(enzymes of the class of hydrolases, which catalyze the hydrolysis of carboxylic acid esters1)

Solubility: insol cold and warm H2O.

Form Supplied in: available from various sources (microorganisms and mammalian) as powders or water suspensions.

Handling, Storage, and Precautions: stable at a pH range 6-10; can be stored at 0-4 °C for months.

Esterase-Catalyzed Hydrolysis.

Hydrolytic enzymes have been accepted in organic synthesis as valuable biocatalysts, since they are commercially available at relatively low price and possess a broad substrate specificity, without necessitating use of expensive cofactors.2 Esterases are such useful enzymes and have been widely used for the preparation of enantiomerically pure chiral compounds, by hydrolytic resolution of racemic esters or asymmetrization of prochiral substrates.3 Well defined experimental procedures for a pig liver esterase-catalyzed saponification have been documented.4 Generally, the enzymatic hydrolysis is carried out in an aqueous buffer, sometimes containing cosolvents,5 at pH 7-9 and keeping the temperature at 20-25 °C. Generally, the molar equivalent NaOH for the hydrolysis is added maintaining the pH constant with an automatic titrator and, after acidification, the product is extracted with organic solvents. Esterases are commercially available from various sources, either microbial or mammalian, and in some instances also crude acetone powders can be used for the same purpose.

Pig Liver Esterase (PLE).

This is the more used carboxylesterase (carboxylic-ester hydrolase, EC, CAS 9016-18-6) which physiologically catalyzes the hydrolysis of carboxylic acid esters to the free acid anion and alcohol.1 PLE is a serine hydrolase which has been widely used for the preparation of chiral synthons and these applications have been fully reviewed.6 An active-site model for interpreting and predicting the specificity of the enzyme has been published.7 In the pioneering studies of the enzyme applications field, PLE was used for the chiral synthesis of mevalonolactone.8 Prochiral 3-substituted glutaric acid diesters are well suited for a PLE-catalyzed asymmetrization, which leads to optically active monoesters (eq 1).9

The asymmetrization of prochiral disubstituted malonates has been enantioselectively realized in the presence of PLE (eq 2).10

The asymmetric hydrolysis of several cyclic meso-diesters has been accomplished and optically pure monoesters have been obtained.11 A classical example is the hydrolysis of dimethyl cis-4-cyclohexene-1,2-dicarboxylate, which affords the corresponding nearly optically pure half ester, a versatile synthon for various chiral cyclohexane derivatives (eq 3).12

The PLE-catalyzed asymmetric hydrolysis of meso-1,3-cis-3,5-cis-1,3-diacetoxy-5-benzyloxycyclohexane afforded (1S,3S,5R)-1-acetoxy-5-benzyloxycyclohexan-3-ol, which could be used as chiral building block for the synthesis of the compactin lactone moiety and quinic acid (eq 4).13

Organometallic meso-diesters can be asymmetrized as well to the corresponding half ester, as shown for an (arene)tricarbonylchromium diester (eq 5).14

The resolution of racemic esters is catalyzed by PLE in a highly enantioselective fashion.3,6 Several interesting applications of this method are available. The hydrolysis of trans-bicyclo[2.2.1]heptane diesters has been studied to ascertain the structural requirements for the PLE hydrolysis.15 A bulky tricyclodecadienone ester can be resolved by an highly enantioselective reaction (eq 6).16

The resolution procedure applies to racemic organometallic esters17 and to the esters of a thianucleoside, for the preparation of pure enantiomers of an antiviral agent (2,3-dideoxy-5-fluoro-3-thiacytidine) (eq 7).18

PLE has usually been applied to the enantioselective preparation of optically active compounds, but its use can be extended to chemo- or regioselective hydrolyses. A continuous process for the separation of a cis/trans unsaturated ester was realized using immobilized PLE (eq 8).19

The chemoselective hydrolysis of an acetoxy group in the presence of a g-lactone ring has been reported in the presence of PLE (eq 9).20 In a benzylpenicillin, PLE catalyzes the chemoselective hydrolytic opening of the b-lactam ring, the methoxycarbonyl moiety remaining uneffected (eq 10).21

Dimethyl malate presents two ester functions a and b with respect to a hydroxy group, and PLE is able to regioselectively discriminate between these two moieties.22

Acetone Powder Containing Esterase Activity.

