Bio‐Orthogonal Conjugation of a Cationic Metalloporphyrin to BSA and HSA via “Click” Chemistry

Abstract

In this study, we present a convenient method for the labelling of tyrosine residues on bovine serum albumin (BSA) and human serum albumin (HSA) and report for the first time their subsequent bio‐orthogonal conjugation with porphyrins via “click” chemistry. We demonstrate that these serum proteins can be labelled with an alkyne‐diazonium heterobifunctional linker and can then undergo chemo‐selective bio‐orthogonal conjugation with a water‐soluble azido metalloporphyrin via “click” chemistry to yield protein‐conjugates that retain their photodynamic properties. In our hands, this method was found to be highly reproducible, scalable, and tuneable which allows for the production of bioconjugates where the porphyrin‐protein conjugate not only retains an ability to generate singlet oxygen but possess an enhanced relative singlet oxygen quantum yields relative to the porphyrin alone. Furthermore, we have investigated the photochemical properties of these conjugates through photospectrometric techniques and have determined that the porphyrin macrocycles remain appreciably photostable under light irradiation. Our phototoxic protein‐photosensitizer‐conjugates show excellent photodynamic activity against a human colorectal adenocarcinoma cancer cell line (HT‐29) with cell viabilities of 7.7±0.5 % (IC50 8.76±2.14 μM) and 1.7±1.9 % (IC50 8.48±5.11 μM) for BSA and HAS, respectively, when irradiated with 20 J cm−2 of white‐light. Importantly, neither of the conjugates was found to possess any significant “dark” toxicity even at concentrations of 100 μM. Furthermore, the natural fluorescent properties of the bioconjugates allowed for the determination of cellular uptake in vitro via fluorescence microscopy thus highlighting the potential theranostic applications of these unique protein‐drug‐conjugates.

Porphyrins—valuable pigments of life

“…Porphyrins and metallo-porphyrins are considered as promising labelling tools (Ethirajan et al, 2011;Rabiee et al, 2020;Sandland et al, 2021;Jin et al, 2023). Additionally, porphyrin derivatives were employed in PDT of tumours and as labels for the detection of cancer cells (Tian et al, 2020;Chen et al, 2021;Park et al, 2021;Shi et al, 2021).…”

“Click” for precise photodynamic therapy

“…These reactions are generally biocompatible and can be applied for a wide range of biomolecules, including peptides, proteins, saccharides, DNAs, and small molecular inhibitors. Using this synthetic methodology, bioconjugates of various classes of photosensitisers based on phthalocyanines, [33][34][35][36] porphyrins, 37,38 boron dipyrromethenes (BODIPYs), 39,40 aggregation-induced emission (AIE) dyes, 41 ruthenium(II) complexes 42,43 or benzo[a]phenothiazinium dyes 44 have been synthesised for PDT application. In particular, antibody-photosensitiser conjugates can also be prepared similarly, which are highly promising towards precise PDT.…”