Phosphorescent Ruthenium(II) and Iridium(III) Complexes as Bioimaging Reagents and Bioorthogonal Probes


Student thesis: Doctoral Thesis

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Award date15 Mar 2017


The visualization and tracing of the distribution and dynamics of biomolecules and organelles in live cells have been greatly enhanced by the use of luminescent or luminogenic biological probes. In view of their attractive phosphorescence properties, structural diversity, and potent biological activity, d6 transition metal complexes have emerged as attractive models for the construction of bioprobes and biosensors that exhibit high specificity and sensitivity for target species. In this thesis, the design of ruthenium(II) polypyridine complexes and iridium(III) cyclometalates as bioimaging reagents and bioorthogonal probes has been described, with a focus on the investigation of their structure-property relationships, biomolecular binding, bioconjugation, cellular uptake, imaging, and therapeutic applications.

An ideal live cell imaging reagent is typically characterized by efficient cellular internalization, high organelle-targeting ability, and biocompatibility. As the lipophilicity of transition metal complexes substantially influences their uptake, localization, and cytotoxicity, the structural modification of these complexes with polar substituents is expected to perturb both their hydrophobic character and cellular imaging properties. In Chapter 2, the synthesis and characterization of four phosphorescent biscyclometalated iridium(III) ethylenediamine (en) complexes functionalized with a polar ester or carboxylate group [Ir(N^C)2(en)]n(X) (n = +1, X = Cl- , HN^C = methyl 4-(2-pyridyl)benzoate Hppy-COOMe (1a) and methyl 2-phenyl-4-quinolinecarboxylate Hpq-COOMe (2a); n = -1, X = Li+, HN^C = 4-(2-pyridyl)benzoate Hppy-COO- (1b) and 2-phenyl-4-quinolinecarboxylate Hpq-COO- (2b)) are reported. Photoexcitation of these complexes led to intense and long-lived greenish-yellow to yellow emission of spin-forbidden metal-to-ligand charge-transfer (3MLCT) (dπ (Ir) → π*(N^C)) and intraligand (IL) (π → π*) (N^C) character. Protonation of the carboxylate complexes 1b and 2b resulted in emission quenching and lifetime shortening. Nonspecific binding of the pq complexes 2a and 2b to the protein bovine serum albumin (BSA) caused hypsochromic shifts in their emission maxima and a substantial increase in emission intensity. Cellular uptake and laser-scanning confocal microscopy (LSCM) results revealed that the ester complexes 1a and 2a were efficiently taken up by human cervix epithelioid carcinoma (HeLa) cells through energy-requiring pathways and subsequently localized in endosomes and mitochondria, respectively. Interestingly, they displayed good biocompatibility in the dark, but became considerably cytotoxic upon photoirradiation owing to the generation of singlet oxygen. In contrast, the carboxylate complexes 1b and 2b existed as an anionic form in physiological pH and hardly entered live cells because of limited membrane permeability, as proven by intense emission surrounding the plasma membrane of the cells. They exhibited negligible cytotoxicity in the dark or upon irradiation as the cells remained viable for an incubation period of 24 hours. By virtue of the low cytotoxicity and strongly emissive nature of the hydrophilic ppy-COO- complex 1b, the complex was applied as a visualization reagent for in vivo imaging studies using zebrafish (Danio rerio) as an animal model.

Cell-surface glycoconjugates play an important role in multiple pivotal cellular processes, including cell-cell interactions and cell adhesions. Imaging the metabolic recycling processes of these membrane-associated biomolecules within live cells through the combined use of the bioorthogonal chemical reporter strategy and luminescent bioorthogonal probes is anticipated to gain valuable insights into cellular metabolism. In Chapter 3, the synthesis and characterization of four phosphorescent ruthenium(II) polypyridine complexes functionalized with a dibenzocyclooctyne (DIBO) or amine moiety [Ru(N^N)2(L)](PF6)2 (L= 4-(13-N-(3,4:7,8-dibenzocyclooctyne-5-oxycarbonyl) amino-4,7,10-trioxa-tridec anyl-aminocarbonyl-oxy-methyl)-4’-methyl-2,2’-bipyridine bpy-DIBO, N^N = 2,2’-bipyridine bpy (1a) and 1,10-phenanthroline phen (2a); L= 4-(13-amino-4,7,10-trioxa-tridecanylaminocarbonyl-oxy-methyl)-4’-methyl-2,2’¬bipyridine bpy-NH2, N^N = bpy (1b) and phen (2b)) are reported. These complexes were 3MLCT emitters and displayed intense and long-lived orange-red emission upon photoexcitation. The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction of the DIBO complexes 1a and 2a with benzyl azide in MeOH were studied. Since the azide-reactive DIBO complexes were hardly permeable across the plasma membrane of live cells, they were used to label and visualize N-azidoglycoconjugates located on the surface of Chinese hamster ovary (CHO)-K1 and human lung adenocarcinoma (A549) cells that were pretreated with 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine (Ac4ManNAz). LSCM images of the Ac4ManNAz-pretreated cells labeled by the DIBO complexes revealed that the cell-surface glycoconjugates were partitioned into endosomal, Golgi, and lysosomal compartments during their dynamic recycling within cells. The biolabeling and cellular uptake efficiency of the DIBO complexes 1a and 2a were cell-line dependent, as revealed by flow cytometry and inductively coupled plasma-mass spectrometry (ICP-MS). Additionally, the complexes displayed good biocompatibility toward the Ac4ManNAz-pretreated cells in the dark, but exhibited photoinduced cytotoxicity owing to the generation of singlet oxygen, highlighting the therapeutic value of the SPAAC labeling technique in combination with the use of photocytotoxic reagents.

