Perfluoro-tert-butyl Homoserine Is a Helix-Promoting, Highly Fluorinated, NMR-Sensitive Aliphatic Amino Acid: Detection of the Estrogen Receptor·Coactivator Protein–Protein Interaction by ¹⁹F NMR

Abstract

Highly fluorinated amino acids can stabilize proteins and complexes with proteins, via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions, via selective detection by 19F NMR and the absence of native 19F signals in biological contexts. However, the potential applications of 19F NMR in probing biological processes are limited both by the strong propensities of most highly fluorinated amino acids for the extended conformation and by the relatively modest sensitivity of NMR spectroscopy, which typically constrains measurements to mid-micromolar concentrations. Herein, we demonstrate that perfluoro-tert-butyl homoserine exhibits a propensity for compact conformations, including α-helix and polyproline helix (PPII), that is similar to that of methionine. Perfluoro-tert-butyl homoserine has 9 equivalent fluorines that do not couple to any other nuclei, resulting in a sharp singlet that can be sensitively detected rapidly at low micromolar concentrations. Perfluoro-tert-butyl homoserine was incorporated at sites of leucine residues within the α-helical LXXLL short linear motif of estrogen receptor (ER) co-activator peptides. A peptide containing perfluoro-tert-butyl homoserine at the i+3 position of the ER coactivator LXXLL motif exhibited Kd = 2.2 μM for the estradiol-bound estrogen receptor, similar to that of the native ligand. 19F NMR spectroscopy demonstrated the sensitive detection (5 μM concentration, 128 scans) of peptide binding to ER and of inhibition of protein-protein interaction by the native ligand or by the ER antagonist tamoxifen. These results suggest diverse potential applications of perfluoro-tert-butyl homoserine to probe protein function and protein-protein interfaces in complex solutions.

Hydrolysis, polarity, and conformational impact of C-terminal partially fluorinated ethyl esters in peptide models

“…Fluorine-containing groups can be incorporated into biopolymers by various approaches, including those that utilize biosynthesis [ 5 – 7 ], enzymatic conversion [ 8 ], chemical synthesis [ 9 – 10 ], and ligation reactions [ 11 ]. Depending on the research target, 19 F NMR measurements can be used to study ligand–protein [ 12 ] and protein–protein interactions [ 13 ]; membrane proteins [ 14 – 16 ] and membrane-associated peptides [ 17 – 18 ]; equilibria among conformations of RNA [ 19 ], DNA [ 20 ], and peptide nucleic acids (PNA) [ 21 ]; and many others. Particularly recent is the development of peptide-based contrast agents for 19 F imaging [ 22 ].…”

Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine-Thiol Displacement Reaction (FTDR)

“…The fluorine atom attracted our interest due to its orthogonality from biological molecules, 17 similarity in size to hydrogen, [17][18][19] and the close length of the carbon-fluorine bond to the carbon-hydrogen bond in acetamide, a representative acetylation functional group ( Figure 1A). 19 Fluorinated amino acids have been exploited to label proteins, bringing in minimal perturbations to protein structure and function.…”

“…19 Fluorinated amino acids have been exploited to label proteins, bringing in minimal perturbations to protein structure and function. [17][18]20 Using acetylation for a proof of concept, we first wanted to investigate whether fluorinated acetyl-CoA can hijack the CoA metabolism, and be used by acetyltransferases to label their protein substrates ( Figure 1B). Despite the in vivo toxicity of its pro-metabolite fluoroacetate, 21 only the late-stage metabolite fluorocitrate was found accountable, mostly damaging organ tissues such as the kidney.…”

Synthesis of peptides with cysteine sulfinic acid via the cysteine methoxybenzyl sulfone

“…As an initial test, 4 was incorporated within a model peptide (Ac-GPPXPPGY-NH 2 ) that has been previously employed to quantify the polyproline II helix propensities of both canonical and unnatural amino acids. [43][44][45][46][47][48] Using bulk resulting from β-substitution with the sulfone. [49,50] However, amide coupling proceeded effectively using COMU as a coupling reagent [51] in the presence of catalytic DMAP (Scheme 4).…”