Supercritical fluid chromatographic analysis of polyprenols in Ginkgo biloba L

A supercritical fluid chromatographic (SFC) procedure for the quantitation of three major polyprenols present in the leaves of Ginkgo biloba was developed. In contrast to previously reported high-performance liquid chromatographic (HPLC) methods, the SFC method does not require extensive pre-purification for polyprenol analysis. The SFC analytical procedure described shows a very broad range of linearity and detects many known polyprenol isoprenologs with baseline separation. Dodecaprenol was used as the internal standard. The coefficient of variation of the method was 5.8% for the quantitation of C₈₅, C₉₀ and C₉₅ polyprenols. The SFC assay results showed that the content of polyprenols in ginkgo leaves were higher than the previously published values. In addition, the chromatogram of the highly concentrated leaf extract revealed the presence of an isoprenolog (C₁₂₀) not previously detected by HPLC methods.     

Conjugate addition of (trimethylstannyl)copper reagents to α,β-acetylenic N,N-dimethylamides. Trapping of the intermediates with electrophiles

A study of the reaction of Me₃SnCu·.Me₂S (1) and [Me₃SnCuSPh]Li (2) with α,β - acetylenic N,N-dimethylamides 3 shows (a) that the overall process can be controlled experimentally so as to produce either N,N-dimethyl ($\text{E}$)-(4) or ($\text{Z}$)-3-trimethylstannyl-2-alkenamides (5), (b) that the initially formed intermediate derived from interaction of 2 and 3 is significantly more stable than that obtained by reaction of 2 and α,β-acetylenic esters, and (c) that the intermediate produced by treating 3 with 1 can be trapped with electrophiles other than proton.     

Estrogen receptor microarrays: subtype-selective ligand binding

We present the first example of a nuclear hormone receptor microarray, using for illustration the ligand-binding domains of the two estrogen receptors, ERalpha-LBD and ERbeta-LBD. The proteins are printed and allowed to attach to aldehyde slides; the efficiency of attachment depends on whether the LBD is liganded with agonists (low attachment) versus liganded with antagonists or unliganded (high attachment). This suggests that attachment is orientation specific and involves principally a single lysine residue. The attached ERs retain good ligand-binding activity that can be assessed using an estradiol-fluorophore conjugate, and specific and ER subtype-selective binding of ligands can be determined conveniently in competitive binding assays. This powerful new, high-throughput technique to study ligand binding to ER-LBDs can be extended to other nuclear hormone receptors and adapted to assay the recruitment of coregulator proteins.     

A synthetic strategy for the preparation of cyclic peptide mimetics based on SET-promoted photocyclization processes

A novel method for the synthesis of cyclic peptide analogues has been developed. The general approach relies on the use of SET-promoted photocyclization reactions of peptides that contain N-terminal phthalimides as light absorbing electron acceptor moieties and C-terminal alpha-amidosilane or alpha-amidocarboxylate centers. Prototypical substrates are prepared by coupling preformed peptides with the acid chloride of N-phthalimidoglycine. Irradiation of these substrates results in the generation of cyclic peptide analogues in modest to good yields. The chemical efficiencies of these processes are not significantly affected by (1) the lengths of the peptide chains separating the phthalimide and alpha-amidosilane or alpha-amidocarboxylate centers and (2) the nature of the penultimate cation radical alpha-heterolytic fragmentation process (i.e., desilylation vs decarboxylation). An evaluation of the effects of N-alkyl substitution on the amide residues in the peptide chain showed that N-alkyl substitution does not have a major impact on the efficiencies of the photocyclization reactions but that it profoundly increases the stability of the cyclic peptide.     

Diaquabis(1,3‐propanediamine‐κ2N,N′)nickel(II) bis(sulfanilate)

The title compound, [Ni(C₃H₁₀N₂)₂(H₂O)₂](C₆H₆NO₃S)₂, contains alternating layers of sulfanilate anions and di­aqua­bis(1,3‐propane­di­amine)­nickel(II) cations. The Ni atom lies on an inversion centre and is hexacoordinated by the 1,3‐propane­di­amine ligands, which function as N,N′‐bidentate ligands, and the water mol­ecules, which are in a trans arrangement. The sulfanilate anions are arranged in layers, with the sulfonate and amine groups directed towards opposite sides of the layer. The structure is stabilized by a network of hydrogen bonding between the O and N atoms of the sulfanilate anions, the water mol­ecules, and the N atoms of the 1,3‐propane­di­amine ligands.     

Adsorption of NH3 onto activated carbon prepared from palm shells impregnated with H2SO4

Adsorption of ammonia (NH3) onto activated carbons prepared from palm shells impregnated with sulfuric acid (H2SO4) was investigated. The effects of activation temperature and acid concentration on pore surface area development were studied. The relatively large micropore surface areas of the palm-shell activated carbons prepared by H2SO4 activation suggest their potential applications in gas adsorption. Adsorption experiments at a fixed temperature showed that the amounts of NH3 adsorbed onto the chemically activated carbons, unlike those prepared by CO2 thermal activation, were not solely dependent on the specific pore surface areas of the adsorbents. Further adsorption tests for a wide range of temperatures suggested combined physisorption and chemisorption of NH3. Desorption tests at the same temperature as adsorption and at an elevated temperature were carried out to confirm the occurrence of chemisorption due to the interaction between NH3 and some oxygen functional groups via hydrogen bonding. The surface functional groups on the adsorbent surface were detected by Fourier transform infrared spectroscopy. The amounts of NH3 adsorbed by chemisorption were correlated with the contents of elemental oxygen present in the adsorbents. Mechanisms for chemical activation and adsorption processes are proposed based on the observed phenomena.     

Protein profiles and identification of high performance liquid chromatography isolated proteins of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures.     

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.     

Dynamic identification of phosphopeptides using immobilized metal ion affinity chromatography enrichment, subsequent partial beta-elimination/chemical tagging and matrix-assisted laser desorption/ionization mass spectrometric analysis

The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified beta-elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time-dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol-tagged derivatives simultaneously by their mass difference (-20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data.     

Synthesis of PI3K inhibitor GDC-0077 via a stereocontrolled N-arylation of α-amino acids

An efficient synthesis of PI3K inhibitor GDC-0077, featuring two consecutive Cu-catalyzed CN coupling reactions, is reported. The described synthetic route involves a chemoselective Ullmann-type coupling of a chiral difluoromethyl-substituted oxazolidinone, a Cu-catalyzed N-arylation of l-alanine with high stereochemical integrity, and a high-yielding final amide bond formation step to produce GDC-0077 in >99.5 area % HPLC purity.