Lipase amino acid esters

The advantages of the methoxyethyl ester over some of the other water-solubilizing esters are that many of the amino acid esters are crystalline and thus easily purified, are cleaved with a number of readily available lipases, and are useful for the synthesis of A-linked glycopeptides. ... 

Dynamic Kinetic Resolution Synthesis of a Fluorinated Amino Acid Ester Amide by a Continuous Process Lipase-mediated Ethanolysis of an Azalactone... 

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]. 

Immobilized enzymes are currently the object of considerable interest. This is due to the expected benefits over soluble enzymes or alternative technologies. The number of applications of immobilized enzymes is increasing steadily [5]. Occasionally, however, experimental investigations have produced unexpected results such as a significant reduction or even an increase in activity compared with soluble enzymes. Thus, cross-linked crystals of subtihsin showed 27 times less activity in the aqueous hydrolysis of an amino acid ester compared to equal amounts of soluble enzyme [6]. On the other hand, in the application of hpo-protein lipase in the solvent-mediated synthesis of esters there was a 40-fold increase in activity using immobihzed or otherwise modified enzyme preparations as compared to enzyme powder [7]. 

Kawashiro, K., Kaiso, K., Minato, D., Sugiyama, S., and Hayashi, H. (1993) Lipase-catalyzed peptide synthesis using Z-amino acid esters as acyl donors in aqueous water-miscible organic solvents. Tetrahedron, 49,4541-4548. 

JY Houng, ML Wu, ST Chen. Kinetic resolution of amino acids esters catalyzed by lipases. Chirality 8 418-422,1996. 

Serine proteases have the same machinery as lipases and esterases but do not have the common fold. They all hydrolyze peptides, and for this purpose have recognition sites, for the amine and acid sides of the peptide bond to be cleaved, determining their amino acid specificity. They can catalyze the hydrolysis, transesterification, and synthesis of amino acid esters. 

Kanerva, L.T., Csomos, P., Sundholm, O., Bernath, G., and Fiilop, F. (1996) Approach to highly enantiopure P-amino acid esters by using lipase catalysis in organic media. Tetrahedron Asymmetry, 7, 1705-1716. 

Lipase Amino acid surfactants Ester bond formation between free fatty acids and hydroxyls of arginine esterified to glycerol Infante et al., 2009... 

Selective hydrolysis of esters is a well-established procedure for the resolution of chiral carboxylic acids. Enzymes such as hydrolases, lipases and proteases are utilized. Due to their high selectivity for (5)-amino acids, proteases have been widely used in the selective transformations of amino acids and their derivatives. a-Chymotrypsin- a serine protease -catalyzes not only the hydrolysis of amide bonds, but also the cleavage of various esters, including a-alkyl-a-amino acid esters. One application is the synthesis of (5)-a-[ C]-methyltryptophan The anion synthesized by LDA-deprotonation of M-benzylidene tryptophan methyl ester (T) was alkylated with [ CJmethyl iodide to obtain the methyl A-benzylidene derivative 2, which was hydrolyzed under acidic conditions. Subsequent selective cleavage of the ester group with a-chymotrypsin provided the enantiomerically pure (5)-amino acid 3 in 33% radiochemical yield. 

This ester was developed to impart greater hydrophilicity in C-terminal peptides that contain large hydrophobic amino acids, since the velocity of deprotection with enzymes often was reduced to nearly useless levels. Efficient cleavage is achieved with the lipase from R. niveus (pH 7, 37°, 16 h, H2O, acetone, 78-91% yield)... 

Lipase-catalyzed methanolysis of racemic N-benzyloxycarbonyl (Cbz) amino acid trifluoroethyl esters carrying aliphatic side chains afforded the L-methyl esters and the D-trifluoromethyl esters (Figure 6.16). The released alcohol (CF3CH2OH) is a weak nucleophile that cannot attack the ester product. The nucleophilidty of the leaving group is depleted by the presence of an electron-withdrawing group [63]. 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

Fig. 16. Binding pocket of the lipase from P. aeruginosa for the acid part of 2-methyldecanoic acid p-nitrophenyl ester showing the geometric position of amino acids 160-163 which were randomized in cassette mutagenesis (49).

An elegant method to suppress the undesired spontaneous hydrolysis of a 5(47/)-oxazolone in aqueous media uses a lipase-catalyzed alcoholysis reaction. Of particular importance is the synthesis of /crt-leucine, a non-proteinogenic a-amino acid that has found widespread use both as a chiral auxiliary and as a component of potentially therapeutic pseudopeptides. Racemic 4-/ert-butyl-2-phenyl-5(47/)-oxazolone 238 was submitted to Mucor miehei catalyzed alcoholysis using butanol as a nucleophile. Addition of a catalytic amount of triethylamine promoted in situ racemization. In this way, the enantiomericaUy pure butyl ester of (5)-A-benzoyl-/ert-leucine 239 was obtained in excellent yield (Scheme 7.75). 

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. 

Several synthetic applications are given in Scheme 16 (40). The hydrogenation of a /3-keto ester on a 6-100-kg scale is used for the synthesis of tetrahydrolipostatin, a pancreatic lipase inhibitor developed at Hoffmann-LaRoche Company (1c). Tartaric acid is the best chiral modifier a-amino acids or a-hydroxy acids are not satisfactory. The source... 

---