Reexamination of Cyclodextrin-Induced Conformational Enantiomerism of Bilirubin in Aqueous Solution

A cyclodextrin (CDx)-induced conformational enantiomerism of bilirubin IX(alpha) in dianionic form (BR^2-) was reinvestigated to establish the mechanism for complexation of BR^2- with native CDxs, such as (alpha)-, (beta)-, and (gamma)-CDxs, in aqueous solution. Since previous studies have lacked NMR examinations, we concentrated our effort on measuring the 1H NMR spectra to determine the structure of the BR^2--CDx complex, where BR^2- takes the (M)-helix conformation. Circular dichroism (CD) spectroscopy was also utilized. 1H NMR measurements including ROESY suggest the shallow penetration of a hydrophobic part of BR^2- into the CDx cavity. 1H NMR data also show adsorption of the BR^2- dianion on the outside wall of the CDx cavity. Although BR^2- is adsorbed on the cavities of heptakis(2,3-di-O-methyl)and heptakis(2,6-di-O-methyl)-(beta)-CDxs, no enantiomerism occurred when these O-methylated (beta)-CDxs were used in place of native CDxs. In contrast, heptakis(6O-methyl)-(beta)-CDx exhibits a definite (-)-to-(+) bisignate CD spectrum due to preferencial formation of (M)-helix BR^2-. These results are interpreted in terms that the BR2- dianion is adsorbed on the outside wall of the asymmetrically twisted CDx cavity, and is anchored through two-point hydrogen bonding between the CO2group(s) of BR^2- and the OH groups at the 2- and 3-positions of the CDx.