Photofunctional Rhenium(I) and Iridium(III) Polypyridine Complexes as Photocytotoxic Agents and Bioorthogonal Crosslinkers for Peptide Stapling and Construction of Nanosized Materials


Student thesis: Doctoral Thesis

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Award date13 Sept 2022


Luminescent biological probes have been utilized to visualize cellular compartments and biological species in complex biological environments, providing valuable insights into complex subcellular structures and biochemical processes. Due to their appealing phosphorescence features and potent biological activity, d6 transition metal complexes have emerged as promising candidates for the development of biological probes with high specificity and sensitivity for their target species. This thesis describes the design of photofunctional tricarbonylrhenium(I) polypyridines and cyclometalated iridium(III) polypyridines for bioconjugation, bioimaging, and therapeutic applications.

Perylene derivatives such as perylene diimide (PDI) and benzoperylene monoimide (BPMI) are intriguing building blocks for functional molecular and supramolecular systems. Fluorescent PDI and BPMI derivatives have been modified with transition metal centers for different applications such as photocytotoxic agents for photodynamic therapy, photocatalysts for water oxidation, and building units for nanofibers. However, these transition metal complexes are π-conjugated to the perylene moieties, and the systems are reduced to one single chromophore/luminophore unit. Thus, the favorable photophysical and photochemical characteristics of the original inorganic and organic units cannot be effectively utilized. To date, luminescent transition metal complexes appended with a PDI or BPMI moiety via a non-conjugated linker have not been designed as bioimaging and phototherapeutic agents. In Chapter 2, the synthesis and characterization of a rhenium(I) perylene diimide (PDI) complex [Re(Ph2-phen)(CO)3(py-SS-PDI-TEG)](CF3SO3) (py-SS-PDI-TEG = 3-(2-((2-(N-tetraethyleneglycol-perylene-3,4,9,10-tetracarboxyl-diimidyl)ethyl)dithio)ethyl)aminocarbonylpyridine; Ph2-phen = 4,7-diphenyl-1,10-phenanthroline (1a)), its PDI-freecounterpart [Re(Ph2-phen)(CO)3(py-SS-boc)](CF3SO3) (py-SS-boc = 3-(2-((2-tert-butyloxycarbonylaminoethyl)dithio)ethyl)aminocarbonylpyridine) (1b), and threebenzoperylene monoimide (BPMI) complexes [Re(N^N)(CO)3(py-CONH-C3-BPMI)](CF3SO3) (py-CONH-C3-BPMI = 3-(3-(1,3-dioxo-1H-peryleno[1,12-efg]isoindol-2(3H)-yl)propyl)aminocarbonylpyridine; N^N = 3,4,7,8-tetramethyl-1,10-phenanthroline Me4-phen (2a), Ph2-phen (3a), and 2,2’-biquinoline biq (4a)), and their BPMI-free counterparts [Re(N^N)(CO)3(pyridine)](CF3SO3) (N^N = Me4-phen (2b), Ph2-phen (3b), and biq (4b)) are described. Non-conjugated linkers were used in all the complexes to avoid any direct electronic communication between the inorganic and perylene components. Upon irradiation, all the complexes exhibited intense greenish-yellow to red emission similar to the free ligands py-SS-PDI-TEG and py-CONH-C3-BPMI. The utilization of the PDI complex in thiol-sensing was demonstrated by using spectrofluorometry and electrospray ionization mass spectrometry. Selectivity assays showed high selectivity of the complex toward sulfhydryl-containing compounds, including glutathione (GSH), L-cysteine (Cys), 2-mercaptoethanol (2-ME), and dithiothreitol (DTT). Furthermore, the PDI complex was utilized as a phosphorogenic sensor for intracellular thiols in live cells. Cell-based assays showed that the PDI and BMPI complexes were non-cytotoxic in the dark with [Re] < 15 μM and incubation time = 3 h. However, when the complex-treated cells were irradiated at 365 nm for 15min, the cell viability dropped to < 10%, with IC50 values ranging from 0.27 to 18.21 μM.

The inverse electron-demand Diels–Alder (IEDDA) [4+2] cycloaddition of 1,2,4,5-tetrazine with strained dienophiles such as cyclooctynes has been extensively utilized in bioorthogonal labeling and imaging of specific biomolecules. Common fluorophore-tetrazine probes display fluorescence quenching, and their fluorescence is reinstated upon reaction with cyclooctynes due to the conversion of the quenching tetrazine unit into a nonquenching pyridazine derivative. Recently, fluorogenic probes with doubly functionalized bioorthogonal motifs have been designed for peptide stapling and bioimaging. Despite these interesting studies, luminescent transition metal complexes appended with two bioorthogonal reactive groups have not been explored. The incorporation of two tetrazine units into iridium(III) complexes is anticipated to generate versatile phosphorogenic bioorthogonal probes for bis-cyclooctynylated substrates with a linker of an appropriate length. In Chapter 3, the synthesis and characterization of three novel phosphorogenic bioorthogonal probes derived from iridium(III) complexes featuring two tetrazine units [Ir(dptz)2(N^N)](Cl) (Hdptz = 3,6-diphenyl-1,2,4,5-tetrazine; N^N = ethylenediamine en (1), Ph2-phen (2), and biq (3)) are reported. The photophysical behaviors and biological labeling applications of the complexes were studied. The complexes were weakly emissive due to the two quenching tetrazine units. Upon reaction with (1R,8S,9s)-bicyclo[6.1.0]non-4-yne (BCN) substrates, the reaction mixtures exhibited emission enhancement due to the formation of bis-pyridazine conjugates. Importantly, the reaction of a peptide with two BCN reactive units and complex 3 led to the formation of a cyclized peptide as the major product. Also, the cellular uptake, localization, and bioorthogonal labeling property of the complexes were studied using HeLa cells with or without an exogenous substrate 1,13-bis-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonylamino)-4,7,10-trioxatridecane (bis-BCN). The complexes displayed intense and organelle-specific staining upon bioorthogonal labeling of bis-BCN within the cells. Additionally, the photocytotoxicity of the complexes was manipulated through a bioorthogonal approach.

