Organic solvents, lipase reaction

You carry out a reaction in an organic solvent. The reaction is catalysed by a lipase immobilised by adsorption on a porous support. The reaction virtually stops long before equilibrium is reached and you suspect that this can be due to the formation of an acidic reaction product. What can you do to increase the buffering capacity of the system ... 

C. S. Chen and C. J. Sih, Enantioselective biocatalysis in organic solvents. Lipase catalyzed reactions, Angew. Chem. 1989,... 

Ghanem, A. The Utility of cyclodextrins in lipase-catalyzed transesterification in organic solvents enhanced reaction rate and enantioselectivity. Org. Biomol. Chem., 2003, 1, 1282 -1291. 

Although reaction routes are influenced by organic solvents, some enzymes retain their activity [11]. For example, in organic solvents, lipases [12] esterify the saccharides nearly quantitatively, but in aqueous solutions ester hydrolysis prevails. 

For bench-scale synthesis of borage or ev iing primrose oil-based structured lipids, 297-300 mg oil were mixed with 115 mg EPA and/or 120 mg DHA. The mole ratio was 1 1 for oil to EPA or DHA. The reactions were conducted in 3 mL of hexane or in the absence of any organic solvent. Lipases from Candida antarctica (Novozym-435) or Pseudomonas sp. (PS-30) were added to the reaction mixture and incubated in an orbital shaker at 37 C for 24 h at 250 rpm. 

The use of organic solvents as reaction media for biocatalytic reactions can not only overcome the substrate solubility issue, but also facilitate the recovery of products and biocatalysts as well. This technique has been widely employed in the case of lipases, but scarcely applied for biocatalytic reduction processes, due to the rapid inactivation and poor stability of redox enzymes in organic solvents. Furthermore, all the advantages for nonaqueous biocatalysis can take effect only if the problem of cofactor dependence is also solved. Thus, bioreductions in micro- or nonaqueous organic media are generally restricted to those with substrate-coupled cofactor regeneration. 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

The report from Sheldon and co-workers was the second publication demonstrating the potential use of enzymes in ionic liquids and the first one for lipases (Entry 13) [43]. They compared the reactivity of Candida antarctica lipase in ionic liquids such as [BMIM][PFg] and [BMIM][BF4] with that in conventional organic solvents. In all cases the reaction rates were similar for all of the reactions investigated alcoholysis, ammoniolysis, and per hydrolysis. 

Of course, the influence of organic solvents on enzyme enantioselectivity is not limited to proteases but it is a general phenomenon. Quite soon, different research groups described the results obtained with lipases [28]. For instance, the resolution of the mucolytic drug ( )-trans-sobrerol (11) was achieved by transesteriflcation with vinyl acetate catalyzed by the lipase from Pseudomonas cepacia adsorbed on celite in various solvents. As depicted in Scheme 1.3 and Table 1.5, it was found that t-amyl alcohol was the solvent of choice in this medium, the selectivity was so high ( >500) that the reaction stopped spontaneously at 50% conversion giving both +)4rans-sobrerol and (—)-trans-sobrerol monoacetate in 100% optical purity [29]. 

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. 

Ganske and co-workers reported that lipase-catalyzed acylation of a glucose derivative proceeded smoothiy in a mixed soivent of [bmim][BF4] with r-BuOH, while no reaction took place in [bmim][BF4] (Fig. 12). These results taught us that a mixed soivent system of IL with organic solvent may be a good solution if the desired reaction did not take piace in a pure IL solvent. 

Lipase is an enzyme which catalyzes the hydrolysis of fatty acid esters normally in an aqueous environment in living systems. However, hpases are sometimes stable in organic solvents and can be used as catalyst for esterifications and transesterifications. By utihzing such catalytic specificities of lipase, functional aliphatic polyesters have been synthesized by various polymerization modes. Typical reaction types of hpase-catalyzed polymerization leading to polyesters are summarized in Scheme 1. Lipase-catalyzed polymerizations also produced polycarbonates and polyphosphates. 

Several enzymes like lipases, esterases, and dehydrogenases have been active in hydrophobic environments. Thermodynamic water activity is a good predictor of the optimal hydration conditions for catalytic activity [51]. Enzyme preparation can be equilibrated at a specific water activity before the reaction [52]. When water concentration is very low, enzyme is suspended in the solid state in the water-immiscible organic solvent [46]. Enzymes are easily recovered after the reaction by the method of filtration. 

