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

(2S,4R)and (2S,4S)-perfluoro-tert-butyl 4hydroxyproline were synthesized (as Fmoc-, Boc-, and free amino acids) in 2−5 steps. The key step of each synthesis was a Mitsunobu reaction with perfluoro-tert-butanol, which incorporated a perfluoro-tert-butyl group, with nine chemically equivalent fluorines. Both amino acids were incorporated in model α-helical and polyproline helix peptides. Each amino acid exhibited distinct conformational preferences, with (2S,4R)perfluoro-tert-butyl 4-hydroxyproline promoting polyproline helix. Peptides containing these amino acids were sensitively detected by F NMR, suggesting their use in probes and medicinal chemistry. F amino acids have unique properties, due to the hydrophobicity and electronegativity of fluorine and the magnetic properties of the sensitive spin-1/2 F nucleus. The special nature of fluorine has led to broad interest in the incorporation of fluorinated amino acids in small molecules, peptides, and proteins, for applications in medicinal chemistry, in the design of stabilized proteins, and in NMR and MRI approaches to protein detection and imaging. Fluorinated amino acids allow the specific, quantitative detection of individual species in complex media, including the observation of protein synthesis and folding in living E. coli cells, due to the low background of fluorine in most environments. The sensitivity of fluorine NMR probes depends on the concentration of the molecule, the coupling patterns of the F nuclei to other fluorines and to hydrogens in the molecule, and the number of chemically equivalent fluorines in the molecule. For probes to be employed biologically, it is ideal to be able to work at the lowest concentration of molecule possible due to the typical nanomolar protein concentrations and affinities observed in protein−protein interactions. The most sensitive protein-based probes of biological activity employed to date have utilized aryl trifluoromethyl groups, which exhibit sharp singlets by F NMR of intensity 3 (one aryl-CF3 group) or 6 (two symmetrically related, chemically equivalent aryl-CF3 groups). 35−37 In contrast, in the most widely used direct analogue of a canonical α-amino acid, hexafluoroleucine (6 fluorines), the two trifluoromethyl groups are diastereotopic, exhibiting separate resonances, and each is coupled to the methine hydrogen, resulting in reduced peak intensity due to each trifluoromethyl group existing as a doublet (four total peaks observed). We previously described the synthesis of tetrapeptides containing the novel amino acids (2S,4R)and (2S,4S)perfluoro-tert-butyl 4-hydroxyproline (Figure 1). Peptides with these amino acids were prepared by the method of proline editing, in which a hydroxyproline amino acid within a fully synthesized peptide is site-specifically modified via reaction on solid phase to incorporate novel functionalities. The perfluoro-tert-butyl group of these amino acids, in contrast to most other fluorinated amino acids, exhibits fluorines that are singlets by F NMR, here with nine chemically equivalent fluorines that are uncoupled to any other nuclei. The unique magnetic nature of the perfluoro-tert-butyl group, combined with the large chemical shift dispersion of 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 addition to the magnetic properties of the perfluoro-tertbutyl group, in general, tert-butyl groups are broadly attractive in medicinal chemistry due to functional group symmetry and the consequent reduced conformational entropy penalty upon target binding. However, in amino acids and peptides, tertbutyl ethers, the most easily installed tert-butyl groups, are not stable to standard acidic cleavage/deprotection reactions. 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. Perfluoro-tert-butyl hydroxyproline residues in model AcTYXN-NH2 peptides, X = either 4R-perfluoro-tert-butyl hydroxyproline or 4S-perfluoro-tert-butyl hydroxyproline, exhibited structural effects of proline substitution that were consistent with previously observed stereoelectronic effects in 4-substituted prolines, in which conformational preferences are dependent on the stereochemistry and the electron-withReceived: April 18, 2014 Published: May 28, 2014 Note