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. 2025 Jul 28;147(31):28002–28014. doi: 10.1021/jacs.5c07710

Breaking Bonds with Short-Wave Infrared Light: BODIPY Photocages for Two-Photon Activation in the 900–1500 nm NIR-II Window

Yagmur Aydogan-Sun †, Alexandra Egyed ‡, Arthur H Winter ‡,*, Josef Wachtveitl †,*
PMCID: PMC12333369  PMID: 40720589

Abstract

Photocages enable light-triggered cargo release in biological systems, but their excitation is often restricted to UV/visible wavelengths, where tissue penetration is limited. Two-photon excitation (2PE) offers a solution by allowing near-infrared (NIR) or short-wave infrared (SWIR) activation within biological windows of maximal tissue transparency. While photocaging in the first biological window (650–950 nm) has been demonstrated, applications in the second biological window (1000–1350 nm) remain unexplored. Here, we investigate the two-photon absorption (2PA) properties of 11 BODIPY photocages featuring single-photon absorption spanning 450–750 nm, focusing on 3- and 5-position substitutions to identify key motifs that enhance 2PA in the 900–1500 nm range. We find that strong charge transfer character and increased vibrational freedom can relax symmetry-related selection rules, significantly enhancing 2PA. Cross sections (δ) exceeded 4000 GM at 900 nm for a bis­(styryl)-BODIPY with carbazole units and reached 1110 GM at 1240 nm for its monostyryl analog. Two additional B-methylated molecules with improved uncaging quantum yields were synthesized, yielding uncaging action cross sections (δΦu) up to 5.8 GM at 900 nm and around 1 GM at 1300–1400 nm. Notably, these modifications preserve the core photophysical properties of BODIPY, making them ideal for molecular engineering. These findings highlight key design principles for efficient 2P-activatable photoactuators operating in the NIR-II biological window and show that heterolytic C–O bond cleavage can be triggered by two SWIR photons carrying as little as 20 kcal/mol each.

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Introduction

Light is an advantaged exogenous trigger in numerous research and application fields due to its precise spatiotemporal control and noninvasive nature. Since light serves as a temporal trigger, light-sensitive molecular platforms must be specifically designed to induce a certain response upon irradiation. This requires extensive photochemical engineering to tailor molecular systems for specific applications. Over the past few decades, many photoactivatable compounds have been developed and categorized into various classes, including photoswitches, , photocatalysts, , fluorophores and many more.

Among these, photocages stand out for their high potential in biomedical applications, as they are designed to initially mask and then release a specific functional unit. Typically, photocages consist of two main components: a chromophore, which acts as the light-sensitive core and a leaving group (LG), which serves as the functional key component. When exposed to light, the LG is released and its functionality is restored. While the leaving group can be chosen based on the intended functionality, the chromophore is selected based on its chemical and optical properties. Combining various chromophore and LG units offers a wide range of potential applications, such as monitoring biological activity, − controlled drug release, − photodynamic therapy , and many more.

Although these applications are highly promising, they are not yet broadly applicable in organisms due to several significant limitations. A key obstacle for clinical usage is the excitation wavelength of most photocages, which falls within the UV/vis range, where penetration into biological tissue is generally limited. For effective biomedical applications, an ideal photocage should combine high uncaging efficiency with excitation wavelengths in the NIR range, where phototoxicity is minimal and tissue transparency is optimal. Especially, the first (650–950 nm) and second (1000–1350 nm) biological windows are particularly advantageous due to their high tissue transparency. However, optimizing specific photocage properties without compromising others remains a considerable challenge. For instance, shifting the excitation toward longer wavelengths typically requires extending the π-system or incorporating push–pull groups. Unfortunately, such structural modifications often introduce new nonradiative, decay pathways, which can shorten the excited state lifetime, reducing the efficiency of slower processes such as fluorescence or uncaging. ,

Nevertheless, progress on this field continues, as recent work from our group and others − showed that structural modifications on photocages can significantly shift their 1-photon excitation (1PE) wavelength into the first biological window. In the same study, we demonstrated that increased conformational rigidity can enhance the uncaging efficiency of red-shifted BODIPYs. Despite these advances, the benefits diminish as excitation wavelengths are shifted further into the red spectrum, where the decreasing energy gap between the ground and first excited state leads to a drastic increase in nonradiative transitions. , Certainly, the energy gap law implies that there must be a limit for the long-wavelength excitation of photocages, where the uncaging might be completely lost. Consequently, while extending 1PE into the second biological window seems to be unrealistic, accessing it via 2PE is more feasible as it only requires a 1PA between 500 and 675 nm.

BODIPY is a strong candidate for this purpose, as its core chromophore naturally absorbs around 500 nm and further structural modifications can extend its 1PA up to 700 nm. However, a key drawback of BODIPY’s core is its inherently weak two-photon absorption (2PA) in the S1 transition, which in the past has led to its incorporation into energy-transfer systems with 2PA antennas to enhance 2P excitability. − To date, only a few studies have explored BODIPY derivatives for 2PA applications within the second biological window , with some studies focusing on aza-BODIPYs, , which are well suited for optical power-limiting applications.

In this study, we systematically investigate a series of BODIPY derivatives with structural modifications at the 3- and 5- positions to identify structural motifs that enhance 2PA, particularly in the second biological window. A total of 11 molecules with different substitution patterns were examined. For easier comparison, the molecules are grouped into three categories based on their structural features (see Figure ). Initially, comparisons are made within each group, followed by a broader comparison between the different groups. We explore how structural features influence steady-state properties. To gain deeper insights, we performed ground-state optimizations and TD-DFT calculations of frontier orbital transitions using CAM-B3LYP/def2-SVP, which showed best agreement with experimental BODIPY data in a benchmark section of our recent publication. Solvent effects were included via the polarizable continuum model (PCM)** within the Gaussian package. − These calculations were used to evaluate conformational flexibility, charge transfer characteristics and transition symmetries. For investigating the impact of structural modifications on excited-state dynamics and photochemical efficiency, femtosecond transient absorption measurements were conducted on selected molecules from each group. Additionally, time-correlated single-photon counting (TCSPC) measurements were performed under both 1PE and 2PE conditions to determine whether the excitation wavelength affects the fluorescence lifetime.

