Candida antarctica lipase CALB

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-... 

Racemization of the remnant substrate in a DKR process can be performed either spontaneously or by the employment of a chemo- or biocatalyst, which must be compatible with the reaction conditions used for the KR reaction. In the case of sec-alcohols, most of successful DKRs have been carried out by the use of ruthenium complex catalysts, soluble in the organic reaction media, which promote racemization through redox processes. The first examples describe the resolution of 1-phenylethanol (rac-1) by the combination of a rhodium catalyst (Rh2(OAc) ) with Pseudomonas fluorescens lipase [22], although more effective results were afforded by Backvall and coworkers [23], who developed the DKR of the same substrate and derivatives catalyzed by Candida antarctica lipase (CALB) and a ruthenium complex (Shvo s catalyst, 2 (Figure 14.2)), affording excellent conversions and enantiomeric excess (ee) values [24]. 

In recent years, a great variety of primary chiral amines have been obtained in enantiomerically pure form through this methodology. A representative example is the KR of some 2-phenylcycloalkanamines that has been performed by means of aminolysis reactions catalyzed by lipases (Scheme 7.17) [34]. Kazlauskas rule has been followed in all cases. The size of the cycle and the stereochemistry of the chiral centers of the amines had a strong influence on both the enantiomeric ratio and the reaction rate of these aminolysis processes. CALB showed excellent enantioselec-tivities toward frans-2-phenylcyclohexanamine in a variety of reaction conditions ( >150), but the reaction was markedly slower and occurred with very poor enantioselectivity with the cis-isomer, whereas Candida antarctica lipase A (GALA) was the best catalyst for the acylation of cis-2-phenylcyclohexanamine ( = 34) and frans-2-phenylcyclopropanamine ( =7). Resolution of both cis- and frans-2-phenyl-cyclopentanamine was efficiently catalyzed by CALB obtaining all stereoisomers with high enantiomeric excess. 

The use of an enzyme in a cascade using nanoencapsulation has also been demonstrated [23]. In this case, the dynamic kinetic resolution (DKR) of secondary alcohols was achieved with an acidic zeolite and an incompatible enzyme, Candida antarctica lipase B (CALB) (Scheme 5.8). 

The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Among them, two commercially available Upases, Candida antarctica lipase B (CALB trade name, Novozym-435) and Pseudomonas cepacia lipase (PCL trade name. Lipase PS-C), are particularly useful because they have broad substrate specificity and high enantioselectivity. They display satisfactory activity and good stability in organic media. In particular, CALB is highly thermostable so that it can be used at elevated temperature up to 100 °C. 

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]. 

Biphasic systems consisting of ionic liquids and supercritical CO2 showed dramatic enhancement in the operational stability of both free and immobilized Candida antarctica lipase B (CALB) in the catalyzed kinetic resolution of rac- -phenylethanol with vinyl propionate at 10 MPa and temperatures between 120 and 150°C (Scheme 30) 275). Hydrophobic ionic liquids, [EMIM]Tf2N or [BMIM]Tf2N, were shown to be essential for the stability of the enzyme in the biotransformation. Notwithstanding the extreme conditions, both the free and isolated enzymes were able specifically to catalyze the synthesis of (J )-l-phenylethyl propionate. The maximum enantiomeric excess needed for satisfactory product purity (ee >99.9%) was maintained. The (S)-l-phenylethanol reactant was not esterified. The authors suggested that the ionic liquids provide protection against enzyme denaturation by CO2 and heat. When the free enzyme was used, [EMIM]Tf2N appeared to be the best ionic liquid to protect the enzyme, which... 

Since then, the process has been extended to a wide variety of lactones of different size and to several lipases, as recently reviewed [93-96]. Interestingly, large-membered lactones, which are very difficult to polymerize by usual anionic and coordination polymerizations due to the low ring strain, are successfully polymerized by enzymes. Among the different lipases available, that fi om Candida antarctica (lipase CA, CALB or Novozym 435) is the most widely used due to its high activity. An alcohol can purposely be added to the reaction medium to initiate the polymerization instead of water. The polymerization can be carried out in bulk, in organic solvents, in water, and in ionic liquids. Interestingly, Kobayashi and coworkers reported in 2001 the ROP of lactones by lipase CA in supercritical CO2... 

