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. 2025 Nov 24;16:10342. doi: 10.1038/s41467-025-65244-7

Biocompatible decarboxylative coupling enabled by N-hydroxynaphthalimide esters

Hongze Liao 1,#, Zhiyou Su 1,#, Jia Zhang 1,#, Shuai Liu 2, Meng-Jun Fan 3, Shu-Ping Wang 1, Fan Sun 1, Weihua Jiao 1, Lei Li 1, Hong-Rui Zhu 1, Yong-Jun Zhou 1, Zhizhen Zhang 2, Hou-Wen Lin 1,4,✉
PMCID: PMC12644656  PMID: 41285715

Abstract

This study develops a catalyst-free, light-induced decarboxylative coupling utilizing N-hydroxynaphthalimide esters (NHNI esters) as multifunctional reagents, acting as photosensitizers, oxidants, and redox-active esters. In cell lysates, this method efficiently enabled Csp3-Csp3 coupling reactions with silyl enol ethers or Michael acceptors. Substrates with donor-acceptor (D-A) frameworks are found to significantly enhance reaction efficiency, with photophysical characterization showing that efficient D-A framework promotes the formation of charge transfer state (CT), thus improving coupling efficiency. The system was further demonstrated in peptide labeling and DNA-encoded library (DEL) technology for the introduction of diverse alkyl groups. Notably, the successful application of this methodology in live cells is confirmed through confocal microscopy and HRMS, providing an efficient tool for chemical modification in biological systems.

Subject terms: Synthetic chemistry methodology, Chemical modification

Achieving selective and precise modification of macromolecules under biocompatible conditions (such as aqueous solutions, mild temperatures, and neutral pH) remains a significant technical challenge. Here, the authors develop a photoinduced coupling reaction based on N-hydroxy naphthalimide esters, which is effective in biologically relevant conditions.

Introduction

Chemically modified biomacromolecules (such as proteins, nucleic acids, and carbohydrates) play a crucial role in chemical biology1,2, with wide applications in disease diagnostics and high-throughput drug screening3,4. However, achieving selective and precise modification of these macromolecules under biocompatible conditions (such as aqueous solutions, mild temperatures, and neutral pH) remains a significant technical challenge2,5. In particular, performing efficient conjugation reactions in complex cellular environments continues to be highly demanding. Recently, light-induced bio-conjugation reactions have garnered attention due to their mildness, non-invasiveness, and spatiotemporal controllability6–10. Nevertheless, current photocatalytic techniques face issues such as poor biocompatibility, high redox potentials, and sensitivity to catalyst loading in complex biological environments, limiting their practical application scope and efficiency11–13. Therefore, developing visible light-induced coupling reactions that not only perform efficiently under biocompatible conditions but are also compatible with complex cellular environments remains a critical challenge.

Naphthalimide has been widely used in optoelectronic materials and biological imaging probes due to its strong visible-light absorption and excellent biocompatibility in live-cell environments14–16. In particular, those bearing donor–acceptor (D–A) frameworks exhibit efficient intersystem crossing (ISC), generating long-lived triplet excited states that have been reported to display room-temperature phosphorescence under specific conditions17–19. These triplet states possess strong oxidative potential and can initiate single-electron transfer (SET) processes with suitable reductants20, enabling diverse visible-light-driven transformations under mild conditions. Collectively, these photophysical properties suggest that naphthalimide-based chromophores could participate in visible-light-induced radical processes under biologically relevant conditions.

Among redox-active esters, N-hydroxyphthalimide (NHPI) esters have been widely employed in visible-light-induced decarboxylative functionalization. Nevertheless, these reactions typically require additional metal-based photocatalysts or preassembled electron donor–acceptor (EDA) complexes to facilitate single-electron transfer (SET) processes, which limits their compatibility with complex biological environments21–23. Notably, Mendoza and co-workers demonstrated that NHPI esters could be applied in DNA-encoded library (DEL) chemistry using exogenous NADH as a reductant to promote Giese-type alkylation in aqueous media24. While this strategy supports biomolecular conjugation in aqueous settings, its reliance on external reductants and incompatibility with cellular contexts remain significant limitations.

