Insights into the activation patterns of 1,2-dithiolane unit in biofunctional molecules

Domain: biology
Published: 2026-03-13
Editorial outcome: minor_revision

Abstract
The cyclic five-membered disulfide 1,2-dithiolane, known for its moderate reactivity and ease of preparation, holds significant promise as a recognition unit in probe design and drug development. However, potential limitations in activation specificity—most notably the diminished selectivity toward thioredoxin reductase (TrxR) caused by nonspecific reactions with abundant low-molecular-weight (LMW) thiols such as glutathione (GSH)—raise concerns about its reliability in biological settings. Here we systematically evaluate the activation behavior of 1,2-dithiolane by synthesizing a panel of prodrugs and fluorescent probes incorporating either amine- or hydroxyl-linked cargoes. Our results reveal that TrxR-mediated selective activation of the 1,2-dithiolane unit is achievable when the cargo is incorporated within an amine-based framework. In contrast, hydroxyl-linked conjugates undergo rapid cleavage by physiological GSH levels, resulting in a pronounced loss of TrxR selectivity. Generally, the recognition site 1,2-dithiolane, the linker unit and the leaving group in a cargo coordinate to determine the selectivity activated by TrxR. Overall, this study resolves ambiguities in previous reports, reconciles conflicting observations, and provides new conceptual guidance for the use of the 1,2-dithiolane scaffold in the design of biofunctional molecules.

Introduction
Preservation of redox homeostasis represents an essential biological process governing a myriad of physiological functions.1, 2, 3, 4 The thioredoxin (Trx) system plays a key role in maintaining cellular redox homeostasis and represents a major thiol-dependent antioxidant system.5 Within this system, thioredoxin reductase (TrxR) serves as the upstream redox enzyme that couples NADPH-derived reducing power to Trx, thereby enabling dithiol–disulfide exchange reactions in downstream effector proteins.5, 6 Notably, mammalian TrxRs comprise a unique class of selenoproteins characterized by the presence of a catalytically essential selenocysteine (Sec) residue within their C-terminal redox-active motif. Emerging evidence implicates the dysregulation of TrxR in the pathogenesis of multiple disease states, particularly autoimmune disorders and cancer, positioning this enzymatic system as a highly promising therapeutic target for pharmacological intervention.6, 7, 8, 9, 10

Given the essential role of TrxR in biological processes, advancing techniques to monitor and respond to its dynamic behavior is key to deepening our understanding of cellular physiology. Recent advances have yielded diverse activity-based fluorescent probes for TrxR imaging in cells and in vivo.11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Three principal triggering motifs are incorporated into these molecules: (i) a linear diselenide unit,16, 19 (ii) a 1,2-thiaselenane ring,17, 20 and (iii) a 1,2-dithiolane moiety11, 12, 13, 14, 15, 18 (Fig. 1A). Although linear disulfides are used as TrxR-responsive motifs, in vitro assays conducted under subphysiological GSH concentrations (≤10 μM vs 1–10 mM intracellularly) can obscure TrxR-specific activation by competing thiols, compromising their ability to model native enzyme behavior.21, 22 We previously developed five 1,2-dithiolane-based fluorescent probes (the TRFS series) for cellular and in vivo imaging, as well as for rapid screening of TrxR inhibitors (Fig. 1B).11, 12, 13, 14, 15 Furthermore, motivated by the well-documented upregulation of TrxR in tumors,6, 8, 9 we reported two prodrugs specifically designed to target TrxR for cancer therapy (Fig. 1C).23, 24 All our molecules are constructed with amine-based cargoes (Fig. 1B, C), displaying favorable selectivity for TrxR (nM concentration) over GSH (mM concentration). Activation paradigms for 1,2-dithiolane-based molecules (e.g., TRFS-green) involve a TrxR-initiated cascade reaction sequence comprising disulfide reduction, intramolecular cyclization, and cargo release (Fig. 1D).11 In contrast, the probe Fast-TRFS variant achieves direct fluorescence uncaging through a simplified disulfide cleavage mechanism (Fig. 1E).15 Some fluorescent probes detect TrxR through covalent binding.25, 26 However, these compounds substantially inhibit the enzyme, potentially leading to unintended biological consequences.26, 27 Moreover, considering the low nanomolar abundance of TrxR in cells, covalent probe strategies that rely on stoichiometric enzyme engagement can increase the risk of false-positive or misleading signals. Our evaluations demonstrate a clear correlation between the activation of biomolecular tools based on the 1,2-dithiolane framework and TrxR activity, with cargoes consisting mainly of amine-based drugs or fluorophores. However, recent structural analyses reveal that conjugation of 1,2-dithiolane to a fluorogenic phenolic moiety induces paradoxical activation profiles, wherein promiscuous low-molecular-weight (LMW) thiol-mediated triggering overrides enzymatic specificity.28 This work calls into question our previous conclusion that TRFS-green is selective for TrxR and concludes based on the probe examined in their study that the apparent non-selective reactivity of 1,2-dithiolane–based molecules stems from the intrinsic chemistry of the ring rather than from variations in the conjugated cargo or linker. To address this inconsistency, we focus on two key mechanistic questions: (i) whether LMW thiols (e.g., GSH) can mediate disulfide bond reduction within 1,2-dithiolane scaffolds, and (ii) the structural determinants governing cargo release versus retention following LMW thiol–mediated cyclic disulfide cleavage. Accordingly, systematic structure–activity relationship (SAR) studies are essential for elucidating the activation thermodynamics and selectivity constraints of 1,2-dithiolane systems bearing distinct cargoes, which support that the recognition site 1,2-dithiolane, the linker unit and the leaving group in a cargo coordinate to determine the selectivity triggered by TrxR (Fig. 2).

In this study, we dissect the SAR underlying cargo-dependent activation of 1,2-dithiolane systems. Using hydroxyl-containing drugs—including (+)-camptothecin (CPT), SN38, gemcitabine (Gem), and paclitaxel (PTX)—we conducted in vitro kinetic assays to assess the reactivity of corresponding prodrugs (S-CPT, S1-SN38, S2-SN38, S1-Gem, S-PTX; Fig. 3) toward GSH and TrxR. Notably, carbonate (−O−C(O)−O−)-linked conjugates were highly susceptible to non-specific activation by physiological GSH, abolishing TrxR selectivity, highlighting a limitation of linker chemistry rather than the cyclic disulfide trigger. Heterogeneous activation profiles were observed across TrxR-treated prodrugs, exemplified by S-PTX’s aberrant stability. We further synthesized prodrugs and probes with amine-containing cargoes (S1-DOX, S2-DOX, S-Cou, S1-Cou; Fig. 3) to examine TrxR selectivity. Our findings demonstrate a clear difference in reactivity between molecules featuring a carbamate (−NH−C(O)−O−) linker and those with a −O−C(O)−O− linker. Cellular evaluation of TRFS-green confirmed its reliability as a TrxR-responsive fluorescent probe, and comparative analysis revealed a clear dichotomy: −NH−C(O)−O− linkers confer TrxR specificity, whereas −O−C(O)−O− linkers lead to promiscuous activation. Taken together, these findings resolve prior inconsistencies in 1,2-dithiolane tools and establish design principles for enhancing TrxR specificity and functionality. Key results are summarized in Fig. 2, with details in the Results section.

Results
2.1. Linker- and cargo-dependent design of 1,2-dithiolane prodrugs and probes

In cellular redox environments, reduction-activated “trigger–cargo” systems have been designed to release a variety of cargoes, including fluorophores bearing hydroxyl or amine functionalities, as well as therapeutic compounds.29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 The previously reported TRFS probes employed aniline-based fluorophores as reporter cargoes (Fig. 1B). In prodrug systems, critical functional groups within drug scaffolds are well defined, typically involving a fixed molecular scaffold and a specific chemical bond linking the cargo to the triggering unit. In our earlier investigations of two prodrugs incorporating the 1,2-dithiolane unit, −NH−C(O)−O− linkages were used for conjugation (Fig. 1C). This design choice was dictated by the requirement that the amine functionality remain accessible to ensure intracellular biological activity, despite the presence of additional functional groups within the drug scaffold.40, 41, 42 Conversely, for drugs where the hydroxyl group serves as the critical active site, using a −O−C(O)−O− linker is essential to fully explore the properties of 1,2-dithiolane derivatives. However, this strategy was not extensively explored in our prior work. To explore the influence of hydroxyl-based leaving groups in 1,2-dithiolane-based prodrugs, we synthesized five prodrugs—S-CPT, S1-SN38, S2-SN8, S1-Gem, and S-PTX (Fig. 3). Each features a uniform recognition site structure, the 1,2-dithiolane unit, with varying alcohol types as leaving groups: primary (Gem), secondary (PTX), and tertiary (CPT and SN38). The distinction between S1-SN38 and S2-SN38 lies in the specific hydroxyl group protected by the recognition unit: S1-SN38 masks the tertiary hydroxyl group at the C20 position of SN38, whereas S2-SN38 masks the phenolic hydroxyl group at the C10 position. S1-SN38, previously reported by us as a control compound, was re-evaluated in this study.43 To minimize the risk of false-positive signals arising from thiol-induced attack on −O−C(O)−O− linkers, three corresponding control molecules—PC-CPT, C-SN38, and C-PTX—were also synthesized. To further elucidate reactivity differences between amine- and hydroxyl-based prodrug designs, doxorubicin (DOX)-based prodrugs, S1-DOX and S2-DOX, were prepared to assess TrxR selectivity. S1-DOX features a 1,2-dithiolane unit directly conjugated to the aliphatic amine of DOX via a −NH−C(O)−O− linkage. In contrast, S2-DOX incorporates a self-immolative spacer between the recognition unit and DOX, a design commonly employed to facilitate intramolecular reactions, including electron-transfer cascades within aromatic systems.44, 45 Replacement of sulfur atoms with selenium in S1-DOX and S2-DOX afforded Se1-DOX and Se2-DOX, respectively, enabling further insight into rate-limiting steps in the intramolecular cyclization process. An inactive control compound, C-DOX, corresponding to S1-DOX, was synthesized and employed in cytotoxicity assays. In addition, Se-NBL, a selenium-containing analogue of TRFS-red,14 was synthesized. Finally, the probes S-Cou and S1-Cou were prepared to evaluate their TrxR selectivity and to determine whether 1,2-dithiolane-based probes derived from aniline exhibit reactivity profiles comparable to those of the TRFS series.

