The pigmentation of hippopotamus sweat after perspiration is observed. The colorless viscous sweat of the hippopotamus gradually turns red within a few minutes. In spite of the interesting and wonderful phenomenon, the responsible pigment had remained to be elucidated because of it instability. The red solution of the crude extracts of the sweat turns brown with decomposition of the pigment when the solution is subjected to the following conditions: violent acidification, addition of strong bases, standing for a long time at room temperature, addition of abundant organic solvent, and concentration to dryness. Therefore, special precaution to isolate the pigment was required. Gel filtration of the red solution through Sephadex G-15 and G-25 with H_2O indicated the presence of three pigments (orange, red, and brown) in the crude extracts. Then, to each orange and red solution was added an anion exchange resin QAE Sephadex A-25 (0.2M phosphate buffer, pH6.1), and the resin was quickly washed and eluted with NaCl / buffer to afford a comparably concentrated bright red and orange solutions. For NMR analyses, the samples were eluted with deuterated buffer solution. For MS analyses, the samples were eluted with TEA-HCO_2H buffer. In order to obtain further information on the structure, the unstable red pigment 1 was converted to a stable derivative through several steps. X-ray analysis of this derivative suggested that the structure of 1 should be the highly oxidized fluorene. The UV spectra of the red (1) and the orange (2) pigments suggested that they have the same chromophore. Taking these data into consideration, the structures of 1 and 2 were elucidated to be substituted fluorene diquinones. The tautomeric structures of these pigments were also studied by chemical synthesis and by the model studies. These studies will provide a useful information about the role of hippopotamus sweat and the coloring mechanism.
