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ARTICLE
Received 00th January 20xx,
Accepted 00th January 20xx
Restricted suitability of BODIPY for caging in biological applications based on singlet oxygen generation
Theo Rodat, †a Melanie Krebs, †a Alexander Döbber, a Björn Jansen, a Anja Steffen-Heins, b Karin Schwarz b and Christian Peifer *a
Recent studies report the boron-dipyrromethene (BODIPY) moiety to be interesting for caging applications in photopharmacology based on its response to irradiation with wavelengths in the biooptical window. Thus, in a model study we investigated the meso-methyl-BODIPY caged CDK2 inhibitor AZD5438 and aimed to assess usability of BODIPY as photoremovable protecting group in photoresponsive kinase inhibitor applications. Photochemical analysis and biological characterisation in vitro revealed significant limitations of the BODIPY-caged inhibitor concept regarding solubility and uncaging in aqueous solution. Notably, we provide evidence for BODIPY-caged compounds generating singlet oxygen/radicals upon irradiation followed by photodegradation of the caged compound system. Consequently, instead of caging, a non-specific induction of necrosis in cells suggests potential usage of BODIPY derivatives for photodynamic approaches.
Introduction
The boron-dipyrromethene (BODIPY) moiety is known for applications in biological chemistry such as dye agent1, fluorescence label2,3 or photosensitiser2,4–7. More recently, BODIPY has been investigated in the field of photopharmacology as a photoremovable protecting group (PPG) towards caging approaches (Table 1). The BODIPY moiety responds to wavelengths above 500 nm within the biooptical window, suggesting it to be highly suitable for the caging of inhibitors in biological chemistry. In line with this notion, Umeda et al. described a BODIPY-caged histamine that can be photo- released via irradiation of 500 nm in 2014.8 Herein, the histamine leaving group was linked to the boron atom. Further studies connected a meso-methylhydroxy-BODIPY either directly or via a carbamate linker to a leaving group.9,10 In 2017, Slanina et al. evaluated the influence of substituents attached to the meso- methyl-BODIPY group, but without providing further biological validation.11 Moreover, Sitkowska et al. stated a halide substituted BODIPY linked to the leaving group via a carbamate linker with fast uncaging, but some stability restrictions in aqueous solution.12 Similar to this approach, two publications from Kawatani et al. (boron linked leaving group) and Peterson et al. (red light single photon PPG) in 2018 were reported.13,14 In 2019, Toupin et al. published the use of a photocleavable BODIPY moiety combined with singlet oxygen generation for an increased efficacy in cancer cell apoptosis.15
a. Institute of Pharmacy, Kiel University, Gutenbergstraße 76, 24118 Kiel (Germany).
b. Institute of Human Nutrition and Food Science, Division of Food Technology, Kiel University, Heinrich-Hecht-Platz 10, 24118 Kiel (Germany).
† These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available
1. Selected cargos caged with BODIPY-moieties in meso position.
I) Goswami et al.10 II) Sitkowska et al.12 III) Rubinstein et al.9 IV) Peterson et al.13
V) Toupin et al.15
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Table 1. Comparison of current publications evaluating BODIPY as PPG for caging.
