Kinetic Resolution of Unnatural and Rarely Occurring Amino Acids: Enantioselective Hydrolysis of l/-Acyl Amino Acids Cataly zed by Acylase Ir

Acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC 3.5.1.14, from porcine kidney and the fungus Aspergi l lus) is a broadly appl icable nzymatic atalyst for the kinetic resolut ion of unnatural and rarely occurring a-amino acids. I ts enantioselectivi ty for the hydrolysis of N-acyl L-a-amino acids is nearly absolute, yet i t accepts ubstrates having a wide range of structure and functional i ty. This paper reports the init ial rates of enzyme-catalyzed hydrolysis of over 50 N-acyl amino acids and analogues, the stabi l i t ies of the enzymes in aqueous and aqueous/organic solut ions, and the effects of dif ferent acyl groups and metal ions on the rates of enzymatic hydrolysis. E,leven a-amino and a-methyl a-amino acids were resolved on a 2-29-9 scale. Crude Land o-amino acid products had general ly ' >90% ee. The uti l i ty of resolved amino acids as chiral synthons was i l lustrated by the preparation of (R)and (S)-l-butene oxide and the diastereoselective (cis:trans, 7-8:1 ) iodolactonization of three 2-amino-4-alkenoic a id derivatives. Acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC 3.5.1.14) catalyzes the enantioselective hydrolysis of N-acyl L-amino acids (eq 1).+7 Acylase I enzymes isolated from porcine ?o'" fl acyrase | ?oo:o'* fl .5rA*, *,An,*"* + ,/-"A*, (1) kidney f i*ol and the fungus ,trp,rrgit tu, ,o ioo, are commercial ly avai lable, inexpensive, stable in solut ion, and high in specific activity. Here we describe the use of these enzymes as catalysts for the kinetic resolution of a-amino acids. We report the range of substrates accepted by each enzyme, factors influencing the activi t ies and stabi l i t ies of the enzymes, and methods for the preparative-scale resolution of representative compounds. Both land o-amino acid products were obtained with high (general ly >90Vo) enantiomeric excesses. Amino acids are important as biomedically active compoundss and as chiral direct ing auxi l iar iese'10 and chiral synthonse'r l-13 in (l) This research was supported by the National Institutes of Health, Grant GM 30367, and by a grant from Firmenich, SA. (2) NIH Train ing Grant Trainee, 1984-1985 (Grant 5-T32-GM-07598). (3) Deutsche Forschungsgemeinschaft Postdoctoral Fellow, I 986I 987. (4) Greenstein, J. P.; Winitz, M. Chemistry of the Amino Acids; Wiley: New York, l96l ; Vol . 2, pp 1753-1767. (5) Greenstein, J. P. Methods Enzymol.1957, 3,554-570. (6) Birnbaum, S. M.; Levintow, L.; Kingsley, R. B.; Greenstein, J. P. .r. Biol. Chem. 1952, 194,455-470. (7) (a) Chibata, I.;Tosa, T.; Sato, T.; Mori, T. Methods Enzymol.1976, 44,'146-7 59. (b) Immobilized Enzymes'. Research and Deuelopment: Chibata, L, Ed. ;Wi ley: New York, 1978;pp 168-178. (c) Tosa, T. ; Mor i , T. ; Fuse, N.; Chibata, L Biotechnol. Bioeng. 1967,9,603-615. (8) (a) Barrett, G. C., Ed. Chemistry and Biochemistry of the Amino Acids:Chapman and Hall: London, 1985. (b) Wagner,l.;Musso, H. Angew. Chem., Int. Ed. Engl.1983, 22,816-828. (c) Montreuil, J. In Comprehensiue Biochemistry; Neuberger, A., van Deenen, L. L. M., Eds.; Elsevier: Amsterdam, The Nether lands, 1982;Vol . l98, Part I I , pp l -188. (d) Pais, M.; Jarreau, F.-X. In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, l97l;Vol. l, pp l2'l-157. (e) Amino Acids, Pept., Proteins (Spec. Period. Rep.) 1976, 8, 326-327. (0 Davies, J. S. In Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1977; Vol. 4, pp l-27. (g) Amino Acids, Pept., Proteins (Spec. Period Rep.) 1983, t4,432-502. (9) Coppola, G. M.; Schuster, H. F. Asymmetric Synthesis'. Constuction of Chiral Molecules Using Amino Acids: Wiley: New York, 1987. (10) (a) Evans, D. A. In Asymmetric Synthesis; Morrison, J. D., Ed.; Academic: Or lando, FL, 1984; Vol . 