Development of a 1,3a,6a-triazapentalene derivative as a compact and thiol-specific fluorescent labeling reagent

Fluorescent organic molecules are widely used in imaging, sensing, and optoelectronics, but commonly used fluorophores are often large and can perturb the biological activity of small molecules or affect protein interactions when appended near binding sites. There is a strong need for compact fluorescent labeling reagents that minimize perturbation. The authors introduce the 1,3a,6a-triazapentalene (TAP) scaffold as a compact, highly fluorescent chromophore whose emission can be tuned by C2 substituents. They aim to develop the most compact thiol-reactive TAP derivative lacking a 2-phenyl group, enabling selective labeling of small bioactive molecules and visualization in cells without compromising activity.

Prior work established the importance of fluorescent probes for biological studies and technology (refs 1–11) and demonstrated chemical biology strategies using fluorescent labeling to probe targets (refs 12–14). The TAP scaffold was introduced as a compact, highly fluorescent chromophore synthesized via a click–cyclization–aromatization cascade (ref 15) and applied to various probes and functional systems (refs 16–24). Emission wavelength tuning in TAPs correlates with the inductive effect at C2, enabling yellow/red derivatives smaller than conventional dyes (ref 25). Reaction-based turn-on probes are advantageous for chemoselective bioimaging (refs 26–30). Human serum albumin structure (ref 31) and macropinocytosis for cell-penetrating peptides (refs 32–33) provide biological context. Captopril is a small ACE inhibitor (refs 34, 38) whose quantification has been studied (refs 35–37), but fluorescence imaging inside cells is lacking due to labeling-induced activity loss and nonspecific intracellular thiol-reactive probes.

