The Study Of Lipase-catalyzed Synthesis Of Lauroyl Mannose In Organic Solvent

Saccharide fatty acid esters, which have important physiological activity, have been widelyused as nonionic surfactants in food, cosmetic, and pharmaceutical industries. The lipase-catalyzedsynthesis of the esters through reverse hydrolysis (condensation reaction) was carried out in organicsolvent with low water content, and under reduced pressure and /or in the presence of a desiccant,which was adopted to remove water, one of the condensation products, from the reaction system toshift the reaction toward synthesis.In this context, lauroyl mannose was synthesized through condensation of lauric acid andmannose using an immobilized lipase Chirazyme L-2, c.-f. C2 in acetone in the presence ofmolecular sieves. The conditions for lipase-catalyzed synthesis of lauroyl mannose were optimizedafter evaluating the separation method for lauroyl mannose. Furthermore, the mathematic model forpredicting the equilibrium conversion for the synthesis of lauroyl mannose was proposed.The dehydration mechanism of molecular sieves for lipase-catalyzed reaction systems inorganic solvent was firstly studied. The adsorption isotherms of water onto various molecular sieveswere obtained in three different kinds of organic solvents, acetonitrile, 2-methyl-2-propanol, and2-methyl-2-butanol, respectively, in the absence or presence of the substrates, mannose, sucrose andlauric acid. The adsorption isotherms of water onto various molecular sieves, in the presence ofboth mannose and lauric acid, were similar to that onto molecular sieves in the absence of thesubstrates, which just lined between the isotherm of molecular sieves when one substrate alone,lauric acid or mannose, existed. The dehydration ability of 3A molecular sieve is better than 4Amolecular sieve, and the dehydration ability of 5A molecular sieve is too weak to be not appropriatefor the lipase-catalyzed synthesis.Analyse condition for the thin Layer Chromatography – TLC was confirmed, 3 μl of thereaction mixture were applied to silica gel G plates and hexane: ethyl acetate (1:1, v/v) was used asmobile phase. The reaction mixture was detected by spraying with 5% sulfuric acid ethanol andheated at 120°C for 20 min. The retention factor( Rf,) of the mono-lauroyl mannose di-lauroylmannose and its isomer is 0.72,0.56,0.38,respectively.Then,the condition of TLC is applied to silicagel column chromatography for separating and purifying the lauroyl mannose: 5ml of the reactionmixture was applied to silica gel column(60-100mesh)and hexane: ethyl acetate (1:1,v/v) was usedas mobile phase. The flow rate was 18ml/h, the eluent, 1tube/10min, was collected and the sampletubes were detected with TLC, collected the products. High performance liquid chromatography(HPLC) was used to analyse the lauroyl mannose. Chromatogram with high resolution was obtainedusing the column of Zorbax SB-C18 (5μm , 250 ×4. 6 mm) with a UV detector at 210nm and themobile phase of methanol: water 90:10v/v at a flow rate of 1ml/min at 30°C.The separated productsof the lauroyl mannose which were monolauroyl mannose and two kinds of dilauroyl mannose inacetone, were identified by electrospray ionization Mass spectrometry.The condition for lipase-catalyzed synthesis of lauroyl mannose in organic solvent wasoptimized. Acetone was confirmed as the lipase-catalyzed reaction solvent, and 3A molecular sieveas desiccant. The molar ratio of lauric acid to mannose was 3:1, and the amount of molecular sievewas 80mg/mL. The condensation of mannose and lauric acid was conducted with vigorouslyshaking in a water bath at 50 degree for 72 hours. The conversion of the lauroyl mannose was48.8%. The effect of the addition method of molecular sieves was also studied. And the conversion60% of the lauroyl mannose was achieved by batch addition of molecular sieves. The characters oflauroyl mannose were also studied, the hydrophilic lipophilic balance (HLB) of the mono-lauroylmannose di-lauroyl mannose and its isomer were 15.16, 15.13and15.12, respectively. The criticalmicelle concentration of the dilauroyl mannose isomer was 3.22×10-5mol/L.The reaction equilibrium constant of monolauroyl mannose and dilauroyl mannose weredetermined by HPLC. And the adsorption isotherm of water on 3A molecular sieve in acetone wasalso determined. The mathematic model was proposed for predicting the equilibrium conversion forthe synthesis of lauroyl mannose, which based on the apparent reaction equilibrium constant, theadsorption isotherm of water on the molecular sieve, the solubility of mannose in the solvent, andthe mass balance with respect of water.Validity of the model was examined for the synthesis of monolauroyl mannose, dilauroylmannose I and its isomer dilauroyl mannose II in acetone with molecular sieves 3A1/16. Thepredicted conversions for total mannose esters and dilauroyl mannose I agreed well with theexperimental values except for the cases where the molar ratio of the lauric acid to the mannose washigh. The predicted conversions for monolauroyl mannose was higher than the experimental values,while the predicted conversions for dilauroyl mannose II was lower than the experimental values, itindicated that the probability of lauric acid reacting with monolauroyl mannose to produce dilauroylmannose II would be high in acetone in the presence of molecular sieve 3A.The optimized technology for the synthesis of lauroyl mannose obtained with HPLC was quitesimilar to that optimized with silica gel column chromatography. The same parameters for synthesiswere obtained by the analysis methods, the molar ratio of lauric acid to mannose was 3:1, and theamount of molecular sieve was 80mg/mL.