FLUORESCENT CHEMOAFFINITY LABELING - POTENTIAL APPLICATION OF A NEW AFFINITY LABELING TECHNIQUE TO GLUCOCORTICOID RECEPTORS

A new, general methodology for affinity labeling is proposed. We call this method chemo-, or chemically activated, affinity labeling (CAL) since the addition of a specific chemical is required to initiate the affinity labeling process. Chemoaffinity labeling thus combines control over the timing of covalent bond formation, a distinguishing feature of photoaffinity labeling (PAL), with the advantages of the relatively stable and specific “reactive” functional groups found in conventional, or electrophilic, affinity labeling (EAL). Our chemical initiator for CAL is o-phthalaldehyde (OPTA), which rapidly reacts with primary amines and thiols to give l-(alkylthio)-2-alkylisoindoles in high yield [Simons, S. S., Jr., & Johnson, D. F. (1978) J. Org. Chem. 43,2886-2891], Since these isoindoles are intensely fluorescent [Simons, S. S., Jr., & Johnson, D. F. (1978) Anal. Biochem. 90, 705-725], this CAL reaction wIII usually result in fluorescent chemoaffinity (FCA) labeling, or FCAL. In order to obtain a covalent receptor-steroid complex, we have prepared three hydrolyt-ically stable glucocorticoid derivatives which should participate in this CAL reaction. All three steroids exhibit high affinity for the glucocorticoid receptors from rat liver hepatoma tissue culture (HTC) cells and induce the enzyme tyrosine aminotransferase (TAT). In model studies with OPTA and an added amine or thiol, each steroid gives a good yield of the desired isoindole, two of which are strongly fluorescent and display good stability and response to changes in solvent polarity. Thus, these synthetic steroids show excellent promise for being effective FCA labels for glucocorticoid receptors. OPTA is also a new cross-linking reagent, and cross-linked protein-isoindole derivatives readily display energy transfer from tryptophan to isoindole. © 1979, American Chemical Society. All rights reserved.