Spatiotemporally controlled drug release via a click-release system utilizing mono-alkyl-hydroxylamine and cyclooctyne chemistry

Domain: chemistry
Published: 2026-01-16
Editorial outcome: major_revision

Abstract
The advancement of bioorthogonal bond-breaking chemistry requires precise spatiotemporal control over chemical reactions. In this study, we introduce a click-release strategy based on the reaction between mono-alkyl-hydroxylamine and cyclooctyne (COT), enabling rapid and nearly complete payload release. We demonstrate that mono-alkyl-hydroxylamine and hydroxylamine react with COT to form nitrone and oxime, respectively, facilitating a versatile and efficient release mechanism. By conjugating mono-alkyl-hydroxylamine with responsive cleavable groups, we convert its inherent reactivity into an on-demand activation system. In vivo, this strategy is applied in a 4T1 mouse breast tumor model, showing significant tumor inhibition compared to the parent anticancer drug. Additionally, in local anesthesia, the anesthetic effect could be modulated by light exposure, allowing for repeated activation. This click-release system combines fast kinetics, high release efficiency, and precise spatiotemporal control, offering promising applications in chemical biology and drug delivery.

Introduction
Click chemistry has fostered an unprecedented synergy between chemistry and biology, revolutionizing research paradigms in the life sciences through its exceptional reaction selectivity and efficiency1–6. Initially celebrated for its robust, bioorthogonal covalent bond formation, click chemistry has empowered precise biomolecule labeling, visualization, and manipulation within complex biological systems7–9. More recently, the advent of the click-release strategies—wherein a rapid click reaction is directly coupled to a subsequent bond-cleavage event—has expanded the toolkit for in situ payload liberation10–13, driving transformative implications in bioimaging14, regulation of the activity of nucleic acids and enzymes15–19, drug delivery20–26, and beyond27–30. In particular, the tetrazine (Tz)—triggered decaging of trans-cyclooctene (TCO)—caged molecules has attracted extensive attention owing to its extraordinary reactivity and biocompatibility31,32, fueling applications ranging from tumor imaging33,34 and immunomodulation35–37 to prodrug therapies38–43, one of which has advanced into phase II clinical trials44.

Despite these remarkable achievements, a critical need persists for bioorthogonal platforms that deliver not only rapid reaction kinetics and high release efficiency but also exquisite spatiotemporal control. The intrinsic always-on reactivity of traditional bioorthogonal handles limits their potential for precise activation and localization. Recent efforts—such as Wang’s concentration-promoted click—release strategy that enriches reactants at defined sites45, along with the methods reported by Devaraj46,47 and Pu34,48 that employ photo-controlled or enzymatic deprotection of dihydrotetrazine (DHTz) precursors to generate Tz—have begun to surmount these challenges. Despite these advancements, reported strategies remain relatively scarce and predominantly focus on modifications of the tetrazine scaffold. Consequently, the development of controllably activatable bioorthogonal reaction platforms remains highly desirable and represents a crucial frontier in the field.

In pursuit of a more versatile solution, recent work by Kim and colleagues has exploited the ring strain–promoted hydroamination of N,N-dialkylhydroxylamines with alkynes to generate N-oxides49 that can be reductively triggered by boron reagents50. Similarly, Ji’s group has accelerated decaging kinetics via silicon substitution in boronic acid systems, thereby enhancing prodrug activation51. Yet, the intrinsic instability and perpetual reactivity of boron reagents have hindered precise spatiotemporal modulation in complex settings.

In this work, we expand the chemistry of ring strain–assisted hydroamination by showing that mono-alkyl-hydroxylamine reacts directly with cyclooctyne (COT) to form a hydroxyaminooctene intermediate. When a leaving group is installed at the propargyl position of COT, this intermediate undergoes rapid 1,4-elimination, releasing the payload and generating a nitrone octene—a bioorthogonal bond-cleavage reaction with fast kinetics and near-quantitative efficiency (Fig. 1a). Capitalizing on the synthetic versatility of hydroxylamine, we developed a “caged” design in which its click activity is masked via a carbamate linkage. This dormant state can be activated by specific stimuli—elevated glutathione (GSH), enzymatic action, or light—enabling precise spatiotemporal control over the release process (Fig. 1b). The utility of this stimuli-responsive platform is demonstrated through two complementary in vivo applications: tumor-targeted prodrug activation triggered by the reductive tumor microenvironment in a 4T1 breast cancer model, and repeatable, on-demand local anesthesia controlled by 405 nm light. Together, these results establish a versatile click–release system for spatially and temporally controlled bioorthogonal chemistry (Fig. 1c).

Results
Hydroxylamine and mono-alkyl-hydroxylamine hydroamination reactions with COT
Building on the proposed click-release platform design, we initially investigated the fundamental reactivity between hydroxylamine derivatives and COT. Our mechanistic hypothesis proposed that enamine N-oxides, formed via retro-Cope elimination between hydroxylamine and COT, could undergo proton transfer at the nitrogen center, yielding nitrone through N-alkyl-hydroxyl-enamine intermediates. Notably, this proton transfer requires a transferable proton on the hydroxylamine nitrogen—a feature inherently absent in N,N-dialkylhydroxylamines, which are restricted to enamine N-oxide formation (Fig. 2a,b).

To test this hypothesis, we systematically examined the reactivity of both mono-alkyl-hydroxylamine and hydroxylamine with COT, characterizing the resulting products. As expected, the initially formed N-oxides underwent proton transfer, leading to the production of nitrones and oximes, respectively (Fig. 2c). Quantitative analysis by HPLC revealed that when COT 1a reacted with mono-alkyl-hydroxylamine 2c in a mixed aqueous solution (50% ACN/PBS) at room temperature, 81% conversion was achieved within 30 min, reaching nearly 99% conversion after 2 h (Fig. 2d). In contrast, hydroxylamine 2a showed significantly lower reactivity under identical conditions, with only 41% conversion after 30 min (Fig. 2e). This reduced reactivity is attributed to the weaker nucleophilicity of hydroxylamine compared to its N-alkylated counterpart. The absence of alkyl substituents in hydroxylamine fail to sufficiently stabilize the nitrogen cation generated during retro-Cope elimination, thus increasing the activation barrier. Importantly, both hydroxylamine and mono-alkyl-hydroxylamine participated in reactions with efficiencies that meet stringent click chemistry criteria.

Expanding our investigation, we evaluated the hydroamination reactivity of a series of hydroxylamine derivatives with COT (Supplementary Fig. 1A-D). Given the ease with which mono-alkyl-hydroxylamine can be modified, we functionalized them with various groups at the nitrogen or oxygen positions. Our results revealed that modifications substituting the proton on the oxygen atom (compounds 2f and 2g) or sterically shielding the nucleophilic nitrogen (compounds 2h and 2i) completely abolished reactivity (Fig. 2f). Conversely, mono-alkyl-hydroxylamines bearing various alkyl substituents successfully reacted with COT 1a, yielding the corresponding click products. HPLC analyses demonstrated a clear correlation between steric parameters and reaction efficiency. Derivatives 2b and 2c, with minimal steric hindrance, exhibited the highest click reactivity, with over 80% conversion within 30 min. In contrast, increasing steric bulk progressively diminished the reaction rate, with compound 2a (lacking alkyl substitution) also exhibiting suboptimal reactivity. Notably, the tert-butyl substituted derivative 2e displayed the lowest click reactivity among those tested, with only 14.4 ± 2.9% conversion within 30 min (Fig. 2g).

Bioorthogonal mono-alkyl-hydroxylamine/COT bond-cleavage chemistry
Following the establishment of the reactivity of our click reaction, we focused on its potential application in triggered molecular release. As previously noted, the click reaction between mono-alkyl-hydroxylamine and COT results in the formation of an N-alkyl-hydroxyl-enamine intermediate. We hypothesize that this intermediate facilitates a spontaneous 1,4-elimination process upon introduction of a leaving group at the propargylic position, ultimately leading to the development of a bioorthogonal cleavage chemistry.

To validate this concept, we synthesized COT 3a using p-nitroaniline as a model caging molecule and examined its reaction with mono-alkyl-hydroxylamine 2c in a 20% ACN/PBS solution at room temperature. As expected, this reaction led to the successful release of p-nitroaniline, with a release yield of up to 97.9%. In contrast to the reduction of N-oxides formed from N,N-dialkylhydroxylamine and COT by boric acid to release caged molecules, our findings reveal that mono-alkyl-hydroxylamine can directly function as a bioorthogonal scissor to cleave caged molecules tethered to COT, eliminating the need for an additional reduction step (Fig. 3a,b).

To further validate this mechanism, we monitored the reaction progress using 1H NMR spectroscopy. To capture the reaction midpoint, we selected the less reactive mono-alkyl-hydroxylamine derivative 2e (Fig. 3c). Within 10 minutes of mixing 25 mM of substrate 3a with 50 mM of 2e in DMSO-d6, the formation of nitrone 3d was observed by 1H NMR spectroscopy, which captured the alkene proton generated from the elimination reaction (two addition isomers were observed), along with the quantitative release of the payload, p-nitroaniline (3c). As the reaction progressed, the gradual disappearance of 3a corresponded with the increasing release of p-nitroaniline. After 1 h, only trace amounts of 3a remained, and by 12 h, complete consumption of 3a was observed with full payload liberation. Notably, the elimination intermediate 3b was not detectable, suggesting that once formed, it rapidly eliminates to release p-nitroaniline. Furthermore, the absence of any non-releasing byproducts confirms the excellent regioselectivity of the click reaction, ensuring nearly complete payload release.

To further characterize the decaging chemistry, we conducted a systematic evaluation of both the reaction kinetics and substrate scope. A series of probes (4b and 4c), bearing representative nitrogen- and oxygen-based leaving groups, were synthesized via carbamoylation or Mitsunobu reactions. As anticipated, the fluorescence of both coumarin and naphthalimide was effectively quenched in these caged compounds (Fig. 4a). When incubated in PBS at 37 °C, neither 4b nor 4c exhibited any fluorescence over 1 day (Supplementary Fig. 2A, B), confirming their stability.

Upon testing the reactivity of 4b in a 20% DMSO/PBS solution at room temperature, we observed the rapid consumption of 50 μM 4b in the presence of 300 μM of hydroxylamine 2c. This reaction led to the formation of product 4d, accompanied by a significant increase in fluorescence intensity at 438 nm, and the formation of nitrone 4a (Fig. 4b). Quantitative HPLC analysis revealed that the disappearance of 4b was nearly stoichiometrically matched by the formation of 4d, achieving a release efficiency as high as 96% (Fig. 4c). The absence of detectable elimination intermediates further emphasizes the rapidity of the release process. By monitoring the time-dependent increase in fluorescence at 438 nm, we determined a rate constant (k2) of 1.8 ± 0.1 M−1s−1 at room temperature, which increased to 4.7 ± 0.1 M−1s−1 at 37 °C (Fig. 4d and Supplementary Fig. 3A, B).

Under similar conditions, probe 4c exhibited exceptional reactivity. In the presence of 2c, 4c was almost completely consumed within 10 minutes, releasing 4e and generating nitrone 4a (Fig. 4e). The corresponding fluorescence increase at 556 nm allowed us to calculate a rate constant of 39.1 ± 6.1 M−1s−1 (Fig. 4f and Supplementary Fig. 3C). Expanding this analysis to additional bond types, we synthesized 4f, which, upon treatment with 2c, was converted to nitrone 4g within 30 min, accompanied by the loss of a fluoride ion as confirmed by LC–MS (Fig. 4g and Supplementary Fig. 4). The rate constant for this reaction, monitored via the nitrone absorption peak at 300 nm, was determined to be 10.1 ± 1.0 M−1s−1 (Supplementary Fig. 5A, B).

These results confirm the robustness of our bioorthogonal bond-breaking chemistry, which efficiently cleaves a wider range of bonds, including carbamate, ether, and others. The elimination step occurs rapidly, with the overall release rate being primarily governed by the initial click reaction. Moreover, the high regioselectivity of the reaction between hydroxylamine and COT ensures near-complete payload liberation. Notably, payload-bearing COT derivatives show high stability against biologically relevant nucleophiles like glutathione (GSH), cysteine (Cys), and dimethylamine (DMA), as previously confirmed25.

Effectiveness of click-release strategy in cellular environments
Encouraged by our findings, we next validated the efficacy and bioorthogonality of our click–release strategy within cellular environments, applying it to both bioorthogonal fluorescence imaging and prodrug activation.

For fluorescence imaging, we first incubated 4T1 breast cancer cells with the caged probes 4b and 4c at a concentration of 20 μM for 60 min; under these conditions, no fluorescence signal was observed, confirming that the probes remain inert in the absence of a trigger. Following this, subsequent treatment with mono‑alkyl‑hydroxylamine 2c at 100 μM resulted in a pronounced increase in fluorescence—blue for 4b and green for 4c—as visualized by confocal microscopy (Fig. 5a,b). This result indicates that the ether and carbamate bonds in these probes are efficiently cleaved in the cellular milieu via the click–release mechanism, leading to robust activation of the fluorophores.

