Beta-hydroxynorleucine: separation of its isomers and biological studies

Separation of the four isomers of beta-hydroxynorleucine was accomplished by partition column chromatography and asymmetric enzymatic hydrolysis of the N-chloroacetyl derivatives. From these, the corresponding N-chloroacetyl derivatives were made. The purity and configuration of each isomer of the free acid and N-chloroacetylated derivative were ascertained by: (a) paper chromatography in five solvent systems, (b) elemental analysis, (c) Van Slyke nitrous acid determination of alpha-carbonyl carbon, and (d) Van Slyke ninhydrin determination of alpha-carbonyl carbon, and (e) optical rotation. Comparison of the rate of enzymatic hydrolysis by hog renal acylase I of the N-chloroacetyl derivative of the L-isomers of each diastereomer showed that the acyl B isomer is a better substrate than the acyl A isomer, where A denotes the faster moving diastereomer and B denotes the slower moving diastereomer in a defined chromatographic solvent system. Microbiological assay using Lactobacillus casei in a system selected for screening for possible antitumor activity indicated that while none of the isomers as free amino acids had any growth inhibitory action, the N-acylated isomers showed modest but significant activity. The N-chloroacetyl derivative of the D-enantiomorph of diastereomer B exhibited the greatest growth inhibitory activity, showing about twice the activity of the other three isomers.