Candida antarctica lipase alcohols

Aqueous solutions are not suitable solvents for esterifications and transesterifications, and these reactions are carried out in organic solvents of low polarity [9-12]. However, enzymes are surrounded by a hydration shell or bound water that is required for the retention of structure and catalytic activity [13]. Polar hydrophilic solvents such as DMF, DMSO, acetone, and alcohols (log P<0, where P is the partition coefficient between octanol and water) are incompatible and lead to rapid denaturation. Common solvents for esterifications and transesterifications include alkanes (hexane/log P=3.5), aromatics (toluene/2.5, benzene/2), haloalkanes (CHCI3/2, CH2CI2/I.4), and ethers (diisopropyl ether/1.9, terf-butylmethyl ether/ 0.94, diethyl ether/0.85). Exceptionally stable enzymes such as Candida antarctica lipase B (CAL-B) have been used in more polar solvents (tetrahydrofuran/0.49, acetonitrile/—0.33). Room-temperature ionic liquids [14—17] and supercritical fluids [18] are also good media for a wide range of biotransformations. 

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). 

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-... 

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... 

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]. 

Tertiary alcohols are very unreactive toward hydrolases. There are, however, exceptions. Some enzymes have a certain amino acid motif located in the oxyanion binding pocket that allows the docking of space-demanding alcohols such as tertiary ones into the acylated enzyme [104]. One such hydrolase is Candida antarctica lipase A (CALA), which has been found to catalyze the acylation of the tertiary 2-phenyl-3-butyn-2-ol rac-110 by vinyl acetate in organic solvents. Thus efficient resolution of 110 was achieved in isooctane at room temperature (Scheme 4.34) [105]. 

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]. 

The integration of a catalyzed kinetic enantiomer resolution and concurrent racemization is known as a dynamic kinetic resolution (DKR). This asymmetric transformation can provide a theoretical 100% yield without any requirement for enantiomer separation. Enzymes have been used most commonly as the resolving catalysts and precious metals as the racemizing catalysts. Most examples involve racemic secondary alcohols, but an increasing number of chiral amine enzyme DKRs are being reported. Reetz, in 1996, first reported the DKR of rac-2-methylbenzylamine using Candida antarctica lipase B and vinyl acetate with palladium on carbon as the racemization catalyst [20]. The reaction was carried out at 50°C over 8 days to give the (S)-amide in 99% ee and 64% yield. Rather surpris-... 

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). 

M. Woudenberg-van Oosterom, F. van Rantwijk, and R. A. Sheldon, Regioselective acylation of disaccharides in tert-butyl alcohol catalyzed by Candida antarctica lipase, Biotechnol. Bioeng., 49 (1996) 328-333. 

Primary alcohols have been successfully used as substrates for lipases. Monterde et. Al60 reported the resolution of the chiral auxiliary 2-methoxy-2-phenylethanol 1 via Candida antarctica lipase B (CAL-B)-catalyzed acylation using either vinyl acetate (R=H) or isopropenyl acetate (R= CH3) as acyl donor (cf. fig. 8) and the alkoxycarbonylation using diallyl carbonate as the alkoxycarbonylation agent in THF at 30 °C (cf. fig. 9). 

Various lipases and esterases have been used for the enantioselective esterification of alcohols and hydrolysis of esters. For example, Burkholderia cepacia lipases (PS, Amano Enzyme Inc.) and Candida antarctica lipase (CAL, Novozymes) have been widely used for its wide substrate specificities, high activities and chemo, regio and enantioselectivities. Fundamentals and some selected applications are shown in this section. The origins and abbreviations of lipases introduced here are as follows. 

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... 

In addition, Itoh and coworkers have reported that acylation of the alcohol was accomplished by three types of enzymes Candida Antarctica lipase (CAL, Novozym 435), lipase QL Alcalgenes sp.), and lipase PS Pseudomonas cepacia). Scheme 10.5. The desired acetate showed extremely high enantioselectivity, but no reaction took place when lipase (CRL, Candida rugosa) or Procine liver lipase (PPL) was used as the catalyst in the ionic liquid (Table 10.3). 

