Assembly of a pentasaccharide repeating unit corresponding to extracellular polysaccharide S-88
Having achieved success in synthesizing L-glycosyl fluoride from the corresponding D-sugars, our focus now shifts towards the application of this method in the synthesis of intricate oligosaccharides obtained fromPseudomonas ATCC 31554 extracellular polysaccharide (S-88). S-88 is a representative gellan which shows potential in food, chemical, and pharmaceutical industries due to its functional characteristics.[22] Furthermore, recent discoveries have revealed the probiotic potential of gellan oligosaccharides derived from the hydrolysis of S-88, as they have the capability to modulate gut flora in a manner that promotes human health.[23] S-88 is composed of a pentasaccharide repeating unit made up of a backbone of →4)-α-L-mannopyranosyl (L-Manp )-(1→3)-β-D-glucopyranosyl (Glcp )-(1→4)-β-D-glucuronic acid (Glcp A)-(1→4)-β-D-Glcp -(1→ and a branch of α-L-rhamnopyranosyl (Rhap ) appended to C3-OH of D-Glcp at the reducing end.[24] The occurrence of L-Manp moiety and of D-Glcp A residue linked to the sterically hindered C4-OH of D-Glcp makes S-88 an attractive, but synthetically challenging target. The pursuit of comprehending the intricate structure and functionalities of S-88 has driven our motivation to engage in chemical syntheses of S-88 oligosaccharides. Herein, we report the synthesis of a pentasaccharide fragment (7 ) corresponding to the repeating unit of extracellular polysaccharide S-88. The synthesis features the successful construction of challenging glycosidic linkage by which D-Glcp A is appended to D-Glcp and the efficient installation of L-mannosyl moiety with L-mannosyl fluoride as the glycosyl donor. L-mannosyl fluoride was prepared from D-galactose via a head-to-tail inversion strategy.
Highly efficient incorporation of uronic acid residues is crucial for assembly of oligosaccharides containing such units. Typically, two strategies are employed to accomplish this objective.[25] The first approach involves the postglycosylation oxidation strategy, wherein an oligosaccharide backbone is constructed before converting a sugar into its uronic acid form through oxidation. Despite additional protecting group manipulations required, the advantages of this approach lie in the higher reactivity of non-oxidized building blocks than the corresponding carboxylate counterparts, deceased side reactions associated with epimerization α to carboxylate, and β-elimination leading to the formation of 4-deoxy-hex-4-enopyranuronic acid.[26] The second is preglycosylation oxidation strategy, that is, directly using a uronic acid-based building block as glycosylating donor and acceptor. Its strength is to prevent oxidation event on a complex setting. However, the presence of carboxylate substituent imposes challenges to glycosylation reaction due to the withdrawing-electron effect of carboxylate resulting in decreased reactivity of the building blocks as glycosyl donor and acceptor.[27] Mindful of these considerations, we designed two retrosynthetic plans for the target pentasaccharide7 . As depicted in Scheme 3, we envisioned that the target molecule 7 could be obtained from the fully protected pentasaccharide 8 by a global deprotection. Glycan 8in turn was planned to assemble by glycosylation of L-mannopyranosyl fluoride 9 with tetrasaccharide either 10 or11 . Compound 9 could be traced back to readily available 1-phenyl-2-(β-D-C -galactossyl) ethanone12 [18]. Fluoride 9 is designed to possess a benzoyl at C2-OH to ensure the formation of 1,2-trans
Scheme 3 Retrosynthetic analysis of pentasaccharide fragment (7 ) of extracellular polysaccharide S-88
Scheme 4 Synthesis of L-mannosyl fluoride 9
Reagents and conditions: (a) NaBH4, MeOH, ice bath; (b) 2,6-lutidine, Tf2O, CH2Cl2, 70% over two steps; (c) MeONa, MeOH; (d) PhCH(OMe)2, CSA, 40°C, CH3CN; (e) BnBr, KOH, 18-Crown-6, THF, 72% over three steps; (f) OsO4, 2,6-lutidine, NaIO4, 1,4-dioxane/H2O; (g) NaBH4, MeOH, 73% over two steps; (h) BzCl, DMAP, pyridine; (i)p -TsOH·H2O, CH2Cl2/MeOH, 79% over two steps; (j) TBSCl, DMAP, pyridine; (k) BzCl, DMAP, pyridine; (l) 70% HF·pyridine, CH3CN, 76% for three steps; (m) TEMPO, BAIB, CH2Cl2/H2O, 93%; (n) Selectfluor, KF·2H2O, Ag2CO3, acetone/H2O, 88%. CSA = Camphorsulfonic acid; THF = Tetrahydrofuran; TBSCl =tert -Butyldimethylsilyl chloride.
