Amperometric enzyme electrode for theophylline.

An amperometric biosensor for theophylline, based on the recently isolated enzyme theophylline oxidase, is described. The enzyme is entrapped, together with a ferricytochrome C cofactor, within a polymeric (Nafion) coating. The anodic detection (at +0.4 V versus Ag-AgCl) is facilitated by the addition of a redox-mediating hexacyanoferrate(III) ion. The influence of various experimental variables is described. The limit of detection is 2 x 10(-6) mol dm-3 theophylline, with linearity prevailing up to 3 x 10(-4) mol dm-3. The fast response and wash times permit rapid flow-injection measurements, with a frequency of 180 samples h-1 and a relative standard deviation of 3.0-4.0%. Prospects of using this electrode for clinical diagnostics are discussed.     

Intrazeolite photooxidations of electron-poor alkenes

The inability of the intrazeolite environment to influence the regiochemistry of addition of singlet oxygen to electron-poor olefins is reported. This divergent behavior with respect to electron-rich alkenes is rationalized with a multicomplexation model that emphasizes the importance of intrazeolite substrate-cation binding.     

Nanometer-scale ion aggregates in aqueous electrolyte solutions: guanidinium sulfate and guanidinium thiocyanate

Neutron diffraction experiments and molecular dynamics simulations are used to study the structure of aqueous solutions of two electrolytes: guanidinium sulfate (a mild protein conformation stabilizer) and guanidinium thiocyanate (a powerful denaturant). The MD simulations find the unexpected result that in the Gdm2SO4 solution the ions aggregated into mesoscopic (nanometer-scale) clusters, while no such aggregation is found in the GdmSCN solution. The neutron diffraction studies, the most direct experimental probe of solution structure, provide corroborating evidence that the predicted very strong ion pairing does occur in solutions of 1.5 m Gdm2SO4 but not in 3 m solutions of GdmSCN. A mechanism is proposed as to how this mesoscopic solution structure affects solution denaturant properties and suggests an explanation for the Hofmeister ordering of these solutions in terms of this ion pairing and the ability of sulfate to reverse the denaturant power of guanidinium.     

Photophysics of ruthenium polypyridyl complexes formed with lacunary polyoxotungstates with iron addenda

The interactions between luminophore [Ru(bpy)3]2+, and the lacunary Dawson heteropolyanions, [P2W17O61(FeOH2)]7-, [P2W17O61(FeBr)]6- and [P2W17O61]10- were investigated using a combination of photophysics, optical and Raman spectroscopy. Extensive quenching of the excited state of [Ru(bpy)3]2+ was observed in each case. Quenching is attributed to the formation of association complexes between [Ru(bpy)(3)]2+ and the heteropolyanions in which the charge on the heteropolyanions is fully compensated for by the ruthenium polypyridyl species. The interaction appears to be principally electrostatic in nature producing [Ru(bpy)3]3.5[P2W17O61(FeOH2)], [Ru(bpy)3]3[P2W17O61(FeBr)] and [Ru(bpy)3]5[P2W17O61]10-. The association constants for formation of the clusters were obtained from photophysical studies and surprisingly, despite the electrostatic nature of the interaction, there was no correlation between the charge on the polyoxometallate and the association constant. In particular, the unsubstituted lacunary, [P2W17O61]10-, showed considerably weaker association compared to the transition metal substituted lacunaries, in spite of its 10- charge. Difference absorption spectroscopy revealed a new transition at ca. 480 nm for each of the cluster complexes. From resonance Raman spectroscopy the origin of this transition was found to involve the polyoxometallate. Unlike previously reported adducts, the cluster complexes formed were not luminescent. In all cases the cluster complexes exhibit remarkable photostability, with no photodecomposition or photo-induced ligand exchange reactions evident in acetonitrile, under conditions where [Ru(bpy)3]2+ alone exhibits considerable photolability.     

