An Isotope‐Coded Fluorogenic Cross‐Linker for High‐Performance Target Identification Based on Photoaffinity Labeling

A photoaffinity labeling (PAL)‐based method for the rapid identification of target proteins is presented in which a high‐performance chemical tag, an isotope‐coded fluorescent tag (IsoFT), can be attached to the interacting site by irradiation. Labeled peptides can be easily distinguished among numerous proteolytic digests by sequential detection with highly sensitive fluorescence spectroscopy and mass spectrometry. Subsequent MS/MS analysis provides amino acid sequence information with a higher depth of coverage. The combination of PAL and heterogeneous target‐selecting techniques significantly reduces the amount of time and protein required for identification. An additional photocleavable moiety successfully accelerated proteomic analysis using cell lysate. This method is a widely applicable approach for the rapid and accurate identification of interacting proteins.     

Highly Stable trans‐Cyclooctene Amino Acids for Live‐Cell Labeling

trans‐Cyclooctene groups incorporated into proteins via non‐canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans‐cyclooct‐2‐ene isomers ( 1 a , b ). We further show that the axially connected isomer has a half‐life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped‐flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.     

Photo-cross-linkers incorporated into g-protein-coupled receptors in Mammalian cells: a ligand comparison

Capturing the right ligand at the right spot: A well-balanced system for non-natural amino acid mutagenesis allows the ligand binding sites of a class?II G-protein coupled receptor to be mapped and distinct binding domains to be identified for different ligands in the native environment of mammalian cells.     

Hybrid peptides: expanding the beta turn in peptide hairpins by the insertion of beta-, gamma-, and delta-residues

The beta turn segment in designed peptide hairpins has been expanded by the insertion of beta-, gamma- and delta-amino acids at the i+2 position. The model octapeptides Boc-Leu-Phe-Val-DPro-Ac6c-Leu-Phe-Val-OMe (1), Boc-Leu-Phe-Val-DPro-beta3-Ac6c-Leu-Phe-Val-OMe (2), and Boc-Leu-Phe-Val-DPro-Gpn-Leu-Phe-Val-OMe (3) have been shown to adopt beta hairpin conformations in methanol by the observation of key diagnostic nuclear Overhauser effects. Boc-Leu-Val-Val-DPro-delta-Ava-Leu-Val-Val-OMe (4) adopts a beta hairpin conformation in crystals; this is stabilized by three cross-strand hydrogen bonds as demonstrated by X-ray diffraction. The canonical C10 turn in an alpha-alpha segment is expanded to C11, C12, and C13 turns in alpha-beta, alpha-gamma, and alpha-delta segments, respectively. The crystal structures of Piv-LPro-beta3-Ac6c-NHMe (5) and Boc-Ac6c-Gpn-Ac6c-OMe (6) reveal intramolecularly hydrogen-bonded C11 and C12 conformations, respectively. Computer modeling of octapeptide sequences that contain centrally positioned hybrid-turn segments, by using turn parameters derived from the structures of peptides 5 and 6, establishes the stereochemical acceptability of the beta hairpins in the cases of peptides 2 and 3. Accommodation of omega-amino acids into the turn segments is achieved by the adoption of gauche conformations around the backbone C--C bonds.     

The structure of hormaomycin and one of its all-peptide aza-analogues in solution: syntheses and biological activities of new hormaomycin analogues

Four new aza-analogues of hormaomycin 1, a secondary metabolite with interesting biological activities produced by Streptomyces griseoflavus, were synthesized and subjected to preliminary tests of their antibiotic activity to provide new insights into the structure-activity relationship studies of this class of compounds. The solution structures of hormaomycin 1 and its aza-analogue 2 a were determined by NMR spectroscopy. The data exhibited a reasonably rigid conformation for both molecules, stabilized by stacking interactions between the aromatic moieties attached to the ring and the side chain. According to NMR-spectral data the aza-analogue epi-2 a has a rather different conformation and indeed shows no antibacterial activity whatsoever.     

A Designed Three‐Stranded β‐Sheet in an α/β Hybrid Peptide

The incorporation of β‐amino acid residues into the antiparallel β‐strand segments of a multi‐stranded β‐sheet peptide is demonstrated for a 19‐residue peptide, Boc‐LV^βFV^DPGL^βFVVL^DPGLVL^βFVV‐OMe (BBH19). Two centrally positioned ^DPro–Gly segments facilitate formation of a stable three‐stranded β‐sheet, in which β‐phenylalanine (^βPhe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR‐derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well‐defined three‐stranded β‐sheet structure in solution. Cross‐strand interactions between ^βPhe3/^βPhe17 and ^βPhe3/Val15 residues define orientations of these side‐chains. The observation of close contact distances between the side‐chains on the N‐ and C‐terminal strands of the three‐stranded β‐sheet provides strong support for the designed structure. Evidence is presented for multiple side‐chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three‐stranded β‐sheet structures, which in turn influences the conformational interconversion between type I′ and type II′ β‐turns at the two ^DPro–Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc‐LV^βFV^DPGL^βFVV‐OMe (BBH10), which has been previously characterized as a type I′ β‐turn nucleated hairpin, is shown to favour a type II′ β‐turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.     

Conformations of alpha-aminobutyric acid in the gas phase.

A laser-ablation molecular-beam Fourier transform microwave (LA-MB-FTMW) spectrometer has been successfully applied to the structural study of alpha-aminobutyric acid. Three neutral conformers have been identified in the gas phase by comparing their experimental rotational and 14N nuclear quadrupole coupling parameters with those predicted by ab initio calculations at the MP2/6-311++G(d,p) level. The most stable conformer is stabilized by a bifurcated amine-to-carbonyl hydrogen bond (N--HO=C) and a cis-COOH group, and the side-chain adopts a configuration with a torsion angle tau(C(gamma)-C(beta)-C(alpha)-C') of about 180 degrees. The second most stable conformer exhibits the same configuration for the amino acid skeleton but adopts a different orientation for the side chain with tau(C(gamma)-C(beta)-C(alpha)-C') approximately -60 degrees. In the third conformer an intramolecular hydrogen bond is established between the hydroxyl group and the nitrogen atom (NH--O), with a side-chain orientation similar to that of the most stable conformer.     

Solubilizing sodium fluoride in acetonitrile: synthesis, molecular structure, and complexation behavior of bis(organostannyl)methyl-substituted crown ethers

The synthesis of the crown-ether-substituted bis(organostannyl)methanes Ph(3)SnCH(2)Sn(Ph(2))-CH(2)-[16]crown-5 (1) and Ph(2)ISnCH(2)Sn(I)(Ph)-CH(2)-[16]crown-5 (2) is reported. Both compounds have been characterized by elemental analyses, (1)H, (13)C, (19)F, and (119)Sn NMR spectroscopy, and in the case of compound 2 also by electrospray ionization mass spectrometry. Single-crystal X-ray diffraction analysis revealed for the aqua complex 2.H(2)O trigonal-bipyramidal-configured tin atoms with intramolecular Sn(1)-O(1) and Sn(2)-O(1W) distances of 2.555(2) and 2.440(3) A, respectively. The water molecule is trapped in a sandwich-like fashion between the crown ether oxygen atoms O(2) and O(4) and the Sn(2) atom. NMR spectroscopy unambiguously proved the ability of compound 2 in acetonitrile to overcome the high lattice energy of sodium fluoride and to complex the latter under charge separation.