Conformational analysis of quinine and its pseudo enantiomer quinidine: a combined jet-cooled spectroscopy and vibrational circular dichroism study

Laser-desorbed quinine and quinidine have been studied in the gas phase by combining supersonic expansion with laser spectroscopy, namely, laser-induced fluorescence (LIF), resonance-enhanced multiphoton ionization (REMPI), and IR-UV double resonance experiments. Density funtional theory (DFT) calculations have been done in conjunction with the experimental work. The first electronic transition of quinine and quinidine is of π-π* nature, and the studied molecules weakly fluoresce in the gas phase, in contrast to what was observed in solution (Qin, W. W.; et al. J. Phys. Chem. C2009, 113, 11790). The two pseudo enantiomers quinine and quinidine show limited differences in the gas phase; their main conformation is of open type as it is in solution. However, vibrational circular dichroism (VCD) experiments in solution show that additional conformers exist in condensed phase for quinidine, which are not observed for quinine. This difference in behavior between the two pseudo enantiomers is discussed.     

Enantioselective Artificial Metalloenzymes Based on a Bovine Pancreatic Polypeptide Scaffold

Site creation : Enantioselective artificial metalloenzymes have been created by grafting a new active site onto bovine pancreatic polypeptide through the introduction of an amino acid capable of coordinating a copper(II) ion. This hybrid catalyst gave good enantioselectivities in the Diels–Alder and Michael addition reactions in water (see scheme) and displayed a very high substrate selectivity.

     

Tuning the spin-transition properties of pyrene-decorated 2,6-bispyrazolylpyridine based Fe(II) complexes

Two 2,6-bispyrazolylpyridine ligands (bpp) were functionalized with pyrene moieties through linkers of different lengths. In the ligand 2,6-di(1H-pyrazol-1-yl)-4-(pyren-1-yl)pyridine (L1) the pyrene group is directly connected to the bpp moiety via a C-C single bond, while in the ligand 4-(2,6-di(1H-pyrazol-1-yl)pyridin-4-yl)benzyl-4-(pyren-1-yl)butanoate (L2) it is separated by a benzyl ester group involving a flexible butanoic chain. Subsequent complexation of Fe(II) salts revealed dramatic the influence of the nature of the pyrene substitution on the spin-transition behaviour of the resulting complexes. Thus, compound [Fe(L1)(2)](ClO(4))(2) (1) is blocked in its high spin state due to constraints caused by a strong intermolecular π-π stacking in its structure. On the other hand, the flexible chain of ligand L2 in compounds [Fe(L2)(2)](ClO(4))(2) (2) and [Fe(L2)(2)](BF(4))(2)·CH(3)CN·H(2)O (3) prevents structural constraints allowing for reversible spin transitions. Temperature-dependent studies of the photophysical properties of compound 3 do not reveal any obvious correlation between the fluorescence of the pyrene group and the spin state of the spin transition core.     

Physicochemical and interfacial investigation of lipid/polymer particle assemblies

A model study was investigated to develop colloidal supramolecular assemblies consisting of particles coated with lipid layers. The interactions between monodisperse sulfate-charged poly(styrene) submicrometer particles and zwitterionic/cationic lipid vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-3-trimethylammonium propane were considered. The influence of relevant experimental parameters on the final associations was examined by quasi-elastic light scattering to point out some new phenomena occurring in these colloidal systems. The major role of electrostatic interactions as driving forces to control the organization between cationic lipids and oppositely charged poly(styrene) particles was clearly evident, whereas this influence was less pronounced when considering the zwitterionic lipids. The characterization of these original complex assemblies was completed by a thorough study of the surface modification. The combination of zeta potential measurements, X-ray photoelectron spectroscopy analyses, and microscopy observations proved that the envisioned model can really correspond to polymer particles surrounded by lipids.     

A Process to Produce Penicillin G Acylase by Surface-Adhesion Fermentation Using <Emphasis Type="Italic">Mucor griseocyanus</Emphasis> to Obtain 6-Aminopenicillanic Acid by Penicillin G Hydrolysis

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V _max, and the Michaelis constant, K _M, were determined using the Michaelis–Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0–10 mM). K _M and V _max were determined to be 3.0 × 10⁻³ M and 4.4 × 10⁻³ mM/min, respectively.     