To extend the system for in vivo imaging applications, we synthesized compound 5a, designed to shift the emission to the near‑infrared (NIR) region (Fig. 5c). Compound 5a displayed effectively quenched fluorescence and remained non‑fluorescent when incubated in 20% DMSO/PBS at 37 °C for 24 hours (Supplementary Fig. 6). In contrast, when 5a was used at 50 μM in the presence of 2c at 250 μM, we observed a robust increase in fluorescence intensity at 620 nm, peaking within 1 h with a decaging rate constant (k2) of 3.1 ± 0.2 M−1s−1 (Fig. 5d and Supplementary Figs. 7, 8). Consistently, when 5a (20 μM) and 2c (100 μM) were co‑incubated with 4T1 cells, significantly enhanced red fluorescence was detected after 60 min (Fig. 5e), confirming efficient release of the NIR fluorophore. To further evaluate whether this click-release chemistry is sufficiently biocompatible to proceed within live cells, we synthesized COT-chloroalkane 5d (Supplementary Fig. 9A). 4T1 cells expressing HaloTag-PS were incubated with 5d (50 μM) for 1 h. After washing with PBS to remove extracellular 5d, the cells were treated with hydroxylamine 2c (100 μM) for 30 min. A significant increase in blue fluorescence was observed (Supplementary Fig. 9B, C), confirming not only the cell permeability of both reactants but, critically, their successful intracellular reaction.

Building upon these imaging studies, we applied the click–release strategy to bioorthogonal prodrug activation. Doxorubicin (DOX) was conjugated to cyclooctynol via a carbamate bond to generate the prodrug 5c, effectively masking its cytotoxicity. HPLC monitoring revealed that, upon treatment with 2c, 5c was rapidly consumed within 1 h, concomitant with the release of DOX. The click–release process proceeded with a rate constant of 4.8 ± 0.3 M−1s−1, and the overall release yield was determined to be 98.2 ± 0.7% (Fig. 5f,g and Supplementary Fig. 10A, B).

Finally, we evaluated the antiproliferative activity of prodrug 5c in combination with 2c using 4T1 cells. Mono‑alkyl‑hydroxylamine 2c alone was non‑toxic, even at concentrations up to 1 mM. In contrast, prodrug 5c alone exhibited markedly reduced cytotoxicity compared to free DOX. Importantly, when cells were co‑treated with 5c and 2c, the cytotoxic effect was effectively restored, with the inhibitory activity on cell proliferation approaching that of free DOX (Fig. 5h). This result strongly suggests that 5c is efficiently activated in the cellular environment via the click–release strategy.

Spatiotemporally controllable click-release chemistry
In Fig. 2f, we demonstrate that acylation of the nitrogen atom in mono-alkyl-hydroxylamine effectively shields its reactivity. Exploiting this property, we linked various responsive bond-breaking groups to hydroxylamine via carbamate bonds to block its clickability (i.e., Clickability OFF). Upon exposure to a specific stimulus, the blocked clickability is restored (Clickability ON), thereby enabling precise control over the timing and location of the click–release event. To this end, we developed a controlled activation strategy by designing and synthesizing three hydroxylamine derivatives with distinct activation mechanisms: thiol-responsive 6a, photo-activated 6b, and nitroreductase (NTR)-responsive 6c. In each case, the clickability of these compounds was effectively masked until triggered.

To demonstrate specificity, we first activated compound 6a by incubation with 2 mM glutathione (GSH) and subsequently performed a click reaction with probe 4b. Under these conditions, more than 98% of product 4d was released after 2 h, whereas in the absence of GSH, less than 5% of 4d was generated (Fig. 6a). Similarly, to verify that 6c could be activated, we treated it with an aqueous solution of sodium sulfite as a mild reducing agent. The results confirmed that the clickability of 6c was restored, thereby enabling the release of the caged 4d from COT 4b.

Given that endogenous GSH concentrations in cancer cells typically range from 1 to 10 mM—substantially higher than the extracellular levels in normal cells (generally below 10 µM)52—we investigated whether compound 6a could be activated by the GSH-rich milieu present in tumor cells. When 6a (100 µM) and probe 4c (10 µM) were incubated with 4T1 cells, confocal microscopy revealed prominent green fluorescence after 2 h, indicating successful activation. In contrast, pretreatment of cells with the thiol-reactive reagent N-ethylmaleimide (NEM) (500 µM for 1 h) to deplete endogenous GSH resulted in only weak fluorescence upon subsequent incubation with 6a and 4c (Fig. 6b).

To further validate the in vivo efficacy of our strategy, compound 6a and probe 5a were co-injected intratumorally into 4T1 tumor-bearing mice to assess GSH-triggered activation localized within the tumor microenvironment. Significant fluorescence was observed at the tumor site for over 7 h, whereas the control group that received only probe 5a exhibited no detectable fluorescence at any time point (Fig. 6c). These results suggest that the clickability of 6a is efficiently activated in the high GSH environment of tumor hypoxic regions, thereby turning on fluorescence via the click–release mechanism.

Building upon the successful demonstration of bioorthogonal imaging, we extended our strategy to prodrug activation to further showcase the versatility of our spatiotemporally controlled click–release system. Cytotoxicity assays in 4T1 cells confirmed the effectiveness of the approach: 6a alone shows no impact on cell viability, even at concentrations as high as 300 μM. Encouragingly, after its combination with 5c, the cytotoxicity exhibited a greater inhibitory effect on the cancer cell line than free DOX, indicating that this prodrug has been effectively activated within the cells (Fig. 6d and Supplementary Fig. 11). These results underscore the potential of our click–release strategy for precise, spatiotemporal control over prodrug activation in complex biological environments.

To illustrate that our strategy enables control not only over the reaction location via endogenous stimuli but also over the reaction timing via exogenous triggers, we evaluated the photo-activated system. Under 405 nm LED light, the clickability of compound 6b was activated, allowing it to react with probe 4b and achieve more than 98% release of product 4d after 100 min. In contrast, in the absence of light, only trace amounts of 4d were observed (Fig. 6e and Supplementary Fig. 12A-C). Moreover, when 6b and probe 4c were co-incubated with 4T1 cells, confocal microscopy revealed significant green fluorescence in the light-exposed group, whereas no fluorescence enhancement was observed in the group without light (Fig. 6f).

Overall, through simple chemical modification we effectively blocked the clickability of mono-alkyl-hydroxylamine and then reactivated it using specific stimuli—either endogenous (e.g., GSH, NTR) or exogenous (e.g., light). This spatiotemporally controllable click–release strategy thus offers precise regulation over the timing and location of molecular release, broadening its potential applications in targeted drug delivery and bioorthogonal imaging.

GSH-triggered bioorthogonal prodrug for targeted cancer treatment
To further validate the therapeutic potential of our spatiotemporally controlled click–release strategy, we evaluated its in vivo efficacy using a GSH-triggered bioorthogonal prodrug in a 4T1 mouse breast tumor model. Building on our encouraging in vitro results, 4T1 cells were subcutaneously implanted into mice, which were then randomly assigned into five groups (n=5 per group). Once tumor volumes reached 60–100 mm³ (typically after 7 days), treatment commenced with administrations every other day for a total of four injections.

For treatment, the prodrug 5c (2.4 mg/kg, DOX-equivalent) was pre-mixed with 6a (17 mg/kg) and administered via a single tail vein injection. In parallel, a positive control group received free DOX (2 mg/kg), while separate control groups were treated with 5c alone (2.4 mg/kg) or with PBS (100 μL). Throughout the experimental period, tumor growth and body weight were monitored daily until day 12. When the tumor volume in the PBS group approached 1500 mm³, the animals were euthanized, and tumors along with major organs were collected for analysis (Fig. 7a).

As illustrated in Fig. 7b,d, mice treated with 6a alone exhibited tumor growth comparable to that of the PBS group, and prodrug 5c by itself showed only a modest inhibitory effect. Notably, the combined administration of 5c and 6a resulted in a significant suppression of tumor growth, outperforming both the negative control and the free DOX group. Histological analysis via H&E staining also revealed that the tumor tissues from the group receiving the combination of 6a and 5c exhibited significantly higher levels of necrosis and apoptosis. Importantly, no significant changes in body weight were observed in any treatment group during the study period, demonstrating the safety of the strategy, which was further supported by H&E staining of the collected vital organs, including the heart, liver, spleen, lungs, and kidneys (Fig. 7b-e and Supplementary Fig. 13).

To directly confirm that the bioorthogonal click–release reaction occurs at the tumor site, we conducted a pharmacokinetic (PK) study comparing free DOX (2.0 mg/kg) with a prodrug system consisting of compound 5c (2.4 mg/kg DOX equivalent) and the GSH-responsive activator 6a (17 mg/kg), both administered via intravenous injection. Doxorubicin concentrations in plasma, tumor, heart, and liver were quantified using UPLC-MS/MS at multiple time points. In contrast to free DOX, the 6a + 5c group showed markedly reduced drug levels in non-target tissues (e.g., heart and liver) and in the plasma, while maintaining comparable tumor concentrations across all time points (Supplementary Fig. 14). These results provide compelling evidence that the click-release reaction is preferentially triggered within the tumor microenvironment by elevated endogenous GSH, thereby effectively reducing off-target exposure. While this study establishes spatially selective prodrug activation in vivo, further validation in expanded cohorts and extended therapeutic studies will be essential to confirm long-term safety and efficacy.

Photo-triggered bioorthogonal prodrug for local anesthesia
In contrast to the GSH-activated system that relies on endogenous stimuli, this section explores the use of an exogenous trigger to demonstrate the conceptual versatility of our click–release strategy. As a proof-of-concept, we designed a photo-triggered prodrug system for local anesthesia. We note that the anesthetic effect could, in principle, be achieved through more straightforward direct uncaging approaches; however, the present design serves to illustrate the modularity of the platform and the decoupling of the activation trigger from the payload structure. In this system, the conventional local anesthetic tetracaine was conjugated to COT via a carbamate bond to mask its anesthetic activity, yielding the prodrug 7a. In vitro studies under 405 nm LED irradiation, in the presence of the photoactivator 6b, showed that 7a released tetracaine with an 89.4 ± 2.4% yield within 30 min (Fig. 8a).

To facilitate in vivo application, 7a and 6b were incorporated into a thermosensitive hydrogel formulation (P407-(6b+7a). This formulation remains liquid at room temperature for ease of injection and gels at body temperature, thereby prolonging drug retention at the administration site.

For in vivo evaluation, the P407-(6b+7a) formulation was injected into the plantar surface of rat paws. The local anesthetic effect was quantified by stimulating the footpad with a Touch Test sensory evaluator, with subsequent measurement of withdrawal responses to calculate the maximum peak effect (MPE) and duration of nerve block (see “Methods” for details). Notably, administration of 7a alone produced no anesthetic effect, confirming that the activity of tetracaine was effectively masked. However, upon exposure to 405 nm LED light 10 min post-injection, the prodrug was activated, yielding an anesthetic effect comparable to that of free tetracaine (Fig. 8b).

The anesthetic effect was further modulated by adjusting the light intensity and exposure duration. Irradiation at 200 mW cm−2 for 2 min produced a nerve block lasting 26.3 ± 7.5 min, while increasing the irradiance to 300 mW cm−2 extended the duration to 33.8 ± 7.5 min. Prolonging the exposure at 300 mW cm−2 to 5 min further extended the block to 48.8 ± 7.5 min (Fig. 8c).

To address clinical needs for prolonged pain relief, we also evaluated the potential for repetitive on-demand local anesthesia. After an initial light-triggered nerve block, subsequent exposures were administered once the previous effect had subsided. Over the course of five cycles, each exposure produced a measurable anesthetic effect (Fig. 8d); however, the duration of the block gradually decreased with successive triggers—likely due to drug depletion—with up to four effective activations observed (Fig. 8e).

To assess the safety of the drug delivery system, rats were euthanized at 4 and 14 days following the administration of free tetracaine, prodrug 7a, or the combination treatment (7a+6b). Footpads were harvested for subsequent histological analysis. Consistent with the known inflammatory responses associated with local anesthetic injections53, mild to moderate inflammation was observed in all treatment groups at day 4. By day 14, inflammation had largely resolved in all cohorts. Importantly, the 7a+6b combination group did not demonstrate exacerbated toxicity compared to the other treatment arms, thus supporting the favorable safety profile of this delivery system (Supplementary Fig. 15).

These findings collectively demonstrate that our photo-triggered bioorthogonal prodrug system provides precise temporal control over the delivery of local anesthetics using an exogenous light stimulus. This approach not only distinguishes itself from endogenous glutathione (GSH)-triggered systems but also presents a tunable method for on-demand pain management with potential clinical applications.

Methods
Chemical synthesis
Synthesis protocol and additional characterization can be found in the Supplementary Information.

HPLC monitoring of hydroamination between COT 1a and hydroxylamine
HPLC analysis: COT 1a (1 mM) and hydroxylamine (3 mM) were dissolved in 50% ACN/PBS, and the obtained solution was incubated at room temperature. An aliquot was taken from the mixture at intervals for HPLC analysis. The conversion was determined based on the HPLC data.

Reaction kinetics studies
The rate of this click-release reaction is almost entirely dependent on the rate of the first click step because the release step is too fast to monitor the intermediates. Therefore, the overall rate of click release was measured in this experiment.

Reaction progress was monitored either by the increase in fluorescence intensity (for payload release) or by the rise in UV absorbance (for nitrone formation). The conversion C at time t was calculated from the measured signal St as C = (St - S0) / (Smax - S0) where S0 is the initial baseline signal and Smax is the signal at reaction completion. The concentration of each reactant at time t was then determined as [A] = [A]₀(1–C) and [B] = [B]₀(1–C).