The synthesis of 1,2-dithiolan-4-ol, 1,2-diselenolan-4-ol, and derivative 1 was performed according to our previously reported procedures,15, 24 whereas intermediates 2 and 3 were prepared via analogous methodologies. Utilizing CPT as the starting material, S-CPT and C-CPT were synthesized with moderate isolated yields of 40% and 50%, respectively. S1-SN38 was obtained from SN38 through a multistep synthetic sequence involving regioselective functionalization, disulfide coupling, and purification, achieving an overall yield of 22%. S2-SN38 and C-SN38 were generated via direct nucleophilic substitution of SN-38 with intermediates 1 and 2, yielding 20% and 30%, respectively. The preparation of S1-Gem, S-PTX, C-PTX, S1-DOX, S2-DOX, Se1-DOX, Se2-DOX, C-DOX, and Se-NBL followed synthetic routes analogous to S2-SN38, with S1-Gem exhibiting a diminished yield of 8% and other compounds demonstrating yields spanning 10–50%. S-Cou was synthesized using a strategy paralleling the TRFS-green methodology,11 while S1-Cou was prepared by adapting the Fast-TRFS protocol.15 Fig. 3 outlines the specific steps, and detailed synthesis procedures and characterization of all final products are provided in the Supplementary Information.

2.2. Hydroxyl cargoes promote GSH-mediated activation of 1,2-dithiolane prodrugs

CPT and its derivative SN38 exhibit intrinsic fluorescence.36, 46, 47, 48, 49 We observed significant changes in the fluorescence properties of S-CPT, S1-SN38, and S2-SN38 compared to their parent drugs (i.e., CPT and SN38). As shown in Fig. 4A, upon reaction with GSH (1 mM), the fluorescence intensity of S-CPT (10 μM) increased by 4.7-fold within 10 minutes. A comparable outcome was also observed when TrxR (50 nM) was used as the bioactive reducing agent, resulting in a 4.3-fold increase in fluorescence intensity (Fig. 4B). We subsequently conducted fluorescence assays with S1-SN38 (10 M), which also displayed an “OFF to ON” fluorescence behavior. Upon reaction with GSH (1 mM) or TrxR (50 nM) (Fig. 4C, D), the fluorescence intensity increased by 3.6-fold and 5.2-fold, respectively. Upon incubation with GSH (1 mM) or TrxR (50 nM), the emission wavelength of S2-SN38 (10 M) shifted from 427 nm to 545 nm within 10 minutes, with a near-complete shift observed (Fig. 4E, F). Collectively, preliminary fluorescence data indicate that the prodrug molecules (i.e., S-CPT, S1-SN38, and S2-SN38) exhibit a lack of TrxR selectivity, regardless of whether the tertiary hydroxyl group from CPT, the hydroxyl group from SN38, or the phenolic hydroxyl group is involved. Fig. 4G illustrates the proposed activation mechanism of hydroxyl-linked 1,2-dithiolane molecules, exemplified by S-CPT, which was later confirmed by HPLC analyses. Compared to linear disulfide bonds, cyclic disulfide linkages exhibit a more efficient quenching effect on the fluorescence of CPT.36 This property has important implications for the design of small-molecule fluorescent probes based on cyclic disulfides, as it allows for the development of sensors with improved signal-to-noise ratios.

Prior to evaluating prodrug activation via HPLC, we assessed the stability of the prodrug molecules under test conditions. The results demonstrated that all prodrugs exhibited substantial stability in PBS buffer (pH = 7.4) at 37 ℃ (Supplementary Fig. 1). The stability of 1,2-dithiolane-class prodrugs was further assessed in serum-containing culture medium. All prodrug candidates remained well‑stabilized under this condition except for S2–SN38, which exhibited pronounced instability (Supplementary Fig. 2). S2–SN38 underwent rapid degradation in serum-containing medium, with approximately 90% loss within ~20 minutes; this rapid serum‑mediated decomposition does not affect the conclusions drawn from experiments conducted in PBS buffer but indicates that S2–SN38 is unsuitable for applications in biological systems. As shown in Fig. 5A–5D, HPLC analysis reveals that, upon reaction with GSH (1 mM), the percentage of cargoes released from S-CPT (10 µM) was 46% within 10 minutes, while S1-SN38 (10 µM) released 17%, S2-SN38 (10 µM) released 90%, S1-Gem (50 µM) released 70%, and S-PTX (20 µM) released 36%. Upon reaction with TrxR (50 nM), the percentage of cargo release was 48% for S-CPT (10 µM), 10% for S1-SN38 (10 µM), 55% for S2-SN38 (10 µM), 34% for S1-Gem (50 µM), and 0% for S-PTX (20 µM). Interestingly, the release kinetics of CPT from its prodrug, S-CPT, upon reaction with GSH, demonstrates that cyclic disulfides act as much more efficient triggering units than linear disulfides. Specifically, when the linear disulfide-based prodrug CPT-S is exposed to 100 equivalents of GSH, it takes over 60 minutes to achieve ~50% of the maximum drug release.36 In contrast, the corresponding cyclic disulfide prodrug, S-CPT, reaches the same release level in approximately 10 minutes. This striking difference in release dynamics highlights the enhanced efficiency of cyclic disulfides in facilitating rapid prodrug activation. Such an acceleration of drug release could be particularly beneficial for the development of prodrug strategies aimed at achieving fast, controlled activation in therapeutic applications. The control molecules (i.e., C-CPT, C-SN38, and C-PTX) did not release the parent drugs upon incubation with GSH or TrxR (Supplementary Fig. 4). This observation indicates that the cargo release is attributed to thiol-induced intramolecular cyclization through disulfide bond reduction by GSH or TrxR, rather than a false-positive result due to nucleophilic attack on the −O−C(O)−O− linkage by these reagents. Recent studies employing the –O–C(O)–O– motif for the selective recognition of endogenous redox species, while resisting interference from GSH, lend mechanistic support to the present work.50, 51 Specifically, GSH-triggered activation of –O–C(O)–O–-based 1,2-dithiolane prodrugs is primarily driven by disulfide bond cleavage and subsequent intramolecular cyclization. Interestingly, GSH (1 mM) can activate S-PTX (20 µM), whereas TrxR (50 nM) cannot. Notably, the comparison between 1 mM GSH and 50 nM TrxR is not intended to imply equivalence, but rather to reflect their physiologically relevant abundance. These conditions were deliberately chosen to recapitulate this intrinsic biological disparity, rather than to achieve stoichiometric comparability.

We then present the residual amounts of various prodrug molecules, which further elucidate their interactions with GSH or TrxR. Mechanistically, the activation of prodrugs involves a cascade reaction, where the interaction with reductants does not necessarily correlate with cargo release. As shown in Fig. 5E–5H, HPLC analysis indicated that, following reaction with GSH (1 mM), the remaining percentages at 10 minutes were 33% for S-CPT (10 µM), 68% for S1-SN38 (10 µM), 0% for S2-SN38 (10 µM), and 0% for S1-Gem (50 µM). Upon reacting with TrxR (50 nM), the remaining percentages for S-CPT (10 µM), S1-SN38 (10 µM), S2-SN38 (10 µM), and S1-Gem (50 µM) were recorded as 40%, 70%, 16%, and 30%, respectively. These results demonstrate that the molecules (i.e., S-CPT, S1-SN38, S2-SN38, and S1-Gem) are capable of responding to both GSH and TrxR; however, an analysis of the cargo release data reveals that the reduction of prodrugs does not fully translate into the corresponding cargoes, as evidenced by the fact that the percentage decrease in prodrugs exceeds the percentage of cargo released. The liquid chromatography PDA absorption peaks of the control compounds C-CPT, C-SN38, and C-PTX remained unchanged (Supplementary Fig. 4). Additionally, the results demonstrated that GSH (1 mM) rapidly degrades the disulfide bond in S-PTX (20 µM), resulting in 52% of S-PTX (20 µM) remaining after 10 minutes of reaction. In the TrxR (50 nM) reaction system, 99% of S-PTX (20 µM) remained after 10 minutes. Subsequently, the concentrations of each reactant were adjusted to further validate the accuracy of the observed result and the general behavior of S-PTX. Comparable results were obtained: after a 30-minute reaction with GSH (5 mM), S-PTX (10 M) was nearly fully degraded, whereas in the presence of TrxR (75 nM) for 1 hour, 90% of S-PTX (10 M) remained intact (Supplementary Fig. 5).

To elucidate the molecular basis of TrxR-mediated activation, we conducted computer-aided docking simulations for four prodrugs—S-CPT, C-CPT, S-PTX, and C-PTX. All compounds yielded favorable docking scores (< −5.0 kcal/mol), suggesting energetically feasible binding interactions (Supplementary Fig. 6). Notably, SN-CPT adopted a binding pose positioning its 1,2-dithiolane ring toward the C-terminal Sec residue, with sulfur atoms at 3.6 Å and 3.9 Å from the selenium atom—distances consistent with potential Se–S interactions. In contrast, C-CPT, although its cyclopentane CH₂ carbons were similarly positioned (3.9 Å and 4.5 Å), lacks the reactive disulfide motif and therefore cannot undergo redox activation. Although S-PTX localized near the surface-exposed catalytic site adjacent to the C-terminal Sec residue, its 1,2-dithiolane sulfur atoms were positioned 9.5 Å and 9.8 Å away from the Sec selenium atom—too distant for efficient Se–S interaction—thus explaining its poor TrxR-mediated activation efficiency (for C-PTX, the cyclopentane CH₂ carbons were 7.4 Å and 7.5 Å from Sec). These findings underscore that TrxR-mediated prodrug activation depends not only on binding affinity but also critically on the precise spatial alignment of the reactive disulfide moiety relative to the Sec active site. Collectively, our docking analysis provides mechanistic insight into the differential activation profiles of these prodrugs and emphasizes the dual importance of molecular recognition and geometric complementarity in the rational design of TrxR-selective therapeutics.