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Overview of the molecules’ absorption spectra (upper panel) and their respective structures (lower panel). The molecules are categorized into three distinct groups based on structural similarities: I) The first group comprises molecules with one or two fluorene-like substituents. II) The second group includes molecules featuring two styryl substituents with varying donor or acceptor groups. III) The last group consists of molecules incorporating rigid substituents.

Results and Discussion

Figure provides an overview of the investigated structures and their steady-state absorption in chloroform. The lowest energy absorption bands of the molecules span a range from 580 nm up to 710 nm, making them suitable candidates for 2PA within the second biological window.

The first group of molecules features at least one styryl-like substituent, where the phenyl moiety is replaced by a fluorene analogue. In the upper panel of group I) monosubstituted molecules are shown, while the lower panel displays their symmetric disubstituted counterparts. Among these, mono-FL-BODIPY and di-FL-BODIPY feature fluorene (FL) substituents. In contrast, the other group members have one of the carbon atoms in the five-membered fluorene ring replaced by a heteroatom. These modifications result in dibenzofuran (DBF) moieties in mono-DBF-BODIPY and di-DBF-BODIPY and ethyl-carbazole (EC) moieties in mono-EC-BODIPY and di-EC-BODIPY. The electron-donating ethyl-carbazole moieties in mono-EC-BODIPY and di-EC-BODIPY induce the strongest red shift among the group, followed by the fluorene-substituted mono-FL-BODIPY and di-FL-BODIPY. In contrast, the dibenzofuran moieties in mono-DBF-BODIPY and di-DBF-BODIPY lead to more blue-shifted absorption, suggesting a reduced electron delocalization. One possible explanation is that the inductive effect of oxygen outweighs its mesomeric effect, giving oxygen an overall electron-withdrawing character. Another contributing factor could be the differing substitution patterns. In the heteroatom-containing molecules, the heteroatoms are positioned para to the styryl unit, whereas in mono-FL-BODIPY and di-FL-BODIPY, the fluorene units are substituted in the meta position. This difference in alignment could further account for the enhanced electron delocalization within the fluorene containing molecules compared to the more blue-shifted mono-DBF-BODIPY and di-DBF-BODIPY.

The second group of molecules consists of three BODIPYs, each containing double substituted styryl moieties with distinct functional groups at the para-position of the phenyl ring. One of these molecules, TFA-TFA-BODIPY, features trifluoromethane (TFA) groups on both styryl moieties, which are typically strong electron-withdrawing groups and may function as electron acceptors. In another molecule, OMe-OMe-BODIPY, the trifluoromethane units are replaced by methoxy (OMe) groups, which are strong electron donors in the para position. This substitution enhances electron delocalization and induces a more pronounced red-shift in absorption. The final compound in this group, TFA-OMe-BODIPY, integrates both characteristics by incorporating a trifluoromethane group on one styryl moiety and a methoxy group on the other. Intuitively, TFA-OMe-BODIPY would be expected to exhibit strong push–pull behavior, leading to enhanced electron delocalization. However, in fact, the absorption properties suggest that the extent of delocalization falls between TFA-TFA-BODIPY and OMe-OMe-BODIPY.

The third group includes only two members, both based on more rigidly constructed BODIPYs. In these molecules, the substituents at the lower part are fused to the BODIPY core through hexane rings, reducing their flexibility and thereby minimizing additional deactivation pathways. Despite their similar structural concept, the two molecules differ in some key aspects. In rigid-BODIPY-1, the boron atom is substituted with methyl groups, while in rigid-BODIPY-2, the boron atom is fluorinated. Additionally, the two molecules feature different leaving groups at the meso-methyl position: rigid-BODIPY-1 bears a benzeneacetic acid, whereas rigid-BODIPY-2 contains a smaller acetic acid. Another significant difference is the additional methoxyphenyl substituents of rigid-BODIPY-2, which are absent in rigid-BODIPY-1. These significant differences in rigid-BODIPY-2 result in a stronger overall red-shift compared to rigid-BODIPY-1.

Two-Photon Excitation Spectra

The two-photon excitation (2PE) spectra were acquired using the two-photon induced fluorescence (2PIF) method in a relative manner. The integrated fluorescence intensity of each molecule was recorded at different excitation wavelengths, alongside the reference compound Styryl 9M. Data analysis followed the methodology described by Albota et al., using reference values for Styryl 9 M from Makarov et al. Further details on the experimental setup are available in the Supporting Information. The 2PE spectra span from 900 to 1500 nm, covering the entire S1 transition and partially extending into the higher-lying S2 transition. This lower limit was chosen based on the relatively red-shifted fluorescence of the molecules (see Figure S9) to prevent stimulated emission, which could be induced by high laser intensities and cause deviations from the intrinsic 2PA response.

Before analyzing the different molecular groups, we provide a brief overview of the molecular features that influence 2PA efficiency and how they apply to BODIPYs. Similar to 1PE, symmetry-based selection rules such as the Laporte rule also apply to 2PE, but in an inverted form due to the involvement of two photons. While 1PA transitions require a change of parity between ground and excited state, which means that the orbitals need to transform differently under inversion, 2P transitions are allowed when both states have the same parity. However, these rules only strictly apply to centrosymmetric molecules.

For molecules with lower symmetry, such as the BODIPYs studied here, the Laporte rule no longer holds. Instead, one must determine the molecular point group and its irreducible representations for each orbital, which describe how the orbitals transform under symmetry operations. A 2P transition is symmetry-allowed if the direct product of the ground state, the excited state, and the relevant tensor component transforms as the totally symmetric representation. This analysis requires the use of character tables and group theory, as the 2PA strength depends on how specific components of the second-order transition tensor transform and which are symmetry-allowed. As a general rule, the lower the molecular symmetry, the more tensor components contribute to the transition. Nevertheless, the complementary character of 1PA and 2PA usually holds: transitions that are weak in 1PA tend to be stronger in 2PA and vice versa.

