C. W. Hale, G. G. Newton, E. Abraham
May 1, 1961
Citations
0
Influential Citations
48
Citations
Journal
The Biochemical journal
Abstract
Cephalosporin C, an acidic antibiotic produced by a Cephalosporium sp., has been shown to have the structure (I) (Abraham & Newton, 1961; Hodgkin & Maslen, 1961). When a preparation of cephalosporin C which had been kept overnight in a pyridine-acetate buffer was chromatographed in the same buffer on an anion-exchange resin (Amberlite IR-4B), a substance which showed activity against Staphylococcus aureus was present in the first fractions from the column. This substance, named cephalosporin CAI was well resolved from cephalosporin C itself. When the eluted cephalosporin C was concentrated and rechromatographed later under similar conditions, a further quantity of cephalosporin CA appeared. It thus seemed that cephalosporin CA was being formed from cephalosporin C in aqueous pyridine-acetate. Further experiments, which have been briefly reported by Abraham & Newton (1958) and by Hale, Abraham & Newton (1958), showed that the new active substance was the product of a reaction between cephalosporin C and pyridine. When cephalosporin C was kept in 2M-pyridineacetate at pH 7 and at temperatures varying from 370 to 700, the activity of the solution against Staph. aureus rose to a value that was several-fold greater than the original one and then declined. Typical changes in activity are shown in Fig. 1. Electrophoresis on paper showed that this rise in activity was associated with the formation of a compound (cephalosporin CA) that showed no net charge at pH 7-0. The compound was not formed when the pyridine buffer was replaced by the acetates of a number of other bases, including ammonia, trimethylamine, piperidine, or by pyridinium methyl bromide, aniline or N-dimethylaniline. It was formed more readily in aqueous pyridine at pH 7 than at pH 5 and its yield increased with the concentration of pyridine to a maximum at about 2M. Further work showed that * Present address: Radiochemical Centre, Amersham, Bucks. a family of compounds could be produced by allowing cephalosporin C to react with different heterocyclic tertiary bases. The specificcompound obtained with pyridine will be referred to as cephalosporin CA (pyridine). On a preparative scale, cephalosporin CA (pyridine) was separated from remaining cephalosporin C, and from acidic degradation products formed during the reaction, by chromatography on Dowex 1X8 (acetate form). Cephalosporin CA (pyridine) passed rapidly through the column and was obtained as an amorphous solid on freezedrying the eluate. This product contained acetate and was extremely hygroscopic. For further purification it was adsorbed on Dowex 50X8 and eluted by stirring the resin with water and adding aqueous ammonia until the pH rose to 7 0. The resulting product was obtained as a non-hydroscopic white powder with an activity of approximately 120 u./mg. against Staph. aureus and