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. 2024 Mar 18;146(12):8417–8424. doi: 10.1021/jacs.3c14197

Light in a Heartbeat: Bond Scission by a Single Photon above 800 nm

Marina Russo †, Hana Janeková †, Debora Meier ‡, Melanie Generali ‡, Peter Štacko †,*
PMCID: PMC10979397  PMID: 38499198

Abstract

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Photocages enable scientists to take full control over the activity of molecules using light as a biocompatible stimulus. Their emerging applications in photoactivated therapies call for efficient uncaging in the near-infrared (NIR) window, which represents a fundamental challenge. Here, we report synthetically accessible cyanine photocages that liberate alcohol, phenol, amine, and thiol payloads upon irradiation with NIR light up to 820 nm in aqueous media. The photocages display a unique chameleon-like behavior and operate via two distinct uncaging mechanisms: photooxidation and heterolytic bond cleavage. The latter process constitutes the first example of a direct bond scission by a single photon ever observed in cyanine dyes or at wavelengths exceeding 800 nm. Modulation of the beating rates of human cardiomyocytes that we achieved by light-actuated release of adrenergic agonist etilefrine at submicromolar concentrations and low NIR light doses (∼12 J cm–2) highlights the potential of these photocages in biology and medicine.

Introduction

Photocages capitalize on the biorthogonality and high spatiotemporal resolution of light as a trigger to seize control over substrate activity.1 Exemplified by their success in activating proteins,2,3 nucleotides,4,5 drugs,6,7 and other biologically relevant molecules,8−10 photocages have gained significant traction in photoactivated chemotherapy11−16 (PACT), an emerging approach complementary to photodynamic therapy (PDT). Yet, to clinically establish photocages alongside PDT, it is critical to shift their absorption into the near-infrared, tissue-transparent window (NIR; 650–900 nm) without compromising the release efficacies.17,18

Although remarkable advances in the field of photocages have been recorded in the past decades (Figure 1A),1,19 the discovery of novel scaffolds for organic photocages has historically relied mostly on a serendipity, and their development remains far from being an accomplished task.20 The challenges are even more daunting for NIR photocages as the excitation energy provided by NIR photons is low, excited states are short-lived, and the application demands compatibility with aqueous media.

Figure 1.

Figure 1

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(A) State-of-the-art NIR photocages. (B) The concept of uncaging from Cy7 is based on direct bond scission. (C) Isolobal analogy of known photocages with a hitherto elusive analogous photocage based on the Cy7 scaffold.

In 2015, Šolomek et al. postulated a design strategy to predict new photocage scaffolds drawing on the Zimmerman’s effect and, at that time, known photocages.21 The concept relies on the accumulation of electron density in the excited state in a position adjacent to the carbon-payload bond (Figure 1C, denoted by arrows). Mixing of the filled orbital with the adjacent antibonding orbital weakens the carbon-payload bond and facilitates departure of the payload. Such shifts of electron density can be predicted by simple Hückel calculations of the frontier molecular orbital (HMO) coefficients. This approach was supported by the emergence of BODIPY-based photocages,22,23 which underwent impressive optimization in terms of efficacies and absorption properties.24−27 Reports of xanthene-,28 porphyrin-,29 and, most recently also,30 rhodamine-derived photocages emerged, all isolobally analogous chromophores. Yet, analogous photocages derived from heptamethine cyanine (Cy7) dyes that operate via heterolytic bond cleavage from the excited state have remained elusive despite sharing the same orbital topology (Figure 1C).

The Cy7-based uncaging reported to date relies exclusively on 1O2-mediated fragmentation of the Cy7 scaffold followed by thermal solvolysis of the intermediates to liberate the payloads of only a limited scope (Figure 1A). Schnermann and co-workers harnessed photooxidative cleavage of Cy7 to release phenols through a cascade of reactions.15,31−33 Recently, we and the group of Feringa simultaneously reported uncaging of carboxylic acids from Cy7 dyes,34,35 which was eventually experimentally confirmed to proceed through a related mechanism, i.e., only in the presence of singlet oxygen (1O2).35 The uncaging efficacies consequently strongly depend on the concentration of both the photocage and oxygen in the system, and the slow thermal steps limit their application in studies of fast processes. Aware of these drawbacks and convinced about the generality of the HMO approach, we leveraged it in Cy7 scaffolds.