In general, as discussed by Pawlicki et al., two terms can contribute to the overall 2PA cross-section of a molecular system: the dipolar term and the two-photon term. 2PA occurs via a virtual state, where the first photon perturbs the electronic structure of the molecule. The two-photon term accounts for the contribution of real excited states lying near this virtual state, thereby enhancing the transition. The dipolar term, by contrast, represents a direct transition from ground to excited state mediated by allowed dipole coupling. While the two-photon term dominates in centrosymmetric molecules, the dipolar term is typically stronger in noncentrosymmetric systems like the herein presented BODIPYs.

Nonetheless, symmetry alone often does not fully explain 2PA behavior in low-symmetry systems, as transition rules can often be relaxed by symmetry-breaking through conformational changes, vibrational coupling and charge-transfer characteristics. As a result, accurate prediction of 2PA efficiency in low-symmetry systems is not straightforward and often requires empirical validation, as presented in this study.

The 1PE and 2PE spectra of group I) molecules with various fluorene analogues are illustrated in Figure . In general, across all molecules the 2PA maxima and shoulders associated with the S1 state align well with twice the wavelength of their respective 1PA peaks. However, there are still remarkable differences among these molecules, especially between the single- and double-substituted variants.

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1PA and 2PA spectra are shown for A) mono- and di-DBF-BODIPY, B) mono- and di-FL-BODIPY, and C) mono- and di-EC-BODIPY. 2PA cross sections are represented by dots connected with lines, and the corresponding 1PA spectra are shown as filled, dashed lines in a transparent color. The left axis indicates 2PA cross sections with 2PE wavelengths on the bottom axis, while the right axis shows extinction coefficients for the 1PA spectra with 1PA wavelengths on the top. In panels B and C, the 2PA cross-section axes are split with a tilded line to accommodate large differences between S1 and S2 transitions.

A comparison of the 2PA cross sections of the molecules with the electron-donating ability of their substituents reveals a direct correlation: the 2PA cross sections increase with stronger electron-donating substituents. This trend is evident when moving from the moderately electron-donating dibenzofuran-substituted molecules (mono-DBF-BODIPY and di-DBF-BODIPY) and fluorene-substituted molecules (mono-FL-BODIPY and di-FL-BODIPY) to the more strongly electron-donating ethyl-carbazole-substituted molecules (mono-EC-BODIPY and di-EC-BODIPY). This was further confirmed by computed orbital density distributions (Figures S5 and S6), which show significant shift in electron density from the substituents to the BODIPY core, particularly in the case of ethyl-carbazole substituents. At first glance, one might expect that double substitution would lead to significantly higher 2PA cross sections due to charge-transfer contributions from both substituents. However, the observed values for the S1 transition in mono- and disubstituted molecules are surprisingly similar. Yet, a closer look at the 2PE spectra reveals key differences: in monosubstituted molecules, the 2PA maximum closely aligns with the 1PA maximum of the S1 transition, whereas in disubstituted molecules, this 0–0 transition becomes weakened in the 2PE spectra. This behavior indicates the influence of vibronic coupling and symmetry-based selection rules, effects that have previously been shown to notably influence the shape of 2PE spectra of various systems, including noncentrosymmetric molecules. −

These differences in 2PA can partially be explained by differences in vibrational modes and molecular symmetry of the compounds. Monosubstituted molecules inherently lack symmetry elements and thus belong to the C1 point group, in which all 2PA tensor components are formally allowed. Geometry optimizations further show that monosubstituted molecules (Figure S1) exhibit greater structural flexibility, enabling good planarization and enhanced vibrational coupling. In contrast, double substitution, particularly in para-substituted di-DBF-BODIPY and di-EC-BODIPY (Figure S2), reduces vibrational degrees of freedom due to steric hindrance. The optimized structures show a tilted alignment of the substituents in disubstituted molecules, imposing structural constraints that reduce planarization and may restrict certain vibrational modes that could otherwise enhance 2PA.

While the disubstituted molecules can generally be assigned to the C2 point group, the out-of-plane alignment of the substituents can further reduce the point group to C1. However, for certain molecular orientations in solution, the C2 point group can still be relevant. For instance, the S1 transition involves orbitals with different symmetries: the LUMO exhibits a mirror-symmetric distribution along the vertical axis, whereas the HOMO contains a nodal plane along the same axis, leading to mismatched orbital symmetry. This reduces the number of tensor elements that can contribute to the transition, resulting in lower 2PA efficiency. However, strong vibrational features can partially relax these restrictions, while strong electron-donating groups can enhance 2PA by promoting charge transfer character. This effect is evident in the ethyl-carbazole containing molecules. Mono-EC-BODIPY exhibits higher cross sections (1110 GM at 1240 nm) than di-EC-BODIPY (655 GM at 1270 nm) despite having only one donor. This enhancement may stem from the fact that in mono-EC-BODIPY, two orbitals, HOMO and HOMO–1, contribute to the S1 transition to LUMO, both exhibiting a strong charge-transfer character, whereas for di-EC-BODIPY only HOMO contributes to the S1 transition.

For the S2 transition, it should be noted that it cannot be fully resolved within the measurement window. Based on energy differences from theoretical calculations, corrected by experimental spectra, the S2 transition is expected to lie between 415 and 460 nm in the 1PA spectrum, corresponding to 830 to 920 nm in the 2PE spectra for nearly all molecules. As a result, for most molecules, the S2 transition is situated at the edge of the measurement window or even beyond its lower limit, preventing a complete analysis. However, despite this limitation, the significantly steeper rise in 2PA cross sections at the onset of the S2 transition for disubstituted molecules strongly suggests that they exhibit substantially stronger 2PA cross sections at this transition compared to their monosubstituted counterparts.

This enhancement likely arises from the nature of the orbitals involved. The S2 transition primarily involves HOMO–1 to LUMO transition, where both orbitals share a mirror-symmetric distribution, allowing more tensor components of the disubstituted molecules to contribute to the transition. Additionally, the charge transfer character of the S2 transition is much stronger than the S1 transition, further enhancing the 2PA cross sections. Consequently, while disubstitution does not significantly enhance 2PA in the S1 transition due to reduced symmetry overlap and conjugation, it plays a crucial role in the S2 transition, where the electronic and symmetry characteristics are more favorable for efficient 2PA.

