Lipase-catalyzed reactions approach

The use of MALDI-MS for the measurement of low molecular mass compounds is widely accepted now [61], but quantification remains problematic. The main problem is the inhomogeneous distribution of the analytes within the matrix [62]. This leads to different amounts of ions and therefore to different signal intensities at various locations of a sample spot. The simplest and most effective way to overcome this problem is the use of an appropriate internal standard [63]. The use of deuterated compounds with a high molecular similarity to the analyte as internal standards leads to a linear correlation between relative signal intensities and relative amount of the compound to be quantified (Fig. 4b) [64]. Using this approach it is possible to quantitate substrates and products of enzyme catalyzed reactions. Two examples were shown recently by Kang and coworkers [64, 65]. The first was a lipase catalyzed reaction which produces 2-methoxy-N-[(lR)-l-phenylethyl]-acetamide (MET) using rac-a-... 

Lipase catalyzed reactions take place in the neat oil or in a nonpolar (usually hydrocarbon) solvent. The efficiency depends on the amount of water, solvent (if present), temperature, and ratio of reactants. A factorial approach can be used to optimize the conditions (32). In interesterification reactions, 1,3-specific enzymes give control over product composition that is not possible using chemical catalysts. For example, starting with SOS and OOO, chemical interesterification produces aU eight possible isomers (see Table 5). Enzymatic interesterification does not exchange fatty acids at the sn-2 position, and it will result in only two additional molecular species, OOS and SOO. In more realistic situations, chemical and enzymatic interesterification may produce the same or a similar number of molecular species, but in different proportions (31). 

Recently [63], we studied the behavior of two-enzyme system catalyzing two consecutive reactions in a macroheterogeneous medium (modified Lewis cell). The system consisted of lipase-catalyzed hydrolysis of trilinolein and subsequent lipoxygenation of liberated fatty acids (Fig. 3). Our approach compared the kinetic behavior of coupled enzymes in the Lewis cell with the sequential study of separated phenomena presented before ... 

In an alternative approach to prepare the chiral side chain of captopril (14) and zofenopril (18), the lipase-catalyzed stereoselective esterification of racemic 3-benzoylthio-2-methylpropionic acid (19) (Fig. 8B) in an organic solvent system was demonstrated to yield i -(+)-methyl ester (20) and unreacted acid enriched in the desired S-( )-enantiomer (19) [45], Using lipase PS-30 with toluene as solvent and methanol as nucleophile, the desired S-(-)-(19) was obtained in 37% reaction yield (theoretical max. 50%) and 97% e.e. Substrate was used at 22-g/ liter concentration. The amount of water and the concentration of methanol supplied in the reaction mixture was very critical. Water was used at 0.1 % concentration in the reaction mixture. More than 1% water led to the aggregation of enzyme in the organic solvent, with a decrease in the rate of reaction which was due to... 

In particular, the use of hydroxynitrile lyase has proved to be a general and reliable method for obtaining a-hydroxy nitriles of both configurations [22]. An interesting approach is the Upase- and baseacyl-cyanohydrin for a synthetic DKR [23]. This is a combination of two reaction systems the dynamic, base-catalyzed equiUbrium between acetone cyanohydrin, acetone, HCN, aldehyde and a racemic cyanohydrin and the lipase-catalyzed enantioselective and irreversible acylation of the hydroxyl group. The combination yields the... 

A more elegant approach towards lipase-catalyzed synthesis of chiral polymers is to take advantage of the intrinsic ability of lipases to discriminate between ena-tiomers. Chiral polymers can be formed when one of the two enantiomers of a chiral monomer preferentially reacts during the polymerization reaction. This is referred to as a kinetic resolution polymerization (KRP) and allows the optically active polymer to be directly procured from a racemic mixture, albeit in a maximum of 50% yield. 

While the polymerization of optically inactive AA-BB and AB monomers under DKR conditions leads to chiral polyesters, these approaches always result in limited molecular weights since a condensation product is formed that needs to be effectively removed. A solution for this would be to use the eROP of lactones, where no condensation products are formed during polymerization. In principle, the eROP of lactones can lead to very high MW polyesters (>80kgmol 1) [57]. Addition of a methyl substituent at the ro-position of the lactone introduces a chiral center. Peeters et al. conducted a systematic study of substituted e-caprolactones which revealed that monomers with a methyl at the 3-, 4-, or 5-position could be polymerized enantioselectively while a methyl at the 6-position (a-methyl-e-caprolactone, 6-MeCL) could not [58]. The lack of reactivity of the latter monomer in a Novozym 435-catalyzed polymerization reaction was attributed to the formation of S-secondary alcohol end-groups. These cannot act as a nucleophile in the propagation reaction since the lipase-catalyzed transesterification is highly R-selective for secondary alcohols. 