Nanosized hydrogels are recognized as hydrophilic three-dimensional polymer networks with a hydrodynamic diameter in the subnanometer range. They have been shown to function as potential drug delivery carriers for various low molecular chemotherapeutics, peptides, RNAs, and DNAs because of their distinctive characteristics such as good stability and high drug loading capacity. Poly(ethylene glycol) (PEG) is the most common macromonomer for the design of hydrogels due to its inert, nonreactive, and nondegradable properties. In Chapter 4, the design of nanosized iridium(III) hydrogels (Ir-gels) through the IEDDA reaction of a branched-chain PEG polymer containing four BCN units with an iridium(III) bis-tetrazine complex [Ir(dptz)2(Ph2-phen)](Cl) reported in Chapter 3 is described. Functionalization of the crosslinked hydrogels with an iridium(III) complex endowed the hydrogels with phosphorescence properties. Fluorescent BODIPY (BDP) dyes of different sizes were encapsulated in the Ir-gels to investigate live cell imaging and cargo delivery applications. Upon irradiation at 350 nm, all the Ir-gels exhibited intense and long-lived yellow emission originating from the triplet metal-to-ligand charge-transfer (3MLCT) excited state. Upon irradiation at 640 nm, an arrow lower-energy emission band with a much shorter lifetime was observed, which is assigned to the fluorescence of BODIPY inside the hydrogels. Upon incubation for 2 h, while the BDP dyes encapsulated in the Ir-gels were localized in the lysosomes of HeLa cells, free BDP dyes were accumulated in the endoplasmic reticulum. Upon incubation for an additional 4 h in fresh medium, while the small BDP dye was still encapsulated in the Ir-gels, the BDP dyes of larger sizes were released from the Ir-gels. Additionally, the Ir-gels displayed good biocompatibility toward the cells in the dark and exhibited negligible photoinduced cytotoxicity, highlighting the potential of Ir-gels as an intracellular delivering tool with phosphorescence behavior and low photocytotoxicity for biomedical applications.

Numerous reports have reported that many cancer cells have elevated matrix metalloproteinases (MMPs) levels, which are closely implicated in tumor growth, invasion, and metastasis. The most common MMP-related anticancer approach was the development of MMP-sensitive materials for stimuli-responsive drug release. These MMP-responsive systems can effectively control drug accumulation and drug release at the tissue, cellular, and intracellular levels. Since bioactive substrates can be readily encapsulated in hydrogels by simply blending them in the precursor solution, stimuli-responsive hydrogels have been utilized as drug carriers in tumor therapy. However, hydrogels for simultaneous tumor cell-specific lysosome imaging and photodynamic therapy have been rarely reported. In Chapter 5, the construction of an MMP-sensitive PEG-peptide hydrogel through the IEDDA reaction of a branched-chain PEG polymer containing four tetrazine units with an MMP-sensitive peptide containing two BCN units in both ends and an iridium(III) bis-tetrazine complex [Ir(dptz)2(Ph2-phen)](Cl) reported in Chapter 3 is described. Owing to the elevated MMPs levels in cancer cells, the cleavable peptide for MMP-2/9 in the hydrogel was gradually degraded, and the crosslinked iridium(III) complex was released from the hydrogel. The released iridium(III) complex fragments were accumulated in the lysosomes of MCF-7 and MDA-MB-231 breast cancer cells after 24 h incubation. A lower amount of the iridium(III) complex fragments was accumulated in HEK-293T cells due to their low level of released MMPs. Upon irradiation at 450 nm for 1 h, the viability of MDA-MB-231 cells dropped to < 10%. Furthermore, the released lysosome-targeting iridium(III) conjugate showed inhibitory effects on cell migration and invasion of MDA-MB-231 cells.

In conclusion, the works described in this thesis illustrate how the interesting photophysical and photochemical properties of tricarbonylrhenium(I) polypyridines and cyclometalated iridium(III) polypyridines can be exploited for the construction of innovative bioimaging reagents, photocytotoxic agents, and biocompatible hydrogels for a range of biological applications including bioconjugation, bioimaging, and therapeutic applications.

    Research areas

  • Bioimaging reagent, Bioorthogonal, Hydrogel, Iridium compounds, Luminescent probes, Nanomaterials, Peptide Stapling, Photocytotoxic agents, Rhenium compounds