A Highly Ecient Approach to the Synthesis of Complex α -Glycosyl Phosphosaccharides

: A highly efficient and stereoselective approach to the synthesis of biologically important and complex α -glycosyl phosphosaccharides (GPSs) has been disclosed, employing direct gold(I)-catalyzed glycosylation of the weakly nucleophilic phosphoric acid acceptors. The broad substrate scope is demonstrated with more than 45 examples, including glucose (Glc), xylose, glucouronatose, galactose (Gal), mannose (Man), rhamnose (Rha), fucose (Fuc), 2-N 3 -2-dexoxymannose (ManN 3 ), 2-N 3 -2-dexoxyglucose (GlcN 3 ), 2-N 3 -2-dexoxygalactose (GalN 3 ) and unnatural carbohydrates. Moreover, the glycosyl phosphotriester prepared herein was successfully applied to the one-pot synthesis of a GPS from Leishmania donovani , and an effective preparation of a trisaccharide diphosphate of GPS fragments from Hansenula capsulate via iterative elongation strategy is realized.


Introduction
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 ( Figure 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 ( Figure 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 bis-thiourea, the armed GPSs proceed an SN-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.
Nevertheless, the resulted phosphate anions render product incompatible with follow-up or late-stage chemical modifications to increase molecular complexity and diversity 20 . The alternative approach employing phosphoramidite displays great success in installation of phosphoester, yet is rarely applied to synthesis of anomeric GPS probably due to issues of diastereo-selectivity and undesired oxidative cleavage reaction (Scheme 1B) 2 [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 glyconjugates 33 , along with other natural products 34 , by exploiting the compatibility with oxygen-containing functionalities 35  the glycosylation of exceedingly poor nucleophile of phosphoric acid remains elusive, which entails mild conditions free from competitive nucleophilic species. We herein 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 oxygen-containing 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 (Scheme 1D). Moreover, the anomeric effect of phosphoric acid in the glycosylation reaction is also exploited to direct the αselectivity under the present weakly acidic condition 38,39 .

Results and Discussions
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 good yield (88%) and α-selectivity (α/β = 2.8/1, entry 1). Thus, detailed optimizations were subsequently conducted by tuning reaction temperature, solvent and additives. As depicted in Table 1, lowering temperature was not effective (83%, α/β = 2/1, entry 2); varying the anion in the catalysis with -OTf diminished α-selectivity (entry 3); switching to solvent of ether and additive of Ph3P=O did not provide satisfactory results though they were effective in the case of alcohol acceptor (entry 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 α-one, but compromise the overall yield (62%, entry 7). In contrast, heterogenous acidic H + resin did not affect the diastereoselectivity (entry 8). Hence, the homogeneous Lewis acid of gold(I) catalysis was anticipated to activate the armed glycosyl phosphate under an elevated temperature. Indeed, after complete glycosylation of 2a at 0 o C for 0.5 h, keeping the mixture at 60 o C for 2 h in non-coordinating ClCH2CH2Cl (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 o C and 95 o 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 an excellent yield of 93% (entry 12).

Table 1. Optimizations of the Reaction Conditions a
Next, we wondered whether this gold (I)-catalyzed glycosylation strategy was amenable to various glycosyl donors outfitted with different protecting groups or configurations (Table 2). First, donor 1b, of which the BnOCH2 moiety is omitted compared to 1a, delivered the expected xylosyl phosphate 3b in good yield and diastereoselectivity (78%, 11/1) by utilizing the aforementioned protocol. Replacing BnOCH2 moiety with electron-withdrawing groups such as AcOCH2 and COOMe 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 higher temperature of 100 o C with a good yield of 87%.

Table 3. The reaction scope of various acceptors of phosphoric acids
Furthermore, one of phosphoglycopeptides (4zb), which are found in parasites, was concisely assembled via glycosylation of threonyl phosphate 2g in a stereospecific manner, albeit in a low yield of 60% because of limited solubility of 2g in DCE 48 (Scheme 2).  generated a trisaccharide 8 consisting of two phosphotriester funcitonalities in good yield (70%), which was subsequently converted to trisaccharide donor 9 in a procedure similar to that for 7. As a late-stage chemical modification on this trisaccharide, a third glycosylation reaction between donor 9 and 3azidopropanol was performed to install a linker with the two present phosphotriesters intacted. Finally, the resulting trisaccharide was globally deprotected under mild conditions to afford an amino-linker tethered trisaccharide diphosphate 10.

Conclusion
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,