4‐Formyl‐N‐Methylpyridinium‐Mediated N‐Terminal Cysteine Modification/Removal Facilitates One‐Pot Multiplex Peptide Ligation

ABSTRACT

The chemical synthesis of proteins with site‐specific modifications remains a fundamental challenge in chemical biology. One‐pot peptide ligation strategies have emerged as powerful tools to enhance synthetic efficiency, primarily relying on N‐terminal cysteine (Cys) protection. However, current Cys deprotection conditions require various reagents or pH adjustments during the reaction, rendering downstream processing cumbersome. Here, a visible‐light‐mediated deprotection strategy using 2‐(N‐methylpyridinium‐4‐yl)‐thiazolidine (4‐NMP‐Thz) as a novel N‐terminal Cys‐protecting group is reported. This reaction, catalyzed by [Ru(bpy)₃]Cl₂ at physiological pH (6.0–8.0), enables smooth one‐pot multi‐segment peptide assembly. The strategy demonstrates complete orthogonality to native chemical ligation (NCL) and desulfurization conditions, eliminating the requirement for intermediate purification or pH adjustment. This methodology was used to facilitate an efficient one‐pot synthesis of a 400‐amino acid (aa) glycosylated MUC1 glycoprotein bearing 40 O‐glycosyl modifications that is difficult to prepare using previously reported techniques. The 400‐aa MUC1 significantly enhanced antigenic immunogenicity compared with shorter MUC1 glycopeptides. This streamlined approach establishes a robust platform for the construction of complex post‐translationally modified proteins.

A thiazolidine‐type photoprotecting group, 4‐NMP‐Thz, was flexibly introduced into peptide segments and shown to be fully compatible with native chemical ligation‐desulfurization, and Ru(II)‐mediated photocleavage. This strategy enables smooth one‐pot multiplex peptide ligation. The utility of this approach was demonstrated by the streamlined synthesis of MUC1 tandem‐repeat polypeptides, highlighting its potential for the chemical synthesis of complex proteins.

1. Introduction

Chemically synthesized proteins overcome the inherent limitations associated with biological expression systems by enabling precise installation of post‐translational modifications and atomic‐level control over protein structure [1, 2, 3, 4, 5]. This approach typically employs a modular assembly strategy, where synthetic peptides prepared via solid‐phase peptide synthesis (SPPS) are coupled through highly chemoselective ligation reactions [6, 7, 8, 9, 10]. Among these strategies [11, 12, 13, 14, 15], native chemical ligation (NCL) is a robust and reliable method, facilitating the conjugation of unprotected peptides via N‐terminal cysteine (Cys) and C‐terminal thioester functionalities while maintaining exceptional structural fidelity [16, 17, 18, 19]. However, subsequent purification relies on reverse‐phase high‐performance liquid chromatography (HPLC), resulting in significant reductions in peptide recovery and an increase in preparation time due to time‐consuming lyophilization steps. In contrast, one‐pot ligation significantly enhances synthetic efficiency by eliminating the requirement for intermediate purification, streamlining the reaction, and improving overall yield compared with stepwise approaches [20, 21]. Among one‐pot ligation strategies, temporary protection of the N‐terminal amine or thiol of Cys residues is a common approach, as these groups are typically incorporated as protected building blocks during SPPS (Scheme 1A). For instance, acetamidemethyl (Acm) [22], trifluoroacetamidemethyl (Tfacm) [23], 6‐nitroveratryl (NV) [24], 7‐(piperazin‐1‐yl)‐2‐(methyl)quinolinyl (PPZQ) [25] and picolyl group (Pic) [26] for the temporary protection of thiol, the 4‐(dimethylamino)‐phenacyloxycarbonyl (Mapoc) [27], p‐boronobenzyloxycarbonyl (Dobz) [28], allyloxycarbonyl (Alloc) [29] and 9‐fluorenylmethyloxycarbonyl (Fmoc) [30] for the temporary protection of amines have been developed and utilized by various groups in one‐pot peptide ligations.

