Stereoselective gold(I)-catalyzed approach to the synthesis of complex α-glycosyl phosphosaccharides

Glycosyl phosphosaccharides represent a large and important family of complex glycans. Due to the distinct nature of these complex molecules, efficient approaches to access glycosyl phosphosaccharides are still in great demand. Here, we disclose a highly efficient and stereoselective approach to the synthesis of biologically important and complex α-glycosyl phosphosaccharides, employing direct gold(I)-catalyzed glycosylation of the weakly nucleophilic phosphoric acid acceptors. In this work, the broad substrate scope is demonstrated with more than 45 examples, including glucose, xylose, glucuronate, galactose, mannose, rhamnose, fucose, 2-N3-2-deoxymannose, 2-N3-2-deoxyglucose, 2-N3-2-deoxygalactose and unnatural carbohydrates. Here, we show the glycosyl phosphotriester prepared herein was successfully applied to the one-pot synthesis of a phosphosaccharide from Leishmania donovani, and an effective preparation of a trisaccharide diphosphate of phosphosaccharide fragments from Hansenula capsulate via iterative elongation strategy is realized.

G lycosyl phosphosaccharides (GPSs) represent a large and important family of complex glycans, which are ubiquitously distributed in bacteria, yeasts, protozoan parasites and animals, and exhibit numerous bio-functions including bacterial infections, cell adhesive, immunoresponse, and antimicrobial ( Fig. 1b) [1][2][3][4][5] . The GPSs consist of anomeric glycosyl phosphates in which the anomeric position of one constituent glycan was linked to another one mainly by α-type phosphodiester linkage (Fig. 1a). In the process of carbohydrate metabolism, the constituent glycosyl phosphates (GPs) are significant intermediates 6 . Synthetically, protected GPs have been utilized as effective glycosyl donor reagents 7 , and under the catalysis of bisthiourea, the armed GPSs proceed an S N -2 glycosylation pathway 8 . Despite the significance of GPSs, efficient approaches to access GPSs are rather limited: isolation from cell culture hardly affords homogeneous sample and chemical synthesis remains challenging due to the character of complex structure. Accordingly, efficient methods of constructing homogeneous GPSs and GPs are still in great demand.
One-step and direct glycosylation of phosphate acceptors with glycosyl donors represents a convergent and concise method for the synthesis of α-GPSs, which does not require oxidation transformation (Fig. 2C). This glycosylation strategy to access α-GPSs was previous realized by utilizing glycosyl trichloroacetimidate as donor and the weak nucleophile of phosphoric acid as acceptor under the action of strong acid 24 , and later, by a panel of donors with different leaving groups (e.g., SPh, 3-methoxypyridin-2-yl, pentenyloxy) [25][26][27][28][29][30][31] . The efficiency of these glycosylation reactions with phosphates as acceptors remains unmet: (1) the yield was deteriorated when strong acid was used to realize α-stereoselectivity 24 , (2) stoichiometric base was applied to preserve the entity of GPs, but leading to poor 1,2-cis stereoselectivity 28 , (3) only a handful of complex disaccharide GPSs were accessed by using the direct glycosylation strategy with phosphate anion as acceptor 25 .
Catalytic glycosylation methods have emerged as an appealing approach to the synthesis of carbohydrates, which feature less promoter and waste, and high efficiency 32 . Among those, alkynylphilic gold(I) catalysis has been extensively applied in the syntheses of numerous complex glycans and glycoconjugates 33 , along with other natural products 34 , by exploiting the compatibility with oxygen-containing functionalities 35 . Especially, glycosyl donor with ortho-alkynylbenzoate as leaving group first introduced by Yu and coworkers can glycosylate a variety of acceptors 36,37 . Nevertheless, the glycosylation of exceedingly poor nucleophile of phosphoric acid remains elusive, which entails mild conditions free from competitive nucleophilic species.