2.3. Amino cargoes bias activation toward TrxR in 1,2-dithiolane prodrugs

To clarify the impact of leaving groups in 1,2-dithiolane prodrugs and probes, we evaluated the reactivity of −NH−C(O)−O−-linked molecules carrying amine-based drugs or fluorophores toward LMW thiols and TrxR. Exploiting DOX’s intrinsic fluorescence and aliphatic amine, we designed a fluorogenic “trigger-cargo” system using 1,2-dithiolane as the trigger and DOX as the cargo. The fluorescence of DOX is significantly quenched by the incorporated triggering unit (Supplementary Fig. 7). As shown in Fig. 6, S1-DOX (10 μM), S2-DOX (10 μM), Se1-DOX (10 μM), and Se2-DOX (10 μM) all exhibit fluorescence release upon reduction with Dithiothreitol (DTT) (1 mM). The increases in fluorescence were 8.8-fold (12 h), 17-fold (12 h), 7.1-fold (3 h), and 47-fold (5 h), respectively. It is of note that the fluorescence increase is assessed at different time points, reflecting the distinct plateau phases of fluorescence release for each molecule. DTT, a potent disulfide-reducing agent, provides a theoretical maximum for comparison with actual experimental results. In the presence of TrxR (50 nM), the fluorescence enhancements observed for each molecule were as follows: S1-DOX (10 μM), 6.5-fold increase; S2-DOX (10 μM), 4.4-fold increase; Se1-DOX (10 μM), 1.4-fold increase; and Se2-DOX (10 μM), 1.4-fold increase. Following a 24-hour incubation with GSH (5 mM), S1-DOX (10 M) showed no change, whereas S2-DOX (10 M) showed an increase of 1.6-fold, Se1-DOX (10 M) by 1.3-fold, and Se2-DOX (10 M) by 2.4-fold. Consequently, fluorescence data revealed that both S1-DOX and S2-DOX effectively circumvent GSH-induced interference, even under high GSH conditions (5 mM, corresponding to a prodrug-to-GSH molar ratio of 1:500). The fluorescence release of S1-DOX in the presence of TrxR was comparable to that observed with DTT (8.8-fold for DTT vs. 6.5-fold for TrxR), indicating that DOX can be released through the reducing action of TrxR. While S2-DOX also exhibits enzyme selectivity for TrxR, its release efficiency is markedly lower than that with DTT (17-fold for DTT vs. 4.4-fold for TrxR), emphasizing the differences among various leaving groups. Moreover, both control molecules, Se1-DOX and Se2-DOX, demonstrated resilience against degradation by GSH and TrxR, highlighting the intricate nature of the cascade reaction mechanisms involved. Se-NBL, as a selenium-containing analogue of TRFS-red, exhibits similar characteristics (Supplementary Fig. 8).14 Cytotoxicity assays of the DOX-based prodrugs revealed that free DOX exhibited the highest anticancer potency among the three tested tumor cell lines (A549, HeLa, and HepG2). S1-DOX displayed activity ranking second to DOX, while S2-DOX, Se1-DOX, Se2-DOX, and C-DOX showed significantly reduced activity compared to S1-DOX (Supplementary Fig. 9). Remarkably, Se2-DOX exhibited negligible cytotoxicity under all tested conditions, indicating poor activation of the prodrug even within the complex redox microenvironment of living cells. These findings highlight a fundamental limitation of five-membered cyclic diselenide-based delivery systems: despite their higher susceptibility to mild reductive stimuli compared to disulfides, the dominant intramolecular cyclization following bond cleavage markedly hampers efficient cargo release. Notably, the dynamic equilibrium between bond cleavage and molecular reconstitution may also apply to analogous cyclic disulfide systems, offering mechanistic insights that could inform the rational design of next-generation redox-responsive prodrugs and probes.

Building on the observation that S1-DOX exhibits remarkable cytotoxicity in tumor cells and undergoes efficient fluorescence release upon activation by TrxR, we conducted an in-depth investigation into its activation properties. As shown in Fig. 7A, a dose-dependent response to TrxR was observed. S1-DOX (10 μM) exhibited resistance to interference by GSH (5 mM) and did not respond to other LMW thiols, such as cysteine (Cys, 5 mM), or to bioactive substances like NADPH (200 μM) (Fig. 7B, C). HPLC analysis showed that approximately 30% of DOX was released over a 24-hour period in the presence of TrxR (Supplementary Fig. 10). To achieve optimal experimental validation, we implemented CRISPR/Cas9-mediated genome editing to generate a stable TrxR1-knockout (KO) model in human colorectal carcinoma HCT-116 cells.52 Subsequent western blot analysis demonstrated comparable TrxR1 expression profiles between parental HCT-116 cells and their non-targeting control (NC) counterpart (HCT116-NC), while exhibiting complete absence of TrxR1 protein expression in the engineered HCT116-TrxR1-KO clone (Supplementary Fig. 11). Cytotoxicity assays revealed that S1-DOX exerted comparable inhibitory effects across three cell lines (HCT116, HCT116-NC, and HCT116-TrxR1-KO), with IC₅₀ values consistently around 0.6 M (Fig. 7D). This observation indicates that its cytotoxic activity is not solely dependent on TrxR-mediated activation. As noted, carbamate-linked 1,2-dithiolane derivatives can be activated not only by TrxR but also, to a limited extent, by an intact GSH system, as exemplified by TRFS-green.28 Given the relatively low TrxR-mediated release efficiency of S1-DOX (~30% at 24 h, versus ~80% within 8 h for other 1,2-dithiolane-based prodrugs23, 24), even in cells with intact TrxR1, this modest enzymatic activation may still suffice to accumulate cytotoxic levels of the active drug, with additional reduction pathways potentially contributing to overall toxicity. In TrxR1-deficient cells, intrinsic non-TrxR reductive systems, such as the GSH pathway, can be upregulated compensatorily,53, 54 enhancing S1-DOX reduction. Over prolonged incubation (24 h), these alternative pathways may generate sufficient active compound to induce substantial cytotoxicity. As a result, the TrxR-dependent component of prodrug activation is effectively masked in conventional cytotoxicity assays.

Molecular docking simulations revealed that both S1-DOX and Se1-DOX exhibit favorable binding affinities toward TrxR, with calculated binding energies of −5.981 and −6.007 kcal/mol, respectively (Supplementary Fig. 12). The selenium atom of the Sec residue is positioned 3.7 Å and 4.5 Å from the sulfur atoms of S1-DOX, and 3.8 Å and 4.0 Å from the selenium atoms of Se1-DOX, suggesting spatial proximity conducive to Se–S or Se–Se interactions at the Sec-containing redox center. These results indicate that both prodrugs can stably engage TrxR, enabling potential redox exchange between their disulfide/diselenide motifs and the enzyme’s active selenol–thiol pair. However, only S1-DOX undergoes efficient drug release, whereas Se1-DOX remains largely inert. This divergence likely arises from the higher nucleophilicity of the selenol intermediate derived from Se1-DOX, which favors reoxidation to a stable diselenide rather than proceeding through intramolecular cyclization required for payload liberation (as elaborated in the Discussion section).

2.4. Aniline scaffolds dictate structure-dependent TrxR-selective activation of 1,2-dithiolane fluorescent probes

The aniline-based fluorescent probe S-Cou was used to evaluate its selectivity toward TrxR. As shown in Fig. 8B–8D, S-Cou exhibited a robust 30-fold fluorescence increase after a 60 min reaction with DTT (1 mM). When S-Cou (10 μM) was incubated with TrxR (50 nM) for 3 h, an 18-fold fluorescence enhancement was observed. By contrast, incubation with GSH (5 mM) for 3 h resulted in only a low 4.5-fold increase in fluorescence. This TrxR selectivity is further substantiated by the minimal interference observed from other bioactive molecules. For instance, Cys (5 mM) induced only a modest 3-fold fluorescence increase, while NADPH (200 μM) led to a mere 2-fold enhancement in fluorescence (Supplementary Fig. 13). In addition, we investigated the resistance of TRFS-green to activation by high concentrations of GSH, as prior studies have indicated a 1:100 reaction ratio between TRFS-green and GSH.11 Our results revealed that when TRFS-green (10 M) was incubated with TrxR (50 nM) for 3 hours, the fluorescence intensity increased by 7-fold. In contrast, exposure of TRFS-green (10 M) to GSH (5 mM) under the same conditions led to only a 1.2-fold increase in fluorescence intensity (Supplementary Fig. 14). These observations underscore the pronounced selectivity of TRFS-green for TrxR over GSH. To further explore the properties of a probe incorporating a urea linker analogous to that in Fast-TRFS, we synthesized S1-Cou and assessed its reactivity. Upon exposure to DTT (1 mM), S1-Cou (10 M) demonstrated a strikingly rapid fluorescence release, exhibiting a 37-fold enhancement within just 5 minutes, indicative of an efficient and fast-acting disulfide cleavage mechanism (Fig. 8F). In stark contrast, when S1-Cou (10 μM) was incubated with either TrxR (50 nM) or GSH (5 mM), the fluorescence response was minimal, showing only a 2.1-fold increase with TrxR and no detectable change with GSH (Fig. 8G, H). This stark difference suggests that the disulfide bond in S1-Cou remains highly resistant to reduction by these bioactive cellular reducing agents. Notably, the serendipitous discovery of Fast-TRFS catalyzed our ongoing efforts to design long-wavelength (> 600 nm) probes operating via similar fluorescence mechanisms for TrxR detection. However, despite significant effort, extending this mechanism to longer-wavelength emitting probes has proved challenging, and achieving this goal remains elusive.

