Candida antarctica lipase synthesis

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. 

The complete transformation of a racemic mixture into a single enantiomer is one of the challenging goals in asymmetric synthesis. We have developed metal-enzyme combinations for the dynamic kinetic resolution (DKR) of racemic primary amines. This procedure employs a heterogeneous palladium catalyst, Pd/A10(0H), as the racemization catalyst, Candida antarctica lipase B immobilized on acrylic resin (CAL-B) as the resolution catalyst and ethyl acetate or methoxymethylacetate as the acyl donor. Benzylic and aliphatic primary amines and one amino acid amide have been efficiently resolved with good yields (85—99 %) and high optical purities (97—99 %). The racemization catalyst was recyclable and could be reused for the DKR without activity loss at least 10 times. 

Burkholderia cepacia lipase (Amano PS) 2,2 -Bis(diphenylphosphino)-1,1 -binaphthyl Candida antarctica lipase B Diversity-oriented synthesis... 

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... 

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 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. 

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],... 

Lozano, P., G. Villora, D. Gomez, A.B. Gayo, J.A. Sanchez-Conesa, M. Rubio and J.L. Iborra, Membrane Reactor with Immobilized Candida Antarctica Lipase B for Ester Synthesis in Supercritical Carbon Dioxide, Journal of Supercritical Fluids, 29, 121-128 (2004). 

In tandem with the use of supercritical carbon dioxide, in ester synthesis employing Candida Antarctica lipase B, adsorbed on silica gel, as the esterification catalyst under minimum water conditions.50... 

Irimescu, R., Furihata, K., Hata, K., Iwasaki, Y., and Yamane, T. 2001. Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of l,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol. J. Am. Oil Chem. Soc., 78, 285-289. 

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). 

Figure 24.2. Synthesis of positionally labeled, symmetrically structured TAGs of the MLM-type constituting MCFA at the end-positions, and EPA or DHA at the midposition, by a chemoenzymatic approach based on immobilized Candida antarctica lipase (CAL).

Aryl-l,4-dihydropyridine-3,5-dicarboxylates are widely studied due to their use in the treatment of cardiovascular diseases. Most of these compounds are synthesized using the Hantzsch method (Section 4.2.3.4.2) but this is less suitable for the synthesis of unsymmetrical or chiral derivatives. Enzymatic desymmetrization of bis(ethoxycarbonyl-methyl)-l,4-dihydropyridine-3,5-dicarboxylates, using Candida antarctica lipase B, can generate enantiopure 1,4-dihy-dropyridines in reasonable to high yields with good enantiomeric selectivity <2000TA4559>. 

Gotor-Femandez, V., Busto, E., and Gotor, V. 2006. Candida antarctica lipase B An ideal biocatalyst for the preparation of nitrogenated organic componnds. Advanced Synthesis Catalysis, 348 797-812. 

Lozano P, Villora G, Gomez D, Gayo AB, Sanchez-Conesa JA, Rubio M, and Iborra JL. Membrane reactor with immobilized Candida antarctica lipase B for ester synthesis in supercritical carbon dioxide. J. Supercrit. Fluids 2004 29(1-2) 121-128. 

A total synthesis of 1,3-dideoxynojirimycin starting from cyclopentadiene was proposed by Johnson et al. [206]. Photooxidation of cyclopentadiene and reductive workup with thiourea generates c/ -cyclopent-2-ene-l,4-diol, which is monoacylated with high enantioselectivity (>99% ee) with isoprenyl acetate and Candida antarctica lipase B (Novo Nordisk SP 435) to give 461. After silylation of 461 and subsequent treatment with KOH and oxidation, enantiomerically pure enone 462 is obtained [207]. 

Immobilized forms or reticulated crystals of Candida antarctica lipase are effective biocatalysts for the synthesis of pure enantiomers utilized as anti-inflammatory agents. For example, one route for produetion of the 5-isomers of 2-aryl propionic acids (ibuprofen, naproxen, ketoprofen, and flurbprofen) involves enantioselective hydrolysis of the corresponding raeemie esters. Arroyo has indicated that an immobilized form of C. antarctica lipase (fraetion B) is used to mediate the selective acetylation of a diol to form the 5-enantiomer of a monoaeetate (Fig. 5), which is further proeessed to obtain an antifungal agent. ... 

The application of ionic liquids in lipase biocatalysis has not remained entirely restricted to CaLB, PcL or CrL. Other lipases have been used in ionic liquids for ester synthesis such as Candida antarctica lipase A (CaLA) [15,16], Thermomyces lanuginosus lipase [17] (TLL), Rhizomucor miehei lipase (PmL), Pseudomonas fluorescens lipase (PJL) [18], Pig pancreas lipase (PpL) [17] and Alcaligenes sp. lipase (A5 L) [16]. 

Lozano P, Piamtongkam R, Kohns K, de Diego T, Vaultier M, Iborra JL (2007) Ionic liquids improve dtroneUyl ester synthesis catalyzed by immobilized Candida antarctica lipase B in solvent-free media. Green Chem 9 780-784... 

Fig. 8.1 Experimental set-up of the recirculating enzymatic membrane reactor used for the synthesis of butyl propionate from vinyl propionate and 1-butanol catalysed by Candida antarctica lipase B in supercritical carbon dioxide and supercritical carbon dioxide/ionic liquid biphasic system [17]...

The synthesis of butyl butyrate was also successfully carried out in [bmim ] [NTfj-J/scCOj biphasic systems using free Candida antarctica lipase B as catalyst... 

Fig. 8.4 Initial reaction rate (bars) and selectivity (points) exhibited by free Candida antarctica lipase B for butyl propionate synthesis in supercritical carbon dioxide and in four different ionic liquids/supercritical carbon dioxide systems. The reaction conditions were r=50°C, vinyl propionate 150 mM and 1-butanol 100 Mm [38]...

Romero MD, Calvo L, Alba C et al (2005) Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in supercritical caibon dioxide. J Supercrit Fluids 33 77-84... 

Lozano P, De Diego T, Carrie D et al (2001) Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis. Biotechnol Lett 23 1529-1533... 

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