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• Copper ions are essential metal ions for life [1–3]. Through redox cycling between monovalent copper (Cu⁺) and divalent copper (Cu²⁺), they play catalytic roles in critical biological processes such as metabolism, proliferation, growth, and respiration [2,4–11]. Concurrently, clinical studies have demonstrated that dysregulation of copper-dependent redox activity is closely associated with pathological conditions including cancer [7], inflammatory immune responses [12,13], major neurodegenerative diseases [14], and hereditary disorders of copper metabolism [15,16]. Owing to the intracellular reducing environment, Cu⁺ predominates over Cu²⁺ within cells [17]. Consequently, fluorescence-based detection techniques targeting Cu⁺ have achieved significant advancements [18–27]. In contrast, Cu²⁺ exists at lower concentrations in living cells [17]. Additionally, Cu²⁺ exhibits paramagnetic properties, enabling strong fluorescence signal quenching through electron or energy transfer pathways [28,29]. Consequently, most Cu²⁺-responsive probes operate via turn-off mechanisms [28,29]. That severely limited the visualization of Cu²⁺ and created substantial obstacles for investigating intracellular Cu²⁺ distribution, coordination interactions with proteins, and its correlations with various pathological factors.

  Driven by the discovery of metal ion-mediated covalent modification of proteins by small molecules, chemists have developed activity-based sensing (ABS) strategies to address the pressing need for detecting intracellular metal ions and their responses to exogenous/endogenous stimuli [17,30,31]. These approaches leverage metal ion-triggered covalent reactions to achieve the spatial anchoring of probes through conjugation with proteins proximal to metal ion sites [17,30,31]. By leveraging Cu⁺/Cu²⁺-directed acyl imidazole strategies, scientists developed probes CD649 and CD649.2 for intracellular labile copper pools (Fig. 1) [17,30]. Upon Cu⁺/Cu²⁺coordination, the probes trigger Lewis acid activation of the acyl imidazole moiety, enabling covalent capture of proximal protein nucleophilic residues [30]. This mechanism allows for the spatial retention of the fluorophore while liberating fluorescence-quenching Cu²⁺ ions [17]. Therefore, this approach enables the real-time tracking of dynamic fluctuations in intracellular labile copper pools [17]. Notably, the CD649.2 exhibited dual selectivity for copper ions and the divalent oxidation state [17].

  Figure 1

  

  Figure 1.  Selective copper-responsive activity-based protein labeling.

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  Although progress has been made in ABS-based copper ion probes, current strategies predominantly employ the acyl imidazole core framework, with a notable absence of alternative usable core structures. To address this limitation and explore alternative structural frameworks, we have developed a Cu²⁺-directed covalent labeling strategy by using thiophosphoryl pyridinol/pyrimidinol as a reactive and dual Cu²⁺-recognizing group. It reacts with nucleophilic lysine under alkaline conditions [32]. Both sulfur atom of thiophosphoryl and pyridine/pyrimidine rings are widely recognized as potent Cu²⁺ chelators and have been extensively utilized in probe design [33–35]. Coordination to the Lewis acidic Cu²⁺ center enhances the electrophilicity of the thiophosphoryl moiety, thereby inducing covalent conjugation with proteins (Fig. 1). This mechanism enables detection of Cu²⁺ fluctuations in labile copper pools within living cells under exogenous stimuli.

  To develop Cu²⁺-responsive ABS probes capable of proximal protein labeling, we designed target compounds Q1–Q5 featuring a coumarin fluorophore, a diethylene glycol spacer, and thiophosphoryl pyridinol/pyrimidinol that integrate covalent anchoring and Cu²⁺-coordination functionalities. The designed compounds were successfully synthesized via a two-step reaction routine (Fig. 2).

  Figure 2

  

  Figure 2.  Synthesis of Q1–Q5. Reagents and conditions: (ⅰ) 2-(2-aminoethoxy)ethanol, 2-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), Et₃N, N,N-dimethylformamide (DMF), room temperature (RT). (ⅱ) PSCl₃/POCl₃, R-OH, Et₃N, MeCN, 0 ℃-RT.

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  Next, we compared the activity of Q1–Q5 under Cu²⁺-catalyzed conditions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis| (SDS-PAGE) fluorescence imaging showed that compounds with ortho-positioned nitrogen (Q3, Q4 and Q5) exhibited activity, whereas those lacking ortho-nitrogen (Q1, Q2) showed negligible activity (Fig. 3A). Furthermore, introduction of additional nitrogen atoms at either the ortho (Q4) or para position (Q5) enhanced activity compared to Q3 (Fig. 3A). Comparative analysis of Q3 and Q3a revealed that replacing the sulfur atom with an oxygen atom (Q3a) abolished the Cu²⁺-catalyzed enhancement of labeling activity (Fig. 3B). These results demonstrated the critical importance of the ortho-positioned nitrogen atoms and sulfur atom.

