Candida antarctica lipase, enzymatic reactions

Both chiral compounds have been prepared by enantioselective reduction of ethyl-5-oxohexanoate 71 and 5-oxohexanenitrile 72 by Pichia methanolica SC 16116. Reaction yields of 80%-90% and more than 95% EEs were obtained for each chiral compound. In an alternate approach, the enzymatic resolution of racemic 5-hydroxy-hexane nitrile 73 by enzymatic succinylation was demonstrated using immobilized lipase PS-30 to obtain (S)-5-hydroxyhexanenitrile 69 in 35% yield (maximum yield is 50%). (S)-5-Acetoxy-hexanenitrile 74 was prepared by enantioselective enzymatic hydrolysis of racemic 5-acetoxyhexanenitrile 75 by Candida antarctica lipase. A reaction yield of 42% and an EE of more than 99% were obtained [96]. 

In polyester synthesis via ring-opening polymerizations, metal catalysts are often used. For medical applications of polyesters, however, there has been concern about harmful effects of the metallic residues. Enzymatic synthesis of a metal-free polyester was demonstrated by the polymerization of l,4-dioxan-2-one using Candida antarctica lipase (lipase CA). Under appropriate reaction conditions, the high molecular weight polymer (molecular weight = 4.1 x 10" ) was obtained. 

A combination of an enzymatic kinetic resolution and an intramolecular Diels-Alder has recently been described by Kita and coworkers [23]. In the first step of this domino process, the racemic alcohols ( )-8-55 are esterified in the presence of a Candida antarctica lipase (CALB) by using the functionalized alkenyl ester 8-56 to give (R)-8-57, which in the subsequent Diels-Alder reaction led to 8-58 in high enantioselectivity of 95 and 91 % ee, respectively and 81 % yield (Scheme 8.15). In-... 

An interesting example of biocatalysis and chemical catalysis is the synthesis of a derivative of y-aminobutyric acid (GABA) that is an inhibitor for the treatment of neuropathic pain and epilepsy (Scheme 10.4). The key intermediate is a racemic mixture of cis- and trons-diastereoisomer esters obtained by a hydrogenation following a Horner-Emmons reaction. The enzymatic hydrolysis of both diaste-reoisomers, catalyzed by Candida antarctica lipase type B (CALB), yields the corresponding acid intermediate of the GABA derivative. It is of note that both cis- and trans-diastereoisomers of the desired enantiomer of the acid intermediate can be converted into the final product in the downstream chemistry [10]. 

Scheme 4.15 Examples of promiscuous enzymatic reactions conducted with the oxyanion hole of Candida antarctica lipase B (a) the aldol reaction [104] (b) the conjugate addition reaction (Michael addition) [105] (c) the epoxidation reaction [106],...

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

It is generally stated that biocatalysis in organic solvents refers to those systems in which the enzymes are suspended (or, sometimes, dissolved) in neat organic solvents in the presence of enough aqueous buffer (less than 5%) to ensure enzymatic activity. However, in the case of hydrolases water is also a substrate and it might be critical to find the water activity (a ) value to which the synthetic reaction (e.g. ester formation) can be optimized. Vahvety et al. [5] found that, in some cases, the activity of Candida rugosa lipase immobihzed on different supports showed the same activity profile versus o but a different absolute rate. With hpase from Burkholderia cepacia (lipase BC), previously known as lipase from Pseudomonas cepacia, and Candida antarctica lipase B (CALB) it was found that the enzyme activity profile versus o and even more the specific activity were dependent on the way the enzyme was freeze dried or immobihzed [6, 7]. A comparison of the transesterification activity of different forms of hpase BC or CALB can be observed in Tables 5.1 and 5.2, respectively. 

Waste oils, from restaurants and household disposals and being creating serious problems of environmental control and food safety, have been considered as good raw material for biodiesel production. Immobilized Candida antarctica lipase was found to be effective for the methanolysis of waste oil. A three-step methanolysis protocol could be used to protect lipase from inactivation by methanol. Compared with one-step reaction, it needs a longer time to reach the reaction equilibrium. So, efforts should be made to increase enzymatic reaction rate. Reports on the enhancement of the activity of certain enzymes by applying ultrasonic irradiation on the enzymes led us to investigate its effects on the enzymatic transesterification of waste oil to biodiesel in a solvent free system. 

