4-Phosphothiophen-2-yl alanine: a new 5-membered analogue of phosphotyrosine

Polyclonal antibodies raised against 4-phosphothiophen-2-yl alanine 2a, a novel five-membered ring analogue of phosphotyrosine, showed high selectivity for phosphotyrosine and no cross-reactivity with other phosphorylated amino acids. Western blots showed that the polyclonal was similarly effective, but different in selectivity, to a commercially available monoclonal antibody

Radical functionalization of unsaturated amino acids: synthesis of side-chain-fluorinated, azido-substituted, and hydroxylated amino acids

A range of enantiomerically pure protected side-chain-fluorinated amino acids has been prepared (13 examples) by treatment of protected amino acids containing unsaturated side chains with a combination of Fe(III)/NaBH4 and Selectfluor. The modification of the conditions by replacement of Selectfluor with NaN3 allowed the preparation of side-chain azido-substituted amino acids (five examples), which upon catalytic hydrogenation gave the corresponding amines, isolated as lactams (four examples). Radical hydration of the unsaturated side chains leading to side-chain-hydroxylated protected amino acids has also been demonstrated

31P NMR spectroscopy demonstrates large amounts of phosphohistidine in mammalian cells

Protein phosphorylation plays a key role in many cellular processes but there is presently no accurate information or reliable procedure to determine the relative abundance of many phosphoamino acids in cells. At pH ≤ 8, phosphohistidine is unstable compared to the extensively studied phosphoserine, phosphothreonine and phosphotyrosine. This study reports the absolute quantitative analysis of histidine phosphorylation of proteins from a human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. The method was designed to minimize loss of the phosphohistidine phosphoryl group. Phosphohistidine was determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 20 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 μmol/g and 68 μmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. After tryptic digest of proteins from the16HBE14o- cell lysate, the phosphohistidine signal was abolished and increasing phosphoserine/phosphothreonine signal was observed, which has implications for mass spectrometry investigations. The 31P NMR spectroscopic analysis not only highlights the abundance of phosphohistidine, which likely reflects its importance in mammalian cells, but also provides a way of measuring and comparing levels of phosphorylated amino acids in cells

Chemical tools for study of phosphohistidine: generation of selective Τ‐phosphohistidine and Π‐phosphohistidine antibodies

Non-hydrolysable stable analogues of τ-phosphohistidine (τ-pHis) and π-pHis have been designed aided by electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both τ-pHis and π-pHis conjugates in the form of a BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by 31P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the generated pHis antibodies generated using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated using the phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated using the pyridyl amino amide 13 were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis

Quantitation of phosphohistidine in proteins in a mammalian cell line by 31P NMR

There is growing evidence to suggest that phosphohistidines are present at significant levels in mammalian cells and play a part in regulating cellular activity, in particular signaling pathways related to cancer. Because of the chemical instability of phosphohistidine at neutral or acid pH, it remains unclear how much phosphohistidine is present in cells. Here we describe a protocol for extracting proteins from mammalian cells in a way that avoids loss of covalent phosphates from proteins, and use it to measure phosphohistidine concentrations in human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. Phosphohistidine is determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 15 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 μmol/g and 68 μmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. Phosphorylation is largely at the N3 (tele) position. Typical tryptic digest conditions result in loss of most of the phosphohistidine present, which may explain why the amounts reported here are greater than is generally seen using mass spectroscopy assays. The results further strengthen the case for a functional role of phosphohistidine in eukaryotic cells

Structure elucidation of dimethylformamide-solvated alkylzinc cations in the gas phase