  Figure 3

  

  Figure 3.  (A) In-gel fluorescence image and relative fluorescence intensity of HeLa cell lysates treated by Q1-Q5 (50 µmol/L) with or without CuCl₂ (250 µmol/L). (B) In-gel fluorescence image and relative fluorescence intensity of HeLa cell lysates treated by Q3/Q3a (50 µmol/L) with or without CuCl₂ (250 µmol/L). (C) In-gel fluorescence image and relative fluorescence intensity of HeLa cell lysates treated with Q5 (50 µmol/L) and different metal ions (250 µmol/L). Cell lysate (0.5 g/L) was preincubated with 250 µmol/L metal ions for 30 min (Fe²⁺ and Cu⁺ groups supplemented with 1000 µmol/L TCEP as a reducing agent), followed by incubation with 50 µmol/L compounds at 20 ℃ for 2 h. In-gel fluorescence for SDS-PAGE was scanned by ChemiDoc MP and relative fluorescence intensity was analyzed in ImageJ. Error bars denote standard deviation (SD, n = 5). ****P < 0.0001. ns: no significance.

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  To evaluate the Cu²⁺ selectivity of Q3–Q5, we performed fluorescent labeling assays using HeLa cell lysate as a proteomic substrate in vitro. The lysate was pre-incubated with various metal ions (250 µmol/L each) under physiological buffer conditions (50 mmol/L HEPES, pH 7.4) for 30 min. For the Fe²⁺ and Cu⁺ conditions, tris(2-carboxyethyl)phosphine (TCEP, 1000 µmol/L) was included as a reducing agent. Following the metal ion pretreatment, the samples were incubated with Q3–Q5 (50 µmol/L) for 2 h at 20 ℃. As shown in Fig. S1 (Supporting information), subsequent fluorescent scanning analysis of SDS-PAGE separated samples demonstrated that Q3 (Fig. S1A), Q4 (Fig. S1B), and Q5 (Fig. 3C) exhibited Cu²⁺-dependent covalent labeling of lysate proteins, with significantly higher efficiency than observed for other metal ions. Notably, even under conditions where Cu⁺ concentration is tenfold higher than that of Cu²⁺, Q5 remains inactive (Fig. S2 in Supporting information), demonstrating its high selectivity for Cu²⁺.

  Next, we examined the effects of various conditions on Cu²⁺-dependent covalent labeling of proteins, including Cu²⁺ concentration and reaction time, as well as pH value and competition with copper chelators. As shown in Figs. S3 and S4 (Supporting information), the protein labeling of Q5, catalyzed by Cu²⁺ ions, exhibits dependence on Cu²⁺ concentration and reaction time, aligning well with the intended design (Figs. S3A and B). Further experimental results indicate that Q5 labels proteins more readily under alkaline conditions (Fig. S3C). Additionally, Cu²⁺ chelator tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA, Fig. S4B) inhibits the Cu²⁺-dependent protein labeling of Q5 in a concentration-dependent manner (Fig. S4A).

  To delineate the underlying mechanism, we first evaluated the effect of Cu²⁺ on the hydrolytic activity of thiophosphoryl pyrimidinol. To circumvent potential interference from the coumarin moiety, we modified the Q5 by replacing the coumarin group with a butynyl group, yielding the redesigned compound R5 (Fig. 4A). Hydrolysis kinetics of R5 (1 µmol/L) were subsequently analyzed in HEPES buffer (pH 8.0). High performance liquid chromatography (HPLC) monitoring revealed slow hydrolysis of R5 under a mild alkaline condition, with ~30% degradation observed within 92 min (Fig. 4B). Notably, Cu²⁺ (5 µmol/L) dramatically accelerated this process, as evidenced by detectable hydrolysis within 2 min and complete degradation within 92 min (Fig. 4B). Liquid chromatography-mass spectrometry (LC-MS) analysis identified two hydrolysis products: pyrimidinol (m/z 97.0401 [M + H]⁺, Fig. 4C) eluting at 2.9 min (Fig. S5 in Supporting information), and a thiophosphate intermediate (m/z 243.0028 [M − H]⁻, Fig. 4D) eluting at 5.9 min (Fig. S5), corresponding to the hydrolysis of the thiophosphoryl pyrimidinol fragment. These results suggest that Cu²⁺ promotes the hydrolytic activation of the thiophosphoryl pyrimidinol unit, thereby potentiating the compounds’ covalent protein-labeling activity.