Here, we report a family of Cy7 photocages that can release a broad palette of payloads including amines, phenols, alcohols, and thiols with tissue-penetrating NIR light (Figure 1B). Their applicability is illustrated in human cardiomyocytes by light-induced modulation of their beating rate. The photocages exhibit a chameleon-like behavior and operate through two distinct uncaging mechanisms. We finally provide evidence that direct uncaging from Cy7 dyes with a single near-infrared photon at wavelengths >800 nm is a viable strategy. Besides, further reinforcing the generality of the HMO design strategy, this pathway absolves Cy7 photocages of the reliance on oxygen and slow secondary steps and instill them as strong contenders in the pursuit of PACT.

Results and Discussion

We synthesized the photocages 1a–1h conveniently in four steps using the Zincke chemistry protocol (Scheme 1).36 The starting alcohol 2 was reacted with bis(4-nitrophenyl) carbonate to provide activated carbonate 3 in 85% yield. The payloads were subsequently introduced in excellent yields (up to 89%) by nucleophilic substitution of 3 with the corresponding alcohols, thiol, or amines in the presence of DIPEA or DMAP as auxiliary bases. The reaction of the pyridines 4a–4g with 2,4-dinitrophenyl tosylate or triflate (DNP-OTs or DNP-OTf) in acetonitrile provided the corresponding Zincke salts 5a–5g, which were subsequently transformed into the desired photocages 1a–1k containing diverse payloads in a one-pot reaction with the terminal heterocycles 6a–6c and AcOK as a base. Carbamate-containing photocages 1e–1h were observed by 1H NMR spectroscopy as mixtures of two rotamers, which coalesced into a single spectrum at an elevated temperature. In contrast, carbamate analogues derived from primary amines remained elusive, likely due to abstraction of the N–H under the mildly basic reaction conditions.

Scheme 1. Synthesis of the Photocages.

Scheme 1

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(i) DMAP, bis(4-nitrophenyl)carbonate, MeCN. (ii) Amine/alcohol/thiol payload, MeCN or DMSO, 80 °C. (iii) DNP-OTs or DNP-OTf, MeCN, 40 °C. (iv) 6a–6c, AcOK, MeCN, rt. Caged payloads are depicted in red. Counter anions are omitted for clarity.

Reported in ref (35).

Photocages 1a–1k in PBS (pH 7.4, 10 mM, 20% DMSO) display a strong absorption located in the center of the NIR region at λmax = 809–818 nm, matching the emission of commercial diode lasers and the isosbestic point of the hemoglobin-deoxyhemoglobin system (∼810 nm).37 The compounds exhibit weak emission (ΦF < 0.02) with Stokes shifts of ∼500 cm–1, comparable to similar red-shifted cyanines.38 While 1f and 1g are fully soluble in aqueous media, some derivatives showed propensity to aggregate (c > 10–5 M) depending on lipophilicity of the payload. DMSO was therefore employed as a cosolvent in UV–vis studies to facilitate direct comparison between derivatives 1a–1h.

Irradiation of 1a in CD3OD at 810 nm (∼300 mW cm–2) for 24 h under ambient conditions led to the complete loss of the green color typical for Cy7 dyes accompanied by liberation of the 4-fluorophenol (pFP) payload observed by 1H and 19F NMR (Figure S27). Complete destruction of the Cy7 scaffold and the production of ketone 7 (vide supraScheme 2) were consistent with the previously reported 1i operating via the 1O2-mediated uncaging mechanism.39,40 In strong contrast, irradiation of 1a with NIR light under the exclusion of oxygen leads to its clean conversion into a new Cy7 species with concurrent uncaging of pFP as evidenced by 1H and 19F NMR spectroscopies (Figure 2A,B, depicted in green and red, respectively). The signal of the proton at the reaction center experiences a significant upfield shift from 5.98 to 4.59 ppm, consistent with the presence of a less electron-deficient residue in this position, i.e., a formal substitution of the payload by methanol. The anticipated structure 8 that originates from trapping the putative carbocation by CD3OD was further supported by HRMS (Figure 2C). To unequivocally establish its identity, we synthesized 8 independently and confirmed that its 1H NMR and UV–vis spectra perfectly match those produced by the irradiation of 1a. We observed uncaging under oxygen-free conditions also for other payload functionalities (Table 1, e.g., 1b, 1c, and 1e), wheareas no uncaging or formation of 8 was observed for the control samples kept in the dark (Figures S27–S36). These results are in strong contrast to the behavior of previously reported 1i and provide compelling evidence that the payload from 1a is released by a direct C–O bond scission, with intermediary formation of a carbocation at the cyanine residue. Both 1a and its des-methoxy analogue 1j release pFP when irradiated under oxygen-free conditions with comparable efficiencies of 91 and 93%, respectively, demonstrating that the electron-donating substituents are not instrumental to the direct uncaging mechanism (Figures S44 and S45). The quantum yield of the direct uncaging pathway (Φhet) from 1a in CD3OD was determined to be (6.8 ± 0.7) × 10–4, which is comparable to the BODIPY photocages that operate at wavelengths blue-shifted by ∼300 nm,23 and approximately an order of magnitude higher than the first generation of their red-shifted analogues.24 The uncaging cross section of 1a (εΦ ∼ 80 M–1 cm–1) sits comfortably in the range desired for biological application.19