The only exception, where the S2 transition is fully resolved within the measurement window, is di-EC-BODIPY, where the transition is estimated to occur around 500 nm in the 1PA spectrum. In fact, this transition manifests as strong absorption (1510 GM) near 1050 nm in the 2PE spectrum, slightly red-shifted compared to twice the 1PA peak at 1000 nm. Such a redshift could be attributed to differences in the relative contributions of Franck–Condon and Herzberg–Teller terms in 1P and 2P transitions, as previously discussed by Lin et al. Another transition of di-EC-BODIPY appears around 900 nm, reaching up to 4000 GM and can be attributed to the S3 transition. This transition is calculated to occur around 940 nm and involves a multitude of contributing mirror-symmetric orbitals (Figure S6). However, the observed high 2PA cross sections at both 900 and 1050 nm cannot be solely attributed to 2PA. In fact, power dependency measurements (Figures S11–13) reveal significant deviations from a quadratic response, even at low intensities. This suggests that, at these wavelengths, di-EC-BODIPY might exhibit additional strong excited-state absorptions or other nonlinear effects.

Overall, group I) molecules exhibit moderate to high 2PA cross sections across both the first and second biological windows. The single-substituted chromophores show unexpectedly good performance in the second biological window, while the double-substituted molecules exhibit comparable values in this range, but outperform in the first biological window. More specifically, the single-substituted chromophores reach their highest 2PA cross sections at the 1PA maximum of the S1 transition. In contrast, the double-substituted chromophores exhibit strong 2PA at the 1PA shoulders of the S1 transition and achieve their highest values at shorter excitation wavelengths around 900 nm, attributed to transitions to higher-lying states such as the S2 or S3.

In Figure the 1PE and 2PE spectra of group II) molecules with various styryl substituents are shown. Here as well, the 2PA maxima and shoulders associated with the S1 transition align well with twice the wavelength of their respective 1PA. In the following, we will highlight the impact of donor and acceptor groups on the 2PA properties of these molecules.

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1PA and 2PA spectra of A) TFA-TFA-BODIPY, B) TFA-OMe-BODIPY and C) OMe-OMe-BODIPY. The measured 2PA cross sections are represented by dots connected with lines, and the corresponding 1PA spectra are shown as filled, dashed lines in a transparent color. The left axis indicates 2PA cross sections with 2PE wavelengths on the bottom axis, while the right axis shows extinction coefficients for the 1PA spectra with 1PA wavelengths on the top.

In group II) molecules, the substituents align within the same plane of the BODIPY core (Figure S3), in contrast to the partially tilted geometry observed in group I). This improved planarity enhances π-conjugation and orbital overlap. As in group I) molecules, the S1 transition involves a mirror-symmetric LUMO and a nodal-containing HOMO along the vertical axis of BODIPY (Figure S7). The planar alignment of the substituents, combined with their greater conformational flexibility, likely improves electronic coupling and orbital overlap, potentially explaining the slightly stronger 2PA cross sections of the S1 transition in group II) molecules compared to disubstituted molecules in group I). However, like group I), group II) molecules exhibit their strongest 2PA in the S1 transition at one of the pronounced shoulders of the 1PA spectrum. However, at the 1PA maximum (0–0 transition) of the S1 transition, the extent of 2PA varies between the molecules. The strongest 2PA at the 1PA maximum, relative to the shoulder, is observed in the push–pull molecule TFA-OMe-BODIPY, closely followed by OMe-OMe-BODIPY, while the lowest values appear in TFA-TFA-BODIPY. The strong 2PA of TFA-OMe-BODIPY at the 1PA maximum can be attributed to its dipolar character, where electron density is primarily shifted from the methoxy substituent to the BODIPY core (Figure S7). Since the TFA-substituted side shows minimal electron density redistribution, the S1 transition in TFA-OMe-BODIPY behaves more like the monosubstituted molecules in group I) rather than the symmetrically double-substituted molecules. As a result, TFA-OMe-BODIPY is best described by the C1 point group, whereas the symmetric molecules TFA-TFA-BODIPY and OMe-OMe-BODIPY can be assigned to C2.

The symmetry reduction in TFA-OMe-BODIPY relaxes the selection rules, allowing more 2PA tensor components to contribute to the transition. In contrast, the symmetric C2 systems are restricted to a smaller subset of tensor elements, which may explain the reduced 2PA observed in TFA-TFA-BODIPY. For OMe-OMe-BODIPY, however, the strong electron-donating ability of the methoxy groups along with the high flexibility of the substituents, enable a strong 2P transition to the S1 state despite the symmetry.

For the S2 transition, our calculations suggest that it is expected to occur between 420 and 445 nm in the 1PE case, corresponding to 840 to 890 nm in the 2PE case. While the main contributing orbitals are HOMO–1 and LUMO in TFA-OMe-BODIPY and OMe-OMe-BODIPY, in TFA-TFA-BODIPY the dominant orbitals are HOMO–2 and LUMO. However, comparing the symmetry of the contributing orbitals, reveals that OMe-OMe-BODIPY and TFA-TFA-BODIPY both exhibit mirror-symmetry along the vertical axis, which supports coupling via symmetric tensor components. In contrast, TFA-OMe-BODIPY shows asymmetric orbital contributions due to its dipolar character and C1 symmetry.

Overall, group II) molecules exhibit high 2PA cross sections in the second biological window and moderate values in the observed range of the first biological window. The 2PA values of TFA-TFA-BODIPY and TFA-OMe-BODIPY are similar in magnitude, with TFA-TFA-BODIPY displaying slightly higher values for the S1 transition. Interestingly, the push–pull effect in TFA-OMe-BODIPY becomes more apparent in the 1PA maximum of the S1 transition and at the S2 transition (around 900 nm), where it outperforms TFA-TFA-BODIPY. However, among these group members, the strong electron-donating character of the methoxy groups in OMe-OMe-BODIPY results in superior 2PA performance. Remarkably, OMe-OMe-BODIPY even slightly surpasses di-EC-BODIPY (see Table for more detailed values), the most efficient 2P absorbing, disubstituted molecule from group I), in the second biological window.

1. Overview of the Molecule’s Characteristic 1PA and 2PA Steady-State Properties and Their Excited-State Lifetimes τ f Determined from TCSPC Transients (Figure S10)­ .

