Photoaffinity analogues of methotrexate as probes for dihydrofolate reductase structure and function.

Previous work from our laboratory [1] has established the efficient syntheses of lysine (APA-Lys)* and ornithine (APA-Orn) analogues of the folate antagonist drug methotrexate (MTX). Both analogues are potent inhibitors of dihydrofolate reductase (DHFR) , the intracellular target for MTX and other antifolate drugs. Reaction of APA-Lys and APA-Orn with dansyl chloride furnishes the corresponding dansyl derivatives [2, 3]. These two fluorescent compounds, like their precursors, are effective inhibitors of D H F R isolated from several sources. They also exhibit an enhancement of their fluorescence and a shift in fluorescence maxima to a lower wavelength when bound to D H F R [4]. In addition, the dansyl derivative of APA-Lys competes effectively with MTX for transport into intact L1210 cells, both MTX-sensitive and -resistant, and becomes bound to intracellular D H F R [5]. These results are consistent with conclusions drawn from X-ray crystallographic analyses of inhibitor complexes of DHFR, which suggest that the y-carboxylate of the glutamate moiety of MTX plays a minor role in the binding of MTX to D H F R [6-8]. As a complement to fluorescent probes, the technique of photoaffinity labeling has been widely used in the study of molecular interactions in biological systems (for a review, see Ref. 9). An advantageous property of photoaffinity labeling probes over chemically reactive affinity probes is that it allows the study of both competitive, reversible as well as covalent, irreversible interactions with a single ligand and its receptor. This technique was applied recently to folate-dependent enzymes with the synthesis of 2'azidoaminopterin [10]. However, this compound was incorporated into Escherichia coli D H F R with an efficiency of only 0.5%. The present study describes the syntheses and inhibitory potencies of four photoaffinity analogues of MTX. These include N~-(4-amino-4-deoxy-10-methyl pteroyl)-N~-(4'-azidosalicylyl)-IMysine (APA-ASA-Lys) , the corresponding ornithine analogue (APA-ASA-Orn) and their iodinated counterparts (APA-I-ASA-Lys and APA-I -ASA-Orn) . A preliminary account of these syntheses has appeared [11,+]. In addition, data on the photoaffinity labeling of murine L1210 D H F R with APA-[t25I]ASA-Lys are described.