In this work, we disclose a stereoselective and general approach to the synthesis of α-GPs and α-GPSs via a gold(I)-catalyzed glycosylation method with glycosyl ortho-alkynylbenzoate as donor and weakly nucleophilic phosphoric acid as acceptor. While the alkynylphilicity of gold(I) catalysis has been widely investigated and applied, the Lewis acid property when oxygencontaining functionalities is activated by gold(I) remains much less explored. Herein, both the alkynylphilicity and weak acidity of gold(I) catalyst are capitalized on to initiate the glycosylation reaction and promote epimerization to α-anomer, respectively (Fig. 2D). Moreover, the anomeric effect, which is conferred by the weak nucleophile of phosphoric acid in the glycosylation reaction, is also exploited to direct the α-selectivity under the present weakly acidic condition 38,39 . The present gold(I)-catalyzed glycosylation method facilitates the efficient synthesis of more than 45 complex glycosyl phosphates, one-pot synthesis of GPS from Leishmania donovani, and iterative elongation of a trisaccharide diphosphate of GPS fragments from Hansenula capsulate. Mechanistic studies are investigated to indicate the dual role of Ph 3 PAuNTf 2 in the glycosylation-epimerization process, in which Ph 3 PAuNTf 2 not only triggers the glycosylation reaction, but also subsequently promotes the epimerization of the α/β products to enrich the α one.

Results
Reaction optimization. To test the validity of this proposal, tetrabenzyl glucoside 1a was examined with a structurally simple acceptor of phosphoric acid dibenzyl ester 2a (Table 1). Initial experiment gave promising results with a good yield of α/β mixture (88%) and α-selectivity (α/β = 2.8/1, entry 1). Thus, detailed optimizations were subsequently conducted by tuning reaction temperature, solvent and additive. As depicted in Table 1, lowering temperature was not effective (83%, α/β = 2/1, entry 2); replacing the anion of Ph 3 PAuNTf 2 with − OTf diminished α-selectivity (entry 3). The ether solvent and additive of Ph 3 P = O, which direct α-selectivity when alcohols are used as acceptors, did not lead to satisfactory results in the present reaction (entries 4-6) 11 . Gratifyingly, the diastereoselective ratio was raised to 10/1 by an added HOTf (0.1 equiv.), which is supposed to thermodynamically equilibrated β-anomer to the αone, but compromise the overall yield (62%, entry 7). The strong and heterogenous acidic H + resin did not result in an improvement in diastereoselectivity as that of HOTf (entry 8). Then, the homogeneous and weak Lewis acid of gold(I) catalysis under an elevated temperature was anticipated to trigger the epimerization to enrich α-anomer and maintain a high yield. Indeed, after complete glycosylation of 2a at 0°C for 0.5 h, keeping the mixture at 60°C for 2 h in non-coordinating ClCH 2 CH 2 Cl (DCE) for anomerization produced the α-anomer in good selectivity (α/ β = 9/1) without deteriorating yield (87%, entry 9). Further elevation of temperature (75°C and 95°C) resulted in a drop of yield or decomposition of product (entry 10, 11). Fortunately, increasing the equivalence of donor 1a to 1.5 relative to acceptor 2a (1.0 equiv.) reached an exceptional ratio of 16/1 in 62% yield which was calculated based on donor (entry 12). Interestingly, decreasing the equivalence of donor to 1.0 relative to acceptor (1.0 equiv.) significantly lowered the yield and α-selectivity (entry 13). While prolonging the reaction time for epimerization from 2 h to 3 h gave higher α-selectivity of 20/1, the yield dropped to 57% (entry 14). Notably, the yields of α/β mixtures were herein reported owing to that the α/β mixture was not separable by utilizing flash silica gel column chromatography, and the yields were calculated based on the donor which was used in excess (1.5 equiv.).
Reaction scope of donors. Next, we wondered whether this gold (I)-catalyzed glycosylation strategy was amenable to various glycosyl donors outfitted with different protecting groups or configurations (Fig. 3). First, xylosyl donor 1b, of which the BnOCH 2 moiety is omitted compared to 1a, delivered the expected xylosyl phosphate 3b in a good diastereoselectivity (11/1) and 55% yield of α/β mixture calculated based on the donor (1.5 equiv.) by utilizing the aforementioned protocol. The electron-withdrawing groups such as AcOCH 2 of 6-O-acetyl-glucosyl donor 1c and COOMe of glucuronic acid methyl ester donor 1d may greatly reduce the reactivity of donor, thereby causing difficulty in epimerization. Luckily, the 6-O-Ac product (3c) could be reached in a diastereoselective ratio of 12/1 merely at room temperature, of which α-selectivity is presumably dominated by anomeric effect 38,39 . For the other one equipped with COOMe, the product (3d) can be epimerized to enrich α-anomer (9.7/1) under a higher temperature of 100°C with a yield of 58%.
