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The fraction of sp3‐hybdrised carbons (Fsp3) in drug candidates and other biologically relevant compounds has been proposed to be an important physicochemical consideration within medicinal chemistry. Currently available small‐molecule probes for Raman imaging are not optimised for biological applications. We sought to improve upon the highly Raman‐active, yet rigid, lipophilic, and aggregative, bisarylbutadiyne (BADY) motif by introducing Fsp3 character. BADY analogues were designed, efficiently synthesised, and analysed by mass spectrometry – revealing a statistical correlation between compound aggregation and cLogP, but no correlation with Fsp3. However, X‐ray crystallography demonstrated that the modifications to the BADY structure had a marked effect on crystal packing, suggesting Fsp3 may still be important in reducing intermolecular stacking, and thus aggregation, of BADY. Cellular imaging by stimulated Raman scattering (SRS) microscopy allowed comparison of the intracellular distribution of the tags, guiding the design of future Raman probes. [ABSTRACT FROM AUTHOR]

Peptide stapling is an effective strategy to stabilise α-helical peptides, enhancing their bioactive conformation and improving physiochemical properties. In this study, we apply our novel diyne-girder stapling approach to the MDM2/MDMX α-helical binding region of the p53 transactivation domain. By incorporation of an unnatural amino acid to create an optimal i , i + 7 bridge length, we developed a highly α-helical stapled peptide, 4, confirmed via circular dichroism. This diyne-girder-stapled peptide demonstrated enhanced helicity and nanomolar binding affinity for MDM2, as assessed by fluorescence polarisation. Crucially, peptide 4 exhibited strong selectivity for MDM2, with approximately 100-fold reduced affinity for MDMX. Molecular modeling and docking studies suggested that this selectivity arose from diminished hydrophobic interactions at the MDMX binding site, driven by the diyne-girder's constrained geometry. The use of the diyne-girder, a unique feature amongst stapled peptide analogues, for cellular visualisation using Raman spectroscopy in the "cell-silent" region was explored. This capability potentially offers a novel method for tracking stapled peptides in biological systems without the need for large fluorophores. Overall, peptide 4 represents a promising tool for probing MDM2 activity and a valuable addition to the arsenal of peptide-based therapeutic strategies., Competing Interests: A patent application [(GB) patent application no: 2219576.2] has been submitted on this work. D. T. H. is a consultant for Triana Biomedicines. There are no other conflicts to declare., (This journal is © The Royal Society of Chemistry.)