In this work, we establish a catalyst-free, visible-light-induced decarboxylative coupling using N-hydroxynaphthalimide (NHNI) esters as multifunctional reagents, efficiently enabling Csp3–Csp3 coupling reactions in cell lysates (Fig. 1a). Notably, substrates with donor-acceptor (D-A) frameworks significantly enhance reaction efficiency, and photophysical characterization reveals that the D-A framework promotes charge transfer, improving coupling outcomes. The robust reactivity of NHNI esters in biological environments, without the need for external reductants, highlights their practical utility and biocompatibility.

Fig. 1. Blueprint of biocompatible decarboxylative coupling of NHNI esters and condition optimization.

Fig. 1

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a A blueprint for the decarboxylative coupling using NHNI esters as photosensitizers, oxidants, and redox-active esters. Under blue LED irradiation at r.t. in cell lysates, NHNI esters absorb light and undergo ISC to generate an excited triplet state, which then participates in SET with a reductant to form an alkyl radical. This radical subsequently couples suitable partners to produce the final product. This catalyst-free reaction is compatible with cell environments, performed at high dilution, and applicable to peptide and DNA derivatives. b Condition optimization for the decarboxylative alkylation reaction. The efficiency was examined with three alkyl substituents (with or without D-A frameworks) and in various reaction environments, including PBS, DMSO, DMSO/PBS buffer, Tris buffer, and cell lysates (E. coli (BL21) and human epithelial cells (HEK293)). Control experiments were performed with different LED wavelengths (390 nm and 423 nm), varying NHNI ester concentrations (0.1 mmol and 0.05 mmol) and different reaction time. The results highlight the high efficiency of this reaction in biocompatible environments, particularly in cell lysates, with yields up to 91%. r.t., room temperature, NI naphthalimide, NHNI ester N-hydroxynaphthalimide ester, NADH nicotinamide adenine dinucleotide, ISC intersystem crossing, SET single-electron transfer, D-A donor-acceptor, PBS phosphate-buffered saline, DMSO dimethyl sulfoxide, Tris tris(hydroxymethyl)aminomethane, TMS trimethylsilyl, E. coli (BL21) Escherichia coli BL21 strain, HEK293 human embryonic kidney 293 cells.

Results and discussion

Along these lines, we envision that N-hydroxy naphthalimide (NHNI) esters can overcome these limitations owing to their intrinsic visible-light absorption and favorable excited-state properties. Upon photoexcitation, NHNI esters are expected to undergo intersystem crossing (ISC) to access oxidizing triplet states (E1/2* = +1.6 V vs. SCE, detail in Table S3), which could engage in single-electron transfer (SET) with reductive partners such as silyl enol ethers or even endogenous NADH. The resulting radical anions may undergo decarboxylation to generate alkyl radicals capable of coupling with electron-deficient acceptors (For detailed mechanism, See Figs. S15, S19). This mechanism suggests the potential for establishing a catalyst-free, visible-light-induced alkylation strategy that operates under aqueous and even cellular conditions. Such a system could provide a broadly applicable platform for biocompatible radical conjugation, with potential utility in DNA-encoded library synthesis, peptide modification, and live-cell labeling.

To validate this approach, NHNI esters 1, 2, and 3—synthesized from cyclohexanecarboxylic acid, isonipecotic acid, and gemfibrozil, respectively—were reacted with excess silyl enol ether 4 under visible light in Tris buffer (pH 7.5) at room temperature. These heterogeneous reactions, carried out in opaque suspensions, smoothly yielded the target ketones 5, 6, and 7 with yields of 52%, 81%, and 87%, respectively. All three NHNI esters reached optimal yields after 6 h, with shorter reaction times (1 or 3 h) resulting in lower yields. Notably, ester 3 exhibited the fastest reaction rate, reaching 75% conversion within 1 h, whereas esters 1 and 2 required 6 h for complete conversion (Fig. 1b). The high efficiency of these reactions can be attributed to the “on-water” model: vigorous stirring of silyl enol ether 4 in water produces microdroplets that aggregate with NHNI esters through hydrophobic interactions, forming high-concentration reactive clusters that lower the activation energy of the redox reaction25–28. Moreover, when organic co-solvents were introduced, these clusters were disrupted, weakening hydrophobic interactions and significantly reducing the yields. Compared to previous work on the coupling of NHPI esters and silyl enol ethers, which employed PPh3/NaI29 or Ir catalysts30 under homogeneous conditions and required over 10 h, our approach offers a more efficient reduction in activation energy, as demonstrated by the significantly shorter reaction time. This phenomenon further underscores the advantages of this catalyst-free system in aqueous media, highlighting its potential for efficient coupling reactions under biocompatible conditions.

