Enzymatic synthesis Candida antarctica

Ionic liquids are promising nonaqueous solvents for the dissolution of carbohydrates and they have been used in several studies on enzymatic sugar ester synthesis. Candida antarctica lipase B was the best enzyme for synthesis of glucose esters in a two-phase system containing an ionic liquid and rm-butanol [31]. Recently, the use of supersaturated sugar solutions [32] and ultrasonic treatment [33] has been reported to make sugar ester synthesis in ionic liquids even more efficient. 

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 multipolymer enzymatic resolution of soluble polymer-supported alcohols 42 and 43 was achieved using an immobilised lipase from Candida Antarctica (Novozym 435). The R-alcohol was obtained in enantiomerically pure form (>99% ee) after its cleavage from the poly(ethylene) glycol (PEG) scaffold . The achiral hydantoin- and isoxazoline-substituted dispirocyclobutanoids 47 were produced using both solution and solid-phase synthesis <00JOC3520, OOCC1835>. 

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]. 

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)... 

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],... 

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],... 

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). 

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>. 

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]...

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... 

Since they already had a good synthesis of racemic ester, a late stage in the synthesis, an enzymatic hydrolysis looked a good bet. In the event hydrolysis with Candida antarctica lipase was superlative hydrolysis gave only the required enantiomer and stopped cleanly at 50% conversion. Various supported versions were tried and the best was the Boehringer L-2 preparation in which the enzyme is covalently bound to a cross-linked resin making it stable in aqueous solution. In the pilot plant 76 kgm of racemic methyl ester 230 react in 2.5 hours with 6 kg of supported enzyme. Benzyl chloroformate in CH2C12 is added and the unreacted ester (7 )-230,... 

Many of the polymerizations presented in this book proceed in organic solvents. To enhance the stability of enzymes in these solvent systems and to ensure efficient recovery of the biocatalysts the enzymes are commonly immobilized. Chapter 2 reviews some of the new trends of enzyme immobilization on nanoscale materials, while Chapter 3 sheds light on some new approaches to improve the commercial immobilization of Candida antarctica lipase B - the biocatalyst most often employed in enzymatic polymer synthesis. 

In this work we have shown that it is possible to use an ezyme to catalyze the polycondensatioh reaction to form polyamides. The large number of polyamides that have been made indicate diat lipases from Candida antarctica and Mucor miehei are rather nonspecific and can be used generally for polyamide synthesis. In a particular example, a water-soluble polyamide has been produced from dimethyl adipate and ethylene triamine via this enzyme-catalyzed reaction at 50-110°C. The enzymatic polymerization is easy to do and... 

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). 

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

Enzymatic synthesis of a methacrylamide-type polyester macromonomer was reported (80,81). In the polymerization of 12-hydroxydodecanoic acid in the presence of 11-methacryloylaminoundecanoic acid using lipase CC or Candida antarctica lipase (lipase CA) as catalyst, the polymerizable group was quantitatively incorporated into terminal of the poljmier chain. 

Enzymatic synthesis of fV-acylalanine is possible and has been demonstrated recently. Candida antarctica lipase (CAL) has been widely used for the amidation of esters and amines. Izumi and coworkers [75] prepared a N-lauroyl-3-alanine homologous series in organic media, where the enzymatic amidation of 3-amino-propionitrile and P-alanine ethyl ester reacted with methyl caproate or methyl laurate by immobilized C. antarctica lipase as follows ... 

Radzi, S., M. Basri, A. Salleh, A. Arif, R. Mohammad, M. B. Abdul Rahman, and R. N. Z. R. Abdul Rahman. 2005. High Performance Enzymatic Synthesis of Oleyl Oleate Using Immobilised Lipase from Candida Antarctica. Electronic Journal of Biotechnology 8 (3) 291-298. 

Romero, M. D., L. Calvo, C. Alba, M. Habulin, M. Primozic, and Z. Knez. 2005. Enzymatic Synthesis of Isoamyl Acetate with Immobilized Candida Antarctica Lipase in Supercritical Carbon Dioxide. Journal of Supercritical Fluids 33 (l) 77-84. 

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