Lipases activated acyl donors

Once bearing some substituents, the decrease of polarity of the sucrose derivatives makes them soluble in less-polar solvents, such as acetone or tert-butanol, in which some lipases are able to catalyze esterifications. Unlike proteases, which necessitate most often the use of an activated acyl donor (such as vinyl or trifluoroethyl esters), lipases are active with simple esters and even the parent carboxylic acids in the presence of a water scavenger. The selectivity of the lipase-catalyzed second esterification is specific for OH-6 allowing the synthesis of mixed T,6 -diesters.123,124 For some lipases, a chain-length dependence on the regiochemistry was observed.125 Selectively substituted monoesters were thus prepared and studied for their solution and thermotropic behavior.126,127 Combinations of enzyme-mediated and purely chemical esterifications led to a series of specifically substituted sucrose fatty acid diesters with variations in the chain length, the level of saturation, and the position on the sugar backbone. This allowed the impact of structural variations on thermotropic properties to be demonstrated (compare Section III.l).128... 

Most of lipase-catalyzed acylations of sugars in organic solvents have been reported as transesterification rather than esterification reactions. The displacement of the equilibrium towards products has been accomplished by using activated acyl donors [58] such as 2,2,2-trichloroethyl esters and, more often, enol esters. The use of enol esters, such as a vinyl or an isopropenyl ester, was, in fact, first reported in lipase-catalyzed reactions with sugars [59]. In the reaction, an unstable enol is liberated which instantaneously tautomerizes to the corresponding aldehyde or ketone, making the reaction irreversible. 

Several techniques of displacing the reaction equilibrium to reach a quasi-irreversible situation have been used previously. For a review of these, see Faber and Riva [119]. The techniques of using activated acyl donors when resolving chiral alcohols afford a more or less irreversible acylation step in the reaction mechanism since the first product is designed to be a poor nucleophile or is supposed to tautomerize or otherwise leave the re tion system (Scheme 3). Some examples of acyl donors frequently used include 2-haloethyl, cyanomethyl, oxime, and enol esters. The rates of the acyl transfer reactions of racemic 2-octanol with various esters catalyzed by porcine pancreatic lipase were one to two orders of magnitude faster when activated esters were used compared with methyl or ethyl al-kanoates [120]. 

In 1992, Oda et al. reported a one-pot synthesis of optically active cyanohydrin acetates from aldehydes, which were converted to the corresponding racemic cyanohydrins through transhydrocyanation with acetone cyanohydrin, catalyzed by a a strongly basic anion-exchange resin [46]. The racemic cyanohydrins were acetylated by a lipase from P. cepacia (Amano) with isopropenyl acetate as the acyl donor. The reversible nature of the base-catalyzed transhydrocyanation enabled continuous racemization of the unreacted cyanohydrins, thereby effecting a total conversion (Figure 4.21). 

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. 

DKR of secondary alcohol is achieved by coupling enzyme-catalyzed resolution with metal-catalyzed racemization. For efficient DKR, these catalyhc reactions must be compatible with each other. In the case of DKR of secondary alcohol with the lipase-ruthenium combinahon, the use of a proper acyl donor (required for enzymatic reaction) is parhcularly crucial because metal catalyst can react with the acyl donor or its deacylated form. Popular vinyl acetate is incompatible with all the ruthenium complexes, while isopropenyl acetate can be used with most monomeric ruthenium complexes. p-Chlorophenyl acetate (PCPA) is the best acyl donor for use with dimeric ruthenium complex 1. On the other hand, reaction temperature is another crucial factor. Many enzymes lose their activities at elevated temperatures. Thus, the racemizahon catalyst should show good catalytic efficiency at room temperature to be combined with these enzymes. One representative example is subtilisin. This enzyme rapidly loses catalytic activities at elevated temperatures and gradually even at ambient temperature. It therefore is compatible with the racemization catalysts 6-9, showing good activities at ambient temperature. In case the racemization catalyst requires an elevated temperature, CALB is the best counterpart. 

The natural substrates of lipases are triglycerides and, in an aqueous environment, lipases catalyze their hydrolysis into fatty acids and glycerol. In anhydrous media, lipases can be active in the reverse reaction [19]. In fact, in the acylation step, acids, lactones, (cyclic) carbonates [20, 21], cyclic amides [22, 23], (cyclic) thioesters [24, 25], and cyclic phosphates [26] have been found to act as suitable acyl donors. In the deacylation step, apart from water, lipases also accept alcohols [27], amines [28, 29], and thiols [30] as nucleophiles although the specificity of lipases is lower for amines and thiols than for water and alcohols [31]. 