Reference Leaving group Linker
Activation wavelength
Biological evaluation
Umeda et al.8 Histamine Boron linked 500 nm Ca2+ rise in HeLa cells
Goswami et al.10 Acetic acids Ester 500 nm Compound integration of Drosophilia S2 cells
Slanina et al.11
Different carbon acid analogues
Mostly ester 507 nm none
Sitkowska et al.12 Amine cargos Carbamate 520-560 nm none
Peterson et al.13
Mostly carbon acid analoguesMostly ester 635 nm Compound integration of HeLa cellsKawatani et al.14 Capsaicin analogue Boron linked 500 nm Intracellular Ca2+ dynamics in CaTM3
Toupin et al.15 CTSB inhibitor Ester 532 nm
present work AZD5438 Methylamine 519/525 nm
CTSB
Cell viability and flow cytometry of MDA-MB- 231 and MCF-10A
CDK2/cyclin E1 kinase Panc89 cell proliferation
Caspase 3/7-mediated apoptosis
Based on our platform towards photoresponsive kinase inhibitors16,17 and the above-mentioned findings, we became interested in investigating whether BODIPY would be suitable for caging applications using a potent protein kinase inhibitor. Protein kinases mediate key roles in nearly all important processes in eukaryote cells like proliferation, migration or apoptosis. Thus, alterations of kinase activities including gain-of-function in relevant signal transduction cascades are leading to uncontrolled cell proliferation or reduced apoptosis, most importantly in the development of cancer.18,19 As an example for highly critical kinases, the cyclin-dependent kinases (CDK) are essential for a functional cell cycle and transcription regulation.20,21 For instance, CDK2 in complex with cyclin E1 regulates G0 to G1 and G1 to S phase transition.22 Thus, altering the cell cycle activity via kinase inhibitors targeting CDK2 may be a powerful strategy against cancer.23,24 However, influencing such essential functions in the cell like proliferation can cause crucial impact in normal cell physiology, and may thus cause fatal side effects in patients. Therefore, actually effective CDK2 inhibitor candidates are often not being developed further due to issues detected in clinical studies.21 Examples include the effective CDK2 inhibitor AZD5438 of which clinical development was discontinued based on the appearance of severe side effects.25–27 Regarding kinase inhibitors, several approaches of photochemistry and photopharmacology have been reported so far to overcome the lack in specificity of these biologically effective compounds.28–32 Besides caging16,17,33–38 further applications use photoswitchable moieties including an azobenzene switch introduced in a BRAFV600E inhibitor to manage the BRAF paradox.39–41 Another approach of regulating the VEGFR2 activity was achieved by using Axitinib as a photoresponsive kinase inhibitor.42
However, the wavelengths used to uncage PPGs is frequently not within the biooptical window.35,36 UV light only has a penetration range of millimetres in biological tissue and can additionally cause
cellular apoptosis or DNA damages.43–47 Against this background, the BODIPY moiety would provide significant advantages towards caging as it responds to higher wavelengths within the biooptical window, thus allowing deeper tissue penetration of light.
In the present study, we aimed to use the in vivo actually effective clinical candidate CDK2 inhibitor AZD5438 for caging with the BODIPY group. Our hypothesis was that the caged compound would be biologically inactive, but the potent activity of the leaving group AZD5438 could be spatially and temporally controlled by irradiation with light of approximately 500 nm. This approach would probably provide a highly useful caged CDK2 inhibitor system, which could for example be employed as photopharmacological tool.
Results and discussion
Design, synthesis and photochemical characterisation
To determine a suitable position at the inhibitor structure for the functionalisation with BODIPY as PPG, we initially performed molecular modelling using the x-ray defined ligand complex of AZD5438 in CDK2 with cyclin E1 (PDB: 4FKO). Herein, the ligand AZD5438 forms key H-bonds via the cyclic guanidine moiety towards LEU83, an amino acid residue located in the hinge region of the kinase ( 2). Thus, the binding mode suggested the NH function to be suitable for caging. In contrast, modelling the BODIPY-caged AZD5438 into the active site of CDK2 revealed no plausible binding mode basically due to significant sterical clashes ( 2C). Furthermore, over the course of our studies towards caged kinase inhibitors, the NH moiety of the AZD5438 pyrimidine-anilino core structure was found to be a feasible leaving group.48
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2. Molecular modelling of CDK2 inhibitor AZD5438 and the respectively caged compound 3 in the ATP binding pocket of CDK2/cyclin E1 (PDB: 4FKO). (A) Overview of CDK2 kinase structure with AZD5438 as ligand bound within the ATP pocket. (B) Binding mode of AZD5438 addressing H-bonds (yellow dotted lines) to the hinge region LEU83 within the ATP pocket. (C) Superposition of BODIPY-caged AZD5438 (compound 3) in the CDK2 ATP binding pocket analogous to the original binding mode of the ligand. Caged compound 3 cannot bind into the binding
pocket due to significant sterical clashes (red dotted lines).
