Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5'-azido-salicylamido)-undecanoic acid (5' ASU) and its acetyl ester (Ac5' ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5' ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34 x 10(-7) M, evidenced a slightly higher affinity than that reported for C16-C20 fatty acids. Consistent with the binding curve, 5' ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 microM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of 125I-5' ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5' ASU and Ac5' ASU respectively. In turn, irradiation of L-FABP incubated with 5' ASU or Ac5' ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
The regioselective synthesis of pyrido[2,3‐b]‐ and [3,4‐b]pyrazine derivatives II by the Hinsberg reaction is reported starting from 2,3 and 3,4‐diaminopyridine and excess of pyruvic acid or ethyl pyruvate as reactants. Good yields (higher than 90%) were obtained for pyrido[2,3‐b]pyrazine derivatives at room temperature using anhydrous methanol and chloroform as solvents which promote regioselective reactions to give 2‐methylpyrido[2,3‐b]pyrazin‐3(4H)‐one (3a) and 3‐methylpyrido[2,3‐b]pyrazin‐2(1H)‐one (4a) respectively. On the other hand, when the same procedure was applied to 3,4‐diaminopyridine results were not so encouraging for the formation of 2‐methylpyrido[3,4‐b]pyrazin‐3(4H)‐one (3b) and 3‐methylpyrido[3,4‐b]‐pyrazin‐2(1H)one (4b). Kinetic studies were performed in aqueous buffers (pH ‐0.89 to 11.5) and different organic solvents trying to improve yields and achieve regioselectivity. The course of the reactions was followed by uv spectrophotometry being those with ethyl pyruvate 2 to 800 times faster than with pyruvic acid. This investigation involves the kinetics and mechanism of this reaction studying its factibility when π‐deficient substrates are used and its regioselectivity according to the position of the pyridine nitrogen atom.
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