SCHEME 1.

N‐terminal Cys protecting groups used in one‐pot peptide ligation strategies. (A) N‐terminal Cys protecting strategies enable one‐pot ligation. (B) Strategies for N‐terminal thiazolidine (Thz) deprotection. (C) This study: 4‐NMP‐Thz‐assisted one‐pot multi‐step ligation strategy for protein synthesis.

Compared with these protecting groups for single amine or thiol, commercially available Boc‐L‐thiazolidine‐4‐carboxylic acid (Boc‐Thz‐OH) is the most widely used Cys surrogate in one‐pot ligation (Scheme 1B). At pH 4.0, the Thz is deprotected by aldehyde scavengers such as methoxyamine [31, 32], 2‐amino‐5‐methoxy‐N′, N′‐dimethylbenzohydrazide (AMDBH) [33], or O‐benzylhydroxyl‐amine [34]. After deprotection, the reaction buffer pH requires adjustment to ∼7.0 to facilitate the subsequent NCL reaction. Moreover, Thz removal can be achieved with heavy metals, such as excessive Cu or catalytic Pd [35, 36]. Alternatively, the photochemical “traceless removal” of the Thz functionality provides an appealing solution for N‐terminal Cys release under physiological pH, featuring rapid reactivity, orthogonality to traditional Cys protecting groups, and negligible workup requirements. Despite their utility, photolabile protecting groups (PPGs) such as PPZQ, Mapoc, and Nv‐type encounter limitations resulting from their structural complexity. PPZQ preparation proceeds through eight steps, and its installation on peptides lacks thiol‐specific reactivity, further restricting its application. Mapoc and Nv‐type are typically incorporated during SPPS, and the nitro group of Nv‐type is incompatible with NCL desulfurization conditions. Moreover, cleaving these PPGs typically necessitates high‐energy ultraviolet (UV) irradiation (λ = 300∼400 nm), posing risks of biomolecular damage and exhibiting limited penetration capability. Therefore, an easily accessible Thz scaffold PPG that can be readily introduced via building blocks or late‐stage modification, while being cleavable under NCL reaction conditions in the presence of visible light and compatible with NCL desulfurization conditions, remains elusive.

In this report, the utility of a photocleavable Thz scaffold, 2‐(N‐methylpyridinium‐4‐yl)‐thiazolidine (4‐NMP‐Thz), is demonstrated for N‐terminal Cys protection in one‐pot ligation (Scheme 1C). Readily prepared 4‐NMP‐Thz is incorporated into peptides either as a presynthesized building block or via late‐stage modification. Crucially, the photoredox deprotection conditions for 4‐NMP‐Thz are fully compatible with NCL, eliminating the requirement for buffer exchange. Furthermore, traceless 4‐NMP‐Thz removal is achieved within 30 min of exposure to visible light across a broad pH range (6.0–8.0) in aqueous buffers. Notably, 4‐NMP‐Thz is fully compatible with NCL desulfurization. These combined features enable the efficient one‐pot assembly of large, complex proteins, and the robustness of this approach is exemplified through the one‐pot synthesis of histone H3.2 and glycosylated MUC1 containing 400 amino acids (aa) with Tn (GalNAcα‐O‐Thr) and STn (Neu5Acα2‐6GalNAcα‐O‐Thr) antigens, employing orthogonal peptide modification, NCL desulfurization, and photolytic deprotection. Relevant biological assays indicate that the synthesized MUC1 significantly increased the immunogenicity of the antigenic protein. This strategy is practical and convenient, and is expected to facilitate the broad application of 4‐NMP‐Thz in protein chemical synthesis and biomolecule modification.

