Irreversible Inhibition of High‐Affinity [³H]Kainate Binding by a Novel Photoactivatable Analogue: (2′S,3′S,4′R)‐2′‐Carboxy‐4′‐(2‐Diazo‐1‐Oxo‐3,3,3‐Trifluoropropyl)‐3′‐Pyrrolidinyl Acetate

Abstract

A photolabile trifluoromethyldiazoketone derivative of kainate (KA), (2 'S,3 'S,4'R)-2 '-carboxy-4 '-(2diazo-1 -oxo-3,3,3-trifluoropropyl)-3 '-pyrrolidinyl acetate (DZKA), was synthesized and evaluated as an irreversible inhibitor of the high-affinity KA site on rat forebrain synaptic plasma membranes (SPMs). In the absence of UV irradiation, DZKA preferentially blocked [ 3H]KA binding with an 1050 of 0.63 1iM, a concentration that produced little or no inhibition at AMPA or NMDA sites. At 100~sM, however, DZKA inhibited [ 3HI AMPA and L-[3H}glutamate binding by -50%. When examined electrophysiologically in HEK293 cells expressing human KA (GIuR6) or AMPA (GIuR1) subtypes, DZKA acted preferentially at KA receptors as a weak agonist. DZKA also exhibited little or no excitotoxic activity in mixed rat cortical cultures. Irreversible inhibition was assessed by pretreating SPMs with DZKA (50 1iM) in the presence of UV irradiation, removing unbound DZKA, and then assaying the reisolated SPMs for radioligand binding. This protocol produced a selective and irreversible loss of -50% of the [ 3H]KAsites. The binding was recoverable in SPMs pretreated with DZKA or UV alone. Coincubation with Lglutamate prevented the loss in [3H]KA binding, suggesting that the inactivation occurred at or near the ligand binding site. These results are consistent with the action of DZKA as a photoaffinity ligand for the KA site and identify the analogue as a valuable probe for future investigations of receptor structure and function. Key Words: Excitatory amino acids-Kainate receptor-Photoaffinity ligand. J. Neurochem. 68, 1503Neurochem. 68, -1510Neurochem. 68, (1997.L-Glutamate participates in several activities within the CNS, including fast synaptic signaling, long-term potentiation, development, synaptic plasticity, and neuronal pathology (for review, see Cotman et al., 1995), through the activation of various excitatory amino acid (EAA) receptors. These receptors have been functionally divided between those that gate ion channels (ionotropic) and those linked to second messenger systems (metabotropic), such as the cyclic AMP or inositol phosphate/diacylglycerol pathways (Collingridge and Watkins, 1994;Nakanishi and Masu, 1994;Schoepp, 1994;Cotman et al., 1995). Tonotropic receptors are further subdivided into Nmethyl-D-aspartate (NMDA), kainate (KA), and aamino -3 -hydroxy -5 -methyl -4 -isoxazole propionate (AMPA) classes based on agonist selectivity.

Aryldiazirine-Modified Pyroglutamates: Photoaffinity Labels for Glutamate

“…36 The stereochemistry of this compound was confirmed by comparison of 1 H NMR chemical shift data for related compounds, 35 and by NOESY analysis, which clearly indicated the exo-orientation of the arylmethyl residue (see Scheme 2). 36 This product was elaborated to pyroglutamate 6 by acid-catalysed deprotection, ester hydrolysis, oxidation and esterification in good overall yield; in this compound, the 13 C NMR and 19 F NMR characterisation data (see Table 1) of the diazirine unit of 6 was consistent with that of its simpler precursors. 37 In contrast to the successful alkylations, however, mercury-lead exchange of 2f with lead tetraacetate, followed by attempted Pinhey arylation of lactam 4, 38 was not successful.…”

“…We have previously demonstrated the utility of aryldiazirines as photaffinity labels readily activated by laser irradiation, 10 and have become interested in the application of this approach for the determination of binding site information for excitatory amino acid (EAA) receptors. Azidoderived photoaffinity labels have recently been investigated for the NMDA receptor 11,12 and KA receptors, [13][14][15][16][17] and there has been a recent report of a selective mGluR1 radioligand, 18 but there are no reports of the incorporation of diazirine systems into EAA receptor agonists. Reports by Hatanaka 19 and Brunner 20 indicated that 3-methoxyphenyldiazirines 1a could be thallated and further reacted with a variety of electrophiles under mild conditions, thereby permitting introduction of an aryldiazirine unit intact into a substrate and avoiding a lengthy linear synthesis.…”

“…13 C NMR and19 F NMR Chemical Shift and Coupling Constants for the CF 3 Unit in some Trifluoromethyldiazirines…”