Spectroscopic investigation of an antifertility agent gossypol and an ionophore antibiotic, Lasalocid A

Abstract

The present thesis has as its objective the study of the structure and interaction of two biologically active, polyfunctional organic molecules. Spectroscopic studies on gossypol and lasalocid A form the subject matter of the thesis. Both compounds have been the focus of intensive research in recent years. Gossypol, a naturally occurring polyphenolic binaphthyl, holds the promise of being the most economical, reversible, and safe male antifertility agent to date. Lasalocid A (X 537A), a polyether antibiotic, has extensive applications in membrane biology as a carrier ionophore, and is used widely in veterinary science and medicine. The spectroscopic techniques employed in this study are principally circular dichroism (CD), nuclear magnetic resonance (NMR), and fluorescence. The thesis is divided into two parts:

Section I: Studies on gossypol Section II: Studies on lasalocid A

Chapter I – Introduction Chapter I provides:

a brief introduction to low molecular weight polyfunctional natural products that arise as secondary metabolites, an overview of their structural diversity, biological activities, and usefulness as experimental tools, a brief survey of various approaches to fertility regulation in males, a detailed discussion on the emergence of gossypol as a major lead in male contraception, covering:

discovery and distribution structure and activity biological effects site and mechanism of action interactions with macromolecules and membranes

Chapters II–V present the results from the gossypol studies.

Chapter II – CD Studies on Gossypol The scope of CD in studies of chiral molecules is briefly reviewed. This chapter describes:

the CD characteristics of (+)-gossypol, the naturally occurring enantiomer, solvent effects on its CD spectra, interactions of (+)-gossypol with:

proteins, peptides, small amine containing molecules.

These studies establish (+)-gossypol as a useful CD probe.

Chapter III – Optical Resolution of Gossypol This chapter explains:

why resolving racemic gossypol to obtain ( )-gossypol has been an important goal, general strategies for resolving chiral aldehydes, the optical resolution of racemic gossypol achieved in the present study.

Several chiral amines proved successful resolving agents, including:

3 aminoalcohols, amino sugars (2 amino 2 deoxyhexoses), the tetrapeptide Boc Lys Pro Aib Lys NHMe.

All methods involved reverse phase HPLC on an achiral column. A comparison with other recently reported methods shows the present approach is particularly attractive for large scale separations.

Chapter IV – Structure of Gossypol–Amine Adducts This chapter discusses:

earlier attempts to characterise gossypol–amine adducts, comparable adducts formed by other ortho hydroxy aldehydes, such as:

salicylaldehyde, 2 hydroxy 1 naphthaldehyde, pyridoxal phosphate.

The ¹H NMR, ¹³C NMR, and UV spectra of gossypol–amine adducts obtained here strongly support the formation of Schiff base structures.

Chapter V – Comparative Interactions of Gossypol Forms This chapter studies whether the three forms of gossypol:

(+)-gossypol ( )-gossypol (+)-racemic gossypol

show differences in interaction with biological systems. Key findings:

Fluorescence quenching studies on bovine serum albumin (BSA) and human serum albumin (HSA) show no significant differences in binding constants among the three forms. However, induced CD of racemic gossypol under certain conditions suggests differences in binding modes of the two enantiomers. Displacement of bilirubin from its albumin binding site shows subtle enantiomeric effects. Differences were also observed in:

gossypol induced membrane fluidity changes in liposomes, uncoupling of oxidative phosphorylation in rat liver mitochondria.

Section II – Studies on Lasalocid A Chapters VI–VIII present the lasalocid A studies.

Chapter VI – Introduction to Lasalocid A Provides an introduction to:

conformational studies on lasalocid A, its diverse biological effects, its importance as a membrane active ionophore.

Chapter VII – CD and NMR Studies on Lasalocid A Goals:

Re examine the assignment of long wavelength CD bands. Investigate the conformation of lasalocid A in aqueous media. Study complexation with organic diamines.

Key findings:

Lasalocid A exhibits aggregation in aqueous media. The ionophore forms symmetric diamine complexes, useful as models for “sandwich complexes.” A detailed CD assignment is presented.

Chapter VIII – Aggregation Studies This chapter illustrates methods for probing molecular size:

fluorescence polarization, NMR, gel filtration.

Experiments under various conditions show:

Lasalocid A forms aggregates in aqueous solution. An aggregation number of ~30 is suggested. Aggregated forms may also exist at the membrane–water interface.

Chapter IX – Summary of Major Findings The final chapter summarises:

insights into gossypol’s interactions, enantiomeric behaviour, and protein binding, successful optical resolution methods, characterization of gossypol–amine Schiff bases, conformational and aggregation properties of lasalocid A in various environments.