2. Results and Discussion

2.1. NMP‐Based PPG for N‐Terminal Cys Protection

To design PPG for N‐terminal Cys protection that enables one‐pot peptide ligation, two criteria must be satisfied: (1) the PPG must be readily installable via SPPS or late‐stage modification; (2) it must undergo traceless removal under mild conditions, with deprotection reagents fully compatible with one‐pot NCL and subsequent desulfurization steps. To address these challenges, we explored the 4‐NMP‐Thz group for N‐terminal Cys protection. Notably, pioneering studies by Falvey, Boncella, and coworkers demonstrated that N‐alkylpicolinium‐based PPGs have been used to cage carboxylic acids and amines [37, 38, 39]. Recently, Pic‐derived moieties were shown to serve as effective protecting groups for diverse functional groups, including thiols, amines, carboxylic acids, hydroxyl groups, and phosphoryl groups, with applications in peptide side‐chain and α‐amino groups protection during SPPS. Crucially, the Pic group is efficiently cleaved by photoredox catalysis via spin‐center‐shift C−X bond cleavage (see Supporting Information page S36 for the proposed mechanism, Scheme S1) [26, 40, 41], enabling TFA/piperidine‐free peptide synthesis [42, 43]. We hypothesized that the 4‐NMP‐Thz scaffold would fulfill all the aforementioned criteria. Therefore, this study was initiated to expand the toolkit for N‐terminal Cys protection and one‐pot protein ligation.

Initially, (4R)‐N‐Boc‐2‐(N‐methylpyridinium‐4‐yl)‐thiazolidine‐4‐carboxylic acid (1, Boc‐4‐NMP‐Thz‐OH) is readily synthesized from Cys via three steps at a 10‐gram scale (Supporting Information page S5). Boc‐4‐NMP‐Thz‐OH 1 is introduced to peptides by SPPS, providing the desired peptide 2a with an N‐terminal 4‐NMP‐Thz (Scheme 2A). Furthermore, 4‐NMP‐Thz is also introduced by late‐stage modification. As shown in Scheme 2B, peptide 3 was treated with commercially available 4‐formyl‐N‐methylpyridinium benzenesulfonate (NMP‐a) in guanidinium hydrochloride buffer (6 M GND•HCl, 200 mM Na₂HPO₄, pH 4.0) at 37 °C for 1 h, providing the desired peptide 2a in quantitative yield with excellent selectivity. Installation during SPPS ensures precise incorporation but may reduce coupling efficiency with sterically hindered residues. In contrast, late‐stage modification provides greater flexibility and is applicable to the N‐terminal Cys of fully synthesized peptide fragments to achieve complete conversion. Crucially, NMP‐a exhibits exceptional selectivity, modifying only the N‐terminal Cys while leaving all other residues, including internal Cys residues, unmodified (Supporting Information pages S15 and S19) [44]. This enables efficient and selective N‐terminal Cys protection in proteins.

SCHEME 2.

Installation strategies for 4‐NMP‐Thz. (A) Incorporation of 4‐NMP‐Thz by SPPS. Conditions of SPPS: Boc‐4‐NMP‐Thz‐OH (1, 2.0 equiv), DIC (2.2 equiv), Oxyma (2.5 equiv), DMF, 45°C, 2 h. (B) Late‐stage installation and scope of N‐methylpyridinium substrates. Standard reaction conditions: peptide (2 mM, 1.0 equiv), NMP‐a (5.0 equiv), 6 M GND•HCl, 200 mM Na₂HPO₄, pH 4.0, 37°C, 60 min. ^aLiquid chromatography–mass spectrometry (LCMS) yield was estimated via UV absorption (230 nm) as the modified peptide product peak versus the sum of all peptide peaks (analyzed by MS) of the crude reaction mixture. ^bIsolated yields were provided in brackets. ^cThe reaction was performed at pH 3.0.