The main advantage in using crude homogenates or acetone powders of organs such as liver is to have a cheap source of different enzymes. If one of these is desired for a specific substrate, the crude enzymatic mixture can be used with some advantage, compared to the purified enzymes. Pig liver acetone powder (PLAP), together with other extracts, is commercially available or can be prepared from fresh pig liver.23 PLAP has been used for the enantioselective hydrolysis of the racemic acetate of trans-2-phenylcyclohexanol (eq 11).24

The acetates of 1-arylalkan-1-ols were successfully resolved by acetone powders (PLAP and goat liver acetone powder, GLAP) containing esterase activity (eq 12).25

An interesting application of the esterase activity of horse liver acetone powder (HLE) has been the enantioselective hydrolysis of racemic lactones. The powder proved to be more effective than PLE in this hydrolysis, from which the unreacted lactone was recovered with high enantiomeric excess. The process seems more effective for d and medium size lactones (eq 13).26

Cholesterol Esterase.

This enzyme (EC; CAS 9026-00-0) physiologically catalyzes the hydrolysis of cholesterol esters, monoacylglycerols, and vitamin esters.27 It has also been used for several cyclic and noncyclic substrates with variable enantioselectivity.28 The resolution of racemic esters has been reported29 and an interesting example is the application to the racemic acetate of an hemiacetal (eq 14).30

Acyl Cholinesterases.

Acetylcholinesterase (AChE; EC; CAS 9000-81-1) is the serine esterase which catalyzes the hydrolysis of acetylcholine and possesses an esteratic site,31 and which is responsible for unspecific hydrolyses of several substrates. Also, butyrylcholinesterase (EC; CAS 9001-08-5) has been sometimes used for asymmetric hydrolysis of esters.32 Acetylcholinesterase has been used for the hydrolysis of noncyclic substrates and the results have shown satisfactory enantioselectivity.32a,33 The enzyme from electric eel seems especially well suited to the hydrolysis of cyclic diols.34 The asymmetrization of cis-3,5-diacetoxycyclopent-1-ene to (3R)-acetoxy-(5S)-hydroxycyclopent-1-ene (eq 15)35 and the preparation of an optically active triol monoacetate starting from the triacetate of 1,3,6-trihydroxycyclohept-4-ene (eq 16)36 are good examples of successful reactions catalyzed by acetylcholinesterase.

Other Esterases.

Other less common esterases have been sometimes used for biocatalytic applications in organic synthesis.37 The enzymatic approach can be the method of choice for the preparation of optically pure drugs, although sometimes special enzymes have to be prepared for this aim. By cloning a carboxylesterase into a microorganism, high level production of the esterase is made possible for the production of 2-(aryloxy)propionates and (S)-naproxen.38 The esterase activity of rabbit plasma has been used for a chemoselective hydrolysis of a methylthiomethyl ester.39 An esterase from Candida lypolitica has been used for the resolution of a tertiary a-substituted carboxylic acid ester.40 Recently, a carboxyl esterase of molecular weight 30 000 (Esterase 30 000) has been introduced for the asymmetric hydrolysis of diesters. A cyclopropyl malonate has been hydrolyzed by the esterase and the unreacted diester was recovered nearly optically pure (eq 17).41 Diethyl 3-hydroxyglutarate, a substrate which is asymmetrized with modest enantioselectivity with PLE or other enzymes,9e,f has been enantioselectively hydrolyzed in the presence of Esterase 30 000 (eq 18).42