Organozinc iodides, useful for the synthesis of nonproteinogenic amino acids, are investigated in the gas phase by a combination of electrospray (ESI)-MS/MS, accurate ion mass measurements, and infrared multiphoton dissociation (IRMPD) spectroscopy employing a free electron laser. ESI allowed the full characterization of a set of dimethylformamide (DMF)-solvated alkylzinc cations formed by formal loss of F in the gas phase. Gas phase ion structures of the organozinc cations were identified and optimized by computations at the B3LYP/6-311G** level of theory. The calculations indicate that the zinc cation in gas phase alkylzine-DMF species preferentially adopts a tetrahedral coordination sphere with four ligands, namely the alkyl group, any internal coordinating group, and DMF (the number of which depends on the number of internal coordinating groups present). Besides the sequential loss of coordinated DMF, collision-induced dissociation (CID) patterns demonstrate that the zinc-DMF interaction in tetrahedral four-coordinate mono-DMF-zinc complex ions can be even stronger than covalent bonds. The IRMPD spectra of the alkylzine-DMF species examined show a rich pattern of indicative bands in the range of 1000-1800 cm(-1). All major features of the recorded IRMPD spectra are consistent with the computed IR spectra of the respective gas phase ion structures predicted by theory, allowing identification and assignment

Evidence for the role of tetramethylethylenediamine in aqueous Negishi cross-coupling: synthesis of nonproteinogenic phenylalanine derivatives on water

The structure of the alkylzinc−tetramethylethyl-enediamine (TMEDA) cluster cation 3 has been determined in the gas phase by a combination of tandem mass spectrometry, infrared multiphoton dissociation (IRMPD) spectroscopy, and DFT calculations. Both sets of experimental results establish the existence of a strongly stabilizing interaction of TMEDA with the zinc cation. High-level DFT calculations on the alkylzinc−TMEDA cluster cation 3 allowed the identification of two low energy conformers, each featuring a four-coordinate zinc atom with a bidentate TMEDA ligand, and internal coordination from the carbonyl group of the Boc group to zinc. The experimental IRMPD spectrum is reproduced with an appropriately weighted combination of the IR spectra of the two conformers identified by theory. DFT calculations on the structure of the alkylzinc halide 2 with coordinated TMEDA using the PCM model of water solvent suggest that TMEDA can promote ionization of the zinc−iodine bond in organozinc iodides under aqueous conditions, providing a credible explanation for the role of TMEDA in stabilizing the carbon−zinc bond. Reaction of the serine-derived iodide 1 with aryl iodides "on water", promoted by nano zinc in the presence of PdCl2(Amphos)2 (5 mol %) and TMEDA, leads to the formation of protected phenylalanine derivatives 4 in reasonable yields. In the case of ortho-substituted aryl iodides and aryl iodides that are solids at room temperature, conducting the reaction at 65 °C gives improved results. In all cases, the product 5 of reductive dimerization of the iodide 1 is also isolated

Gas-phase study of new organozinc reagents by IRMPD-spectroscopy, computational modelling and tandem-MS

An extensive set of organozinc iodides, useful for Negishi-type cross-coupling reactions, are investigated as respective cations after formal loss of iodide in the gas phase. Firstly, two new alkylzinc compounds derived from Tyrosine (Tyr) and Tryptophan (Trp) are closely examined. Secondly, the influence of specific protecting groups on the subtle balance between intra- and intermolecular coordination of zinc in these reagents is probed through trifluoroacetyl (TFA)-derivatized alkylzinc compounds. Finally, the influence of the strongly coordinating bidentate ligand N,N,N′,N′-tetramethylethylenediamine (TMEDA) on the structure of alkylzinc cations is further explored in order to better understand the stability of the respective complexes towards water. A combination of electrospray (ESI)-MS/MS, accurate ion mass measurements, infrared multiple-photon dissociation (IRMPD) spectroscopy and computational modelling allowed the full characterisation of all dimethylformamide (DMF)-solvated and TMEDA-coordinated alkylzinc cations in the gas phase. The calculations indicate that the zinc cation in gas-phase alkylzinc-DMF or TMEDA-complex ions preferentially adopts a tetrahedral coordination sphere with four ligands. Additionally, conformers with only three binding partners bound to zinc but with effectively combined hydrogen-bond interactions are also found. Collision induced dissociation (CID) patterns demonstrate that the zinc-DMF interaction in tetrahedral four-coordinate mono-DMF-zinc complex ions as well as the interaction between TMEDA and zinc in the corresponding complex ions is even stronger than typical covalent bonds. In most cases, all major features of the IRMPD spectra are consistent with only a single major isomer, allowing secured identification and assignment