylphosphoryl chloride afforded the arabinofuranosyl phosphate 84 as a single a anomer (Scheme 14).29 Figure 11 BnO BnO O OO O BnO OH OBn 83 Cl P O BnO O N-methylimidazole 80% O BnO O P O O OBn 84 Scheme 14 We have found that the selectivity of the glycosylation with phosphate 84 very much depended on the nature of the glycosyl acceptor as well as the amount of TMSOTf as activator. The reaction of phosphate 84 with acceptors 16, 46, 48, and 85 in the presence of a catalytic amount of TMSOTf yielded predominantly b-arabinofuranoside (a:b, < 1:3), while with acceptors 18, 86, 87 and 88, the aarabinofuranosides were the major products (a:b, > 2:1) (Figure 11). In the case of the coupling between phosphate 84 and acceptor 85, increasing the amount of TMSOTf from catalytic amount to 1 equiv resulted in the reversal of the anomeric selectivity from b-arabinofuranoside (a:b 1:3) to a-arabinofuranoside (a:b 6:1). We have also observed that the b-arabinoside 89 could be treated with 1.5 equiv of TMSOTf under normal glycosylation conditions, and this resulted in its complete stereochemical inversion of the anomeric centre to give the a-arabinoside 90 (Scheme 15). Synlett 2002, No. 1, 16–25 ISSN 0936-5214 O O O TMSOTf (1.5 equiv) BnO OBn 89 BnO 88 CH2Cl2, 0 °C BnO OBn 90 Scheme 15 We have so far demonstrated that glycosyl phosphates are useful glycosyl donors, which can be used for the stereoselective synthesis of glycosides. Their applications complement the existing glycosylation methods available in glycosylation chemistry. References © Thieme Stuttgart · New York (1) Hanessian, S. Total Synthesis of Natural Products: The ‘Chiron’ Approach; Pergamon Press: Oxford, 1983. (2) Nicolaou, K. C.; Mitchell, H. J. Angew. Chem. Int. Ed. 2001, 40, 1576. (3) (a) Varki, A. Glycobiology 1993, 3, 97. (b) Raymond, A. D. Chem. Rev. 1996, 96, 683. (c) Sears, P.; Wong, C.-H. Chem. Commun. 1998, 1161. (d) Simanek, E. E.; McGarvey, G. J.; Jablonowski, J. A.; Wong, C.-H. Chem. Rev. 1998, 98, 833. (e) Sears, P.; Wong, C.-H. Angew. Chem. Int. Ed. 1999, 38, 2300. (4) Koenigs, W.; Knorr, E. Ber. Dtsch. Chem. Ges. 1901, 34, 957. (5) (a) Toshima, K.; Tatsuta, K. Chem. Rev. 1993, 93, 1503. (b) Boons, G. J. Tetrahedron 1996, 52, 1095. (c) Davis, B. G. J. Chem. Soc., Perkin Trans 1 2000, 2137. (6) (a) Caputto, R.; Leloir, L. F.; Cardini, C. E.; Paladini, A. C. J. Biol. Chem. 1950, 184, 333. (b) Leloir, L. F. Science 1971, 172, 1299. (c) Heidlas, J. E.; Williams, K. W.; Whitesides, G. M. Acc. Chem. Res. 1992, 25, 307. (7) Inazu, T.; Hosokawa, H.; Satoh, Y. Chem. Lett. 1985, 297. ACCOUNT Glycosylation Based on Glycosyl Phosphates as Glycosyl Donors (8) (a) Ueki, M.; Inazu, T. Bull. Chem. Soc. Jpn. 1982, 56, 204. (b) Ueki, M.; Inazu, T. Chem. Lett. 