Herein, the starting material 3-ethyl-2,4-dimethylpVyierwroAlreticleaOs nlainne electron-rich aromatic compound waDsOI: r1e0a.1c0t3e9d/D0PwPi0th00972C- chloracetylchloride in dry dichloromethane. After addition of dry triethylamine and borontrifluoriddiethyletherate as Lewis acid the reaction was left stirring for 1 h at 50°C. A Finkelstein reaction exchanged chloride for iodide, in which sodium iodide was added to a solution of 1 in dry THF and the mixture was refluxed for 45 min to yield 2, which was used in the following step providing a better
Taken together, our modelling data suggested the BODIPY caged AZD5438 to be biologically inactive, whereas uncaging by irradiation with light of ca. 500 nm wavelength could cleave the BODIPY cage restoring the potent CDK2 inhibition of AZD5438. To prove this hypothesis, we set out to synthesise and subsequently perform photochemical characterisation of the caged compound. Synthesis of the key BODIPY caging group 1 was achieved according to literature49,50 based on a Friedel-Crafts-acylation (Scheme 1).
Scheme 1. Convergent synthesis of caged compound 3. The BODIPY precursor 1 was synthesized by a Friedel-Crafts-acylation.49,50 The 8-iodomethyl BODIPY 2 was achieved
based on a Finkelstein reaction.50
3. Uncaging of 3 under irradiation with 525 nm in DMSO solution. 25 µM of
3 were irradiated with 6.5 mW/cm² per well in a white 96-well plate. Concentrations
of reaction products were quantified by HPLC. Error bars indicate SD (N = 3).
leaving group. In turn, using a SN reaction in dry DMF substitution of the benzylic BODIPY position by the deprotonated NH position of AZD5438 at -35 °C yielded key compound 3 (Scheme 1). Notably, all reactions were performed under argon atmosphere and were protected from light.
Next, we performed photochemical characterisation in terms of the uncaging process of compound 3. Determination of UV spectra revealed significant absorption of 3 at a wavelength of 525 nm (S12). Thus, we initially irradiated a 25 µM solution of 5 in DMSO with light at this wavelength and monitored the uncaging reaction progress by HPLC analysis
The BODIPY uncaging reaction was considered to proceed via a cationic transition state, analogously to the coumarin caging group, although the detailed mechanism is still under discussion.9,12 However, in our experiments, by irradiation of 3 in aprotic DMSO solution, the decline of the BODIPY moiety in compound 3 could be clearly detected via adsorption at 544 nm (to avoid artefacts, a higher wavelength was chosen, see S13). While under the DMSO conditions, uncaging of 3 yielded AZD5438 as determined by LC/MS analysis, we could, in contrast, not detect a main BODIPY derivative, neither by UV adsorption nor by LC/MS analysis (S17). Interestingly, the situation turned out to be different under aqueous uncaging conditions and HPLC quantification could not be realised. Compared to the situation in DMSO, an aqueous solution of caged compound 3 was not quite stable even without irradiation and immediately after irradiation, the caged compound declined under the detection limit while the free inhibitor AZD5438 was released, but not in an equimolar manner compared to 3. Instead, irradiation resulted in a complex product mixture indicating nonspecific degradative reactions.