Moreover, a broad range of NMP substrates was examined to evaluate the protection–deprotection efficiency of individual substrates. Relocation of the formyl group from C‐4 (NMP‐a) to C‐3 (NMP‐b) of pyridine had a negligible impact on reactivity, whereas placement at C‐2 (NMP‐c) slightly reduced efficiency, likely because of steric interference from the N‐methyl substituent. Subsequently, substituent effects at the 2‐position of 4‐formyl‐N‐methylpyridinium were examined (Scheme 3B). An electron‐donating group (NMP‐d) showed minimal influence, while electron‐withdrawing groups (NMP‐e, f) markedly reduced the yield. The removal of the N‐methyl group had a negligible effect, as 4‐formylpyridine (Py‐g) displayed reactivity comparable to that of NMP‐a. These results indicate that electron‐withdrawing groups at the pyridine 2‐position reduce the electron density of the N‐methylpyridinium core, hindering the key dehydration step.

SCHEME 3.

Photocatalytic deprotection of 4‐NMP‐Thz. (A) Optimization of deprotection conditions. Standard conditions: peptide (2 mM, 1 µmol), PC1 (5 mol%), MPAA (25.0 mM), and TCEP (50.0 mM) in GND buffer (0.5 mL, pH 7.0), under irradiation by blue LED (455 nm, 40 W) at 25°C for 30 min. (B) Water‐soluble photocatalysts are employed. (C) Scope of N‐methylpyridinium substrates and their photolytic cleavage ^aYields were determined by analyses of reverse‐phase HPLC. ^bIsolated yields were provided in brackets. ^cThe reaction was performed at pH 4.0. MPAA = 4‐mercaptophenylacetic acid, TECP = (tris(2‐carboxyethyl) phosphine, GSH = glutathione, Asc = ascorbic acid.

Afterward, the deprotection conditions for 4‐NMP‐Thz were systematically investigated (Scheme 3A). Upon irradiation with 455 nm blue LED, deprotection was performed in GND buffer (pH 7.0) using 5 mol% Ru(bpy)₃Cl₂ (Scheme 3B, PC1, E_1/2 = −1.33 V) [45], 25 mM MPAA, and 50 mM TCEP for 30 min, providing the desired peptide 3 quantitatively (Scheme 3A, Entry 1). MPAA, previously shown to be essential for Pic group removal and widely used in NCL, was selected as the reductant. With the organic photocatalyst Eosin Y (PC2, E_1/2 = −1.06 V) [46], conversion decreased moderately (Entry 2). In contrast, riboflavin (PC3, E_1/2 = –0.29 V) [47] resulted in no reaction (Entry 3). Considering the reduction potential of N‐methylpyridinium is −1.00 V [37], PC1 was selected for its superior reducing capability. Conversion slightly decreased under basic or mildly acidic conditions, while phosphate‐buffered saline (pH 7.0) showed no effect (Entries 4–6). In reductant screening, ascorbic acid (Asc) enabled quantitative deprotection at pH 4.0, whereas glutathione (GSH) was ineffective (Entries 7 and 8). Replacing blue light with a compact fluorescent lamp (CFL, 40 W) required 1 h of irradiation to achieve comparable conversion (Entry 9); lower‐power sources showed reduced efficiency (Supporting Information page S32, Table S3). Green light completely inhibited the reaction (Entry 10), consistent with the characteristic excitation wavelength of PC1. Control experiments confirmed PC1, light, and MPAA are critical for deprotection (Entry 11). Meanwhile, orthogonal deprotection studies have shown that 4‐NMP‐Thz is fully compatible with traditional Cys protecting groups, such as Thz and Acm, enabling its orthogonal application in more complex synthetic strategies (Supporting Information pages S33, S34). In addition, experiments employing β‐thiol Val [48] and β‐thiol Leu [49] confirmed the compatibility of the methodology with established desulfurization‐based junction expansion approaches, highlighting its potential application to a broader range of non‐cysteine ligation sites (Supporting Information pages S91–S96).