Molecule εmax/cm–1 M–1 Φ f /% δS1,max/GM δS1,shoulder/GM δ900 nm/GM τ f /ns
mono-DBF-BODIPY 88000 80 145 (1180 nm) 100 (1100 nm) 10 3.98
mono-FL-BODIPY 92500 73 195 (1210 nm) 125 (1120 nm) 90 3.92
mono-EC-BODIPY 111500 58 1110 (1240 nm) 440 (1140 nm) 145 3.98
di-DBF-BODIPY 71500 19 110 (1330 nm) 160 (1230 nm) 200 3.78
di-FL-BODIPY 58000 24 75 (1370 nm) 200 (1260 nm) 445 3.26
di-EC-BODIPY 108000 18 310 (1430 nm) 655 (1270 nm) 4125 3.16
TFA-TFA-BODIPY 107500 28 115 (1280 nm) 310 (1180 nm) 55 3.80
TFA-OMe-BODIPY 73000 22 230 (1320 nm) 310 (1230 nm) 200 3.30
OMe-OMe-BODIPY 110000 15 500 (1330 nm) 720 (1240 nm) 415 3.53
rigid-BODIPY-1 87000 64 40 (1290 nm) 65 (1200 nm) 10 5.27
rigid-BODIPY-2 81000 37 150 (1350 nm) 220 (1240 nm) 95 5.24
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The steady-state properties include the extinction coefficient εmax at their S1 maximum as well as their fluorescence quantum yield Φf. The 2PA cross sections are provided at three distinct wavelengths corresponding to the 1PA maximum δS1,max, 1PA shoulder δS1,shoulder and transitions to higher states around 900 nm δ900 nm.

In Figure the 1PE and 2PE spectra of group III) molecules featuring rigid substituents are presented. As observed in the previous groups, the 2PA maxima and shoulders match well with twice the wavelength of the respective 1PA peaks. This section will explore the influence of rigid modifications on the 2PA properties of these molecules.

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1PA and 2PA spectra of A) rigid-BODIPY-1 and B) rigid-BODIPY-2. The measured 2PA cross sections are represented by dots connected with lines, and the corresponding 1PA spectra are shown as filled, dashed lines in a transparent color. The left axis indicates 2PA cross sections with 2PE wavelengths on the bottom axis, while the right axis shows extinction coefficients for the 1PA spectra with 1PA wavelengths on the top.

Both rigid molecules demonstrate low 2PA cross sections for the S1 transition, comparable to di-DBF-BODIPY and di-FL-BODIPY, which can be attributed to the similarly low electron delocalization of the hexane moieties in the rigid substituents (Figure S8). Similar to other disubstituted molecules, rigid-BODIPY-1 and rigid-BODIPY-2 undergo a S1 transition from the asymmetric HOMO to the mirror-symmetric LUMO orbital. This results in an overall asymmetric transition, reducing the contribution of symmetric 2PA tensor components. Additionally, the restricted geometry prevents effective vibronic mode contributions to the 2P-excited transitions. Furthermore, the rigid geometry influences the ground-state conformation, so that rigid-BODIPY-2 and rigid-BODIPY-1, similar to di-DBF-BODIPY and di-EC-BODIPY, display a tilded alignment of their bulky substituents (Figure S4), which probably reduces the effective C 2 symmetry. Interestingly, the methoxyphenyl groups in the upper part of rigid-BODIPY-2 do not contribute to the orbitals involved in the S1 transition. However, they might play a significant role in vibronic coupling and thus potentially enhancing the 2PE transition. Thus, the primary cause of the strong redshift in rigid-BODIPY-2 is not its additional methoxyphenyl groups, but rather the substitution on the boron atom, where the fluorination induces a stronger redshift compared to the methylation in rigid-BODIPY-1.

Similar to all other disubstituted molecules, the S2 transition mainly involves two mirror-symmetric orbitals, which should favor the 2P transition. The S2 transition is calculated to occur around 940 nm for rigid-BODIPY-1 and 885 nm for rigid-BODIPY-2. While the calculated S2 transition of rigid-BODIPY-1 falls within the range of the measurement window, a distinct maximum is not yet visible until 900 nm, with only a slight slope increase. In contrast, rigid-BODIPY-2 exhibits a sharp rise toward 900 nm and already shows 10-fold increase in 2PA cross-section compared to rigid-BODIPY-1. This pronounced difference is attributed to the nature of the contributing orbitals. In rigid-BODIPY-1, the S2 transition predominantly involves excitations from HOMO–1 and HOMO–2 to LUMO, with all contributing orbitals being localized on the rigid substituents. Whereas in rigid-BODIPY-2, the dominant excitation involves electron density redistribution from the methoxyphenyl groups in HOMO–2 to LUMO, which extends through the BODIPY core toward the rigid substituents. This strong charge-transfer character of the S2 transition is the main reason for the strong 2PA enhancement in rigid-BODIPY-2.

In conclusion, the rigid molecules exhibit low electron redistribution for the S1 transition and lack conformational flexibility. However, rigid-BODIPY-2 contains two additional methoxyphenyl substituents, which may enhance vibronic coupling and thereby increase the 2PA cross sections compared to rigid-BODIPY-1. Furthermore, fluorination in rigid-BODIPY-2 appears to be more effective in inducing an overall redshift of the S1 transition and likely contributes to the improved 2PA performance compared to rigid-BODIPY-1.

A comparison of the 2PA properties across all molecule groups reveals intriguing insights into structural motifs that enhance 2PA. Generally, single-substituted molecules exhibit similar or slightly higher 2PA values at their S1 transition compared to their double-substituted counterparts, as seen in group I) molecules. One contributing factor is the quadrupolar character of double-substituted molecules, where substituents push electron density toward the BODIPY core in a push–pull-push fashion. This arrangement seems to be less effective for the S1 transition as the involved orbitals lack a common symmetry and thus reduce symmetric 2PA tensor elements. However, strong electron-donating substituents can loosen symmetry considerations as observed in OMe-OMe-BODIPY, which achieves remarkably high 2PA cross sections. Similarly, strong vibrational coupling can enhance 2PA, particularly at the 1PA shoulder of the S1 transition, where all double-substituted molecules exhibit higher values than at the weakened 1PA maximum. Asymmetric double-substitution such as in TFA-OMe-BODIPY leads to a stronger dipolar character, similar to single-substituted molecules. Unlike quadrupolar arrangements, dipolar configurations do not negatively impact the S1 transition and even enhance 2PA cross sections, as seen in mono-EC-BODIPY. In general, a dipolar substitution already enables strong two-photon excitation of the S1 transition making these molecules highly effective within the second biological window. However, double-substituted molecules cover a broader excitation range in the second biological window, offering more versatile excitation schemes while reducing the 2PA peak values. Additionally, they exhibit significantly higher 2PA values in the first biological window, making them suitable for applications in both biological windows.

