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A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-α-Fmoc-l-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R = ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z = Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the Kd values were found to be in the low micromolar range (0.5−3.0 μM). Compound 5j was subsequently labeled with ^99mTc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Re-analogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [⁹⁹TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the {M(NNC5H4NHx)2} core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M{CH3N(CH2CH2S)2}(NNC5H4N)(NHNC5H4N] (M = ⁹⁹Tc (5a), Re (5b)), [Re{C5H3N-2,6-(CH2S)2}(NNC5H4N)(NHNC5H4N)]·CH2Cl2·0.5MeOH (7), [Re{SCH2CH2)2O} (NNC5H4N)(NHNC5H4N)] (8), and [Re{(SCH2CH2)2S}(NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3{NNC5H3NH(CO2R)} {NHNC5H3N(CO2R)}] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3{NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)} {NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)}]·2HCl (14·2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2{NNC5H3N(CO2Me)}{NHNC5H3N(CO2Me)}] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.

A tridentate single amino acid chelate (SAAC) derived from N-α-Fmoc-l-lysine was incorporated within a short peptide sequence using an automated peptide synthesizer. Novel derivatives of the chemotactic peptide fMLF were prepared such that the SAAC and its Re complex were selectively placed between a terminal glycine amino acid and the targeting fMLF sequence. The products, which were synthesized in parallel, were characterized by mass spectrometry and multi-NMR spectroscopy. The latter technique demonstrated that the structures of the targeting portions of the peptides are the same in the SAAC and Re−SAAC derivatives. The affinities of the reported compounds for the formyl peptide receptor were subsequently determined using flow cytometry and were found to be comparable to that of the parent peptide. The results of this work demonstrate the feasibility and numerous benefits of using the SAAC system to prepare peptide-targeted Tc(I) and Re(I) radiopharmaceuticals.