The affinity of the binding domain of FK-506 was determined in the absence of the constraints imposed by its macrocyclic framework. Investigations into the mechanism of action of the immunosuppressant FK-506 have provided insights into cytoplasmic signal transduction. FK-506 binds to the cytosolic protein FKBP12 (FK binding protein, molecular weight 12,000), and the structure of this complex has been determined by X-ray and NMR methods, showing approximately half of the FK-506 macrocycle is deeply buried within FKBP12 while the other half protrudes and is exposed to solvent. FKBP12 plays a role analogous to cyclophilin (the binding protein for cyclosporin), and both are well-characterized immunophilins. A composite surface of FK-506 and FKBP12 binds to and inhibits the protein phosphatase calcineurin, interrupting signal transduction and mediating immunosuppression. We separated the immunophilin binding region of FK-506 from the macrocyclic framework constraints by total synthesis of compound 2 (the binding region) and determined its ability to displace a radiolabeled FK-506 derivative from isolated FKBP12. The synthesis of compound 2 allowed affinity measurement without macrocyclic constraints. We reasoned the macrocyclic framework (protruding from FKBP12) restricts binding domain element positions, affecting affinity. Schreiber et al. replaced FK-506's non-binding portion with a 'scaffolding domain' (506BD), observing a 20-fold affinity drop. FK-506's affinity for FKBP12 was Kd=2×10⁻¹¹ M (n=8, ±2×10⁻¹² M), while compound 2's was Kd=1×10⁻⁹ M (n=4, ±2.4×10⁻¹⁰), a 50-fold drop. Compound 2 is anticipated to inhibit FKBP rotamase activity. Moderate remaining affinity in compound 2 may result from binding region features (stereoelectronic control of the anomeric hydroxyl group, A1,3 strain interactions) that constrain conformation without the macrocycle. Removing the macrocycle likely allows substituted cyclohexyl moiety motion relative to the rest of the binding region. Simplifying the binding domain and designing alternative biologically active scaffolding frameworks is ongoing.