1. (a) Junge, W. In Methods of Enzymatic Analysis; Bergmeyer, H. U. Ed.; Verlag Chemie: Weinheim, 1984; Vol. IV, p 2. (b) Enzyme Handbook; Schomburg, D.; Salzmann, M., Eds.; Springer: Berlin, 1991; Vol. III.
2. Jones, J. B. T 1986, 42, 3351.
3. (a) Boland, W.; Frössl, C.; Lorenz, M. S 1991, 1049. (b) Santaniello, E.; Ferraboschi, P.; Grisenti, P.; Manzocchi, A. CRV 1992, 92, 1071.
4. Eberle, M.; Missbach, M.; Seebach, D. OS 1990, 69, 19.
5. (a) Guanti, G.; Banfi, L.; Narisano, E.; Riva, R.; Thea, S. TL 1986, 27, 4639. (b) Björkling, F.; Boutelje, J.; Hjalmarsson, M.; Hult, K.; Norin, T. CC 1987, 1041. (c) Lam, L. K. P.; Hui, R. A. H. F.; Jones, J. B. JOC 1986, 51, 2047.
6. (a) Ohno, M.; Otsuka, M. OR 1989, 37, 1. (b) Zhu, L.-M.; Tedford, M. C. T 1990, 46, 6587. (c) Jones, J. B. PAC 1990, 62, 1445.
7. Toone, E. J.; Werth, M. J.; Jones, J. B. JACS 1990, 112, 4946.
8. Huang, F.-C.; Hsu Lee, L. F.; Mittal, R. S. D.; Ravikumar, P. R.; Chan, J. A.; Sih, C. J.; Caspi, E.; Eck, C. R. JACS 1975, 97, 4144.
9. (a) Mohr, P.; Waespe-&SSbreve;ar&cbreve;evi&cgrave;, N.; Tamm, C.; Gawronska, K.; Gawronski, J. K. HCA 1983, 66, 2501. (b) Brooks, D. W.; Palmer, J. T. TL 1983, 24, 3059. (c) Francis, C. J.; Jones, J. B. CC 1984, 579. (d) VanMiddlesworth, F.; Wang, Y. F.; Zhou, B.-N.; DiTullio, D.; Sih, C. J. TL 1985, 26, 961. (e) Mohr, P.; Rösslein, L.; Tamm, C. HCA 1987, 70, 142. (f) Santaniello, E.; Chiari, M.; Ferraboschi, P.; Trave, S. JOC 1988, 53, 1567. (g) Andruszkiewicz, R.; Barrett, A. G. M.; Silverman, R. B. SC 1990, 20, 159. (h) Chênevert, R.; Desjardins, M. TL 1991, 32, 4249.
10. (a) Schneider, M.; Engel, N.; Boensmann, H. AG(E) 1984, 23, 66. (b) Björkling, F.; Boutelje, J.; Gatenbeck, S.; Hult, K.; Norin, T. TL 1985, 26, 4957. (c) Luyten, M.; Müller, S.; Herzog, B.; Keese, R. HCA 1987, 70, 1250. (d) De Jeso, B.; Belair, N.; Deleuze, H.; Rascle, M.-C.; Maillard, B. TL 1990, 31, 653. (e) Fadel, A.; Canet, J.-L.; Salaün, J. SL 1991, 60.
11. (a) Schneider, M.; Engel, N.; Hönicke, P.; Heinemann, G.; Görisch, H. AG(E) 1984, 23, 67. (b) Sabbioni, G.; Shea, M. L.; Jones, J. B. CC 1984, 236. (c) Gais, H.-J.; Lukas, K. L.; Ball, W. A.; Braun, S.; Lindner, H. J. LA 1986, 687. (d) Naemura, K.; Takahashi, N.; Chikamatsu, H. CL 1988, 1717. (e) Zemlicka, J.; Craine, L. E.; Heeg, M.-J.; Oliver, J. P. JOC 1988, 53, 937. (f) Brion, F.; Marie, C.; Mackiewicz, P.; Roul, J. M.; Buendia, J. TL 1992, 33, 4889. (g) Hutchinson, E. J.; Roberts, S. M.; Thorpe, A. J. JCS(P1) 1992, 2245.
12. Kobayashi, S.; Kamiyama, K.; Iimori, T.; Ohno, M. TL 1984, 25, 2557.
