Orthogonally Protected Artificial Amino Acid as Tripod Ligand for Automated Peptide Synthesis and Labeling with [^99mTc(OH₂)₃(CO)₃]⁺

Abstract

1,2-Diamino-propionic acid (Dap) is a very strong chelator for the [^99mTc­(CO)₃]⁺ core, yielding small and hydrophilic complexes. We prepared the lysine based Dap derivative l-Lys­(Dap) in which the ε-NH₂ group was replaced by the tripod through conjugation to its α-carbon. The synthetic strategy produced an orthogonally protected bifunctional chelator (BFC). The -NH₂ group of the α-amino acid portion is Fmoc- and the -NH₂ of Dap are Boc-protected. Fmoc-l-Lys­(Dap­(Boc)) was either conjugated to the N- and C-terminus of bombesin BBN(7–14) or integrated into the sequence using solid-phase peptide synthesis (SPPS). We also replaced the native lysine in a cyclic RGD peptide with l-Lys­(Dap). For all peptides, quantitative labeling with the [^99mTc­(CO)₃]⁺ core at a 10 μM concentration in PBS buffer (pH = 7.4) was achieved. For comparison, the rhenium homologues were prepared from [Re­(OH₂)₃(CO)₃]⁺ and Lys­(Dap)-BBN(7–14) or cyclo-(RGDyK­(Dap)), respectively. Determination of integrin receptor binding showed low to medium nanomolar affinities for various receptor subtypes. The IC₅₀ of cyclo-(RGDyK­(Dap­[Re­(CO)₃])) for α_vβ₃ is 7.1 nM as compared to 3.1 nM for nonligated RGD derivative. Biodistribution studies in M21 melanoma bearing nude mice showed reasonable α_vβ₃-integrin specific tumor uptake. Altogether, orthogonally protected l-Lys­(Dap) represents a highly versatile building block for integration in any peptide sequence. Lys­(Dap)-precursors allow high-yield ^99mTc-labeling with [^99mTc­(OH₂)₃(CO)₃]⁺, forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with excellent in vivo characteristics