2.5. TrxR drives fluorescence release of structurally distinct S-Cou and TRFS-green in cells

We next assessed the specificity of two 1,2-dithiolane-based fluorescent probes, S-Cou and TRFS-green, for TrxR activation in cellular environments. Of note, the TrxR-selective fluorescence imaging capacity of TRFS-green has been questioned,28 prompting a comprehensive re-evaluation of its cellular behavior in this study. As depicted in Fig. 9A, the fluorescence signal of S-Cou remained exceptionally weak after 4 hours of probe incubation, attributed to the misalignment between the fluorescence emission wavelength of S-Cou and the detection range of the confocal microscope. To corroborate these findings, we conducted parallel validation studies employing another fluorescence microscopy in conjunction with AFC-SS—a structurally analogous, well-characterized thiol-activated probe.55 Fluorescence image analysis revealed robust intracellular fluorescence generation by S-Cou (Supplementary Fig. 15), confirming its metabolic activation capability. Flow cytometric analysis indicated an approximately 30% decrease in S-Cou fluorescence intensity in HCT116-TrxR1-KO cells compared with HCT116-NC cells (Fig. 9B; Supplementary Fig. 16), indicating that the intracellular activation of S-Cou is closely associated with TrxR activity. Confocal microscopy showed marked attenuation of TRFS-green fluorescence in HCT116-TrxR1-KO cells (Fig. 9C), with flow cytometric analysis quantifying an approximately 80% diminution in signal intensity relative to HCT116-NC cells (Fig. 9D). To rigorously establish TrxR selectivity of TRFS-green, we employed TRi-1—a highly selective TrxR1 inhibitor.56 Dose-response studies showed concentration-dependent suppression of TRFS-green fluorescence (Fig. 9E, F), with 2 μM TRi-1 producing an approximately 30% reduction in signal intensity. By comparison, the same concentration of the reference TrxR inhibitor auranofin (AF) resulted in an approximately 45% decrease in fluorescence (Fig. 9F). A representative gating strategy used for all flow cytometry experiments in this study is shown in Supplementary Fig. 17. The concentration of TRi-1 was selected based on its inhibitory effect on TrxR activity in HCT116 cells (Supplementary Fig. 18).

Therefore, these systematic variations in chemical and biological reductants, along with comprehensive time-course and endpoint analyses, highlight a crucial observation: under our experimental conditions, 1,2-dithiolane-based prodrugs and probes—particularly those with amine-based drugs or fluorophores as cargoes—demonstrate a distinct selectivity for TrxR. Even at elevated concentrations (i.e., 5 mM), LMW thiols (e.g., GSH and Cys) did not activate these biofunctional agents. Cellular fluorescence imaging and flow cytometric analysis further strengthen the foundation of our conclusions.

Methods
4.1. Chemical synthesis and drug release
Full details of chemical synthesis, drug release experiments, and compound characterization are described in the Supplementary Information.

4.2. Materials and instrumentations
All chemicals and reagents were used as received from commercial suppliers unless otherwise specified. Solvents were purified and dried using standard procedures prior to use. DMSO, Dulbecco’s modified Eagle’s medium (DMEM), GSH, NADPH, Cys, DTT, and GSSG were purchased from Sigma-Aldrich (St. Louis, USA). S3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin, and streptomycin were obtained from Sangon (Shanghai, China). TRi-1 was synthesized according to the method described in the published study.56 Two independent batches of recombinant rat TrxR1 were used in this study. The first batch was kindly provided by Dr. Jianqiang Xu (Dalian University of Technology, China), and the second batch was provided by Prof. Chao Zhang (Wannan Medical College, China). The following antibodies were used: TrxR1 antibody (Abcam, catalog no. ab124954, dilution 1:2000) and GAPDH antibody (Abcam, catalog no. ab8245, dilution 1:5000).

Mass spectrometry (MS) was performed on a Bruker Daltonics Esquire 6000 or a Shimadzu LCMS-2020 mass spectrometer. Flow cytometry was performed using an ACEA Biosciences NovoCyte flow cytometer and a BD FACSVerse flow cytometer. Confocal fluorescence imaging was carried out on a Leica SP8 confocal microscope. HPLC analyses were conducted using a Shimadzu system equipped with an SPD-M20A photodiode array detector. Fluorescence spectroscopy measurements were carried out using a Thermo Scientific Lumina fluorescence spectrometer. The acquired fluorescence spectra were analyzed using Thermo Scientific Lumina software (version 4.3, build 1). Microplate-based assays were conducted on a BioTek Cytation 5 multimode microplate reader.

4.3. Cell viability assay
The cytotoxicity of DOX HCl, S1-DOX, S2-DOX, Se1-DOX, Se2-DOX, and C-DOX was assessed in A549, HepG2, and HeLa cells using the MTT assay. For this, 8 × 10³ cells were seeded into 96-well plates and incubated for 12 hours to allow attachment. The cells were then treated with varying concentrations of the prodrugs for 48 hours. Following treatment, the medium was discarded, and 100 μL of fresh medium containing MTT (0.5 mg mL⁻¹) was added to each well and incubated for an additional 4 hours at 37 °C. Subsequently, 100 μL of extraction buffer (10% SDS, 5% isobutanol, and 0.1% HCl) was added to each well, and the cells were further incubated for 8 hours at 37°C. The absorbance was measured at 570 nm using a microplate reader.

The cytotoxicity of S1-DOX was evaluated in HCT116, HCT116-NC, and HCT116-TrxR1-KO cells using the CCK-8 assay. Briefly, 5000 cells per well were seeded in 96-well plates and allowed to adhere overnight. The cells were then treated with serial dilutions of S1-DOX (eight concentrations, starting from 20 μM with a fourfold dilution factor). Blank wells received complete medium without drug, while control wells were treated with an equivalent volume of DMSO. After 24 h of incubation, 10 μL of CCK-8 reagent was added to each well and the plates were further incubated at 37 °C for 2 h. The absorbance at 450 nm was measured using a Cytation 5 microplate reader (BioTek). The cell growth inhibition rate was calculated according to the formula: Growth inhibition rate = (AB value of control - AB value of test) / (AB value of control - AB value of blank) × 100%.

4.4. Cell lines and culture conditions
HeLa, HepG2, A549, and HCT116 cell lines were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (HeLa, SCSP-504; HepG2, SCSP-510; A549, SCSP-503; HCT116, SCSP-5076). HeLa, HepG2, and A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 U mL⁻¹ penicillin–streptomycin, and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. HCT116, HCT116-NC, and HCT116-TrxR1-KO cells were cultured in RPMI-1640 medium. The HCT116-NC and HCT116-TrxR1-KO cell lines were generated by Prof. Chao Zhang.52

HeLa, HepG2, A549 and HCT116 cell lines were authenticated by STR profiling prior to use. HCT116-NC and HCT116-TrxR1-KO cell lines were generated by genetic editing based on the parental HCT116 cell line. The identity of these derived cell lines was verified by confirmation of their parental origin and by validation of the intended genetic modifications, rather than by independent STR profiling.

4.5. Live cell imaging and flow cytometry-based fluorescence quantification assay
For both TRFS-green and S-Cou, HCT116, HCT116-NC, and HCT116-TrxR1-KO cells were treated with the specified concentrations (10 M) of probes for the indicated time periods (4 h). Fluorescence images were captured using a Leica SP8 laser scanning confocal microscope. For TRFS-green, fluorescence intensity in each group was quantified using a Novocyte flow cytometer with NovoExpress 1.2.4 software. For S-Cou, fluorescence intensity in each group was measured using a BD FACSVerse flow cytometer (San Diego, CA, USA) with FlowJo 10.9 software. In the TRi-1 inhibition assay, HCT116 cells were treated with the specified concentrations of TRi-1 (negative control: equivalent volume of DMSO; positive control: auranofin (AF)) for 4 hours, followed by an additional 4-hour incubation with TRFS-green (10 μM). Fluorescence images were obtained using the Leica SP8 laser scanning confocal microscope, and the fluorescence intensity in each group was quantified using the Novocyte flow cytometer with NovoExpress 1.2.4 software.

4.6. Assessment of TrxR1 enzyme activity in TrxR1-KO and TRi-1 inhibitor-mediated suppression of cellular TrxR1 activity
For the assessment of TrxR1 cellular enzyme activity, cells were seeded in cell culture dishes. Once cell confluence reached over 90%, the cells were trypsinized, centrifuged, and collected. Proteins were then extracted and quantified using the BCA method. TrxR1 activity in the aforementioned cells was measured using an insulin endpoint enzyme activity assay. A total of 20 μg of cellular protein was incubated with 50 μL of a buffer containing 100 mM Tris-HCl (pH 7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and 1.3 μM Trx protein at 37°C for 1 hour. The reaction was terminated by adding 200 μL of 6 M guanidine hydrochloride containing 1 mM DTNB (pH 8.0). Absorbance (AB) was measured at 412 nm using a Cytation 5 microplate reader (BioTek). A standard curve was generated using rat liver TrxR1 protein (218.53 U/mg, Sigma), diluted across a series of concentrations, and the TrxR1 enzyme activity in each sample was calculated based on this standard curve.

For the TRi-1 inhibition assay, cells were treated with TRi-1 for 4 hours (starting at 10 μM, with 2-fold dilutions for 6 concentrations). Total cellular protein extraction and TrxR1 activity assays were performed as described above. The same amounts of DMSO were added to the control experiments, and the TrxR1 inhibitory rate was calculated using the following formula: TrxR1 inhibitory rate = [1 - (AB value of sample - AB value of blank ) / (AB value of control - AB value of blank )] × 100%.

