Post-synthetic modification of near-infrared absorbing cyclodextrin-encapsulated cyanine dyes for controlling absorption wavelength, stability, and singlet oxygen generation

Abstract

In this study, near-infrared absorbing rotaxane-type cyanine dyes exhibit high tolerance to various chemical reactions, which is attributed to the encapsulation effect of their cyclic molecules. As a result, rotaxane dyes can be post-modified on the host α-cyclodextrin or guest cyanine skeleton to adjust their solubility, absorption wavelength, stability, and singlet oxygen generation ability. The guest modification product obtained via the Heck reaction demonstrates a red shift of its absorption wavelength owing to the extended conjugation system. Moreover, the products of host modification through the methylation and benzylation of all cyclodextrin hydroxyl groups not only become lipophilic and show extended absorption, but also exhibit higher photooxidation tolerance, lower singlet oxygen generation rate, and increased singlet oxygen tolerance, indicating their potential applicability as highly durable dyes. Furthermore, the outstanding singlet oxygen tolerance of these dyes enables their use in long-life singlet oxygen generators, in which the total amount of singlet oxygen increases. This work demonstrates that an intrinsically unstable near-infrared cyanine dye can be used as a synthetic intermediate by stabilizing it via α-cyclodextrin encapsulation, allowing the post-modification of various properties of cyanine dyes toward the higher-order near-infrared-absorbing materials with complex functionalities and diverse utilities.

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1. Introduction

Near-infrared (NIR) dyes are widely used in various applications, such as solar cells, optical filters, and photochemical sensors, owing to their efficient NIR-absorbing properties.^1–4 In recent years, they have been also used in biomedical fields, including prodrugs, bio-imaging probes, and ¹O₂ generators for photodynamic therapy because of their minimal invasion, deep tissue penetration, and low interference from biomolecular autofluorescence.^5–11 However, depending on the application, each dye must possess certain optical properties, stability, hydrophilicity/hydrophobicity, and reactivity. Developing chemical transformation methods for adjusting the physical and chemical properties of NIR dyes may enhance their utility and application potential. However, dyes react with various redox and acid–base reagents owing to their inherently narrow gaps between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) (i.e. high HOMO and/or low LUMO levels). The low stability of dyes, which prohibits various chemical transformations after the formation of dye backbones, and their short lifetimes limit their practical utilization in materials. Accordingly, improving the chemical tolerance of core backbones of NIR dyes may enable their late-stage direct chemical transformation for achieving desirable properties and the development of dyes with long lifetimes to expand their application range.

Rotaxane formation has been utilized as an effective strategy for the stabilization of functional moieties,^12–15 including NIR dyes.^16–21 Anderson et al.²² reported 1,1′,3,3,3′,3′-hexamethylindotricarbocyanine as a heptamethine chromophore threaded through α-cyclodextrin (Cy7⊂α-CD), which was prepared by reacting glutaconaldehyde acetate with a tetramethyl indolium salt in the presence of α-CD in aqueous potassium acetate. The rotaxane structure enhanced photostability and formed a nanoring-rotaxane supramolecule, which promoted energy transfer as a light-harvesting system.²³ In recent years, we have found that various functional guest molecules can be encapsulated by methylated CD to improve their physical properties, such as luminescence^24–27 and electronic conductivity.^28,29 In particular, we have successfully stabilized guest molecules through the encapsulation of methylated α-CD and clearly demonstrated the effectiveness of rotaxane structures in controlling the reactivity of guest molecules, including the discovery of new chemical reactivities.^30–33

In this study, we focused on a rotaxane structure for improving the chemical tolerability of NIR dyes under harsh reaction conditions, demonstrating that the encapsulated structure can be used as a synthetic intermediate for post-synthetic molecular transformation. The post-modification on the host or guest moieties of these chemically stable rotaxane dyes not only improves their photooxidation tolerance and ¹O₂ tolerance but also allows the adjustment of their absorption wavelength, solubility, and ¹O₂ generation rate (Fig. 1).

2. Results and discussion

2.1 Synthesis of various encapsulated cyanine dyes

First, a rotaxane-type cyanine dye (Cy7⊂α-CD) was obtained in a 17% yield via the method previously reported by Anderson et al.²² In addition, Br-Cy7⊂α-CD with effective coupling reaction points was obtained from 5-bromo-1,2,3,3-tetramethyl-3H-indol-1-ium iodide using the same method. Afterwards, Cy7⊂α-CD and Br-Cy7⊂α-CD were post-modified with the guest cyanine or host α-CD moieties.