13. Suemune, H.; Matsuno, K.; Uchida, M.; Sakai, K. TA 1992, 3, 297.
14. Malézieux, B.; Jaouen, G.; Salaün, J.; Howell, J. A. S.; Palin, M. G.; McArdle, P.; O'Gara, M.; Cunningham, D. TA 1992, 3, 375.
15. Klunder, A. J. H.; van Gastel, F. J. C.; Zwanenburg, B. TL 1988, 29, 2697.
16. Klunder, A. J. H.; Huizinga, W. B.; Hulshof, A. J. M.; Zwanenburg, B. TL 1986, 27, 2543.
17. Alcock, N. W.; Crout, D. H. G.; Henderson, C. M.; Thomas, S. E. CC 1988, 746.
18. Hoong, L. K.; Strange, L. E.; Liotta, D. C.; Koszalka, G. W.; Burns, C. L.; Schinazi, R. F. JOC 1992, 57, 5563.
19. Klibanov, A. M.; Siegel, E. H. Enzyme Microb. Technol. 1982, 4, 172.
20. Wang, Y.-F.; Sih, C. J. TL 1984, 25, 4999.
21. Jones, M.; Page, M. I. CC 1991, 316.
22. Papageorgiou, C.; Benezra, C. JOC 1985, 50, 1144.
23. (a) Adachi, K.; Kobayashi, S.; Ohno, M. C 1986, 40, 311. (b) Seebach, D.; Eberle, M. C 1986, 40, 315.
24. Whitesell, J. K.; Lawrence, R. M. C 1986, 40, 318.
25. Basavaiah, D.; Raju, S. B. SC 1991, 21, 1859.
26. (a) Fouque, E.; Rousseau, G. S 1989, 661. (b) Guibé-Jampel, E.; Rousseau, G.; Blanco, L. TL 1989, 30, 67.
27. Rudd, E. A.; Brockman, H. L. In Lipases; Borgström, B.; Brockman, H. L., Eds.; Elsevier: Amsterdam, 1984; p 185.
28. Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A. JOC 1991, 56, 2656.
29. Chenault, H. K.; Kim, M.-J.; Akiyama, A.; Miyazawa, T.; Simon, E. S.; Whitesides, G. M. JOC 1987, 52, 2608.
30. Chênevert, R.; Desjardins, M.; Gagnon, R. CL 1990, 33.
31. Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 253, 872.
32. (a) Dropsy, E. P.; Klibanov, A. M. Biotechnol. Bioeng. 1984, 26, 911. (b) Aragozzini, F.; Valenti, M.; Santaniello, E.; Ferraboschi, P; Grisenti, P. Biocatalysis 1992, 5, 325.
33. Santaniello, E.; Canevotti, R.; Casati, R.; Ceriani, L.; Ferraboschi, P; Grisenti, P. G 1989, 119, 55.
34. Danishefsky, S. J.; Cabal, M. P.; Chow, K. JACS 1989, 111, 3456.
35. Deardorff, D. R.; Matthews, A. J.; McMeekin, D. S.; Craney, C. L. TL 1986, 27, 1255.
36. Johnson, C. R.; Senanayake, C. H. JOC 1989, 54, 735.
37. Senanayake, C. H.; Bill, T. J.; Larsen, R. D.; Leazer, J.; Reider, P. J. TL 1992, 33, 5901.
38. Mutsaers, J. H. G. M.; Kooreman, H. J. RTC 1991, 110, 185.
39. Kamal, A. SC 1991, 21, 1293.
40. Yee, C.; Blythe, T. A.; McNabb, T. J.; Walts, A. E. JOC 1992, 57, 3525.
41. Fliche, C.; Braun, J.; Le Goffic, F. SC 1991, 21, 1429.
42. Monteiro, J.; Braun, J.; Le Goffic, F. SC 1990, 20, 315.

Enzo Santaniello, Patrizia Ferraboschi & Paride Grisenti

Università di Milano, Italy

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