Phosphorogenic Rhenium(I) and Iridium(III) Polypyridine Complexes as Bioorthogonal Probes and Bioconjugation Reagents

Abstract

Luminescent transition metal complexes with a d⁶ electronic configuration have emerged as promising candidates for the development of intracellular sensors, bioimaging reagents, and photocytotoxic agents. This is due to their intrinsic photophysical and biological properties, including large Stokes shifts, long-lived excited states, excellent photostability, and controllable cellular uptake and localization, which can be easily manipulated by modifying the polypyridine ligands with diverse functional groups. This thesis delineates the design of phosphorogenic rhenium(I) and iridium(III) polypyridine complexes as bioorthogonal probes and bioconjugation reagents, thereby enriching the toolkit for important bioimaging and phototherapeutic advancements.

Bioorthogonal chemical reactions are invaluable tools for labeling biomolecules in living systems. Nitrones belong to a relatively new bioorthogonal system that has demonstrated high versatility and applicability. These compounds show high reactivity toward strained cyclooctynes via the strain-promoted alkyne–nitrone cycloaddition (SPANC) reaction. Solutions of transition metal complexes containing a nitrone moiety are weakly emissive, but exhibit intense and long-lived emission upon addition of strained alkynes, such as (1R,8S,9s)-bicyclo[6.1.0]nonyne (BCN), due to the conversion of the quenching nitrone unit into a non-quenching isoxazoline moiety. In Chapter 2, the design, synthesis, and characterization of four cyclometalated iridium(III) polypyridine complexes functionalized with two nitrone units [Ir(N^C)₂(bpy-(nitrone)₂)](PF₆) (bpy-(nitrone)₂ = 4,4’-bis((methyl(oxido)imino)methyl)-2,2’-bipyridine; HN^C = 2-phenylpyridine (Hppy) (1), 2-phenylquinoline (Hpq) (2), 2-phenyl-4-quinolinecarboxylate (Hpqe) (3), and benzo[a]phenazine (Hbp) (4)) are described. Upon photoexcitation, these complexes exhibited very weak greenish-yellow to red emission in solutions and low singlet oxygen (¹O₂) generation under ambient conditions. The emission quenching was attributed to an efficient non-radiative decay pathway via the low-lying T₁/S₀ minimum energy crossing point (MECP), as determined by computational analyses. However, upon reaction with BCN substrates, the complexes displayed significant emission enhancement and efficient ¹O₂ photosensitization. Notably, the complexes showed a higher reaction rate toward a bis-cyclooctyne derivative, 1,13-bis-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonylamino)-4,7,10-trioxatridecane (bis-BCN), compared with its monomeric counterpart. The complexes exhibited high photocytotoxicity in bis-BCN-pretreated HeLa cells, which was ascribed to the enhanced ¹O₂ photosensitization upon elimination of the nitrone-associated quenching pathway. The exceptional crosslinking capabilities of the complexes enabled the development of functional nanosized hydrogels and stapled/cyclized peptides with intriguing photophysical, photochemical, and biological attributes.

There has been growing interest in the development of bioorthogonal dissociation reactions for the targeted delivery of functional payloads, such as drugs and fluorophores, in living systems. The bioorthogonal chemistry involving tetrazines and isonitriles has garnered attention due to the structural compactness of the isonitrile group that minimizes perturbation to biological environments. The reaction entails the inverse electron-demand Diels–Alder [4+1] cycloaddition of 3-isocyanopropyl (ICPr) or 3-isocyanopropyl-1-carbamoyl (ICPrc)-caged compounds with tetrazines, followed by rapid expulsion of N₂, tautomerization, and hydrolysis to release the desired phenol or amine cargo from the ICPr/ICPrc protecting group. In Chapter 3, the design, synthesis, and characterization of three tricarbonylrhenium(I) polypyridine complexes incorporating a tetrazylmethyl (TzMe) group [Re(N^N)(CO)₃(py-OCH₂-Tz-^tBu)](CF₃SO₃) (py-OCH₂-Tz-^tBu = 3-(tert-butyl)-6-((pyridin-3-yloxy)methyl)-1,2,4,5-tetrazine; N^N = 4,4’-dimethyl-2,2’-bipyridine (Me₂-bpy) (1a), 1,10-phenanthroline (phen) (2a), and 4,7-diphenyl-1,10-phenanthroline (Ph₂-phen) (3a)) are presented, together with the TzMe-free analogues [Re(N^N)(CO)₃(py-OH)](CF₃SO₃) (py-OH = 3-hydroxypyridine; N^N = Me₂-bpy (1b), phen (2b), and Ph₂-phen (3b)). Upon photoexcitation, the TzMe complexes exhibited weak greenish-yellow emission in solutions under ambient conditions. The aqueous and acidic buffer solutions of the complexes demonstrated significant emission enhancement upon incubation with 3-isocyanopropyl benzylcarbamate (ICPrc-Bn) due to the elimination of the quenching TzMe group. The TzMe complexes served as phosphorogenic probes for profluorophore and prodrug activation, enabling the controlled release of umbelliferone (also known as 7-hydroxycoumarin), fluorescein, and doxorubicin from their respective ICPr/ICPrc-modified derivatives in HeLa cells. This precise activation allowed the simultaneous liberation of the rhenium(I) 3-hydroxypyridine complex and the functional payload, thereby supporting combined bioimaging and phototherapeutic applications.

N-Terminal cysteine (N-Cys) residues have been employed for site-specific labeling of peptides and proteins due to several key factors. These include the unique reactivity of the 1,2-aminothiol group and the low natural abundance of N-Cys residues. Additionally, the modification of N-Cys residues causes minimal structural perturbation, thereby preserving the native conformation of the biomolecule. Thioesters have conventionally been utilized in ligation reactions with N-Cys residues of peptides via native chemical ligation (NCL). The process involves transesterification and an S → N acyl shift to yield a native amide bond. It is anticipated that the incorporation of a thioester group into transition metal complexes can generate luminescent probes for N-Cys-containing biomolecules. In Chapter 4, the design, synthesis, and characterization of four cyclometalated iridium(III) polypyridine complexes bearing a thioester group [Ir(N^C)₂(bpy-COSBn)](PF₆) (bpy-COSBn = S-benzyl 4’-methyl-2,2’-bipyridine-4-carbothioate; HN^C = Hpq (1a), 2-(1-naphthyl)benzothiazole (Hbsn) (2a), 1-(benzo[b]-thiophen-2-yl)isoquinoline (Hiqbt) (3a), and 6-(benzo[b]thiophen-2-yl)phenanthridine (Hbtph) (4a)), and their ester counterparts [Ir(N^C)₂(bpy-COOMe)](PF₆) (bpy-COOMe = methyl 4’-methyl-2,2’-bipyridine-4-carboxylate; HN^C = Hpq (1b), Hbsn (2b), Hiqbt (3b), and Hbtph (4b)) are reported. Upon photoexcitation, the complexes displayed orange-red to near-infrared (NIR) emission in solutions under ambient conditions. The thioester complexes were utilized for the development of intracellular cysteine (Cys) sensors and Cys-activatable photosensitizers for cancer-targeted photodynamic therapy (PDT). Through the efficient reaction of complex 3a with several N-Cys-modified tumor-targeting peptides, photofunctional iridium(III)–peptide conjugates with high ¹O₂ generation efficiencies were prepared. These conjugates showed a notable specificity for MDA-MB-231 cells compared with MCF-7 and HEK-293 cells, leading to targeted photocytotoxicity against this triple-negative breast cancer cell line.

Date of Award	2 Oct 2025
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Kam Wing Kenneth LO (Supervisor)