The hydroxyl groups of host α-CD were methylated or benzylated via the S_N2 reaction (Scheme 1a). The alkylation and silylation of hydroxyl groups on CD-based rotaxane structures are frequently performed to control the affinity (solubility) of cyclic molecules.^34,35 In this study, Cy7⊂α-CD was reacted with CH₃I or BnBr in dimethylformamide (DMF) in the presence of a strong base (NaH) producing Cy7⊂Me₁₈α-CD or Cy7⊂Bn₁₈α-CD, respectively, in which 18 hydroxyl groups of α-CD in Cy7⊂α-CD were replaced with methoxy or benzyloxy groups in high yield.³⁶ Notably, the NIR dye skeleton is susceptible to basic reagents, and Cy7 was degraded after NaH exposure (Supplementary Fig. S20). These results suggested a high chemical tolerance of the CD-encapsulated Cy7⊂α-CD.

After host modification, Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD exhibited high lipophilicity and solubility in a wide range of organic solvents, such as CHCl₃ (Fig. 2, right).³⁷ In contrast, unmodified Cy7⊂α-CD is soluble only in water or highly polar solvents, such as DMF and methanol owing to the high hydrophilicity of native α-CD (Fig. 2, left). The high lipophilicity of Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD can be advantages for the further chemical transformations of NIR dyes in organic solvents tailored for particular applications. Thus, the alkylation of Cy7⊂α-CD can be considered the initial step for functionalizing NIR dyes via chemical modification rather than simply adjusting their solubility.

The guest modification of the rotaxane-type cyanine dye was performed using Br-Cy7⊂α-CD. Prior to the guest modification procedure, the host was methylated as described above to improve its solubility in organic solvents and produce Br-Cy7⊂Me₁₈α-CD. Subsequently, the cross-coupling reaction of Br-Cy7⊂Me₁₈α-CD with styrene in DMF in the presence of Pd(dba)₂/P(o-tol)₃ at 100 °C generated Sty-Cy7⊂Me₁₈α-CD with a 53% yield (Scheme 1b). In contrast, Cy7 decomposed in DMF after 1 h at 100 °C in the presence of K₂CO₃. In particular, Br-Cy7⊂Me₁₈α-CD exhibited high chemical stability under the Heck reaction conditions because of the encapsulation (Scheme 1c, Supplementary Fig. S21).

2.2 Shift of absorption wavelength

The absorption maximum wavelengths of Cy7, Cy7⊂α-CD, Cy7⊂Me₁₈α-CD, and Cy7⊂Bn₁₈α-CD in MeCN are 740, 768, 774, and 781 nm, respectively (Fig. 3). The observed red shift of the absorption wavelength after encapsulation and modification can be attributed to the less polar environment around the cyanine backbone. Cyanines as charged molecules exhibit negative solvatochromism, which induces a red shift of the absorption peak in low-polarity solvents.³⁸ As a result, the cyanine backbone in Cy7⊂α-CD demonstrated red-shifted absorption owing to the lower polarity of the inner hydrophobic environment of α-CD than that of Cy7 surrounded by solvent molecules (MeCN). Moreover, the alkylation of cyclic molecule on Cy7⊂α-CD promoted hydrophobic substitutions around the cyanine backbone, resulting in slight red shifts of the absorption wavelengths of Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD. The modified cyclic molecule effectively protected the cyanine backbone to inhibit the solvation effects of the cyanine dyes. Thus, Cy7⊂Me₁₈α-CD exhibited weaker solvatochromism than Cy7, as indicated by the absorption spectra recorded in solvents with different polarities (Supplementary Fig. S22).

Next, the effects of guest structures on the absorption spectra were investigated. Although the absorption spectra of Cy7⊂Me₁₈α-CD and Br-Cy7⊂Me₁₈α-CD are similar, the guest modification product obtained during a cross-coupling reaction (Sty-Cy7⊂Me₁₈α-CD) shows a large red shift of the absorption peak (Fig. 4), which is attributed to the extended conjugation of the styryl moieties of the cyanine dye. As a result, the maximum absorption wavelength of Sty-Cy7⊂Me₁₈α-CD is 823 nm, which is approximately 80 nm longer than that of Cy7 (740 nm), confirming the validity of guest modification for controlling the dye optical properties.

2.3 Photooxidation tolerance studies

The highly encapsulated cyanine dyes are protected from reactive species owing to the steric effects of the rotaxane structures, which increase the dye lifetime. Herein, the degradation rates of Cy7, Cy7⊂α-CD, Cy7⊂Me₁₈α-CD, and Cy7⊂Bn₁₈α-CD were determined in the presence of light and oxygen as follows. A cyanine dye solution (MeCN, 5 μM) was irradiated at a wavelength above 650 nm (200 mW/cm²) for 120 min with air bubbling to monitor the decomposition of each dye by recording its absorption spectra (Fig. 5). The half-lives (τ) of Cy7 and Cy7⊂α-CD determined under the reaction conditions assuming the first-order degradation reaction were 22.4 and 45.0 min, respectively. In contrast, Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD hardly degraded under the same conditions, indicating that host modification dramatically improved the photooxidation tolerance of the dyes.