Bioorthogonal probes with activatable emission offer various advantages in biolabeling and bioimaging because they can minimize background signals, eliminate stringent washing steps, and enable highly sensitive detection. Since these probes are modified with a bioorthogonal functional group that also acts as an emission quencher, they exhibit emission turn-on upon specific reactions with target compounds. Although functional groups such as azide and tetrazine have been conjugated to fluorescent and phosphorescent compounds to generate these probes, there is still a very strong demand for new bioorthogonal turn-on probes with high reactivity and staining selectivity. In Chapter 4, the synthesis and characterization of four phosphorogenic ruthenium(II) polypyridine N-methyl nitrone complexes [Ru(N^N)2(bpy-nitrone-Me)](PF6)2 (bpy-nitrone-Me = 4-((methyl(oxido) imino)methyl)-4’-methyl-2,2’-bipyridine; N^N = bpy (1), 4,4’-bis(n-butylaminocarbonyl)-2,2’-bipyridine bpyC4 (2), phen (3), and 4,7-diphenyl-1,10-phenanthroline Ph2-phen (4)) and an N-phenyl nitrone complex [Ru(bpy)2(bpy-nitrone-Ph)](PF6)2 (5) (bpy-nitrone-Ph = 4-((phenyl(oxido)imino)methyl)-4’-methyl-2,2’-bipyridine) are reported. These complexes were extremely weakly emissive due to quenching by the nitrone unit, but exhibited significant emission enhancement upon the strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction with bicyclo[6.1.0]nonyne (BCN)-modified substrates. The reactivity and phosphorogenic responses of these complexes toward (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH) in MeOH and BCN-modified BSA in aqueous buffer were investigated. Importantly, the modification of nitrone with a dicationic ruthenium(II) polypyridine unit at the α-C position and a phenyl ring at the N-position resulted in significantly accelerated reaction kinetics, which are substantially greater (up to ca. 278 fold) than those of other acyclic nitrone-BCN systems. Also, the cellular uptake, localization, cytotoxicity, and bioorthogonal labeling property of the complexes were studied using HeLa cells with or without pretreatment with the exogenous substrate N-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl)-1-aminodecane (BCN-C10), which contains a BCN group and a decyl chain. The complexes displayed intense and organelle-specific staining upon bioorthogonal labeling of BCN-C10 within the cells. The regioselective cellular staining was highly dependent on the distribution of BCN-C10 within the cells, and the bioorthogonal reactivity and lipophilicity of the complexes. Additionally, the in situ generation of the more lipophilic isoxazoline adduct in the cytoplasm resulted in increased cytotoxicity, highlighting a new approach to apply the SPANC labeling technique in drug activation.

The inverse electron-demand Diels-Alder (IEDDA) reaction of a 1,2,4,5-tetrazine derivative with a dienophile (i.e., a strained alkene or alkyne) is the fastest bioorthogonal reaction, which favors biolabeling of target species that exist at a low concentration in cells. Various tetrazine derivatives have been conjugated to fluorescent and phosphorescent compounds to generate bioorthogonal turn-on probes, where the tetrazine unit acts as both a bioorthogonal functional group and emission quencher, and the quenching mechanism is generally photoinduced electron transfer (PeT), Förster resonance energy transfer (FRET), and through-bond energy transfer (TBET) in nature. However, the construction of these bichromophoric sensing systems requires laborious design efforts to modulate the quenching of luminophore by the tetrazine unit. Thus, a more direct approach is to develop a sensory system comprising a single luminophore that has a tetrazine-derived molecular structure. The system exhibits emission turn-on upon the structural conversion of the built-in tetrazine motif into a pyridazine or dihydropyridazine derivative. In Chapter 5, the synthesis and characterization of three phosphorogenic biscyclometalated iridium(III) pyridyl-tetrazine complexes [Ir(N^C)2(py-Tz-Me)](PF6) (py-Tz-Me = 3-(2-pyridyl)-6-methyl-1,2,4,5-tetrazine; HN^C = 2-(2,4-difluorophenyl)pyridine Hdfppy (1), 2-phenylpyridine Hppy (2), and 2-phenylquinoline Hpq (3)) and a pyridyl-pyridazine complex [Ir(pq)2(py-pzBCN-Me)](PF6) (3-pz) (py-pzBCN-Me = 1-methyl-6,6a,7,7a,8,9-hexahydro-4-(2-pyridinyl)-5H-cyclopropa[5,6]cycloocta [1,2-d]pyridazine-7-methanol) are reported. The reactivity and phosphorogenic responses of the tetrazine complexes toward BCN-OH in aqueous solutions were investigated. The results revealed that the direct coordination of a 3,6-disubstituted tetrazine to a cationic iridium(III) center can serve as a novel design strategy to enhance the tetrazine reactivity and construct bioorthogonal turn-on probes for dienophile-modified substrates. Although the tetrazine complexes were very weakly emissive, the chemoselective ligation of the complexes with cyclooctynes significantly changed the π-conjugation and thus triplet excited-state properties, resulting in the formation of highly emissive pyridazine products. Also, their application as phosphorogenic probes for specific endogenous biomolecules in live cells was demonstrated through the selective visualization of genetically encoded HaloTag protein with the use of a BCN-modified chloroalkane derivative.