The second-order rate constant k was obtained by fitting the time-dependent concentration data to the integrated rate Equation 1.
kt = 1 / ([B]₀ - [A]₀) * ln ([A]₀[B] / [B]₀[A])
where [A]₀ and [B]₀ are the initial concentrations (mmol/mL), and t is the reaction time (s). The value of k is reported in M−1s−1.

Fluorescence-based monitoring (for 2c with COT derivatives 4b, 4c, and 5b): Stock solutions of hydroxylamine 2c and the corresponding COT derivative (50 mM in DMSO) were diluted with PBS to the desired PBS/DMSO ratio and final concentration, affording separate solutions of the COT derivative (solution A) and hydroxylamine 2c (solution B). Solutions A and B were rapidly mixed in equal volumes. After a brief initial delay, fluorescence intensity at the characteristic emission wavelength was recorded using a fluorescence spectrophotometer. Measurements were repeated at fixed time intervals until the signal reached a plateau. The kinetic trace (fluorescence intensity vs. time) was converted to concentration versus time as described above, and the second-order rate constant was extracted from the fit to equation (1).

UV-based monitoring (for 2c with COT 4f): Separate acetonitrile solutions of COT 4f and hydroxylamine 2c were prepared at the desired concentration. The two solutions were rapidly combined, and after a set delay, the UV absorbance at 300 nm was measured with a UV spectrophotometer. Absorbance readings were taken at regular intervals until no further increase was observed. The absorbance-time profile was converted to concentration-time data and fitted to equation (1) to obtain the second-order rate constant.

HPLC-based monitoring (for 2c with COT 5c): The release of doxorubicin (DOX) from COT 5c upon reaction with hydroxylamine 2c was quantified by HPLC. Aliquots withdrawn at designated time points were analyzed to determine the concentration of released DOX. The resulting release profile (Fig. 5f) was treated as described above, and the second-order rate constant was calculated from the fit to Eq. (1).

Stability assessment
Stability of fluorescent Probes 4b, 4c, and 5a: The stability of probes 4b, 4c, and 5a was evaluated by monitoring changes in their fluorescence intensity. Each compound was dissolved in 20% DMSO/PBS to a final concentration of 20 μM, and the solution was incubated at 37 °C for 24 h. Fluorescence intensity at the characteristic emission wavelength of the parent fluorophore was recorded at 0 h and 24 h.

Fluorescence or HPLC‑based monitoring of stimulus‑triggered payload release from probe 4b
The release of payload 4d from probe 4b under various stimuli was monitored by fluorescence spectroscopy. Stock solutions (50 mM in DMSO) of the probe (4b) and each trigger compound (6a, 6b, or 6c) were prepared. Appropriate dilutions were made to obtain the desired working concentrations in a mixed solvent system. The release was initiated by combining the diluted solutions of probe 4b and the respective trigger compound.

For activation by glutathione (GSH), a 50 mM GSH stock solution in PBS was added to the mixture containing compound 6a to a final concentration of 5 mM. The reaction mixture was then incubated at 37 °C. For photo‑triggered release, the mixture containing compound 6b was directly exposed to 405 nm LED light at room temperature. For activation by sodium dithionite, a 50 mM Na2S2O4 stock solution in PBS was added to the mixture containing compound 6c to a final concentration of 5 mM, followed by incubation at 37 °C.

In all cases, payload release was verified either in real-time by tracking the fluorescence increase at 438 nm using a fluorescence spectrophotometer, or by quantitative HPLC analysis of samples taken at designated time points.

Preparation of P407-(6b+7a)
At 0 °C, dissolve 4 g of P407 in 16 mL of PBS to prepare a 20% P407 solution. Dissolve compound 7a in a small amount of ethanol, then add an equivalent molar amount of 2 M HCl. After completely removing the solvent by reduced pressure evaporation, the obtained 7a hydrochloride is dissolved in P407 aqueous solution. Next, it is mixed with an appropriate amount of 6b in DMSO solution to prepare a hydrogel formulation. The final concentrations are 50 mM for 7a and 60 mM for 6b, with 10% DMSO included. The formulation is typically stored at a temperature of 0–4 °C (it will form a gel at room temperature or 37 °C).

Cell culture
The 4T1 cells (Product No. CC-Y2004) used in this study were purchased from Shanghai EK-Bioscience Biotechnology Co., Ltd. (Shanghai, China). Cells were cultured in RPMI 1640 medium (Jiangsu Ketu Biotechnology Co., Ltd.; Cat. No. RG-CE-4) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO2.

Cytotoxicity assay
4T1 cells in the logarithmic growth phase were seeded on 96-well plates at a density of 5 × 103 cells per well. Following a 24-h incubation at 37 °C in a 5% CO2 atmosphere, the cells were treated with the tested compounds at different concentrations for an additional 24 h. Subsequently, the cells were washed twice with PBS and treated with 10% CCK-8 (Keygen BioTECH; Cat. No. KGA1606-500) for 1 h at 37 °C. The absorbance at 450 nm was then recorded using a microplate reader. Cell viability was assessed by calculating the ratio of experimental wells (after subtracting background) to control wells (also after subtracting background).

Cell imaging studies
Briefly, after incubating 4T1 cells in a confocal culture dish for 24 h, the culture medium is replaced with a culture medium solution containing the desired concentration of the compound. After the set incubation time, stain with Hoechst 33342 for 10 minutes if needed. Then, detection and image capture are carried out using CLSM (Olympus Corporation, Japan).

Stable 4T1 cell lines expressing the HaloTag protein from the pHaloTag-PS plasmid (where PS denotes the HSV-TK poly(A) signal) were generated as follows. Briefly, 4T1 cells were seeded in 6-well plates at a density of 5.0 × 105 cells per well in 2 mL of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Cells were cultured in a humidified incubator at 37 °C with 5% CO2 until they reached ~80% confluency. For transfection, each well was treated with a complex formed by combining 2.0 µg of the pHaloTag-PS plasmid (purchased from Beijing Solarbio Science & Technology Co., Ltd.) with 6 µL of Lipofectamine 3000 transfection reagent in 100 µL of Opti-MEM I reduced serum medium, according to the manufacturer’s protocol. After 36 h of transfection, cells were washed three times with 1 mL PBS. Subsequently, 400 µL of serum-free RPMI 1640 medium containing the respective test compounds was added to each well for further treatment.

The detailed procedure for the confocal experiments using 5d and 2c is as follows: To unequivocally demonstrate the intracellular occurrence of the click-release reaction, 4T1 cells stably expressing HaloTag-PS were employed. Briefly, the cells were incubated with compound 5d (50 µM) for 1 h to facilitate covalent labeling of the intracellular HaloTag-PS. Following two extensive washes with PBS to remove any unbound compound, the cells were treated with the trigger molecule 2c (100 µM) for 30 min. The successful intracellular reaction was subsequently visualized by confocal microscopy, as indicated by a pronounced increase in blue fluorescence. For control experiments, a negative control group was treated with compound 5d alone (without 2c), and a positive control group was treated with the covalent control compound 5d-1 under identical incubation conditions.

Animal
All animal procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of China Pharmaceutical University under the approved protocol (No. 2024-09-150). The protocol permitted a maximum tumor volume of 2000 mm3, a limit that was not exceeded in any experiment. Euthanasia was performed when tumor size reached this permitted maximum or if animals became moribund due to severe weight loss or unhealing ulceration. Female BALB/c mice (4–6 weeks old) were purchased from Nanjing Huimiaoxin Biotechnology Co., Ltd. (Nanjing, China). Male Sprague–Dawley (SD) rats (7–8 weeks old, 200–250 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were housed under specific pathogen-free (SPF) conditions with a 12 h light/12 h dark cycle, a temperature of 18–23 °C, and 50 ± 10% relative humidity, with free access to food and water. In this study, only one sex was used per model (female mice for tumor studies, male rats for pharmacokinetic studies) to minimize variability within each experimental system. Data are therefore reported disaggregated by sex as appropriate for each experiment. The overall animal numbers for individual experiments are provided in the corresponding figure legends and source data files.

In vivo imaging studies
The mice bearing subcutaneous 4T1 tumors were randomly assigned to two groups, each consisting of three individuals (n=3). One group received intratumorally injection of 5a (2 mg/kg) as the control group, while the other group received intratumorally injection of 5a + 6a (6a: 15 mg/kg; 5a: 2 mg/kg). In vivo fluorescence images of subcutaneous 4T1 tumor-bearing mice were obtained at specified intervals (0, 0.5, 1 and 7 h) after injection, utilizing the IVIS near-infrared fluorescence imaging system.

In vivo anticancer efficacy
The mice were allowed a 7-day acclimation period to the experimental conditions before being randomly assigned to the experimental groups. In this study, a 4T1 mouse breast tumor model was established by subcutaneously injecting 5 × 106 4T1 cells into BALB/c female mice. Once the tumor volume reached around 60–100 mm³, the BALB/c mice with 4T1 tumors were utilized for the in-vivo experiment. Dissolve the compound in a solution containing 5% DMSO and 5% Tween 80. The mice were intravenously injected at Day 0, 2, 4 and 6 with PBS (100 μL), DOX·HCl (2 mg/kg), 6a (17 mg/kg), 5c (2.4 mg/kg), or 6a + 5c (6a: 17 mg/kg; 5c: 2.4 mg/kg). Starting from the first day of administration, counted as day 0, tumor size was measured daily, and the weight of the mice was recorded. Tumor volume was calculated using a formula (volume = width2 × length × 0.52).

Pharmacokinetics and tissue targeting studies
To establish the 4T1 subcutaneous murine breast cancer model, 5 × 106 4T1 cells were injected into the right leg muscle of mice. Upon reaching a tumor volume of ~500 mm3, the 4T1 tumor-bearing mice were randomly divided into two groups (12 mice per group) and intravenously administered either DOX•HCl (2 mg/kg) or a combination of 6a (17 mg/kg) and 5c (2.4 mg/kg). At 2, 4, 8, and 24 h post-injection, mice were euthanized, and plasma, liver, heart, and tumor tissues were collected. These samples were then stored at −80 °C until analysis by UPLC-MS/MS (SHIMADZU-LCMS-8060NX).

Internal standard: Diclofenac sodium; Parent ion: M=296.0000; Daughter ion 1: M=250.0000, Q1=−14.0 V, CE=−24.0, Q3=−16.0 V; Daughter ion 2: M=214.0000, Q1=−35.0 V, CE=−21.0, Q3=−30.0 V.
DOX·HCl; Parent ion: M=544.0000; Daughter ion 1: M=321.0000, Q1=−28.0 V, CE=-28.0, Q3=−22.0 V; Daughter ion 2: M=397.0000, Q1=−20.0 V, CE=−14.0, Q3=−26.0 V; Daughter ion 3: M=361.0000, Q1=−20.0 V, CE=−29.0, Q3=−12.0 V.

Evaluating the effects of local anesthesia
Groups of SD rats were kept in an environment with a 12-h light/12-h dark cycle. Under brief isoflurane-oxygen anesthesia, a solution of the compound was injected into the plantar region of the hind paw of the rat. At specified intervals, neurobehavioral assessments were conducted by applying stimuli to the rat’s foot pad using Touch Test sensory evaluators (filaments designed to exert a target force of 180 g), while observing the rat’s motor reactions, such as foot withdrawal. No withdrawal after six trials were defined as complete nerve block or 100% MPE (Maximum peak effect). The duration of the nerve block was defined as the period during which the nerve block exceeded 50% of the MPE. In photo-triggered anesthesia experiments, the hind paw of the rat with injected formulations were irradiated by the LED at designed intensity for 2–5 min under brief isoflurane–oxygen anesthesia. The LED light source had a diameter of 2.5 cm, allowing it to fully cover the entire foot pad.

Histopathological analysis
Upon completion of the antitumor efficacy study, mice from all experimental groups were euthanized. Major vital organs (heart, liver, spleen, lungs, and kidneys), along with tumors, were collected for histopathological evaluation. In the local anesthesia study, rats were euthanized at 4 and 14 days following the administration of free tetracaine, prodrug 7a, or the combination treatment (7a+6b), and the injected hind foot pads were harvested for analysis. All histopathological processing and staining were performed by Nanjing Youmeng Biotechnology Co., Ltd.