The significant differences in reaction efficiency among NHNI esters can be attributed to their varying singlet-triplet conversion efficiencies. Naphthalimide moieties, due to their π-π* transition characteristics, typically exhibit a large singlet-triplet energy gap (ΔE_ST), resulting in low intersystem crossing (ISC) efficiency31,32. However, the incorporation of a donor-acceptor (D-A) framework can effectively reduce the ΔE_ST by introducing intramolecular charge transfer (ICT) states, thereby enhancing singlet-triplet conversion efficiency33–35. As shown in Fig. 2b, the emission spectrum of ester 1 displays both fluorescence and phosphorescence at approximately 420 nm and 540 nm, respectively, though the phosphorescence ratio remains relatively low (Fig. 2c). In contrast, esters 2 and 3 exhibit sequentially increasing phosphorescence ratios, despite their photoluminescence spectra being similarly shaped. Additionally, the phosphorescence lifetimes of all three esters were extended to sub-second durations (Fig. 2d), which is essential for facilitating efficient bimolecular redox reactions. These experimental results indicate a strong correlation between the increased phosphorescence ratios, extended lifetimes in esters 2 and 3, and their higher reaction yields, suggesting that photophysical properties play a pivotal role in determining reaction efficiency.

Fig. 2. Photophysical properties and molecular structures of NHNI esters.

Fig. 2

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a Chemical structures of NHNI esters 1, 2, and 3. b Prompt PL spectra of NHNI esters 1, 2, 3 at 77 K. c Delayed PL spectra (10 ms) at 77 K. d Average phosphorescence lifetimes (τP) of NHNI esters 1, 2, 3. e Highest occupied and lowest unoccupied NTOs of NHNI esters based on time-dependent DFT calculations for the S1 state, with calculated dipole moments (Δμ) shown in the inset. The structural and photophysical properties of NHNI esters 1, 2, and 3 illustrate the relationship between singlet-triplet conversion efficiency and reaction yields. The D-A framework in esters 2 and 3 enhances phosphorescence and singlet-triplet conversion efficiency, contributing to improved reactivity under biocompatible conditions. All spectral intensities are plotted in arbitrary units (a.u., arb. units). Colors in panels b–d correspond to NHNI esters 1 (blue), 2 (green), and 3 (purple). NHNI ester N-hydroxynaphthalimide ester, NBoc tert-butoxycarbonyl, PL photoluminescence, τP phosphorescence lifetime, Φp phosphorescence quantum yield, Φf fluorescence quantum yield, NTO natural transition orbital, Δμ(S0–S1) change in dipole moment between S0 and S1 states, D–A donor–acceptor.

To further validate these findings, we conducted TDDFT calculations. The results revealed that in ester 1, the highest occupied natural transition orbital (NTO) completely overlaps with the lowest unoccupied NTO, indicating a localized transition and resulting in low singlet-triplet conversion efficiency. In contrast, for esters 2 and 3, the highest occupied NTO is confined to the carboxyl group, while the lowest unoccupied NTO is delocalized over the naphthalimide moiety, forming a distinct charge-transfer (CT) state (Fig. 2e). The introduction of this CT state effectively reduces ΔE_ST, significantly improving the singlet-triplet conversion efficiency. As the spatial separation between orbitals increases in esters 2 and 3, their phosphorescence-to-fluorescence ratios also gradually increase. Additionally, the donor-acceptor (D-A) framework enhances the dipole moments in the CT states of 2 and 3, stabilizing them in highly polar solvents26,36,37. These computational results are highly consistent with the experimental data, revealing the intricate relationship between molecular structure and photophysical properties, and indicating that NHNI esters with efficient donor-acceptor (D-A) framework facilitate the formation of charge-transfer (CT) states, thereby enhancing their suitability for efficient decarboxylative fragmentation.