Consequently, this strategy was extended to a variety of donors including Gal, Man, ManN 3 , Rha, Fuc, GlcN 3 , GalN 3 and unnatural carbohydrates (Fig. 3). For the production of those with highly anomeric effect and dominated by steric hindrance, such as galactosyl phosphate (3e and 3e'), mannosyl phosphate x weakly acidic condition x high D-selectivity x post-modifications available x broad substrate scope and good yield xsuitable for one-pot elongation

D) This work:
A) The H-phosphonate method:  (3i), an operationally simple procedure at room temperature was effective to access desired products in highly diastereoselective manners. However, switching to phenyl tetrabenzyl-1-thio-mannoside which was preactivated with p-TolSCl and AgOTf, followed by addition of 2a, gave only byproduct 3p derived from intramolecular cyclization between 2-O Bn and anomeric position (please see SI) 40 . Among those, 3 g represents constituent unit derived from capsule polysaccharide of Neisseria meningitidis A 41 . The straightforward syntheses of extremely unstable diphenyl phosphate 3e' 8 and 3 f' demonstrate the utility of this strategy for directly glycosylating weakly nucleophilic phosphoric acid. In comparison, only 7% yield was obtained when galactosyl trichloroacetimidate was used as donor in the presence of TMSOTf (0.1 equiv.) at 0°C. Moreover, 3 f can be obtained on a 1.2 g scale with low catalytic loading of Ph 3 PAuNTf 2 (0.5 mol%) albeit in slightly decreased diastereoselectivity (17/1) and yield (58%) 42 . The deprotected form of 3 f might serve as replacement therapy for the disease of congenital disorder of glycosylation type Ia which is under clinical trial 43 . Interestingly, 1,2-cis αfucosyl phosphate 3i was obtained even with neighboringparticipating Bz situated at 2-O, demonstrating the strong anomeric effect in the case of phosphoric acid as acceptor. However, the diastereoselectivity can be reversed by adding extra base of iPr 2 NEt (0.2 equiv.) to form phosphate anion as acceptor.
Because of the distinct nature of N 3 substituent in comparison with OBn, formation of 1,2-cis α-D-glycosamine glycosidic bond remains elusive in the case of alcohols as acceptors [14][15][16][17] . Although the azido substituted glycosyl phosphates are resistant to epimerization at high temperature, donors of tribenzyl GlcN 3 and GalN 3 underwent smoothly coupling reactions with good diastereoselective ratios of 8.6/1 (3j) and 7.3/1 (3k) which might find utility in the syntheses of Lipid A or other phosphosaccharides 2,44 Derivatization and mimicking of natural glycosyl phosphates emerge as attractive tools to elucidate molecular mechanism of glycosyltransferases and discover novel therapeutic reagents 45,46 . Herein, a panel of fluorine-substituted α-GPs (3l-3o) were readily assembled via gold(I)-catalyzed glycosylation approach. Notably, the highly α-selective outcomes are in stark contrast to the reported results of fluorine-directed glycosylation with alcohol acceptors 47 .
Global deprotection reactions. Although the prepared glycosyl phosphotriesters are rather labile toward acid, the corresponding anions readily prepared through hydrogenolysis exhibit remarkable inertness toward acid. Thus, the deprotection sequence for these glycosyl phosphotriesters could be carried out through a two-step protocol: removal of benzyl group of benzyl phosphate and global deprotection of other protecting groups (Fig. 6). The phosphotriester 3a and 3e were hydrogenated in the presence of Et 3 N to produce quantitatively intermediate compounds of glycosyl phosphomonoester, which were then subjected to global deprotection of all other benzyl groups even in the presence of acidic HCOOH to afford cleanly the product 5 and 6. Similarly, the glycosyl phosphodiester 4c were directly deprotected under the acidic hydrogenation condition to furnish 7 in excellent yield (90%). For the benzoyl protected 4d, one additional step of deacetylation by using hydrazine after hydrogenation reaction was required to release the bis-glycosyl phosphoester 8 in high yield (89%).