Statistics and reproducibility
All quantitative data represent the mean ± SD of ≥3 independent replicates. Statistical analyses were performed in GraphPad Prism 9, with significant differences evaluated by one-way ANOVA followed by Tukey’s test. Data visualization was done using Origin 2022 and GraphPad Prism 9.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The work describes a new kind of a cageable click to release system using cyclooctynes that react with caged hydroxylamines. In general, bioorthogonal chemistry can suffer from off target reactions, and so mechanisms to control where such reactions occur are needed. The work is thorough and should be of interest to the wider community. However, I found several issues that need to be addressed before being reconsidered for publication.\n\nA major issue is the lack of certain controls. The mechanism of the release strategy relies initially on a nucleophilic attack of the COT by the hydroxylamine. This necessarily makes one question the biorthogonality and whether COT or the many COT derivatives react with biological nucleophiles like amines and especially thiols. Its actually well known that cyclooctynes can react with thiols and this leads to side reactions. Thus, it would be important for the authors to rigorously show how their caged COT derivatives respond to high concentrations of thiols and amines in physiologically relevant buffer conditions.\n\nThe imaging in Figure 5a and 5d can be of higher quality and could be magnified to make the details clearer. As it is now, its extremely unclear and blurry.\n\nIn the conclusions the authors should discuss the drawbacks and potential future direction for improvements of their technology. One drawback is the rate of reaction, which is lower than the faster tetrazine/TCO click to release reactions. Also, the use of very strong nucleophiles like hydroxylamines might have drawbacks in vivo as would the use of cyclooctynes which are known to undergo side reactions with thiols. Some more discussion here would be good. There are likely other areas for improvement that the authors may wish to discuss.\n\nMinor points:\nRegarding the statement “along with the methods reported by Devaraj and Pu that employ photocontrolled or enzymatic deprotection of dihydrotetrazine (DHTz) precursors to generate Tz—have begun to surmount these challenges.” The Devaraj lab has also described enzymatic uncaging in a preprint that was published before the work of Pu. I suggest the authors cite this work as well. See: https://chemrxiv.org/engage/chemrxiv/article-details/65b6ca2e9138d23161ee5b4e\n\nAlso, I’m not sure this is a fair comment: However, the necessity for the challenging synthesis and modification of tetrazines, coupled with the inherent instability of tetrazines or DHTz themselves. . . clearly the author’s system will suffer from the same challenges and unless the authors do a head to head comparison of stability, this statement be removed. I also will point out that the references cited don’t necessarily support the authors claim, particularly that of the instability of DHTz’s which are actually very stable structures in comparison to tetrazines.", "reviewer_2": "The authors reported the discovery and in situ drug-release application of a new click-to-release system based on mono-alkyl-hydroxylamine and cyclooctyne. Further caging the hydroxylamino group with conditionally cleavable groups enables the spatiotemporally controlled drug release.The noteworthy results are the spontaneous cleavage of the payload at the propargylic position through N-alkyl-hydroxyl-enamine intermediates.Although the original click reaction between the alkylhydroxylamines and strained alkynes was reported in 2021(J. Am. Chem. Soc. 2021, 143, 5616−5621) and the cick-release reactions employing enamine N-oxides produced by the alkyl-hydroxylamine and strained alkyne under reductive conditions was developed(Chem 2022, 8, 2260–2277; ACS Cent. Sci. 2021, 7, 631−640). The authors have mentioned the previous work, and emphasized their spontaneous elimination of the payload at the propargylic position of the N-alkyl-hydroxyl-enamine intermediate from mono-alkyl-hydroxylamine, which didn't require additional reduction step. However, in order to achieve the spatiotemporal control of the payload release, the authors caged the amino group on the alkylhydroxylamine so that the self-elimination can be controlled by the on-demand uncaging the hydroxylamino group. You see, what's the adavantage of the spationally controllable click-release systeme utilizing the mono-alkyl-hydroxylamine and cyclooctyne over the bioorthogonal click and release reactions employing N,N-dialkylenamine N-oxides and diboron reagents or reducing conditons?", "reviewer_3": "• What are the noteworthy results?\nIn this manuscript, Guo, Zheng and coworkers describe a click-to-release reaction involving mono-alkyl-hydroxylamines and cyclooctynes. The method builds on retro-Cope Elimination work from the labs of Kim, with modification of using a secondary (rather than tertiary) hydroxylamine in combination with a cyclooctyne bearing a propargylic leaving group. The cycloaddition is efficient provided sufficient concentrations of reactants, and release kinetics are rapid. The authors characterize the kinetics (rates in the range k2 ~10E1), and demonstrate the release of varied payloads. Reactions are demonstrated to be successful in vivo and under cell culture conditions.\n\n• Will the work be of significance to the field and related fields? How does it compare to the established literature? If the work is not original, please provide relevant references.\nWhile the reaction chemistry is related to prior art, the addition of release chemistry to the retro-Cope reaction is an intriguing addition that will be appreciated by the chemical biology community.\n\nKinetics are a challenge for the described work, as they lag significantly behind the click-to-release methodologies involving TCO and tetrazine, and we are left wondering what niche this new method will find for the field. The cellular experiments in Figure 5 are described as “confirming intracellular activation”, but there is no evidence to whether the reactants are even cell permeable, as reactions could take place extracellularly and deliver payload which subsequently becomes internalized.\nSuch “on cell” demonstrations are of limited value relative to demonstrations where one reactant is clearly localized to a cellular target (e.g. via a covalent bond). In Figure 6, while it can be concluded that disulfide reduction takes place intracellularly, there is no way to conclude that the cycloaddition takes place inside a cell. For the in vivo experiment described in Fig 6c, there is no evidence that uncaging takes place with spatial control. The uncaging could take place anywhere within the animal, followed by enhanced retention in cancerous tissue. This is a known effect for other dyes used in NIR imaging (review: PMC10827512).\n\nIn vivo studies appear to indicate that the combination of 6a + 5c is more effective than doxirubicin itself. This potentially significant finding warrants additional study to higher statistical significance. The concluding statement from this section that “These in vivo findings confirm that our GSH-triggered bioorthogonal click–release strategy effectively activates the prodrug within the tumor microenvironment, leading to targeted cancer therapy with enhanced efficacy and minimal systemic toxicity” needs to be reconsidered, as there is no evidence that the click reaction takes place at the tumor.\n\nFinally, the authors use light to uncage a hydroxylamine (6b), which can then be used to uncage a fluorophore in vitro, or tetracaine on the plantar surface of rat paws. These uncaging experiments are successful, but it is unclear why the authors pursue a double uncaging experiment, when tetracaine could have just been as easily have been directly caged and photouncaged.\n\nSummary: I find this to be interesting chemistry. Applications are limited by modest reaction rates that are going to limit in vivo and cellular applications. Biological experiments were performed “on cells” and in vivo at high concentrations without hard evidence for spatial control.\n\n• Does the work support the conclusions and claims, or is additional evidence needed?\nyes\n\n• Are there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\nThe major flaw, described above, relates to the interpretation of spatial control.\n\n• Is the methodology sound? Does the work meet the expected standards in your field?\nyes\n\n• Is there enough detail provided in the methods for the work to be reproduced?\nyes"}, "author_response": "Re: Revised article manuscript\nManuscript ID: NCOMMS-25-30990-T\nTitle: \" Spatiotemporally Controlled Drug Release via a Novel Click-Release System\nUtilizing Mono-alkyl-hydroxylamine and Cyclooctyne Chemistry \"\nAuthor(s): Xiaowei Xu, Xidan Tong, Yangfei Shi, Xin Wang, Yanzhao Chen, Yuanan\nWang, Mingxin Cheng, Shuanglong Chen, Weiwei Guo and Yueqin Zheng\nWe sincerely appreciate the reviewers' valuable comments. We have addressed each\npoint in the detailed, point-by-point responses below and made corresponding revisions\nto the manuscript. The critiques are presented in regular black text, followed by our\nresponses in blue italics. Changes made in the revised manuscript are highlighted in\nyellow.\nReviewer: 1\nComments: The work describes a new kind of a cageable click to release system using\ncyclooctynes that react with caged hydroxylamines. In general, bioorthogonal\nchemistry can suffer from off target reactions, and so mechanisms to control where such\nreactions occur are needed. The work is thorough and should be of interest to the wider\ncommunity. However, I found several issues that need to be addressed before being\nreconsidered for publication.\nResponse: Thank you very much for your positive feedback and valuable suggestions.\nWe have made revisions based on your comments; please see the details below.\n1. A major issue is the lack of certain controls. The mechanism of the release strategy\nrelies initially on a nucleophilic attack of the COT by the hydroxylamine. This\nnecessarily makes one question the biorthogonality and whether COT or the many COT\nderivatives react with biological nucleophiles like amines and especially thiols. Its\nactually well known that cyclooctynes can react with thiols and this leads to side\nreactions. Thus, it would be important for the authors to rigorously show how their\ncaged COT derivatives respond to high concentrations of thiols and amines in\nphysiologically relevant buffer conditions.\nResponse: We thank the reviewer for raising this essential concern regarding the\nbioorthogonality of our click-release system. To address this, we systematically\nevaluated the stability of payload-bearing COT derivatives (e.g., 4b) under biologically\nrelevant conditions. Specifically, 4b (50 μM) was incubated at 37°C in 20%\nDMSO/PBS buffer with high concentrations of nucleophilic species: glutathione (GSH,\n3 mM), cysteine (Cys, 3 mM), and dimethylamine (DMA, 3 mM). After 24 hours,\n4b\nexhibited no significant degradation in any condition (Figure S10A), confirming the\nbioorthogonality of payload-bearing COT derivatives for biological applications.\nFor comparison, 1a (50 μM) showed significant degradation specifically in GSH,\nwith only 76.1 ± 2.7% remaining (Figure S10B), while LC-MS analysis identified a\nmajor thiol-addition product (Figure S10C), consistent with literature (Bioconjugate\nChem. 2012, 23, 392–398.). This differential stability is mechanistically explained by\nthiol-yne reactions are believed to proceed via radical pathways\n(Macromolecules 2010, 43, 4113–4119.), where the propargylic electron-withdrawing\ngroup in COT derivatives destabilizes radical intermediates, increases the activation\nbarrier, and reduces thiol reactivity—distinguishing our payload-bearing COT from\nconventional strained cyclooctynes (e.g., DIBO, DIBAC, BCN) that exhibit greater\nsusceptibility to thiol addition.\nFigure S10. (A) The residual amount of 4b (50 μM) was quantified over 24 hours under the following\nconditions: (i) 20% DMSO/PBS (37 °C), (ii) with 3 mM dimethylamine (DMA), (iii) with 3 mM cysteine (Cys),\n(iv) with 3 mM glutathione (GSH). (B) Residual amounts of 1a (50 µM) quantified over 24 hours under the\nsame conditions as in (A). (C) LC-MS monitoring of 1a at 24 h observed the formation of a GSH adduct under\ncondition (iv).\nWe have revised the relevant sections of the text as follows.\nNotably, payload-bearing COT derivatives demonstrate exceptional stability against\nbiologically relevant nucleophiles, including glutathione (GSH), cysteine (Cys), and\ndimethylamine (DMA). This high level of stability, confirmed by the negligible\ndegradation of 4b after 24 hours of incubation, highlights the excellent\nbioorthogonality of the system (Figure S10A). In contrast, conventional strained\ncyclooctynes such as BCN 1a exhibit significant susceptibility to nucleophilic attack51,\nparticularly from thiols, as evidenced by GSH-induced degradation and formation of\nthiol-adducts (Figure S10B-C). This differential reactivity is mechanistically attributed\nto the electron-withdrawing propargylic moiety in COT scaffolds, which destabilizes\nradical intermediates52 and elevates the activation barrier for thiol–yne reactions,\nthereby minimizing undesired off-target interactions in biological environments.\n2. The imaging in Figure 5a and 5d can be of higher quality and could be magnified to\nmake the details clearer. As it is now, its extremely unclear and blurry.\nResponse: We thank the reviewer for this constructive suggestion regarding image\nclarity. In response, we have replaced the original images in Figures 5a and 5d with\nhigher-resolution versions and magnified them to enhance detail visibility.\n3. In the conclusions the authors should discuss the drawbacks and potential future\ndirection for improvements of their technology. One drawback is the rate of reaction,\nwhich is lower than the faster tetrazine/TCO click to release reactions. Also, the use of\nvery strong nucleophiles like hydroxylamines might have drawbacks in vivo as would\nthe use of cyclooctynes which are known to undergo side reactions with thiols. Some\nmore discussion here would be good. There are likely other areas for improvement that\nthe authors may wish to discuss.\nResponse: We appreciate the reviewer's thoughtful comments. We have significantly\nrevised the Discussion section to address the drawbacks of our approach, as well as\nhighlight potential future improvements and applications.\nWe have revised the Discussion section of the original manuscript as follows:\nDISCUSSION\nOver the past decade, significant advances have been made in the development of\nclick–release chemistries for controlled drug delivery. However, conventional\nplatforms remain limited by their inherent “always-on” reactivity, which limits\nspatiotemporal precision and increases the risk of off-target activation. In this study,\nwe introduce a distinct bioorthogonal bond-cleavage strategy based on the reaction\nbetween mono-alkyl- hydroxylamine and COT derivatives. By structurally masking the\nhydroxylamine moiety with stimuli-responsive groups, we achieved conditional\nactivation triggered by either endogenous signals (e.g., elevated GSH) or exogenous\nstimuli (e.g., light), establishing a versatile and modular platform for spatiotemporally\nregulated bioorthogonal activation.