Riboflavin as a bioorthogonal photocatalyst for the activation of a PtIV prodrug

Abstract

Encouraging developments demonstrate that few transition metal and organometallic catalysts can operate in a bioorthogonal fashion and promote non-natural chemistry in living systems by minimizing undesired side reactions with cellular components. These catalytic processes have potential for applications in medicinal chemistry and chemical biology. However, the stringent conditions of the cell environment severely limit the number of accessible metal catalysts and exogenous reactions. Herein, we report an unorthodox approach and a new type of bioorthogonal catalytic reaction, in which a metal complex is an unconventional substrate and an exogenous biological molecule acts as a catalyst. In this reaction, riboflavin photocatalytically converts a Pt^IV prodrug into cisplatin within the biological environment. Due to the catalytic activity of riboflavin, cisplatin-like apoptosis is induced in cancer cells under extremely low doses of light, potentially preventing systemic off-target reactions. Photocatalytic and bioorthogonal turnover of Pt^IV into Pt^II species is an attractive strategy to amplify the antineoplastic action of metal-based chemotherapeutics with spatio-temporal control.

Scheme 1. Transition-metal complex acting as a substrate and its bioorthogonal activation by riboflavin that functions as a photocatalyst.

Scheme 2. Light-induced reduction of cis,cis,trans-[Pt(NH₃)₂(Cl)₂(O₂CCH₂CH₂CO₂)₂]^2– (1) promoted by riboflavin (Rf) in MES buffer.

Fig. 1. (a) Absorption spectrum of riboflavin (Rf) and cis,cis,trans-[Pt(NH₃)₂(Cl)₂(O₂CCH₂CH₂CO₂)₂]^2– (1) in aqueous solution. (b) Rf-catalysed photoreduction of 1 in the MES buffer (18 mM, pH 6) monitored by ¹H NMR. Spectra were obtained for a MES/D₂O (9 : 1) solution of 120 μM 1 and 50 μM Rf upon t = 0 s, 30 s, 1 min, and 2.5 min of 460 nm light irradiation (2.5 mW cm^–2). ¹H NMR signal labelling: Pt–OCOCH₂CH₂CO₂ ^–, Pt–OCOCH₂ CH₂CO₂ ^–, methyl groups of Rf isoalloxazine ring, and free O₂CCH₂CH₂CO₂ ^–.

Fig. 2. Proposed mechanism for the photocatalytic activation of 1 by Rf. (a) Computed structure and frontier molecular orbitals (DFT:PBE0/def2-SVP) of a selected 1-RfH₂ adduct. Intermolecular H-bonds in 1-RfH₂ are highlighted with magenta lines (top). Isodensity surfaces are plotted with the isovalue of 0.02 e^– bohr^–3. Atoms color code: Pt grey, Cl green, O red, N blue, C pale brown (1) or yellow (Rf), H white. (b) Rf absorbs 460 nm photons to generate the triplet excited state (³Rf*), which oxidizes two MES molecules to give the reduced species RfH₂/RfH^–. Next, complex 1 forms stable adducts with either RfH₂ (shown in Fig. 2a) or RfH^– and undergoes photoreduction and elimination reactions upon absorption of more photons, liberating cytotoxic Pt^II species and regenerating the Rf catalyst.

Fig. 3. Antiproliferative activity in human prostate cancer PC-3 cells. (a) Percentage cell viability of PC-3 cells following treatment with Rf, 1, Rf/1 and cisplatin with and/or without light activation (460 nm, 0.36 J cm^–2). (b) Fluorescence microscopy images showing the effects of Rf/1 on PC-3 cells upon light irradiation. (A and B) Untreated PC-3 cells, (C and D) Rf/1 (30 : 120 μM) in the dark, (E and F) Rf/1 (30 : 120 μM) activated by 460 nm light and (G and H) cisplatin (120 μM) in the dark. Top row: cell nuclei (green); bottom row: apoptotic cells (red). (c) Quantification of early apoptotic PC-3 cells (Annexin V+/SYTOX–) treated by Rf (30 μM), Rf/1 (30 : 120 μM) and cisplatin (120 μM) with and/or without light activation. Cell viability and flow cytometry data are presented as mean ± SEM of at least three independent measurements. ***P < 0.001, **P < 0.01, ns = non-significant by two-way ANOVA followed by Bonferroni's test (a) or by one-way ANOVA followed by Tukey's test (c).