Counter-current distribution studies on streptomycin; the tautomerism of streptomycin.

The simplicity and reproducibility which characterize the conversion of streptomycin to malt01 (1) by heating in dilute alkali have made this reaction the basis of a frequently used analytical method for streptomycin (2). When used in these laboratories as a method of assaying streptomycin preparations, however, the malt01 reaction consistently gave lower yields per unit of potency with pure streptomycin trihydrochloride or its calcium chloride double salt than with impure preparations of lower activity. This observation suggested the possibility that the cruder concentrates contained other malt01 precursors less active than streptomycin, a supposition which has been confirmed by the isolation of mannosidostreptomycin’ (5, 6). In a preliminary communication (7) it was remarked that the broad distribution curves obtained by the application of the Craig counter-current distribution technique (8, 9) to solid streptomycin preparations assaying approximately 400 units per mg. could be considered to indicate the presence of at least three maltol-producing entities (cf. Curve B (7) ). The distribution studies herein described were originally undertaken as part of the effort to isolate the hypothetical third streptomycin-like substance, but they have now shown that this third fraction does not represent such an entity and have instead yielded evidence that streptomycin exists in several tautomeric modifications, the relative proportions of which depend on the pH. These tautomers differ in the ratio of distribution between some immiscible solvent pairs and are at least partially separable by adsorption on alumina. In order to concentrate the different components, the crude mixture of streptomycin (400 to 500 units per mg:) was fractionated by liquid chroma-