Synthesis and application of 5'-azido-[3,6-3H2]-naphtyl-phthalamic acid, a photo-activable probe for auxin efflux carrier proteins

Auxin transport is essential for the initiation and maintenance of spatially organized patterns of plant cell division, cell enlargement and differentiation. Hence, auxin carrier proteins are considered to play a fundamental role in auxin action. To unravel the molecular properties of auxin efflux carrier proteins specific probes must develop allowing identification, purification and microsequencing of auxin carrier proteins. Polar auxin transport is specifically and reversibly inhibited by 1-N-naphthylphthalamic acid (NPA), a compound which binds to auxin carrier proteins. We therefore aimed to develop a photoaffinity probe for auxin carrier proteins. We have synthesized a photo-activatable, tritiated azido-analogue of NPA, 5'-azido-[3,6-3H2] NPA. We describe here in detail the synthesis and characterization of 5'-azido-[3,6-3H2] N PA and its precursor compounds. We demonstrate the successful application of this compound to photoaffinity label specific proteins with apparent molecular masses of 23 kDa, 24 kDa and 31 kDa from Arabidopsis thaliana plasma membranes.