Biological evaluation
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4. Kinase assay and cell proliferation assay of CDK2 inhibitor AZD5438, caged 3, and BODIPY derivative 5. (A) Dose-response curve of 3 without irradiation (squares, green dotted line, IC50 = 398 nM), 3 with 519 nm irradiation (rhomb, green line, IC50 = 258 nM) and AZD5438 as positive control (circles, black line, IC50 = 1.9 nM) on a CDK2/cyclin E1 system. (B) Dose-response curve of 5 without irradiation (purple squares), 5 with 519 nm irradiation (purple rhombs) and AZD5438 as positive control (circles, black line) on a CDK2/cyclin E1 system. Compound 5 as well as 5 upon irradiation followed no dose-dependent inhibition. Kinase assay was performed with a final concentration of 10 µM ATP and irradiated for ten minutes, 6.5 mW/cm² per Well. (C) Dose-response curve of 3 without irradiation (squares, green dotted line, IC50 > 10 µM), 3 with 525 nm irradiation (rhombs, green line, IC50 = 0.03 µM) and AZD5438 as positive control (circles, black line, IC50 = 0.9 µM) in a Panc89 cell proliferation assay. (D) Dose- response curve of 5 without irradiation (squares, purple dotted line, IC50 > 10 µM), 5 with 525 nm irradiation (rhombs, purple line, IC50 = 0.02 µM) and AZD5438 as positive control (circles, black line, IC50 = 0.9 µM) in a Panc89 cell proliferation
assay. Well plate was irradiated for two minutes, 8.9 mW/cm² per Well.
However, since AZD5438 is partly released under irradiation with 525 nm, we decided on further biological in vitro evaluation of 3 in an enzymatic CDK2/cyclin E1 assay and towards CDK2 dependent Panc89 cells51 . Accordingly to our initial hypothesis, when tested without irradiation in the enzymatic CDK2/cyclin E1 assay, caged 3 proved to be significantly less active compared to AZD5438. In contrast, the potent CDK2 inhibitor AZD5438 was determined with an IC50 value of 1.9 nM, which is in accordance to data from literature25 ( 4A). A similar situation was detected in our cellular assay, where caged 3 was initially less active without irradiation compared to AZD5438 ( 4C).
Unexpectedly, when we irradiated caged compound 3 within the enzymatic assay, the results showed only minor inhibition of CDK2 activity. The biological activity of the irradiated 3 was actually comparable to the situation without irradiation and in sharp contrast to the potency of the native AZD5438 ( 4A). Potential explanations may again include degradation under the aqueous assay conditions (in contrast to uncaging determined in DMSO, see 3). However, to our great astonishment, the irradiation of 3 in the cellular Panc89 assay with 525 nm resulted in a very strong biological activity (IC50 = 0.026 µM). This efficacy is significantly higher compared to the potent CDK2 inhibitor AZD5438 itself
5. Biological evaluation of 3 and 5 on Panc89 cells. (A) Caspase 3/7 assay of 3 under irradiation (squares, green line, no caspase activity) and 5 (rhombs, purple line, no caspase activity or necrosis) and AZD5438 as positive control (circles, black line, dose-dependent caspase activity). (B) Fluorescence microscopy of Panc89 treated with 5. Nucleus was stained with Hoechst 33342 (blue) and compound distribution of 5 is coloured in green. Scale bar represents a length of 20 µm (C) Fluorescence microscopy of Panc89 treated with DMSO, AZD5438, 3 or 5 under controlled light conditions. Scale bar at the lower right represents a total length of
70 µM.
(IC50 = 0.91 µM, 4C). Thus, our results indicate a bioactive role of the BODIPY moiety upon irradiation, which is in line with further recent reports.6,52,53 This substantial increase in potency could not be explained with additive effects of light or temperature increase due to irradiation (control experiments S21). To further investigate this unexpected effect, we synthesised the proposed hydroxylated BODIPY cleavage product 5 without any inhibitor attached (Scheme 2).
Having compound 5 in hand, we next tested its biological activity in the CDK2 kinase assay (no inhibition, 4B) and cellular Panc89 proliferation assay with and without irradiation by light of 525 nm ( 4D). Interestingly, the inhibition of the Panc89 cells following treatment with 5 and irradiation with light (IC50 = 0.022 µM) is similar to the results obtained by performing the assay using compound 3 (IC50 = 0.026 µM). These results suggest the irradiated BODIPY moiety and not the potent inhibitor AZD5438 to be mainly responsible for the strong inhibition of PANC89 cell proliferation in the assay.