Scheme 2. Suggested Photooxidative (Left) and Direct (Right) Uncaging Mechanisms That Operate in Cy7 Photocages.

Scheme 2

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Figure 2.

Figure 2

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(A) Photouncaging of pFP payload from 1a via the direct pathway including a cationic intermediate, which is trapped by the solvent to give 8. (B) 1H (left) and 19F (right) NMR spectra of 1a, 1a irradiated at 810 nm in oxygen-free conditions, pFP for 44 h, and the photoproduct 8 (from bottom to top). (C) ESI-HRMS of the identical sample of 1a irradiated at 810 nm in the absence of oxygen showing the clean formation of 8.

Table 1. Photophysical Properties of the Selected Photocages.

photocage λabs/nma λem/nmb εa,c Φox × 105a,d yieldaer/%e,g yielddeg/%f,g
1a 809 843 117 300 4.5 ± 0.1 47 ± 2 88 ± 1 (100)
1b 809 839 120 900 3.4 ± 0.3 44 ± 2 92 ± 4 (46)
1c 811 843 94 200 5.8 ± 0.9 63 ± 3 103 ± 3 (100)
1d 808 844 115 300 2.2 ± 0.2 40 ± 7 n.d.
1e 810 847 100 400 2.7 ± 0.2 44 ± 2 67 ± 8 (26)
1f 818 854 117 800 2.3 ± 0.2 48 ± 2 n.d.
1g 816 856 85 000 2.5 ± 0.1 55 ± 5 n.d.
1hb 808 836 44 600 0.6 ± 0.1 41 ± 3 n.d.
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a

Determined in PBS (pH 7.4, 10 mM) with 20% DMSO.

b

Determined in methanol.

c

Molar absorption coefficient, εmax/mol–1 dm3 cm–1.

d

Absolute quantum yields of photooxidative decomposition.

e

Chemical yield of the payload uncaging with 820 nm light in CD3OD under ambient conditions.

f

Chemical yield of the payload uncaging with 810 nm light in CD3OD under oxygen-free conditions, corrected for the conversion (in parentheses).

g

Determined by 1H NMR spectroscopy. Average and standard deviations of the mean from at least three independent samples are given. n.d. stands for not determined.

We subsequently followed uncaging by UV–vis absorption spectroscopy (Figure 3A–C). Irradiation of 1a–1h in PBS (pH 7.4, 10 mM, 20% DMSO) at 820 nm (∼40 mW cm–2) for 20–40 min under ambient conditions was accompanied by depletion of the cyanine absorption band, and in the case of 1a–1c, a concurrent minor blue-shift (∼3–9 nm) of the absorption maxima (see Figures S11–S13). The former is attributed to photooxidation of the cyanine scaffold,35,40 whereas the latter suggests a concomitant formation of a new cyanine species that bears a less electron-accepting residue in the C4′ position, i.e., 8.38 As anticipated, the photoxidation process was inhibited under oxygen-free conditions in both MeOH and PBS, but a blue-shift was observed, indicating that oxygen is not vital for the latter process (Figures S22–S26). LC-HRMS analysis of these samples detected the expected photoproduct formed by trapping the putative carbocation by MeOH or water. The quantum yields of the photooxidation (Φox) process summarized in Table 1 are comparable to other Cy7 dyes.38

Figure 3.

