Enzymic and chemical transformations of the side chain of cephalosporin C

摘要

2532 J.C.S. Perkin IEnzymic and Chemical Transformations of the Side Chain of Cephalo-sporin CBy P. Mazzeo and A. Romeo,* lstituto di Chimica Farmaceutica dell'Universit5, Centro di Studio per la Chimicadel Farmaco del CNR, Roma, ItalyThe synthesis of 7- (5-oxoadipamido)cephalosporanic acid by treating cephalosporin C with D-amino-acid oxidaseis described. The same compound is obtained by a non-enzymic transamination reaction between pyridoxal!phosphate and cephalosporin C.ENZYMES able to act on cephalosporin C include p-lactamases,l which open the 3-lactam ring, and acetyl-esterases,2 which hydrolyse the acetic ester function.However, enzymes with acylase activity able to detachthe D-a-aminoadipoyl chain are not known.We describe here the oxidative deamination of theD-a-aminoadipoyl chain by D-amino-acid oxidase frompig kidney.This enzyme is known to transformD-a-amino-acids into a-keto-acids, but its specificity islimited and D-a-aminoadipic acid is reported not to bea suitable ~ubstrate.~By use of a high enzyme-substrate ratio we havetransformed cephalosporin C into an acid productidentified as 7- (5-oxoadipamido)cephalosporanic acid(I) on the basis of analytical and spectroscopic data(yield 10-20~0).The same compound (I) has been prepared by a non-enzymic transamination reaction involving treatmentwith pyridoxal 5-phosphate ; by employing a reagent-cephalosporin C ratio of 5 , a 33 yield was obtained.EXPERIMENTALM.p.s were taken with a Kofler hot-stage apparatus.U.V.spectra were measured for solutions in ethanol with aCary 14 spectrometer. 1.r. spectra were recorded forpotassium bromide discs with a Perkin-Elmer 521 spectro-meter. N.m.r. spectra were determined for solutions inhexadeuterioacetone with a Varian A-60 D instrument, withtetramethylsilane as internal standard. Mass spectroscopicL. D. Sabath, M. Jago, and E. P. Abraham, Biochenz. J . ,1965, 96, 739.J . d'A. Jeffery, E. P. Abraham, and G. G. F. Newton,Biochem. J . , 1961, 81, 591.analysis was performed a t 70 eV with an A.E.I. MS12spectrometer. Optical rotations were recorded at ca. 20"with a Schmidt-Haensch polarimeter.Reaction of Cephalosporin C with D-Amino-acid Oxidase .-The potassium salt of cephalosporin C (680 mg, 1.5 mmol),D-amino-acid oxidase (50 mg) (Boehringer ; crystallinesuspension in 1.8M-ammonium sulphate), and catalase (2mg) in phosphate buffer ( 0 .1 ~ ; pH 6.2; 30 ml) underoxygen were stirred a t 27" for 26 h. The mixture was thensaturated a t 0' with ammonium sulphate, and, afterremoval of precipitated enzyme by centrifugation, acidifiedto pH 3 with B~-hydrochloric acid and extracted with ethylacetate. The extract was washed with saturated ammoniumsulphate solution, dried (Na,SO,), and evaporated to leavematerial (124 mg), which crystallized from ethyl acetate-hexane to give 7- (5-oxoadi~amido)ce~halos~oranic acid (I),m.p. 163-164' (decomp.), Lx. 258 nm (E 5600), a, +lolo(c 1 in EtOH), vmBX. 3290, 1745, 1730, 1710, 1685, 1650, 1530,and 1230 cm-l, m/e 414 (Mf), 6 5-87 (lH, q, J 5 and 9 Hz,exchangeable with D,O), 3.45 and 3-80 (2H, ABq, JAB18 Hz, S-CH,), 4.83 and 5.18 (2H, ABq, JAB 13 Hz, CH,*OAc),and 2-03 p.p.m. (3H, s, Ac) (Found: C, 46-45; H, 4.85;N, 6.95; S, 7-95.Cl,H1,N,O,S requires C, 46.35; H, 4.4;N, 6.75; S, 7.75).Reaction of Cephalosporin C with Pyridoxal5-Phosphate.-A solution of the potassium salt of cephalosporin C (2.27 g,5 mmol) and sodium pyridoxal 5-phosphate (6-75 g, 25mmol) in phosphate buffer (0.1~; pH 6-2; 80 ml) was keptat room temperature for 16 h. The mixture was thensaturated a t 0" with ammonium sulphate, acidified to pH 3with 2~-hydrochloric acid, and extracted with ethylacetate. The extract was washed with saturated ammoniumsulphate solution, dried (Na,SO,) , and evaporated to leave aproduct (690 mg), which, after crystallization from ethylacetate-hexane, was identical with the acid (I) alreadyobtained.2/386 Received, 21st February, 197213 R. B. hlorin and B. G. Jackson, Fortschr. Chem. org.4 A. E. Bender and H. A. Krebs. Biochem. J . , 1950, 46, 210.7-H), 5.17 (lH, d, J 5 Hz, 6-H), 7*88'(1H, d, J 9 Hz, NH,Naturstofle, 1970, 28, 365