To further improve reaction yields and validate their effectiveness under biocompatible conditions, we screened various reaction parameters for NHNI esters (see Table S1 for detail). Initial light source screening indicated that a wavelength of 390 nm provided better reaction efficiency, while 423 nm also supported the reaction, leading us to select broad-spectrum blue light as the optimal light source. NHPI esters, lacking visible-light absorption, did not undergo reaction. Subsequently, screening of reaction media revealed that pure water, Tris buffer, and PBS buffer were all capable of supporting the reaction. Under biocompatible conditions, we employed E. coli (BL21 strain) and human epithelial cells (HEK293) lysates as reaction media. The results demonstrated smooth progress in both cell lysate environments, confirming the broad applicability of the reaction in biological systems. Furthermore, under low concentration conditions (0.05 mmol), NHNI ester 3 completed the reaction in E. coli lysate within just 30 minutes. In contrast, photocatalytic systems, which struggle to achieve efficient bimolecular single-electron transfer reactions in low-concentration and complex biological environments, could not match this level of efficiency38. NHNI esters, however, maintained high efficiency under these same conditions, underscoring their unique advantage. Notably, oxygen inhibition of the reaction efficiency was consistent with a triplet-state reaction mechanism, as oxygen quenches triplet-state intermediates. Through systematic optimization of the light source, reaction medium, and environmental conditions, we confirmed the high coupling efficiency of NHNI esters under biocompatible conditions, especially at low concentrations, providing strong evidence of their suitability for application in complex biological systems.

To verify the broad substrate compatibility of NHNI esters, we conducted decarboxylative coupling reactions under two conditions: Condition A simulated biocompatible environments using E. coli (BL21 strain) cell lysates, while Condition B involved large-scale preparation in Tris buffer at higher concentrations. The experimental results demonstrated that a variety of primary alkyl carboxylic acid derivatives were efficiently converted to their corresponding ketones, including alkyl chains (8, 11), alkenes (9), alkynes (10), benzyl (12-14), ethers (15), amide (16), esters (17), halides (18) and protected amine (54). Additionally, secondary radical precursors, both in cyclic alkanes (5, 6, 21, 50) and acyclic chains (20), were successfully transformed. Tertiary sites also effectively generated quaternary centers in systems such as bridgehead (23), cyclic (53), and chain (7, 22) structures (Fig. 3a). The application of this reaction system in the late-stage alkylation of natural products and pharmaceuticals was validated by the successful modification of biotin (25), drimanic acid (26), dehydrocholic acid (27), and etientic acid (28). Specifically, NHNI esters containing donor-acceptor (D-A) frameworks showed significantly higher reaction yields under biocompatible conditions, particularly in large-scale aqueous preparations. The incorporation of D-A frameworks effectively enhanced intramolecular charge-transfer (ICT) properties, thereby improving singlet-triplet conversion efficiency and significantly increasing yields. Moreover, aryl ketone derivatives with varying electronic or steric properties also exhibited full compatibility with this method (29-48) (Fig. 3b). However, alkyl ketone-derived silyl enol ethers failed to react under the same conditions, presumably due to an incompatibility in redox potentials.

Fig. 3. Substrate scope for the biocompatible decarboxylative coupling of NHNI ester with silyl enol ethers.

Fig. 3

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a NHNI esters without D-A framework. b Silyl enol ethers. c NHNI esters with D-A framework. The reactions were conducted under Condition A (E. coli (BL21 strain) cell lysate) with NHNI esters (0.05 mmol) and silyl enol ether (0.1 mmol), and under Condition B (Tris buffer) with NHNI esters (0.2 mmol) and silyl enol ether (0.4 mmol), using blue LEDs, and r.t. for 0.5–6 h. Yields were determined by 1H NMR using an appropriate internal standard. NI naphthalimide, NHNI ester N-hydroxynaphthalimide ester, TMS trimethylsilyl, r.t. room temperature, D–A donor–acceptor, Boc tert-butoxycarbonyl, Fmoc fluorenylmethoxycarbonyl, Cbz benzyloxycarbonyl, Ac acetyl.