The applied catalytic system consisted of a Ru-Noyori-type racemization catalyst 1 (Fig. 12b) and Novozym 435. This catalyst combination tolerates a wide range of acyl donors, and it was expected that it would allow the use of bifunctional acyl donors for the formation of polycondensates. Before the start of the reaction, the monomer mixture showed the expected diastereomer ratio of (S,S) R,R) R,S) of 1 1 2 of the 1,4-diol employed. After 30 h of reaction the (5,5)-enantiomer almost completely disappeared, whereas the ratio of [R,R)- to (/ , 5)-monomer was ca. 3 1 (R S ca. 7 1). At a hydroxyl group conversion of 92% after 70h, no further conversion was observed and a final ratio of R,R) to R,S) of 16 1 (R S ca. 33 1) was obtained. Unfortunately, the molecular weights of the polymer were moderate at best (Mw = 3.4kDa) and Novozym 435 had to be added every few hours to compensate for the activity loss of the lipase. This suggests that Ru-catalyst 1 and Novozym 435 are not fully compatible. 

Anthonsen, T. and Hoff, B. (1998) Resolution of derivatives of 1,2-propanediol with lipase B from C antarctica. Effect of substrate structure, medium, water activity and acyl donor on enantiomeric ratio. Chem. Phys. Lipids, 93, 199-207. 

The resolution of chiral amines via lipase-catalyzed enantioselective acylation is now a major industrial process, but interest in adopting ionic liquid reaction media has been surprisingly scant. Interestingly, acids could be used as the acyl donor (Figure 10.15) rather than the usual activated ester in a range ofionic liquids. CaLB was employed as the biocatalyst, and water was removed to shift the equilibrium toward the product [130, 131]. The highest rates were found in [BMMIm][TfO], [EMIm][TfO], and [EMIm][BF4]. 

Figure 11.5 Influence of thermodynamic water activity, aw, on the hydrolytic ( ), alcoholytic ( ) and total (A) activity for the Candida antarctica lipase -catalyzed acylation of 2-pentanol with methyl propanoate as acyl donor.

Racemic l-azido-3-aryloxy-2-propanols 35 was resolved by the lipase-catalyzed kinetic resolution using different acyl donors to access to the enantiomers in optically pure form.66 The reduction of the azide group can afford the l-amino-3-aryloxy-2-propanols, which is present in numerous biologically active compounds such as [3-adrenolytic drugs (/ -blockers) used in the treatment of angina pectoris, hypertension and other cardiac diseases. 

Reaction rates by POS-PVA lipase gradually increased from C4 to C8, reached a plateau between C8 and 02, and then an activity decrease was observed, probably owing to a substrate-size feature limitation. From this set of results, caprylic acid (C8) maybe considered the best acyl donor for the synthesis of butyl esters in heptane by POS-PVA lipase. 

The dynamic cyanohydrin system was next challenged with lipase-catalyzed transesterification resolution using different operational conditions. Thus, different lipases, organic solvents, additives, and acyl donors were evaluated. Isopropenyl acetate 26 was chosen and used as acyl donor because its reaction produces acetone as by-product, which does not interfere in the reaction and the NMR spectra. Molecular sieve 4 A was also added in the dynamic resolution process to control the water activity. The lipase preparation PS-C I was chosen in the resolution process since it expressed the highest lipase activities for both the substrate structure and the enantiomeric selectivities. Different organic solvents were also... 

This first section wiU study different regjoselective processes of several types of nucleosides depending on the lipase used. Application of biotransformations over these compounds has acquired great importance in order to prepare new derivatives with interesting pharmacological activities. Two lipases, namely, Candida antarctica type B (CALB) and Pseudomonas cepacea, free (PSL) or Pseudomonas cepacea, immobilized (PSLC), are selective towards one of the two hydroxyl groups of different 2 -deoxynucleosides. Thus, it is possible to prepare the acylated compounds in S -position with CALB [10], whereas PSL is selective towards the secondary hydroxyl group [11]. Vinyl or oxime esters can be used as acyl donors. 

The work from Sheldon s group [10] was the first to present the use of ionic liquids in the enzymatic synthesis of esters. Since then, there have been many reports on biosynthesis of esters in ionic liquids. De los Rios et al. [64,65] synthesised a wide range of aliphatic organic esters, commonly used in the perfumery, flavour and pharmaceutical industries, by transesteriflcation from vinyl esters and alcohols catalysed by free CaLB in different 1,3-dialkylimidazolium-based ILs (Fig. 7.2). They analysed the effects of the alkyl chain lengths of the acyl donor and the alcohol. The optimum (C6 for acyl donor and C4 for alcohol) chain lengths were found because the activity decreased with further increase in alkyl chain length. The authors attributed the enzyme behaviour to a substrate modulation mainly due to the different affinity of the lipase towards the different substrates and steric hindrance and denaturalisation by small alcohol molecules. 