With these optimized conditions, a series of NMP‐Thz derivatives was examined. As shown in Scheme 3C, the position of the formyl group had minimal impact on deprotection, with C‐2, C‐3, and C‐4 derivatives (2a–2c) exhibiting comparable reactivity. The electronic nature of the substituents on the pyridine ring exerts a pronounced influence on the reaction. Electronic effects of pyridine substituents significantly influenced the reaction: electron‐donating (methoxy 2d) or conjugating groups (chloro 2e) at C‐2 enabled efficient deprotection, whereas strong electron‐withdrawing substituents (ester 2f) completely inhibited it. Notably, the unmethylated variant 2g achieved quantitative conversion only under acidic conditions (pH 4.0; Supporting Information page S36). This aligns with the authors’ prior finding that pyridine ring protonation (E_1/2 = –1.19 V) dramatically elevates the reduction potential relative to neutral analogs (E_1/2 = –2.19 V) [26]. Collectively, these results establish a practical framework for designing one‐pot, N‐terminal Cys–directed NCL strategies.

2.2. Case 1: Synthesis of Histone H3.2 C110A Mutant With NCL and Desulfurization

To further exploit the potential of the 4‐NMP‐Thz PPG, this N‐terminal Cys “caging/uncaging” strategy was applied to the synthesis of challenging proteins. Efficient photolysis of the 4‐NMP‐Thz group in standard NCL buffer with catalytic PC1 renders 4‐NMP‐Thz a promising Cys‐specific temporary protecting group, facilitating large protein synthesis via a tandem one‐pot NCL–photolysis strategy. In this context, histone H3.2, a central architectural component that organizes DNA into chromatin, was prepared using this strategy. H3.2 was divided into four segments: the peptide thioester 4 (1–24), thioester segments 5 (25–74) and 6 (75–109), each featuring an N‐terminal 4‐NMP‐Thz‐protected Cys, and the C‐terminal peptide 7 (110–135) (Scheme 4A). The 4‐NMP‐Thz‐appended segments 5 and 6 were efficiently synthesized via a one‐pot, two‐step reaction involving late‐stage modification with NMP‐a followed by thioesterification (Supporting Information pages S41–S43).

SCHEME 4.

4‐NMP‐Thz–assisted one‐pot strategy for the synthesis of histone H3.2 C110A mutant. (A) Sequence and retrosynthetic plan of protein H3.2. (B) NMP‐Thz–assisted one‐pot synthesis of protein H3.2. Condition of NCL: MPAA (25 mM), TCEP (50 mM), GND buffer, pH 7.0, 25°C, 3–8 h. Condition of uncaging: PC1 (5 mol%), MPAA (25 mM), TCEP (50 mM), GND buffer, pH 7.0, 25°C, 30 min. Condition of desulfurization: protein (1 mg mL⁻¹), GSH (40 mM), VA‐044 (40 mM), TCEP (100 mM), GND buffer, pH 7.0, 37°C, 3 h. (C) Crude analytical HPLC traces (λ = 230 nm) obtained during the iterative synthesis of protein H3.2. *non‐peptide. (D) Analytical HPLC profile of the purified protein H3.2. (E) LCMS (ESI) spectrum of purified protein H3.2.