Building on these encouraging results, we further investigated the reactivity of D-A framework substrates, focusing on NHNI esters derived from α-hydroxy acids and amino acids (Fig. 3c). All these substrates demonstrated efficient reactions under biocompatible conditions, fully showcasing the broad applicability of this method. The results also revealed a direct correlation between reaction efficiency and the strength of the D-A characteristics of the substrates. Substrates with strong D-A characteristics, such as phenoxy-protected lactic acid (56), significantly outperformed their methoxy-protected counterparts (55) in terms of reaction yield, while shortening reaction times from 4 h to 1 h. Similarly, among α-amino acids with various carbamate protecting groups, those with fluorenylmethyloxycarbonyl (Fmoc) protection (59) achieved the highest yields. Particularly, the trimethoxyphenylacetic acid (52), possessing strong D-A character, demonstrates low reaction yields. Mass spectrometry analysis revealed that this is due to the high stability of the benzyl radical, which undergoes self-coupling and hydrogen abstraction, leading to byproduct formation (Fig. S17). This biocompatible alkylation method not only successfully handled relatively unstable substrates, such 1-aminocyclobutane carboxylic acid (60) and methionine (64), but also produced the desired β-amino esters (69, 70) from silyl ketene acetals, yielding valuable target products. Although the loss of stereochemical information of chiral amino acids is an inherent consequence of the free-diffusing (out-of-cage) mechanism, these results demonstrate that D-A framework substrates enhance intramolecular charge-transfer (ICT) properties, significantly improving intersystem crossing (ISC) efficiency, thereby directly increasing reaction yields.

Classical photocatalytic alkyl coupling reactions often compromise protein structures due to the strong redox characteristics of the catalysts, resulting in poor biocompatibility and rendering them unsuitable for alkylation modifications of peptides or proteins4,7,8,39. In contrast, the pronounced donor-acceptor (D-A) characteristics of peptide derivatives, combined with our developed biocompatible alkylation reactions, allow efficient reactions with these large-molecule substrates in complex biological systems. When dipeptide derivatives were reacted in cell lysates, they were efficiently converted into their corresponding ketones (71, 72), resulting in a diastereomeric mixture. Furthermore, we successfully coupled proteinogenic amino acids (73, 75, 77) with silyl enol ether 4 on solid-phase resin, introducing ketone groups and further demonstrating the broad applicability of this method for late-stage peptide modifications (Fig. 4). This reaction not only exhibited high precision and regioselectivity in peptide labeling but also showed strong potential for extension to protein substrates. As a result, this technique provides an effective tool for targeted biomacromolecule modifications, holding significant promise for applications in chemical biology.

Fig. 4. Biocompatible decarboxylative coupling of peptides in solid-phase synthesis.

Fig. 4

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Three peptides 73, 75, 77 containing carboxyl groups were synthesized on solid-phase resin. The carboxyl groups were in situ activated and converted to NHNI esters, followed by reaction with silyl enol ether 4 in 50% DMSO/Tris buffer. After the reaction, the peptides were cleaved from the resin to yield 74, 76 and 78 bearing ketone functional groups. This demonstrates the applicability of solid-phase synthesis for decarboxylative alkylation of peptides, providing a general method for peptide modification. NHNI ester N-hydroxynaphthalimide ester, TMS trimethylsilyl, DMSO dimethyl sulfoxide, SPPS solid-phase peptide synthesis.

The Giese reaction, which involves radical addition to α,β-unsaturated carbonyl compounds (Michael acceptors), offers significant potential for late-stage modification of biomacromolecules, particularly for site-specific labeling and coupling22. Building on the results that NHNI esters could engage in efficient radical coupling with silyl enol ethers in cell lysates under visible light, we sought to explore whether this reactivity could be extended to Michael acceptors. Additionally, inspired by the study from Chowdhury et al., which employed exogenous NADH to enable NHPI-mediated Giese-type coupling in aqueous media24, we hypothesized that endogenous NADH in cell lysates might also serve as a compatible reductant for NHNI esters—eliminating the need for added reductants or engineered EDA complexes. Given the strong oxidizing character of photoexcited NHNI esters, we further reasoned that these esters might enable more efficient decarboxylative alkylation than NHPI-based systems, particularly under mild, aqueous, and catalyst-free conditions. This would further demonstrate the versatility and biocompatibility of the NHNI ester platform.

