Synthesis of Chrysogeside B from Halotolerant Fungus Penicillium and Its Antimicrobial Activities Evaluation

Chrysogeside B, a natural cerebroside, was efficiently synthesized from commercial feedstocks. The bioassays showed that compounds 4, 5 and 6 exhibited enhanced biological activities compared Chrysogeside B. Further studies revealed that free hydroxyl groups and glycosidic bond have significant impact on the antimicrobial activities. The synthesis of Chrysogeside B and analogues designed to allow identification of the features of this glycolipid required for recognition by tested bacteria and Hela cells is described.

3 diethyl ether and THF were freshly distilled after dried by Lithium aluminum hydride. Unless otherwise stated, all reagents were commercially available and were used without purification.
Organic solutions were concentrated under reduced pressure on a rotary evaporator or an oil pump.

2-Hexyl-2,3-dihydrofuran 7
n-Butyllithium solution (54.00 mL of a 2.4 M solution in hexane, 129.60 mmol) was added dropwise to a solution of anhydrous THF and 2.3-dihydrofuran (9.69 g, 120.00 mmol) at -78 °C in the atmosphere of nitrogen. The solution was stirred for 30 min at -78 °C, and then warmed to 0 °C and maintained for 2 h. Then the reaction mixture was allowed to cool to -30 °C, 1-iodohexane (14.46 mL, 96.00 mmol) was added and stirred for 2 h. The resulting mixture was warmed to room temperature. After the reaction was completed by TLC detection, saturated aqueous ammonium chloride (50 mL) was added to quench the reaction, and the product was extracted with ethyl ether (3 × 50 mL). The combined organic phase was washed by saturated sodium chloride solution (3 × 100 mL), dried over sodium sulfate and concentrated under vacuum to get colorless transparent oil (14.80 g) as compound 7, which was directly used in the next step.

(E)-4-methyldec-3-en-1-ol 8
Methylmagnesium bromide solution (68.00 mL of 3 M in toluene, 204.00 mmol) was added to a mixed solution of bis(triphenylphosphine)nickel(II) dichloride (2.23 g, 3.40 mmol) and anhydrous toluene under the protection of nitrogen, and then the mixture was stirred at room temperature for 30 min. After compound 7 (14.80 g) was added, the solution was heated to reflux for 1 h. 1 When the reaction was completed based on TLC detection, saturated aqueous ammonium chloride solution (50 mL) was added to quench the reaction, and the product was extracted with ethyl ether (3 × 50 mL). The combined organic phase was washed by saturated sodium chloride solution (3 × 100 mL), dried over sodium sulfate and concentrated under vacuum. The residue was purified using silica gel chromatography (8% ethyl acetate in hexanes) to give a colorless transparent oil (13.00 g, 80% yield for two steps), which was confirmed as (E)-4-methyldec-3-en-1-ol 8 by 1 H NMR. The NMR spectra of obtained compound 8 matched completely with those for the known product. 1  (E)-1-Iodo-4-methyldec-3-ene 9 Iodine (10.28 g, 40.50 mmol) was added in batches to a solution of imidazole (4.09 g, 60.00 mmol), triphenylphosphine (12.59 g, 48.00 mmol) indichloromethane (250 mL) at room temperature. Then the mixed solution was allowed to cool to 0 °C under stirring. The compound 8 (6.80g, 40.00 mmol in DCM 40mL) was dropped during 30 min. Then the reaction solution was warmed to room temperature, and stirred for 2 h. 1 After the reaction was completed by TLC detection, the resulting mixture was concentrated to 20 mL under vacuum, and the residue was purified using silica gel chromatography (10% ethyl acetate in hexanes) to give the dark red oil. Then the obtained dark red oil was dissolved in 50 mL of ethyl ether, washed by saturated sodium thiosulfate solution (3 × 50 mL), dried over sodium sulfate, and concentrated under vacuum to give (E)-1 -iodo-4-methyldec-3-ene 9 as a colorless transparent oil (9.86 g, 88% yield). The NMR spectra of obtained compound 9 matched completely with those for the known product. 1

α-D-Glucopyranose pentabenzoate21
α-D-Glucose (2.00 g, 11.12 mmol) in pyridine (24.00 mL) was cooled to 0 °C, benzoyl chloride (4.00 mL, 69.00 mmol) was added dropwise in 10 min, and then the solution was warmed to room temperature and was stirred over 16 h. After the reaction was completed by TLC detection, the solution mixture was diluted with ethyl acetate, washed with hydrochloric acid (1 M) (3 × 50 mL), water (3 × 50 mL), dried over sodium sulfate, and concentrated under vacuum. 6 Recrystallizing the residue with solvents acetone and water gave the compound 21 as a white solid (5.55 g, 71% yield).

2,3,4,6-Tetra-O-benzoyl-α-D-glucopyranosyl bromide 22
Compound 21 (4.00 g, 5.71 mmol) was dissolved in DCM (20 mL) and cooled to 0 °C. To this solution hydrobromic acid (10 mL, 33% in acetic acid) was added, and was stirred at room temperature for 6 h. After the reaction was completed by TLC detection, the mixed solution was diluted with diethyl
After the reaction was completed by TLC detection, the reaction mixture was filtered through Celite, and solid was rinsed with DCM. Organic phase was combined together, dried over sodium sulfate and concentrated under vacuum. 6 The residue was purified using silica gel chromatography (25% ethyl acetate in hexanes) to give the compound 27 as a white solid (1.65 g, 84% yield for two steps).

(2R,3E)-2-Hydroxynonadec-3-enoic acid 34
Lithium hydroxide (1.72 g, 103 mmol) and compound 33(1.38 g, 4.05 mmol, in THF 32 mL)in order was add to a solution of methanol/water (10 mL/20 mL) at 0 °C. Then the solution was warmed to room temperature, and stirred for 4 h. After the reaction was completed by TLC detection, hydrochloric acid (1 M) was added to adjust pH 1-2, and the product was extracted with ethyl ether (3 × 50 mL).The combined organic phase was washed by saturated sodium chloride solution (3 × 100 mL), dried over sodium sulfate and concentrated under vacuum to get colorless transparent oil (1.10 g), which was directly used in the next step.

(2R,3E)-2-Acetoxynonadec-3-enoic acid 35
Acetic anhydride (1.00 mL) was added to a mixed solution of compound 34 (1.10 g, 3.00 mmol), DCM (30 mL), pyridine (7.5 mL) at room temperature and stirred for overnight. After the reaction was completed by TLC detection, hydrochloric acid (1 M) was added to adjust pH 1-2, and the product was extracted with DCM (3 × 50 mL).The combined organic phase was washed by water (3 × 100 mL), dried over sodium sulfate and concentrated under vacuum to get colorless transparent oil (1.05 g), which was directly used in the next step.