4.7. Molecular docking simulation
Molecular docking simulations were conducted using AutoDock Vina (v1.1.2) to predict the binding modes and affinities of the compounds toward TrxR1 (PDB ID: 3QFA). The protein structure was prepared by removing water molecules and co-crystallized ligands, followed by the addition of polar hydrogens and Kollman charges using AutoDock Tools (v1.5.7).91, 92 The lowest-energy docking pose for each ligand was selected for visualization and analysis in PyMOL (v3.1.6.1), with particular attention to measuring the distance between the ligand’s disulfide ring and the selenium atom of Sec498 to validate the binding geometry.

4.8. Statistics analysis
Statistical analyses were performed using Origin 2019 (OriginLab). Data are presented as mean ± SD. Comparisons among multiple groups were conducted using one-way ANOVA. A P value of less than 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a very signifcant paper in the TrxR field. The authors have effectively addressed an earlier report (published in this journal) that largely aimed to refute their (and others') studies in the field:\nA recent report by Felber et al. raised questions about previous work by the authors of the current paper. In their study, Felber and colleagues reported that the 1,2-dithiolane-based probe SS50-PQ demonstrated non-selective reductive activation. They inferred that 1,2-dithiolane-based probes, including SS50-PQ and the TRFS series (developed by the authors of the current paper), are intrinsically unable to selectively report on TrxR activity. This conclusion attributed the lack of selectivity to the inherent properties of the 1,2-dithiolane scaffold, regardless of the attached cargo or chemical linkage.\nThe authors of the current paper offer a different scientific stance, proposing that the enzyme selectivity of 1,2-dithiolane-based probes is largely determined by the molecular components surrounding the core scaffold, specifically the leaving group and the linker.\nThe current study designed five 1,2-dithiolane-based prodrugs (S-CPT, S1-SN38, S2-SN38, S1-Gem, and S-PTX) with hydroxyl-based drugs as leaving groups. Their findings confirmed that these prodrug molecules, featuring primary, secondary, and tertiary alcohols, as well as phenolic hydroxyls, were broadly susceptible to activation by a range of cellular reductants, including both GSH and TrxR. This aligns with the non-selective activation observed in the Felber paper when hydroxyl functionalities are involved67.\nIn contrast, 1,2-dithiolane molecules incorporating amino groups (such as S-Cou and S1-DOX) consistently showed results aligned with the authors' previously reported findings, demonstrating resistance to interference from LMW thiols (e.g., GSH) while retaining sensitivity to TrxR activation. This behavior is similar to that of their TRFS probes and prodrugs like S-Gem and S1-Puro.\nThe Felber report also questioned whether TRFS-green, a probe developed by the current authors, is exclusively activated by TrxR, suggesting that other intracellular proteins like Grx might also activate it13. In response, the current study re-evaluated TRFS-green's cellular behavior through experiments including TrxR1 knockout cells and chemical inhibition using TRi-1 (a selective TrxR1 inhibitor) and auranofin. The results collectively confirmed the reliability of TRFS-green as a high-selectivity probe for TrxR.\nThe current paper acknowledges that a previous study (by their collaborators) showed TRFS-green could be a substrate for the complete bacterial Trx system (TrxR/Trx) and the GSH system (GR, GSH, and Grx), but was not activated by GSH, GR, or bacterial TrxR alone. They clarify that this observation does not contradict their mammalian TrxR findings because bacterial TrxR has a single N-terminal active site, while mammalian TrxRs possess a C-terminal active site with a characteristic GCUG motif, which they suggest plays a key role in TRFS-green activation in mammalian systems.\nThe authors acknowledge that small-molecule probes, ligands, or inhibitors typically cannot achieve absolute selectivity, and this challenge extends to TRFS probes. They recognize that other species in cells must also contribute to the activation of TRFS probes but emphasize that TrxR plays the predominant role, especially supported by studies using TrxR-overexpressing and knockout cell lines.\nIt would be helpful for the authors to further (or more strongly) expound on why the lack of \"absolute selectivity\" is not a major issue in this field.\nOverall this work addresses a major controversy in the field initiated by Felder et al. and is enthusiatically recommended for publication.", "reviewer_2": "I recommend this manuscript for publication as it serves as a response to previous concerns (Nat Commun 2022, 13, 1754. DOI: 10.1038/s41467-022-29136-4), which is highly valuable for scientific discourse. The authors have placed the response to previous concerns in the Discussion section. I suggest considering moving it earlier, perhaps to the Introduction, to make the contentious points clearer to readers from the outset. This manuscript presents a systematic and in-depth investigation into the structure–activity relationship of 1,2-dithiolane-based probes and prodrugs, with a particular focus on how linker chemistry governs the selectivity balance between thioredoxin reductase (TrxR) and glutathione (GSH). The study is well motivated and clearly articulated, addressing a challenge in the field, namely the inconsistent activation patterns of 1,2-dithiolane-containing molecular systems that have been reported in previous literature. The authors present compelling evidence demonstrating that carbamate linkers confer high selectivity toward TrxR while conferring resistance to GSH at physiologically relevant concentrations. In contrast, carbonate linkers are susceptible to nonspecific activation by endogenous GSH, thereby compromising enzyme-specific response. This insight is not only conceptually significant but also offers a convincing mechanistic rationale for previously conflicting observations. Furthermore, the work establishes a valuable design principle applicable to the development of next-generation redox-responsive tools, including fluorescent probes and prodrugs. The novelty of the findings, the rigor of the experimental approach, and the broad implications for biochemical tool development make this work highly significant and of wide potential interest to the research community.\nDespite these strengths, there remain several issues that merit clarification or further validation before the conclusions can be fully accepted. My primary concern relates to the prodrug S1-DOX. While the manuscript indicates that S1-DOX is responsive to TrxR activation in vitro, the actual release efficiency of the parent compound appears relatively modest. Specifically, after 24 hours of incubation with TrxR, only around 30% of the parent drug is released. By contrast, earlier studies on related 1,2-dithiolane-based prodrugs (J. Med. Chem. 2023, 66, 3250–3261; Angew. Chem. Int. Ed. 2018, 57, 6141–6145) have reported release efficiencies of approximately 80% under comparable in vitro conditions. Importantly, those compounds were also shown to retain TrxR-dependent activation in cellular environments, at least to a measurable degree. This discrepancy raises questions about the robustness of S1-DOX activation and whether it can genuinely serve as a TrxR-selective prodrug in more complex biological systems. To strengthen the study, I recommend that the authors conduct additional cell-based assays to directly evaluate the TrxR selectivity of S1-DOX and clarify whether the in vitro limitations also translate to cellular contexts. Without such validation, the broader applicability of the design principle for carbamate-linked prodrugs remains uncertain.\nA second point that requires clarification concerns the TRFS-green probe. This probe is described as being responsive to both the TrxR and GSH systems in cell-free assays (Nat Commun, 13, 1754); however, in HCT-116 cells with TrxR knockout, a dramatic loss of fluorescence intensity was observed. This apparent discrepancy warrants a more detailed explanation. Why does TRFS-green fail to respond to the GSH system in the cellular context, despite exhibiting dual responsiveness in vitro?\nBeyond these major points, there are also minor but noteworthy issues in the manuscript that should be corrected to improve accuracy and presentation. For example, the mechanism diagram in the TOC is concise but not clear enough. It is recommended to simplify the mechanism diagram from Fig. 10 and feature it in the TOC. The structural formulas of NBL and Se-NBL in Fig. 3 contain errors and should be carefully revised. Similarly, in Fig. 6D the labels do not fully correspond with the descriptions provided in the main text, which could lead to confusion for readers. These inconsistencies in figures and legends should be carefully corrected to maintain clarity and precision.", "reviewer_3": "Previous research has utilized 1,2-dithiolane-based probes for detecting thioredoxin reductase (TrxR). A notable limitation of this approach is its susceptibility to interference from low-molecular-weight (LMW) thiols, which can lead to erroneous readings. To address this inherent constraint, the authors strategically engineered the molecular architecture to achieve a distributary response, effectively diverting signals from LMW thiols and thereby enabling specific activation and detection of TrxR. The findings of this manuscript lay a critical foundation for future research aimed at distinguishing between LMW thiols and TrxR in biological environments using molecular probes. However, a significant portion of the current manuscript lacks clarity and requires further elaboration and revision.\n1. The manuscript lacks critical reference compounds—for example, those systematically varying the linker of the responsive group (e.g., carbamate versus carbonate) while keeping the released cargo unchanged. Whether such modifications yield a discernible pattern in the activation signal remains an important unresolved question warranting further investigation.