To validate this hypothesis, we performed the reaction between NHNI ester 3 and acrylate acceptor using E. coli (BL21 strain) cell lysates as the reaction medium, utilizing the naturally abundant NADH in the cells as the reducing agent40. Under blue light irradiation, the decarboxylative coupling product 79 was successfully generated with high efficiency. Additionally, when we used ester S49, which lacks a donor-acceptor (D-A) framework, as the reactant, the reaction performed poorly in aqueous conditions, but the introduction of DMSO as a co-solvent enabled successful formation of the target product 89. Notably, when the cell lysates were exposed to air for 24 h, fully oxidizing the NADH, the reaction failed to produce the desired ketone product. Adding three equivalents of NADH to the PBS buffer successfully restored the reaction, confirming NADH’s critical role as a reductant (see Table S2 for detail). To further confirm the direct excitation of NHNI esters is the primary mechanism driving the reaction, we explored replacing NADH with silyl enol ether to investigate this cascade reaction and successfully obtained the three-component coupling product (Fig. S21). Importantly, the corresponding NHPI esters exhibited comparable reactivity in organic solvents, but lost activity in aqueous buffer and afforded only trace products in cell lysates, whereas NHNI esters retained high reactivity and clean conversions, underscoring their superior biocompatibility (see Table S4 for detail). UV-vis spectra with S43 and NADH (Fig. S20) also indicated a weak EDA effect. While the EDA effect and NADH photoexcitation pathway cannot be fully excluded based on these results, we propose that the direct excitation of NHNI esters plays a crucial role in driving the reaction. Unlike the reaction between NHNI esters and silyl enol ethers, the Giese-type reaction proceeded smoothly in the presence of air, possibly due to the strong reductive environment provided by NADH, which protected the triplet-state intermediate of the NHNI esters from oxygen quenching. This feature significantly enhances the potential of this method for biomacromolecule coupling reactions.

When evaluating the efficiency of Csp3-Csp3 coupling reactions between multiple NHNI esters and various Michael acceptors, we set up two reaction conditions: Condition A simulated biocompatible environments using cell lysates to assess the applicability of NHNI esters in complex biological systems, while Condition B involved 50% DMSO in Tris buffer with three equivalents of NADH for large-scale preparation at higher concentrations. As expected, NHNI esters with D-A frameworks, such as derivatives from amino acids (70-84), peptides (85) and hydroxy acids (87-88) exhibited excellent to good reaction efficiency in cell lysates, achieving the desired yields (Fig. 5a). Unlike previously reported decarboxylative coupling based on EDA complexes, which primarily focused on alkyl-substituted carboxylic acids24,41, this method provides an alternative chemical tool for the decarboxylative coupling of α-heteroatom carboxylic acids. Similarly, tertiary (89-93), secondary (94-94) and primary (97-101) alkyl carboxylic acid derivatives exhibited broad adaptability under both conditions (Fig. 5b). The system showed high compatibility with various Michael acceptors, including ethyl ester (102), sterically hindered unsaturated esters (103, 104, 112) and unsaturated alkenes containing sulfone (105, 111), amide (106, 110), ketone (107), and cyano (108) groups, further underscoring the broad applicability and potential value of this biocompatible coupling reaction (Fig. 5c).

Fig. 5. Substrate scope for the biocompatible decarboxylative coupling of NHNI ester with Michael acceptors.

Fig. 5

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a NHNI esters with D-A framework. b NHNI esters without D-A framework. c Michael acceptors. The reactions were conducted under Condition A (50% DMSO/ (E. coli (BL21 strain) cell lysate) with NHNI esters (0.1 mmol) and Michael acceptors (0.1 mmol), and under Condition B (50% DMSO/Tris buffer) with NHNI esters (0.5 mmol), Michael acceptors (1 mmol) and NADH (1.5 mmol), using blue LEDs, air, and r.t. for 0.5–4 h. Yields were determined by 1H NMR using an appropriate internal standard. NADH, nicotinamide adenine dinucleotide; EWG, electron-withdrawing group. aYield obtained in a gram-scale reaction. NHNI ester N-hydroxynaphthalimide ester, D-A donor–acceptor, NADH nicotinamide adenine dinucleotide, EWG electron-withdrawing group, r.t. room temperature, DMSO dimethyl sulfoxide, Tris tris(hydroxymethyl)aminomethane, E. coli (BL21) Escherichia coli BL21 strain.

