Fmoc-diethylglycine

1,2-Diamino-propionic acid (Dap) is a very strong chelator for the [(99m)Tc(CO)(3)](+) core, yielding small and hydrophilic complexes. We prepared the lysine based Dap derivative l-Lys(Dap) in which the ε-NH(2) group was replaced by the tripod through conjugation to its α-carbon. The synthetic strategy produced an orthogonally protected bifunctional chelator (BFC). The -NH(2) group of the α-amino acid portion is Fmoc- and the -NH(2) 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 [(99m)Tc(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(OH(2))(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 IC(50) of cyclo-(RGDyK(Dap[Re(CO)(3)])) for α(v)β(3) 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(v)β(3)-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 (99m)Tc-labeling with [(99m)Tc(OH(2))(3)(CO)(3)](+), forming small and hydrophilic complexes, which in turn leads to peptide radiopharmaceuticals with excellent in vivo characteristics.

Prothymosin alpha (ProTα) is a highly conserved mammalian polypeptide (109 amino acids in man) exerting in vitro and in vivo immunoenhancing activities. Recently, our team has developed a 99mTc-radiolabeled derivative of the C-terminal bioactive decapeptide of ProTα ([99mTc]C1) and employed it in in vitro studies, the results of which support the existence of binding sites on human neutrophils that recognize [99mTc]C1, intact ProTα as well as the C-terminal decapeptide of ProTα and presumably involve Toll-like receptor 4. In the present work, [99mTc]C1 was administered to Swiss albino mice with experimentally-induced inflammation for in vivo biodistribution and imaging studies, in parallel with a suitable negative control, which differs from [99mTc]C1 only in bearing a scrambled version of the ProTα decapeptide. The biodistribution data obtained with [99mTc]C1 demonstrated fast clearance of radioactivity from blood, heart, lungs, normal muscle, and predominantly urinary excretion. Most importantly, slow clearance of radioactivity from the inflammation focus was observed, resulting in a high ratio of inflamed/normal muscle tissue (9.15 at 30min post injection, which remained practically stable up to 2h). The inflammation-targeting capacity of [99mTc]C1 was confirmed by imaging studies and might be attributed to neutrophils, which are recruited at the inflamed areas and bear binding sites for [99mTc]C1. In this respect, apart from being a valuable tool for further studies on ProTα in in vitro and in vivo systems, [99mTc]C1 merits further evaluation as a radiopharmaceutical for specific imaging of inflammation foci.

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide with intracellular functions associated with cell proliferation and apoptosis and an extracellular role associated with immunopotentiation. The N-terminal fragment [1-28], which is identical with the immunostimulating peptide thymosin α1 (Tα1), was earlier considered as the immunoactive region of the polypeptide; however, recent data suggest that ProTα may exert a discrete immunomodulating action through its central or C-terminal region, via targeting Toll-like receptor- 4 (TLR4). In this work, a derivative of the C-terminal fragment ProTα[100-109] (ProTα-D1) that can be radiolabeled with (99m)Tc was developed. The biological activity of the non-radioactive (185/187)rhenium-complex of this derivative ([(185/187)Re]ProTα-D1, structurally similar with [(99m)Tc]ProTα-D1) was verified through suitable in vitro bioassays on human neutrophils. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [(99m)Tc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and ProTα[100-109], as well as by a "prototype" TLR4-ligand (lipopolysaccharide from Escherichia coli). Overall, our results support the existence of ProTα-binding sites on human neutrophils, recognizing [(99m)Tc]ProTα-D1, which might involve TLR4. [(99m)Tc]ProTα-D1 may be a useful tool for conducting further in vitro and in vivo studies, aiming to elucidate the extracellular mode of action of ProTα and, eventually, develop ProTα-based immunotherapeutics.