Synthesis and coordination of hybrid phosphinoferrocenes with extended donor pendants.

Combining a phosphinoferrocene fragment with extended multidonor moieties affords novel, flexible multidonor pro-ligands. This contribution describes the synthesis of two structurally similar functional phosphines, PhPfcNHC(O)CHPPh (1) and PhPfcNHCHCHPPh (2, fc = ferrocene-1,1'-diyl), and their coordination behaviour towards Pd(II). The former amidophosphine reacts with [PdCl(MeCN)] to produce the chelate complex [PdCl(1-κ,')] as a mixture of and isomers, which convert into bis-chelate [PdCl(PhPfcNC(O)CHPPh-κ,',)] upon reacting with a strong base (KO-Bu).

Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II.

Quantum and molecular mechanics (QM/MM) and QM-only (cluster model) modeling techniques represent the two workhorses in mechanistic understanding of enzyme catalysis. One of the stringent tests for QM/MM and/or QM approaches is to provide quantitative answers to real-world biochemical questions, such as the effect of single-point mutations on enzyme kinetics. This translates into predicting the activation energies to 1-2 kcal·mol accuracy; such predictions can be used for the rational design of novel enzyme variants with desired/improved characteristics.

Flow cytometric determination of cell cycle progression via direct labeling of replicated DNA.

The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry.

Efficient allele conversion in mouse zygotes and primary cells based on electroporation of Cre protein.

Cre-loxP recombination system is a powerful tool for genome engineering. One of its applications is found in genetic mouse models that often require to induce Cre recombination in preimplantation embryos. Here, we describe a technically simple, affordable and highly efficient protocol for Cre protein delivery into mouse zygotes by electroporation.

Novel ferrocenyl functionalised phosphinecarboxamides: synthesis, characterisation and coordination.

The synthesis of two novel ferrocenyl-substituted phosphinecarboxamides, FcNHC(O)PH (1; Fc = ferrocenyl) and FcCHNHC(O)PH (2), is reported. These two primary phosphines were obtained by the reaction of aminoferrocene with sodium 2-phosphaethynolate in the presence of a proton source or, directly, from aminomethylferrocene hydrochloride and sodium 2-phosphaethynolate. Their ability to act as ligands was probed via reactions of 1 with rhodium(iii) and ruthenium(ii) precursors.

Synthesis and Structural Characterisation of an N-Phosphanyl Ferrocene Carboxamide and its Ruthenium, Rhodium and Palladium Complexes.

[((Diphenylphosphanyl)amino)carbonyl]ferrocene (1) has been synthesized and coordinated to light platinum metals, ruthenium, rhodium and palladium, in diverse coordination modes. Specifically, compound 1 was used to prepare the following phosphane complexes, [(η -mes)RuCl (1-κP)], [(η -C Me )RhCl (1-κP)], trans-[PdCl (1-κP) ], and [(L )PdCl(1-κP)] (mes=mesitylene, L =[2-(dimethylamino-κN)methyl]phenyl-κC ). They were subsequently converted into cationic O,P-chelate complexes by halide abstraction with AgClO and into neutral O,P-chelate complexes by deprotonation with potassium tert-butoxide.

The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity.

Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches.

Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificities.

Unlabelled: Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates.

Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism.

Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity.

Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations.