Patent GB-785098-A (GB)
Title: Separation of the antibiotic spiramycin into its components
IPC: C07G11/00
Filed: 1956-01-02
Abstract: Spiramycin, an antibiotic produced by the fermentation of Streptomyces ambofaciens NRRL 2430 according to the parent Specification, and having three constituents named spiramycins I, II and III, is separated into the pure constituents by (a) treatment in a Craig counter-current distribution apparatus, (b) chromatography or alumina, (c) fractional crystallization from an aromatic solvent or (d) a combination of (c) with (a) or (b).  In treatment (a) the organic phase is an aromatic hydrocarbon such as benzene or a chlorinated hydrocarbon solvent such as dichloroethane, or mixtures thereof, and the aqueous phase consists of a salt solution buffered to pH 6 to 7, the two phases being mutually saturated.  The crude spiramycin is dissolved in a small amount of the organic phase just sufficient to go into one or up to four of the first cells of the counter-current distribution apparatus.  100 c.c. of aqueous phase are introduced into each of the cells of the Craig apparatus disposed in a chamber at 24 DEG  C. 100 c.c. of spiramycin solution in the organic phase is introduced into the first cell, shaken and decanted, and the organic phase transferred to the second cell.  A further 100 c.c. of spiramycin solution is then introduced into the aqueous phase in the first cell.  After agitation and decanting, the organic phase is transferred from the second cell to the third cell and that from the first cell into the second cell.  The fractionation is continued this way introducing 100 c.c. of organic phase into the first cell after each decanting operation. After 59 transfers in series have been effected, the contents of each cell are collected, the aqueous phase is made alkaline at pH 9 to 10 by means of sodium hydroxide, agitated and the organic phase decanted.  The aqueous phase is given a further extraction with the organic phase and the organic phases are combined.  The organic phases having a common constituent in the pure state are collected and the solvent removed in vacuo.  In the case of spiramycin II and III, further purification is obtained by crystallization from an aromatic hydrocarbon solvent.  For chromatography on alumina, the crude spiramycin is dissolved in an aromatic hydrocarbon, e.g. benzene, or ether and spiramycin I which remains on the column after washing with the same solvent as is used for dissolution, is eluted with benzene-acetone, benzene-alcohol, or by alcohol or acetone.  Spiramycin II and spiramycin III are separated by adsorbtion on alumina using as solvent a mixture of cyclohexane and an aromatic hydrocarbon such as benzene or a mixture of cyclohexane and ether.  Spiramycin II remains on the column and is eluted with ether followed by a mixture of ether and acetone. Fractional crystallization from an aromatic hydrocarbon such as benzene, enables spiramycin I to be separated from spiramycin II and III.  Base I is further purified and bases II and III separated into their components and also purified by counter-current distribution or by chromatography on alumina.  The Rf value of spiramycin 1 is 0.04, of spiramycin II is 0.15 and of spiramycin III is 0.22.  Spiramycin sulphate is produced by dissolving a particular component in methanol adding sulphuric acid to pH 5.5 and pouring into ether.  Other salts specified are the hydrochloride, nitrate, perchlorate and phthalate.