Biradicals based on PROXYL containing building blocks for efficient dynamic nuclear polarization in biotolerant media

A versatile strategy for synthesizing tailored peptide based biradicals is presented. By labeling the protected amino acid hydroxyproline with PROXYL via the OH functionality and using this building block in solid phase peptide synthesis (SPPS), the obtained peptides become polarization agents for DNP enhanced solid-state NMR in biotolerant media. To analyze the effect of the radical position on the enhancement factor, three different biradicals are synthesized. The PROXYL spin-label is inserted in a collagen inspired artificial peptide sequence by binding through the OH group of the hydroxyproline moieties at specific position in the chain. This labeling strategy is universally applicable for any hydroxyproline position in a peptide sequence since solid-phase peptide synthesis is used to insert the building block. High performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses show the successful introduction of the spin label in the peptide chain and electron paramagnetic resonance (EPR) spectroscopy confirms its activity. Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) experiments performed on frozen aqueous glycerol-d₈ solutions containing these peptide radicals show significantly higher enhancement factors of up to 45 in ¹H→¹³C cross polarization magic angle spinning (CP MAS) experiments compared to an analogous mono-radical peptide including this building block (ε ≈ 14). Compared to commercial biradicals such as AMUPol for which enhancement factors > 100 have been obtained in the past and which have been optimized in their structure, the obtained enhancement up to 45 for our biradicals presents a significant progress in radical design.