First, peptides 6 (1.0 equiv) and 7 (1.1 equiv) were subjected to the first NCL at pH 7.0, completing within 3 h to afford the desired peptide 8 (Scheme 4B). Subsequently, 4‐NMP‐Thz deprotection was initiated by adding 1.0 M TCEP (pH 7.0, 50.0 µL) and PC1 (5 mol%), followed by irradiation with blue light (455 nm, 40 W) for 30 min. After adding extra TCEP, the second NCL was conducted with fragment 5 (1.1 equiv). The ligation time was extended to 8 h because of steric hindrance from the thioester peptide [50]. After photolysis of the N‐terminal 4‐NMP‐Thz group, peptide 4 (1.1 equiv) was added, and the third NCL proceeded smoothly. HPLC monitoring indicated high efficiency at each step, with no significant byproducts observed (Scheme 4C). After acidification, the MPAA was removed from the reaction mixture by extraction with precooled diethyl ether. Subsequently, the synthesis was completed by a metal‐free desulfurization [51], affording histone H3.2 C110A mutant (12) in 18% isolated yield across six steps. The final product was validated by analytical HPLC (Scheme 4D), SDS‐PAGE (Supporting Information page S49, Figure S106), and LCMS (ESI) (Scheme 4E). These results indicate that 4‐NMP‐Thz has significant potential to enable efficient protein synthesis via one‐pot ligation strategies.

2.3. Case 2: Exploration of the Photo‐Uncaging Activity Via the One‐Pot Synthesis of MUC1 Tandem Repeats

Recent advances in chemical protein synthesis have enabled the assembly of increasingly large and complex protein targets through multisegment ligation strategies [52, 53, 54, 55]. To further demonstrate the generality of the one‐pot NCL strategy, this approach was applied to the synthesis of MUC1 tandem repeats (TRs), a transmembrane glycoprotein that functions as a significant tumor‐associated antigen and therapeutic target [56, 57]. Compared with MUC1 on normal cells, tumor‐associated MUC1 exhibits substantially altered glycosylation, particularly in its N‐terminal extracellular domain, which contains 20–125 TRs of APDTRPAPGSTAPPAHGVTS and is densely decorated with truncated O‐linked glycans [58, 59]. Currently, the longest chemically synthesized MUC1 constructs reported comprise only five repeats with 100 aa [60, 61, 62, 63]. To recapitulate the native mucin architecture and evaluate the scalability of our approach toward large proteins, the synthesis of an MUC1 domain consisting of 20 repeats (400 aa) bearing 40 O‐glycosylation sites was targeted. This was achieved through a one‑pot, multi‑step NCL strategy using a 20‐aa motif bearing two O‑glycosylations (Scheme 5A). Accordingly, the synthetic target encompasses the key structural and biological features of MUC1 and constitutes an exceptional synthetic challenge because of its extraordinary length and high O‐glycosylation density.

SCHEME 5.

4‐NMP‐Thz–assisted one‐pot strategy for the synthesis of MUC1 TRs bearing the STn antigen. (A) Sequence and retrosynthetic plan of MUC1 TRs. (B) Synthesis of MUC1 TR 13 and thioester 14. (C) 4‐NMP‐Thz‐assisted one‐pot synthesis of MUC1 TRs. Condition of NCL: MPAA (25 mM), TCEP (50 mM), GND buffer, pH 7.0, 25°C, 2 h. Condition of uncaging: PC1 (5 mol%), MPAA (25 mM), TCEP (50 mM), GND buffer, pH 7.0, 25°C, 30 min. Condition of desulfurization: protein (1 mg mL⁻¹), GSH (40 mM), VA‐044 (40 mM), TCEP (100 mM), GND buffer, pH 7.0, 37°C, 3 h. (D) Crude analytical HPLC traces (λ = 230 nm) obtained during the iterative synthesis of MUC1 TRs. (E) Analytical HPLC profile of the purified 100‐aa MUC1 TR 17. (F) LCMS (ESI) spectrum of purified 100‐aa MUC1 TR 17.

