The isobutyl side chain is a highly prevalent hydrophobic group in drugs, and it notably constitutes the side chain of leucine. Its replacement by a hexafluorinated version containing two CF3...
Structural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.
We report the direct incorporation of the hexafluoroisobutyl group on a chiral glycine Schiff base complex mediated by 1,8diazabicyclo[5.4.0]undec-7-ene (DBU). The fluoroalkylation involves 2-(bromomethyl)-1,1,1,3,3,3-hexafluoropropane reagent, which generates in situ hexafluoroisobutylene (HFIB), and reacts then with the enolate through a tandem allylic shift/hydrofluorination process. We showed that the use of neutral organic base DBU generates in situ an original DBU•HF salt, which preserves the fluoride nucleophilicity and acts as a fluorinating agent. This fluoride salt promotes the hydrofluorination of the pentafluorinated alkene overcoming the usual fluoride βelimination observed with α-CF 3 -vinyl reagents. With alkali metal bases, by contrast, the hydrofluorination is disfavored and the pentafluorinated alkene intermediate is obtained predominantly. This study highlights the critical role of the fluoride counter ion to preserve its nucleophilicity. The protocol is amenable to multidecagram scale synthesis of enantiopure (S)-and (R)-5,5,5,5',5',5'-hexafluoroleucine and their N-Fmoc or N-Boc derivatives in good overall yield.
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