Kinetic resolution technology has also been applied to the duloxetine problem (Scheme 14.13). In this case, chloroketone 41 was converted to racemic alcohol 43 using sodium borohydride. The racemate was then treated with vinyl butanoate in hexanes, in the presence of catalytic immobilized Candida antarctica Lipase B (CALB). The reaction was stopped after reaching 50% conversion, leading to the isolation of the desired (5)-chloroalcohoI 43a, as well as the (Zf)-ester 45 in good yields and excellent enantiomeric excesses. Chloroalcohol 43a was converted to duloxetine (3) via the... 

Chemoenzymatic polymerizations have the potential to further increase macro-molecular complexity by overcoming these limitations. Their combination with other polymerization techniques can give access to such structures. Depending on the mutual compatibility, multistep reactions as well as cascade reactions have been reported for the synthesis of polymer architectures and will be reviewed in the first part of this article. A unique feature of enzymes is their selectivity, such as regio-, chemo-, and in particular enantioselectivity. This offers oppormnities to synthesize novel chiral polymers and polymer architectures when combined with chemical catalysis. This will be discussed in the second part of this article. Generally, we will focus on the developments of the last 5-8 years. Unless otherwise noted, the term enzyme or lipase in this chapter refers to Candida antarctica Lipase B (CALB) or Novozym 435 (CALB immobilized on macroporous resin). 

The activity of three ester spHtting enzymes, Candida antarctica lipase B (CALB), Mucor miehei lipase (MML) and esterase, towards the carbonate surfactant was studied. While CALB and esterase were found to catalyze the hydrolysis of the carbonate bond, MML showed no activity. 

Genetic engineering. The X-ray structures are known for many hydrolases, allowing for modeling of the substrate in the active site as well as structurally based, random or rational protein mutation to magnify or invert enantioselectivity. An example of the latter is provided by the rational design of a mutant of Candida antarctica lipase (CALB), which, instead of the wild-type R-selectivity, displayed... 

Although lipases from Pseudomonas are usually the catalysts of choice for primary alcohols, 2-(2-furyl)-propan-l-ol (Scheme 4.8 7 n = 0 with instead of S) actually gives a higher E (E = 20) with Candida antarctica lipase (CALB) than it does with Pseudomonas sp. lipase (PSL) (E = 2) on acylation with vinyl acetate in pentane [78]. 

The rather complex furylvinylcarbinol derivative 76 shown in Scheme 4.28 was required in enantiopure form as a key intermediate in the synthesis of the natural product cneorin. The carbinol moiety is heavily substituted with sterically demanding groups. Therefore attempts to resolve the furylvinylcarbinol with CALB or lipase PS-II led to very slow reactions. However, the rarely used enzyme Candida antarctica lipase A (CALA), which is known to act on sterically hindered substrates offers an alternative. Thus acylation of the furylvinylcarbinol 76 with 2,2,2-trifluoroethyl butanoate catalyzed by CALA (immobilized on celite with sucrose at pH 7.9) furnished the enantiomerically enriched propanoate of S-76 and R-76 (Scheme 4.28) [90]. Small-scale experiments gave E > 300. 

Kanerva et al. have also reported DKR of cyanohydrins [22]. In particular, they obtained very good results with Candida antarctica lipase A (CAL-A) as the catalyst for the KR of a variety of substrates for which other enzymes such as CALB or PS-C do not give good results (Scheme 5.9) [22a]. 

