Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.

(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.

Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.

Electrostatic Contribution to 19F Chemical Shifts in Fluorotryptophans in Proteins.

DFT calculations indicate that the 19F chemical shifts of aromatic rings containing single fluorine substituents are sensitive to the electric fields and electric field gradients at the position of the fluorine atom. The present work explores whether long-range structure restraints can be gained from changes in 19F chemical shifts following mutations of charged to uncharged residues. 19F chemical shifts of fluorotryptophan residues were measured in two different proteins, GB1 and the NT* domain, following mutations of single asparagine residues to aspartic acid. Four different versions of fluorotryptophan were investigated, including 4-, 5-, 6-, and 7-fluorotryptophan, which were simultaneously installed by cell-free protein synthesis using 4-, 5-, 6-, and 7-fluoroindole as precursors for the tryptophan synthase present in the S30 extract. For comparison, the 1H chemical shifts of the corresponding nonfluorinated protein mutants produced with 13C-labeled tryptophan were also measured. The results show that the 19F chemical shifts respond more sensitively to the charge mutations than the 1H chemical shifts in the nonfluorinated references, but the chemical shift changes were much smaller than predicted by DFT calculations of fluoroindoles in the electric field of a partial charge in vacuum, indicating comprehensive dielectric shielding by water and protein. No straightforward correlation with the location of the charge mutation could be established.

Through-Space Scalar 19F-19F Couplings between Fluorinated Noncanonical Amino Acids for the Detection of Specific Contacts in Proteins.

Fluorine atoms are known to display scalar 19F-19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. We show that fluorinated noncanonical amino acids positioned in the hydrophobic core or on the surface of a protein can be linked by scalar through-space 19F-19F (TSJFF) couplings even if the 19F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF3 groups, O-trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that 19F-19F TOCSY experiments are sufficiently sensitive to detect TSJFF couplings between 2.5 and 5 Hz in the 19 kDa protein PpiB measured on a two-channel 400 MHz NMR spectrometer with a regular room temperature probe. A quantitative J evolution experiment enables the measurement of TSJFF coupling constants that are up to five times smaller than the 19F NMR line width. In addition, a new aminoacyl-tRNA synthetase was identified for genetic encoding of N6-(trifluoroacetyl)-l-lysine (TFA-Lys) and 19F-19F TOCSY peaks were observed between two TFA-Lys residues incorporated into the proteins AncCDT-1 and mRFP despite high solvent exposure and flexibility of the TFA-Lys side chains. With the ready availability of systems for site-specific incorporation of fluorinated amino acids into proteins by genetic encoding, 19F-19F interactions offer a straightforward way to probe the spatial proximity of selected sites without any assignments of 1H NMR resonances.

Genetic Encoding of N6-(((Trimethylsilyl)methoxy)carbonyl)-l-lysine for NMR Studies of Protein-Protein and Protein-Ligand Interactions.

Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N6-(((trimethylsilyl)methoxy)carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.

In vitro anti-bacterial and time kill evaluation of binuclear tricyclohexylphosphanesilver(I) dithiocarbamates, {Cy3PAg(S2CNRR')}2.

Four binuclear phosphanesilver(I) dithiocarbamates, {cyclohexyl3PAg(S2CNRR')}2 for R = R' = Et (1), CH2CH2 (2), CH2CH2OH (3) and R = Me, R' = CH2CH2OH (4) have been synthesised and characterised by spectroscopy and crystallography, and feature tri-connective, µ2-bridging dithiocarbamate ligands and distorted tetrahedral geometries based on PS3 donor sets. The compounds were evaluated for anti-bacterial activity against a total of 12 clinically important pathogens. Based on minimum inhibitory concentration (MIC) and cell viability tests (human embryonic kidney cells, HEK 293), 1-4 are specifically active against Gram-positive bacteria while demonstrating low toxicity; 3 and 4 are active against methicillin resistant S. aureus (MRSA). Across the series, 4 was most effective and was more active than the standard anti-biotic chloramphenicol. Time kill assays reveal 1-4 to exhibit both time- and concentration-dependent pharmacokinetics against susceptible bacteria. Compound 4 demonstrates rapid (within 2 h) bactericidal activity at 1 and 2 × MIC to reach a maximum decrease of 5.2 log10 CFU/mL against S. aureus (MRSA).

µ3-Chlorido-µ2-chlorido-(µ3-pyrrolidine-1-carbo-dithio-ato-κ4S:S,S':S')tris-[(tri-ethyl-phosphane-κP)copper(I)]: crystal structure and Hirshfeld surface analysis.

The title trinuclear compound, [Cu3(C5H8NS2)Cl2(C6H15P)3], has the di-thio-carbamate ligand symmetrically chelating one CuI atom and each of the S atoms bridging to another CuI atom. Both chloride ligands are bridging, one being µ3- and the other µ2-bridging. Each Et3P ligand occupies a terminal position. Two of the CuI atoms exist within Cl2PS donor sets and the third is based on a ClPS2 donor set, with each coordination geometry based on a distorted tetra-hedron. The constituents defining the core of the mol-ecule, i.e. Cu3Cl2S2, occupy seven corners of a distorted cube. In the crystal, linear supra-molecular chains along the c axis are formed via phosphane-methyl-ene-C-H⋯Cl and pyrrolidine-methyl-ene-C-H⋯π(chelate) inter-actions, and these chains pack without directional inter-actions between them. An analysis of the Hirshfeld surface points to the predominance of H atoms at the surface, i.e. contributing 86.6% to the surface, and also highlights the presence of C-H⋯π(chelate) inter-actions.

A triclinic polymorph of tri-cyclo-hexyl-phosphane sulfide: crystal structure and Hirshfeld surface analysis.

The title compound, (C6H11)3PS (systematic name: tri-cyclo-hexyl-λ5-phosphane-thione), is a triclinic (P-1, Z' = 1) polymorph of the previously reported ortho-rhom-bic form (Pnma, Z' = 1/2) [Kerr et al. (1977 ▸). Can. J. Chem. 55, 3081-3085; Reibenspies et al. (1996 ▸). Z. Kristallogr. 211, 400]. While conformational differences exist between the non-symmetric mol-ecule in the triclinic polymorph, cf. the mirror-symmetric mol-ecule in the ortho-rhom-bic form, these differences are not chemically significant. The major feature of the mol-ecular packing in the triclinic polymorph is the formation of linear chains along the a axis sustained by methine-C-H⋯S(thione) inter-actions. The chains pack with no directional inter-actions between them. The analysis of the Hirshfeld surface for both polymorphs indicates a high degree of similarity, being dominated by H⋯H (ca 90%) and S⋯H/H⋯S contacts.