Design and synthesis: To remove the 2-phenyl group and maintain photostability, the authors identified a ketone as a stable C2 substituent and designed a C2 vinyl ketone to enable thiol 1,4-addition and fluorescence change upon deconjugation. Starting from 2-methoxycarbonyl-TAP (4), hydrolysis and conversion to a Weinreb amide (5) (two steps, 87% overall) were followed by nucleophilic addition. Vinylmagnesium bromide or 1-propenylmagnesium bromide gave undesired 1,4-addition products (6a, 20%; 6b, 48%). Using isobutenylmagnesium bromide provided the desired vinyl ketone 2a (TAP-VK1) in 84% yield, as the β-dimethyl group sterically blocked undesired additions. Thiol labeling in organic solvent: Reactions were performed in dichloromethane (0.2 M) using thiol (1.0 equiv), TAP-VK1 (2a, 1.2 equiv), and tetramethylguanidine (TMG). With ethanethiol (7a), either 1.0 equiv TMG or 5 mol% TMG gave 1,4-adduct 3a in 95% and 87% isolated yields, respectively. Fluorescence properties in CH2Cl2: 2a λabs 409 nm, λem 574 nm, ΦF 0.023; 3a λabs 389 nm, λem 521 nm, ΦF 0.19. Time-course monitoring (excitation 370 nm in CH2Cl2) showed green emission within 5 min and maximal intensity at 50 min. Other nucleophiles (ethylamine, ethanol, ammonium acetate) did not react. Substrate scope in CH2Cl2: Aliphatic thiols: 7b (1-dodecanethiol) 86% yield, λem 519 nm, Φ 0.14; 7c (allylthiol) 88%, 525 nm, 0.34; 7d (benzylthiol) quantitative, 517 nm, 0.16; 7e (1,3-propanedithiol; 2.2 equiv 2a) 86%, 536 nm, 0.13 (bis-adduct); multifunctional thiols reacted selectively at sulfur: 7f (alcohol) quantitative, 532 nm, 0.18; 7g (amine; in water, base-free) 96%, 471 nm, 0.23; 7h (triazole) 73%, 521 nm, 0.27; 7i (secondary thiol) 82%, 521 nm, 0.27; 7j (protected amino acid) quantitative, 521 nm, 0.12; 7k (protected dipeptide) 91%, 540 nm, 0.17. Several aromatic thiols did not react. Aqueous labeling: Optimal conditions for free peptides/protein were 0.1 M sodium phosphate buffer (NaPB), pH 7.0, room temperature, without additives. Glutathione (7l) reacted with 2.0 equiv 2a; crude indicated near-quantitative conversion, but isolated yield after RP silica gel purification was 47% due to adsorption. An octapeptide (7m) containing diverse residues was labeled at cysteine with 20 equiv 2a in 0.1 M NaPB pH 7.0 for 6 h, giving quantitative conversion; HPLC showed only product and excess 2a. Human serum albumin (single free Cys) was cleanly labeled under similar buffer conditions. Cell imaging model with peptide: A water-soluble peptide (7n) containing an R8 cell-penetrating sequence was labeled in 1 mM NaPB (pH 7.0) with 2.0 equiv 2a for 3 h (71% conversion to 3n by HPLC). The reaction mixture was diluted into serum-free DMEM and applied directly to A549 cells. Confocal imaging (excitation 405 nm, emission 490 nm) after 18 h showed cytoplasmic fluorescence for 10 μM and stronger for 25 μM 3n without washing. Controls (2a alone, peptide 7n alone, untreated) showed no staining. Excess common thiol-labeling dyes (coumarin-maleimide, NBD-Cl, ABD-F) caused cell surface adsorption and damage under similar conditions. Captopril labeling and ACE activity: Captopril (7o) was labeled with 1.5 equiv 2a and 1.1 equiv TMG in CH2Cl2 to yield TAP-VK1-captopril (30) in 82% as a 4:1 diastereomeric mixture at the methyl-bearing center. ACE inhibitory activity was assayed (Dojindo ACE Kit-WST); 30 retained potent inhibition similar to captopril, whereas fluorescein-labeled captopril lost activity. HPLC of assay solutions indicated no retro-thia-Michael formation of free captopril. Captopril imaging: MBEC4 vascular endothelial cells were treated with 50 μM 30 in serum-free DMEM at 37 °C; confocal imaging at 24 h showed intracellular cytoplasmic fluorescence. Controls with 50 μM captopril (7o) or 50 μM 2a showed weak to negligible fluorescence. Competition with 100 μM 7o reduced intracellular fluorescence of 30, suggesting ACE-associated accumulation. Co-localization with anti-ACE immunostaining confirmed distribution overlap. Comparative labeling with coumarin (80), NBD (90), and ABD (100) showed that NBD- and ABD-labeled captopril did not show intracellular fluorescence; coumarin-labeled captopril entered cells but was weaker and more nonspecific than 30. Photostability under continuous 405 nm excitation: 30 T1/2 = 76.9 min vs coumarin analog 80 T1/2 = 41.5 min. Computations: DFT (ωB97XD/6-311+G(d,p)) and TD-DFT with PCM (CH2Cl2) and VEM approach were used to analyze excitation/emission and charge-transfer character; the emission blue-shift upon thiol addition is attributed to decreased charge transfer due to termination of C2 conjugation. Cell culture and imaging details: A549 and MBEC4 cells were maintained in DMEM with 10% FBS. For imaging, cells were incubated in serum-free DMEM containing compounds, fixed with 4% PFA, stained with propidium iodide for nuclei, and imaged on ZEISS LSM700. For ACE co-localization, anti-ACE primary and Alexa Fluor 555 secondary antibodies were used.