DNA-encoded library (DEL) technology is a powerful tool for discovering bioactive molecules by screening vast compound libraries. However, efficiently introducing functional groups while preserving the integrity of the DNA tag remains a significant challenge42. Conventional decarboxylative and hydrogen alkylation reactions, particularly for introducing high C(sp3) content building blocks, often suffer from harsh reaction conditions and poor biocompatibility, making it difficult to meet DEL technology’s requirements for efficiency, cost-effectiveness, and operational simplicity43,44. Given the notable advantages of NHNI ester coupling reactions in terms of biocompatibility and reaction rate, we further explored their application in DEL technology. In our experiments, nine different types of NHNI esters successfully reacted with α, β-unsaturated amide-tagged DNA, efficiently introducing diverse alkyl groups (114-122) under biocompatible conditions (Fig. 6). This strategy not only improves the practicality of DEL chemistry in constructing complex molecular structures and facilitating high-throughput screening but also establishes a mechanistic basis for potential applications in more biologically relevant environments, including on-cell DEL systems4,45.

Fig. 6. On-DNA decarboxylative coupling of NHNI esters.

Fig. 6

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Reactions were performed in 50% DMSO/cell lysate with NHNI esters (0.05 mmol) and DNA-encoded library headpieces 123 (0.05 mmol) bearing α,β-unsaturated amide tags, using the endogenous NADH in the cell lysate as the reductant. The reactions were carried out under blue LED irradiation, at room temperature (r.t.) for 0.5 h. NHNI ester N-hydroxynaphthalimide ester, DMSO dimethyl sulfoxide, NADH nicotinamide adenine dinucleotide, r.t. room temperature, Boc tert-butoxycarbonyl.

To verify the feasibility of applying this methodology in living cells, we investigated the decarboxylative coupling of NHNI ester 3 with silyl enol ether 4 in live HUVEC cells (Fig. 7)46. The cells were pre-treated with compounds 3 and 4, followed by blue LED irradiation at room temperature for 0, 5, 10, and 15 min. Confocal microscopy, with an excitation wavelength of 405 nm, revealed a gradual decrease in fluorescence associated with NHNI ester 3, indicating its consumption during the reaction. After 15 min of irradiation, the cells were lysed, and the resulting lysate was analyzed by high-resolution mass spectrometry. The mass spectrum confirmed the formation of the ketone 7 (Fig. S24), providing further support for the successful decarboxylative coupling reaction in vitro. These results provide strong evidence for the applicability of this reaction in living cells and highlight the potential of this approach for studying light-induced coupling reactions in live cell environments.

Fig. 7. Decarboxylative coupling of NHNI ester 3 with silyl enol ether 4 in live cells.

Fig. 7

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a Reactions were performed in HUVEC cells with NHNI esters 3 (5 µmol) and silyl enol ether 4 (15 µmol) followed by blue LED irradiation at r.t. for 0, 5, 10, or 15 minutes. b UV-vis absorption spectrum of NHNI esters 3 (5 µmol), ketone 7 (5 µmol) and NI (5 µmol). c Representative confocal images of HUVEC cells treated with NHNI ester 3 and silyl enol ether 4. The cells were incubated with ester 3 and ether 4 for 30 min at 37 °C, then irradiated with blue LEDs for 0, 5, 10, or 15 min. Experiments were independently repeated three times with consistent results. Scale bar: 50 μm. NHNI ester N-hydroxynaphthalimide ester, NI naphthalimide, r.t. room temperature, UV–vis ultraviolet–visible, HUVEC human umbilical vein endothelial cell.