  Figure 4

  

  Figure 4.  (A) The binding of Cu²⁺ to thiophosphorylated pyrimidinol promotes R5 hydrolysis or reacts with Cbz-Lys. (B) Effect of CuCl₂ (5 µmol/L) on the hydrolytic activity of R5 (1 µmol/L) in HEPES buffer (pH 8.0). Error bars denote SD (n = 5). (C, D) High-resolution mass spectrometry (HRMS) spectra of R5 (1 µmol/L) hydrolysis intermediates mediated by Cu²⁺ (5 µmol/L). (E) HRMS spectra of the product from the Cu²⁺-catalyzed reaction between R5 (5 µmol/L) and Cbz-Lys (0.5 µmol/L).

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  Lysine is highly nucleophilic and predominantly located on protein surfaces, making it an accessible target for covalent reagents and probes [31,36,37]. To examine Cu²⁺-dependent lysine modification by R5, we conducted LC-MS analyses of R5 (5 µmol/L) and Cbz-Lys (0.5 µmol/L, Fig. 4A) reaction in the presence of Cu²⁺ (25 µmol/L) (Fig. S6A in Supporting information). The product detected (m/z 519.1982 [M − H]⁻, Fig. 4E) was confirmed as R5K (a thiophosphorylated lysine adduct) by comparison with a synthetic standard (Fig. S7 in Supporting information). Critically, no R5K formation was observed under Cu²⁺-free conditions, even after 12 h of incubation during which R5 had fully been hydrolyzed (Fig. S6B in Supporting information). Collectively, these mechanistic studies demonstrate that Cu²⁺ binding enhances the reactivity of the thiophosphorylated pyrimidinol moiety, promoting either hydrolysis or lysine conjugation (Fig. 4A).

  Building on the Cu²⁺ selectivity of the compounds, we further investigated their ability to detect changes in intracellular Cu²⁺ levels. Among the tested compounds, Q5 exhibited the highest Cu²⁺-catalyzed activity in vitro (Fig. 3A). As shown in Figs. S8 and S9 (Supporting information), further cellular experiments and stability assays confirmed the low cytotoxicity of Q5 toward HeLa cells (50% cytotoxic concentration (CC₅₀) = 75 ± 0.41 µmol/L, Fig. S8) and its slow hydrolysis under physiological conditions (phosphate buffered saline (PBS), pH 7.4, 37 ℃, Fig. S9). These results prompting the selection of Q5 for monitoring intracellular Cu²⁺ fluctuations. To validate its responsiveness, intracellular Cu²⁺ levels were modulated by pre-incubation with Cu(gtsm) (2 µmol/L) (a membrane-permeable Cu²⁺ ionophore, Fig. S10 in Supporting information) to elevate Cu²⁺ pools or treatment with TETA (100 µmol/L) (triethylenetetramine, a high-affinity Cu²⁺ chelator, Fig. S10) to deplete endogenous Cu²⁺, followed by incubation with Q5 (1 µmol/L). Live-cell fluorescence imaging revealed distinct Cu²⁺-dependent responses. Cu(gtsm)-treated cells showed markedly enhanced fluorescence compared to untreated control (Figs. 5A and B). TETA-treated cells exhibited significant fluorescence attenuation (Figs. 5A and B). The trend that cellular fluorescence intensity changes of Q5 is similar to that of the Cu²⁺ probe N-aminofluorescein (Fig. S11 in Supporting information) [38–40]. These results conclusively demonstrate that Q5 serves as a reliable probe for monitoring the changes of labile Cu²⁺ levels in living cells. Subcellular co-localization experiments demonstrated that Q5 localizes to endoplasmic reticulum, lysosomes, and mitochondria (Fig. S12 in Supporting information).