Practical aspects of fast HPLC separations for pharmaceutical process development using monolithic columns

A large number of samples can be generated during pharmaceutical process development. Fast separation for these samples is usually challenging due to the complexity of sample matrix, which requires high efficiency as well as high speed. Monolithic columns (E. Merck, Germany) were investigated as a possible tool for reducing separation time in reversed-phase HPLC without significantly sacrificing efficiency or resolution. Both van Deemter plots and separations of alkyl benzenes and in-process samples showed that monolithic columns were suitable for fast separations without significantly compromising resolution. Practical parameters including the pressure drop, retention factor, selectivity, and tailing factor of monolithic columns (Chromolith type) were compared to those of conventional YMC 150 mm × 4.6 mm (3-μm particles) and 250 mm × 4.6 mm (5-μm particles) packed columns. The batch-to-batch reproducibility of the 100 mm × 4.6 mm Chromolith columns from five randomly ordered batches was also compared to the 250 mm × 4.6 mm YMC particle-packed columns. Fast and efficient separations of complicated process samples including crude drug substances, reaction mixtures, and crystallized mother liquors were demonstrated for both monolithic columns and conventional packed columns. The analysis times were decreased by three to seven times on the coupled monolithic columns, while maintaining the comparable resolution to typical 5-μm particle-packed 250 mm × 4.6 mm columns.     

Utilization of hydrogen bonds to stabilize M-O(H) units: synthesis and properties of monomeric iron and manganese complexes with terminal oxo and hydroxo ligands

Non-heme iron and manganese species with terminal oxo ligands are proposed to be key intermediates in a variety of biological and synthetic systems; however, the stabilization of these types of complexes has proven difficult because of the tendency to form oxo-bridged complexes. Described herein are the design, isolation, and properties for a series of mononuclear Fe(III) and Mn(III) complexes with terminal oxo or hydroxo ligands. Isolation of the complexes was facilitated by the tripodal ligand tris[(N'-tert-butylureaylato)-N-ethyl]aminato ([H(3)1](3-)), which creates a protective hydrogen bond cavity around the M(III)-O(H) units (M(III) = Fe and Mn). The M(III)-O(H) complexes are prepared by the activation of dioxygen and deprotonation of water. In addition, the M(III)-O(H) complexes can be synthesized using oxygen atom transfer reagents such as N-oxides and hydroxylamines. The [Fe(III)H(3)1(O)](2-) complex also can be made using sulfoxides. These findings support the proposal of a high valent M(IV)-oxo species as an intermediate during dioxygen cleavage. Isotopic labeling studies show that oxo ligands in the [M(III)H(3)1(O)](2-) complexes come directly from the cleavage of dioxygen: for [Fe(III)H(3)1(O)](2-) the nu(Fe-(16)O) = 671 cm(-1), which shifts 26 cm(-1) in [Fe(III)H(3)1((18)O)](2-) (nu(Fe-(18)O) = 645 cm(-1)); a nu(Mn-(16)O) = 700 cm(-1) was observed for [Mn(III)H(3)1((16)O)](2-), which shifts to 672 cm(-1) in the Mn-(18)O isotopomer. X-ray diffraction studies show that the Fe-O distance is 1.813(3) A in [Fe(III)H(3)1(O)](2-), while a longer bond is found in [Fe(III)H(3)1(OH)](-) (Fe-O at 1.926(2) A); a similar trend was found for the Mn(III)-O(H) complexes, where a Mn-O distance of 1.771(5) A is observed for [Mn(III)H(3)1(O)](2-) and 1.873(2) A for [Mn(III)H(3)1(OH)](-). Strong intramolecular hydrogen bonds between the urea NH groups of [H(3)1](3-) and the oxo and oxygen of the hydroxo ligand are observed in all the complexes. These findings, along with density functional theory calculations, indicate that a single sigma-bond exists between the M(III) centers and the oxo ligands, and additional interactions to the oxo ligands arise from intramolecular H-bonds, which illustrates that noncovalent interactions may replace pi-bonds in stabilizing oxometal complexes.     

