Synthesis of novel compounds for the study of biomolecules using NMR spectroscopy

Abstract

This thesis presents an investigation into the synthesis of compounds viz., bifunctional chelators that could be used as lanthanide shift reagents and fluorinated amino acids that could be used as probes in the study of biomolecules using nuclear magnetic resonance spectroscopy.

Initially, syntheses of acyclic multidentate ligands like DTPA that could function as lanthanide shift reagents were investigated. A DTPA derivative with a cysteine side arm that could bind to biomolecules via a disulfide bond was synthesised. The lanthanum complex of this ligand was synthesised but the NMR spectrum of this coordination compound showed a complex spectrum due to the high conformational flexibility of the compound. Hence the synthesis of other non cyclic ligands with more stereochemically rigid structures, incorporating more stereo centers in the triamine pentaacetic acid backbone was investigated employing dialkylation and reductive amination methods. However, these acyclic ligands could not be synthesised due to steric hindrance in the substrates.

Research was then directed towards the synthesis of another class of bifunctional chelators possessing a polyaza polycarboxylic macrocycle as in TETA. A TETA derivative with a cyclam backbone with three of the four substituents being an acetic acid moiety and the fourth substituent being a cysteine moiety was synthesised. The ytterbium complex of this ligand however was presumed to exist in a dynamic equilibrium with the uncomplexed ligand. This calls for significant structural modification of the ligand for it to be successfully used as a lanthanide shift reagent.

Synthesis of fluorinated amino acids were investigated that can be incorporated into proteins. A stereoselective synthesis of (S)-y-fluoroleucine was accomplished. An HPLC assay was developed to determine the enantiomeric purity of the amino acid. Gratifyingly, preliminary studies showed that this unnatural amino acid was incorporated significantly in its hydrazide form into the protein via cell free protein synthesis. Development of a simple and straightforward general synthetic route for the synthesis of fluorinated amino acids using N-phthaloylvaline and N-phthaloylleucine and the corresponding hydrazides were investigated. The bromo and chloro substituted analogues were synthesised but the synthesis of the corresponding fluoro substituted analogues failed due to competing intramolecular and elimination side reactions favoured under the reaction conditions.