Lipases amine acylation

BASF successfully developed a process, which is operated on a rnulti-thousand-ton scale, for the resolution of chiral primary amines by lipase-catalyzed acylation... 

Pencillin G acylase from E. coli is functionally, but not structurally, related to lipases. The enzyme would find wider use if it could be rendered tolerant of low-water media, which is the kind of problem that ionic liquids were expected to solve. It was found, however, that a covalently immobilized penicillin acylase, PGA 450, required aw = 0.8, which also was the minimum in toluene, to stay active in the ionic liquids [BMIm][BF4], [OMIm][BF4], and [BMIm][PF6] [67]. In a simple amine acylation test reaction (Figure 10.4), PGA 450 was somewhat less active in ionic liquids than in toluene. 

Similarly, the DKR of chiral amines can, in principle, be achieved by combining the known amine resolution by lipase-catalyzed acylation [23] with metal-cat-... 

Another important difference between (dynamic) kinetic resolution of alcohols and amines is the ease with which the acylated product, an ester and an amide, respectively, is hydrolyzed. This is necessary in order to recover the substrate enantiomer which has undergone acylation. Ester hydrolysis is generally a facile process but amide hydrolysis, in contrast, is often not trivial. For example, in the BASF process [28] for amine resolution by lipase-catalyzed acylation the amide product is hydrolyzed using NaOH in aq. ethylene glycol at 150 °C (Fig. 9.18). In the case of phenethylamine this does not present a problem but it will obviously lead to problems with a variety of amines containing other functional groups. 

In order to broaden the scope we also examined [30] a combination of lipase-catalyzed acylation with penicillin acylase-catalyzed hydrolysis (deacylation). Good results (high enantioselectivity in the acylation and smooth deacylation) were obtained, with a broad range of both aliphatic amines and amines containing an aromatic moiety, using pyridylacetic acid ester as the acyl donor (Fig. 9.21). 

The first chemoenzymatic synthesis of organoselenium containing amines was recently reported by Andrade and Silva (Figure 14.7) [10]. Compounds containing a selenium atom have important antioxidant and anti inflammatory activities. Lipase mediated acylation of amine 13 gave the corresponding chiral amides 14 and amines 13 with excellent enantioselectivity (up to 99% ee). 

Some groups have reported on their search for less reactive acylating agents, to suppress noncatalyzed chemical acylation and increase product enantiomeric excess. Irimescu and Kato carried out an enantioselective lipase catalyzed acylation of 1 phenylethylamine and 2 phenyl 1 propylamine by reacting the amines with carbox ylic acids in a nonsolvent system or in ionic liquids (Figure 14.9). The reaction equilibrium was shifted toward amide synthesis by the continuous removal of the... 

The kinetic resolution of l,l-diethoxyethyl(hydroxymethyl)phosphinate racial possessing chirality at the phosphorus atom was achieved via a lipase-catalyzed acylation. The product 269 was transformed into the corresponding amine 270,... 

A third reason to use organic solvents is that other features of the reaction require it to be an acylation and not a hydrolysis. For example, some dynamic kinetic resolutions require an alcohol substrate because organometal-lic complexes racemize secondary alcohols, but not esters. The substrate must be an alcohol for the dynamic kinetic resolution to proceed. In another example, lipase- and esterase-catalyzed hydrolysis of amides to amines is too slow for practical use. However, the lipase-catalyzed acylation of amines in... 

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

In recent years, a great variety of primary chiral amines have been obtained in enantiomerically pure form through this methodology. A representative example is the KR of some 2-phenylcycloalkanamines that has been performed by means of aminolysis reactions catalyzed by lipases (Scheme 7.17) [34]. Kazlauskas rule has been followed in all cases. The size of the cycle and the stereochemistry of the chiral centers of the amines had a strong influence on both the enantiomeric ratio and the reaction rate of these aminolysis processes. CALB showed excellent enantioselec-tivities toward frans-2-phenylcyclohexanamine in a variety of reaction conditions ( >150), but the reaction was markedly slower and occurred with very poor enantioselectivity with the cis-isomer, whereas Candida antarctica lipase A (GALA) was the best catalyst for the acylation of cis-2-phenylcyclohexanamine ( = 34) and frans-2-phenylcyclopropanamine ( =7). Resolution of both cis- and frans-2-phenyl-cyclopentanamine was efficiently catalyzed by CALB obtaining all stereoisomers with high enantiomeric excess. 