As illustrated in Scheme 5B, TR fragment 13 was prepared by Fmoc–SPPS, and the corresponding thioester 14 was obtained via a one‐pot two‐step transformation (Supporting Information pages S51 and S52). The one‐pot five‐segment ligation was performed as outlined in Scheme 5C. Peptides 13 (1.0 equiv) and 14 (1.05 equiv) were first dissolved in NCL buffer and allowed to react at pH 7.0 for 2 h to complete the first ligation. Upon completion, the N‐terminal 4‐NMP‐Thz protecting group was removed by photolysis, affording product 15 within 30 min. Iterative cycles of ligation with thioester 14 and photolysis enabled the sequential assembly of peptide segments, culminating in the fourth ligation, affording glycopeptide 16. After removing MPAA via extraction, metal‐free desulfurization was performed, affording the 100‐aa MUC1 fragment 17 (comprising five TRs) in 28% isolated yield across eight steps. Analytical HPLC confirmed a highly efficient conversion at each synthetic step (Scheme 5D). The identity and molecular weight of compound 18 were unambiguously verified by analytical HPLC (Scheme 5E) and LCMS (ESI) (Scheme 5F).

To improve overall synthetic efficiency, a convergent strategy was adopted for the assembly of the full‐length protein. As shown in Scheme 6A, fragment 17 was converted into the corresponding fragments 18 and 19 through C‐terminal thioester formation and photolytic removal of the N‐terminal 4‐NMP‐Thz protecting group, respectively. Without purification, fragments 18 and 19 were directly mixed and subjected to NCL at pH 7.0 and 25°C for 5 h, affording the 200‐aa MUC1 protein 20 comprising 10 TRs. Subsequently, fragment 20 was processed through the same thioester‐formation and photolysis, followed by NCL, to furnish 400‐aa MUC1 23 consisting of 20 TRs in 26% isolated yield after size‐exclusion chromatography (SEC) purification. Final photolysis and desulfurization afforded the 400‐aa MUC1 core domain 24 bearing 40 O‐glycosyl modifications, representing the largest fully synthetic MUC1 TR construct reported to date. The purity and molecular weight of protein 24 were confirmed by analytical HPLC (Scheme 6B) and SDS–PAGE analysis (Scheme 6C). A slight band broadening was observed for the synthesized MUC1 24, which is attributable to its extensive O‐glycosylation and sialylation.

SCHEME 6.

Synthesis of multiple MUC1 TRs and evaluation of biological activity. (A) 4‐NMP‐Thz‐assisted one‐pot synthesis of 400‐aa MUC1 (20 TRs) bearing the STn antigen. Condition of NCL: MPAA (25 mM), TCEP (50 mM), GND buffer, pH 7.0, 25°C, 5 h. Condition of uncaging: PC1 (5 mol%), MPAA (25 mM), TCEP (50 mM), GND buffer, pH 7.0, 25°C, 30 min. Condition of desulfurization: protein (1 mg∙mL⁻¹), GSH (40 mM), VA‐044 (40 mM), TCEP (100 mM), GND buffer, pH 7.0, 37°C, 3 h. (B) Analytical HPLC of a crude trace of 23, and SEC‐purified profiles of 23 and 24 (λ = 230 nm). (C) SDS‐PAGE of 400‐aa MUC1 (20 TRs) 24. (D) Structures of synthesized MUC1 TRs. (E) ELISA analysis of the interaction between the commercial anti‐CA15‐3/Gatipotuzumab and chemically synthesized MUC1 TRs. (F) Evaluation of MCF‐7 cell adhesion in the presence of chemically synthesized MUC1 TRs. (G) Immunization schedule for BALB/c mice. (H) ELISA analysis of antisera from mice immunized with chemically synthesized MUC1 TRs. In Scheme 6E,F, data are presented as mean ± SD (n = 3), while in Scheme 6H, data are presented as mean ± SD (n = 5 mice). Error bars indicate SD. Statistical significance was analyzed using an unpaired two‐tailed Student's t‐test in GraphPad Prism. Differences were considered statistically significant at **p < 0.01, ***p < 0.001, and **p < 0.0001.