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]. 

Oosterom, M.W., F. Rantwijk, and R.A. Sheldon, Regioselective Acylation of Disaccharides in Terf-Butyl Alcohol Catalyzed by Candida antarctica Lipase, Biotechnol. Bioeng. 49 328-333 (1996). 

Candida antarctica Lipase, trifluoroethyl levulinate, THF, 40"C, 4 days, 65-83% yield. The method was used for the selective protection of the primary alcohol of the galactose saccharide. ... 

Sinha et al. [7] for the first time reported a highly efficient and recyclable combination of Candida antarctica lipase B (CAL-B) and nentral ionic liqnid [hmim] [Br] for metal-free activation in the chemoselective oxidation of aryl alcohols. Initial study was carried out using 4-methoxyphenyl propanol, as substrate, and HjOj at 40°C in the presence of CAL-B/[hmim][Br], thereby providing aldehyde in 90% yield after 16 h. Increasing the reaction temperature to 60°C significantly brought down the reaction time from 16 to 8 h (Scheme 14.7). 

Triglycerides can be converted to the corresponding amides using C. antarctica, with ammonia in fe/t-butyl alcohol.228 Olive oil gave a 90% yield of oleamide in 72 h at 60°C. Industrial reactions of this type are run at 200°C. The enzyme can also be used to convert oleic acid to oleamide in 90% yield in tert-amyl alcohol containing two equivalents of mbutyl alcohol. The intermediate butyl ester is formed in situ. Candida antarctica lipase can also be used to prepare monoesteramides from dimethyl succinate... 

In our study, we conducted the enzyme-catalyzed methanolysis of rapeseed oil using Novozym 435, a well-known nonspecific lipase. Novozym 435 facilitates reactions between a wide variety of alcohols and is also a remarkably heat-tolerant enzyme [6, 8], Watanabe et al. [9] previously reported that immobilized Candida antarctica lipase was inactivated in the presence of more than half the stoichiometric amount of methanol against total fatty acids in the oil. This disadvantage was surmounted by the utilization of three-step methanolysis, in which only one third of the total amount of methanol was added in each stage [7, 9]. 

PEO = poly(ethylene oxide), PBE = poly(benzyl ether), CALB -Candida antarctica lipase B, PVA = poly(vinyl alcohol), HEMA = hydroxyethyl methacrylate, EVAL = ethylene-vinyl alcohol copolymer, PAA = poly(acrylic acid), PMMA = poIy(methyl methacrylate)... 

Using the Candida antarctica lipase (CAL) in SCCO2 kinetically resolves racemic secondary alcohol 31 (Scheme 50). " The optical purity of the product is continuously tunable by changing the CO2 pressure. The enantiomeric ratio decreases from 50 to 10 as the CO2 pressure varies from 80 to 190 bar. 

Fig. 7. Synthesis of chiral intermediates for anti-Alzheimer drug enzymatic resolution of racemic secondary alcohols by Candida antarctica lipase.

The same reactions also take place in ionic liquids (BMIM -X, BIMIM = 1-butyl-3-methylimidazolium X = PFs, BF4). It was found that the anion X has a huge influence on the stereoselectivity of the resolution reactions in BMIM PFg are up to six time more enantioselective than in common organic solvents, whereas in BMIM BF4 racemic products are obtained. They tentatively attributed this lack of selectivity to the miseibility of BMIM BF4 with water. An extension of the same chemistry to supercritical carbon dioxide has also been reported by Kielbasinski and co-workers. They found that Candida antarctica lipase was reactive for the transformation. The enantioselectivity of the reaction (up to 88% ee for unreacted alcohol 144) was strongly dependent on the pressure and the substrate. 

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