The first example of chemoenzymatic DKR of allylic alcohol derivatives was reported by Williams et al. [37]. Cyclic allylic acetates were deracemized by combining a lipase-catalyzed hydrolysis with a racemization via transposition of the acetate group, catalyzed by a Pd(II) complex. Despite a limitation of the process, i.e. long reaction times (19 days), this work was a significant step forward in the combination of enzymes and metals in one pot Some years later, Kim et al. considerably improved the DKR of allylic acetates using a Pd(0) complex for the racemization, which occurs through Tt-allyl(palladium) intermediates. The transesterification is catalyzed by a lipase (Candida antarctica lipase B, CALB) using isopropanol as acyl acceptor (Scheme 5.19) [38]. 

TIL Thermomyces lanuginosus lipase, RdL Rhizopus delemar lipase, RnL Rhizopus niveus lipase, MmE Mucor miehei esterase, PsL Pseudomonas sp. lipase, MmL Mucor miehei lipase, RoL Rhizopus orvzae lipase, CaLA Candida antarctica lipase A, CaLB Candida antarctica lipase B, PLE Pig liver esterase, EP Enteropeptidase, PKA Porcine kidney acylase, CE Cholesterol esterase Figure 8.1 (S)-Selective enzyme hits from hydrolase screening. ... 

The same concept is applicable to allylic alcohols, ketones, or ketoximes. Enol acetates or ketones were successfully converted in multi-step reactions to chiral acetates in high yields and optical yields through catalysis by Candida antarctica lipase B (CALB, Novozyme 435) and a ruthenium complex. 2,6-Dimethylheptan-4-ol served as a hydrogen donor and 4-chlorophenyl acetate as an acyl donor for the conversion of the ketones (Jung, 2000a). 

Recent studies in the pharmaceutical field using MBR technology are related to optical resolution of racemic mixtures or esters synthesis. The kinetic resolution of (R,S)-naproxen methyl esters to produce (S)-naproxen in emulsion enzyme membrane reactors (E-EMRs) where emulsion is produced by crossflow membrane emulsification [38, 39], and of racemic ibuprofen ester [40] were developed. The esters synthesis, like for example butyl laurate, by a covalent attachment of Candida antarctica lipase B (CALB) onto a ceramic support previously coated by polymers was recently described [41]. An enzymatic membrane reactor based on the immobilization of lipase on a ceramic support was used to perform interesterification between castor oil triglycerides and methyl oleate, reducing the viscosity of the substrate by injecting supercritical CO2 [42],... 

Csajagi et al. (2008) recently demonstrated the enantioselective acylation of racemic alcohols in a continuous flow bioreactor, using Candida antarctica lipase B (CaLB) 167. Employing a packed-bed reactor, containing 0.40 g of enzyme 167, and pumping a solution of rac-phenyl-1-ethanol 119 (10 mg ml-1) in hexane THF vinyl acetate 168 (2 1 1) at a flow rate of 100 gl min-1 (at 25 °C), the authors found the reactor reached steady state after 30 min of operation. Under the aforementioned conditions, the... 

The application of enzymes as catalysts in organic chemistry is closely linked to their immobilization. Indeed, many enzymes are only available in an immobilized form. The immobilized enzymes can be used as received, greatly easing their application. Numerous of these readily available immobilized enzymes are now the working horses of biocatalysis. This has even led to the incorrect use of the abbreviation of an enzyme name for a specific enzyme preparation, that is CALB for the immobilized form of Candida antarctica lipase B on cross-linked polymethacrylate (also known as Novozym 435). Vice versa the commercial name of an enzyme preparation-Amano PS-has taken the place of the enzyme (Burkhdderia cepacia lipase on dextrin or diatomaceous earth). Surprisingly, often no attention is paid to the fact that the enzyme is immobilized [1]. 

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. 

Inhibition of lipases, both by the substrate or the product, has been observed. In alcoholysis of methyl propanoate with n-propanol catalyzed by Candida antarctica lipase B (CALB), the alcohol was found to inhibit the enzyme resulting in a deadend complex [21]. Phosphate- and phosphonate-conlaining inhibitors are known to inhibit proteases. Studies of the inhibition of CALB have shown inhibition by diethyl p-nitrophenyl phosphate. The inactivation of the enzyme was caused by covalent binding of diethyl p-nitrophenyl phosphate in the active site [22]. 

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