Semipreparative separation of intact hopanoids from Zymomonas mobilis

Hopanoids are an important class of molecules that play a structural and physiological role in the membrane processes of prokaryotic and plant cells. Studies on the function of hopanoids require milligram quantities but have been limited by current procedures for isolation and characterization: most separations have isolated only derivatized compounds of hopane in microgram quantities. Our method employs aminopropyl bonded-phase solid-phase extraction columns with sequential elution and silica semipreparative HPLC with isocratic elution. When applied to freeze-dried cells of Zymomonas mobilis, the procedure separated the three major hopanoids present (bacteriohopanetetrol, bacteriohopanetetrol-glucosamine, and bacteriohopanetetrol-ether) from the other lipid classes. Using the solid-phase extraction column, a fraction containing an average of 3.3 mg of bacteriohopanetetrol per gram dry weight of cells was recovered without any detectable impurities along with a fraction that contained a mixture of the other two hopanoids. After this mixture was subsequently purified by semipreparative HPLC, approximately 4.7 mg of bacteriohopanetetrol-glucosamine and 3.6 mg of bacteriohopanetetrol-ether per gram dry weight of cells could be separated and recovered.