1982, 45. (9) Inazu, T.; Yamanoi, T. Chem. Lett. 1989, 69. (10) Yamanoi, T.; Inazu, T. Chem. Lett. 1990, 849. (11) Singh, G.; Tranoy, I. Carbohydr. Lett. 1998, 3, 79. (12) (a) Ramage, R.; Hopton, D.; Parrott, M. J.; Kenner, G. W.; Moore, G. A. J. Chem. Soc., Perkin Trans 1 1984, 1357. (b) Ramage, R.; Hopton, D.; Parrott, M. J.; Richardson, R. S.; Kenner, G. W.; Moore, G. A. J. Chem. Soc., Perkin Trans 1 1985, 461. (13) Vankayalapati, H.; Singh, G.; Tranoy, I. Chem. Commun. 1998, 2129. (14) Hashimoto, S.; Honda, T.; Ikegami, S. J. Chem. Soc., Chem. Commun. 1989, 685. (15) Vankayalapati, H.; Singh, G.; Tranoy, I. Tetrahedron: Asymmetry 2001, 12, 1373. (16) Plante, O. J.; Andrade, R. B.; Seeberger, P. H. Org. Lett. 1999, 1, 211. (17) Montreuil, J. Adv. Carbohydr. Chem. Biochem. 1980, 37, 157. (18) (a) Barresi, F.; Hindsgaul, O. In Modern Methods in Carbohydrate Synthesis; Khan, S. H.; O’Neill, R. A., Eds.; Harwood Academic: Amsterdam, 1996, 251. (b) Gridley, J. J.; Osborn, H. M. J. Chem. Soc., Perkin Trans 1 2001, 1471. (19) (a) Kim, G.; Stork, G. J. Am. Chem. Soc. 1992, 114, 1087. (b) Stork, G.; La Clair, J. J. J. Am. Chem. Soc. 1996, 118, 274. (c) Barresi, F.; Hindsgaul, O. J. Am. Chem. Soc. 1991, 113, 9376. (d) Barresi, F.; Hindsgaul, O. Synlett 1992, 759. (e) Barresi, F.; Hindsgaul, O. Can. J. Chem. 1994, 72, 1447. (f) Ennis, S. C.; Fairbanks, A. J.; Tennant-Eyles, R. J.; (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) 25 Yeates, H. S. Synlett 1999, 1387. (g) Ito, Y.; Ogawa, T. J. Am. Chem. Soc. 1997, 119, 5562. (h) Lergenmuller, M.; Nukada, T.; Kuramochi, K.; Dan, A.; Ito, Y.; Ogawa, T. Eur. J. Org. Chem. 1999, 1397. (i) Ito, Y.; Ohnishi, Y.; Ogawa, T.; Nakahara, Y. Synlett 1998, 1102. Yamanoi, T.; Nakamura, K.; Takeyama, H.; Yanagihara, K.; Inazu, T. Bull. Chem. Soc. Jpn. 1994, 67, 1359. Boger, D. L.; Honda, T. J. Am. Chem. Soc. 1994, 116, 5647. Vankayalapati, H.; Singh, G. Tetrahedron: Asymmetry 2001, 11, 125. (a) Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293. (b) Duus, J. Æ.; Gotfredsen, C. H.; Bock, K. Chem. Rev. 2000, 100, 4589. (a) Garegg, P. J.; Henrichson, C.; Norberg, T. Carbohydr. Res. 1983, 116, 162. (b) Dasgupta, F.; Garegg, P. J. Acta. Chem. Scand. 1989, 43, 471. Vankayalapati, H.; Singh, G. unpublished results. (a) Kobertz, W. R.; Betozzi, C. R.; Bednarski, M. D. Tetrahedron Lett. 1992, 33, 737. (b) Hung, S.-C.; Lin, C.C.; Wong, C. H. Tetrahedron Lett. 1997, 38, 5419. (c) Saleh, T.; Rousseau, G. Synlett 1999, 617. Vankayalapati, H.; Singh, G. Tetrahedron Lett. 1999, 40, 3925. Vankayalapati, H.; Singh, G. J. Chem. Soc., Perkin Trans.1 2000, 2187. Li, Y.; Singh, G. Tetrahedron Lett. 2001, 42, 6615. Synlett 2002, No. 1, 16–25 ISSN 0936-5214 © Thieme Stuttgart · New York