Lipase B from Candida antarctica

The one-pot dynamic kinetic resolution (DKR) of ( )-l-phenylethanol lipase esterification in the presence of zeolite beta followed by saponification leads to (R)-l phenylethanol in 70 % isolated yield at a multi-gram scale. The DKR consists of two parallel reactions kinetic resolution by transesterification with an immobilized biocatalyst (lipase B from Candida antarctica) and in situ racemization over a zeolite beta (Si/Al = 150). With vinyl octanoate as the acyl donor, the desired ester of (R)-l-phenylethanol was obtained with a yield of 80 % and an ee of 98 %. The chiral secondary alcohol can be regenerated from the ester without loss of optical purity. The advantages of this method are that it uses a single liquid phase and both catalysts are solids which can be easily removed by filtration. This makes the method suitable for scale-up. The examples given here describe the multi-gram synthesis of (R)-l-phenylethyl octanoate and the hydrolysis of the ester to obtain pure (R)-l-phenylethanol. 

Classic resolntion has been performed by formation of diastereomeiic salts which could be separated. In a series of synthetic steps and when resolution is one step, it is of utmost importance that the correct chirality is introduced at an early stage. When a racemate is subject to enzyme catalysis, one enantiomer reacts faster than the other and this leads to kinetic resolution (Figure 2.2c). Results of hydrolysis using lipase B from Candida antarctica (CALB) and a range of C-3 secondary butanoates are shown in Table 2.1. 

Table 2.1 Enantiomeric ratios (E) obtained in hydrolysis of butanoates la-15a and transesterifrcation of alcohols lb-9b and 13b-15b using 2-chloroethyl butanoate as acyl donor and lipase B from Candida antarctica (CALB) as catalyst. For significance of and R2 see Figure 2.9. F-values were determined from chromatographically measured ee and ee at several degrees of conversion and using the computer program E K calculator version 2.03. Note that the stereopreference of CALB changes from (R) to (S) when R2=F, ClandBr.

Hansen, T.V., Waagen, V., Partali, V., Anthonsen, H.W. and Anthonsen, T. (1995) Cosolvent enhancement of enantioselectivity in lipase-catalysed hydrolysis of racemic esters. A process for production of homochiral C-3 building blocks using lipase B from Candida antarctica. Tetrahedron Asymmetry, 6, 499-504. 

Lundhaug, K., Overbeeke, P., Jongejan, J. and Anthonsen, T. (1998) Organic co-solvents restore the inherently high enantiomeric ratio of lipase B from Candida antarctica in hydrolytic resolution by relieving the enatiospecrfic inhibition of product alcohol. 

In the first approach, a maleimide-functionalized polystyrene of 40 repeat units (P DI = 1.04) was covalently coupled to the sulfur atom of a reduced disulfide bridge on the surface of the lipase B from Candida antarctica (CALB) (Figure 6.11a) [33]. TEM studies of the reaction mixture in water/THF revealed well-defined enzyme fibers with a length of several micrometers. The fibers consisted of bundles of rods, of which the smallest had diameters between 25 and 30 nm. These diameters were in... 

Studies of the ability of the lipase B from Candida antarctica (CAL-B) to catalyse the enantioselective aminolysis of esters by cis- and firms-2-phenylcycloalkanamines (54 n = 1, 3, 4) have been followed up by molecular modelling approaches in order to probe the lipase-catalysed aminolysis mechanism. CAL-B possesses a typical serine-dependent triad, so it was possible, with access to an X-ray crystal structure of CAL-B, to model a series of phosphonamidates (55 n = 1, 3, 4) as analogues of the tetrahedral intermediate (TI) resulting from attack of the amine on the carbonyl of the acyl-enzyme. The results suggested as the most plausible intermediate for the CAL-B-catalysed aminolysis a zwitterionic TI resulting from the direct His-assisted attack of the amine on to a C=0 group of the acyl-enzyme.80... 

Steytler et al. (1991) Continuous Transesterification of lauric acid with butanol Lipase B from Candida antarctica... 

An example for the application of enzymatic kinetic resolutions with high E values in natural product synthesis is the chemoenzymatic synthesis of the northern half of epothilones (also see Sect. 4.1). Various lipases and esterases could be found with outstanding enantioselectivity (up to >100) among these were lipase B from Candida antarctica, a lipase from Burkholderia cepacia, a lipase from Pseudomonas sp., and a lipase from Streptomyces diastochromogenes, all affording the desired (S)-configurated alcohol with >99% enantiomeric excess (Fig. 4) [65],... 

The components of the modular biohybrid amphipiles were constmcted by the specific covalent coupling of a 2,2 6, 2//-terpyridine-maleimide linker to a variety of enzymes, (the lipase B from Candida antarctica (CALB) and a protein, the bovine serum albumin (BSA)), using the same methodology developed for the construction of covalent biohybrids.272 Once functionalized, a series of and homo-enzyme-enzyme and hetero enzyme-enzyme and enzyme-protein assemblies are in principal readily accessible via the... 

