Enzymatically Regioselective Synthesis Of Lauryl Mannoses

Saccharide fatty acid esters (SFAEs) are nonionic, digestible, absorbable, biodegradable, and nontoxic amphiphilic derivatives, which have been widely used as surfactants and emulsifiers in food, cosmetic, and pharmaceutical industries. The physicochemical properties of SFAE depends on the fatty acid, the sugar moiety, the degree of substitution, and its chemical structure. Enzyme-catalyzed condensation is superior to the chemical synthesis because of the direct use of unmodified substrates, and moderate reaction conditions. But both of them have low regioselectivities in SFAE synthesis.In this study, regioselective synthesis of lauryl mannoses catalyzed by Novozym 435 was achieved and the physicochemical properties of lauryl mannoses were sudied. Based on the batch reactor study, the reaction rules and key factors influencing the mono- and diesters regioselective syntheses were investigated. Different reaction systems were designed and built for the selective synthesis of mono- or dilauryl mannoses, and the optimum reaction parameters for these systems were determined. The physicochemical properties of lauryl mannoses, such as water solubilities, interfacial properties and Millard activities were studied.The components of reaction solution were analyzed and separated. The reaction solution was analyzed by HPLC using a Sunfire-C18 column (4.6×250 mm, Waters, USA) eluted with methanol/water (95:5, v/v) at 1 ml/min, and detected by a Waters 2420 evaporative light scattering detector (ELSD). ELSD conditions were optimized at drift-tube temperature 45 oC; sprayer temperature 36 oC; carrier gas pressure 20 psi; and gain 1. The reaction solution was applied to a silica gel column (100 to 200 mesh, 100×600 mm) and then eluted with the mixture of n-hexane/ethyl acetate/methanol by a linear programming. The elution gradients were n-hexane/ethyl acetate (50/50, v/v, 4000 ml), n-hexane/ethyl acetate (40/60, v/v, 3000 ml), n-hexane/ethyl acetate/methanol (20/70/10, v/v, 4000 ml), n-hexane/ethyl acetate/methanol (20/40/40, v/v, 3000 ml) and 100 % methanol (2000 ml). The flow rate of the eluent was 10 ml/min, and the eluent, 1 tube/10 min, was collected and monitored by thin-layer chromatography (TLC). Fractions containing the same product were combined together. The concentrated solution was applied to a preparative HPLC with ELSD and a Sunfire-C18 column (5μm, 19×150 mm, Waters, USA) to purify the products. The methanol/water eluant (90:10, v/v), was used at the flow rate of 8.0 ml/min. The loading amount was 200μl. The purified products were identified by FT-IR, MS, 1H NMR and 13C NMR. And they were 6-O-lauryl mannose (ME), 3,6-di-O-lauryl mannose (D 3,6), 4,6-di-O-lauryl mannose (D 4,6), and 1,6-di-O-lauryl mannose (D 1,6).The batch reactor was used to synthsize lauryl mannose. The optimum parameters were achieved by single factor experiments. The optimum parameters for monolauryl mannose were as follow: mannose (75 mmol/L), the molar ratio of lauric acid to mannose 5:1, lipase (15 g/L), and 3 ? molecular sieves (10 g/L) in 5 ml acetone at 50°C for 72 h; the optimum parameters for dilauryl mannoses: mannose (75 mmol/L), the molar ratio of lauric acid to mannose 5:1, lipase (15 g/L), and 3 ? molecular sieves (80 g/L) in 5 ml acetone at 50°C for 120 h. Highest equilibrium conversions were 33 % and 21 % for monoester and diesters, respectively.A novel Circulating fluidized bed bioreactor (CFBBR) was employed for continuous and selective synthesis of monlauryl mannoses by lipase-catalyzed condensation of D-mannose and lauric acid. The highest equilibrium conversion of monoesters of 30 % (the average yield of monolauryl mannose was 59.26 g/(d·L)) for first 5 days and 25 % for after 2 days with no diester detected all the time were achieved. And the optimum parameters were as follow: lauric acid (80 mmol/L), saturated mannose, feed rate (1.0 ml/min), lipase (6 g), expansion rate of the bed (1.6) at 50 oC for 7.5 days.A novel biphasic system, simultaneous reaction-extraction system (SRE), consisting of two immiscible organic solvents, acetonitrile and n-hexane, was employed for the selective synthesis of dilauryl mannoses by lipase-catalyzed condensation of D-mannose and lauric acid. The highest equilibrium conversion of diesters of 51 % and the total conversion of lauryl mannoses of 76 % were achieved at the n-hexane/acetonitrile ratio of 1:1, the molar ratio of lauric acid to mannose of 4:1, 60 g/L molecular sieves and 5 g/L lipase at 50 oC and 150 rpm for 72 hour in 15 ml SRE system.At 25 oC, monolauryl mannose was higher in the solubility and lower in hydrophile-lipophile balance (HLB) value than dilauryl mannnoses. The paremeters, critical micelle concentration (CMC),γCMC, maximal surface excess (Γmax), minimum area per molecule (Amin), critical packing parameter (CPP) and the Gibbs free energy of adsorption (ΔGm) showed that dilauryl mannoses would favor the formation of micelles.The foamabilities, foam stabilities, emulsifying activities and emulsion stabilities of different concentrations of lauryl mannoses at 25 oC were compared. Monolauryl mannose was better in foamabilities, emulsifying activities, emulsion stabilities. The foaming property and emulsifying property would be better while mixing monoester and diester together.Maillard reaction activities of mannose with L-cysteine and lauryl mannoses with L-cysteine were evaluated by headspace solid phase microextraction (HS-SPME) method combined with GC/MS. 1,6-di-O-lauryl mannose exhibited the lowest maillard reaction activity, compared with 6-O-lauryl mannose, 3,6-di-O-lauryl mannose and 4,6-di-O-lauryl mannose.