As evident from Fig. 8.4, an increase in the selectivity has been observed in IL/ scCOj biphasic systems media (>99.5%) with respect to scCO assayed alone (95%). These results could be explained by the use of water-immiscible ILs which have a specific ability to reduce water activity in the enzyme microenvironment. The synthetic activity of the immobilized lipase in IL/scCO biphasic systems is lower than that in scCO assayed alone. Similar results were found by Mori et al. [40] in IL/ hexane biphasic systems. These authors reported that the enzymatic membranes prepared by simple adsorption of CaLB onto the surface were more reactive than membranes prepared with ILs. As can be observed in Fig. 8.4, the initial reaction rate in the assayed IL/scCO biphasic systems increased in the following sequence [bdimim ][PF ]<[bmim ][PFg ]<[bmim ][NTfj ]<[omim ] [PF ], which was practically in agreement with flie activity sequence reported by these authors using free Candida antarctica lipase B in homogeneous ionic liquid systems ([bmim ] [PF ]<[bdmim+][PFg ]<[bmim+][NTfj ]<[omim ][PF ]), with the exception of [bmim [PF ] and [bdimim+][PFg ]. These results were explained taking into account that biotransformation occurs within the ionic liquid phase, so substrates have to be transported from scCOj to the ionic liquid phase. The mechanism of substrate transport between the ionic liquid and the supercritical carbon dioxide could be by three consecutive steps diffusion of the substrates through the diffusion... 

Loupy and co-workers [68] have studied the effectiveness of microwave irradiation in increasing the enzymatic affinity and selectivity of supported lipases in esterification and transesterification reactions under dry media conditions (see Scheme 37). The esterification and transesterifications of racemic 1-phenylethanol 64 were studied in a temperature range of 70-100 °C. The lipases considered were the Pseudomonas cepacia lipase (LP) and Candida Antarctica lipase (SP-435). The initial rates and enantiomeric ratios E were significantly enhanced under microwave irradiation. Even so, in cases where classical conditions showed poor reaction, complete conversion could be achieved with increased reactivity under microwave conditions. This was largely attributed to the exclusion of the volatile by-products from the equilibrium. More importantly, the supported enzymes showed good stability and could be reused three more times in the reactions under study without loss of activity. 

Aliphatic polyesters were also synthesized via an enzymatic polymerization of dicarboxyiic acids and glycols in a solvent-free system [69, 70]. The lipase from Candida antarctica (lipase CA) provided an efficient catalysis of the polymerization under mild reaction conditions, despite the presence of a heterogeneous mixture of the monomers and catalyst. The ahphatic chain length of the monomers had a major effect on both the polymer yield and molecular weight typically, a molecular weight in excess of 1 x Da was obtained when the reaction was conducted under reduced pressure. However, the addition of a small amount of adjuvant proved effective for polymer production when both monomers were solid at the reaction temperature [71]. 

Stmctured triacylglycerols (see Chapter 6) of the type CGC (where C represents caprylic acid, and G represents GLA) have been prepared in a two-step process. In the first stage the triacylglycerol GGG is prepared from glycerol, GLA, and Candida antarctica lipase. This is then subjected to acidolysis with caprylic acid or interesterification with ethyl caprylate in the presence of Rhizopus delemar lipase. A typical reaction product contains 53% caprylic acid and 47% GLA with CCG (58%) and CGG (29%) as the major triacylglycerols (Kawashima etal., 2001). This particular work was carried out only on a laboratory scale but there are many reports of this type of enzymatic process being carried out on a kilogram scale. At present such compounds are probably too expensive to be incorporated into functional foods and are more likely to be used in pharmaceutical preparations. 

Efficient enzymatic conversion can be achieved even though most of the reactants are present as solids, provided that there is a liquid phase in which the reaction can occur. This approach has been successfully used for carbohydrate ester synthesis with synthesis of glucose esters of fatty acids between C12 and C18 as typical examples [34]. It is important that the substrates dissolve during the reaction, and often the products precipitate as they are formed, which can be an advantage due to a favourable effect on the equilibrium position. Candida antarctica lipase B is an efficient catalyst in this system and solvents used (in moderate amounts) include ethyl methyl ketone, acetone or dioxane. In order to increase the ester yield, water formed in the reaction can be removed by azeotropic distillation and the solvent (e.g. ethyl metyl ketone) can after condensation be dried by pervaporation, giving a practically useful complete process [35]. 