Amano G and Lipozyme RM IM) [20], The highest yield of terpinyl acetate was obtained by using Candida rugosa type VII. Near cero yield of terpinyl acetate was obtained with Amano PS and Amano G. An esterification up to of 53.0% was obtained under optimized conditions in continuous operation using acetic anhydride as acyl donor and Candida rugosa lipase as enzyme at 10 MPa and 50°C for 1.5 h. However, the enzyme activity decreased up to 50% after 10.5 h repeated esterification in a batch. 

Optically active tricyclic isoxazolidine 105, a precursor to (-)-rosmarinecine, was prepared from racemic hydroxy-nitrone 103 through a hydrolase-catalyzed kinetic resolution (KR). In particular, Candida antarctica lipase, fraction B (CAL-B), effectively catalysed the KR of ( )-103 in the presence of the acyl donor 104 in MeCN at 0-5 °C. The generated (R)-ester underwent a fast intramolecular 1,3-DC to 105 under the reaction conditions <05CC2369>. 

Wallace and Morrow used halogenated alcohols, such as 2,2,2-trichloroethyl, to activate the acyl donor and thereby improve the polymerization kinetics [53, 56], They also removed by-products periodically during reactions to further shift the equilibrium toward chain growth instead of chain degradation. They copolymerized bis(2,2,2-trichloroethyl) tmns-3,4-epoxyadipate and 1,4-butanediol using porcine pancreatic lipase as the catalyst. After 5 days, an enantioenriched polyester with Mw = 7900 g mol-1 and an optical purity in excess of 95% was formed (Scheme 4.6). 

In the formation of carboxylic esters in an anhydrous organic solvent, its hydrophobicity and the water activity have a major influence on the reaction [30, 36, 134l Hence, the organic solvent used can significantly influence the selectivity of a lipase-catalyzed enantiotopos- or enantiomer-differentiating reaction. Furthermore, the acyl donor may influence reactivity and selectivity. 

The use of an active carbon cloth (Kynol AGG 507-15) with a large surface (1500 m g ) has also been reported for the immobilization of a lipase from Burkholderia cepacia (Amano PS, enzyme immobilized on Gelite) [27]. The system has been studied for the KR of several sec-alcohols with vinyl acetate as the acyl donor, using conventional solvents in a batch system (Figure 17.2a). A comparison of the catalyst coated and uncoated by an IL was made, showing that the activity and stability for reuse were preserved only for the system coated with [EMIM][NTf2]. Gonversions of 50% were obtained, with values of 98% ee for this system, with toluene as the solvent... 

As shown in Scheme 3.2, the relative rate of the enantioselective acylation of ( )-2-octanol, catalyzed by porcine pancreatic lipase (PPL), was one to two orders of magnitude faster when activated esters were used as acyl donors instead of nonactivated methyl or ethyl alkanoates. 

For some enzymes, such as Pseudomonas sp. lipase (PSL), the liberated acid does not present any problems, but others like CRL are more sensitive and require more protection. For instance, when acetic anhydride is used, the Uberated acetic acid may lead to a decrease of the pH in the micro-environment of the enzyme, thus leading to a depletion of activity and selectivity. The CRL-catalyzed resolution of the bicyclic tetrachloroalcohol shown in Scheme 3.5, using acetic anhydride as acyl donor, initially proceeded with only moderate selectivity (E = 18). Addition of a weak inorganic or (preferably) organic base such as 2,6-lutidine which functions... 

A generally applicable method for the preparation of optically active epoxides makes use of a lipase-catalyzed resolution of halohydrins bearing the halogen in the terminal position (Scheme 3.10). Pseudomonas sp. lipase-catalyzed acylation of racemic halohydrins affords a readily separable mixture of (f )-halohydrin and the corresponding (5)-ester in good to excellent optical purities [193, 194]. Treatment of the latter with base leads to the formation of epoxides with no loss of optical purity. A semiquantitative comparison of the reaction rate obtained with different acyl donors using substrates of this type revealed that they were in the order ethyl... 

Lipases are able to catalyse the acylation of alcohols in addition to the hydrolysis of esters. For acylations, the reactions are typically carried out in low-water systems (water activity (a ) < 1)), to minimize hydrolysis, and with a suitably reactive acyl donor to ensure high rates of reaction and efficient conversions. Suitable acyl donors include oximes, vinyl esters and anhydrides (Scheme 4.5). 

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