Lipase-catalyzed polyester synthesis has received considerable interest [31] due to the harsh conditions used in traditional chemical polymerization (> 200 C), and has also been subjected to reaction in ILs. Both CaLB and PcL lipases have been found to catalyze polyester synthesis in [BMIM][BF4] and [BMIM][PF6], but this approach does not seem to offer any advantages over state-of-the-art lipase-mediated polyester synthesis [32, 33] [Eq. (3)]. Still, the polydispersity index of the polymers formed in ILs were remarkably affected, giving values close to 1, which indicate a very narrow molecular weight range compared to material prepared by conventional polymerization processes [33]. 

Another workup approach has been to use the inherent phase behavior of oil-water-surfactant systems to separate product from remaining reactants and from surfactant. A Winsor III system made with a branched-tail phosphonate surfactant was used as reaction medium for lipase-catalyzed hydrolysis of trimyristin. The enzyme resided almost exclusively in the middle-phase microemulsion together with the surfactant. The products formed, 2-myristoylglycerol and sodium myristate, partitioned into the excess hydrocarbon and water phases, respectively, and could easily be recovered [129]. A similar procedure was used for cholesterol oxidation using cholesterol oxidase as catalyst [130]. 

Syntheses of aliphatic polyesters by fermentation and chemical processes have been extensively studied in a viewpoint of biodegradable materials. Recently, another approach of their production has been performed by using an isolated lipase or esterase as catalyst via nonbiosynthetic pathways under mild reaction conditions. Lipase and esterase are enzymes which catalyze hydrolysis of esters in an aqueous environment in living systems. Some of them can act as a catalyst for the reverse reactions, esterifications and transesterifications, in organic media (1-5). These catalytic actions have been expanded to enzymatic synthesis of polyesters. Figure 5 represents three major reaction types of lipase-catalyzed polymerization leading to polyesters (6-14,73). 

Another approach of enzymatic synthesis of sugar-containing polyesters was demonstrated (123). Lipase CA-catalyzed reaction of sucrose or trehalose with an excess of divinyl adipate produced 6,6 -diacylated product having vinyl esters at both ends, which was employed as monomer in the enzymatic polycondensation with various glycols, jdelding linear polyesters with Mw up to 2.2x 10. ... 

Lipase-Catalyzed Hydrolysis Goto and coworkers [63] proposed an approach for carrying out enzymatic reactions in water-in-IL microemulsion. 

Another demonstration of a continuous flow operation is the psi-shaped microreactor that was used for lipase-catalyzed synthesis of isoamyl acetate in the 1-butyl-3-methylpyridinium dicyanamide/n-heptane two-phase system [144]. The chosen solvent system with dissolved Candida antarctica lipase B, which was attached to the ionic liquid/n-heptane interfacial area because of its amphiphilic properties, was shown to be highly efficient and enabled simultaneous esterification and product removal. The system allowed for simultaneous esterification and product recovery showed a threefold reaction rate increase when compared to the conventional batch. This was mainly a consequence of efficient reaction-diffusion dynamics in the microchannel system, where the developed flow pattern comprising intense emulsification provided a large interfacial area for the reaction and simultaneous product extraction. Another lipase-catalyzed isoamyl acetate synthesis in a continuously operated pressure-driven microreactor was reported by the same authors [145]. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. Controlling flow rates of both phases reestablished a parallel laminar flow with liquid-liquid boundary in the middle of the microchannel and a separation of phases was achieved at the y-shaped exit of the microreactor (Figure 10.25). The microreactor approach demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations and has proven more effective and outperformed the batch operation, which could be attributed to the favorable mass and heat transfer characteristics. 

During the development of the one-pot reaction sequence for the case in Fig. 8.40, it is important to avoid diol formation from over-hydrogenation, thus the loading of hydrogenation catalysts should be optimized. Suppression of the consecutive hydrogenation to diols is essential in the one-pot approach where, in the presence of an enzyme, lipase-catalyzed acetylation of the isomeric alcohols would result in undesired mixtures of acetylated products. 

The lipase-catalyzed transesterifications described earlier are in general equilibrium reactions and a few methods have been proposed to make the reaction irreversible or to slow down the back reaction [120,121]. One approach consists of the use of trifluoroethyl esters as acylating reagents (Scheme 20). In this way trifluoroethanol is produced and the reverse reaction is slow, as the fluorinated alcohol is less nucleophile than ethanol [122]. 

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