First and Stereoselective Synthesis of an α-(2→5)-Linked Disaccharide of 3-Deoxy-d-manno-oct-2-ulosonic Acid (Kdo)

Resistance of bacterial pathogens toward antibiotics has revived interest in lipopolysaccharide (LPS) motifs as potential therapeutic targets. The LPS of several pathogenic Acinetobacter strains comprises a 4,5-branched Kdo trisaccharide containing an uncommon (2→5)-linkage. In this contribution the first stereoselective glycosylation method for obtaining an α-Kdo-(2→5)-α-Kdo disaccharide in good yield is highlighted. The synthetic approach used for accessing this linkage type will allow for future studies of the immunoreactivity associated with this unique bacterial Kdo inner core structure.


manno-oct-2-ulopyranosid)onate (8)
NaH (0.18 g, 4.60 mmol) was added in small portions at 0 °C to a solution of 6 (0.35 g, 1.15 mmol) in anhydrous DMF (14 mL). After complete addition the mixture was stirred at 0 °C for 30 min. Then, BnBr (0.55 mL, 4.60 mmol) was added dropwise and after 3 h at ambient temperature the mixture was cooled to 0 °C and diluted with anhydrous CH 2 Cl 2 . Anhydrous MeOH (1.0 mL) was added dropwise and after completed addition the mixture was immediately subjected to extraction with CH 2 Cl 2 and aq.
To improve the yield, 6 (22.0 mg, 0.072 mmol) was dissolved in anhydrous DMF (1.0 mL) and treated with NaH (17 mg, 0.431 mmol) at ambient temperature for 30 min. After dropwise addition of BnBr (51 µL, 0.431 mmol), the mixture was stirred for 5 h before it was cooled to 0 °C and diluted with anhydrous CH 2 Cl 2 . MeOH (0.1 mL) was added dropwise followed by immediate extraction with CH 2 Cl 2 and aq. NaHCO 3 . The organic phase was further washed with brine, dried (MgSO 4 ), filtered and concentrated. Column chromatography (SiO 2 , toluene/EtOAc 15:1) provided benzyl ester 8 (13.3 mg, 33%) and methyl ester 7 (16.1 mg, 46%) as a mixture. Thus, by increasing the amount of NaH and BnBr from 2 eq. to 3 eq./ free hydroxyl group and performing deprotonation with NaH at ambient temperature instead of 0 °C monobenzylation could be avoided and the yield was increased from 59% to 79%.
S-6 0.75 mmol) and anhydrous DMF (1.05 mL, 13.55 mmol) were added successively and the mixture was kept at 60 °C for 14 h. The mixture was cooled to rt, then diluted with EtOAc and washed sequentially with 1 M aq. HCl, aq. NaHCO 3 , aq. Na 2 S 2 O 3 (5 w/w%) and brine. The organic layer was dried (MgSO 4 ), filtered over Celite and concentrated. The crude mixture was purified by column chromatography (SiO 2 , toluene/EtOAc 9:1→5:1) yielding 12 (0.04 g, 8%) as the faster migrating compound followed by 11 (0.21 g, 52%). Yields were calculated for 2 steps and were based on the relative amounts of 7 and 8 in the mixture.
To obtain the methyl ester 11 from benzyl ester 12, the latter compound (22 mg, 0.036 mmol) was treated with a 0.1 M methanolic sodium methoxide (0.43 mL, 0.043 mmol) in anhydrous MeOH (2 mL) under argon for 2 h. Neutralization of the solution by addition of ion exchange resin DOWEX 50 (H + form), filtration and concentration of the filtrate gave a residue which was subjected to elution on silica gel (toluene/EtOAc 5:1) affording the methyl ester 11 (19 mg, 98%) S-7
After cooling to -60 °C, BF 3 .Et 2 O (11.9 µL, 0.058 mmol, ~ 46% BF 3 basis) was added dropwise and the mixture was kept at -60 °C for 15 min. The mixture was warmed up to -40 °C quickly and kept at this temperature again for 15 min. The procedure was repeated at a temperature of -20 °C and -10 °C.
After 15 min at each temperature a sample was taken, which

S-11
was immediately added to a cold solution of triethylamine in CH 2 Cl 2 . TLC analysis after each step showed, that the donor had been converted already at -60 °C (Fig. S4). Thus, a much higher reactivity was observed than for the acetylated pendant described recently. S2 The reaction mixture was diluted with CH 2 Cl 2 and washed with aq. sat. NaHCO 3 solution, aq. Na 2 S 2 O 3 (5 w/w%) solution and water successively. The organic layer was dried (MgSO 4 ), filtered and concentrated. The crude product was purified by column chromatography (SiO 2 , toluene/EtOAc 40:1) affording 18 (15.9 mg, 70%) in sufficient purity for further reactions.
After addition of aq. NaHCO 3 (5 mL) the mixture was filtered over a pad of Celite and rinsed with CH 2 Cl 2 . The filtrate was subjected to phase separation and the organic phase was thoroughly washed successively with aq. Na 2 S 2 O 3 (5 w/w%) and water. The organic phase was dried (MgSO 4 ), filtered and concentrated. Purification by column chromatography (SiO 2 , toluene/EtOAc 50:1) provided 18 (11.7 mg, 74%).

Addition of base
The addition of triethylamine as base (1.5 eq/ eq donor 16) during glycosylation drastically reduced the reactivity of the donor. With BF 3 .Et 2 O as promotor, the temperature had to be raised to 0°C to get conversion. With Cp 2 ZrCl 2 /AgClO 4 as promotor couple no reaction took place in the presence of triethylamine even after 2 d. For determination of the anomeric configuration of 19, latter compound (ca. 6 mg) was dissolved in anhydrous pyridine (2 mL) and treated with acetic anhydride (0.2 mL) at ambient temperature over night. Excessive reagent was destroyed at 0 °C by addition of anhydrous MeOH (1 mL) and after 10 min the mixture was coevporated with toluene (3x). Column chromatography (SiO 2 , n-hexane/EtOAc 1:1) of the crude residue afforded the known methyl (propan-2-yl 4,5,7,8-tetra-O-acetyl-3-deoxy-α-Dmanno-oct-2-ulopyranosid)onate S2 verifying the α-configuration of 19.
During addition, the color of the solution turned orange and finally to a pale yellow. After complete addition the mixture was stirred at -30 °C for further 5 min. Then, the solution was poured onto an icecold aq. sat. NaHCO 3 solution (10 mL) and was quickly extracted with EtOAc (40 mL). The aqueous phase was once again washed with EtOAc (10 mL) and the combined organic phases were washed successively with ice-cold aq. Na 2 S 2 O 3 (5 w/w%) solution and brine. Drying (MgSO 4 ), filtration and swift concentration afforded a crude product which was purified by column chromatography (SiO 2 , nhexane/

S-17
MeOH and the solvent was removed under reduced pressure. The residue was dissolved in water (1.0 mL) and was treated with 0.1 M aq. NaOH (1.0 mL) at 0 °C for 2 h and at ambient temperature for 4 h. The solution was made neutral by adding DOWEX 50 resin (H + form), followed by filtration and freeze-drying of the filtrate. The residue was desalted on a PD-10 column (H 2 O) to afford 24 as a colorless amorphous solid (5.6 mg, ca. 90%; minor impurity present; Fig. S7).