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Design and Synthesis of Fluorescent and Antibiotic Conjugates of Vitamin B₁₂ and Cobinamide

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Date

2025

Authors

Fredericksen, Jessica

Supervisor

Brasch, Nicola
Seale, Brent

Item type

Thesis

Degree name

Doctor of Philosophy

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Publisher

Auckland University of Technology

Abstract

The essential micronutrient vitamin B₁₂ has shown considerable promise as a delivery vehicle for antibiotics into bacteria. Novel antibiotic therapies using this ‘Trojan horse’ approach have considerable potential in the battle against antibiotic resistance. The goals of this research were 1) to synthesise highly fluorescent derivatives of vitamin B₁₂ and its naturally occurring analogue cobinamide, and 2) to synthesise vitamin B₁₂ conjugates of the widely used antibiotics gentamycin and ciprofloxacin. Vitamin B₁₂ has its own specific and active uptake pathway within bacterial cells. Fluorescent conjugates were designed to function as tools to follow the uptake, transport and cellular localisation of vitamin B₁₂ and cobinamide in bacteria. Although the synthesis of fluorescent CNCbl derivatives is well-documented, no fluorescent derivatives of CNCbi have been reported to the best of our knowledge. Furthermore, practically all existing vitamin B₁₂ derivatives of fluorophores exhibit significant fluorescence quenching. The key focus of this work was to synthesise vitamin B₁₂ and cobinamide conjugates with high fluorescent quantum yields. A range of fluorescent derivatives of CNCbl and CNCbi incorporating NBD-X, sulfo-Cyanine5 and Cyanine7 have been successfully synthesised in high purities (≥90%) and very good to reasonable yields. These conjugates were designed to retain their binding capacity to the Btu uptake system (including BtuB, BtuF and BtuCD transport proteins) for CNCbl/CNCbi uptake in the gram-negative bacterium Escherichia coli. Fluorescence analysis established high quantum yields (>0.20) for CNCbl/CNCbi conjugates incorporating sulfo-Cyanine5 and Cyanine7 fluorophores. An exciting finding from this research was that the conjugation of the near infrared emitting Cyanine7 to CNCbl and CNCbi resulted in no observable fluorescence quenching. Significant fluorescence quenching was observed for the conjugates that incorporated the NBD-X fluorophore. Solution studies between the free components suggested a dynamic quenching mechanism. For the conjugates, where the donor-acceptor distance is constrained, the significant spectral overlap between NBD-X emission and the CNCbl absorbance suggests Förster Resonance Energy Transfer (FRET) is a likely contributor, though other mechanisms such as Photoinduced Electron Transfer (PeT) cannot be excluded. Recently a vitamin B₁₂ conjugate of the antibiotic ampicillin was shown to be 500 times more effective than ampicillin itself in killing E coli. Once robust synthetic approaches were developed for synthesising the fluorescent CNCbl conjugates, these procedures were used to inform the synthesis of two antibiotic conjugates of CNCbl. Novel CNCbl-linker-antibiotic conjugates were synthesised incorporating the antibiotics gentamicin and ciprofloxacin. The conjugation of ciprofloxacin to CNCbl was achieved at two sites - the carboxylic acid and the secondary amine functionalities of ciprofloxacin. Site-selective conjugation of gentamicin via an amine is challenging, as multiple reactive amines are available for conjugation. However, conjugation of gentamicin to CNCbl was determined to occur selectively at one amine site using both LC-MS and ¹H NMR spectroscopy. The purification of CNCbl-linker-antibiotic conjugates was achieved by reversed-phase C18 chromatography and required isocratic conditions and the collection of small fractions to achieve purities of >95%. This was reflected in lower product yields (9-40%); however, conjugates were isolated for analysis and are suitable for biological testing. These conjugates will allow others to investigate the uptake and efficiency of CNCbl/CNCbi delivery system as a Trojan horse strategy to deliver antibiotics into bacteria - a relatively unexplored and promising area of medicinal chemistry.

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http://hdl.handle.net/10292/19902

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