Synthesis of a liposome incorporated 1-carboxyalkylxanthine-phospholipid conjugate and its recognition by an RNA aptamer

RNA or DNA aptamers have received much attention in recent literature as therapeutic agents and chromatographic matrices, however, their use in analytical methodologies is relatively unexplored. We describe here investigations aiming to combine this promising technology with versatile liposomes in a competitive assay format. Thus, a phospholipid derivative of an unsymmetrical 1,3-disubstituted xanthine (1-carboxyethyl-3-methylxanthine-DPPE) was prepared for incorporation into the lipid bilayers of dye-encapsulating liposomes. Its synthesis and characterization using GC-MS, ¹H NMR, and HPLC are described. Equilibrium filtration experiments using enzyme linked immunosorbent assays (ELISAs) were completed to assess the affinity for theophylline of an unmodified RNA aptamer and one that had been modified on the 3′ end with biotin. A dissociation constant (K_d) for theophylline with the unmodified RNA aptamer of 0.9 μM and biotinylated aptamer of 1.0 μM was determined which showed that this modification did not affect the aptamer's affinity using this technique. The observed K_d values correlated well to the previously reported value of 0.6 μM. Experiments were also carried out in a competitive manner with the prepared 1-carboxypropyl-3-methylxanthine intermediate, and the final 1-carboxypropyl-3-methylxanthine-DPPE conjugate once it had been incorporated into the bilayers of liposomes. The K_d value for 1-carboxypropyl-3-methylxanthine was approximately 2.7 μM. Finally, successful binding to theophylline-analog-tagged liposomes in a competitive assay format was shown versus liposomes prepared without the tag.     

2,2′-Bis((di-tert-butylphosphino)methyl)-1,1′-biphenyl (ditbi): a bulky analogue of bisbi. The crystal structure of [Rh2Cl2(1,5-cod)2(μ-ditbi)]

Diphosphine 2,2′-bis(di-tert-butylphosphino)methyl)-1,1′-biphenyl (ditbi) is synthesised by the addition of to 2,2′-bis(bromomethyl)-1,1′-biphenyl, followed by deprotection with diethylamine. Treatment of [Rh₂Cl₂(1,5-cod)₂], with ditbi gives [Rh₂Cl₂(1,5-cod)₂(μ-ditbi)] (2) as confirmed by its X-ray crystal structure determination. Hydroformylation of 1-hexene using [Rh(acac)(CO)₂]/ditbi as catalyst gave n- and iso-heptanal in a ratio of 1:1.     

Infrared spectra of a model phenol-amine proton transfer complex in nanoconfined CH3Cl

The vibrational spectra of a model phenol-amine proton transfer complex dissolved in CH3Cl solvent confined in a 12 A radius spherical hydrophobic cavity were calculated using mixed quantum-classical molecular dynamics simulations. The reaction free energy of the proton transfer complex was varied in order to explore the contributions to the vibrational absorption band from product and reactant species. The vibrational spectra of the model proton transfer complex resulted in motionally narrowed spectral linewidths with two distinct peaks for products and reactants in cases where the system undergoes chemical exchange. It was found that the n=1 and n=2 vibrational excited states combine to form diabatic states such that the spectra have contributions from both n=0 --> n=1 and n=0 --> n=2 transitions. A strong relationship between the instantaneous vibrational frequency and a collective solvent coordinate was found that assists in understanding the origin of the spectral features.     

In-silico guided discovery of novel CCR9 antagonists

Antagonism of CCR9 is a promising mechanism for treatment of inflammatory bowel disease, including ulcerative colitis and Crohn’s disease. There is limited experimental data on CCR9 and its ligands, complicating efforts to identify new small molecule antagonists. We present here results of a successful virtual screening and rational hit-to-lead campaign that led to the discovery and initial optimization of novel CCR9 antagonists. This work uses a novel data fusion strategy to integrate the output of multiple computational tools, such as 2D similarity search, shape similarity, pharmacophore searching, and molecular docking, as well as the identification and incorporation of privileged chemokine fragments. The application of various ranking strategies, which combined consensus and parallel selection methods to achieve a balance of enrichment and novelty, resulted in 198 virtual screening hits in total, with an overall hit rate of 18%. Several hits were developed into early leads through targeted synthesis and purchase of analogs.