\n2. In Figure 5, when comparing the activation efficiency of 1 mM GSH with that of 50 nM TrxR, does this imply that these concentrations are deemed comparable or equivalent? Moreover, similar inconsistencies in concentration comparisons appear in multiple sections of the manuscript (e.g., Figures S3, 5, and 6). These non-parallel and inequitable experimental conditions substantially compromise the reliability of the authors’ conclusions.\n3. This manuscript presents a novel insight: beyond the recognition group itself, the overall molecular structure also significantly influences the substrate’s response efficiency. However, the authors have not offered a plausible explanation for the observed phenomena. Computational studies, such as energy calculations and molecular docking simulations, are recommended to elucidate the underlying mechanisms.\n4. The stability of the prodrug molecules should be further assessed in serum-containing cell culture medium rather than PBS buffer.\n5. The limited number of time points chosen by the authors in Figure 5 renders it challenging to generate a reliable fitting curve based on the provided data."}, "author_response": "Point-by-point Response to Reviewers\nReviewer #1 (Remarks to the Author):\nThis is a very signifcant paper in the TrxR field. The authors have effectively addressed an earlier\nreport (published in this journal) that largely aimed to refute their (and others’) studies in the field:\nA recent report by Felber et al. raised questions about previous work by the authors of the current\npaper. In their study, Felber and colleagues reported that the 1,2-dithiolane-based probe SS50-PQ\ndemonstrated non-selective reductive activation. They inferred that 1,2-dithiolane-based probes,\nincluding SS50-PQ and the TRFS series (developed by the authors of the current paper), are\nintrinsically unable to selectively report on TrxR activity. This conclusion attributed the lack of\nselectivity to the inherent properties of the 1,2-dithiolane scaffold, regardless of the attached cargo or\nchemical linkage.\nThe authors of the current paper offer a different scientific stance, proposing that the enzyme\nselectivity of 1,2-dithiolane-based probes is largely determined by the molecular components\nsurrounding the core scaffold, specifically the leaving group and the linker. The current study\ndesigned five 1,2-dithiolane-based prodrugs (S-CPT, S1-SN38, S2-SN38, S1-Gem, and S-PTX) with\nhydroxyl-based drugs as leaving groups. Their findings confirmed that these prodrug molecules,\nfeaturing primary, secondary, and tertiary alcohols, as well as phenolic hydroxyls, were broadly\nsusceptible to activation by a range of cellular reductants, including both GSH and TrxR. This aligns\nwith the non-selective activation observed in the Felber paper when hydroxyl functionalities are\ninvolved. In contrast, 1,2-dithiolane molecules incorporating amino groups (such as S-Cou and\nS1-DOX) consistently showed results aligned with the authors’ previously reported findings,\ndemonstrating resistance to interference from LMW thiols (e.g., GSH) while retaining sensitivity to\nTrxR activation. This behavior is similar to that of their TRFS probes and prodrugs like S-Gem and\nS1-Puro.\nThe Felber report also questioned whether TRFS-green, a probe developed by the current authors,\nis exclusively activated by TrxR, suggesting that other intracellular proteins like Grx might also\nactivate it. In response, the current study re-evaluated TRFS-green’s cellular behavior through\nexperiments including TrxR1 knockout cells and chemical inhibition using TRi-1 (a selective TrxR1\n1 / 13\ninhibitor) and auranofin. The results collectively confirmed the reliability of TRFS-green as a\nhigh-selectivity probe for TrxR.\nThe current paper acknowledges that a previous study (by their collaborators) showed TRFS-green\ncould be a substrate for the complete bacterial Trx system (TrxR/Trx) and the GSH system (GR,\nGSH, and Grx), but was not activated by GSH, GR, or bacterial TrxR alone. They clarify that this\nobservation does not contradict their mammalian TrxR findings because bacterial TrxR has a single\nN-terminal active site, while mammalian TrxRs possess a C-terminal active site with a characteristic\nGCUG motif, which they suggest plays a key role in TRFS-green activation in mammalian systems.\nThe authors acknowledge that small-molecule probes, ligands, or inhibitors typically cannot\nachieve absolute selectivity, and this challenge extends to TRFS probes. They recognize that other\nspecies in cells must also contribute to the activation of TRFS probes but emphasize that TrxR plays\nthe predominant role, especially supported by studies using TrxR-overexpressing and knockout cell\nlines. It would be helpful for the authors to further (or more strongly) expound on why the lack of\n“absolute selectivity” is not a major issue in this field.\nOverall, this work addresses a major controversy in the field initiated by Felder et al. and is\nenthusiatically recommended for publication.\nResponse: We sincerely thank the reviewer for the positive and encouraging comments on our\nwork. We fully agree that achieving absolute selectivity in small-molecule redox probes is inherently\nchallenging—and, to some extent, conceptually unrealistic. Thiol-based redox systems, including\nTrxR/Trx, Grx/GSH/GR, and related redoxin networks, are deeply interconnected and often exhibit\noverlapping substrate specificities and redox potentials. Consequently, small-molecule probes are\nhard to display absolute exclusivity toward a single biomolecule under complex cellular conditions.\nInstead, the practical goal is to achieve functional selectivity—that is, a probe’s ability to\npredominantly and reproducibly reflect the activity of the intended target under relevant conditions.\nIn this sense, “selectivity” should be viewed as a contextual and quantitative property rather than an\nabsolute one. Functionally selective probes are not only sufficient but also ideally suited for\nmonitoring TrxR activity and associated biological processes in situ. Moreover, our study goes\nbeyond defending the selectivity of TRFS probes; it establishes a broader conceptual framework for\nunderstanding structure–selectivity relationships in 1,2-dithiolane-based systems. We further expand\n2 / 13\non these points in the revised Discussion (page 19, highlighted in yellow), emphasizing that the\nabsence of absolute selectivity does not diminish the biological value or interpretability of\nTRFS-green and related probes. Together, our results highlight a more nuanced understanding that\nTrxR selectivity is conditional, tunable by molecular structure, and thus represents a meaningful and\npractical design principle for future probes and prodrugs.\nReviewer #2 (Remarks to the Author):\nI recommend this manuscript for publication as it serves as a response to previous concerns (Nat\nCommun 2022, 13, 1754. DOI: 10.1038/s41467-022-29136-4), which is highly valuable for\nscientific discourse. The authors have placed the response to previous concerns in the Discussion\nsection. I suggest considering moving it earlier, perhaps to the Introduction, to make the contentious\npoints clearer to readers from the outset. This manuscript presents a systematic and in-depth\ninvestigation into the structure–activity relationship of 1,2-dithiolane-based probes and prodrugs,\nwith a particular focus on how linker chemistry governs the selectivity balance between thioredoxin\nreductase (TrxR) and glutathione (GSH). The study is well motivated and clearly articulated,\naddressing a challenge in the field, namely the inconsistent activation patterns of\n1,2-dithiolane-containing molecular systems that have been reported in previous literature. The\nauthors present compelling evidence demonstrating that carbamate linkers confer high selectivity\ntoward TrxR while conferring resistance to GSH at physiologically relevant concentrations. In\ncontrast, carbonate linkers are susceptible to nonspecific activation by endogenous GSH, thereby\ncompromising enzyme-specific response. This insight is not only conceptually significant but also\noffers a convincing mechanistic rationale for previously conflicting observations. Furthermore, the\nwork establishes a valuable design principle applicable to the development of next-generation\nredox-responsive tools, including fluorescent probes and prodrugs. The novelty of the findings, the\nrigor of the experimental approach, and the broad implications for biochemical tool development\nmake this work highly significant and of wide potential interest to the research community.\nResponse: We sincerely appreciate the reviewer’s insightful suggestion and positive comments. we\nhave briefly introduced the debated viewpoints in the Introduction (page 4, highlighted in yellow)\nwhile retaining a more detailed discussion in the Discussion section. Also, This revision enhances the\n3 / 13\noverall logical flow of the manuscript and enables readers to more clearly grasp the scientific\nmotivation and controversy underlying our work.\nDespite these strengths, there remain several issues that merit clarification or further validation\nbefore the conclusions can be fully accepted. My primary concern relates to the prodrug S1-DOX.\nWhile the manuscript indicates that S1-DOX is responsive to TrxR activation in vitro, the actual\nrelease efficiency of the parent compound appears relatively modest. Specifically, after 24 hours of\nincubation with TrxR, only around 30% of the parent drug is released. By contrast, earlier studies on\nrelated 1,2-dithiolane-based prodrugs (J. Med. Chem. 2023, 66, 3250–3261; Angew. Chem. Int. Ed.\n2018, 57, 6141–6145) have reported release efficiencies of approximately 80% under comparable in\nvitro conditions. Importantly, those compounds were also shown to retain TrxR-dependent activation\nin cellular environments, at least to a measurable degree. This discrepancy raises questions about the\nrobustness of S1-DOX activation and whether it can genuinely serve as a TrxR-selective prodrug in\nmore complex biological systems. To strengthen the study, I recommend that the authors conduct\nadditional cell-based assays to directly evaluate the TrxR selectivity of S1-DOX and clarify whether\nthe in vitro limitations also translate to cellular contexts. Without such validation, the broader\napplicability of the design principle for carbamate-linked prodrugs remains uncertain.