Alkenes as a transition-metal-mediated activating group. (a) (i) Allyl carbamate is a well-studied substrate with palladium (Pd) or ruthenium (Ru) as the catalyst. The activated alkene is attacked by an external nucleophile to eliminate a free amine. (ii) Pentenoic sec-amides are shown to form stable cyclized products with Ru photocatalysis. (b) Possibilities for an amide bond cleavage reaction via cyclization mechanism. The imine intermediate can be hydrolyzed after cyclization. Addition of a leaving group at the allyl position allows dual release of substrates possibly via β-elimination. (c) Design elements involved in the dual release strategy.

(a) Substrate scope; carbamate (A), tertiary amides (B–D), secondary amides (E, F), and dual-release model substrates (G–I) were used to survey the uncaging reaction. (b) Efficiency of the cleavage reaction under different conditions was assessed by 1H NMR spectroscopy (Table SI2; values). Metal complexes used in the study are (Table SI1); Zn(II): ZnSO4·7H2O, Mg(II): MgCl2·6H2O, Fe(II): FeSO4·7H2O, Fe(III): Fe2(SO4)3·9H2O, Ce(IV): Ce(NH4)2(NO3)6, Cu(I): CuSO4·5H2O + THPTA, Cu(II): CuSO4·5H2O, Ru(III): RuCl3·3H2O, Rh(II): Rh2(AcO)4, Ag(I): Ag2CO3, Pd(II): Na2[PdCl4], Pt(II): K2[PtCl4], Au(I): AuCl, Au(III): Na[AuCl4]. (c) 1H NMR spectroscopy for the uncaging of the substrate (I) in the presence of Na[AuCl4]. The reaction possibly generates a cyclized intermediate that undergoes hydrolysis to release morpholine. The allyl leaving group allows simultaneous release of PFP. General procedure for determining conversions by 1H NMR spectroscopy: substrates and metal salts/complexes were dissolved in MeOD: D2O(1:4) at 37 °C. The reactions were transferred to an NMR spectroscopic tube and measured at specific time points (2–24 h). Conversions were calculated based on the relative ratios of methylene peaks resulting from the starting material and the released amine product.

(a) (i) Mechanistic possibility involving an alkene as a directing group for Au(III) to activate a carbonyl group towards hydrolysis. (ii) Model substrate with a thiol directing group failed to release free amine, eliminating the possibility of such a mechanism. (b) Phenyl amide with its lone pair in conjugation with the ring did not show any reaction, while the benzyl amide results in complete release of the free amine, suggesting that the carbonyl group should initially act as a nucleophile through its C–O resonance form. (c) Naphthalimide-based fluorogenic probes (QF1–4) were designed to study the cleavage efficiency of the Au(III) for uncaging alkene-containing molecules. The caged naphthalimide derivatives exhibited high stability in solution, and their quenched fluorescence could be reactivated upon removal of the caging group (λex = 445 nm, λem = 535 nm). (d) Representative example with QF4 demonstrating the increase in fluorescence intensity during the time course of the uncaging reaction with Na[AuCl4]. (e) Determined half-time for the uncaging reaction for QF1–4. QF3 showed no increase of fluorescence under standard reaction conditions. (f) HPLC trace showing the formation of F4 from QF4 upon treatment with Na[AuCl4] at 37 °C. Time = 0 h is the time point recorded prior to the addition of the Au salt.

