Synthesis of fluorinated leucines, valines and alanines for use in protein NMR

Abstract

Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various 13C/15N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such...

Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis

“…Remarkably, the 19 F chemical shift dispersion for CF-2F-C-HETs was ~20 ppm, much higher compared to protein samples with 19 F labeled aromatic amino acids 100 . We observed the CH 2 19 F signals in a range of −220 to −240 ppm, in agreement with solution 19 F NMR study by Otting et al 101 , this range being very different compared to the C 19 F 3 range (−65 to −70 ppm). We observed 19 F line widths of ~1000 Hz.…”

“…Next, we implemented the simultaneous incorporation of 13 C and 19 F nuclei at the level of the same amino acid 5,5’-difluoro-L-leucine-5,5’- 13 C 101 (sample termed called CF-2F-C-HETs). This strategic labeling on leucine has two main advantages: (i) we re-introduce two 1 H spins on the methyl sites, to enable efficient 1 H- 13 C polarization transfers to the methyl carbon, (ii) while we keep two fluoromethylated spins for 19 F detection and (iii) we introduce a 13 C isotope directly attached to 19 F spins to shift 13 C signals away from the protonated methyl region and allow recording of 2D 13 C- 1 H or 13 C- 19 F correlations for increased resolution.…”

(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5′-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect

“…The 19 F NMR signals were shown to be well resolved with a dispersion ranging over more than 7 ppm, but no resonance assignments were attempted. 38 In the present work, we used cell-free protein synthesis for high-level incorporation of FLeu1, FLeu2, and diFLeu into the 19 kDa E. coli protein peptidyl−prolyl cis−trans isomerase B (PpiB; UniProt ID P23869). PpiB contains five leucine residues (Figure 2).…”

“…All protein samples were produced by continuous exchange cell-free protein synthesis (CFPS) following an established protocol with modifications to incorporate the fluoroleucine isomer of interest. 38,42 CFPS was conducted at 30 °C for 16 h using 1 mL of inner reaction mixture of S30 cell extract from E. coli BL21(DE3) and 10 mL of outer buffer. The PpiB mutants L28C, L76C, L108C, and L115C were produced using PCR to introduce the requisite codons (TGC for cysteine, GCG for alanine), followed by a second round of PCR to amplify the DNA with eight-nucleotide overhangs for ligation under CFPS conditions.…”

“…This is important, as the incorporation of natural leucine and valine is facile in comparison with their fluorinated analogues, which are increasingly more difficult to install the more fluorine they contain, as observed in this work and previously. 38 Furthermore, the activities of many of the enzymes involved in amino acid biosynthesis are impaired under CFPS conditions, minimizing the chance of amino acid degradation. Accordingly, we found no evidence of "fluorine scrambling," where the fluorine atom would have been transferred to another amino acid.…”