A more direct method for the enantioselective ring opening of unactivated bicyclic /3-lactams has been reported. When lipolase (modified lipase B from Candida antarctica adsorbed on a macroporous resin) was allowed to interact with racemates 126 (n 1 or 2) and 127 (n= 1 or 2) in aqueous diisopropyl ether at 70 °C for 4.5-7 h, the products were the enantiomerically pure ring-opened unsaturated alicyclic /3-amino acids 128 (n = 1 or 2) and 129 (n 1 or 2), and the bicyclic /3-lactams 130 ( = 1 or 2) and 131 (n = 1 or 2) in 45 48% yield with ee 95-99% (Equations 13 and 14) <2004TA2875>. 

An example where a transition metal catalyst is used in combination with an enzyme has been described (Scheme 19.26).207 The racemic alcohol 50 was converted to the (A1)-acetate 51, using a ruthenium catalyst along with Novozym 435 (immobilized Lipase B from Candida antarctica), 3 equivalents of p-chlorophenylacetate in t-BuOH, and 1 equivalent of 1-indanone. The reaction yield was 81% with an optical purity of >99.5% ee. 

Lipase B from Candida antarctica (CALB) has been shown to be an excellent enantioselective biocatalyst for the stereo-selective acylation of racemic alcohols [14, 15]. The most often used commercial preparation of CALB is Novozym 435, where the enzyme is immobilized on a macroporous acrylic resin and the matrix presents about 90% of the total mass. 

Immobilized lipase B from Candida antarctica (CALB) (Novozym 435) was obtained from Novo Nordisk Novozymes. All chemicals and ionic liquids used were of the highest available purity. 

A similar resolution has also been achieved on large scale <20040PD22>. The KR of racemic isoxazoline 312 catalyzed by enzymes was studied. The best result was obtained with lipase B from Candida antarctica (CALB), which hydrolyzed the ethyl ester of (—)-312 to the corresponding monoacid (—)-313. The reaction, which was run in 0.1 M phosphate buffer/acetone at room temperature, spontaneously stopped at 50% conversion to yield monoacid (—)-313 and the residual ester (- -)-312 with ees higher than 99% <2004TA3079>. The C-5 epimer of 312 underwent enantioselective hydrolysis (>99% ee) of the methyl ester linked to C-5 in the presence of the protease proleather (subtilisin Carlsberg), whereas CALB and other lipases were not able to resolve it (Equation 53). 

UppENBERG, J., Hansen, M.T., Patkar, S. and Jones, T.A. (1994) Sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 2, 293-308... 

An ethyl carbonate, prepared and cleaved by conditions similar to those described for a methyl carbonate, was used to protect a hydroxyl group in glucose. Ethyl chlo-roformate in pyridine or CH2CI2/TEA is the most common method of preparation for this carbonate. The carbonate may be prepared by exchange with diethyl carbonate in the presence of a MgLa mixed oxide catalyst. The carbonates of 2-hydroxycar-boxylic acids may also be prepared by the reaction of 2-ethoxy-l-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ). These carbonates can also be cleaved enzymatically with Lipase B from Candida antarctica (phosphate buffer, pH 7, 30-60°C)." ... 

Waste oil was collected from the restaurant in South China University of Technology. The saponification value was 200.3 mg KOH/g, from which, the average molecular weight of the waste oil was known to be 840.2. Novozym 435 (lipase B from Candida antarctica, 164 U/g, limit corresponds to the amount of enzyme that produces 1 pmol methyl oleate from triolein per minute at 35 °C) was kindly donated by Novozymes Co. (Denmark). Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate and methyl heptadecanoate (as an internal standard) were purchased from Sigma (USA). All other chemicals were also obtained commercially and of analytical grade. Ultrasonic irradiation experiments were carried out using an ultrasonic bath (Type NP-B-400-15 Newpower Co. Ltd., China). 

Lipase B from Candida antarctica Smectite group (Laponite, SWy-2 and Kunipia) and organically modified derivatives Oxidation of terpenes [56, 57]... 

In 2005, Azim, Sahoo, and Gross [47] reported the use of immobilized lipase B from Candida antarctica (Novozym 435) as a catalyst for direct formation of amide bonds between dialkyl esters and diamines under mild reaction conditions. Oligoamides were obtained with DP up to 9. The example of the reaction of diethyl allylmalonate and 1,12-dodecanediamine is shown in Scheme 5.2, and the oligoamide structure elucidated by H NMR. 

Fig. 5 Thermal stability of derivatives of lipase B from Candida antarctica obtained by adsorption, incubated in 0.1 M sodium phosphate buffer, pH 7, at 50 or 60 °C...

Pentanol is an intermediate in the synthesis of several potential anti-Alzheimer s drugs, which inhibit a-amyloid peptide release and/or its synthesis (33). The enzymatic resolution of racemic 2-pentanol and 2-heptanol by lipase B from Candida antarctica has been demonstrated (34). 

Patel, R.N., Banerjee, A., Nanduri.V., Goswami, A., Come-zoglu, F.T. (2000) Enzymatic Resolution of Racemic Secondary Alcohols by Lipase B from Candida antarctica, J. Am. Oil Chem. Soc. 77,1015 1019. 

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