Synthesis and application of novel perfluoro-tert-butyl amino acids for the detection of protein activity by 19F NMR and mechanistic insights into polyproline I and polyproline II formation via 4,4-difluoroproline
Date
2017
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
We have developed a series of four novel amino acids containing perfluorotert-
butyl ethers for use in detection of protein and cellular activities by 19F magnetic
resonance. All four amino acids were synthesized, as monomers, for introduction into
peptides. These amino acids contain nine equivalent fluorine atoms which display as a
single peak by 19F NMR and are detectable in nanomolar concentrations in five
minutes. Each amino acid was characterized to determine its structural preferences, in
order to design potential applications for each designed amino acid. ☐ Perfluoro-tert-butyl homoserine demonstrated the ability to form α-helical
structures within a peptide context, and was designed into a series of Estrogen
Receptor binding peptides to target the ER●co-activator protein-protein interaction.
We were able to design a peptide containing perfluoro-tert-butyl homoserine which
bound with similar affinity to the native co-activator ligand. We were also able to
observe the ER●co-activator protein-protein interaction by 19F NMR. ☐ Perfluoro-tert-butyl tyrosine was designed into a series of peptides based on a
high affinity p53 chimeric peptide due to the presence of aromatic residues within the
target binding sequence. While these peptides did not bind with affinity near that of
the native peptide, we were able to detection the perfluoro-tert-butyl tyrosine in 30
seconds by 19F NMR in nanomolar quantities. This amino acid is also being explored
as a potential expressible 19F NMR-based sensor. ☐ 4R-Perfluoro-tert-butyl hydroxyproline and 4S-perfluoro-tert-butyl
hydroxyproline demonstrated opposite conformational preferences, leading to
divergent applications in kinase-sensing activity. Both amino acids were introduced
into kinase recognition motifs and phosphorylation was successfully detected by 19F
NMR. Furthermore, PKA and Akt kinases showed different stereochemical
preferences between the two amino acids, indicating the potential of these amino acids
to differentiate between similar protein kinases. PKA substrates containing 4Rperfluoro-
tert-butyl hydroxyproline were also subjected to phosphorylation in HeLa
cell lysates, which was detectable by 19F NMR, indicating the potential for this
technology to be used in live cell imaging. ☐ Finally, 4,4-difluoroproline was synthesized and examined in proline
oligomers to determine this amino acid’s potential to be used as a probe for cis-trans
isomerization within peptides. 4,4-Difluoroproline was introduced at the beginning,
middle, and end of a nine residue proline oligomer and the interconversion between
PPI and PPII helices was examined by circular dichroism and 19F NMR. These
experiments allowed us to develop a model of PPI helix stabilization as well as a
mechanism of PPI and PPII helix interconversion based on favorable reverse n→π*
and n→π* interactions respectively. These data are also indicative of the potential of
4,4-difluoroproline to be used as a 19F probe of cis-trans isomerization within peptides
and proteins.