Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate

Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.


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Step 2: S15 (16.9g, 0.03 mol) was dissolved in pyridine (40 mL). Benzoyl chloride (7.0 mL, 0.056 mol) were added. The reaction mixture was stirred capped at room temperature overnight at which point excess MeOH was added and stirred. The mixture was concentrated in vacuo and the residue was taken up in EtOAc (150 mL), washed with brine (100 mL), saturated NH 4 Cl solution (100 mL) and brine (100 mL). The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The crude material (20.3 g) was purified by silica gel chromatography using the same protocol used for the purification of S15 to give S16: 18.9 g, 29 mmol, 99+ % yield. TLC: 2: in THF (500 mL) until all solids dissolved, then the solution was purged with argon for 30 minutes.
Step 2: Crude 14 from Step 1 was dissolved in a HOAc/H 2 O solution (4:1, 100 mL) and placed in a heating bath at 63 °C for 1 hour. The mixture was concentrated in vacuo and azeotroped with toluene (3 x 20 mL) and hexanes (2 x 20 mL) to give crude diol 15, which was used as is in the next step.
Step 3: The crude diol 15 was dissolved in pyridine (75 mL Step 1: Triol 20 (6.46g, 6.13 mmol, 1 eq) was azeotroped with acetone (3 x 20 mL). Acetone/2,2dimethoxypropane (4:1, 100 mL) was added, followed by pTsOH•H 2 O (50 mg). The reaction mixture was stirred at room temperature for 20 minutes and then diluted with EtOAc (250 mL). The mixture was washed with saturated NaHCO 3 (100 mL), followed by brine (100 mL). The mixture was then dried over Na 2 SO 4 , filtered, and concentrated. The product was chased with hexane and placed under vacuum to yield (6.5 g) product 21 as a syrup which was used as is in the next step.
Step 2: The substrate 21 from Step 1 (6.1 mmol, 1 eq) was azeotroped with toluene (3 x 10 mL) then dissolved in anhydrous DCM (150 mL) under N 2 . Dess-Martin periodinane (5.17g, 12.2 mmol, 2 eq) was added in 3 portions over 5 minutes. The reaction mixture was stirred at room temperature and became homogeneous within 2 minutes of the addition of each aliquot. After 1.5 hours, wet DCM (1 mL) was added and after 2-3 minutes a small amount of white precipitate began to form. More wet DCM (10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted with DCM (200 mL), filtered through a Celite pad, washed with DCM (100 mL) and brine (200 mL), dried over Na 2 SO 4 , filtered, and concentrated. The crude ketone was used as is in the next step. TLC: 1:1 hexanes:EtOAc, R f 0.62.
Step 3: The crude ketone from Step 2 was taken up in a DCM/MeOH solution (1:1, 150 mL), and NaBH 4 (1.04g, 27.5 mmol, 4.5 eq) was added under N 2 sweep. The reaction mixture was stirred at room temperature for 10 minutes and then concentrated in vacuo to dryness. The residue was partitioned between EtOAc (200 mL) and saturated NH 4 Cl (200 mL). The organic layer was washed with brine (100 mL), dried over Na 2 SO 4 , filtered, and concentrated. The crude product 22 was used as is in the next step. TLC: 1:1 hexanes:EtOAc, R f 0.33.
Step 4: The crude alcohol 22 from Step 3 was taken up in pyridine (24 mL) and cooled to 0 °C in an ice bath. BzCl (2.4 mL, 20.7 mmol) was added, followed by DMAP (10 mg). The mixture was stirred overnight under N 2 and allowed to come to room temperature. Precipitate formed within 15 minutes. The reaction mixture was diluted with EtOAc (200 mL); quickly washed with HCl solution (pH 3, 2 x 100 mL), saturated NaHCO 3 (100 mL), and brine (200 mL); dried over Na 2 SO 4 , filtered, and concentrated. The residue was chased with water (1 x 10 mL) and hexanes (4 x 10 ml) to remove the residual pyridine and EtOAc before proceeding directly to the next step. TLC: 1:1 hexanes:EtOAc, R f 0.82.
Step 2: The crude acetonide from Step 1 (700 mg, 0.239 mmol, 1 eq) was azeotroped with toluene (3 x 5 mL) then taken up in anhydrous DCM (5 mL) under N 2 . Dess-Martin periodinane (202 mg, 0.478 mmol, 2 eq) was added in one portion and stirred at room temperature (monitored by TLC: 2:1:1 heptane:TBME:DCM). After 2.5 hours, wet DCM (4 drops) was added, and at 5.5 hours an additional 10 drops of wet DCM were added. After stirring overnight a final aliquot of wet DCM (1 mL) was added to drive the reaction to completion. Once complete, the reaction mixture was diluted with a MeOH/DCM solution (1:1, 20 mL total) and cooled in an ice bath.
The crude material from the benzoylation reaction was taken up in 80 % HOAc (10 mL) and stirred at 54 °C for 22 hours before concentrating in vacuo and chasing with hexanes (2 x 15 mL). The residue was partitioned between EtOAc (100 mL) and saturated NaHCO 3 . The EtOAc layer was washed with brine (75 mL), dried over Na 2 SO 4 , filtered, and concentrated. Silica gel chromatography (40 g column) using a heptane:EtOAc step gradient (10-50 % EtOAc) gave pure diol 32 (320 mg, 70 % from S22) and a lower R f by-product (20 mg), which was identified by MALDI-MS and NMR as the desired product less one benzoate ester. TLC: 1:1 heptane:EtOAc, R f 0.60.
The mixture was stirred under N 2 overnight. The solution was concentrated then partitioned between EtOAc (100 mL) and saturated NH 4 Cl (100 mL). The aqueous layer was washed with DCM (100 mL), dried over Na 2 SO 4 , filtered, and concentrated. 1 H/ 13 C NMR analysis of the crude material showed a complete loss of Bz groups and a persistence of linker with Cbz group intact. MALDI MS revealed trace amounts of a material consistent with a mono-de-O-benzyl derivative in addition to the desired product.
34 (48 mg, 17.0 µmol, 1 eq) was dissolved in THF (6 mL, inhibitor-free, anhydrous) and transferred to a hydrogenator flask. H 2 O (3 mL, Omni Trace Ultra) was added with stirring. The solution became cloudy. 1N HCl (17 L, 1 eq.) was added, and the reaction mixture was purged with N 2 . 10 % Pd•C (100 mg) was added. The flask was secured to a hydrogenation manifold and purged free of O 2 by repeated evacuation and filling with N 2 followed by the saturation of H 2 using evacuation and filling.
The reaction mixture was stirred at an initial H 2 pressure of 30 psi for 23 hours, then filtered through an Acrodisc CR (25 mm, 0.45 µm PTFE membrane), concentrated, and analyzed by 1 H NMR.
Nearly all aromatic signals were gone, but approximately one Bn group equivalent remained. The reaction mixture was resubjected to the hydrogenation conditions for another 20 hours, filtered through a 0.45 µm PTFE Acrodisc, washing with H 2 O (5 mL). The filtrate was concentrated in vacuo and chased with hexane. Final purification was achieved via size exclusion chromatography using Sephadex G-10 (2.5 cm diameter x 14 cm high, eluted with pH 4 H 2 O, 4 mL fractions).
Carbohydrate-containing fractions were combined and lyophilized to give the desired hexasaccharide antigen 1 as an amorphous white cake (16.2 mg, 67 %).