Total synthesis of tetraacylated phosphatidylinositol hexamannoside and evaluation of its immunomodulatory activity

Tuberculosis, aggravated by drug-resistant strains and HIV co-infection of the causative agent Mycobacterium tuberculosis, is a global problem that affects millions of people. With essential immunoregulatory roles, phosphatidylinositol mannosides are among the cell-envelope components critical to the pathogenesis and survival of M. tuberculosis inside its host. Here we report the first synthesis of the highly complex tetraacylated phosphatidylinositol hexamannoside (Ac2PIM6), having stearic and tuberculostearic acids as lipid components. Our effort makes use of stereoelectronic and steric effects to control the regioselective and stereoselective outcomes and minimize the synthetic steps, particularly in the key desymmetrization and functionalization of myo-inositol. A short synthesis of tuberculostearic acid in six steps from the Roche ester is also described. Mice exposed to the synthesized Ac2PIM6 exhibit increased production of interleukin-4 and interferon-γ, and the corresponding adjuvant effect is shown by the induction of ovalbumin- and tetanus toxoid-specific antibodies.

defined by the MM2 field displays axial/pseudoaxial C3, C4 and C5 substituents and a pseudoequatorial substituent at C2 1 . The approach of the diol 16 from the back portion of the oxocarbenium ion is blocked by C4 and its benzyloxy substituent, whereas the -face is quite crowded. While both O4 and O6 of diol 16 have opportunities in attacking the -face of the oxocarbenium ion from the front portion, O6 has a much wider free area of approach than O4.
The O4 attack may be hindered by the proton attached to the neighboring C2 and may place O6 in an unfavoured position near the proton attached to C5. It is worth mentioning that as a result of the pseudoaxial/pseudoequatorial orientations of the substituents at C2 and C5 of the oxocarbenium ion, their corresponding protons are situated at nearly similar locations with respect to C1 and O5 if 4 H 3 -another likely conformation-is adopted by the oxocarbenium ion.                                                                 Supplementary Figure 109. 13 C and DEPT NMR spectra of compound S17.  The hydrogen multiplicities of carbon peaks were determined using DEPT-90 and DEPT-135 experiments, the spectra of which were herein provided together with the power-gateddecoupled 13 C NMR spectrum.

4-Methylphenyl 2,3,4-tri-O-benzyl-6-O-tert-butyldiphenylsilyl-1-thio--D-
mannopyranoside (7). The mixture of the triol 6 (60 g, 0.12 mol) and benzyl bromide (44.9 mL, 0.38 mol) in DMF (600 mL) was cooled to 0 °C in an ice-bath. NaH (60% oil dispersion, 16.5 g, 0.69 mol) was then added in five portions over 1 h. After gradually warming up to room temperature, the solution was stirred for an additional 2 h. The reaction mixture was poured carefully in ice-water with vigorous shaking, followed by extraction with ethyl acetate. The combined organic layer was washed with cold water and brine. After

4-Methylphenyl 2,3,4-tri-O-benzyl-1-thio--D-mannopyranoside (8). Method A: To
the solution of TBDPS compound 7 (22 g, 27.67 mmol) in CH 2 Cl 2 /MeOH (1/2, 275ml), ptoluenesulfonic acid (PTSA, 5.79g, 30.44 mmol) was added and the solution was stirred at room temperature for 12 h. After the completion of reaction, triethylamine was added to quench the reaction and the solvent was evaporated under reduced pressure. The resulting residue was dissolved in EtOAc and washed successively with saturated solution of NaHCO 3(aq) , water, and brine. After drying over MgSO 4 , the organic layer was concentrated under reduced pressure and the residue was purified by column chromatography (ethyl acetate/hexanes = 1/7) to furnish the 6-alcohol 8 (14.2 g, 92%). Method B: Compound 12 (1 g, 1.64 mmol) and 2-naphthaldehyde (0.27 g, 1.73 mmol) were dissolved in CH 2 Cl 2 (20 mL) and cooled to -78 °C under nitrogen atmosphere. After 5 min, Et 3 SiH (0.39 mL, 2.49 mmol) and temperature was gradually raised to -40 °C over a period of 1 h. After stirring at -40 °C for another hour, the reaction flask was directly moved to an ice-water bath. DMF (15 mL), benzyl bromide (0.58 mL, 4.91 mmol), and sodium hydride (60% oil dispersion, 0.24 g, 10.0 mmol) were subsequently added to the stirring mixture. The reaction mixture was gradually warmed up to room temperature and stirred for 4 h. The temperature was again lowered to 0 °C and water (30 mL) was added slowly. After 5 mins, the aqueous layer was removed by cannulation, DDQ (1.86 g, 8.19 mmol) was introduced to the reaction flask, and the reaction was stirred at room temperature for 15 h. The resulting mixture was filtered through a Celite plug and the filtrate was washed successively with saturated NaHCO 3(aq) and brine. After
After stirring for 5 min, Et 3 consecutively added and the resulting solution was continuously stirred for 48 h. TBAF (1 M in THF, 0.9 mL, 0.9 mmol) was added to the mixture, the reaction flask was warmed up to room temperature, and the solution was stirred for 12 h. The whole mixture was filtered through Celite, the filtrate was diluted with CH 2 Cl 2 (10 mL) and washed with water and brine.

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The resulting mixture was then carefully poured into an ice-cooled saturated solution of ammonium chloride (50 mL) and the target compound was extracted with ethyl acetate. The combined organic layer was washed with water and brine, dried over MgSO 4
The solvent was evaporated under reduced pressure and the resulting crude residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/5) to afford a long chain E/Z olefinic acid mixture.
The mixture was warmed up to room temperature and stirred for an additional 12 h. After S148 filtration through Celite, the organic solution was washed with saturated NaHCO 3(aq) and brine, dried over MgSO 4 , and concentrated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/10) to give compound 29 (354 g, 88%

2-O-Benzoyl-3,4,6-tri-O-benzyl-D-mannopyranose (S14).
To the solution of the thioglycoside 14 (10 g, 15.13 mmol) in acetone (1.0 L) at 0 °C, water (5 ml, 0.30 mol) and NBS (4.04 g, 22.7 mmol) were added and the resulting mixture was stirred at the same S150 temperature for 30 min. The reaction was quenched with 10% Na 2 S 2 O 3(aq) and the solvent was evaporated under reduced pressure. The residue obtained was dissolved in ethyl acetate and washed successively with 10% Na 2 SO 3(aq) and brine. The resulting solution was dried over MgSO 4 and concentrated under reduced pressure. The crude product was purified by using a short silica gel column (ethyl acetate/hexanes = 1/4) to furnish the hemiacetal S14 The solution of compound S14 (3 g, 5.438 mmol) in CH 2 Cl 2 (30 mL) was cooled in an icewater bath and CCl 3 CN (5.43 mL, 54.1 mmol) and diazabicycloundec-7-ene (162 μL, 1.08 mmol) were sequentially added under nitrogen atmosphere. The reaction was gradually warmed up to room temperature and stirred for 5 h. Then, the mixture was diluted with CH 2 Cl 2 (20 mL) and washed with water and brine. After drying over MgSO 4 , the solvent was removed under reduced pressure. The residue was passed through a short silica gel column (ethyl acetate/hexanes = 1/4) to obtain compound 30 (3.71 g, 98%). 1

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The mixture was, then, cooled to 0 °C, diluted with ethyl acetate and quenched with water.
After extraction with ethyl acetate, the combined organic layer was washed with water and brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (ethyl acetate/hexanes = 1/5)  BnO S16 S156