β-Peptides: Influence of Fluorine on Structure, Conformation and Function
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
This thesis examines the synthesis of α-fluoro-β-amino acids, and the influence of the constituent fluorine on the conformation and biological properties of β-peptide derivatives.
Chapter One discusses the unique properties of the C-F bond, and applications of fluorine substitution in organic and medicinal chemistry. This is followed by a review of fluorinated analogues of α-amino acids, and how their incorporation into α-peptides has resulted in profound modifications, such as enhanced thermal and chemical stability, increased affinity for lipid bilayers, stronger self-association and improved biological activity.
Experimental and theoretical data indicate two conformational effects associated with fluoroamides: the F-C-C(O)-N(H) moiety in α-fluoroamides adopts an antiperiplanar conformation, and in N-β-fluoroethylamides a gauche conformation between the vicinal C-F and C-N(CO) bonds is favoured. Chapter Two details the synthesis of a series of fluorinated β-peptides (2.13-2.24) designed to investigate the use of these stereoelectronic effects to control the conformation of β-peptide bonds. X-ray crystal structures were obtained for seven of these compounds and revealed the compounds had the expected conformations: when fluorine was positioned β to a nitrogen a gauche conformation was observed, and when fluorine was positioned α to a C=O group the structure adopted an antiperiplanar conformation. Thus, the strategic placement of fluorine can control the conformation of β-peptide bonds, and hence could be used to direct the secondary structures of β-peptides. The chapter is prefaced by an introduction to β-amino acids and the secondary structures of β-peptides.
Chapter Three outlines the stereoselective synthesis of a series of α-fluorinated-β-amino acids. The synthesis of α-fluoro-β3-amino acids was achieved via direct fluorination of β3-amino acids with LDA and NFSI. The fluorination of N-Boc-protected β3-homophenylalanine, β3-homoleucine, β3-homovaline and β3-homoalanine all proceeded with good diastereomeric excesses (> 85 % de). However, the fluorination of N-Boc-protected β3-homophenylglycine occurred with a lower diastereomeric excess of 66%. Replacement of the Boc amine protecting group of β3-homophenylglycine with Cbz and Bz groups did not alter the stereoselectivity of the fluorination reaction, and substitution with an acetyl amine protecting group reduced the diastereomeric excess to 26%. The stereoselective synthesis of an α-fluoro-β2-homophenylalanine from 3-phenylpropanoic acid is also detailed. Conversion of the acid to the Evan's oxazolidinone followed by enantioselective fluorination and alkylation in high diastereomeric excess, and subsequent amination gave the α-fluorinated β2-amino acid.
Chapter Four describes the enzyme assays carried out to assess the inhibitory activity of α-fluoro-β-amino acids, and the analogous non-fluorinated β-amino acids, against α-chymotrypsin. Both fluorinated and non-fluorinated β-amino acid derivatives were found to be competitive inhibitors of α-chymotrypsin, with Ki values in the low millimolar range. The fluorinated β2-homophenylalanine and β3-homophenylglycine derivatives (2.35, 3.26a, 3.43a and 3.44) were found to be more active against α-chymotrypsin than their non-fluorinated analogues (5.27, 3.24, 3.40 and 3.41), whereas the fluorinated β3-homophenylalanine methyl ester (2S,3S)-2.49 was inactive against α-chymotrypsin although the corresponding non-fluorinated derivative (S)-3.28 was a potent inhibitor.
In Chapter Five a series of N-succinyl-β-amino acids-p-nitroanilides (5.8-5.13), containing both fluorinated and non-fluorinated β-amino acids, were designed and synthesised as possible substrates of α-chymotrypsin. β-Peptides are stable towards proteolytic hydrolysis, but the introduction of fluorine at the α-position in a β-amino acid was proposed to increase the activity of the adjacent amide bond, and thus make the β-peptide more susceptible to protease cleavage. However, the incorporation of fluorine had no influence on the proteolytic stability of compounds 5.8-5.13 as they were all found to be stable towards hydrolysis by α-chymotrypsin. Compounds 5.8, 5.9 and 5.13 were established as reversible competitive inhibitors of α-chymotrypsin
Chapter Six is an experimental chapter and outlines the synthesis, purification and characterisation of the compounds prepared in this thesis.