Development of Photofunctional Rhenium(I) and Iridium(III) Polypyridine Complexes as Bioconjugation Reagents for Peptide Tags and Bioorthogonal Probes for Protein Tag Substrates


Student thesis: Doctoral Thesis

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Award date10 Dec 2021


The visualization and tracing of biomolecules in live cells have been greatly facilitated by specifically designed luminescent biological probes. There has been much interest in the development of bioimaging reagents based on luminescent transition metal complexes with a d6 electronic configuration due to their rich photophysical and photochemical properties, structural diversity, and potent biological activity. In this thesis, the applications of tricarbonylrhenium(I) polypyridine complexes as bioconjugation reagents for peptide tags and cyclometalated iridium(III) polypyridine complexes as bioorthogonal probes for protein tag substrates are described.

Bioconjugation is an indispensable technique for the covalent modification of biomolecules with synthetic modalities and other biomolecules.  It extends the functions of biomolecules and enables the discovery of new drugs, decoration of biomaterials, and design of diagnostic agents for the exploration of complex biological processes.  The π-clamp-mediated cysteine arylation, i.e., the nucleophilic substitution of the cysteine residue of a four amino acid sequence (FCPF) with perfluorobiphenyl (PFBP) derivatives, has been designed for the site-specific cysteine labeling of peptides and proteins in aqueous media.  In Chapter 2, the synthesis and characterization of three tricarbonylrhenium(I) complexes appended with a PFBP moiety [Re(N^N)(CO)3(py-PFBP)](CF3SO3) (N^N = 1,10-phenanthroline (phen) (1), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen) (2), and 4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (3)) for the specific modification of the cysteine residue of π-clamp are described.  Photoexcitation of these complexes led to intense and long-lived green to yellow emission and efficient singlet oxygen (1O2) generation in fluid solution.  Laser-scanning confocal microscopy confirmed that these complexes were localized in the endoplasmic reticulum of human esophageal squamous carcinoma KYSE-510 cells.  Peptides bearing the π-clamp sequence were reacted with complex 3 to afford different photofunctional conjugates.  Time-dependent confocal images showed that the conjugate containing a cell-penetrating peptide (CPP), 3-CPP, was readily internalized into the cells and displayed punctate staining in the mitochondria.  Photocytotoxicity of this conjugate was significant and comparable to the free complex despite its much lower cellular uptake.  Another conjugate 3-DEVD-QSY7 containing a caspase-3/7 cleavable peptide (DEVD) and an emission quencher QSY-7 was prepared and characterized.  This conjugate was weakly emissive due to efficient Förster resonance energy transfer (FRET) but the sample solution displayed emission enhancement upon addition of active caspase-3/7.  The activities of caspase-3/7 during cell apoptosis was monitored using this conjugate.  

Bioorthogonal chemistry is a rapidly emerging field of bioconjugation.  This approach has allowed the detection and visualization of biomolecules in their native environments, enabling the real-time monitoring of the dynamics and functions of these biomolecules in living systems.  Bioorthogonal probes with luminogenic properties offered various advantages in bioimaging because of their highly sensitive detection and minimized background signals without the requirements of stringent washing steps.  Nitrone is one of the potent chemical reporters for the development of luminogenic bioorthogonal probes.  The photoisomerization of the C=N group is known to quench the emission of fluorophores by providing a facile non-radiative deactivation pathway.  The nitrone unit undergoes strained-promoted alkyne-nitrone cycloaddition (SPANC) reaction with strained alkynes to afford non-quenching isoxazoline derivatives.  In Chapter 3, the synthesis and characterization of four cyclometalated iridium(III) polypyridine complexes containing a nitrone moiety [Ir(N^C)2(bpy-nitrone)](PF6) (bpy-nitrone = 4-((methyl(oxido)imino)methyl)-4’-methyl-2,2’-bipyridine; HN^C = 2-phenylbenzothiazole (Hbt) (1), 2-(1-naphthyl)-benzothiazole (Hbsn) (2), dibenzo[f,h]quinoxaline (Hdbq) (3), and dibenzo[a,c]-phenazine (Hdbpz) (4)) are reported.  Upon photoexcitation, these complexes displayed very weak yellow to red emission in solutions with limited 1O2 generation under ambient conditions due to the C=N photoisomerization of the nitrone unit.  However, the complexes exhibited significant emission enhancement and efficient 1O2 generation upon SPANC reaction with strained alkyne-modified proteins.  A strained alkyne-modified benzylguanine BCN-BG was designed as a substrate for SNAP-tag protein.  Confocal images indicated that only CHO-K1 cells expressing cytoplasm-enriched SNAP-tag with pretreatment of BCN-BG displayed significant phosphorescence signal upon incubation with complexes 2 and 3.  Importantly, their photocytotoxicity activity in cells was also substantially increased.  This combination of SNAP-tag and bioorthogonal chemical reporters has allowed the emission and photocytotoxicity turn-on at specific subcellular locations.  

The broad applications of bioorthogonal chemistry have led to strong demands of new chemical reporters.  One remarkable system is the 1,2,4,5-tetrazine moiety that undergoes inverse electron-demand Diels-Alder (IEDDA) reaction with a dienophile such as a strained alkyne and alkene.  The tetrazine unit is an attractive chemical reporter as it quenches the emission of fluorophores through many processes such as FRET, through-bond energy transfer (TBET), and photoinduced electron transfer (PET).  Various tetrazine derivatives have been conjugated to organic fluorophores to afford bioorthogonal fluorogenic probes, which display emission enhancement upon their IEDDA reactions with strained dienophile-modified biomolecules.  In Chapter 4, the synthesis and characterization of four cyclometalated iridium(III) tetrazine complexes [Ir(N^C)2(bpy-Tz-Ph)](PF6) (bpy-Tz-Ph = 4-(6-phenyl-1,2,4,5-tetrazin-3-yl)-4’-methyl-2,2’-bipyridine; HN^C = 2-(2,4-difluorophenyl)pyridine (Hdfppy) (1), 2-phenylpyridine (Hppy) (2), 2-phenylquinoline (Hpq) (3), and methyl 2-phenyl-4-quinolinecarboxylate (Hpqe) (4)) were described.  Photoexcitation of these complexes led to extremely weak green to red emission in fluid solution because of the FRET and PET quenching effect of the tetrazine unit.  The complexes exhibited strong emission enhancement and lifetime extension upon reaction with strained alkyne-modified proteins due to the conversion of the tetrazine unit into non-quenching pyridazine derivatives.  Surprisingly, reaction of the complexes with strained alkene-modified proteins resulted in limited changes in emission intensity and lifetimes because of the PET effect of the resulting dihydropyridazine derivatives.  Interestingly, 1O2 measurement studies indicated that the tetrazine complexes showed significant 1O2 generation quantum yields unlike related organic fluorophores appending a tetrazine unit.  The 1O2 generation of the pyridazine derivatives of the complexes was moderate but that of their dihydropyridazine counterparts remained limited.  Upon incubation of live HeLa cells with strained alkyne derivatives and complex 2, specific labeling of different organelles was demonstrated.  Confocal images of cells expressing HaloTag proteins showed that only cells pretreated with the strained alkyne-modified substrate BCN-C6-Cl displayed significant phosphorescence signals.  Using different dienophiles, the photocytotoxicity activity of complex 2 was demonstrated to be readily modulated by different bioorthogonal reaction partners.