An efficient chemoenzymatic route for the synthesis of optically active substituted indolines has been recently developed (Scheme 7.27), and also the alkoxycarbonyla-tion process is more efficient than the acylation reaction. Different lipases have been tested in the alkoxycarbonylation of these secondary amines, GALA being found to be the best biocatalyst for 2-substituted-indolines, and CALB for 3-methylindoline. The combination of lipases with a variety of allyl carbonates and TBME as solvent has allowed the isolation of the carbamate and amine derivatives in a high level of enantiopurity [51]. 

In a lipase-catalyzed reaction, the acyl group of the ester is transferred to the hydroxyl group of the serine residue to form the acylated enzyme. The acyl group is then transferred to an external nucleophile with the return of the enzyme to its preacylated state to restart the catalytic cycle. A variety of nucleophiles can participate in this process. For example, reaction in the presence of water results in hydrolysis, reaction in alcohol results in esterification or transesterification, and reaction in amine results in amination. Kirchner et al.3 reported that it was possible to use hydrolytic enzymes under conditions of limited moisture to catalyze the formation of esters, and this is now becoming very popular for the resolution of alcohols.4... 

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. 

The reversibility of hydrogen transfer reactions has been exploited for the racemi-zation of alcohols and amines. By coupling the racemization process with an enantioselective enzyme-catalyzed acylation reaction, it has been possible to achieve dynamic kinetic resolution reactions. The combination of lipases or... 

Another possible mechanism for the racemization of amino acid esters involves the in situ, transient, formation of Schiff s bases by reaction of the amine group of an amino acid ester with an aldehyde. Using this approach, DKR of the methyl esters of proline 5 and pipecolic acid 6 was achieved using lipase A from C. ant-arclica as the enantioselective hydrolytic enzyme and acetaldehyde as the racemiz-ing agent (Scheme 2.4). Interestingly, the acetaldehyde was released in situ from vinyl butanoate, which acted as the acyl donor, in the presence of triethylamine. The use of other reaction additives was also investigated. Yields of up to 97% and up to 97% e.e. were obtained [6]. 

Scientists at Huddersfield University in collaboration with Avecia have developed a DKR process involving the combination of immobilized Candida rugosa lipase and an iridium-based racemization catalyst (Scheme 2.30). By using carbonate 62 as the acyl donor, the racemic secondary amine 61 was converted to the corresponding carbamate (R)-63 in high yield and enantiomeric excess [32]. 

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]. 

A prominent example of chemoenzymatic catalysis in bio-organic chemistry is the dynamic kinetic resolution (DKR) of secondary alcohols (Scheme 9) [94, 95] and amines [96-99], In this process, a lipase is employed as an enantioselective acylation catalyst, and a metal-based catalyst ensures continuous racemization of the unreactive enantiomer. 

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

Other similar lipase/esterase resolution processes have been developed such as the use of Bacillus that esterase to produce the substituted propanoic acids that are precursors of non-steroidal anti-inflammatory drags, snch as naproxen and ibuprofen etc., and the formation of chiral amines by Celgene. Other methods start from prochiral precursors and have the advantage that enantioselective synthesis allows the production of particular isomers in yields approaching 100%, rather than the 50% yields characteristic of resolution processes. For instance Hoechst have patented the production of enantiomers using Pseudomonas fluorescens lipase to either acylate diols or hydrolyse diacetate esters. 

Figure 4.5 Empirical selection rule for lipases for preferential acylation of amines. Subtilisin prefers the opposite configuration.

Schering Plough demonstrated the kinetic resolution of a secondary amine (24) via enzyme-catalyzed acylation of a pendant piperidine (Scheme 7.13) [32]. The compound 27 is a selective, non-peptide, non-sulfhydryl farnesyl protein transfer inhibitor undergoing clinical trials as a antitumor agent for the treatment of solid tumors. The racemic substrate (24) does not contain a chiral center but exists as a pair of enantiomers due to atropisomerism about the exocylic double bond. The lipase Toyobo LIP-300 (lipoprotein lipase from Ps. aeruginosa) catalyzed the isobu-tylation of the (+) enantiomer (26), with MTBE as solvent and 2,2,2-trifluoroethyl isobutyrate as acyl donor [32]. The acylation of racemic 24 yielded (+) 26 at 97% and (-) 25 at 96.3% after 24h with an E >200. The undesired enantiomer (25)... 

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]. 

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