Another approach for the enzymatic preparation of 5-ibuprofen has been demonstrated by de Zoete et al. [229]. The enantioselective ammonolysis of ibuprofen 2-chlo-roethyl ester by Candida antarctica lipase (lipase SP435) gave the remaining ester 5-(+) enantiomer in 44% yield and 96% e.e. The enantioselective enzymatic esterification of racemic ibuprofan has also been demonstrated using lipase from Candida cylindraceae [230]. The reaction was carried out in a water-in-oil microemulsion [bis(2-ethyl-hexyl)sulfosuccinate (AOT)/isooctane). The lipase showed high preference for the S-(+) enantiomers of ibuprofen which was esterified and R-(—) enantiomer remained unreacted. The reaction yield of 35% was obtained using n-propanol in the reaction mixture as nucleophile. 

Based on the specific reaction that they catalyze, enzymes have been classified into six groups, three of which have been reported to catalyze or induce polymerization in vitro, namely oxidoreductases, transferases and hydrolases. The latter class includes hpases, the natural role of which is the hydrolysis of fatty acid esters at the cell s water-Upid interface. In organic media, hpases can efficiently catalyze ester bond formation, and so have been used extensively in investigations of the in vitro synthesis of polyester by polycondensation or ring-opening polymerization (ROP), without the need for any cocatalyst. One enzyme that deserves special attention when discussing enzymatic ROP is Candida antarctica Lipase B (CALB). 

In 2004, Ley et al. [45] showed a stereoselective enzymatic synthesis of cis-pellitorine [N-isobutyldeca-(2 ,4Z)-dienamide], a taste-active alkamide naturally occurring in tarragon. The reactants were ethyl ( ,Z)-2,4-decadienoate— the pear ester described before—and isobutyl amine. The reaction is catalysed by lipase type B from Candida antarctica (commercially available), which shows a remarkable selectivity towards the 2 ,4Z ester. The yield was about 80%. 

Other authors have described the lipase-catalyzed chemoselective acylation of alcohols in the presence of phenolic moities [14], the protease-catalyzed acylation of the 17-amino moiety of an estradiol derivative [15], the chemoselectivity in the aminolysis reaction of methyl acrylate (amide formation vs the favored Michael addition) catalyzed by Candida antarctica lipase (Novozym 435) [16], and the lipase preference for the O-esterification in the presence of thiol moieties, as, for instance, in 2-mercaptoethanol and dithiotreitol [17]. This last finding was recently exploited for the synthesis of thiol end-functionalized polyesters by enzymatic polymerization of e-caprolactone initiated by 2-mercaptoethanol (Figure 6.2)... 

The reaction is catalyzed by a variety of both acids and bases but simple bases such as NaOH and KOH are generally used for the industrial production of biodiesel [200, 201]. The vegetable oil feedstock, usually soybean or rapeseed oil, needs to be free of water (<0.05%) and fatty acids (<0.5%) in order to avoid catalyst consumption. This presents a possible opportunity for the application of enzymatic transesterification. For example, lipases such as Candida antarctica B lipase have been shown to be effective catalysts for the methanolysis of triglycerides. When the immobilized form, Novozyme 435, was used it could be recycled 50 times without loss of activity [201, 202]. The presence of free fatty acids in the triglyceride did not affect the enzymes performance. The methanolysis of triglycerides catalyzed by Novozyme 435 has also been successfully performed in scC02 as solvent [203]. 

There are many reports of enzymatic catalysis in scC02 performing hydrolysis, oxidations, esterifications, and franr-esterification reactions. For example, the enzymatic kinetic resolution of 1-phenylethanol with vinyl acetate in scC02 using lipase from Candida antarctica B produces (R)-l-phenyethylacetate in >99% ee (i.e., enantiomeric excess, a measure of how much of one enantiomer is present as compared to the other), as shown in Figure 12.20. 

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