(a) Proposed mechanism for Au(III)-promoted amide cleavage (single-release) from model substrate B′. (b) Minimum energy pathways (MEP) calculated with SMD(H2O)/M06/6-311G(d,p)+SDD(Au) for the amide cleavage (single-release) from model substrate B′ catalyzed by Na[AuCl4] in water. The complete low-energy 5-exo-trig cyclization pathway (in blue) was computed, including all postulated intermediates, while only key transition states and intermediates for the more energetic (i.e., less stable) 6-endo-trig pathway (in orange) were calculated for comparison. The intrinsic activation barriers of the chemically relevant steps are labeled with their associated intrinsic rate constants (kcyc: cyclization; khyd: hydration; krel: amide release). When two chiral centers are generated, only the most stable diastereomer is discussed, irrespective of its absolute configuration. Breaking/forming bonds are represented with green dotted lines. Distances are given in angstrom. Free energies are given in units of kcal mol–1. Asterisks denote steps in which external species such as Na[AuCl4], NaCl, neutral and protonated water clusters, etc., enter or leave the main reaction (see Figure S25 for a more complete depiction); given the intrinsic inaccuracy of calculating the energetics of such hypothetical equilibria, relative energies of charged/neutral species and thus the global thermodynamics of the process should be considered with caution. c, Proposed mechanism for Au(III)-promoted amide and allyl leaving group decaging (dual-release) from model substrate H′ (see Figure S26 for a more complete depiction). The optimized structures of chemically relevant transition states are shown as ball-and-stick models.

(a) Model peptide (R-Y-G-allyl glycine-G-Y-A) bond-cleavage reaction at allyl glycine with Na[AuCl4] at 37 °C in H2O. (b) Mass spectrum data that show the fragments after 30 min. The UV-trace indicates full consumption of the starting material to degraded fragments. The full spectrum is provided in Figure S29.

(a) Au(III) uncages the fluorogenic probe QF4 in cells. HeLa cells were exposed to QF4 in medium for 1 h followed by a wash. Cells were randomly distributed into two conditions: DMSO or Au(III) for 12 h. Confocal image of cells upon treatment shows that the progress of the reaction in cells (green channel). (b) Au(III)-mediated uncaging of drugs in cells: Substrates (PD1, PD2, and ADC1) were used in the study. (c) Toxicity screening of Na[AuCl4] in HeLa cells. Cells were treated with the depicted concentrations for 72 h, and viability was measured by AlamarBlue. (d) ICP-MS analysis of the cellular extracts revealed the intracellular amount of Au after incubation of Na[AuCl4] following several washing steps and lytic treatment. (e, f) HeLa cells were incubated with different concentrations of PD1 or PD2 for 72 h with or without Na[AuCl4] (20 μM, twice a day). (g) Au(III)-mediated drug decaging from an ADC; cysteine-selective and irreversible modification of an internalizing antibody thiomab an MMAE conjugating linker. Deconvoluted ESI–MS mass spectrum of the light-chain confirms the modification. (h) Cell viability of SKBR3 cells (HER++) after treatment with ADC1 and subsequent uncaging efficiency upon treatment with 20 μM Na[AuCl4], twice daily. Toxicity was determined by the AlamarBlue assay. Error bars represent ± standard deviation (n = 3). The statistical significance of the differences between groups was evaluated with the unpaired t test. Statistical results: ns >0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

HCT116 human CRC cells were fluorescently labeled with lipophilic CM-DiI (shown in red) and injected into the perivitelline space (PVS) of 2 days post fertilization (dpf) zebrafish larvae. Zebrafish xenografts were randomly distributed into different treatment groups and were daily treated with DMSO, Na[AuCl4], PD1, PD2, PD1 + Na[AuCl4] and PD2 + Na[AuCl4]. At 4 dpi, zebrafish xenografts were analyzed for cell proliferation, apoptosis, and tumor size. (a) Representative scheme of the Zebrafish xenografts assay. (b) Representative maximum projections of Zebrafish xenografts on where the therapeutic effects of the different treatment conditions were analyzed. (c) Quantification of cell proliferation (mitotic figures). (d) Apoptosis (activated caspase 3 ****P < 0.0001, ***P = 0.003, **P = 0.0019); and (e) tumor size (no. of tumor cells: c, *P = 0.0147). Graphs represent fold induction (normalized values to controls) of Avg ± SEM. The number of xenografts analyzed is indicated in the representative images, each dot represents one xenograft, and the results are from two independent experiments. Statistical analysis was performed using an unpaired t test. Statistical results: ns >0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. All images are anterior to the left, posterior to right, dorsal up, and ventral down. The dashed line represents the tumor area. Scale bar 50 μm.