From a broader perspective, dipolar substitution leads to high 2PA cross sections in the second biological window, while quadrupolar BODIPY molecules exhibit comparable or even higher 2PA values in the first biological window. Based on this classification, we now examine specific structural elements that enhance 2PA efficiency. Styryl substituents in group II) molecules greatly enhance 2PA cross sections, likely due to their increased flexibility, which promotes vibronic coupling and their in-plane alignment with the BODIPY core, which further promotes electron delocalization. These effects are particularly evident, when comparing rigid-BODIPY-2, featuring rigid methoxyphenyl substituents, with OMe-OMe-BODIPY, which contains methoxy-substituted styryl groups. The latter achieves a 3-fold higher 2PA cross sections (720 GM), making it one of the most efficient 2P absorbers in the present study. The highest 2PA in the second biological window is achieved by mono-EC-BODIPY (1110 GM at 1240 nm), closely followed by its double-substituted counterpart di-EC-BODIPY (655 GM at 1270 nm). In the first biological window, di-EC-BODIPY reaches values up to 4000 GM in the observed wavelength range. However, these values are not pure to 2PA and may include ESA contributions or other nonlinear effects. A common feature among these top-performing molecules is the presence of strong electron-donating groups, such as ethyl-carbazole in mono-EC-BODIPY and di-EC-BODIPY or methoxyphenyl in OMe-OMe-BODIPY, which enhance electron delocalization and facilitate 2PA. The critical role of electron-donating groups is particularly apparent in group II) molecules. Here, OMe-OMe-BODIPY, with its superior 2PA properties, significantly outperforms TFA-TFA-BODIPY, which features two electron-withdrawing groups and TFA-OMe-BODIPY, which adopts a dipolar substitution pattern.

In summary, 2PA efficiency depends on a multitude of parameters such as charge transfer characteristics, symmetry, electronic coupling and vibronic interactions. While dipolar molecules excel in the second biological window, quadrupolar molecules dominate in the observed range of the first biological window. The most efficient 2PA active molecules contain electron-donating substituents as well as structural flexibility, allowing enhanced electron delocalization and vibronic coupling.

Excited State Properties and Quantum Yields

As shown in the previous section, all herein presented molecules show moderate to outstanding 2PA cross sections. In the following, we investigate whether the various substitution patterns can alter the excited-state dynamics of the molecules and negatively affect their fluorescence or uncaging efficiency. For this purpose, femtosecond UV/vis transient absorption measurements were conducted for selected molecules from all groups. The time-resolved maps and the resulting lifetime density maps were analyzed using OPTIMUS and are depicted in Figure .

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Transient absorption spectra (left side) and lifetime density maps (right side) of selected molecules (indicated by the name and structure) in chloroform. Experimental settings and details of the analysis procedure are provided in the Supporting Information.

All measured molecules feature long excited-state lifetimes that exceed the measurement window of our transient absorption setup. Additionally, all of them feature a strong negative signal, which is assigned to a mixed contribution of ground state bleaching (GSB) and stimulated emission (SE). Depending on the absorption and emission properties, the wavelength position of the GSB/SE signal can shift, with some being at the boundary of the measurement window. In addition to the strong GSB/SE signal, all molecules show a weaker GSB/SE contribution at lower wavelengths compared to the main band. This weaker GSB/SE signal can be assigned to the characteristic BODIPY shoulder. In mono-DBF-BODIPY, this shoulder overlaps with ESA1 and thus leads to a partial compensation of the positive signal in such a way that ESA1 is split into two main peaks with one thin band around 575 nm and one broader band around 530 nm. In general, for all molecules, ESA1 is located between 550 and 575 nm. Among all molecules, mono-DBF-BODIPY (Figure A) shows the most blue-shifted GSB/SE with the main peak around 600 nm. Thus, its blue-shifted shoulder falls in the range of ESA1. Another important feature missing in mono-DBF-BODIPY is a second positive band, ESA2, which is present in all double-substituted molecules. ESA2 is located between 450 and 500 nm. In molecules with flexible linkers (Figure B,C), ESA1 and ESA2 both show quite broad features, while in the rigid molecules, these bands are way sharper (Figure D,E). An explanation for the sharpening could be the restricted conformational distribution of the rigid substituents. Interestingly, the flexible methoxyphenyl substituents at the upper part of rigid-BODIPY-2 seem to affect the intensity ratio between ESA1 and ESA2. Although in all molecules, both ESAs show similar intensities, in rigid-BODIPY-2 (Figure E), ESA2 has a much stronger contribution than ESA1.

Overall, all molecules show similar and distinct features in the excited-state dynamics. ESA1 and ESA2 seem to be characteristic features of symmetrically substituted molecules in these wavelength regions. On these early femtosecond to picosecond time scales, the substituents do not affect the lifetime of the BODIPY core much, since the decay of the signals at the end of the measurement window is the only dominant feature in the LDMs. As the decay dynamics cannot be fully resolved within the time frame of the transient absorption experiment, additional time-correlated single photon counting (TCSPC) experiments were conducted to analyze the fluorescence lifetime. Transients of representative molecules from each group are presented in Figure . Further experimental settings, data analysis and the single transients of all molecules (Figure S10) are provided in the Supporting Information.

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Fluorescence transients of selected molecules from each group are shown as logarithmic plot of normalized emission against photon arrival time. Experimental settings, details of the analysis procedure and transients of the remaining molecules are provided in the Supporting Information. The obtained fluorescence lifetimes are presented in Table .