Figure 3

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(A) UV–vis absorption (red) and emission (blue) spectra of 1a in PBS and methanol, respectively. (B) Irradiation of 1a in PBS at 820 nm followed by UV–vis spectroscopy every 30 s (blue to red) under ambient conditions. (C) Kinetic trace of the absorbance at 809 nm upon irradiation of 1a at 820 nm (red) and in the dark (blue) under ambient conditions. (D) Chemical yields of the payloads uncaged from selected photocages in the dark (black), upon irradiation at 780 nm under ambient (red) and oxygen-free (blue) conditions.

Uncaging of the fluorogenic 4-methylumbelliferone payload from 1k in aqueous media (PBS/DMSO, 4:1) was then followed by emission spectroscopy (Figures S19–S21). Under ambient conditions, the payload was uncaged at a rate comparable to the depletion of the cyanine band, without the obviously slow, thermal component previously observed for carboxylates.35 Removal of oxygen by purging with Ar (5 min) significantly suppressed the cyanine band depletion (∼14-fold), accompanied by a blue shift of the absorption maxima (8 nm). On the other hand, the rate of uncaging remained relatively fast (3-fold slower) and majority of the payload was still uncaged in the same time frame. The rates of uncaging in these two samples cannot be directly compared because the latter will be slower not only due to suppression of the photooxidation pathway but also because of the inner filter effect of the cyanine photoproduct. However, these results clearly demonstrate that the direct uncaging pathway also operates to a significant degree in aqueous media.

The uncaging from 1a–1h under ambient conditions proceeds with a chemical yield of 40–60%, whereas the yield is significantly higher under oxygen-free conditions and nearly quantitative for 1a–c as determined by 1H NMR spectroscopy (Table 1, Figure 3D). We hypothesized that the lower yield of uncaging 1e in oxygen-free conditions could be due to the released amine payload attacking the putative carbocation, but we found no evidence of such species by LC-HRMS or 1H NMR spectroscopy (Figure S37). No detectable release was observed in the samples kept in the dark, except for 1h, which exhibits a compromised solvolytic stability (∼4% uncaging after 24 h). On the other hand, photocages 1a and 1e displayed excellent stability also in aqueous media (H2O/MeCN, 9:1) and no trace of the uncaged payloads was observed by UPLC after incubation for 4 h in the dark (Figure S49). Interestingly, thiol was liberated from 1c as the corresponding disulfide, likely due to secondary oxidation by ROS or via an electron transfer to Cy7. However, we do not expect this to prevent the application of thiol-releasing Cy7 photocages in a cellular environment as endogenous levels of gluthathione reduce disulfides to thiols.41

Next, we evaluated the toxicity of 1f and 1g and their corresponding photoproducts produced by exhaustive irradiation in cell viability assays on HeLa cells. The compounds exhibited no toxicity after 72 h of exposure at concentrations up to 50 μM (see Figures S52–S53). Their cell-permeable analogues are likely somewhat more toxic as evidenced in the literature.35,42,43

Guided by the ambitions of photocages in biomedical sciences, we sought to demonstrate their applicability in controlling biological processes that extend beyond just cellular death. We therefore synthesized caged etilefrine derivative 1g (Figure 4A). Etilefrine 9, an adrenergic agonist of primarily α1 and β1 receptors, is a cardiac stimulant that is approved as a hypotensive drug.44 We tested 1g and its light-induced activity on spontaneously beating iPSC-derived cardiomyocytes, which were differentiated using a commercial kit and allowed to form a syncytium. The beating frequency under different conditions was quantified using an established protocol using calcium flux fluorescence imaging by Fluo-8AM (Figure 4B, see the Supporting Information for details).45,46 The use of a higher concentration of 1g (800 nM) in comparison to the free etilefrine (400 nM) in control experiments compensated for the moderate chemical yield of uncaging of 9 from 1g (Figure 3D, Table 1). The cardiomyocytes incubated with 1g and subsequently irradiated with NIR light (780 nm, ∼40 mW per well for 5 min) displayed a significant increase in the beating frequency compared to both those kept in the dark and the control experiments (Figure 4C). The magnitude of the increase was comparable to that elicited by free etilefrine, whereas cardiomyocytes incubated with 1g in the dark showed a beating frequency virtually identical to that of the control experiment. Similar effects were observed also in the HL-1 mouse cardiac muscle cell line. While we expect that etilefrine is uncaged for 1g predominantly through photooxidation, we cannot exclude that part of it is uncaged via the direct uncaging pathway, especially given the low concentration of 1g.

