Photofunctional Cyclometalated Iridium(III) Polypyridine Complexes for Bioconjugation, Bioimaging and Therapeutic Applications

具光功能性環金屬化三價銥多吡啶絡合物於生物綴合、生物成像和治療上的應用

Student thesis: Doctoral Thesis

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Award date4 Aug 2020

Abstract

In this thesis, the design of photofunctional cyclometalated iridium(III) polypyridine complexes for bioconjugation, bioimaging, and therapeutic applications is described.

Chemoselective reactions are promising strategies for the covalent attachment of functional entities to biomolecules to create novel constructs for various biological applications. The π-clamp-mediated cysteine arylation, i.e., a site-selective reaction of perfluoroaromatics with the cysteine thiolate of a four-amino acid sequence FCPF, has offered new opportunities for modification of peptides and proteins. The integration of a perfluorobiphenyl (PFBP) group into luminescent transition metal complexes is expected to generate novel biological reagents for bioconjugation, bioimaging, and therapeutic applications. In Chapter 2, the synthesis and characterization of six cyclometalated iridium(III) polypyridine complexes appended with a PFBP moiety [Ir(N^C)2(bpy-PFBP)](PF6) (bpy-PFBP = 4-(S-(perfluoro-(1,1’-biphenyl)-4-yl)-N-mercaptoethylaminocarbonyloxymethyl)-4’-methyl-2,2’-bipyridine; HN^C = 2-phenylpyridine (Hppy) (1a), 2-(4-hydroxymethylphenyl)pyridine (Hppy-CH2OH) (2a), 2-((1,1’-biphenyl)-4-yl)pyridine (Hpppy) (3a), 2-((4’-hydroxymethyl-1,1’-biphenyl)-4-yl)pyridine (Hpppy-CH2OH) (4a), 2-phenylquinoline (Hpq) (5a), and 2-(4-hydroxymethylphenyl)quinoline (Hpq-CH2OH) (6a)) and their PFBP-free counterparts [Ir(N^C)2(bpy-C4)](PF6) (bpy-C4 = 4-(N-n-butylaminocarbonyloxymethyl)-4’-methyl-2,2’-bipyridine; HN^C = Hppy (1b), Hppy-CH2OH (2b), Hpppy (3b), Hpppy-CH2OH (4b), Hpq (5b), and Hpq-CH2OH (6b)) are reported. Upon photoexcitation, all the complexes displayed intense and long-lived greenish-yellow to orange emission in solutions and in low-temperature alcohol glass. Reaction of the PFBP complexes with peptides bearing the FCPF sequence via the π-clamp-mediated cysteine conjugation afforded bioconjugates that exhibited rich photophysical properties. Using complex 3a as an example, the conjugation of complexes to organelle-targeting peptides was demonstrated to be an effective means to modulate their intracellular localization behavior and thereby (photo)cytotoxicity.

The bioorthogonal chemical reporter approach has emerged as a versatile platform for the detection and visualization of biomolecules in their native environments, which facilitates the studies of the dynamics and functions of target biomolecules in living systems. Mesoionic sydnones have recently been discovered as new bioorthogonal reaction partners of alkynes in 1,3-dipolar cycloaddition reaction. Since the polarity of sydnone and its reaction product pyrazole is drastically different, the modification of sydnone with a luminophore displaying environment-sensitive emission behavior is believed to allow the bioorthogonal reaction to be followed by photophysical changes of the probe. In Chapter 3, the design, synthesis, and characterization of three cyclometalated iridium(III) polypyridine complexes containing a sydnone moiety [Ir(N^C)2(bpy-CH2-NHCO-C6H4-sydnone)](PF6) (bpy-CH2-NHCO-C6H4-sydnone = 4-(N-(3-(ψ-5-oxo-1,2,3-oxadiazol-3-yl)phenylcarboxy)aminomethyl)-4’-methyl-2,2’-bipyridine; HN^C = 2-(2,4-difluorophenyl)pyridine (Hdfppy) (1a), Hppy (2a), and methyl 2-phenyl-4-quinolinecarboxylate (Hpqe) (3a)) are described. The sydnone-free analogs [Ir(N^C)2(bpy-CH2-NHCO-C6H5)](PF6) (bpy-CH2-NHCO-C6H5 = 4-(N-(phenylcarboxy)aminomethyl)-4’-methyl-2,2’-bipyridine; HN^C = Hdfppy (1b), Hppy (2b), and Hpqe (3b)) were also prepared for comparison studies. Interestingly, the sydnone complexes showed substantial emission enhancement and lifetime extension upon reaction with strained alkyne derivatives due to the conversion of the dipolar mesoionic ring to a less polar pyrazole unit. The sydnone complexes were utilized to label cyclooctyne-modified proteins and ceramide molecules both in vitro and in live cells. Additionally, the photocytotoxicity of the complexes was manipulated through a bioorthogonal approach.

There has been a rapidly emerging interest in bioorthogonal probes that display fluorogenic or phosphorogenic properties upon the labeling reaction. However, most of these probes developed thus far are limited to azide- and tetrazine-functionalized fluorophore scaffolds, with the emission quenching mechanisms being photoinduced electron transfer, Förster resonance energy transfer, and through-bond energy transfer in most cases. Nitrone, which can selectively undergo strain-promoted alkyne–nitrone cycloaddition reaction with a strained alkyne to give an isoxazoline, has a high potential to function as both a bioorthogonal handle and an emission quencher as photoinduced C=N isomerization is known to provide a facile non-radiative deactivation pathway for fluorescent and phosphorescent compounds. In Chapter 4, the design, synthesis, and characterization of three cyclometalated iridium(III) polypyridine complexes functionalized with a nitrone moiety [Ir(N^C)2(bpy-nitrone)](PF6) (bpy-nitrone = 4-((methyl(oxido)imino)methyl)-4’-methyl-2,2’-bipyridine; HN^C = Hppy (1), Hpq (2), and Hpqe (3)) are reported. Upon photoexcitation, these complexes displayed extremely weak greenish-yellow to red emission in solutions under ambient conditions due to photoinduced isomerization of the nitrone group. However, they exhibited significant emission enhancement upon reaction with strained alkyne derivatives. The direct coordination of the nitrone ligand to the cationic iridium(III) center led to accelerated reaction kinetics. The complexes were used to label BCN-modified proteins and exogenous decane molecules both in vitro and in live cells.