One-pot glycosylation. While one-pot glycosylation protocol emerges as versatile strategy to synthesize complex oligo/polysaccharides of which the units are tethered by acetal linkages 49-51 , this strategy is not applicable to the assembly of conventionally synthesized bis-glycosyl phosphodiester which incorporate reactive functionality of phosphate anion 20 . Gratifyingly, bis-glycosyl benzyl phosphotriesters readily prepared in our system could serve as attractive substrates for one-pot glycosylation, and described in Fig. 7 is an example, in which linker-tethered 9 was assembled in one pot via gold(I)-catalyzed glycosylation reaction and a follow-up orthogonal coupling reaction promoted by NIS and TMSOTf. The chirality of phosphorus atom can be eliminated by converting OBn to O − , generating a single stereomer (10, 97%) derived from phosphosaccharide of Leishmania donovani 1 .
Iterative elongation. Finally, the utility of this gold(I)-catalyzed one-step approach to synthesis of GPSs was further illustrated by iterative elongation of phosphomannosyl fragments from Hansenula capsulate (Fig. 8) 52 . First, condensation of donor 1q and   SPh was converted to ortho-alkynylbenzoate as leaving group via two steps. Next, condensation of 11 and 2i generated a trisaccharide 12 consisting of two phosphotriester funcitonalities in 47% yield based on donor 11 (1.5 equiv.), which was subsequently converted to trisaccharide donor 13 in a procedure similar to that for 11. As a late-stage chemical modification on this trisaccharide, a third glycosylation reaction between donor 13 and 3-azidopropanol was performed to install a linker with the  The epimerization reaction with various acids. c The proposed mechanism. In red are the formed glycosidic bonds. In blue are the phosphoric acid and the phosphate moiety in product. In pink are the reaction temperatures. In yellow is the gold(I) catalysis. In orange is the curve of α/β ratio versus reaction time.
two present phosphotriesters intact. Finally, the resulting trisaccharide was globally deprotected under mild conditions to afford an amino-linker tethered trisaccharide diphosphate 14.
Mechanistic studies. To probe the role of Ph 3 PAuNTf 2 in the epimerization reaction, the epimerization process to enrich αanomer was monitored at elevated temperature (60°C) by HPLC. As depicted in Fig. 9, the ratio of α/β increased along with the time under the catalysis of Ph 3 PAuNTf 2 to give the α/β ratio of 12/1 in approximately 3 h (Fig. 9a). Further prolonging the epimerization time can improve the α/β ratio to 18/1 (entry 2, Fig. 9b). Interestingly, in the presence of sterically hindered base (2,6-di-tertbutyl-4-methylpyridine) which is supposed to not coordinate the gold(I) catalyst, the epimerization process was substantially impeded to give a ratio of 5.8/1 (entry 3, Fig. 9b). Other Lewis acid promoted epimerization afforded the same α/β selectivity as that when Ph 3 PAuNTf 2 was utilized ( Fig. 9b and Supplementary  Fig. 1). On the basis of these results, we speculated the acid was the real specie to trigger the epimerization process, and the gold(I) catalyst probably acted as an acid reservoir which can slowly release acid through possible disproportionation reaction to generate strong acidic Au 3+ at elevated temperature ( Supplementary  Fig. 2) 54 . The strong Lewis acid Au 3+ itself, or the H + which could be generated by reaction of Au 3+ with trace of H 2 O in the reaction system could promote the epimerization process.
In conclusion, we have developed a highly efficient and stereoselective approach to the synthesis of GPSs by employing gold(I)-catalyzed glycosylation of phosphoric acid acceptors. The efficiency of this protocol was demonstrated by its universal application in preparing more than 45 complex GPSs, one-pot synthesis of linker-tethered GPS from Leishmania donovani, and an effective preparation of trisaccharide diphosphate from Hansenula capsulate via iterative elongation. Because of its exceptionally broad substrate scope, high α-selectivity and inertness of phosphotriester toward chemical manipulations in comparison to phosphodiester, this strategy will offer new opportunities to create complex phosphosaccharides and install diverse phosphosaccharides on bioactive molecules.