glycosidic bonds through neighboring group participation. Given the strength and weakness of post- or preglycosylation oxidation strategy, tetrasaccharides either 10 or 11 embedded by GlcAp residue could be constructed by coupling reaction of disaccharide acceptor 13 with either uronic acid-based disaccharide donor 14 or the corresponding non-oxidized donors15 . These disaccharides 13 , 14 , and15 could be disconnected to D-glucosyl thioglycoside16 and L-rhamnosyl trichloroacetimidate (TCAI) 17 . Glucosyl thioglycoside 16 was designed to allow for direct incorporation of rhamnosyl residue at C3-OH while 4,6-O -benzylidene would facilitate the incorporation of carboxylate group and the glycosylation of C4-OH by the hydrolysis of acetal functionality and subsequent differentiation between the primary alcohol and the secondary one. The presence of 2-O -benzoyl (Bz) substituent in 16 could enable anchimerically assisted glycosylation leading to β-glucosidic linkage formation. The incorporation of a spacer 6-amino-hexanoxyl at the reducing end would provide feasibility for conjugation of 7 with biomolecules such as carrier protein. Orthogonal levulinoyl (Lev) group on the disaccharide fragment either 14 or 15 allows for its chemoselective removal and ensuing glycosylation at that site.
Our synthesis began with the preparation of rare L-mannosyl fluoride9 following a head-to-tail switch strategy (Scheme 4). Similar to the synthesis of 6a and 6b , C -galactoside12 was converted to vinyl C -glycoside 18 in 70% yield over two steps. To differentiate the 4-OH and the 6-OH from the 2,3-diol for late-stage equipment of benzoyl group at the C4-OH and the formation of carboxylic acid, 18 was converted into19 in 72% yield over three steps involving deacetylation, the benzylidene protection of 4,6-diol, and the benzylation of 2,3-diol. The oxidative cleavage of C═C double bond in 19 followed by the reduction of aldehyde group led to the introduction of hydroxymethyl at the anomeric position, providing 73% of 20 over two steps. Benzoyl protection of 20 coupled with hydrolysis of benzylidene produced diol 21 in 79% overall yield. Selective benzoyl protection of C4-OH was smoothly realized leading to 22 in 76% overall yield through TBS protection of the primary hydroxy group, the benzoylation of the remaining secondary hydroxy group, and the cleavage of TBS ether with hydrogen fluoride-pyridine complex. Uronic acid23 , prepared by oxidation of 22 in 93% yield, was exposed to Ag2CO3-mediated decarboxylative fluorination to give rise to the required α-L-mannosyl fluoride 9 in 88% yield as the sole product.
Having successfully prepared the L-mannosyl fluoride 9 , we turned to constructing the tetrasaccharide 10 . To this end, disaccharide acceptor 13 was first made. As shown in Scheme 5a,
Scheme 5 Synthesis of disaccharide acceptor 13 as well as donors 14 and 28
Reagents and conditions: (a) TMSOTf, -40 °C, CH2Cl2, 4 Å MS, 87%; (b) Et3SiH, TFA, 5 Å MS, CH2Cl2, 91%, (c) HO(CH2)6N3, NIS, AgOTf, 4 Å MS, -20 °C, CH2Cl2, 70%; (d) BF3·Et2O, -40 °C, CH2Cl2, 4 Å MS, 77%; (e) TCCA, H2O, ice bath, acetone; (f) o -hexynylbenzoic acid, DCC, DMAP, THF, 70% over two steps. TMSOTf = Trimethylsilyl trifluoromethanesulfonate; MS = Molecular sieve; NIS =N -Iodosuccimide; AgOTf = Silver triflate; TCCA = Trichloroisocyanuric acid; DCC = Dicyclohexylcarbodiimide; n -Bu =n -butyl.
Glucose-derived alcohol 16 [27] was glycosylated with Bz-masked L-rhamnosyl TCAI17 [28]. The coupling reaction proceeded smoothly and delivered the desired disaccharide 24 in 81% yield in CH2Cl2 at -40oC under the catalysis of 0.1 equiv of TMSOTf. Reductive ring- opening of the benzylidene in 24 with TFA and Et3SiH in the presence of 5 Å molecular sieves resulted in the formation of 25 with C4’-OH free in 91% yield. Reactivity-based chemoselective glycosylation of 6-azido-1-hexanol[29] with 25 produced disaccharide glycoside 13 in 70% yield under the combined promotion of NIS and AgOTf. The reaction left C4’-OH intact, which is ready for sugar chain elongation at this site.
With 13 in hand, the application of preglycosylation oxidation strategy was first explored in construction of tetrasaccharide10 . We therefore embarked on the preparation of uronic acid-based disaccharide 14 . As outlined in Scheme 5b, BF3·Et2O-catalyzed glycosylation of26 [30] with27 [31] supplied 77% of disaccharide14 in CH2Cl2 in the presence of 4 Å MS. At this stage, the coupling reaction of 14 and13 was explored. As tabulated in Table 1, we were disappointed to find that the reaction under the promotion of NIS with either TfOH or Lewis acids such as AgOTf and TBSOTf supplied the desired tetrasaccharide 10 in the best yield up to 23% (Table 1, entries 1–5). Gold-catalyzed glycosylation of challenging nucleophiles with glycosyl ortho -alkynylbenzoate donors has
Table 1 Optimization of glycosylation reactions of acceptor13 with donor 14 or28