\nResponse: We thank the reviewer for raising this important point regarding S1-DOX. To address the\nconcern, we have evaluated S1-DOX in three cell lines: HCT116 wild-type, HCT116-NC\n(non-targeting control), and HCT116-TrxR1-KO. As shown in Fig. 8E, cytotoxicity assays indicate\nthat S1-DOX exhibits comparable inhibitory effects in all three cell lines, with IC₅₀ values around 0.6\nμM. These results suggest that, although S1-DOX can be activated by TrxR in vitro, its cellular\ncytotoxicity is not entirely TrxR-dependent. Mechanistically, this observation aligns with the known\nreactivity of carbamate-linked 1,2-dithiolane scaffolds, which can undergo reductive activation not\nonly by TrxR but also—albeit less efficiently—by the GSH system, as previously exemplified by\nTRFS-green. As noted, the TrxR-mediated release efficiency of S1-DOX is relatively modest (~30%\nat 24 h, compared with ~80% within 8 h for other 1,2-dithiolane-based prodrugs). Even in cells with\nintact TrxR1, this low-efficiency enzymatic activation may still allow accumulation of cytotoxic\nlevels of the active drug, while additional cellular reduction pathways could further contribute to\noverall cytotoxicity. In TrxR1-deficient cells, intrinsic non-TrxR reductive systems, such as the GSH\n4 / 13\npathway, may be compensatorily upregulated, enhancing S1-DOX reduction. Over prolonged\nincubation (24 h), these alternative pathways can generate sufficient active compound to induce\nsubstantial cytotoxicity. Consequently, the TrxR-dependent contribution to prodrug activation is\nlargely masked under conventional cytotoxicity assays. Relevant results have been highlighted in the\nmain text of the manuscript (page 11, highlighted in yellow).\nA B 50 nM TrxR 25 nM TrxR C\n10 nM TrxR Analytes + S1-DOX\n7.5\n6.0 12\n(1 )\nT\nm\nT\nM\n(5\n)\n0\nn\nR\nM\nR C e le d a u v c a t g io e n : m by o n T o rx th R i ols resistance\nF/\nF0\n1\n3\n4\n.\n.\n.\n5\n0\nF0\n3\nG (5\n)\nS\nm\nH\nM\nL\n(5 )\n-\nm\nCy\nM\ns N\n(2\nM)\nm\n0\n0 5 10 15 20 25 Analytes\nTime (h)\nD E\nAnalytes + S1-DOX 150 HCT116\n)M )M HCT116-NC\nD O X r el e\na\ns e (\n%)\n1 2\n3\n4\n0 0\n0\n0\n(1\nT\nm\nT T\n(50\nrx\nn\nR\nG (5 ) S m H M L (5 ) - m Cy M s N (2 M) 0 A 0 D P H m C ell vi a\nblit y (\n%)1\n3 6\n9\n2\n0 0 0\n0\nHCT116-TrxR1-KO\nAnalytes 0.001 0.01 0.1 1 10\nlog\n10\n([S1-DOX]) (M)\nFig. 8 Activation of S1-DOX mediated by TrxR. (A) Proposed mechanism of S1-DOX activation:\nreduction or cleavage of the 1,2-dithiolane moiety triggers thiol-mediated cyclization, facilitating\nDOX release. (B) Time-dependent fluorescence enhancement of S1-DOX upon incubation with\ndifferent TrxR concentrations (10 nM, 25 nM, and 50 nM). (C) Fluorescence selectivity of S1-DOX\nin the presence of various biological species (24 h). (D) Quantitative analysis of DOX release from\nS1-DOX induced by different biological species (24 h). (E) Comparison of S1-DOX cytotoxicity in\nHCT116, HCT116-NC, and HCT-TrxR1-KO cells (24 h).\nA second point that requires clarification concerns the TRFS-green probe. This probe is described\nas being responsive to both the TrxR and GSH systems in cell-free assays (Nat Commun, 13, 1754);\nhowever, in HCT-116 cells with TrxR knockout, a dramatic loss of fluorescence intensity was\nobserved. This apparent discrepancy warrants a more detailed explanation. Why does TRFS-green\nfail to respond to the GSH system in the cellular context, despite exhibiting dual responsiveness in\nvitro?\nResponse: We thank the reviewer for this insightful comment. TRFS-green is efficiently activated by\n5 / 13\nTrxR in the presence of the cofactor NADPH, whereas activation by the GSH system requires the\ncomplete enzymatic network (GSH, GR, Grx, and NADPH). Notably, the fluorescence enhancement\ninduced by the complete GSH system is significantly lower than that driven by TrxR1. This\nmechanistic nuance explains the dramatic loss of fluorescence observed in TrxR1-knockout cells,\ndespite the probe’s dual responsiveness in simplified in vitro assays. These clarifications have now\nbeen incorporated into the revised manuscript (pages 16 and 17, highlighted in yellow).\nBeyond these major points, there are also minor but noteworthy issues in the manuscript that\nshould be corrected to improve accuracy and presentation. For example, the mechanism diagram in\nthe TOC is concise but not clear enough. It is recommended to simplify the mechanism diagram from\nFig. 10 and feature it in the TOC. The structural formulas of NBL and Se-NBL in Fig. 3 contain\nerrors and should be carefully revised. Similarly, in Fig. 6D the labels do not fully correspond with\nthe descriptions provided in the main text, which could lead to confusion for readers. These\ninconsistencies in figures and legends should be carefully corrected to maintain clarity and precision.\nResponse: We sincerely thank the reviewer for these valuable and detailed comments. All figures\nhave been carefully checked and revised to ensure both accuracy and clarity. Specifically:\n1) The TOC graphic has been redesigned using a simplified mechanistic scheme to enhance clarity\nand better illustrate the core concept;\n2) The chemical structures of NBL and Se-NBL in Fig. 3 have been redrawn and corrected;\n3) The corresponding labels in the main text have been revised to ensure consistency with Fig. 7D\n(previously Fig. 6D) (Page 10, highlighted in green).\nReviewer #3 (Remarks to the Author):\nPrevious research has utilized 1,2-dithiolane-based probes for detecting thioredoxin reductase (TrxR).\nA notable limitation of this approach is its susceptibility to interference from low-molecular-weight\n(LMW) thiols, which can lead to erroneous readings. To address this inherent constraint, the authors\nstrategically engineered the molecular architecture to achieve a distributary response, effectively\ndiverting signals from LMW thiols and thereby enabling specific activation and detection of TrxR.\nThe findings of this manuscript lay a critical foundation for future research aimed at distinguishing\nbetween LMW thiols and TrxR in biological environments using molecular probes. However, a\n6 / 13\nsignificant portion of the current manuscript lacks clarity and requires further elaboration and\nrevision.\nResponse: We sincerely appreciate the reviewer’s constructive and insightful comments. We have\ncarefully revised the manuscript accordingly. Detailed point-by-point responses are provided below.\n1. The manuscript lacks critical reference compounds—for example, those systematically varying\nthe linker of the responsive group (e.g., carbamate versus carbonate) while keeping the released\ncargo unchanged. Whether such modifications yield a discernible pattern in the activation signal\nremains an important unresolved question warranting further investigation.\nResponse: We thank the reviewer for this insightful comment. We fully agree that systematically\nvarying the responsive linker (e.g., carbamate versus carbonate) while keeping the released cargo\nconstant would provide a clearer understanding of how linker chemistry affects activation kinetics\nand signal amplitude. Indeed, we conducted systematic and comprehensive studies addressing this\nissue by fixing the naphthalimide unit as a cargo in our previous work (as illustrated in the figure\nbelow).\n(1) In our earlier work (Nat. Commun., 2019, 10, 2745), we developed a series of\nnaphthalimide-based 1,2-dithiolane derivatives (TRFS1–TRFS8). In that study, while keeping the\noverall cargo structure constant, we systematically examined how variations in the linker and\nrecognition unit affected the reactivity of different probes toward various biological reductants.\nTaking the 1,2-dithiolane–containing derivatives TRFS1–TRFS4 as representative examples: the\nonly structural difference between TRFS-green and TRFS1 is the nature of the linker (carbamate\nversus carbonate). However, TRFS1 was found to be extremely unstable and could not be purified by\ncolumn chromatography, preventing any further analysis of its reductive activation. Similarly,\nTRFS2 also exhibited poor stability. TRFS3 underwent direct disulfide cleavage and rapidly released\nfluorescence, whereas TRFS4 released fluorescence much more slowly due to an intramolecular\ncyclization step that constitutes the rate-limiting process.\n(2) For carbamate-linked systems, two distinct outcomes were observed. When the amine group is\npositioned on the releasable cargo, efficient TrxR-mediated cleavage and activation occur, consistent\nwith our earlier studies on 1,2-dithiolane-based prodrugs and probes. However, when the hydroxyl\ngroup is located on the releasable cargo (as in CPT-Se1, J. Med. Chem., 2021, 64, 17979−17991), no\n7 / 13\nsignificant release of the active species was detected. Although CPT-Se1 features a 1,2-diselenane\nmotif, the observation remains conceptually relevant.\n(3) A representative example is provided by the gemcitabine-based prodrugs S-Gem (Angew.\nChem. Int. Ed., 2018, 57, 6141–6145) and S1-Gem (this work). The key structural distinction lies in\nthe nature of the linker: in S-Gem, the 1,2-dithiolane moiety is connected to an amine group via a\ncarbamate linkage, whereas in S1-Gem, it is attached to a hydroxyl group through a carbonate\nlinkage. Functionally, S-Gem is selectively activated by TrxR and remains largely unresponsive to\nGSH, whereas S1-Gem can be activated by both TrxR and GSH.\nCollectively, these findings demonstrate that both the linker chemistry and the position of the\nleaving group exert profound effects on the activation and release behavior of 1,2-dithiolane-based\nsystems.\nJ. Med. Chem., 2021, 64, 17979−17991\nAngew.Chem. Int.Ed.,2018, 57, 6141 –6145\nNat. Commun., 2019, 10, 2745\nThiswork\n2. In Figure 5, when comparing the activation efficiency of 1 mM GSH with that of 50 nM TrxR,\ndoes this imply that these concentrations are deemed comparable or equivalent? Moreover, similar\ninconsistencies in concentration comparisons appear in multiple sections of the manuscript (e.g.,\nFigures S3, 5, and 6). These non-parallel and inequitable experimental conditions substantially\ncompromise the reliability of the authors’ conclusions.