It is well known that cyanine dyes exhibit ¹O₂ generation properties.^39–41 Meanwhile, cyanine dyes with polymethine chains can degrade through the reaction with ¹O₂ species via undesired side reactions. In particular, controlling the ¹O₂ generation efficiency and ¹O₂ tolerance are important for designing highly photooxidation-resistant dyes and ¹O₂ generators. Therefore, we evaluated the ¹O₂ generation rates of various dyes (Cy7, Cy7⊂α-CD, Cy7⊂Me₁₈α-CD, and Cy7⊂Bn₁₈α-CD) by using 1,3-diphenylisobenzofuran (DPBF) as an ¹O₂ indicator. The absorbance of DPBF at 413 nm decreased during the reaction with ¹O₂. The cyanine dye (5 μM) and DPBF (50 μM) were dissolved in dimethylsulfoxide (DMSO) and irradiated at >650 nm (60 mW/cm²) for 50 min with air bubbling while monitoring the decomposition of DPBF. The first-order half-lives (τ′) of DPBF determined for Cy7, Cy7⊂α-CD, Cy7⊂Me₁₈α-CD, and Cy7⊂Bn₁₈α-CD under the reaction conditions were 7.57, 24.1, 30.4, and 50.2 min, respectively (Fig. 6 and Supplementary Fig. S23). These results indicate that the ¹O₂ generation rate of Cy7 decreased after the α-CD encapsulation and modification with bulky substituents.

Next, we evaluated the degradation rates of the cyanine dyes by exposing their solutions (MeCN, 5 μM) to the ¹O₂ species generated via the thermal decomposition of endoperoxide (EP, 5 mM) at 34 °C. The obtained degradation rates are ranked in the order Cy7 ≈ Cy7⊂α-CD >> Cy7⊂Me₁₈α-CD ≈ Cy7⊂Bn₁₈α-CD (Fig. 7 and Supplementary Fig. S24). Hence, the ¹O₂ tolerance of the cyanine dyes was significantly improved via host modification (Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD). However, the encapsulation on Cy7α-CD was insufficient to efficiently prohibit the reaction of the dyes with ¹O₂, confirming the validity of the host modification of the rotaxane-based cyanine dyes. These results demonstrated that Cy7⊂α-CD decreased the ¹O₂ generation rate; however, its degradation rate in the presence of ¹O₂ was similar to that of Cy7. In contrast, Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD exhibited lower degradation and ¹O₂ generation rates.

2.4 Mechanistic investigation of photooxidation tolerance

Cyanine dyes are generally susceptible to oxidative degradation at their long polymethine chains. Schnermann et al. reported the photooxidation mechanism of Cy7, in which a solution of Cy7 exposed to light at 740 nm under air undergoes a cleavage of the C1–C2 or C3–C4 bonds of the polymethine chain into two carbonyl compounds via dioxetanated intermediates (Fig. 8).⁴² Anderson et al. studied the photooxidation tolerance of Cy7⊂α-CD, which was bleached 3.9 times slower than Cy7 under light irradiation at 752 nm in the presence of air, although the corresponding degradation mechanism has not been identified.²²

Herein, solutions of Cy7 and Cy7⊂α-CD (MeCN, 100 μM) were irradiated at >650 nm (200 mW/cm²) for 2 h, and the resulting degradation products were analyzed via electrospray ionization mass spectrometry (Supplementary Fig. S19). The two expected degradation products, which were cleaved at the C1–C2 and C3–C4 positions, were obtained from Cy7, whereas only one degradation product at the C1–C2 position was observed for Cy7⊂α-CD. This result suggests that the C3–C4 positions do not undergo the cleavage reaction due to the steric effects of the encapsulation with α-CD. Comparing the CPK models of Cy7⊂α-CD, Cy7⊂Me₁₈α-CD, and Cy7⊂Bn₁₈α-CD, it can be concluded that the 18 OMe and OBn groups further increase the encapsulation area of the polymethine moiety (including the C1–C2 and C3–C4 positions) with respect to that of the unmodified α-CD (Fig. 9). These results suggest that the reduced proximity of oxygen molecules, even at the C1–C2 position, is likely attributed to the steric hindrance of Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD rather than to electronic effects.⁴³ Hence, it significantly improves the photooxidation tolerance, decreasing both the ¹O₂ generation rate and degradation rate in the presence of ¹O₂.