\nThe system was validated across multiple in vivo models. In the tumor-targeted\nprodrug delivery study, we demonstrated GSH-triggered activation within the tumor\nmicroenvironment, leading to enhanced doxorubicin accumulation and reduced\nsystemic exposure, as confirmed by UPLC-MS based pharmacokinetic analyses. In the\nlocal anesthesia model, we showed that photocaged hydroxylamine (6b) enabled\nrepeatable, on-demand drug release when activated by light, without observable\ntoxicity. This two-pronged validation highlights the platform’s dual capability for both\nendogenous- and exogenous-triggered therapeutic control.\nDespite these promising results, several challenges remain. First, the reaction\nkinetics of the COT/hydroxylamine system are slower than those of fast Tz/TCO system.\nFuture efforts will focus on modifying the electronic and steric properties of the COT\nscaffold or linkers to accelerate release rates and broaden biological applicability.\nSecond, potential side reactivity of strained alkynes with thiols is a well-known issue.\nOur system addresses this through rational COT design: the incorporation of a\npropargylic electron-withdrawing group reduces radical stability and minimizes\nthiol–yne reactivity. This was confirmed through comprehensive nucleophile stability\ntesting, where 4b exhibited excellent resistance to degradation by GSH and cysteine,\nunlike BCN 1a. Third, although hydroxylamine can be reactive in biological systems,\nour prodrug design ensures that the masked form is biologically inert until activated.\nNonetheless, long-term safety and biodistribution studies of hydroxylamine derivatives\nremain important directions for translational development.\nNotably, the dual-step design of our system also enables spatiotemporal hierarchy\nand amplification. For example, in the local anesthesia model, light-triggered release\nof the reactive hydroxylamine enabled localized, confined activation of the downstream\nCOT-prodrug. Looking forward, this modular activation strategy may be integrated\nwith bioorthogonal cascade amplification systems—such as those previously developed\nby our group ( Angew. Chem. Int. Ed. 64, e202414246 (2025))—wherein a limited\nexogenous trigger can initiate a self-propagating activation loop for enhanced signal\nor drug release in deep or heterogeneous tissues.\nFinally, the demonstrated orthogonality to Tz/TCO reactions (J. Am. Chem. Soc. 143,\n5616-5621 (2021)) enables future multiplexed therapeutic or diagnostic strategies in\ncomplex biological settings. Together with recent reports of dual-click systems in vivo\n(Staudinger and Tz/TCO) (Nat. Biomed. Eng. 9, 686–699 (2025)), our approach\nreinforces the importance of chemically diverse, orthogonal activation platforms for\nnext-generation chemical biology and drug delivery.\nIn summary, we have developed a modular and biocompatible click–release system\nbased on mono-alkyl- hydroxylamine and COT derivatives, capable of spatiotemporally\ncontrolled drug activation through either endogenous or exogenous triggers. This\nplatform was validated in both tumor-targeted prodrug therapy and light-activated\nlocal anesthesia models, demonstrating strong in vivo efficacy, minimal off-target\ntoxicity, and evidence of spatially localized activation. Pharmacokinetic studies\nconfirmed that drug release occurs preferentially at the tumor site, supporting the\npremise of bioorthogonal activation within a physiological context.\nWhile the current system operates with slower kinetics than mainstream Tz/TCO\nplatforms, its tunable controllability, bioorthogonality, and favorable safety profile\nprovide a strong foundation for therapeutic applications where temporal precision and\nstimulus-responsiveness are prioritized. Future directions will focus on enhancing\nreaction kinetics, integrating amplification mechanisms, and conducting extended\nefficacy and safety evaluations across clinically relevant models. This work thus lays a\nversatile chemical foundation for next-generation, conditionally activated drug\ndelivery systems.\nMinor points:\nRegarding the statement “along with the methods reported by Devaraj and Pu that\nemploy photocontrolled or enzymatic deprotection of dihydrotetrazine (DHTz)\nprecursors to generate Tz—have begun to surmount these challenges.” The Devaraj lab\nhas also described enzymatic uncaging in a preprint that was published before the work\nof Pu. I suggest the authors cite this work as well. See:\nhttps://chemrxiv.org/engage/chemrxiv/article-details/65b6ca2e9138d23161ee5b4e\nResponse: Thank you for bringing this important work to our attention. We have now\nadded a citation to this relevant preprint (Devaraj et al., ChemRxiv) in our revised\nmanuscript.\nAlso, I’m not sure this is a fair comment: However, the necessity for the challenging\nsynthesis and modification of tetrazines, coupled with the inherent instability of\ntetrazines or DHTz themselves. . . clearly the author’s system will suffer from the same\nchallenges and unless the authors do a head to head comparison of stability, this\nstatement be removed. I also will point out that the references cited don’t necessarily\nsupport the authors claim, particularly that of the instability of DHTz’s which are\nactually very stable structures in comparison to tetrazines.\nResponse: Thank you for your correction. We have removed the comment regarding the\ninstability of DHTz.\nReviewer #2 (Remarks to the Author):\nThe authors reported the discovery and in situ drug-release application of a new click-\nto-release system based on mono-alkyl-hydroxylamine and cyclooctyne. Further caging\nthe hydroxylamino group with conditionally cleavable groups enables the\nspatiotemporally controlled drug release. The noteworthy results are the spontaneous\ncleavage of the payload at the propargylic position through N-alkyl-hydroxyl-enamine\nintermediates. The authors have mentioned\nthe previous work, and emphasized their spontaneous elimination of the payload at the\npropargylic position of the N-alkyl-hydroxyl-enamine intermediate from mono-alkyl-\nhydroxylamine, which didn't require additional reduction step. However, in order to\nachieve the spatiotemporal control of the payload release, the authors caged the amino\ngroup on the alkylhydroxylamine so that the self-elimination can be controlled by the\non-demand uncaging the hydroxylamino group. You see, what's the adavantage of the\nspationally controllable click-release systeme utilizing the mono-alkyl-hydroxylamine\nand cyclooctyne over the bioorthogonal click and release reactions employing N,N-\ndialkylenamine N-oxides and diboron reagents or reducing conditons?\nResponse: We appreciate the reviewer’s insightful question regarding the advantage of\nour spatiotemporally controllable click-release platform based on mono-alkyl-\nhydroxylamine and cyclooctyne over previously reported bioorthogonal release systems\ninvolving N,N-dialkyl-enamine N-oxides triggered by reductants such as diboron\nreagents. Indeed, we acknowledge the excellent foundational work on enamine N-oxide-\nbased release systems (e.g., Chem 2022, 8, 2260–2277; ACS Cent. Sci. 2021, 7, 631–\n640), which offer redox-responsive activation via selective reduction. Building on Kim's\ngroup seminal work, our research broadens the applications of hydroxylamine-COT\nclick chemistry, enhancing the existing mechanistic understanding and enabling a\nwider range of functionalities. Specifically, our system introduces a fundamentally\ndistinct and advantageous strategy for spatiotemporal control, enabled by the following\nkey innovations:\n1. Intrinsically Triggerable Click–Release Without Need for Exogenous Reducing\nAgents.\nUnlike N-oxide-based systems that rely on exogenous chemical triggers (e.g., diboron\nreagents or reducing environments), our click–release platform employs N-alkyl-\nhydroxyl-enamine intermediates that undergo spontaneous β-elimination of the payload\nat the propargylic position under physiological conditions—no additional chemical\nactivation is required. This property simplifies in vivo applications by eliminating\nreliance on systemically administered triggers, which may have limited bioavailability\nor cause undesired systemic effects.\n2. Modular Spatiotemporal Activation via Caging the Mono-Alkyl-Hydroxylamine.\nAs described in(ACS Cent. Sci. 2021, 7, 631–640), hypoxia has been utilized within\ntumors to enable activation of hypoxia-responsive prodrugs and imaging agents.\nHowever, our approach expands upon this by allowing for spatiotemporally controlled\nreaction through the modular modification of the nitrogen atom of the hydroxylamine\nwith a variety of stimuli-responsive groups (e.g., disulfide for redox-responsiveness, o-\nnitrobenzyl for photocaging), thus broadening the scope of controllable conditions. This\nstrategic design decouples the click reaction and subsequent release, ensuring the\nactive process remains dormant until specifically triggered by a selected endogenous\n(e.g., high GSH) or exogenous (e.g., light) stimulus. This enables high adaptability\nacross a wide range of biological environment.\nThis dual-layered control (bioorthogonality + on-demand activation) is not easily\nachievable with the N-oxide/diboron approach, where the reducing reagent is\nadministered systemically and lacks spatial specificity.\n3. In Vivo Biocompatibility and Application Readiness.\nOur system exhibits excellent biological stability and safety: Mono-alkyl-\nhydroxylamine derivatives exhibited no cytotoxicity even at 1 mM concentration in\ncellular environments. Co-administration in a local anesthesia model revealed no\nobservable toxicity or inflammation; The click partners (COT derivatives) were\ndemonstrated to be exceptionally stable toward biological nucleophiles (GSH, Cys),\nunlike conventional strained alkynes such as BCN, which undergo thiol addition side\nreactions.\nThese features support its safe and selective use in complex in vivo environments,\nwithout off-target activation or byproduct generation—challenges that can limit the\npracticality of some reduction-based systems in living organisms.\n4. Versatility for Multiplexed and Precision Therapy\nOur platform’s orthogonality to Tz/TCO systems (as validated in prior studies, JACS\n2021, 143, 5616–5621) and its compatibility with dual activation schemes (e.g., GSH\n+ light) make it highly attractive for multiplexed applications, such as:\n Spatiotemporally controlled dual-drug delivery in vivo, mediated by dual click-release systems with orthogonal activation.\n Integration into pretargeted antibody–drug conjugates (ADCs), where localized\nactivation dramatically improves therapeutic indices.\nThis modular and adaptable nature is more difficult to achieve with N-oxide systems\nthat depend on global reductive environments or the pharmacokinetics of the reducing\nagents.\nIn summary, our mono-alkyl-hydroxylamine/COT-based click–release system offers:\n Inherently spontaneous release after click reaction (no additional chemistry\nrequired),\n Precise temporal and spatial control via various conditional uncaging,\n Operational simplicity and modularity for versatile therapeutic designs.\nThese features distinguish our approach from prior art and, we believe, provide a\nvaluable and practical advance in the field of bioorthogonal chemistry and drug\ndelivery. We have elaborated on these advantages throughout the revised manuscript,\nparticularly in the Discussion section, where we provide a detailed comparison\nbetween our system and existing N-oxide-based reductive click–release strategies. By\nhighlighting the distinct mechanistic basis, activation controllability, biological\ncompatibility, and translational potential of our approach, we aim to help readers\nappreciate the unique value and broad applicability of this spatiotemporally\ncontrollable click–release platform.\nReviewer #3 (Remarks to the Author):\n• What are the noteworthy results?\nIn this manuscript, Guo, Zheng and coworkers describe a click-to-release reaction\ninvolving mono-alkyl-hydroxylamines and cyclooctynes. The method builds on retro-\nCope Elimination work from the labs of Kim, with modification of using a secondary\n(rather than tertiary) hydroxylamine in combination with a cyclooctyne bearing a\npropargylic leaving group. The cycloaddition is efficient provided sufficient\nconcentrations of reactants, and release kinetics are rapid. The authors characterize the\nkinetics (rates in the range k2 ~10E1), and demonstrate the release of varied payloads.\nReactions are demonstrated to be successful in vivo and under cell culture conditions.\n• Will the work be of significance to the field and related fields? How does it compare\nto the established literature? If the work is not original, please provide relevant\nreferences.\nWhile the reaction chemistry is related to prior art, the addition of release chemistry to\nthe retro-Cope reaction is an intriguing addition that will be appreciated by the chemical\nbiology community.\nResponse: Thank you very much for your positive feedback and valuable suggestions.\nWe have made revisions based on your comments; please see the details below:\nKinetics are a challenge for the described work, as they lag significantly behind the\nclick-to-release methodologies involving TCO and tetrazine, and we are left wondering\nwhat niche this new method will find for the field.\nResponse: We sincerely thank the reviewer for raising this important point regarding\nthe kinetics of our click–release system. We fully acknowledge that while our approach\nachieves near-quantitative release efficiency, the current reaction kinetics are slower\nthan those of the tetrazine–trans-cyclooctene (Tz/TCO) platform, which may limit its\nsuitability for applications requiring fast activation. We have accordingly revised the\nDiscussion section to address this limitation and outline strategies for future\nimprovement.\nDespite these promising results, several challenges remain. First, the reaction\nkinetics of the COT/hydroxylamine system are slower than those of fast Tz/TCO system.\nFuture efforts will focus on modifying the electronic and steric properties of the COT\nscaffold or linkers to accelerate release rates and broaden biological applicability.\nIt is worth noting, however, that reaction kinetics alone do not dictate the utility of\na bioorthogonal reaction, especially in in vivo contexts where factors such as stability,\nbiocompatibility, and controllability are equally, if not more, critical. For example, the\nStaudinger ligation—despite its slow rate constant (k < 0.1 M-1 s-1; J. Am. Chem. Soc.\n2005, 127, 2686)—was effectively applied in in vivo diagnostics by the Pu group (Nat.\nBiomed. Eng. 2025, 9, 686), achieving nanomolar-level detection. Similarly, the widely\nused SPAAC reaction (k < 1 M-1 s-1) has been leveraged for imaging and\nglycoengineering in live animals (Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1821–1826).