Figure 4.

Figure 4

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(A) Uncaging of etilefrine 9 from photocage 1g with light at 780 nm. (B) Representative traces of the total cell fluorescence (TCF) of beating iPSC-derived cardiomyocytes loaded with calcium probe Fluo-8AM and 1g (800 nM) kept in the dark (top) or irradiated at 780 nm (∼40 mW, 5 min, bottom). (C) Observed beating rates of the cardiomyocytes incubated with 1g (800 nM) in the dark (blue) or irradiated with NIR light (red) or with etilefrine (400 nM, cyan) and the control (black). Median, IQR, and 5–95% percentiles are shown.

Contrary to the recent example using the BODIPY scaffold,47 Cy7-caged etilefrine 1g showed no residual activity, suggesting that the charged and bulkier Cy7 scaffold is more efficient at blocking the binding compared to the BODIPY residue. In contrast, we achieved modulation of the beating frequency by direct irradiation of the cardiomyocytes with NIR light instead of irradiating the photocage prior to its incubation with the cells. Our results reaffirm the capacity of Cy7 photocages to directly manipulate delicate biological processes at submicromolar concentration by activation of drug-like molecules containing multiple functional groups at low NIR light doses (∼12 J cm–2), comparable to use in PDT.

Mechanistic Considerations

The presented experimental evidence shows that these photocages can release payloads by two orthogonal mechanisms: self-sensitized photooxidation and direct uncaging through a cationic intermediate (Scheme 2). Since the former is a bimolecular reaction and its Φox depends on the concentration of Cy7, whereas the Φhet of the latter does not, one can find a concentration at which these quantum yields of the competing mechanisms are equal. Below this concentration (or at increasingly hypoxic conditions), the uncaging is dominated by the direct uncaging pathway, whereas above this concentration, the majority of the payload is released via the 1O2-mediated mechanism. We estimated this limit for 1a in aqueous media to c ∼ 1–2 × 10–4 M (see the Supporting Information). Indeed, at sufficiently low starting concentrations of 1a or 1e (c ∼ 2 × 10–4 M in MeOH/H2O), we observed the photoproducts of the direct uncaging, 8, its analogue formed by trapping with water, and the subsequent products of oxidation/elimination even in samples irradiated under ambient conditions (HRMS, Figures S41–S43).

Substituents on the heterocycles and anions are omitted for the sake of clarity.

Yet, the contrasting efficacies of the direct uncaging between (thio)carbonates (1a–1c) and the previously reported carboxylate35 (1i) payloads are puzzling. Since the direct uncaging from the latter cannot be observed, we estimate that it must be at least two orders of magnitude less efficient than for 1a. However, such a drastic difference for comparable payloads (pKas within 2–3 units) extends far beyond the Bro̷nsted dependence of Φrel that has been traditionally observed for other photocages.48 For reference, the Φrel difference of carbonate pairs in BODIPY26 and coumarin48−50 photocages suggests that alternative factors operate here.

We reason that the efficacy of the direct uncaging pathway from Cy7 photocages is governed by the ability of the formed contact ion pair (CIP) to undergo separation before its recombination back to the starting photocage and not by the activation barrier (ΔG‡) of the bond scission in the excited state (Scheme 2). In the case of 1a–1h and 1j and 1k, the loss of CO2 from the liberated anions provides a pathway to perturb the solvent cage around the CIP to drive its escape, whereas no such option likely exists for the carboxylate payload in 1i. While the rate of ion recombination is known to be high in coumarin photocages (krec ∼ 109 s–1),51 it appears that its suppression is crucial for the function of Cy7 photocages. Recently, Winter and co-workers showed that the intramolecular trapping of the cation can increase the efficacy BODIPY photocages, reinforcing our reasoning.25 High significance of the recombination process might also suggest that direct uncaging occurs predominantly from the S1 excited state.