In conventional synthetic transformations, enzymes are normally used in aqueous or organic solvent at moderate temperatures to preserve the activity of enzymes. Consequently, some of these reactions require longer reaction times. In view of the newer developments wherein enzymes can be immobilized on solid supports [183], they are amenable to relatively higher temperature reaction with adequate pH control. The application of MW irradiation has been explored with two enzyme systems namely Pseudomonas lipase dispersed in Hyflo Super Cell and commercially available SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin). 

The reaction mixture is often complicated by condensation of mono-saccharidic molecules yielding unwanted homodisaccharides. This problem is also reduced by the minimum water approach (water activity of ca. 0.7-0.8). The organic solvent at low concentration probably deactivates the enzyme due to structural changes whereas high solvent concentrations with the necessary minimum water cause fixation of the enzyme structure in its active conformation. Unfortunately, glycosidases are rather instable in low-water media. This is a big drawback compared to highly solvent-resistant lipases.94... 

Cull, S. G. Holbrey, J. D. Vargas-Mora, V. et al. Room-temperature ionic liquids as replacements for organic solvents in multiphase bioprocess operations, BiotechnoL Bioeng., 2000, 69(2), 227-233 Lau, R. M. van Rantwijk, F. Seddon, K. R. Sheldon, R. A. Lipase-catalyzed reactions in ionic liquids, Org. Lett., 2000, 2(26), 4189-4191. 

By screening a variety of lipases in organic solvent for their ability to acylate the racemic hydroxynitrile with succinic anhydride, Novozym 435 was found to yield the best results, affording product in 94-95 % ee at conversions of 47 9 % (Scheme 1.34). After optimization, the reaction was successfully run at 22 kg scale. The immobilized catalyst could be easily isolated by filtration and reused. 

Reaction in organic solvent can sometimes provide superior selectivity to that observed in aqueous solution. For example, Keeling et al recently produced enantioenriched a-trifluoromethyl-a-tosyloxymethyl epoxide, a key intermediate in the synthetic route to a series of nonsteroidal glucocorticoid receptor agonist drug candidates, through the enan-tioselective acylation of a prochiral triol using the hpase from Burkholderia cepacia in vinyl butyrate and TBME (Scheme 1.59). In contrast, attempts to access the opposite enantiomer by desymmetrization of the 1,3-diester by lipase-catalysed hydrolysis resulted in rapid hydrolysis to triol under a variety of conditions. 

Secundo, E., Riva, S. and Carrea, G., Effects of medium and of reaction conditions on the enantioselectivity of lipases in organic solvents and possible rationales. Tetrahedron Asymm. 1992, 3, 267-280. 

The lipase-catalyzed resolutions usually are performed with racemic secondary alcohols in the presence of an acyl donor in hydrophobic organic solvents such as toluene and tert-butyl methyl ether (Scheme 1.3). In case the enzyme is highly enantioselective E = 200 or greater), the resolution reaction in general is stopped at nearly 50% conversion to obtain both unreacted enantiomers and acylated enantiomers in enantiomerically enriched forms. With a moderately enantioselective enzyme E = 20-50), the reaction carries to well over 50% conversion to get unreacted enantiomer of high optical purity at the cost of acylated enantiomer of lower optical purity. The enantioselectivity of lipase is largely dependent on the structure of substrate as formulated by Kazlauskas [6] most lipases show... 

In a recent review, some positive attributes of ionic liquids in biocatalysis were discussed 273). An example was given, which compares the enzymatic performance of Pseudomonas cepacia lipase (PCL)-catalyzed reactions as a function of the solvent polarity in both organic and ionic solvents, as shown in Fig. 17. The PCL shows no activity in organic solvents in the polarity range of the ionic liquids, but it is active in the ionic liquids. 

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... 

Lipases are enzymes that catalyze the in vivo hydrolysis of lipids such as triacylglycerols. Lipases are not used in biological systems for ester synthesis, presumably because the large amounts of water present preclude ester formation due to the law of mass action which favors hydrolysis. A different pathway (using the coenzyme A thioester of a carboxylic acid and the enzyme synthase [Blei and Odian, 2000]) is present in biological systems for ester formation. However, lipases do catalyze the in vitro esterification reaction and have been used to synthesize polyesters. The reaction between alcohols and carboxylic acids occurs in organic solvents where the absence of water favors esterification. However, water is a by-product and must be removed efficiently to maximize conversions and molecular weights. 

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