\nWhile the exceptional speed of the Tz/TCO system has understandably made it a\ndominant technology in the field, we believe that our platform provides a distinct and\ncomplementary niche, particularly in applications demanding high bioorthogonality,\ncontrollable activation, and favorable safety profiles. As discussed in our response to\nother reviewer comments, the ability to cage the hydroxylamine partner introduces a\nmodular and stimuli-responsive activation layer—enabling precise spatiotemporal\ncontrol not readily achievable with current “always-on” systems.\nImportantly, we agree that improving the rate of reaction remains a crucial goal.\nAs now elaborated in the revised Discussion, we have identified this as a key direction\nfor future optimization, potentially through structural tuning of the COT scaffold or\nmodification of the linker electronics to facilitate faster elimination kinetics.\nWe have integrated these points into the revised Discussion section (paragraphs\n3–7) of the manuscript, where we discuss both the current limitations and the future\ndirections for enhancing kinetic performance, while positioning our system as a\nvaluable addition to the broader bioorthogonal toolkit.\nThe cellular experiments in Figure 5 are described as “confirming intracellular activation”,\nbut there is no evidence to whether the reactants are even cell permeable, as reactions\ncould take place extracellularly and deliver payload which subsequently becomes internalized.\nSuch “on cell” demonstrations are of limited value relative to demonstrations where one\nreactant is clearly localized to a cellular target (e.g. via a covalent bond).\nResponse: We thank the reviewer for this important and insightful comment regarding\nthe cellular localization of the click–release reaction and the distinction between true\nintracellular activation versus potential extracellular reaction followed by uptake of\nthe released payload. We fully agree that our initial description in the main text—\nstating ““confirming intracellular activation”—was imprecise and could be misleading.\nWe have revised the manuscript text accordingly to avoid overstatement.\nTo directly address the reviewer’s concern and rigorously determine whether the\nreaction occurs intracellularly rather than extracellularly, we conducted additional\ncontrol experiments designed to establish the cell permeability of both reactants and\nthe intracellular nature of the reaction.\nSpecifically, we synthesized a triphenylphosphonium-conjugated hydroxylamine\nderivative (compound 2j), leveraging the well-characterized mitochondrial-targeting\nnature of the triphenylphosphonium (TPP) moiety. 4T1 cells were pre-treated with\ncompound 2j. Detailed experimental methods have been supplemented in the supporting\ninformation, as follows: 4T1 cells were seeded into confocal dishes at a density of 1 ×\n10⁵ cells/mL and allowed to adhere overnight (16 hours). For the experimental group,\ncells were pre-treated with 500 μM of compound 2j (dissolved in DMEM) containing\nHoechst 33342 (diluted 1:2000) for 1 hour. Following this pre-incubation, cells were\nthoroughly washed three times with PBS to remove any extracellular 2j. Next, cells were\ntreated with 40 μM of compound 5a for 1 hour. Subsequently, imaging was performed.\nAs a control group, 4T1 cells were directly treated with 40 μM of compound 5a for 1\nhour, without any prior pre-incubation with 2j. Imaging was then performed.\nFluorescent signals were excited using a 560 nm laser and collected at 620 ± 50 nm\nusing an Olympus FV1200 confocal microscope.\nFigure S19. Intracellular biocompatibility of the reaction between mono-alkyl-\nhydroxylamine and COT in live 4T1 Cells. A) Structure of 2j and 5a. B) 4T1 cells were\npre-treated with 500 μM of compound 2j for 1 hour, followed by three washes with PBS\nto remove extracellular compound. Cells were then incubated with 40 μM of\ncompound 5a for 1 hour before imaging. As a control, a separate group of 4T1 cells\nwas incubated solely with 40 μM of compound 5a for 1 hour before imaging. (Scale bar\n= 20 μm)\nwe have made the following revisions to the relevant discussion in the main text:\nConsistently, when 5a (20 μM) and 2c (100 μM) were co‑incubated with 4T1 cells,\nsignificantly enhanced red fluorescence was detected after 60 minutes (Figure 5d),\nconfirming efficient release of the NIR fluorophore. To further evaluate if this click-\nrelease chemistry possesses sufficient biocompatibility to occur within live cells, we\nsynthesized the mono-alkyl-hydroxylamine 2j (Figure S19A), equipped with a\ntriphenylphosphine mitochondrial targeting group. When 4T1 cells were pre-incubated\nwith 2j for 1 hour, followed by PBS washes to remove extracellular 2j, and then co-\nincubated with 5a, we still observed a significant increase in red fluorescence (Figure\nS19B). This result confirms not only the cell permeability of both reactants but,\ncritically, their successful intracellular reaction.\nIn Figure 6, while it can be concluded that disulfide reduction takes place intracellularly,\nthere is no way to conclude that the cycloaddition takes place inside a cell.\nResponse: We sincerely appreciate the reviewer's insightful critique regarding the\nsubcellular localization of the click reaction. While our permeability studies (in\nresponse to previous comment) confirmed that both hydroxylamine and cyclooctynes\ncan penetrate cells, we acknowledge that the while the click reaction of 6a is indeed\nactivated by the high intracellular GSH concentrations in tumor cells, the cycloaddition\nreaction could potentially occur either intracellularly or extracellularly following\ndisulfide reduction.\nwe have made the following revisions to the relevant discussion in the main text:\nGiven that endogenous GSH concentrations in cancer cells typically range from 1\nto 10 mM—substantially higher than the extracellular levels in normal cells (generally\nbelow 10 µM)54—we investigated whether compound 6a could be activated by the GSH-\nrich milieu present in tumor cells.\nFor the in vivo experiment described in Fig 6c, there is no evidence that uncaging takes\nplace with spatial control. The uncaging could take place anywhere within the animal,\nfollowed by enhanced retention in cancerous tissue. This is a known effect for other\ndyes used in NIR imaging (review: PMC10827512).\nResponse: We thank the reviewer for this valuable comment and appreciate the\nopportunity to clarify our experimental design. We apologize for any confusion caused\nby the lack of detailed methodological description in the original manuscript.\nThe in vivo experiment presented in Figure 6c was specifically designed to\nevaluate whether endogenous glutathione (GSH) within the tumor microenvironment is\nsufficient to activate the caged hydroxylamine and trigger the click–release reaction.\nTo this end, both compound 6a and probe 5a were co-administered via intratumoral\ninjection into established 4T1 tumors. This localized delivery approach ensures that\nboth reactants are spatially confined to the tumor site, thereby excluding the possibility\nof systemic activation or non-specific accumulation that could occur with intravenous\nor intraperitoneal administration. Thus, the observed fluorescence increase is a direct\nconsequence of local, GSH-mediated uncaging and subsequent reaction, not systemic\nredistribution or passive retention.\nwe have made the following revisions to the relevant discussion in the main text:\nTo further validate the in vivo efficacy of our strategy, compound 6a and probe 5a were\nco-injected intratumorally into 4T1 tumor-bearing mice to assess GSH-triggered\nactivation localized within the tumor microenvironment.\nIn vivo studies appear to indicate that the combination of 6a + 5c is more effective than\ndoxirubicin itself. This potentially significant finding warrants additional study to\nhigher statistical significance. The concluding statement from this section that “These\nin vivo findings confirm that our GSH-triggered bioorthogonal click–release strategy\neffectively activates the prodrug within the tumor microenvironment, leading to\ntargeted cancer therapy with enhanced efficacy and minimal systemic toxicity” needs\nto be reconsidered, as there is no evidence that the click reaction takes place at the tumor.\nResponse: We thank the reviewer for this thoughtful and important comment. We fully\nagree that definitively demonstrating tumor-localized activation is critical for\nvalidating the therapeutic specificity of our click–release system. We also appreciate\nthe opportunity to clarify our experimental rationale and further elaborate on the\nsupporting evidence from our pharmacokinetic studies. Our in vivo study was designed\nas a proof-of-concept investigation to evaluate whether endogenous GSH within the\ntumor microenvironment can effectively trigger the click–release reaction between\ncompound 6a (GSH-responsive hydroxylamine precursor) and compound 5c (Dox-\nconjugated COT prodrug), resulting in site-selective activation and enhanced tumor\naccumulation of doxorubicin.\nTo address the reviewer’s concern regarding spatial control and to rule out the\npossibility that uncaging occurred elsewhere followed by passive accumulation in the\ntumor, we conducted comprehensive biodistribution studies comparing the prodrug\nsystem (6a + 5c) versus free doxorubicin. These studies yielded several key findings:\nTumor-specific accumulation of doxorubicin was significantly enhanced in the prodrug\ngroup compared to free Dox at critical time points:\n1)At 4 hours: 210.8 ± 14.4 ng/g (prodrug) vs. 150.7 ± 30.9 ng/g (free Dox)\n2)At 8 hours: 187.9 ± 13.7 ng/g vs. 154.5 ± 11.2 ng/g\n3)At 24 hours: 141.6 ± 30.2 ng/g vs. 111.6 ± 11.6 ng/g\nThese elevated tumor levels strongly support local release of Dox within the tumor,\nrather than systemic activation followed by passive uptake.\nOff-target exposure was markedly reduced in the prodrug group across multiple tissues:\nThese findings provide strong pharmacokinetic evidence that the bioorthogonal\nclick–release reaction predominantly occurs at the tumor site, rather than elsewhere\nin the body, followed by redistribution. The elevated tumor concentrations of Dox\nachieved by the 6a + 5c combination, in contrast to its diminished presence in non-\ntarget tissues, confirm spatially selective activation driven by the high GSH levels\nwithin the tumor microenvironment.\nFigure S21: DOX concentration in tumor (ng/g) at different time points with various\ntreatments (n = 3). Data are presented as mean ± SD (unpaired Student's t-test, *p <\n0.05, NS: not significant).\nFigure S22: DOX concentration in heart (ng/g) at different time points with various\ntreatments (n = 3). Data are presented as mean ± SD (unpaired Student's t-test, *p <\n0.05, NS: not significant).\nFigure S23: DOX concentration in liver (ng/g) at different time points with various\ntreatments (n = 3). Data are presented as mean ± SD (unpaired Student's t-test, *p <\n0.05, **p < 0.01, NS: not significant).\nFigure S24: DOX concentration in plasma (ng/ml) at different time points with various\ntreatments (n = 3). Data are presented as mean ± SD (unpaired Student's t-test, *p <\n0.05, **p < 0.01, NS: not significant).\nwe have made the following revisions to the relevant discussion in the main text:\nThese in vivo findings demonstrate that the combination of 6a and 5c enables\neffective doxorubicin release within the tumor microenvironment, leading to enhanced\ndrug accumulation at the tumor site while significantly reducing systemic exposure. To\ndirectly confirm that the bioorthogonal click–release reaction occurs at the tumor site,\nwe conducted a pharmacokinetic (PK) study comparing free DOX (2.0 mg/kg) with a\nprodrug system consisting of compound 5c (2.4 mg/kg DOX equivalent) and the GSH-\nresponsive activator 6a (17 mg/kg), both administered via intravenous injection.\nDoxorubicin concentrations in plasma, tumor, heart, and liver were quantified by\nUPLC-MS/MS at multiple timepoints. Compared to free DOX, the 6a + 5c group\nshowed significantly lower DOX levels in non-target tissues (e.g., heart and liver) and\nin the plasma (Figures 7f-g, S22-24), but markedly higher concentrations in tumors,\nparticularly at 4 h and 8 h post-injection (Figures 7h, S21). These results provide\ncompelling evidence that the click–release reaction is preferentially triggered within\nthe tumor microenvironment by elevated endogenous GSH. While this study establishes\nspatially selective prodrug activation in vivo, further validation in expanded cohorts\nand extended therapeutic studies will be essential to confirm long-term safety and\nefficacy.\nFinally, the authors use light to uncage a hydroxylamine (6b), which can then be used\nto uncage a fluorophore in vitro, or tetracaine on the plantar surface of rat paws. These\nuncaging experiments are successful, but it is unclear why the authors pursue a double\nuncaging experiment, when tetracaine could have just been as easily have been directly\ncaged and photouncaged.\nResponse: We thank the reviewer for this insightful comment. The rationale behind our\n“double uncaging” design lies in demonstrating the modularity, spatiotemporal\ncontrollability, and therapeutic versatility of our system. Rather than directly\nphotouncaging the drug (e.g., tetracaine), we chose to photouncage a masked\nhydroxylamine precursor (6b), which in turn triggers the click–release activation of a\ndownstream prodrug (7a) via a bioorthogonal bond-cleavage reaction with the COT-\nmodified substrate. This strategy offers several distinct advantages beyond what can be\nachieved by traditional photocaged drugs:\n1).Decoupled activation from payload structure: By placing the photocage on a\ncommon activator (hydroxylamine) rather than on the payload itself, our system allows\nfor universal plug-and-play activation of diverse prodrugs (e.g., anticancer agents,\nanesthetics, fluorophores) bearing COT groups, without the need to reengineer a new\nphotolabile motif for each payload.\n2).Spatiotemporal hierarchy and amplification: The dual-step design introduces a\nlayered activation architecture, wherein an initial exogenous stimulus (e.g., light)\nliberates a reactive intermediate (6b), which subsequently triggers the click–release of\nthe downstream drug. This separation between trigger and release enables precise\nspatial targeting and repeatable, on-demand drug activation, as demonstrated in our\nlocal anesthesia model.\nImportantly, this architecture also lays the groundwork for future integration with\nbioorthogonal cascade amplification systems, such as those previously developed by\nour group (Angew. Chem. Int. Ed. 64, e202414246 (2025)). In such scenarios, an\nexternally applied, low-intensity trigger—limited by tissue penetration or biosafety\nconstraints—could initiate a self-propagating amplification loop that boosts local\nactivator concentrations and enhances drug release in a spatially confined and\nsustained manner. This would enable robust therapeutic effects from minimal initial\ninput, offering a significant advantage for in vivo applications where deep tissue access\nand dosing thresholds are major challenges.