- A compact vinyl ketone TAP derivative, TAP-VK1 (2a), was synthesized (84% yield from Weinreb amide via isobutenylmagnesium bromide) as a thiol-specific fluorescent labeling reagent. - Chemoselectivity: 2a reacts smoothly and selectively with aliphatic thiols via 1,4-addition; alcohols, amines, carboxylates, and triazoles do not react; several aromatic thiols are unreactive. - Turn-on fluorescence: In CH2Cl2, 2a exhibits λabs 409 nm, λem 574 nm, ΦF 0.023; after thiol addition (3a) λabs 389 nm, λem 521 nm, ΦF 0.19 (~10-fold increase). Reaction mixtures turn green within 5 min; fluorescence intensity maximizes at ~50 min. - Broad scope and high yields in CH2Cl2: examples include 7b 86% (519 nm, Φ 0.14), 7c 88% (525 nm, 0.34), 7d quantitative (517 nm, 0.16), 7e 86% (536 nm, 0.13, bis-adduct), 7f quantitative (532 nm, 0.18), 7g 96% (471 nm, 0.23), 7h 73% (521 nm, 0.27), 7i 82% (521 nm, 0.27), 7j quantitative (521 nm, 0.12), 7k 91% (540 nm, 0.17). - Aqueous bioconjugation: In 0.1 M NaPB pH 7.0, free peptides and proteins were labeled at Cys without additives. Glutathione labeling showed near-quantitative conversion (47% isolated due to adsorption). An octapeptide with multiple functional groups was labeled quantitatively and specifically at Cys; HPLC showed no side products. HSA (single free Cys) was cleanly labeled. - Environment-sensitive emission: 2a and peptide adducts are non-emissive in water but become fluorescent in cellular hydrophobic environments, enabling low background in media. - Cellular uptake and imaging: R8-peptide conjugate 3n showed cytoplasmic fluorescence in A549 cells at 10–25 μM without washing; controls (2a, peptide alone) showed none. Common small fluorophores (coumarin-maleimide, NBD-Cl, ABD-F) caused background adsorption and cytotoxicity when used in excess; 2a did not. - Drug labeling without activity loss: Captopril labeled with TAP-VK1 (30) in 82% yield (4:1 d.r.) retained potent ACE inhibitory activity (ACE Kit-WST), whereas fluorescein-labeled captopril lost activity; no retro-Michael release of captopril detected. - Drug imaging: 30 (50 μM) accumulated in MBEC4 cells with cytoplasmic fluorescence; controls (captopril, 2a) gave weak signals; competition with excess captopril reduced fluorescence; co-localization with anti-ACE antibody confirmed targeting. NBD- and ABD-labeled captopril showed no cellular fluorescence; coumarin-labeled captopril showed weaker, more nonspecific distribution. - Photostability: 30 displayed longer fluorescence half-life (T1/2 76.9 min) under 405 nm excitation compared to coumarin analog 80 (T1/2 41.5 min). - TD-DFT analysis attributed emission blue-shift to decreased charge-transfer upon thiol addition due to conjugation termination at C2.

The study addresses the challenge of imaging small bioactive molecules without perturbing function by creating a highly compact, thiol-specific fluorophore based on the TAP scaffold. By eliminating the 2-phenyl group and introducing a vinyl ketone, TAP-VK1 achieves selective thiol 1,4-addition with robust turn-on emission and strong blue-shift, enabling direct visualization of labeled species while minimizing background from unreacted dye. The demonstrated chemoselectivity across diverse thiol substrates, compatibility in both organic and aqueous buffers, and environment-sensitive fluorescence allow direct use of reaction mixtures for cellular studies without purification. Crucially, labeling captopril with TAP-VK1 retained ACE inhibitory activity and enabled intracellular imaging with ACE co-localization, overcoming limitations of conventional small fluorophores that either disrupt activity or introduce nonspecific interactions and cytotoxicity. The combination of compact size, thiol specificity, turn-on behavior, photostability, and biocompatibility underscores TAP-VK1’s relevance for mechanistic studies of small-molecule drugs, peptides, and proteins in cells.

The authors developed TAP-VK1, a most-compact vinyl ketone 1,3a,6a-triazapentalene derivative that selectively labels aliphatic thiols with high yields and large fluorescence turn-on. TAP-VK1 enables efficient peptide and protein labeling in aqueous buffer, cellular delivery and imaging with low background, and preserves the biological activity of very small drugs exemplified by captopril, whose intracellular localization and ACE association were visualized. Compared to commonly used small fluorophores, TAP-VK1 offers superior retention of native behavior and photostability. Future work will focus on increasing intracellular fluorescence (e.g., by introducing hydrophobic substituents to improve emission in aqueous environments) and expanding applications to broader classes of bioactive small molecules and targets.

- Aqueous fluorescence: 2a and its thiol adducts are non-emissive in bulk water; fluorescence arises in hydrophobic environments, which may limit sensitivity until delivered to membranes or vesicles. - Aromatic thiols: Several aromatic thiols did not react with 2a under tested conditions, constraining substrate scope. - Purification losses: Some aqueous peptide adducts (e.g., glutathione) showed reduced isolated yields due to adsorption on reverse-phase silica during purification. - Intracellular brightness: The authors note the need to increase intracellular fluorescence intensity; tuning with hydrophobic substituents is proposed. - Diastereomeric mixtures: Captopril labeling produced a 4:1 diastereomeric mixture; while activity was retained overall, stereochemical effects may matter for other targets.