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One-Carbon Bridge Stapling for use in Peptide-Drug Conjugates



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Cooper, Bethany 


One-Carbon Bridge Stapling for use in Peptide-Drug Conjugates Bethany M. Cooper

Therapeutic peptides lie in between small molecules and therapeutic proteins in terms of their molecular weight and have many advantageous properties, such as high potency and selectivity. Yet one fundamental shortcoming of therapeutic peptides is their poor stability. Two-component peptide stapling (2C-PS) is an incredibly valuable tool used to increase a peptide’s stability and provide a point for further functionalisation. This thesis describes the development of a 2C-PS methodology to mimic a peptides’ disulfide bond, and the synthesis of two peptide-drug conjugates (PDCs) using this methodology.

1.0 The Development of a One-Carbon Bridge Peptide Stapling Methodology

Disulfide bonds are fundamental in many proteins and peptides; they are responsible for determining the tertiary and secondary structure respectively. Unfortunately these bonds are reversible in a cellular environment; installation of a functionalisable one-carbon bridge provides stability and is irreversible. Four peptides were synthesised, and many reaction conditions utilising 2C-PS were screened with staples bearing either an reactive alkynyl or a 1,1-dichloro motif. The stapling using the latter led to observation of the desired stapled peptide.

2.0 The Development of a Colorectal-Targeting Peptide-Drug Conjugate

Peptide-drug conjugates (PDCs) are a class of targeted therapeutics that aim to address adverse events experienced by non-targeting therapeutics. A novel alkynyl bearing staple was synthesised, with stapling yielding a one-carbon bridged colorectal cancer homing peptide. A model reactive oxygen species (ROS) cleavable linker with 7-hydroxycoumarin was analysed using fluorimetry and quencher studies, to confirm hydrogen peroxide dependent release. The same observation was determined for three cytotoxin ROS-cleavable conjugates. Finally, two PDCs were synthesised through a CuAAC reaction with cytotoxin ROS-cleavable linkers. The PDCs were subjected to HPLC hydrogen peroxide cleavage assays that demonstrated rapid cytotoxin release. Cellular assays were used to evaluate the effect of stapled peptides and the PDC on HCT-116 cells. This thesis provides a versatile platform for the development of a novel ROS-cleavable PDC, which can be applied to a range of cancers for specific payload delivery.





Spring, David


Cysteine, Linkers, Payload, Peptide-Drug Conjugates, Peptides, Stapling


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Engineering and Physical Sciences Research Council (2196888)
AstraZeneca EPSRC