  Figure 5

  

  Figure 5.  Fluorescence imaging of labile Cu²⁺ pools in live HeLa cells using Q5. (A) HeLa cells treated with solvent vehicle control, the ionophore Cu(gtsm) (2 µmol/L) for 2 h or the chelator TETA (100 µmol/L) for 12 h. Cells were washed twice with PBS, incubated with Q5 (1 µmol/L) for 1 h, washed twice with PBS, and then imaged. (B) Mean fluorescence intensity of the HeLa cells. Fluorescence intensity of Q5 was determined from experiments performed in triplicate with λ_ex = 445 nm. Mean fluorescence intensity was analyzed in ImageJ. Error bars denote SD (n = 5). Scale bar: 100 µm. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  In summary, we have successfully developed a novel class of Cu²⁺-dependent probes based on activity-driven labeling by thiophosphoryl pyridinol/pyrimidinol. Fluorescence labeling assays in cell lysates demonstrated the high selectivity of compounds for Cu²⁺. Mechanistic studies revealed that Cu²⁺ enhances the hydrolytic activation of the thiophosphoryl pyrimidinol, which catalyzes compounds to react with lysine residues, thereby enhancing protein-targeted labeling efficacy. Intracellular fluorescence imaging experiments demonstrated that Q5 enables the detection of Cu²⁺ fluctuations in living cells under exogenous stimuli. This study provided the thiophosphoryl pyridinol/pyrimidinol as a functional group for detecting labile Cu²⁺ levels in live cells, laying a solid foundation for the broader application of such chemotypes in chemical biology.

  CRediT authorship contribution statement

  Zhiyuan Qi: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Jing Chen: Methodology, Investigation, Formal analysis, Data curation. Wenhao Xie: Writing – review & editing, Data curation. Zhibei Qu: Supervision, Formal analysis. Motonari Uesugi: Writing – review & editing, Writing – original draft, Supervision, Project administration, Formal analysis. Lu Zhou: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  This study was supported by the National Key R&D Program of China (No. 2023YFC3603303), the National Natural Science Foundation of China (No. U24A20524), and Shanghai Municipal Committee of Science and Technology (No. 21TQ016).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.111924.

  
  
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• 

  Figure 1  Selective copper-responsive activity-based protein labeling.

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  Figure 2  Synthesis of Q1–Q5. Reagents and conditions: (ⅰ) 2-(2-aminoethoxy)ethanol, 2-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), Et₃N, N,N-dimethylformamide (DMF), room temperature (RT). (ⅱ) PSCl₃/POCl₃, R-OH, Et₃N, MeCN, 0 ℃-RT.

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  Figure 3  (A) In-gel fluorescence image and relative fluorescence intensity of HeLa cell lysates treated by Q1-Q5 (50 µmol/L) with or without CuCl₂ (250 µmol/L). (B) In-gel fluorescence image and relative fluorescence intensity of HeLa cell lysates treated by Q3/Q3a (50 µmol/L) with or without CuCl₂ (250 µmol/L). (C) In-gel fluorescence image and relative fluorescence intensity of HeLa cell lysates treated with Q5 (50 µmol/L) and different metal ions (250 µmol/L). Cell lysate (0.5 g/L) was preincubated with 250 µmol/L metal ions for 30 min (Fe²⁺ and Cu⁺ groups supplemented with 1000 µmol/L TCEP as a reducing agent), followed by incubation with 50 µmol/L compounds at 20 ℃ for 2 h. In-gel fluorescence for SDS-PAGE was scanned by ChemiDoc MP and relative fluorescence intensity was analyzed in ImageJ. Error bars denote standard deviation (SD, n = 5). ****P < 0.0001. ns: no significance.

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  Figure 4  (A) The binding of Cu²⁺ to thiophosphorylated pyrimidinol promotes R5 hydrolysis or reacts with Cbz-Lys. (B) Effect of CuCl₂ (5 µmol/L) on the hydrolytic activity of R5 (1 µmol/L) in HEPES buffer (pH 8.0). Error bars denote SD (n = 5). (C, D) High-resolution mass spectrometry (HRMS) spectra of R5 (1 µmol/L) hydrolysis intermediates mediated by Cu²⁺ (5 µmol/L). (E) HRMS spectra of the product from the Cu²⁺-catalyzed reaction between R5 (5 µmol/L) and Cbz-Lys (0.5 µmol/L).

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  Figure 5  Fluorescence imaging of labile Cu²⁺ pools in live HeLa cells using Q5. (A) HeLa cells treated with solvent vehicle control, the ionophore Cu(gtsm) (2 µmol/L) for 2 h or the chelator TETA (100 µmol/L) for 12 h. Cells were washed twice with PBS, incubated with Q5 (1 µmol/L) for 1 h, washed twice with PBS, and then imaged. (B) Mean fluorescence intensity of the HeLa cells. Fluorescence intensity of Q5 was determined from experiments performed in triplicate with λ_ex = 445 nm. Mean fluorescence intensity was analyzed in ImageJ. Error bars denote SD (n = 5). Scale bar: 100 µm. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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