2.5 Evaluation of the singlet oxygen generation ability

According to the obtained results, Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD demonstrate extremely high ¹O₂ tolerances, although their ¹O₂ generation rates are lower than that of Cy7. Therefore, while increasing the DPBF:dye ratio from 10:1 to 400:1, cyanine dye solutions (DMSO, 0.25 μM) and DPBF (100 μM) were irradiated at >650 nm (200 mW/cm²) for 23 h with air bubbling. The ¹O₂ generation rate was tracked by monitoring the decomposition of DPBF from the corresponding absorption spectra (Fig. 10 and Supplementary Fig. S25). As a result, Cy7 completely degraded in 2 h, and the DPBF degradation process almost stopped, although the initial ¹O₂ generation rate was larger for Cy7. Meanwhile, the other cyanine dyes (Cy7⊂α-CD, Cy7⊂Me₁₈α-CD, and Cy7⊂Bn₁₈α-CD) continuously generated ¹O₂ even in 8 h, as confirmed by the gradual decrease in the DPBF absorption intensity. Owing to the higher encapsulation area of the host for Cy7⊂Bn₁₈α-CD as compared with that for Cy7⊂Me₁₈α-CD, the initial generation rate of ¹O₂ decreased. After 23 h, approximately ¼ of the initial DPBF amount remained in the presence of Cy7⊂α-CD, whereas Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD completely converted DPBF. These results indicate that Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD can be used not only as highly durable dyes with high photooxidation tolerances, but also as excellent ¹O₂ generators because of their long lifetimes.

3. Conclusion

In this study, the NIR-absorbing rotaxane-type cyanine dyes exhibited high tolerances to various chemical reactions while maintaining the cyanine skeletons, which were attributed to the encapsulation effect of their cyclic molecules. As a result, Cy7⊂α-CD and Br-Cy7⊂α-CD protected by α-CD were post-modified on the host α-CD or guest cyanine skeleton to adjust their solubility, absorption wavelength, stability, and ¹O₂ generation ability. The host modification products, Cy7⊂Me₁₈α-CD, and Cy7⊂Bn₁₈α-CD, demonstrated high lipophilicity and solubility in a wide range of organic solvents. In addition, Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD exhibited increased absorption wavelengths because of the low polarity around the cyanine skeleton caused by the hydrophobic cavity and substitutions of α-CD. The guest modification product, Sty-Cy7⊂Me₁₈α-CD, showed a large red shift of the absorption peak owing to the extended conjugation system. Furthermore, Cy7⊂Me₁₈α-CD and Cy7⊂Bn₁₈α-CD demonstrated higher photooxidation and ¹O₂ tolerances and lower ¹O₂ generation rates than those of Cy7 and Cy7⊂α-CD, indicating their potential applicability as highly durable dyes. The encapsulation of the polymethine portion of the cyanine skeleton effectively prevented oxygen molecules from approaching the polymethine portion. Their outstanding effect on the ¹O₂ tolerance suggested their potential use as long-life ¹O₂ generators.

The various chemical transformations of the dyes for tuning chemical and physical properties were realized by the steric protection of the cyanine skeleton via α-CD encapsulation. The rotaxane structure enabled the utilization of the intrinsically unstable NIR cyanine dye as a synthetic intermediate that could endure severe reaction conditions, such as the S_N2 and coupling reactions. This post-modification method based on rotaxane stabilization can pave the way for a wide range of practical applications of NIR dyes with complex functionalities and diverse utilities.

Supplementary data

Supplementary material is available at Bulletin of the Chemical Society of Japan online.

Funding

This research was supported by the following financial sources: JSPS KAKENHI Grant Numbers 22H02060, JST CREST Grant Number JPMJCR19I2, NEDO Grant Number JPNP21016, “Innovation inspired by Nature” Research Support Program, SEKISUI CHEMICAL CO., LTD., and Ogasawara Foundation.

References

Yoichi Masui

Yoichi Masui received his B.L.A. (2002), M.A. (2004), and Ph.D. (2021) from The University of Tokyo under the supervision of Prof. Makoto Onaka. From 2006, he was an Assistant Professor in Prof. Onaka's team at Graduate School of Arts and Sciences, The University of Tokyo. In 2020, he joined Prof. Terao's team at Graduate School of Arts and Sciences, The University of Tokyo as an Assistant Professor. His current research interests focus on physical and catalytic properties of molecules encapsulated in nanopores

Jun Terao

Jun Terao received his B.Sc. (1994) and Ph.D. degree (1999) from Osaka University. After working as a postdoctoral fellow at Hokkaido University under Professor Tamotsu Takahashi, he joined the Graduate School of Engineering, the Department of Applied Chemistry, Osaka University, as an Assistant Professor. From 2002 to 2003, he worked at the University of Oxford with Professor H. L. Anderson as a postdoctoral fellow. He was promoted to Associate Professor in 2008 in the Graduate School of Engineering, the Department of Energy and Hydrocarbon Chemistry, Kyoto University, now is Professor of Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo. His current research interests focus on the synthesis of functionalized supramolecules

Author notes