Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment

An immunoassay for human alpha(1)-antitrypsin based on affinity-probe capillary isoelectric focusing (AP-CIEF) is described. The method is based on the separation of free and bound labeled antibody fragments by CIEF with laser-induced fluorescence detection. A recombinant Fab' fragment of mouse immunoglobulin G1 (IgG1) against human alpha(1)-antitrypsin was labeled with tetramethylrhodamine on the single cysteine residue at the hinge region. A single pI isomer of the labeled Fab' was purified by IEF in a slab of agarose gel and was then used as the affinity probe for alpha(1)-antitrypsin. The use of recombinant Fab' considerably simplified the labeling process. Although there was some difficulty in the quantification of native alpha(1)-antitrypsin with the affinity probe, carbamylation of the antigen sample by heat treatment with urea made the complex peaks appear reproducibly and more distinct, thus facilitating the identification and quantification of the complex. The system provided an almost linear response to a pure sample of alpha(1)-antitrypsin over a concentration range of 5-1000 ng/mL and the detection limit extended down to around 2 ng/mL. Alpha(1)-antitrypsin in a serum sample was determined using this system to be 1.2 mg/mL which is comparable to the reported value for the protein.     

Enantioselective Robinson‐Type Annulation Reaction Catalyzed by Chiral Phosphoric Acids

Let′s resolve our differences : Implementation of an enantioselective Michael addition followed by an intramolecular aldol reaction catalyzed by two phosphoric acids has enabled the synthesis of cyclohexenone derivatives with excellent enantioselectivities. Prominent kinetic resolution was observed in the latter reaction. Ar=aromatic group, X=H, halogen, Y=H, Me, halogen.

     

Ligand π‐Radical Interaction with f‐Shell Unpaired Electrons in Phthalocyaninato–Lanthanoid Single‐Molecule Magnets: A Solution NMR Spectroscopic and DFT Study

The phthalocyaninato double‐decker complexes [M(obPc)₂]⁰ (M= Y^III, Tb^III, Dy^III; obPc=2,3,9,10,16,17,23,24‐octabutoxyphthalocyaninato), along with their reduced ([M(obPc)₂]^?[P(Ph)₄]⁺; M=Tb^III, Dy^III) and oxidized ([M(obPc)₂]⁺[SbCl₆]^? (M=Y^III, Tb^III) counterparts were studied with ¹H, ¹³C and 2D NMR. From the NMR data of the neutral (i.e., with one unpaired electron in the ligands) and anionic Tb^III complexes, along with the use of dispersion corrected DFT methods, it was possible to separate the metal‐centered and ligand‐centered contributions to the hyperfine NMR shift. These contributions to the ¹H and ¹³C hyperfine NMR shifts were further analyzed in terms of pseudocontact and Fermi contact shifts. Furthermore, from a combination of NMR data and DFT calculations, we have determined the spin multiplicity of the neutral complexes [M(obPc)₂]⁰ (M=Tb^III and Dy^III) at room temperature. From the NMR data of the cationic Tb^III complex, for which actually no experimental structure determination is available, we have analyzed the structural changes induced by oxidation from its neutral/anionic species and shown that the interligand distance decreases upon oxidation. The fast electron exchange process between the neutral and anionic Tb^III double‐decker complexes was also studied.