\nThus, this modular design not only improves current controllability but also opens the\ndoor to next-generation, signal-amplified prodrug systems.\n3).Unified design principle across modalities: In our study, the cancer therapy model\nvalidated endogenous stimulus-triggered activation (via GSH), while the local\nanesthesia model demonstrated exogenous stimulus-based activation (via light). The\nuse of the same core chemistry (click–release via hydroxylamine/COT) in both systems\nunderscores the versatility and unification of our platform, enabling adaptable\ntherapeutic strategies depending on clinical need.\nTherefore, although direct photouncaging of tetracaine would indeed be simpler, our\ngoal was to demonstrate a generalizable activation module capable of mediating\nprecise, bioorthogonal, and temporally controlled drug release across different\nbiomedical applications. We have revised the Discussion section of the manuscript to\nclarify this point and appreciate the reviewer’s suggestion in helping us sharpen the\nconceptual framework of our work.\nNotably, the dual-step design of our system also enables spatiotemporal hierarchy\nand amplification. For example, in the local anesthesia model, light-triggered release\nof the reactive hydroxylamine enabled localized, confined activation of the downstream\nCOT-prodrug. Looking forward, this modular activation strategy may be integrated\nwith bioorthogonal cascade amplification systems—such as those previously developed\nby our group55—wherein a limited exogenous trigger can initiate a self-propagating\nactivation loop for enhanced signal or drug release in deep or heterogeneous tissues.\nSummary: I find this to be interesting chemistry. Applications are limited by modest\nreaction rates that are going to limit in vivo and cellular applications. Biological\nexperiments were performed “on cells” and in vivo at high concentrations without hard\nevidence for spatial control.\nResponse: We sincerely thank the reviewer for their thoughtful summary and\nconstructive feedback. We acknowledge that the current reaction kinetics are slower\nthan those of state-of-the-art systems like Tz/TCO. However, as discussed and now\nfurther clarified in the Discussion section, our system offers unique advantages in terms\nof modularity, biocompatibility, and spatiotemporal controllability, which are\nparticularly valuable in in vivo settings. Moreover, we have outlined future directions\naimed at enhancing reaction rates through structural optimization.\nRegarding spatial control, we agree that this is a critical consideration. To directly\naddress this, we performed pharmacokinetic studies comparing our prodrug system\nwith free Dox. The results showed significantly enhanced tumor-localized drug\naccumulation and reduced systemic exposure, supporting the conclusion that\nbioorthogonal activation occurs preferentially within the tumor microenvironment.\nAdditionally, in our cellular studies, we conducted wash-controlled preloading\nexperiments with a mitochondrial-targeted hydroxylamine to confirm intracellular\nactivation, not merely “on-cell” reactions.\nWe have revised the manuscript to more accurately reflect these points and\nappreciate the reviewer’s comments, which helped us improve the clarity and scientific\nrigor of the work.\n• Does the work support the conclusions and claims, or is additional evidence needed?\nyes\n• Are there any flaws in the data analysis, interpretation and conclusions? Do these\nprohibit publication or require revision?\nThe major flaw, described above, relates to the interpretation of spatial control.\n• Is the methodology sound? Does the work meet the expected standards in your field?\nyes\n• Is there enough detail provided in the methods for the work to be reproduced?\nyes"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have revised the manuscript in a manner that addresses all of my concerns/comments. I would recommend publication.", "reviewer_3": "In a prior evaluation of this manuscript, it was noted that the manuscript describes interesting chemistry, but applications are limited by modest reaction rates that will hinder in vivo and cellular applications. It was further noted that some biological experiments were performed “on cells” while others were conducted in vivo at high concentrations without hard evidence for spatial control. In this revised manuscript, the authors have partially addressed some of the concerns raised previously but some questions remain.\n\nIn their response, they do provide a brief comment on the rate limitation of the rate constant, but further opine that reaction kinetics alone do not dictate the utility of a bioorthogonal reaction, especially in in vivo contexts where factors such as stability, biocompatibility, and controllability are equally, if not more, critical.”\n\nWhile it is of course true that rate constant is not the only important parameter, it is hard to argue that it is not a critical parameter, as bioorthogonal chemistry requiring millimolar concentrations of reagents introduces a host of limitations (toxicity, background, washout in cellular experiments, clearance in animal experiments, etc).\n\nIn response to a comment that it was not shown that reactions are taking place intracellularly, the authors used a triphenylphosphonium-conjugated hydroxylamine derivative (compound 2j) to target the group to the mitochondria. While this experiment suggests that the reaction occurs intracellularly, the authors should note that TPP targeting is non-covalent and reversible, allowing for an extracellular reaction to be possible. In general, the use of covalent intracellular ligands (e.g., SnapTag or HaloTag) is necessary to prove intracellular reactivity. The images provided in the supporting information to support this experiment are of low resolution; higher-resolution images should be provided.\n\nIn the prior review, it was noted that there was insufficient evidence to demonstrate that the click reaction uncages the prodrug at the tumor. In response, the authors carried out biodistribution comparing the prodrug system (6a + 5c) versus free doxorubicin. The authors describe the findings as “strong pharmacokinetic evidence that the bioorthogonal click–release reaction predominantly occurs at the tumor site”, but the data require further interpretation. In Fig S21, slightly higher accumulation is actually observed for the Dox control at 2 hours, and there is no statistical difference at 24 h. At 4 and 8 hours, a statistical difference is observed (p value *), although the difference in tumor uptake is relatively modest. My view of these data is that Dox accumulation is comparable for the uncaging system vs the control, with the benefit of lower background in the release system.\n\nFinally, it was noted that photochemical uncaging experiments were successful; however, it is unclear why the authors pursued a double uncaging experiment in animals when tetracaine could have been directly photouncaged more easily. The authors discuss the need to “(1) Decouple activation from payload structure and (2) provide spatiotemporal hierarchy and amplification... This separation between trigger and release enables precise spatial targeting and repeatable, on-demand drug activation, as demonstrated in our local anesthesia model.” I do not find either of these responses especially compelling. Coupling photochemistry to a kinetically slow seems disadvantaged relative to a simple photochemical uncaging, and more reagents need to be synthesized in the dual uncaging approach. The authors point to signal amplification, as they have described in Angew. Chem. for dihydrotetrazine/TCO chemistry, but it is not clear how that will apply here, and the aforementioned kinetic limitations would seem to counter any advantages for the present system.\n\nAs before, I find this to be an interesting study, but one that is ultimately more limited than current art in the field."}, "author_response": "Re: Revised article manuscript\nManuscript ID: NCOMMS-25-30990A\nTitle: \"Spatiotemporally Controlled Drug Release via a Click-Release System Utilizing\nMono-alkyl-hydroxylamine and Cyclooctyne Chemistry \"\nAuthor(s): Xiaowei Xu, Xidan Tong, Yangfei Shi, Xin Wang, Yanzhao Chen, Yuanan\nWang, Mingxin Cheng, Shuanglong Chen, Hao Hao, Yan Liang*, Weiwei Guo* and\nYueqin Zheng*\nWe sincerely appreciate the reviewers' valuable comments. We have addressed each\npoint in the detailed, point-by-point responses below and made corresponding revisions\nto the manuscript. The critiques are presented in regular black text, followed by our\nresponses in blue italics. Changes made in the revised manuscript are highlighted in\nyellow.\nReviewer #3 (Remarks to the Author):\nIn a prior evaluation of this manuscript, it was noted that the manuscript describes\ninteresting chemistry, but applications are limited by modest reaction rates that will\nhinder in vivo and cellular applications. It was further noted that some biological\nexperiments were performed “on cells” while others were conducted in vivo at high\nconcentrations without hard evidence for spatial control. In this revised manuscript, the\nauthors have partially addressed some of the concerns raised previously but some\nquestions remain.\nIn their response, they do provide a brief comment on the rate limitation of the rate\nconstant, but further opine that reaction kinetics alone do not dictate the utility of a\nbioorthogonal reaction, especially in in vivo contexts where factors such as stability,\nbiocompatibility, and controllability are equally, if not more, critical.”\nWhile it is of course true that rate constant is not the only important parameter, it is hard\nto argue that it is not a critical parameter, as bioorthogonal chemistry requiring\nmillimolar concentrations of reagents introduces a host of limitations (toxicity,\nbackground, washout in cellular experiments, clearance in animal experiments, etc).\nResponse: We thank the reviewer for their valuable feedback. It is indeed true that the\nreaction rate of our current system lags behind that of the established Tz/TCO system,\nwhich may limit its applicability in specific scenarios such as rapid labeling or under\nlow-concentration conditions.\nWe agree that improving the reaction rate will be highly meaningful, and we are\nalready exploring new strategies to accelerate this system. We believe that these\nongoing efforts will yield results suitable for future publications.\nThat said, even at its current rate, our system remains fully applicable to in vivo\nstudies. It is important to note that not all bioorthogonal reactions require extremely\nrapid kinetics; their suitability depends strongly on the specific application scenario.\nFor example, in the 2018 Nature Chemistry report from Wang’s group on a click-release\nstrategy, a relatively moderate second-order rate constant (K₂ ≈ 0.5 M⁻¹·s⁻¹) (Nature\nChem 10, 787–794 (2018)) was intentionally employed to enable target enrichment\nprior to drug release. In such cases, excessively fast reactions could even be detrimental,\nas they might trigger premature release before accumulation at the target site.\nTherefore, while we recognize the importance of kinetics, we emphasize that\nfactors such as spatiotemporal controllability, stability and biocompatibility are\nequally critical, particularly for in vivo applications. Our current work serves as a\nproof-of-concept demonstration of a novel click-release reaction that operates\ncontrollably within living systems. We believe that the unique combination of\ncontrollability and biocompatibility in our system will open new opportunities for\ndiverse biological and therapeutic applications. We fully understand and appreciate\nthe reviewer’s suggestion. Accordingly, we have explicitly emphasized in the revised\nmanuscript that an important future improvement of this reaction will be to further\nenhance its second-order kinetic rate constant. The corresponding revision has been\nmade in the Discussion section as follows:\nDespite these promising results, several challenges remain. First, the reaction kinetics of\nthe COT/hydroxylamine system are slower than those of the fast Tz/TCO system. Future\nefforts will focus on enhancing its reactivity to expand its applicability and practical utility.\nIn response to a comment that it was not shown that reactions are taking place\nintracellularly, the authors used a triphenylphosphonium-conjugated hydroxylamine\nderivative (compound 2j) to target the group to the mitochondria. While this experiment\nsuggests that the reaction occurs intracellularly, the authors should note that TPP\ntargeting is non-covalent and reversible, allowing for an extracellular reaction to be\npossible. In general, the use of covalent intracellular ligands (e.g., SnapTag or HaloTag)\nis necessary to prove intracellular reactivity. The images provided in the supporting\ninformation to support this experiment are of low resolution; higher-resolution images\nshould be provided.\nResponse: We thank the reviewer for this insightful comment. To more definitively\ndemonstrate that the reaction occurs intracellularly, we conducted an additional\nexperiment using 4T1 cells stably expressing HaloTag-PS. This system enables covalent\nintracellular labeling, thereby eliminating the possibility of extracellular reaction.\nSpecifically, the cells were first treated with compound 5d (50 μM) for 1 hour, which\ncovalently binds to HaloTag-PS and is retained within the cells, preventing diffusion\nout of the cellular environment. After extensive washing with PBS twice to remove any\nunbound extracellular compound, the cells were incubated with the trigger 2c (100 μM)\nfor an additional 30 minutes. Subsequent confocal imaging revealed a pronounced\nincrease in blue fluorescence, providing clear evidence that the click-release reaction\nproceeds efficiently inside living cells.\nWe have included these new results and the corresponding discussion in the revised\nmanuscript.\nFigure S19. Intracellular biocompatibility of the reaction between mono-alkyl-hydroxylamine and COT in\nlive 4T1 Cells. A) Structure of 5d and 2c. B) 4T1 cells were pre-treated with 50 μM of compound 5d for 1\nhour, followed by three washes with PBS to remove extracellular compound. Cells were then incubated\nwith 100 μM of compound 2c for 30 minutes before imaging. As a control, a separate group of 4T1 cells\nwas incubated solely with 50 μM of compound 5d for 1 hour before imaging. (Scale bar = 50 μm)\nTo further evaluate whether this click-release chemistry is sufficiently biocompatible to\nproceed within live cells, we synthesized COT-chloroalkane 5d (Figure S19A). 4T1 cells\nexpressing HaloTag-PS were incubated with 5d (50 μM) for 1 hour. After washing with\nPBS to remove extracellular 5d, the cells were treated with hydroxylamine 2c (100 μM)\nfor 30 minutes. A significant increase in blue fluorescence was observed (Figure S19B),\nconfirming not only the cell permeability of both reactants but, critically, their\nsuccessful intracellular reaction.