1288
I. Regla et al. / Tetrahedron: Asymmetry 15 (2004) 1285–1288
was divided into three 20 mL fractions; to each one,
200 mg of kidney acetone powders of beef, hog, and
sheep, were added, respectively, and then incubated at
37 ꢁC for 24 h in an orbital shaker at 250 rpm. After
cooling to 5 ꢁC, the pH was adjusted to 11.0 with 1 M
NaOH under vigorous stirring and the reaction mixtures
was centrifuged separately at 3200g and 5 ꢁC for 15 min.
Each supernatant was acidified to pH 6.3 with 6 M HCl
Acknowledgements
The authors acknowledge Dr. Lainbarry from the
slaughterhouse La Aurora in Ciudad Neza, who sup-
plied the beef, hog, and sheep kidneys, and Dr. Fer-
ꢀ
nando Hernandez of the Dog Control Center,
Iztapalapa, D.F., for supplying the dog kidneys. I. Re-
gla also acknowledges the support received from
DGAPA-PASPA, National Autonomous University of
Mexico (UNAM).
and cooled to 0–5 ꢁC for half an hour. The
L-HPA 12
precipitate was filtered and the mother liquors saved.
The solid was dissolved in 1 M HCl, treated with acti-
vated charcoal, filtered over diatomaceous earth and its
pH again adjusted to 6.3, then filtered, washed with
water and dried at 60 ꢁC until constant weight. The
References and notes
yields of
shown in Table 1. The filtrate from each of the above
samples, containing unreacted -N-Ac-HPA 13, was
L-HPA 12 obtained for different KAPs are
1. Boyer, S. K.; Pfund, R. A.; Portmann, R. E.; Sedelmeier,
G. H.; Wetter, H. F. Helv. Chim. Acta 1988, 71, 337–
343.
2. Chang, C.; Yang, T. Tetrahedron: Asymmetry 2003, 14,
2239–2245.
D
adjusted to pH 2.3 with 10 M HCl, cooled to 0 ꢁC for 2 h,
vacuum filtered, washed with chilled water until the pH
of the washing was between 3 and 4 and dried at 60 ꢁC
3. (a) Hu, Q.; Wang, G.; Wang, X.; Wu, T.; Pan, X.; Chan,
A. S.; Yang, T. Tetrahedron: Asymmetry 2000, 11, 2309;
(b) Williams, R. M.; Sinclair, P. J.; Zhai, D. G.; Chen,
D. M. J. Am. Chem. Soc. 1988, 110, 1547–1557;
(c) Houng, J.-Y.; Chung-L. US Patent 5552317, 1995;
(d) Ben-Ishal, D.; Moshenberg, R. Tetrahedron 1977, 33,
1533–1542; (e) Pulley, S. R.; Hegedus, L. S. J. Am. Chem.
Soc. 1993, 115, 9037–9047; (f) Corey, E. J.; Link, J. O.
J. Org. Chem. 1992, 114, 1906–1908; (g) Hashimoto, C.;
Potier, P. Tetrahedron Lett. 1987, 28, 6062–6069; (h)
Houng, J.-Y.; Wu, M.-L.; Chen, S.-T. Chirality 1996, 8,
418–422; (i) Christine, M.; Serreqi, A. N.; Huang, Q.;
Kazlauscas, R. J. J. Org. Chem. 1994, 59, 2075–2081; (j)
Chen, S.-T.; Huang, W.-H.; Wang, K.-T. Chirality 1994,
6, 572–576.
until constant weight. The yields of
shown in Table 2.
D-N-Ac-HPA 13 are
3.4. Determination of ee of 12 and 13
3.4.1. -Homophenylalanine methyl ester.
L
L-HPA 12
(300 mg, 1.67 mmol) was suspended in anhydrous
methanol (6 mL) and cooled to ꢁ10 ꢁC in an ice-salt
bath. Then, 156 lL (260 mg, 2.18 mmol) of thionyl
chloride were added dropwise and the reaction mixture
kept for 1 h at ꢁ5 to ꢁ10 ꢁC and then at 40–45 ꢁC until
TLC monitoring (7:3 isopropanol–ammonium hydrox-
ide; developed with 0.2% ninhydrine reagent spray)
4. Kiss, A.; Boross, L. Acta Biochim. Biophys Acta Sci. Hung
1980, 15, 29.
indicated the L-HPA had disappeared. The solution was
ꢀ ꢀ
5. (a) Sanchez, R.; Luna, H.; Perez, H. I.; Manjarrez, N.;
then evaporated in a rotatory evaporator. The solid was
dissolved in the minimum amount of cold water, made
alkaline with sodium bicarbonate and extracted with
ethyl acetate (3 · 5 mL), dried, and evaporated to dry-
ꢀ
Solıs, A. Tetrahedron: Asymmetry 2001, 12, 1399–1401; (b)
Basavaiah, D. Arkivoc 2001, 70–82; (c) Kazlauskas, R. J.
J. Am. Chem. Soc. 1989, 111, 4953; (d) Whitesell, J. K.;
Lawrence, R. M. Chimia 1986, 40, 318; (e) Adachi, K.;
Kobayashi, S.; Ohno, M. Chimia 1986, 40, 311.
6. Ware, E. Chem. Rev. 1950, 46, 403–470.
7. Kubota, H.; Nunami, K.; Hayashi, K.; Hashimoto, Y.;
Ogiku, N.; Matsuoka, Y.; Ishida, R. Chem. Pharm. Bull.
Jpn. 1992, 40, 1619–1623.
ness. The
HPLC analysis, on a CHIRALCEL OD column; hex-
ane–isopropanol (90:10). N-Acetyl- -HPA 13 was
L-HPA methyl ester was submitted to chiral
D
treated as above to give the corresponding methyl ester,
which was submitted to chiral HPLC analysis under the
same conditions.
8. Giannis, A.; Henk, T. Liebigs Ann. Chem. 1991, 8, 789–
793.
9. Lin, W.; He, Z.; Zhang, H.; Zhang, X.; Mi, A.; Jiang, Y.
Synthesis 2001, 7, 1007–1009.
10. Doi, E.; Shibata, D.; Matoba, T. Anal. Biochem. 1981,
118, 173–184.
3.5. Enzyme assays and specific activity of
acylase in beef KAP
L-amino-
11. Aldrich Catalog Handbook of Fine Chemicals, Aldrich
Chemical Co.: Milwaukee, 2003–2004; p 1004.
12. Andraco, J.; Smith, D.; Hartung, W. H. J. Pharm. Sci.
1961, 50, 337–340.
13. Walker, J. M. In Methods in Molecular Biology; Walker,
J. M., Ed.; Humana: New Jersey, 1994; Vol. 32, pp
5–8.
To measure L-aminoacylase activity in KAP, a soluble
preparation was made by suspending 200 mg of each
KAP in 10 mL of 0.2 M potassium phosphate buffer
pH 7.5, at 5 ꢁC. The suspension was centrifuged and the
supernatant kept in an ice bath. Protein concentration
was determined with the bicinchoninate method.13