Design, synthesis, and application of a trifluoromethylated phenylalanine analogue as a label to study peptides by solid-state 19F NMR spectroscopy (original) (raw)
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ChemBioChem, 2018
The N-acetyl amino acid methyl esters were prepared by N-acetylation of the amino acids, followed by methylation of the C-termini. Dipeptides were obtained by coupling the Nprotected and C-protected amino acids. Tripeptides were synthesised sequentially either from (i) the C-terminus (starting with the Cprotected methyl ester hydrochloride salt) via N-Boc-protected intermediates (Procedure A, scheme S1), or (ii) the N-terminus (starting with the N-acetyl amino acid) via N-acetyl dipeptides (Procedure B, Scheme S2). Compounds for which no spectroscopic details are provided, were obtained commercially (Sigma Aldrich, AK Scientific). Scheme S1. Sequential synthesis of tripeptides from the C-terminus (Procedure A). Scheme S2. Sequential synthesis of tripeptides from the N-terminus (Procedure B). 1 H and 13 C spectra were recorded on either an Agilent MR 400 MHz NMR spectrometer or an Agilent DD2 500 MHz NMR spectrometer, in either deuterated dimethylsulfoxide (DMSO-d6), deuterated acetonitrile (Acetonitrile-d3) or deuterated methanol (Methanol-d4). Chemical
Chemistry – A European Journal, 2019
Solid‐state 19F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the 19F‐reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non‐hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene‐based dipeptide isostere, Val‐Ψ[(Z)‐CF=CH]‐Gly, and inserted it in the sequence of two well‐studied antimicrobial peptides: PGLa and (KIGAKI)3 are representatives of an α‐helix and a β‐sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for 19F NMR structure analysis.
Journal of Peptide Science, 2007
The conformation, alignment and dynamic behavior of membrane-bound peptides is readily accessible by solid-state 19 F-NMR spectroscopy, but it has been difficult to incorporate suitable 19 F-labelled amino acids into synthetic peptides. To avoid the drawbacks of previously used labels, we have rationally designed and synthesized a novel amino acid that suits all theoretical and practical requirements for peptide synthesis and subsequent 19 F-NMR structure analysis [Mikhailiuk et. al, Angew. Chem. 2006, 118, 5787-5789]. The enantiomerically pure L-form of 3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine (CF 3 -Bpg) carries a CF 3 group that is rigidly attached to the peptide backbone and does not racemize during peptide synthesis. It could be demonstrated for several different peptides that their biological activity is usually not affected by a single label, nor the conformation, as monitored by circular dichroism. Here, we carry out a more detailed structure analysis to evaluate the potential and reliability of CF 3 -Bpg for solid-state NMR, using the well-known α-helical antimicrobial peptide PGLa as a test case. We have collected several orientational constraints from the anisotropic 19 F-19 F dipolar couplings of CF 3 -Bpg in various positions of PGLa embedded in lipid bilayers. These resulting structural parameters are then compared with those previously determined from 4-CF 3 -phenylglycine and 3,3,3-d 3 -alanine labels on the same peptide. The analysis confirms that CF 3 -Bpg does not perturb the α-helical conformation of PGLa. Likewise, the helix alignment is shown to follow the established concentration-dependent pattern in realigning from a surface-bound S-state to an obliquely tilted T-state. Hence, the advantages of CF 3 -Bpg over all previously used 19 F-labeled side chains are evident, as they combine ease of chemical incorporation and peptide purification with high NMR sensitivity and absent background signals, allowing a straightforward analysis of the dipolar splittings with no need for chemical shift referencing without any ambiguity in the sign of the couplings.
Nature Communications
The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.
19 F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-L-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19
Angewandte Chemie-international Edition, 2004
Aspartame 2 and IPy 2 BF 4) were purchased from Aldrich and used without further purification. Peptides 4, 5, and 6 were synthesized on solid support using standard Fmoc chemistry. Methylene chloride was distilled from calcium hydride and sodium metal. Liquid flash chromatography was carried out with Merck silica gel 60 (230-400 mesh) and analytical thin layer chromatography (TLC) was performed on 0.25 mm silica gel coated Kieselgel 60 F264 plates. The spots were visualized with UV light, and staining with nynhidrine followed by heat. NMR spectra were recorded on either a Bruker AMX-400 or AMX-300 spectrometer. Chemical shifts are reported in parts per million (ppm) on the scale, using residual solvent peaks as reference. Reactions were carried out under nitrogen atmosfere. Analytical RP-HPLC was conducted using a C 18 column (LiChrosorb, 250×4 mm). Solvents: A= 0.1% TFA in H 2 O, B= 0.1% TFA in CH 3 CN.