\nIn the prior review, it was noted that there was insufficient evidence to demonstrate that\nthe click reaction uncages the prodrug at the tumor. In response, the authors carried out\nbiodistribution comparing the prodrug system (6a + 5c) versus free doxorubicin. The\nauthors describe the findings as “strong pharmacokinetic evidence that the\nbioorthogonal click–release reaction predominantly occurs at the tumor site”, but the\ndata require further interpretation. In Fig S21, slightly higher accumulation is actually\nobserved for the Dox control at 2 hours, and there is no statistical difference at 24 h. At\n4 and 8 hours, a statistical difference is observed (p value *), although the difference in\ntumor uptake is relatively modest. My view of these data is that Dox accumulation is\ncomparable for the uncaging system vs the control, with the benefit of lower\nbackground in the release system.\nResponse: We thank the reviewer for this insightful comment. We agree that the\ndifference in overall tumor DOX accumulation between the prodrug system (6a + 5c)\nand free DOX is modest, as correctly pointed out. The key pharmacokinetic advantage\nof our system lies in the markedly lower DOX levels observed in non-target tissues (e.g.,\nheart and liver) and in the plasma for the 6a + 5c group compared to free DOX. This\npattern demonstrates that the click-release system is primarily activated within the\ntumor microenvironment, thereby reducing off-target exposure and its associated toxic\nside effects. We have revised the relevant description in the manuscript accordingly to\nmore accurately reflect these findings.\nIn contrast to free DOX, the 6a + 5c group showed markedly reduced drug levels in non-\ntarget tissues (e.g., heart and liver) and in the plasma (Figures S22-24), while\nmaintaining comparable tumor concentrations across all time points (Figure S21). These\nresults provide compelling evidence that the click-release reaction is preferentially\ntriggered within the tumor microenvironment by elevated endogenous GSH, thereby\neffectively reducing off-target exposure.\nFinally, it was noted that photochemical uncaging experiments were successful;\nhowever, it is unclear why the authors pursued a double uncaging experiment in animals\nwhen tetracaine could have been directly photouncaged more easily. The authors\ndiscuss the need to “(1) Decouple activation from payload structure and (2) provide\nspatiotemporal hierarchy and amplification... This separation between trigger and\nrelease enables precise spatial targeting and repeatable, on-demand drug activation, as\ndemonstrated in our local anesthesia model.” I do not find either of these responses\nespecially compelling. Coupling photochemistry to a kinetically slow seems\ndisadvantaged relative to a simple photochemical uncaging, and more reagents need to\nbe synthesized in the dual uncaging approach. The authors point to signal amplification,\nas they have described in Angew. Chem. for dihydrotetrazine/TCO chemistry, but it is\nnot clear how that will apply here, and the aforementioned kinetic limitations would\nseem to counter any advantages for the present system.\nAs before, I find this to be an interesting study, but one that is ultimately more limited\nthan current art in the field.\nResponse: We thank the reviewer for the thoughtful comments and the opportunity to\nclarify our rationale.\nOur work primarily aims to introduce a new controllable click-release chemistry\nand demonstrate its in vivo feasibility through two representative models: an\nendogenous-triggered tumor therapy and an exogenous-triggered local anesthesia. We\nagree that if the goal were solely controllable anesthetic release, direct photouncaging\nwould suffice. However, the anesthesia study was designed as a proof-of-concept to\ndemonstrate that this chemistry can be externally actuated in living tissue, decoupling\nthe trigger from the payload structure and illustrating its generality across application\ncontexts.\nThe dual uncaging design enables platform modularity and precise control. By\nseparating the trigger from the payload, one can readily adapt the same chemistry to\ndifferent external stimuli (light, redox, enzymatic), while maintaining consistent\npharmacological properties. This architecture also allows hierarchical spatiotemporal\ncontrol, where the external stimulus defines the activation site and the click step governs\nthe release profile, enhancing selectivity and minimizing off-target effects.\nRegarding kinetics, we fully acknowledge their importance. The current rate is\nsufficient for controlled in vivo release, and we explicitly state in the revised\nDiscussion that future optimization to improve the second-order rate constant will be pursued.\nFinally, our system is complementary to the tetrazine/TCO pair rather than an\noptimization of it. Unlike tetrazines, which are unstable in highly reducing\nenvironments (e.g., high GSH) (J. Am. Chem. Soc. 2022, 144, 18, 8171–8177) (Angew.\nChem. Int. Ed. 2019, 58, 9043), our hydroxylamine-based chemistry remains stable and\neven benefits from GSH (J. Am. Chem. Soc. 2021, 143, 15, 5616–5621), making it\nadvantageous in reductive tumor microenvironments. These results collectively\nestablish the feasibility and versatility of this new click-release platform and highlight\ndirections for further development.\nThe corresponding revision has been made in the Discussion section as follows:\nCritically, our system serves as a complementary alternative to the established Tz/TCO\npair, founded on a fundamentally different mechanism and unique operational\nprofile. Unlike tetrazines, which suffer from instability in highly reducing environments such\nas those with high GSH concentrations56,57, our hydroxylamine-based chemistry exhibits\nstability and even benefits from GSH48. This inherent advantage positions our platform as\nhighly suitable for deployment in reductive tumor microenvironments."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have made revisions that mostly satisfy earlier critique, and I appreciate their response by carrying out additional experiments.\n\nIn response to an earlier critique, the authors have made a number of clarifying comments. They have included an additional experiment with HaloTag-PS, in order to demonstrate intracellular labeling with a covalently localized ligand. It would be beneficial if the brightfield and DIC images were also included. The intensity of fluorescent labeling is low in these images (Fig S19). Presumably this is due to the low fluorescence of the coumarin dye, and not a reflection of the uncaging chemistry? To demonstrate, it is recommended to run a positive control with direct conjugation of coumarin-halotag to demonstrate that labeling intensities are similar. The authors should also define the \"PS\" in HaloTag-PS and indicate where they obtained the plasmid, etc.\n\nWith respect to the critique about double uncaging with light, as opposed to simple, direct uncaging: the authors have responded by saying \"the anesthesia study was designed as a proof-of-concept to demonstrate that this chemistry can be externally actuated in living tissue, decoupling the trigger from the payload structure and illustrating its generality across application contexts. The dual uncaging design enables platform modularity and precise control…” , which is a lengthy way of saying that double photouncaging is a proof of principle experiment that could have been directly accomplished by simple uncaging. They pivot to mention tetrazine reduction, which is a fair point (and one that has been discussed in the literature, and directly controlled via photocatalysis; see for example JACS 2022, 144, 4, 1647; JACS 2025, 147, 23625). However, this cannot substitute for acknowledging the limitations of their own chemistry. It is recommended for the authors to make clear that their double photouncaging is a proof of principle that could have been more easily carried out by direct uncaging. In the future, I suggest that such proof-of-principle studies not be conducted in animals."}, "author_response": "Re: Revised article manuscript\nManuscript ID: NCOMMS-25-30990B\nTitle: \"Spatiotemporally Controlled Drug Release via a Click-Release System Utilizing\nMono-alkyl-hydroxylamine and Cyclooctyne Chemistry \"\nAuthor(s): Xiaowei Xu, Xidan Tong, Yangfei Shi, Xin Wang, Yanzhao Chen, Yuanan\nWang, Mingxin Cheng, Shuanglong Chen, Hao Hao, Yan Liang*, Weiwei Guo* and\nYueqin Zheng*\nWe sincerely appreciate the reviewers' valuable comments. We have addressed each\npoint in the detailed, point-by-point responses below and made corresponding revisions\nto the manuscript. The critiques are presented in regular black text, followed by our\nresponses in blue italics. Changes made in the revised manuscript are highlighted in\nyellow.\nReviewer #3 (Remarks to the Author):\nThe authors have made revisions that mostly satisfy earlier critique, and I appreciate\ntheir response by carrying out additional experiments.\nIn response to an earlier critique, the authors have made a number of clarifying\ncomments. They have included an additional experiment with HaloTag-PS, in order to\ndemonstrate intracellular labeling with a covalently localized ligand. It would be\nbeneficial if the brightfield and DIC images were also included. The intensity of\nfluorescent labeling is low in these images (Fig S19). Presumably this is due to the low\nfluorescence of the coumarin dye, and not a reflection of the uncaging chemistry? To\ndemonstrate, it is recommended to run a positive control with direct conjugation of\ncoumarin-halotag to demonstrate that labeling intensities are similar. The authors\nshould also define the \"PS\" in HaloTag-PS and indicate where they obtained the\nplasmid, etc.\nResponse: We thank the reviewer for these helpful suggestions. The aim of the cellular\nexperiments was to demonstrate intracellular uncaging and fluorescence activation;\ntherefore, imaging was focused on fluorescence signals, and brightfield or DIC images\nwere not acquired. Following the reviewer’s recommendation, we performed the\nsuggested positive control experiment using direct conjugation of a coumarin–\nHaloTag ligand. The resulting labeling intensity was comparable to that observed after\nuncaging, confirming that the signal level reflects the intrinsic fluorescence of the\ncoumarin dye rather than limitations of the uncaging chemistry. In addition, the\nmanuscript has been revised to define the “PS” in HaloTag-PS and to specify the\nsource of the plasmid..\nThese amendments are detailed in the Methods section:\nStable 4T1 cell lines expressing the HaloTag protein from the pHaloTag-PS plasmid\n(where PS denotes the HSV-TK poly(A) signal) were generated as follows. Briefly, 4T1\ncells were seeded at a density of 5.0 × 105 cells per well in 2 mL of RPMI 1640 medium\nsupplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1 mg/mL\nstreptomycin. Cells were cultured in a humidified incubator at 37 °C with 5% CO until they\nreached approximately 80% confluency. For transfection, each well was treated with a\ncomplex formed by combining 2.0 µg of the pHaloTag-PS plasmid (purchased from Beijing\nSolarbio Science & Technology Co., Ltd.) with 6 µL of Lipofectamine 3000 transfection\nreagent in 100 µL of Opti-MEM I reduced serum medium, according to the manufacturer’s\nprotocol. After 36 hours of transfection, cells were washed three times with 1 mL PBS.\nSubsequently, 400 µL of serum-free RPMI 1640 medium containing the respective test\ncompounds was added to each well for further treatment.\nThe detailed procedure for the confocal experiments using 5d and 2c is as follows: To\nunequivocally demonstrate the intracellular occurrence of the click-release reaction, 4T1\ncells stably expressing HaloTag-PS were employed. Briefly, the cells were incubated with\ncompound 5d (50 µM) for 1 hour to facilitate covalent labeling of the intracellular HaloTag-\nPS. Following two extensive washes with PBS to remove any unbound compound, the\ncells were treated with the trigger molecule 2c (100 µM) for 30 minutes. The successful\nintracellular reaction was subsequently visualized by confocal microscopy, as indicated by\na pronounced increase in blue fluorescence. For control experiments, a negative control\ngroup was treated with compound 5d alone (without 2c), and a positive control group was\ntreated with the covalent control compound 5d-1 under identical incubation conditions.\nSupplementary Fig. 9 Intracellular biocompatibility of the reaction between mono-alkyl-hydroxylamine\nand COT in live 4T1 cells. A) Structures of compounds 5d-1 and 5d. B) Intracellular light-up fluorescence\nactivation via the bioorthogonal reaction. C) 4T1 cells stably expressing HaloTag-PS were pre-treated with\n50 μM compound 5d for 1 h and washed. Cells were then incubated with or without 100 μM trigger 2c for\n30 min prior to imaging, serving as the experimental and negative control groups, respectively. A positive\ncontrol group was treated with 50 μM compound 5d-1 alone under the same conditions. Scale bar = 50 μm.\nImages are representative of three independent biological replicates.\nWith respect to the critique about double uncaging with light, as opposed to simple,\ndirect uncaging: the authors have responded by saying \"the anesthesia study was\ndesigned as a proof-of-concept to demonstrate that this chemistry can be externally\nactuated in living tissue, decoupling the trigger from the payload structure and\nillustrating its generality across application contexts. The dual uncaging design enables\nplatform modularity and precise control…” , which is a lengthy way of saying that\ndouble photouncaging is a proof of principle experiment that could have been directly\naccomplished by simple uncaging. They pivot to mention tetrazine reduction, which is\na fair point (and one that has been discussed in the literature, and directly controlled via\nphotocatalysis; see for example JACS 2022, 144, 4, 1647; JACS 2025, 147, 23625). However,\nthis cannot substitute for acknowledging the limitations of their own chemistry. It is\nrecommended for the authors to make clear that their double photouncaging is a proof of\nprinciple that could have been more easily carried out by direct uncaging. In the future,\nI suggest that such proof-of-principle studies not be conducted in animals.\nResponse: We thank the reviewer for their insightful comments. We have cited the\nrelevant literature on “tetrazine reduction” and clarified in the text that the\nphoto‑triggered anesthesia study serves as a proof‑of‑concept to illustrate the use of an\nexogenous trigger within our platform.\nThe corresponding revision has been made as follows:\nIn contrast to the GSH-activated system that relies on endogenous stimuli, this section\nexplores the use of an exogenous trigger to demonstrate the conceptual versatility of our\nclick–release strategy. As a proof-of-concept, we designed a photo-triggered prodrug\nsystem for local anesthesia. We note that the anesthetic effect could, in principle, be\nachieved through more straightforward direct uncaging approaches; however, the present\ndesign serves to illustrate the modularity of the platform and the decoupling of the activation\ntrigger from the payload structure. In this system, the conventional local anesthetic\ntetracaine was conjugated to COT via a carbamate bond to mask its anesthetic activity,\nyielding the prodrug 7a."}]

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