Kasten, Benjamin B. Ma, Xiaowei Liu, Hongguang Hayes, Thomas R. Barnes, Charles L. Qi, Shibo Cheng, Kai Bottorff, Shalina C. Slocumb, Winston S. Wang, Jing Cheng, Zhen Benny, Paul D. Clickable, Hydrophilic Ligand for <i>fac-</i>[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> (M = Re/<sup>99m</sup>Tc) Applied in an <i>S</i>‑Functionalized α‑MSH Peptide The copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction was used to incorporate alkyne-functionalized dipicolylamine (DPA) ligands (<b>1</b> and <b>3</b>) for <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> (M = Re/<sup>99m</sup>Tc) complexation into an α-melanocyte stimulating hormone (α-MSH) peptide analogue. A novel DPA ligand with carboxylate substitutions on the pyridyl rings (<b>3</b>) was designed to increase the hydrophilicity and to decrease in vivo hepatobiliary retention of <i>fac</i>-[<sup>99m</sup>Tc<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> complexes used in single photon emission computed tomography (SPECT) imaging studies with targeting biomolecules. The <i>fac</i>-[Re<sup>I</sup>(CO)<sub>3</sub>(<b>3</b>)] complex (<b>4</b>) was used for chemical characterization and X-ray crystal analysis prior to radiolabeling studies between <b>3</b> and <i>fac</i>-[<sup>99m</sup>Tc<sup>I</sup>(OH<sub>2</sub>)<sub>3</sub>(CO)<sub>3</sub>]<sup>+</sup>. The corresponding <sup>99m</sup>Tc complex (<b>4a</b>) 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 <i>P</i> analysis compared to an analogous complex with nonfunctionalized pyridine rings (<b>2a</b>). An α-MSH peptide functionalized with an azide was labeled with <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> using both <i>click, then chelate</i> (CuAAC reaction with <b>1</b> or <b>3</b> followed by metal complexation) and <i>chelate, then click</i> (metal complexation of <b>1</b> and <b>3</b> followed by CuAAC with the peptide) strategies to assess the effects of CuAAC conditions on <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> complexation within a peptide framework. The peptides from the <i>click, then chelate</i> strategy had different HPLC <i>t</i><sub>R</sub>’s and in vitro stabilities compared to those from the <i>chelate, then click</i> strategy, suggesting nonspecific coordination of <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> using this synthetic route. The <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup>-complexed peptides from the <i>chelate, then click</i> strategy showed >90% stability during in vitro challenge conditions for 6 h, demonstrated high affinity and specificity for the melanocortin 1 receptor (MC1R) in IC<sub>50</sub> analyses, and led to moderately high uptake in B16F10 melanoma cells. Log <i>P</i> analysis of the <sup>99m</sup>Tc-labeled peptides confirmed the enhanced hydrophilicity of the peptide bearing the novel, carboxylate-functionalized DPA chelate (<b>10a′</b>) compared to the peptide with the unmodified DPA chelate (<b>9a′</b>). In vivo biodistribution analysis of <b>9a′</b> and <b>10a′</b> showed moderate tumor uptake in a B16F10 melanoma xenograft mouse model with enhanced renal uptake and surprising intestinal uptake for <b>10a′</b> compared to predominantly hepatic accumulation for <b>9a′</b>. These results, coupled with the versatility of CuAAC, suggests this novel, hydrophilic chelate can be incorporated into numerous biomolecules containing azides for generating targeted <i>fac</i>-[M<sup>I</sup>(CO)<sub>3</sub>]<sup>+</sup> complexes in future studies. Log P analysis;novel DPA ligand;HPLC;B 16F melanoma cells;peptide;vivo biodistribution analysis;fac;B 16F melanoma xenograft mouse model;IC 50 analyses;metal complexation;MC;1R;CuAAC;serum stability assays;chelate;melanocortin 1 receptor;click strategy;uptake;log P analysis;vivo hepatobiliary retention;MSH;SPECT 2015-12-17
    https://acs.figshare.com/articles/journal_contribution/Clickable_Hydrophilic_Ligand_for_i_fac_i_M_sup_I_sup_CO_sub_3_sub_sup_sup_M_Re_sup_99m_sup_Tc_Applied_in_an_i_S_i_Functionalized_MSH_Peptide/2030133
10.1021/bc5000115.s001