Critical re-evaluation of experimental settings with fluorescent probes for bioorthogonal chemistry with palladium antimony inactivation-resistant catalyst

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.