Nuclear magnetic resonance spectroscopy of bile acids. Development of two-dimensional NMR methods for the elucidation of proton resonance assignments for five common hydroxylated bile acids, and their parent bile acid, 5 beta-cholanoic acid.

D V Waterhous,S Barnes,D D Muccio

doi:10.1016/s0022-2275(20)34280-2

Abstract

The complete 1H nuclear magnetic resonance assignments have been made for the common mono-, di-, and trihydroxy 5 beta-cholanoic acids; lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, and the unsubstituted parent compound, 5 beta-cholanoic acid, by heteronuclear-correlated two-dimensional NMR. The known 13C chemical shifts of these compounds were used to make the proton resonance assignments, and consistency of the carbon and proton assignments was verified by expected changes due to substituent effects. This has led to clarification of previously published 13C NMR resonance assignments. Addition of the 3 alpha, 7 alpha, and 12 alpha hydroxyl substituent effects derived from the mono- and dihydroxycholanoic acids yielded predicted values for proton chemical shifts of the trihydroxy-substituted 5 beta-cholanoic acid, cholic acid, that agreed well with experimental values. It is suggested that the individual substituent effects can be used to predict proton chemical shifts for hydroxycholanic acids containing other combinations of 3 alpha, 7 alpha, 7 beta, and 12 alpha hydroxyl groups.

Highlights

The complete ‘H nuclear magnetic resonance assignments have been made for the common mono, di, and trihydroxy 50-cholanoic acids; lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, and the unsubstituted parent compound, 5P-cholanoicacid, by heteronuclear-correlated two-dimensional NMR
‘H N M R spectroscopy has not been systematically applied to bile acids because most of the 25 or more bile acid proton resonances, apart from the protons geminal to the hydroxyl groups and the methyl group protons, are in a limited chemical shift range of 1.0 to 2.3 ppm
From a comparison of the one-dimensional 'H NMR spectrum of the parent bile acid, 50-cholanoic acid (Fig. 3A), to that of cholic acid and of other biologically significant bile acids (Fig. 3), it is obvious that the resonances of the more than 25 methylene and methine protons that occur in the region from 1.0 to 2.5 ppm extensively over

Summary

Introduction

The complete ‘H nuclear magnetic resonance assignments have been made for the common mono-, di-, and trihydroxy 50-cholanoic acids; lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, and the unsubstituted parent compound, 5P-cholanoicacid, by heteronuclear-correlated two-dimensional NMR. The proton chemical shift assignments of five common hydroxylated bile acids (Fig. l), lithocholic acid (3a-hydroxy-5p-cholanoicacid), chenodeoxycholic acid (3a,7a-dihydroxy-5P-cholanoicacid), ursodeoxycholic acid (3a,7~-dihydroxy-5~-cholanoaiccid), deoxycholic acid (3a,l2a-dihydroxy-5~-cholanoiaccid), and cholic acid (3a,7a,12a-trihydroxy-5~-cholanoaicicd), and their parent, unsubstituted bile acid, 5p-cholanoic acid, were obtained by these 2D N M R techniques. From these data we have determined the effect of hydroxyl substitution at the 3a, 7a,70,and 12a positions and can make predictions of chemical shifts of other related bile acids. Some resolution and clarification of the carbon assignments made by Iida et al (4) have been made as a result of the work presented in this report

Methods

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Discussion

Conclusion