3, pp 2-110. (b) Lutomski , K. A. ; Meyers, A. l. Ibid. pp 213-274. (c) Enders, D. Ibid. pp 275-3a1. (d) Martens, J. Top. Curr. Chem.1984, 125,165-246. o Refcrencc -1 I . h Ref 'e rence J9. 'Refe rencc 24. d Reference 38. " Rcference . l I ' Re ferencc 40 g Ref 'erencc J5. ' Reference 36. ispeci f ic actrv i t \ was deternt ined as descr ibed in Resul ts and Exper i mental Sect ion. /Costs are fbr enzymes as research biochemicals (1988 pr i ce l i s t , S igma Chemica l Co . ) and thus a re upper l im i t s . Approx i mately 1000 U wi l l catalyze the hydrolysis o i i mol of acylt -amino acid per day. organic synthesis. Both oand L-amino acidsr4 and a-methyl amino ac ids have impor tan t uses and a re thus des i rab le i n ho mochiral form Methods for preparing enantiomerically enriched amino ac ids such as f c rn ren ta t i on and enzymat i c ca ta l ys i s , l 5 ( l l ) (a) t -ubel l . W. D.; Rapoport . H. J. Am" Chem. Soc. 1987, 109, 236-239. (b) Kunz, H. ; Lerchen, H. G. Tetrahedron Let t .1987,28,18731876; 1985, 26,525'l-5260. (c) t{ernot, S.; Larcheveque, M.; Petit, Y. Synth. Commun.1986 , 16 . 183-190 . (d ) Hag iwara , l l . ; K imura , K . ; Uda , F I . " r . Chem. Soc. Chem. Commun.1986, 860-861. (e) Mzengeza, S. ; Yang, C. M.; Whitney, R. A. J. Am. Chem. Soc. 1987. 109,276-2'7 '7. (0 Schur ig, V. ; Leyrer, U. ; Wistuba,D. J. Org. Chenr. l986, J l , 242-245. (g) Maurer, P. J. ; Knudson, C. G.; Palkowitz, A. D. ; Rapoport , H. . / . Org. Chem.l985, 50. 325-332. (h) Knudson, C. G.; Rapoport, H. J. Org. Chem. 1983, 48, 2260-2266. (i) Mori, K.: Sasaki, M.; Tamada, S.; Suguro, T.; Masuda, S. Tetrahedron 1979. 3 5. I 601 -l 605. ( l2) Kurokawa, N.; Ohfune, Y. J. Am. Chem. Soc. 1986, 108,6041-6043. (13) Ohfune, Y. ; Kurokawa, N. Tetrahedron Let t .1985,26, 5307-5308. ( 1a) By convention. we refer to the configuration at the a-carbon of amino acids as o or l. The more mmmon, naturally occurring L-amino acids all have the (S) configuration, except for r--cysteine, which, because of priorit izing sulfur over oxygen, has the (R) configuration. (15) (a) Kirk-Othmer Encyclopedia af Chemical Technology,3rd ed.; Wi ley: New York, 1978; Vol . 2, pp 376"410. (b) Considine, D. M., Ed. Chemical and Process Technology Encyclopedia; McGraw-Hill: New York, 1974; pp 93-106. (c) Aida, K. , Chibata, I . , Nakayama, K. , Takinami, K. , Yamada, H., Eds. Biotechnology of Amino Acid Produclion; Kodansha: Tokyo, 1986. (d) Hami l ton, B. K. ; Hsiao, H. Y. ;Swann, W. E. ; Anderson, D. M.; Delente, J. J . Trends Biotechnol . l985, J,64-68. (e) Yamada. H. ; Shimizu, S.; Shimada, H.; Tani, Y.; Takahashi, S.l Ohashi, T. Biochimie 1984, 62,395-399. (l) \ 'amada, H.; Kumagai, H. Pure Appl. Chem. 1978, 50, ll lT-1127 . (g) Yonaha. K.; Soda, K. ln .Aduances in Biochemical Engineering Biotechnology; Fiechter. A., Ed.;Springer-Verlag: Berlin, 1986;Vol. 33, pp 95-130. (h) Chibata, I.; Tosa, T.; Sato, T. Methods Enzy'mol. 1976, 44 .739-746 . 1989 American Chemical Society Table I. Pronerties of Acvlase I porc ine k idney Aspergillus no . o f subun i t s MW (d imer ) op t imum p l l K . ( ch lo roacc t r l -A la ) . m \ ' l Zn2+ pe r subun i t Ko*.1Zn2+;, M l(dirr(Co2+), M essent ia l Cys per subuni t d ipept idase act ivy sp ac t i vy , t U / *g do l l a rs / 1000 [ , ' J 2a 85 500' 7 .0 . -9 .0d