Clickable Hydrophilic Ligand for fac-MI(CO)3+ (M = Re/99mTc) Applied in an S-Functionalized α-MSH Peptide

摘要

The copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (>1 and >3) for fac-[M^I(CO)3]⁺ (M = Re/^99mTc) complexation into an α-melanocyte stimulating hormone (α-MSH) peptide analogue. A novel DPA ligand with carboxylate substitutions on the pyridyl rings (>3) was designed to increase the hydrophilicity and to decrease in vivo hepatobiliary retention of fac-[^99mTc^I(CO)3]⁺ complexes used in single photon emission computed tomography (SPECT) imaging studies with targeting biomolecules. The fac-[Re^I(CO)3(>3)] complex (>4) was used for chemical characterization and X-ray crystal analysis prior to radiolabeling studies between >3 and fac-[^99mTc^I(OH2)3(CO)3]⁺. The corresponding ^99mTc complex (>4a) was obtained in high radiochemical yields, was stable in vitro for 24 h during amino acid challenge and serum stability assays, and showed increased hydrophilicity by log P analysis compared to an analogous complex with nonfunctionalized pyridine rings (>2a). An α-MSH peptide functionalized with an azide was labeled with fac-[M^I(CO)3]⁺ using both click, then chelate (CuAAC reaction with >1 or >3 followed by metal complexation) and chelate, then click (metal complexation of >1 and >3 followed by CuAAC with the peptide) strategies to assess the effects of CuAAC conditionson fac-[M^I(CO)3]⁺ complexation within a peptide framework. The peptides from the click, then chelate strategy had different HPLC tR’s and in vitro stabilities comparedto those from the chelate, then click strategy, suggestingnonspecific coordination of fac-[M^I(CO)3]⁺ using this synthetic route. The fac-[M^I(CO)3]⁺-complexed peptides fromthe chelate, then click strategy showed >90% stabilityduring in vitro challenge conditions for 6 h, demonstrated high affinityand specificity for the melanocortin 1 receptor (MC1R) in IC50 analyses, and led to moderately high uptake in B16F10 melanoma cells.Log P analysis of the ^99mTc-labeled peptidesconfirmed the enhanced hydrophilicity of the peptide bearing the novel,carboxylate-functionalized DPA chelate (>10a′)compared to the peptide with the unmodified DPA chelate (>9a′). In vivo biodistribution analysis of >9a′ and >10a′ showed moderate tumor uptake in a B16F10 melanomaxenograft mouse model with enhanced renal uptake and surprising intestinaluptake for >10a′ compared to predominantly hepaticaccumulation for >9a′. These results, coupled withthe versatility of CuAAC, suggests this novel, hydrophilic chelatecan be incorporated into numerous biomolecules containing azides forgenerating targeted fac-[M^I(CO)3]⁺ complexes in future studies.