Structure-based approach for identification of novel phenylboronic acids as serine-β-lactamase inhibitors

β-Lactamases are bacterial enzymes conferring resistance to β-lactam antibiotics in clinically-relevant pathogens, and represent relevant drug targets. Recently, the identification of new boronic acids (i.e. RPX7009) paved the way to the clinical application of these molecules as potential drugs. Here, we screened in silico a library of ~1400 boronic acids as potential AmpC β-lactamase inhibitors. Six of the most promising candidates were evaluated in biochemical assays leading to the identification of potent inhibitors of clinically-relevant β-lactamases like AmpC, KPC-2 and CTX-M-15. One of the selected compounds showed nanomolar K _i value with the clinically-relevant KPC-2 carbapenemase, while another one exhibited broad spectrum inhibition, being also active on Enterobacter AmpC and the OXA-48 class D carbapenemase.     

Combinatorial discovery of fluorescent pharmacophores by multicomponent reactions in droplet arrays

Fluorescence imaging in clinical diagnostics and biomedical research relies to a great extent on the use of small organic fluorescent probes. Because of the difficulty of combining fluorescent and molecular-recognition properties, the development of such probes has been severely restricted to a number of well-known fluorescent scaffolds. Here we demonstrate that autofluorescing druglike molecules are a valuable source of bioimaging probes. Combinatorial synthesis and screening of chemical libraries in droplet microarrays allowed the identification of new types of fluorophores. Their concise and clean assembly by a multicomponent reaction presents a unique potential for the one-step synthesis of thousands of structurally diverse fluorescent molecules. Because they are based upon a druglike scaffold, these fluorophores retain their molecular recognition potential and can be used to design specific imaging probes.     

Determination of seven arsenic species in seafood by ion exchange chromatography coupled to inductively coupled plasma-mass spectrometry following microwave assisted extraction: method validation and occurrence data

The determination of seven arsenic species in seafood was performed using ion exchange chromatography on an IonPac AS7 column with inductively coupled plasma mass spectrometry detection after microwave assisted extraction. The effect of five parameters on arsenic extraction recoveries was evaluated in certified reference materials. The recoveries of total arsenic and of arsenic species with the two best extraction media (100% H₂O and 80% aqueous MeOH) were generally similar in the five seafood certified reference materials considered. However, because MeOH co-elutes with arsenite, which would result in a positively biased arsenite concentration, the 100% H₂O extraction conditions were selected for validation of the method. Figures of merit (linearity, LOQs (0.019-0.075 mg As kg⁻¹), specificity, trueness (with recoveries between 82% (As(III)) and 104% (As(V) based on spikes or certified concentrations), repeatability (3-14%), and intermediate precision reproducibility (9-16%) of the proposed method were satisfactory for the determination of arsenite, monomethylarsonic acid, dimethylarsinic acid, arsenate, arsenobetaine and arsenocholine in fish and shellfish. The performance criteria for trimethylarsine oxide, however, were less satisfactory. The method was then applied to 65 different seafood samples. Arsenobetaine was the main species in all samples. The percentage of inorganic arsenic varied between 0.4-15.8% in shellfish and 0.5-1.9% at the utmost in fish. The main advantage of this method that uses only H₂O as an extractant and nitric acid as gradient eluent is its great compatibility with the long-term stability of both IEC separation and ICP-MS detection.     

Reversed-phase high-performance liquid chromatographic purification and characterization of the adrenocorticotropin/lipotropin precursor and its fragments

Methods are presented for the effective purification by reversed-phase high-performance liquid chromatography (HPLC) of rat adrenocorticotropin/lipotropin (ACTH/LPH) precursor and its two glycosylated forms. Purification of its NH2-terminal segment from human and porcine pituitaries is presented together with microsequencing data confirming the identity of the purified peptides. The effective separation of various native fragments related to ACTH and beta-LPH from sheep pituitaries is presented. A new putative gamma-MSH hormone has been synthesized and purified by reversed-phase HPLC and tryptic peptide mapping performed to establish the identity of the purified peptide.     

Purification and characterization of recombinant pyrrolidone carboxyl peptidase of Bacillus subtilis.

Bacillus subtilis pyrrolidone carboxyl peptidase (Pcp) overexpressed in Escherichia coli was purified to homogeneity in less than 12 h using ammonium sulphate precipitation and hydrophobic interaction chromatography. The enzyme, which removes amino-terminal L-pyroglutamic acid from peptides, appears to be a tetramer of 25,200 molecular mass subunits. The protein cross-reacted with polyclonal antibodies raised against Pcp from Streptococcus pyogenes. The overexpressed enzyme exhibits an absolute substrate specificity towards N-terminal pyroglutamyl residues with a Michaelis constant of 1.04 mM for L-pyroglutamyl-beta-naphthylamide. The enzyme could be used for the removal of pyroglutamyl residues that block amino termini of proteins and peptides before performing Edman sequential degradation.