The obtained fluorescence lifetimes are presented in Table . As can be seen, the lifetimes of most molecules are similar and vary only slightly between 3.7 and 4.0 ns. The most remarkable differences are present in the rigid constructed molecules. Both rigid-BODIPY-1 and rigid-BODIPY-2 exhibit longer fluorescence lifetimes of 5.24 and 5.27 ns, respectively. This is owing to the fact, that the rigid construction inhibits faster deactivation channels. In addition to measuring the fluorescence lifetimes under 1P excitation, we also determined the fluorescence lifetimes under 2P excitation to investigate whether the excitation wavelength influences the lifetime by altering the excited-state pathways. Remarkably, identical fluorescence lifetimes (Table S1) were observed for all molecules regardless of whether 1P or 2P excitation was used. This indicates that the excited-state behavior is minimally affected by the excitation mechanism and that Kasha’s rule holds. Consequently, it is valid to use the 1-photon fluorescence quantum yields to correct the 2PE spectra, as previously discussed for photocages. More significant differences are observed in the fluorescence quantum yields of the molecules. For the single-substituted molecules of group I), the fluorescence quantum yields range between 58 and 80%. For their double-substituted equivalents, the values vary between 18 and 24%. Group II) molecules with the different donor and acceptor groups vary in a similar range between 15 and 28%. In contrast, the rigid compounds of group III) show higher quantum yields than all other double-substituted molecules. Especially, rigid-BODIPY-1 with its 64% reaches the values of single-substituted molecules. Meanwhile, rigid-BODIPY-2 shows a significantly lower value of 37%.

In summary, it can be stated that mono- and rigid-substitution can elevate the fluorescence quantum yields compared to the double-substituted molecules. However, what about the overall 2-photon fluorescence efficiency, which can be more relevant for applications such as 2-photon fluorescence microscopy? For this purpose, we provided the 2-photon fluorescence action cross-section spectra (see Figure S14), which additionally contain the fluorescence quantum yields. Comparing the 2P fluorescence action cross-section plots reveals that most molecules now show similar values. All double-substituted molecules exhibit action cross sections between 40 and 110 GM for their highest peaks with the only exception di-EC-BODIPY reaching 735 GM at 900 nm. The monosubstituted molecules show slightly higher 2PA cross sections. While mono-FL-BODIPY and mono-DBF-BODIPY exhibit 140 GM and 120 GM, respectively, the highest 2PA action cross-section is still achieved by mono-EC-BODIPY with remarkable 650 GM at 1240 nm. Overall, mono-EC-BODIPY and di-EC-BODIPY seem to be the superior candidates for 2-photon fluorescence applications.

In addition to the 2-photon fluorescence action cross-section plots, the 2-photon uncaging action cross-section of selected molecules were evaluated (see Figure ) using the corresponding 1P uncaging quantum yields. This approach assumes, as a first approximation, that 1P and 2P uncaging quantum yields are equivalent, as we did for the fluorescence action cross-section. However, since the probability of uncaging is much lower than that of fluorescence, uncaging could depend more on the type of excitation. Unfortunately, currently, there is no reliable and standardized approach for a quantitative determination of 2P uncaging quantum yields due to the small excitation volume of 2PE and the low amount of generated photoproducts. So far, 2P uncaging can only be confirmed qualitatively by fluorescence-based imaging, where the leaving group induces a change in fluorescence or a biological response. , Due to the lack of standardized approaches, it is a general practice to use 1P quantum yields for the determination of 2P action cross sections.

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2-Photon uncaging action cross-section plot of the molecules with the best 1P uncaging quantum yields. Values were obtained by multiplying the 2PA cross sections with the corresponding 1P uncaging quantum yields. The 1P uncaging quantum yields of B-Me-mono-EC-BODIPY (0.0084) and B-Me-di-EC-BODIPY (0.0078) were determined by irradiation experiments, which are provided in Figure S15. The values for rigid-BODIPY-1 (0.034) and rigid-BODIPY-2 (0.015) are taken from the indicated references.

As we have identified the molecules with the highest 2PA cross sections, we now tried to boost the uncaging quantum yields for effective 2P uncaging. Thus, two additional compounds with B-methylation were synthesized. In general, B-fluorinated BODIPY photocages, with the exception of rigid-BODIPY-2, have much lower uncaging quantum yields than their B-methylated counterparts. , However, the B-fluorinated BODIPYs are synthetically more accessible, making them more suitable for a large 2PA screening across 11 different molecules. Based on their promising performance, the ethyl-carbazole-substituted compounds mono-EC-BODIPY and di-EC-BODIPY were selected for B-methylation, yielding B-Me-mono-EC-BODIPY and B-Me-di-EC-BODIPY. These, along with rigid-BODIPY-1 and rigid-BODIPY-2, were subjected to more detailed 2P uncaging action cross sections analysis (see Figure ).

Despite the improved 1P uncaging quantum yields of the B-methylated molecules, 0.84% for B-Me-mono-EC-BODIPY and 0.78% for B-Me-mono-EC-BODIPY (see Supporting Information for more details of the irradiation experiments), the resulting 2PA uncaging action cross sections were lower than expected (0.7 GM at 1190 nm and 1.3 GM at 1240 nm, respectively). This reduction can be attributed to the inherently low 2PA cross sections of the B-methylated BODIPYs (see Figure S16). Although the B-methylated carbazole-substituted molecules share the same substituents as the B-fluorinated counterparts, the substitution on the boron seem to play a major role in the 2PA properties. The introduction of methyl groups at the boron slightly reduces electron delocalization, evidenced by a blueshifted absorption of around 30–40 nm compared to the fluorinated molecules. This shift is likely due to the electron-donating effect of the methyl groups, which contrasts the electron-withdrawing effect of fluorine. Furthermore, the methyl groups probably increase the overall steric repulsion with the already bulky carbazole substituents, which can further decrease the planarity of the system and might reduce favorable vibronic coupling. Meanwhile, rigid-BODIPY-1 (2.3 GM at 1200 nm) and rigid-BODIPY-2 (3.3 GM at 1240 nm) exhibit slightly greater values which can be attributed to their higher uncaging quantum yields of 3.45% and 1.5%, respectively. Overall, the B-methylated carbazole-substituted BODIPY derivatives and rigid molecules have comparable uncaging action cross sections as many common two-photon absorbing photocages reported, while their ability to be excited within the second biological window makes them uniquely capable of being activated using NIR or short-wave IR light. However, the simultaneous optimization of both efficient uncaging and 2PA within a single chromophore remains a significant challenge and is another typical example of the persistent bottleneck in the field of photochemistry.