The biological activity of tumor‐associated MUC1 was assessed through antibody‐binding assays, cancer cell adhesion measurements, and in vivo murine immunization studies. Binding affinities between synthetic MUC1 TRs (24–28, Scheme 6D) and commercial anti‐MUC1 monoclonal antibodies, Gatipotuzumab (the humanized version of the murine IgG1 antibody PankoMab, MedChemExpress) and anti‐CA15‐3 (the murine IgG1 antibody, Biorbyt), were evaluated by enzyme‐linked immunosorbent assay (ELISA). The results showed that Gatipotuzumab predominantly recognizes peptide epitopes of MUC1 TRs, with binding affinity increasing with increasing protein length. In contrast, anti‐CA15‐3 exhibits clear specificity toward glycan epitopes and shows strong binding to Tn‐modified MUC1, whereas only weak binding is observed for nonglycosylated MUC1, and sialylation further diminishes binding affinity (Scheme 6E). These binding characteristics clearly demonstrated that anti‐MUC1 antibodies can recognize distinct structural epitopes on the MUC1 molecule, particularly those involving glycosylation.

Tumor‐associated MUC1 is characterized by aberrant glycosylation, which compromises cellular adhesion and thus facilitates tumor metastasis. To assess this effect, a cell adhesion assay was performed using the breast cancer cell line, MCF‐7. As shown in Scheme 6F, STn‐modified MUC1 TR 24 reduced cell adhesion to 21% of that in the control group. These results collectively demonstrate that the inhibitory effect of MUC1 on cancer cell adhesion primarily depends on its glycosylation pattern, rather than the protein backbone. Concurrently, the diminished adhesion reinforces the role of the tumor‐specific STn antigen on the cell surface in promoting tumor immune evasion.

To evaluate the immunogenicity of the synthesized MUC1 TRs, Balb/c mice were immunized with the vaccine with Freund's adjuvant (Scheme 6G) [64]. After three rounds of immunization, serum antibody levels were determined by ELISA. As shown in Scheme 6H, glycosylated MUC1 variants elicited robust immune responses, with antibody titers ranging from 6000 to 30 000, whereas non‐glycosylated MUC1 TR 25 induced a limited response (titer ≈ 1,000). Notably, MUC1 TR 24 bearing the STn antigen elicited the strongest immune response, surpassing that induced by the glycopeptide–BSA conjugate 29. These results indicated that antibody titers increased with the complexity of the MUC1 antigen. Collectively, these results demonstrate that the synthesized MUC1 glycoproteins faithfully recapitulate the key antigenic and functional features of tumor‐associated MUC1, thereby providing a robust and structurally well‐defined platform for antibody profiling and cancer vaccine development, and may enable the future identification of monoclonal antibodies specifically targeting sialylated modifications on tumor‐associated MUC1.

3. Conclusion

In summary, a 4‐NMP‐Thz visible‐light–cleavable temporary protecting group was developed that can be installed into peptides and proteins through multiple routes and removed rapidly and quantitatively under standard NCL conditions. Its high stability and orthogonality to the NCL reaction significantly streamline the assembly of multisegment proteins, enabling efficient one‐pot synthesis of complex targets such as histone H3.2 and tumor‐associated MUC1 variants comprising 100–400 residues. Moreover, synthetic MUC1 produced by this approach retains the functional properties of its native analogs, underscoring the value of precisely defined synthetic proteins for tumor immunology. Overall, 4‐NMP‐Thz provides a robust platform for the synthesis of structurally complex, post‐translationally modified proteins and lays the foundation for future studies probing the structure–function relationships of protein modifications.

Author Contributions

Bingcheng Wei: methodology, data curation, writing – original draft. Xinyao Wang: methodology, data curation. Farong Ye: funding acquisition, writing – original draft. Haozhan Wang: methodology. Gongyu Shi: methodology. Bing Liu: resources. Ping Huang: validation, funding acquisition, resources. Ping Wang: conceptualization, investigation, supervision, funding acquisition, project administration, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: anie73199‐sup‐0001‐SuppMat.pdf

Dedicated to Professor Peter H. Seeberger on the occasion of his 60th birthday.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.