Hence, we reasoned that the direct uncaging pathway should be accelerated by promoting CIP separation, e.g., by increasing the ionic strength. We tested the hypothesis on 1i containing a carboxylate payload that is not released in the absence of oxygen, i.e., via the direct uncaging pathway.35 Indeed, increasing the ionic strength by the addition of LiCl (100 mM) elicited direct, albeit very slow, uncaging of the previously latent carboxylate payload (∼13% after 46 h of irradiation at 810 nm). Like in the case of 1a, the process was accompanied by the formation of a cyanine corresponding to the putative cation trapped by the solvent, as evidenced by NMR and HRMS spectroscopies (see Figures S38–S40). Such a “turn-on” behavior of the direct uncaging pathway by high ionic strength strongly supports the notion that recombination of the produced CIP, instead of the photodissociation step, is indeed the efficacy-limiting step for Cy7 photocages and perhaps for other red-shifted scaffolds (BODIPY, rhodamine) as well. Recently, Feringa and co-workers showed that the efficacy of photocages can be increased by carbocation stabilization.52 While the observed enhancements are impressive, this approach is destined to reach its limits in the context of hydrolytic stability as these parameters are invariably linked. We believe that targeting the recombination pathway, e.g., via geometric reorganization or cascade reactions of the cationic intermediates, provides an alternative productive avenue to empower NIR photocages.

Conclusions

In summary, we report cyanine photocages that are synthetically accessible and liberate alcohol, phenol, amine, and thiol payloads upon irradiation with NIR light up to 820 nm in aqueous media. We showcase their biological utility through modulation of beating rates of iPSC-derived cardiomyocytes by the NIR light-actuated release of adrenergic agonist etilefrine at low light doses. The photocages exhibit a chameleon-like behavior by operating via two orthogonal uncaging mechanisms. The direct uncaging pathway observed here represents the first example of payload liberation from cyanine dyes, which is not reliant on oxygen. On a fundamental level, these observations constitute the piece of the puzzle that cements the previously postulated photocage design theory based on simple Hückel calculations as a generally applicable strategy across all chromophore families. We believe that the valuable lessons learned herein will guide the rational design of NIR photocages and spur the efforts to combat the nonproductive recombination pathways. Indeed, ongoing experiments in our laboratory suggest that strategies targeting the CIP separation significantly increase the photouncaging efficacies and will be reported in due course.

Acknowledgments

We gratefully acknowledge Swiss National Science Foundation (P.Š./PZ00P2_193425), the Department of Chemistry, University of Zurich (M.R./Legerlotz Stiftung, H.J./UZH Candoc), and Novartis Foundation for Medical-Biological Research (P.Š./#22B102) for funding the research project. We thank Prof. Tomáš Šolomek (University of Amsterdam) for invaluable discussions, and Prof. Cristina Nevado, Prof. Karl Gademann, and Prof. Michal Juríček (all from the University of Zurich) for the generous support of our research. We are grateful to Dr. Johannes Schörgenhumer and the High-Throughput Experimental Laboratory for the access to UPLC. D.M. and M.G. acknowledge the Mäxi Foundation and the University of Zurich for funding. Imaging was performed with support of the Center for Microscopy and Image Analysis, University of Zurich.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c14197.

  • Materials, experimental procedures and methods, synthesis, photophysical and photochemical measurements; methodology of biological experiments, NMR spectroscopy, UV-vis absorption and emission spectroscopy, plots of photophysical and photochemical measurements, irradiation setups, biological experiments (PDF)

Author Contributions

§ M.R. and H.J. contributed equally. P.Š., M.R., and H.J. synthesized the intermediates and final photocages. M.R. and H.J. performed the photophysicochemical characterization of the photocages, H.J. performed the mechanistic study, and M.R. performed the biological studies on cardiomyocytes. D.M. and M.G. differentiated and provided the iPSC-derived cardiomyocytes. P.Š., M.R., and H.J. conceived the project, designed the experiments, analyzed the data, and cowrote the manuscript. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Dedication

Dedicated to our friend, professor Petr Klán, on the occasion of his sixtieth birthday.

Supplementary Material

ja3c14197_si_001.pdf (9.4MB, pdf)

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