1. Introduction

Hydrogen sulfide (H₂S), the third endogenous gaseous signaling molecule, plays a critical role as a gasotransmitter and is deeply involved in various physiological and pathological processes [1]. At appropriate concentrations, endogenous H₂S exhibits cytoprotective properties, including antioxidative, anti-apoptotic, and anti-inflammatory effects [2]. However, abnormal H₂S accumulation has been linked to the onset and progression of diseases such as Alzheimer's [3], liver cirrhosis [4], inflammation [5], and cancers [6]. Therefore, accurate monitoring of endogenous H₂S is essential for the early diagnosis and treatment of H₂S-related disorders.

Photoacoustic (PA) imaging is a biomedical technique that combines the high contrast of optical imaging with the deep penetration and spatial resolution of ultrasound [7]. Unlike conventional fluorescence imaging, PA imaging overcomes interference from light scattering and absorption in biological tissues [8], making it more suitable for detecting analytes such as reactive oxygen species (ROS) [9], pH [10], enzymes [11], metal ions [12], and H₂S [13].

However, many PA probes rely on single-wavelength signals, which will be easily affected by probe concentration, tissue environment, and light scattering. To overcome these limitations, ratiometric PA imaging, which employs two distinct wavelength signals for internal calibration, has emerged as a promising method to improve detection reliability and signal-to-sound ratio (e.g., [14,15]. Although several ratiometric PA probes for H₂S detection have been developed (e.g., [16,17], challenges such as synthetic complexity, limited spectral shifts, and insufficient multifunctionality persist, restricting their practical applications. In particular, inadequate modulation of excited-state energy levels limits the ratiometric response and accuracy of current PA probes. Therefore, developing H₂S-responsive ratiometric PA probes with improved energy-level tunability and simplified design is critical for achieving precise, quantitative bioimaging.

Previously, Chen et al. developed a heptamethine cyanine-based probe, HS-CyBz, capable of ratiometric PA sensing of H₂S via a reaction-induced spectral shift. This response is induced through nucleophilic substitution by HS⁻, followed by keto-enol tautomerization, generating distinct absorption shift for dual-wavelength PA detection (Scheme 1) [18]. The probe demonstrated high sensitivity and a linear increase in the PA ratio (PA825/PA775) with increased H₂S concentration, confirming its potential for quantitative detection (Figure 1).

Based on the previous work, the present study investigates how substituents with different electronic properties tune the excited-state energy and PA behavior of the HS-CyBz system. Using DFT and TD-DFT calculations, we gained mechanistic insights into how electronic and structural modulation tune energy levels and PA signal output. The computational results suggest that substituent modification is not an effective standalone strategy for optimizing PA response. Instead, maximizing the difference in π-conjugation between the reactant and product is a more robust and promising design approach for developing efficient PA probes. This strategy offers a rational design principle for constructing next-generation PA probes with enhanced performance and molecular precision.

2. Methods

All computations were performed using the Gaussian 09 suite of programs [19]. Ground-state molecular geometry optimization was conducted using Density Functional Theory (DFT) with the B₃LYP functional and 6-31G(d) basis set. Excited states and excitation wavelengths were calculated using Time-Dependent Density Functional Theory (TD-DFT) with the M06-2X functional.

3. Results and discussion

Firstly, the ground-state molecular geometry of HS-CyBz was optimized via DFT calculations using the B₃LYP functional and 6-31G(d) basis set. To evaluate the accuracy of excitation wavelength calculations, three common TD-DFT functionals (B₃LYP, M06-2X, and PBE) were tested. As shown in Table 1, M06-2X provided the best alignment with the experimental absorption peak (775 nm) and was selected for all subsequent excited-state calculations.

To enhance the PA performance of HS-CyBz, several derivatives were designed by introducing electron-withdrawing groups (EWGs) and electron-donating groups (EDGs) at different positions of HS-CyBz. Specifically, three EWGs, nitro (NO₂), cyano (CN), and aldehyde (CHO), were inserted into the conjugated resonance region to influence the LUMO orbital (Figures 2a and 3a). Meanwhile, three EDGs, dimethylamino (N(CH₃)₂), hydroxyl (OH), and amino (NH₂), were introduced to the phenyl ring, where the HOMO orbital was localized (Figures 2b and 3b). Due to the molecular symmetry of HS-CyBz, substituents were added to both sides of the molecule for synthetic feasibility.

The excitation wavelengths of the substituted HS-CyBz derivatives, and their corresponding Cy-ketone products were calculated (Table 2). The substituent effects were quantified using Hammett constants (σp), where more positive values indicate stronger electron-withdrawing groups (EWGs) and more negative values indicate stronger electron-donating groups (EDGs). The parameter Δλ represents the wavelength shift between reactant and product. Notably, the dimethylamino-substituted (N(CH₃)₂) HS-CyBz exhibited the largest shift (240.45 nm), indicating a substantial narrowing of the HOMO-LUMO gap in the reactant.

However, compared with unsubstituted HS-CyBz and Cy-ketone, of which absorption peak are at 775 nm and 525 nm, respectively, most substituted derivatives demonstrated significantly smaller wavelength shifts, indicating that stronger substituent effect did not translate into PA advantages. The substituents either narrowed the reactant's conjugation leading to a blue shift, or expanded the product's conjugation leading to a red shift, both of which reduced the wavelength shift between reactant and product. The only exception was dimethylamino (N(CH₃)₂), where steric hindrance from the methyl groups weakened conjugation between the amine and phenyl ring, preserving an electronic structure similar to unsubstituted HS-CyBz and Cy-ketone. The distinct absorption wavelengths of HS-CyBz and Cy-ketone arise from differences in their π-conjugation systems. While Cy-ketone exhibits localized conjugation limited to two isolated phenyl rings, HS-CyBz possesses a more extended π-system, resulting in a pronounced red shift in absorption wavelength.

4. Conclusion

In this study, the effects of electron-donating and electron-withdrawing substituents on the photophysical properties of HS-CyBz derivatives was evaluated using TD-DFT calculations. While substituents influenced the excitation energies of both reactant and product forms, strong electron-donating or -withdrawing groups did not consistently enhance the PA signal.

These findings suggest that tuning the substituent’s electronic properties alone may not be an effective strategy to improve the PA signal. Instead, maximizing the difference in the extent of conjugation between the reactant and product, thereby modulating their respective absorption profiles, could be a more reliable approach for optimizing PA contrast in molecular designs.

Acknowledgements

I would like to express my gratitude to all those who offer help for my thesis. First and foremost, my deepest gratitude goes to the stage of Yuanpei Young Scholar program and my supervisors, Prof. Juan Yang and Dr. Runting Fang, for their comprehensive and continuous guidance. They provide me with abundant suggestions and priceless criticisms for my research. My sincere thanks also go to the academic fields. All of the previous research offers me inspiration and encouragement, which accompany me throughout my research process. I feel indebted to my parents, with their mental and material support, I could finish my research very well. Finally, my thanks should be sent to all my classmates for their endless support and warm hearts, making me feel like living in a big united family.