| Enantioselectivity is one of important characterizations displayed by lipasesin organic synthesis, which prominent status in organic chemistry wasenhanced by the foundation and improvement of unaqueous enzymology. Thevast applications of lipases in unaqueous reaction systems results in that moreand more compounds with high enantiopurity can be got in markets, such aschiral drugs, pesticides, liquid crystals and fragrance materials.Some advancement has been achieved in lipase-catalyzed kinetic resolution,but challenges has been existed in this field: the use efficiency of substratesare usually less than 50%, only one of two enantiomers can be obtained inresolution and the catalytic activity of enzymes in organic reaction systemsare very lower than that in the reaction systems with water. In order to resolveaforementioned problems, the work is carried out in this thesis.1. Preliminary investigation on lipase-catalyzed resolution of2-octanol in unaqueous systemWe studied the kinetic resolution of 2-octanol reacting with vinyl octanoatecatalyzed by PSL. In our investigation, it is found that 99% ee (S)-2-octanolcan be obtained by controlling reaction conversion more than 62%.Furthermore an equation C(%)=1/(1.14A+1) for calculation of conversionswas deduced under the experimental conditions. In light of the equation, theconversion is easily detected by GC because the standard compound needn'tbe added to reaction system, and also the quantity of injected samples hasn'tto be cared and that only to be done is to find the peak area ratio A of2-octanol to octan-2-yl octanoate. In another way, the problems thatdemanded to be further investigated were given out, such as low catalyticactivity and moderate enantioselectivity (E≈21) of PSL, and poor yield of(S)-2-octanol. And that (R)-2-octanol with high enantioselectivity can't beobtained in the resolution. When CSL were select as biocatalysis, the lipaseshow excellent enantioselectivity (E≈300) in the aforesaid reaction system,in which the yield of (S)-2-octanol was more than that before. In the researchon reusage of PSL power, the native PSL, the once used PSL and the mixtureof both showed similar activity and enantiolselectivity in resolution if theiroverall enzyme activities were equal. Although the overall yield of chiralcompound is less than 50% and the optically active (R)-2-octanol is hard to beobtained whether PSL or CSL was used in resolution. The primaryexplorations for resolution of 2-octanol lay a foundation and guide us forfurther investigations.2. A way for resolution of 2-octanol by combining double kineticresolution with dynamic kinetic resolutionThe way for resolution of 2-octanol by combining double kinetic resolution(DoKR) with dynamic kinetic resolution (DyKR) is that optically pure(S)-2-octanol and (R)-2-octanol can be obtained in the first resolution and thesecond resolution of DoKR by controlling their conversions, respectively.Moreover, the residual 2-octanol ([R]>[S], ee<8%) after one DoKR can beused as the substrate of next DoKR. And then DoKR is recycled to DyKR byusing residual substrate of DoKR. When PSL with moderateenantioselectivity is used to resolve 2-octanol by combining DoKR withDyKR, 98% ee (S)-2-octanol and 95% ee (R)-2-octanol can be obtained,respectively, and 80% substrate can be converted to enantiopure productsthrough two recycles. If high enantioselective CSL is used in the resolution,then both 99% ee enantiomers of 2-octanol can be got and an overall yield ofmore than 80% can be attained by the way. The way for resolution ofracemates caused both of enantiopure enantiomers to be obtained and a yieldmore than 50% to be achieved, and also extended the applied field of lipaseswith moderate enantioselectivity.3. Enhancement of enzyme activity in organic system by immobilizinglipase on glass wall of reactorIn order to find a desired way to improve the activity of PSL in organicsystems, a several ways were tried by us. It is found that the solvents withlower polarity or the non-polar solvents with great molecules favoredenhancement of enzyme activity, and also did the alkalescenthydrophosphates and especially treatment of lipases with water. However, thebest way for improvement of enzyme activity is to immobilize lipases onglass wall of reactor by keeping a mouth-opened conical flask with littlelipase and water in a rotary incubator at 37℃ and 170r.p.m. for more than10h. The wall-immobilized lipase is readily obtained by facile performanceand need no other special materials as carriers. Using wall-immobilized PSLas biocatalysis in the reaction of 2-octanol with vinyl caprylate, its catalyticactivity is enhanced eight times of equal quantity of PSL power, which can beseen from their conversion during the earlier process of reaction (before 3h).Although the activity of wall-immobilized lipase decreased in its reusage, itcould be renewed by the repeating immobilization. The reason on activeenhancement of wall-immobilized lipases relative to enzyme power in organicsystems is considered that the immobilization and the usage ofwall-immobilized lipase simulate the interfacial activity of lipase by steps.The wall-immobilized means give a facile and effective way for improvementof enzyme activity in unaqueous reaction system. The reactor withwall-immobilized lipase is actually a structure-facile bioreactor.4. The application of wall-immobilized lipase bioreactor in chemioenzymaticsynthesis of chiral propranololsPropranolol is an important antihypertensive drugs, the therapeutic effectresides mainly in (S)-enantiomer, Moreover, (R)-enantiomer is known to actas a contraceptive. In the chemioenzymatic synthesis of chiral propranolol,the wall-immobilized PSL bioreactor was used in the resolution ofl-chloro-3-(l-naphthyloxy)-2-propanol after we optimized the chemicalsynthesis of this intermediate. The activity of wall-immobilized PSL is tentimes of equal quantity of PSL power. If the reaction was stopped atapproximate 50% conversion by dumping reaction mixture out of thisbioreactor, 95% ee (R)-l-chloro-3-(l-naphthyloxy)-2-propanol was recovered,which could be converted to (S)-propranolol and its hydrochloride. And 99%ee (S)-propranolol hydrochloride was obtained by re-crystaling.Synchronously, 98% ee (R)-propranolol hydrochloride could be obtained byan analogous way.5. Understanding the enantioselectivity of PSL and CSL towards twosec-alcoholsIt is the total energy (TE) of acylated lipase docking with substrate and thedistance (dO-C) between alcohol oxygen and serine β-C that corrected mainlywith the enantioselectivity of PSL and CSL towards two sec-alcohols,2-octanol and l-chloro-3-(l-naphthyloxy)-2-propanol. Among intermediates of(R)/(S)-2-octanol docking with acylated PSL (CSL), the TE and dO-C of theformer is always less than that of the latter. Namely, the former is easilyformed, so the enantiomer preferably selected by two lipases is (R)-2-octanol.And also both the ⊿dO-C and ⊿TE of (R)/(S)-2-octanl docking withacylated PSL are less than that of them with acylated CSL. Namely, thedifference in formation between two intermediates of (R)/(S)-2-octanldocking with CSL is more than that of them with PSL. So theenantioselectivity of CSL towards 2-octanol is greater than that of PSLtowards it. In terms of l-chloro-3-(l-naphthyloxy) -2-propanol, it is contrary tothe forementioned case. The (S)-enantiomer is preferably selected by bothlipases, and the enantioselectivity of PSL towards this sec-alcohol is greatrelative to CSL.Based on primary kinetic resolution of 2-octanol, it is found that the way bycombining DoKR with DyKR can be used effectively to obtain two opticallyactive enantiomers with total 80% yield and the means of usingwall-immobilized lipase is a facile and resultful way for enhancement ofenzyme activity in organic systems, which excellent activity is also furtherconfirmed in chemioenzymatic synthesis of chiral propranolol. Finally, thesize and preference of enantioselectivity that two lipases towards twosec-alcohols are analyzed by molecular modeling and theoretical computation. |
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