Synthesis of AX7593, a quinazoline-derived photoaffinity probe for EGFR

[structure: see text] The synthesis of a photoaffinity probe for EGFR is described. O-Alkylation of 4-(meta-azidoanilino)-6-methoxy-7-hydroxy-quinazoline with a protected tetraethyleneglycol linker followed by the attachment of tetramethylrhodamine yielded the fluorescent probe AX7593. Photoaffinity labeling of EGFR by AX7593 (K(b) = 280 nM) was shown to have an efficiency of 34% and to be competitive with the EGFR inhibitors PP2 and AG1478.     

Recursive polynomial curve schemes and computer-aided geometric design

A class of polynomial curve schemes is introduced that may have widespread application to CAGD (computer-aided geometric design), and which contains many well-known curve schemes, including Bézier curves, Lagrange polynomials, B-spline curve (segments), and Catmull-Rom spline (segments). The curves in this class can be characterized by a simple recursion formula. They are also shown to have many properties desirable for CAGD; in particular they are affine invariant, have the convex hull property, and possess a recursive evaluation algorithm. Further, these curves have shape parameters which may be used as a design tool for introducing such geometric effects as tautness, bias, or interpolation. The link between probability theory and this class of curves is also discussed.Communicated by Klaus Höllig.     

Temperature dependence of inhomogeneous broadening: on the meaning of isosbestic points

An isosbestic point in the temperature dependence of an absorption spectrum is commonly viewed as a sign of interconverting chemical or structural species. Here I explore an alternative microscopic origin of temperature-independent absorbance in inhomogeneously broadened spectra: While the equilibrium probability of observing a particular frequency generally changes with the magnitude of thermal fluctuations, the weight of at least one frequency is insensitive to small changes in temperature. The range of temperature accessible to aqueous solutions at ambient pressure is sufficiently small that such insensitivity would appear in experiments as an isosbestic point. This mechanism depends only on the continuity of equilibrium distributions, not on the presence of multiple components. Even the simplest model of a single solute species in a fluctuating environment exhibits isosbestic points, van't Hoff behavior, and, with appropriate dependence of transition frequency on configuration, multiple absorption peaks.     

Immobilization of several multienzyme systems on porous glass beads

Equimolar concentrations of malate dehydrogenase (EC 1.1.1.37) and fumarase (EC 4.2.1.2) and equimolar concentrations of malate dehydrogenase and citrate synthase (EC 4.1.3.7) were simultaneously immobilized to alkylamine porous silica beads with gluteraldehyde. The activity of each enzyme in the two-enzyme immobilized systems was determined and exact concentrations of the free nonimmobilized enzymes were prepared. The activities of the coupled free and coupled immobilized systems were measured, and it was observed that there was a 10-fold enhancement in the catalysis of the immobilized enzymes.     

Glycosyl disulfides: novel glycosylating reagents with flexible aglycon alteration

[reaction: see text] Glycosyl disulfides have been shown for the first time to be effective glycosyl donors. Glucosylation and galactosylation of a panel of representative alcohol acceptors allowed the formation of 28 simple glycosides, disaccharides, and glycoamino acids in yields of up to 90%. As well as providing a novel class of effective glycosyl donors, the ability to easily alter the nature of the aglycon and the ability to differently activate donors that differ only in their aglycon simply through altering conditions lends glycosyl disulfide donors to their use in latent-active reactivity tuning strategies.     

Total internal reflection Raman spectroscopy of barley leaf epicuticular waxes in vivo

The use of total internal reflection (TIR)-Raman spectroscopy to examine cuticular wax layers in vivo at the surface of barley leaves (Hordeum vulgare L. cultivar Pastoral) is demonstrated. The limited penetration depth (40 nm) of the evanescent wave compared to the thickness of the wax layer eliminates problems arising from fluorescence from underlying pigments. Epicuticular wax crystals are observed to be more crystalline than the amorphous wax layer, which is analysed after removal of the wax crystals by cellulose acetate stripping. Carotenoids are detected as trace species in the cuticular waxes.     

Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands

The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.