Nucleophilic participation of reduced flavin coenzyme in mechanism of UDP-galactopyranose mutase

UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(β) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).     

Rhodanine derivatives as novel inhibitors of PDE4

The discovery, synthesis and in vitro activity of a novel series of rhodanine based phosphodiesterase-4 (PDE4) inhibitors is described. Structure-activity relationship studies directed toward improving potency led to the development of submicromolar inhibitors 2n and 3i (IC(50)=0.89 & 0.74 microM). The replacement of rhodanine with structurally related heterocycles was also investigated and led to the synthesis of pseudothiohydantoin 7 (IC(50)=0.31 microM).     

Hyperspectral computed tomographic imaging spectroscopy of vascular oxygen gradients in the rabbit retina in vivo

Diagnosis of retinal vascular diseases depends on ophthalmoscopic findings that most often occur after severe visual loss (as in vein occlusions) or chronic changes that are irreversible (as in diabetic retinopathy). Despite recent advances, diagnostic imaging currently reveals very little about the vascular function and local oxygen delivery. One potentially useful measure of vascular function is measurement of hemoglobin oxygen content. In this paper, we demonstrate a novel method of accurately, rapidly and easily measuring oxygen saturation within retinal vessels using in vivo imaging spectroscopy. This method uses a commercially available fundus camera coupled to two-dimensional diffracting optics that scatter the incident light onto a focal plane array in a calibrated pattern. Computed tomographic algorithms are used to reconstruct the diffracted spectral patterns into wavelength components of the original image. In this paper the spectral components of oxy- and deoxyhemoglobin are analyzed from the vessels within the image. Up to 76 spectral measurements can be made in only a few milliseconds and used to quantify the oxygen saturation within the retinal vessels over a 10-15 degree field. The method described here can acquire 10-fold more spectral data in much less time than conventional oximetry systems (while utilizing the commonly accepted fundus camera platform). Application of this method to animal models of retinal vascular disease and clinical subjects will provide useful and novel information about retinal vascular disease and physiology.     

Structural and spectroscopic studies of a model for catechol oxidase

A binuclear copper complex, [Cu₂(BPMP)(OAc)₂][ClO₄]·H₂O, has been prepared using the binucleating ligand 2,6-bis[bis(pyridin-2-ylmethylamino)methyl]-4-methylphenol (H-BPMP). The X-ray crystal structure reveals the copper centers to have a five-coordinate square pyramidal geometry, with the acetate ligands bound terminally. The bridging phenolate occupies the apical position of the square-based pyramids and magnetic susceptibility, electron paramagnetic resonance (EPR) and variable-temperature variable-field magnetic circular dichroism (MCD) measurements indicate that the two centers are very weakly antiferromagnetically coupled (J = −0.6 cm⁻¹). Simulation of the dipole–dipole-coupled EPR spectrum showed that in solution the Cu–O–Cu angle was increased from 126° to 160° and that the internuclear distance was larger than that observed crystallographically. The high-resolution spectroscopic information obtained has been correlated with a detailed ligand-field analysis to gain insight into the electronic structure of the complex. Symmetry arguments have been used to demonstrate that the sign of the MCD is characteristic of the tetragonally elongated environment. The complex also displays catecholase activity (k _cat = 15 ± 1.5 min⁻¹, K _M = 6.4 ± 1.8 mM), which is compared with other dicopper catechol oxidase models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.