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Critical re-evaluation of experimental settings with fluorescent probes for bioorthogonal chemistry with palladium
Jana Kurcova1, Marie Kudlickova Peskova1,2, Lukas Jeremias1
1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, CZ-613 00, Czech Republic.
Abstract:
Bioorthogonal reactions, both in vitro and in vivo, show great promise in fluorescent proteins labelling and for the development of bioorthogonal anticancer therapies. In this work, we focus on palladium-catalysed deprotection chemistry. Despite its potential, many questions remain unanswered. The most critical issues include the long-term stability of the palladium catalysts in biological milieu, the lack of efficient methods of reaction monitoring etc. Herein, we report the design of palladium catalyst containing phosphorus, arsenic, and antimony ligands. The catalytic activity has been evaluated for up to 7 days in vitro in the presence of bovine serum and for up to 9 h in living cells. Catalyst´s performance was assessed in the presence of cysteine and glutathione, as well as the abiotic thiophenol. The catalytic activity was measured using 14 different fluorescent probes, likely the largest probe library used to date. Furthermore, we have evaluated the cellular retention of fluorescent probes used for the detection of catalysts activity, revealing that most probes or fluorescent products are poorly retained in cells, thereby limiting long-term monitoring of the catalytic activity in physiological conditions in vivo. The results show that triphenylantimony is unparalleled ligand with respect to catalyst longevity under bioorthogonal conditions as well as in the tolerance to thiols. Long-term experiments show that propargylethers are excellent probes in vitro while allylcarbamates are more useful for in vivo studies. Our results also indicate that each probe (or prodrug) - catalyst couple is unique, and a search for a universal catalyst might be futile.
Insights
This study introduces novel palladium catalysts with antimony ligands for bioorthogonal reactions, showing superior longevity and thiol tolerance. Results suggest specific probes are better suited for in vitro or in vivo applications, challenging the idea of a universal catalyst.
Area of Science:
- Bioorthogonal chemistry
- Catalysis
- Chemical biology
Background:
- Bioorthogonal reactions are promising for labeling and anticancer therapies.
- Palladium-catalyzed deprotection chemistry faces challenges in catalyst stability and reaction monitoring in biological settings.
Purpose of the Study:
- To design and evaluate palladium catalysts with phosphorus, arsenic, and antimony ligands for bioorthogonal deprotection.
- To assess catalyst stability, activity, and probe performance in vitro and in vivo.
- To investigate cellular retention of fluorescent probes for long-term monitoring.
Main Methods:
- Design of palladium catalysts with P, As, and Sb ligands.
- In vitro evaluation of catalytic activity for up to 7 days in bovine serum.
- In vivo assessment of catalytic activity for up to 9 hours in living cells.
- Testing catalyst performance with various thiols (cysteine, glutathione, thiophenol).
- Measurement of catalytic activity using 14 different fluorescent probes.
- Evaluation of cellular retention of fluorescent probes and products.
Main Results:
- Triphenylantimony ligands demonstrated unparalleled catalyst longevity and thiol tolerance.
- Catalytic activity was sustained for extended periods in vitro and in vivo.
- Most fluorescent probes and products showed poor cellular retention, limiting long-term in vivo monitoring.
- Propargylethers were effective in vitro probes, while allylcarbamates were more suitable for in vivo studies.
Conclusions:
- Antimony-based palladium catalysts offer improved stability and performance for bioorthogonal reactions.
- Probe selection is critical and context-dependent (in vitro vs. in vivo).
- The development of a universal catalyst-probe system may not be feasible due to unique couple interactions.
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