In conclusion, this study develops a catalyst-free, light-induced decarboxylative coupling reaction based on NHNI esters, achieving efficient reactions with both silyl enol ethers and α, β-unsaturated carbonyl compounds. NHNI esters demonstrate broad substrate compatibility in these reactions, particularly showing significantly enhanced reaction efficiency with substrates containing D-A frameworks. Photophysical characterization reveals that the D-A framework promoted the formation of charge transfer (CT) state, thereby enhancing coupling efficiency—an important innovation of this research. The Giese reactions, induced by either exogenous or endogenous NADH, proceed smoothly in the presence of air, further broadening the scope and applicability of the reaction. Furthermore, this system is successfully applied to the introduction of diverse alkyl groups in peptide labeling and DNA-encoded library (DEL) technology, providing an efficient tool for complex molecular construction. Notably, the efficacy of this methodology in live cells is confirmed by confocal microscopy and HRMS, thereby emphasizing its potential for molecular modifications in complex biological systems. This study not only overcomes limitations of traditional photocatalytic systems but also provides new theoretical foundations and practical applications for molecular modification in chemical biology and drug development.

Methods

General procedure for biocompatible decarboxylative coupling In a 10 mL Schlenk tube equipped with a stirring bar, an NHNI ester (0.05 mmol) and a silyl enol ether (0.1 mmol) were combined with E. coli (BL21 strain) cell lysate (2.0 mL). The reaction mixture was stirred under blue LED irradiation (427 nm) at room temperature for 0.5–2 h. After irradiation, the mixture was extracted with ethyl acetate, and the crude product was purified by thin-layer chromatography, affording the title compounds.

Live-cell confocal imaging HUVEC cells were incubated with NHNI ester 3 (5 μmol) and silyl enol ether 4 (15 μmol) for 30 minutes at 37 °C, followed by irradiation with blue LEDs (3 W) for different time intervals (0, 5, 10, and 15 min). After irradiation, cells were washed with PBS and imaged using a Leica confocal microscope (excitation at 405 nm, emission at 430–480 nm). All confocal imaging experiments were independently repeated three times, and representative images are shown in the figures.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information (26.8MB, pdf)
Reporting Summary (109.1KB, pdf)
Transparent Peer Review file (2.3MB, pdf)

Acknowledgements

We are grateful to the Science and Technology Commission of Shanghai Municipality and Prof Dr. Wei-Jun Zhao (School of Chemistry and Molecular Engineering, East China University of Science and Technology) for assistance with prompt and delayed prompt photoluminescence spectroscopic assistance. This research was funded by the National Key Research and Development Program of China (2022YFC2804100 to H.-W.L); The National Natural Science Foundation of China (22137006 to H.-W.L); The Innovative Research Team of high-level local universities in Shanghai (SHSMUZDCX20212702 to H.-W.L); The Shandong Provincial Natural Science Foundation (ZR2021ZD29 to H.-W.L); the SciTech Funding by CSPFTZ Lingang Special Area Marine Biomedical Innovation Platform (to H.-W.L.), and a Collaborative Research Project (RJYK19-03, to H.-W.L.).

Author contributions

H.L., Z.S., and H.-W.L. conceived and designed the study. Experimental investigations were performed by H.L., Z.S., J.Z., S.L., M.F., S.W., F.S., W.J., L.L., H.-R.Z., and Y.-J.Z. Data analysis and interpretation were carried out by H.L., J.Z., and Z.-Z.Z. The manuscript was written by H.L., Z.S., and J.Z., and reviewed and revised by H.-W.L. Supervision and funding acquisition were provided by H.-W.L.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Data availability

Methods are described in the Supplementary Information. The data that support the findings of this work are available within the paper and Supplementary Information. Data supporting the findings of this manuscript are also available from the corresponding author upon request. Raw confocal microscopy images for Fig. 7c have been deposited in Figshare [10.6084/m9.figshare.30022327].

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Hongze Liao, Zhiyou Su, Jia Zhang.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-025-65244-7.

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Associated Data

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Supplementary Materials

Supplementary Information (26.8MB, pdf)
Reporting Summary (109.1KB, pdf)
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Data Availability Statement

Methods are described in the Supplementary Information. The data that support the findings of this work are available within the paper and Supplementary Information. Data supporting the findings of this manuscript are also available from the corresponding author upon request. Raw confocal microscopy images for Fig. 7c have been deposited in Figshare [10.6084/m9.figshare.30022327].

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