So far, all measurements were conducted in chloroform as the same solvent was used as for the reference Styryl 9 M in order to avoid differences and artifacts caused by the solvent. However, since chloroform is not suitable for biological applications, we performed additional 2PA measurements for the B-methylated compounds in a DMSO/water (8:2 ratio) mixture to better reflect aqueous environments (see Figure S19). For B-Me-mono-EC-BODIPY, the overall magnitude of the 2PA cross sections remain the same as in the chloroform measurements, but the relative ratio of the S1 peaks differ slightly. In contrast, B-Me-di-EC-BODIPY shows no distinct vibrational features and exhibits a 3-fold reduction in 2PA cross sections compared to the values obtained in chloroform. This is attributed to a lack of vibrational coupling, which would also explain the loss in vibrational features. Thus, the choice of solvent seems to affect the vibrational features of the 2PA spectra, particularly in more symmetric and quadrupolar compounds such as B-Me-di-EC-BODIPY. But overall, the 2PA values remain in a reasonable range, making these molecules suitable for biological applications as well.

Conclusion and Outlook

In conclusion, we analyzed 11 BODIPYs in detail with diverse substitution patterns regarding their two-photon absorption properties partially within the first and fully within the second biological window. Our results reveal, that the monosubstituted molecules exhibit moderate to excellent 2PA cross sections in the second biological window due to their dipolar character and greater flexibility, whereas the symmetrically double-substituted molecules demonstrated greater efficiency in the first biological window. Strong electron-donating substituents and significant vibrational coupling can enhance the 2PA cross sections of double-substituted molecules in the second biological window as well. Three molecules, mono-EC-BODIPY, di-EC-BODIPY and OMe-OMe-BODIPY, stand out for their exceptional 2PA cross sections, which we attribute to the presence of strong electron-donating substituents. Among them, mono-EC-BODIPY and di-EC-BODIPY show particularly high potential for two-photon excited uncaging applications, especially within the second biological window. Based on this, their B-methylated counterparts, B-Me-mono-EC-BODIPY and B-Me-di-EC-BODIPY were synthesized due to their higher 1P uncaging quantum yields. While the resulting 2PA uncaging action cross-section lie in the range of many common photocages, their potential of breaking bonds in the second biological window makes them unique. Additionally, the long excited-state lifetimes and high fluorescence quantum yields of BODIPYs make them well-suited for fluorescence-based applications. Furthermore, we showed that despite the various modifications, the fundamental photophysical properties, that make BODIPY exceptional, remain intact. This robustness enables structural optimization to enhance 2PA properties while preserving other beneficial photochemical features of BODIPYs. However, the simultaneous optimization of uncaging and 2PA efficiency still remains a considerable design challenge, where certain trade-offs are often unavoidable.

Recent advances suggest viable strategies to address this fundamental challenge. Several groups have shown that optimizing the uncaging mechanism itself and minimizing nonradiative decay pathways, can allow for red-shifted excitation without compromising uncaging efficiency. ,,,,,− We are confident that further mechanistic insight and design refinement will continue to push the limits forward in this field. One promising direction involves the development of photocages based on intrinsically 2PA-active fluorophores, benefiting from their long excited-state lifetimes and high extinction coefficients. ,, This strategy is typically guided by theoretical calculations as it requires careful placement of the leaving group to enable uncaging. Another elegant approach involves energy-transfer systems that decouple excitation from uncaging. In these dyad or tandem systems, a 2PA-optimized antenna unit absorbs light and transfers the excitation energy to a separate uncaging moiety. This modular approach allows each component to be independently optimized. Our group ,, and many others − have successfully established such design constructs with promising results. Notably, Wang et al. employed a triplet–triplet energy transfer system in which a red-shifted platinum­(II) porphyrin antenna sensitized a BODIPY photocage in an upconversion-like fashion, further expanding the design possibilities.

An important next step in the development of photocages is to enhance their compatibility with biological systems, particularly by improving their solubility in aqueous environments. Significant progress has recently been made in this direction by various research groups. ,,− While the core structure of BODIPYs and many other organic photocage chromophores remain inherently poorly water-soluble, they can still be effectively employed in biological contexts through formulation strategies. , One such approach involves incorporating photocages in liposomes, enabling targeted delivery and controlled release at protein-rich sites. Heckel and coworkers demonstrated that uncaging within such liposomal constructs is feasible, opening the door to membrane-associated photorelease applications.

The BODIPY-based photocages presented in this work are highly adaptable and can be tailored for specific biological applications by modifying the leaving group. In particular, aromatic leaving groups can be leveraged for imaging-guided applications, as they efficiently quench BODIPY fluorescence via photoinduced electron transfer. , Upon photorelease, fluorescence is restored, allowing real-time tracking of the uncaging progress through fluorescence microscopy. Moreover, these molecules absorb within the second biological window and emit within the first, thereby enabling deep-tissue excitation and emission imaging with minimal background interference. These properties make them promising candidates for in vivo applications such as fluorescence-guided uncaging, targeted drug delivery and activity mapping in biological tissues.

Supplementary Material

ja5c07710_si_001.pdf (9.2MB, pdf)

Acknowledgments

J.W. gratefully acknowledges Deutsche Forschungsgemeinschaft (DFG) for the funding of the research training group “CLiC” (GRK 1986, Complex Light-Control) and expresses his sincere gratitude to the European Union for funding the TCSPC laser system through the European Regional Development Fund as part of the Union’s response to the COVID-19 pandemic - REACT-EU, IWB-EFRE-Programm Hessen,\#20008794. AHW thanks the NSF for support (CHE-2349051).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c07710.

  • Synthesis, experimental details, optimized ground-state geometries, frontier molecular orbital transitions including excitation energies and oscillator strengths, emission spectra, TCSPC transients, squared power dependency measurements, 2PE fluorescence action cross-section plots (PDF)

§.

A.E.- Gedeon Richter Plc, P.O. Box 27, 1475 Budapest, Hungary

#.

Y.A.-S. and A.E. contributed equally.

The authors declare no competing financial interest.

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