Facile Approaches For Peptide-metal Carbonyl Complex Using Solid-phase Synthesis

Carbon monoxide（CO）serves as a versatile signaling molecule in vivo.In low concentrations,CO has a beneficial effect on inflammatory disorders,tumor growth,bacterial infections.More recently,CORMs that can be activated by light（photo CORMs）which based on transition metal carbonyl complexes（M-（CO）₃）have been most attractive as they can be applied under better controlled conditions.The described peptides-Photo CORMs conjugates are advantageous due to their great biocompatibility and solubility in aqueous solutions.In further developments,the peptides could maybe also be used for the conjugation of receptors for the specific targeting of the CORM conjugate,so that localized applications,such as in tumors,would become possible.Here we report an efficient solid phase synthetic strategy route for the peptide-Photo CORMs.In the first part of the work,we developed a modular strategy of peptide-Photo CORMs by facile one-pot solid-phase synthetic,which allows the access of peptide-Mn-（CO）₃bioconjugates after a single HPLC purification step.This novel strategy was applied in the synthesis of TAT-Photo CORMs bearing a variety of chelating moieties.All peptide conjugates showed excellent stability in an aqueous buffer in the dark and released CO upon（visible）light activation,indicating that they can serve as viable photo CORMs.Notably,the modular strategy presented herein allows the facile incorporation of substituted heteroaromatic aldehyde derivatives in the late stage,which may help speed up the identification of peptide-based photo CORMs that are responsive to the more physiologically relevant visible or infrared light.This strategy was then applied to synthesis of a glioma targeting peptide-Chlorotoxin（CTX）based peptide-photo CORMs.A series of CTX-Photo CORMs containing fac-[Mn（dpa）（CO）₃]at four different lysine sites were obtained successfully.In order to explore whether CTX targets glioma cells through the specific binding of matrix metalloproteinases-2（MMP-2）which overexpressed on the surface of glioma cells,we developed a facile approach for the expression and purification of the untagged PEX domain of MMP-2 which is the key proteins in the activation process of pro-MMP-2.The yield was established at～6 mg/L of the culture medium,which would greatly facilitate the production and hence the biological study of PEX.In the second part of the work,we explore optimizing the light-response of peptide-Photo CORMs light sources.In order to realize the activation wavelength of peptide-Photo CORMs further moved to the red or near-infrared region,we describe an operationally-simple and efficient one-pot solid-phase synthesis strategy for peptides with 2-phenylazopyridine（azpy）side-chain enabled by the on-resin Mills reaction.Upon conjugation with the Mn–CO moiety,a series of linear or cyclic red-light activatable peptide-based photo CORMs were obtained.While not affecting the intrinsic nature of the peptides,the fac-[Mn（azpy）（CO）₃Br]moiety on the peptide bioconjugates can be safely internalized and intracellular CO release was achieved upon red-light irradiation.And importantly,the targeted/localized CO release enabled by this novel type of peptide-based photo CORM exhibited enhanced cell cytotoxicity.