Perfluoro‐tert‐butyl‐homoserine as a sensitive ¹⁹F NMR reporter for peptide–membrane interactions in solution

Abstract

Fluorine ((19)F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient biological interactions. Here, we evaluate the utility a novel amino acid, L-O-(perfluoro-t-butyl)-homoserine (pFtBSer), that can easily be synthesized and incorporated into peptides and provides greatly enhanced sensitivity over currently used (19)F biomolecular NMR probes. Incorporation of pFtBSer into the potent antimicrobial peptide MSI-78 results in a sharp (19)F NMR singlet that can be readily detected at concentrations of 5 µm and lower. We demonstrate that pFtBSer incorporation into MSI-78 provides a sensitive tool to study binding through (19)F NMR chemical shift and nuclear relaxation changes. These results establish future potential for pFtBSer to be incorporated into various proteins where NMR signal sensitivity is paramount, such as in-cell investigations.

Synthesis of Intrinsically Disordered Fluorinated Peptides for Modular Design of High‐Signal 19F MRI Agents

“…Due to the increased interest in new fluorochemicals for imaging applications, and concerns over toxicity and environmental persistence, these new designs will be studied for broader use in 19 F MRI-based biomedical applications. We note, that with the increased availability of synthetic methods for more highly fluorinated amino acids, [30],[31] even higher SNR may be achievable. Paramagnetic metal chelates as T 1 agents, [32] as well as improvement in data processing such as sparse sampling, [33] may help bring designs of fluorinated materials closer to practical applications in the 19 F MRI field.…”

(2S,4R)- and (2S,4S)-Perfluoro-tert-butyl 4-Hydroxyproline: Two Conformationally Distinct Proline Amino Acids for Sensitive Application in 19F NMR

“…42,43 The perfluoro- tert -butyl group of these amino acids, in contrast to most other fluorinated amino acids, exhibits fluorines that are singlets by 19 F NMR, here with nine chemically equivalent fluorines that are uncoupled to any other nuclei. 37,42,44,45 The unique magnetic nature of the perfluoro- tert -butyl group, combined with the large chemical shift dispersion of 19 F nuclei and the general absence of fluorine in typical biological environments, suggests the possibility of highly sensitive detection of molecules containing this functional group in diverse media.…”

“…In contrast, perfluoro- tert -butyl ethers are not subject to carbocation formation and elimination reactions and thus are expected to be a chemical stable functionality that may be incorporated within small molecules, peptides, and proteins. 44,45 …”

Synthesis and characterization of perfluoro-tert-butyl semifluorinated amphiphilic polymers and their potential application in hydrophobic drug delivery

“…Purification was therefore achieved using a Combi-Flash system equipped with an evaporative light scattering detector (ELSD) while minimizing exposure to vacuum. However, as suggested in a similar synthesis from Yu et al ., 33 purification from large-scale synthesis may be achieved by simple phase separation. The resulting mPEG 1k -OPFtB TRI and mPEG 2k -OPFtB TRI polymers were sparingly soluble in water.…”

“…30-31 The PFtB TRI -OH is achieved by modification a previous literature procedure 33 for small-scale reaction conditions.…”