\nResponse: We thank the reviewer for raising this important point. Comparing 1 mM GSH with 50\n8 / 13\nnM TrxR does not imply that these concentrations are equivalent or directly comparable. Rather, this\nexperimental design reflects their respective physiological abundance levels. Intracellular monothiols,\nsuch as GSH, are typically present at millimolar concentrations (1–10 mM), whereas TrxR exists\nonly at tens of nanomolar levels. Accordingly, these non-parallel experimental concentrations were\nintentionally selected to reproduce this intrinsic biological disparity, rather than to achieve\nstoichiometric equivalence. We have clarified this point in the revised manuscript (page 8,\nhighlighted in yellow).\nIn the carbonate-linked prodrug systems, the GSH concentration was set to 1 mM, while in the\ncarbamate-linked prodrugs and probes, it was increased to 5 mM. This adjustment was made to\ndemonstrate that, under these conditions, carbamate-based molecules are relatively resistant to\nGSH-mediated activation, whereas carbonate-linked molecules are more susceptible to\nGSH-triggered cleavage.\n3. This manuscript presents a novel insight: beyond the recognition group itself, the overall\nmolecular structure also significantly influences the substrate’s response efficiency. However, the\nauthors have not offered a plausible explanation for the observed phenomena. Computational studies,\nsuch as energy calculations and molecular docking simulations, are recommended to elucidate the\nunderlying mechanisms.\nResponse: We thank the reviewer for this insightful comment. We performed computer-aided\ndocking simulations on six prodrug molecules: S-CPT, C-CPT, S-PTX, C-PTX, S1-DOX, and\nSe1-DOX. Based on their reactivity toward TrxR and GSH, these six molecules can be categorized\ninto three groups: (1) S-CPT can be activated by both GSH and TrxR (with C-CPT as a control); (2)\nS-PTX can be activated by GSH but not by TrxR (with C-PTX as a control); (3) S1-DOX can be\nactivated by TrxR but not by GSH (with Se1-DOX as a control). Docking results showed that all\ncompounds exhibited favorable binding scores (< −5.0 kcal/mol), indicating that their binding\ninteractions are energetically feasible. Specifically, S-CPT adopts a binding conformation in which\nits 1,2-dithiolane ring is oriented toward the C-terminal Sec residue, with two sulfur atoms\npositioned 3.6 Å and 3.9 Å from the Sec selenium atom, consistent with potential Se–S interactions.\nIn contrast, C-CPT, although its cyclopentane CH₂ carbons are similarly positioned (3.9 Å and 4.5 Å),\nlacks a reactive disulfide motif and therefore cannot undergo redox activation (Fig. 6). S-PTX, while\n9 / 13\nlocalized near the surface-exposed catalytic site adjacent to the C-terminal Sec residue, has its\n1,2-dithiolane sulfur atoms positioned 9.5 Å and 9.8 Å from the Sec selenium atom, too far for\nefficient Se–S interactions. This observation explains its low TrxR-mediated activation efficiency\n(C-PTX’s cyclopentane CH₂ carbons are 7.4 Å and 7.5 Å from Sec) (Fig. 6). For S1-DOX, the sulfur\natoms are 3.7 Å and 4.5 Å from the Sec selenium atom, whereas for Se1-DOX, the selenium atoms\nare 3.8 Å and 4.0 Å from Sec, indicating spatial proximity favorable for Se–S or Se–Se interactions\nat the Sec-containing redox center (Supplementary Figure 11). These results suggest that both\nprodrugs can stably engage TrxR, allowing potential redox exchange between their\ndisulfide/diselenide motifs and the enzyme’s active selenol–thiol pair. However, only S1-DOX\nachieves efficient drug release, while Se1-DOX remains largely inert. This difference likely arises\nfrom the intrinsic redox preference of the selenol intermediate generated from Se1-DOX, which\nfavors reoxidation to a stable diselenide rather than proceeding through intramolecular cyclization\nfor payload release. Relevant results have been highlighted in the main text of the manuscript (Page\n9, highlighted in yellow; Page 12, highlighted in green).\nWe would also like to emphasize that, beyond the influence of molecular binding affinity and\ncontact patterns with protein targets on activation efficiency, it remains an open question whether the\ndisulfide or diselenide motifs within certain prodrugs or probe molecules can be effectively reduced\nby GSH or other cellular reductants. Experimentally, molecules with specific chemical structures can\nexhibit notable resistance to reduction mediated by GSH or TrxR, as exemplified by S1-Cou. This\nresistance suggests that factors such as molecular microconformation, ring strain, electronic effects,\nand steric hindrance may significantly influence reactivity under physiological reducing conditions.\nObservations of this kind indicate that, although we have gained a preliminary understanding of the\ngeneral mechanisms of target-selective activation, the precise structure–reactivity relationships\nremain largely unexplored. Investigating these questions not only holds theoretical significance but\nalso provides practical guidance for the rational design of prodrugs and activatable probes.\nNevertheless, due to the involvement of multiple kinetic and thermodynamic factors, studies aiming\nto elucidate such structure–activity relationships continue to pose substantial experimental\nchallenges.\n10 / 13\nFig. 6 Molecular docking analysis of prodrugs with TrxR1. (A–D) Molecular docking results of\nS-CPT (A), C-CPT (B), S-PTX (C), and C-PTX (D) with TrxR1 (PDB ID: 3QFA). Chain A and\nchain B of the TrxR1 dimer are shown in aquamarine and lime green, respectively. The -GCUG\nmotif and key residues are highlighted in blue, and the docked compounds are shown in yellow.\nHydrogen bonds and the distances between Sec498 and the disulfide ring are indicated by red and\nyellow dashed lines, respectively. Docking scores (kcal/mol): –5.822 (S-CPT), –6.625 (C-CPT),\n–5.225 (S-PTX), and –6.271 (C-PTX).\nSupplementary Figure 11. Molecular docking analysis of prodrugs and TrxR. (A–B) Molecular\ndocking results of S1-DOX (A) and Se1-DOX (B) with TrxR (PDB ID: 3QFA). Chain A and Chain B\nof the TrxR dimer are shown in Aquamarine and Limegreen, respectively. The GCUG motif and key\namino acid residues are highlighted in blue, while the compounds are shown in yellow. Hydrogen\nbond distances (red dashed lines) and distances between Sec498 and the disulfide ring (yellow\ndashed lines) are indicated. The calculated docking scores are −5.981 kcal/mol (S1-DOX) and\n−6.007 kcal/mol (Se1-DOX).\n4. The stability of the prodrug molecules should be further assessed in serum-containing cell\nculture medium rather than PBS buffer.\nResponse: The stability of the 1,2-dithiolane-class prodrugs was evaluated in serum-containing\nculture medium. All candidates demonstrated good stability under these conditions, with the\n11 / 13\nexception of S2-SN38, which exhibited pronounced instability (Supplementary Figure 2).\nSpecifically, S2-SN38 underwent rapid degradation, with approximately 90% decomposition\noccurring within ~20 minutes. While this rapid serum-mediated breakdown does not affect the\nconclusions derived from experiments in PBS buffer, it highlights that S2–SN38 is unsuitable for\napplications in biological systems. Relevant results have been highlighted in the main text of the\nmanuscript (Page 7, yellow highlight).\nA\nA B C\n27600\n26000 25600\n20700\n19500 19200\n13800\nS-CPT 13000 12800\n6900\n0 0\n0 m I i n ncuba\n2\nt\n0\nio\nm\nn\ni\nt\nn\ni\ntme\n0 mi In ncub\n2\nat\n0\nio\nm\nn\ni\nt\nn\ni\ntme S2-SN38\n0 m In in cuba\n2\nti\n0\no n\nm\nt\ni\ni\ntme\n0 2 R 4 etentio 6 n tim 8 e (min 10 ) 12 14 0 2 Re 4 tention 6 time 8 (min) 10 12 0 2 R 4 etentio 6 n tim 8 e (min 10 ) 12 14\nD E\n68000\n35600\nS1-Gem 51000\n26700\n34000\n17800\n17000\n8900\n20 min 20 min\n0 m\nI\ni\nn\nn\ncubation ti\nme\n0 m\nIn\nin\ncubation ti\nme\n0 4 8 12 16 20 0 2 4 6 8 10\nRetention time (min) Retention time (min)\nSupplementary Figure 2. Stability of the prodrugs S-CPT, S1-SN38, S2-SN38, and S1-Gem,\nS-PTX in serum-containing cell culture medium (containing 10% FBS).\n5. The limited number of time points chosen by the authors in Figure 5 renders it challenging to\ngenerate a reliable fitting curve based on the provided data.\nResponse: We thank the reviewer for this comment. The experiments have been reperformed, and\nadditional time points have been collected for the experiments shown in Fig. 5, allowing for reliable\nkinetic curve fitting. For the control compounds (C-CPT, C-SN38, and C-PTX), which do not exhibit\nreactivity toward either GSH or TrxR, the data from the initial submission were retained and are\npresented in Supplementary Figure 4. The revised figure and corresponding fitted curves are\nincluded in the updated manuscript (Fig. 5). The newly re-evaluated prodrug release and retention\ndata have also been updated in the revised manuscript (pages 7–9, highlighted in green).\n12 / 13\nA B C D\nS1-SN38 + GSH S1-SN38 + TrxR\nS-CPT + GSH S-CPT + TrxR S2-SN38 + GSH S2-SN38 + TrxR S1-Gem + GSH S1-Gem + TrxR S-PTX + GSH S-PTX + TrxR\n45 100 75\nC P T r\nel e a s e (\n%)\n1\n3\n5\nel e a s e (\n%)\n2\n5 7\n5\n0 5\nG e m r\nel e a s e (\n%)\n2\n5\n5\nel e a s e (\n%)\n1\n2\n3\n0\n0\n0\n0 0 0 0\n0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10\nTime (min) Time (min) Time (min) Time (min)\nE F G H\nS1-SN38 + GSH S1-SN38 + TrxR\nS-CPT + GSH S-CPT + TrxR S2-SN38 + GSH S2-SN38 + TrxR S1-Gem + GSH S1-Gem + TrxR 105 S-PTX + GSH S-PTX + TrxR\n100 100 100\ng\n( %) 80\ng\n( %) 75\ng\n( %) 75 ( %)\ng\n90\nn n n n\nai\nni\n60 ai\nni 50\nai\nni 50\nai\nni 75\nm m m m\nR e R e 25 R e 25 R e 60\n40\n0 0\n45\n0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10\nTime (min) Time (min) Time (min) Time (min)\nFig. 5 Reductive responsiveness of S-CPT, S1-SN38, S2-SN38, S1-Gem, and S-PTX. (A–D)\nPercentage of drug release from S-CPT (10 µM), S1-SN38 (10 µM), S2-SN38 (10 µM), S1-Gem (50\nµM), and S-PTX (20 µM) in response to GSH or TrxR. (E–H) Percentage of residual prodrug under\nthe same conditions. Reaction conditions: GSH (1 mM), TrxR (50 nM), and NADPH (200 µM) to\nsupport TrxR catalysis; all reactions conducted at 37 °C.\n13 / 13"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed all my concerns and questions comprehensively. I have no further questions.", "reviewer_3": "The authors have solved my concerns and can be accepted now."}, "author_response": ""}]

Decision letter