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Title: Approaches to fluorine-18 labelling in the formation of peptide radiopharmaceuticals
Author: Smith, Gareth
ISNI:       0000 0004 2715 8887
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2010
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The reader will first be introduced to the principles of positron emission tomography (PET) as a state of the art medical imaging technique. Particular emphasis will be made on the application of the positron emitting isotope fluorine-18 e8F) and how novel binding sites for the insertion of the isotope into biomolecules are currently emerging to overcome the complex formation of C_[18F] bonds. The synthesis of a range of labelled prosthetic groups for the insertion of 18F into peptides was completed. e8F]-fluorobenzaldehyde ([18F]-FBA) was synthesised using three synthetic strategies, including the use of a robotic synthesiser for high activity syntheses. This molecule was subsequently studied in conjugation reactions with a salmon calcitonin derivative formed by solid phase peptide synthesis to include a hydrazine function at the lysine-18 residue. The hydrazine moiety was introduced to the peptide through derivatisation of Fmoc-Iysine-OH with 6-hydrazinonicotinic acid (HYNIC) or 4-hydrazinobenzoic acid (HYBA) in a three-step synthesis. Conjugation of [18F]-FBA to HYNIC-salmon calcitonin and HYBA-salmon calcitonin was studied and produced multiple radiolabelled species as determined by radio-HPLC. The formation of 4-maleimido trimethylammonium triflate as a precursor for 4-fluorophenyl maleimide was completed successfully alongside the formation of a non-radioactive standard. Radiolabelling of this molecule proved unsuccessful and only [18F]-fluoride was returned from reaction mixtures. A thorough study of the formation of e8F]-trifluoroborates was completed to assess the effect of concentration, pH, temperature and the effect of varied fluoride carrier sources on the radiofluorination of arylboronic acids and arylboronate esters. These molecules offer a novel binding site for [18F]-fluoride for the radiolabelling of biomolecules such as peptides and proteins without C_[18F] bond formation. Maximum radiochemical yields in the range of 50%-60% could be achieved in the case of the formation of 4-carboxphenyl-[18F]-trifluoroboate from the corresponding pinacol ester. However these reactions needed to be carried out utilising potassium hydrogen fluoride as a unique source of carrier fluoride and required mM concentrations of boron precursors for the reaction to yield significant radiolabelled product on the range of small molecule boronic acids and boronate esters studied. The formation of cold standard organotrifluoroborates was studied and the use of 19F NMR to attempt to detect mono- and bis- fluorinated intermediates was applied to prove that trifluoroborate formation is a rapid process where no intermediates can be detected between within 2-3 minutes when reacting boronate esters with potassium hydrogen fluoride. Several strategies for the formation of boron containing amino acids were studied to yield precursors suitable for the introduction of boronic acids or boronate esters into peptides through solid phase peptide synthesis. Direct conjugation via amide formation to the side-chain carboxylic acid of Fmoc-Glu-OtBu proved successful when conjugating an aryl boronate ester. The formation of aryl and alkyl boronic acid azide was also shown to be possible for subsequent triazole formation by copper catalyzed reaction with a propargyl derivatised glutamic acid residue {"click" reaction}. A small molecule boronic acid maleimide derivative was synthesised for the sitespecific addition to thiol containing peptides and proteins. This molecule was shown to bind to the free cystiene residue in c{RGDfC) and to a modified construct of the C2A domain of Synaptotagmin {C2Ac}. Radiofluorination of the C2Ac-boron-maleimide derivative was completed to demonstrate the feasibility of kit-based [18F]_ trifluoroborate technology.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available