Since obviously the BODIPY moiety mediated a strong bioactivity in combination with the irradiation of light with 525 nm in cells, we next set out to evaluate this effect in greater detail. First, based on the fluorescence of BODIPY, we used fluorescence microscopy to detect cellular localisation of the compound ( 5B). Herein, decent cellular bioavailability was proven for 5, which is in
accordance with further reports from BODIPY derivatives detecting incubation of cells with BODIPY derivatives under irradiation,
suggesting the BODIPY moiety to show a potential for
Scheme 2 Convergent synthesis of the hydroxylated BODIPY (5). Synthesis was
adapted from literature.49
photodynamic approaches.57–59 In line with this notion, apoptosis and respectively necrosis has been reported to be a common cell
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Journal Name ARTICLE death path for photodynamic agents.60 Thus, we treated Panc89 cells with compounds 3, 5, and AZD5438 with and without irradiation and determined apoptosis rates via a bioluminescence signal of caspase 3/7 activation respectively ( 5A). Herein, AZD5438-treated cells showed a distinct apoptosis according to the mode of action as CDK2 inhibitor. In contrast, when irradiated with light of 525 nm, Panc89 cells treated with both 3 and 5 resulted in significant necrosis. Notably, the BODIPY bearing compounds 3 and 5 do not show detectable cell death without irradiation. Images from the fluorescence microscopy ( 5C) further support these cell proliferation results. Cell density of Panc89 cells is highly decreased after treatment with 3 (-93 %) and 5 (-90 %) under irradiation with light of 525 nm whereas cells treated with DMSO (-14 %) reacted less light-dependent or AZD5438 (+1 %) light-independent, respectively (S21).
Photodegradation
Having clearly established a significant photodynamic effect of BODIPY derivatives 3 and 5, we aimed to elucidate mechanistical aspects of their supposed mode of action. Further evidence showed BODIPY derivatives are also known for creating reactive oxygen species under irradiation.5–7,56 We could demonstrate the considerable formation of short-lived radicals by ESR using the cyclic hydroxylamine TMTH after irradiation of compound 3 at 525 nm (6A). In another test system, both 3 and 5 decreased the absorption maximum of read out signal from 1,3-diphenylisobenzofuran under irradiation, providing further evidence for generating singlet oxygen (6B). In DMSO, singlet oxygen generation as well as photouncaging appeared instantly upon irradiation with +500 nm
6. Photochemical degradation of compound 3 and 5. (A) Electron spin resonance spectrum of the spin probe TMT in the presence of 3 without irradiation (black line) and 3 with 525 nm irradiation (green line) in DMSO, 30 s and intensity of 90 mW/cm² per well. (B) Singlet oxygen assay. 1,3-Diphenylisobenzofuran (DPBF) and compounds were dissolved in DMSO. Absorption of DPBF decreases due to singlet oxygen production of 3 under irradiation (green rhombs) and 5 under irradiation (purple rhombs). 3 without irradiation (green circles) and 5 without irradiation (purple circles) have not generated singlet oxygen. Singlet oxygen quantum yield of 3 (Φ = 0.38) and 5 (Φ = 0.74) upon irradiation was determined with methylene blue (Φ = 0.52).(C) Absorption spectra of an aqueous solution of 0.1 mM 3 over time and under controlled light conditions. In aqueous solution maxima decrease over time. Full deletion at 500 nm absorption was achieved irradiating with
519 nm for 10 minutes, 6.5 mW/cm² per well.
However, compounds 3 and 5 required only one minute of irradiation for a substantial singlet oxygen response in contrast to 15 minutes for uncaging at similar settings. Thus, it seemed that singlet oxygen generation appears preferably during the uncaging process of the BODIPY moiety. By correlating this effect to the observed cytotoxicity in Panc89 cells, we conclude that 3 and 5 produce singlet oxygen upon irradiation, thus severely damaging cells, thereby exceeding the effect of the potent CDK2 inhibitor AZD5438.61,62 Furthermore, the proposed radical mechanism provides a possible explanation for the observation of non-successful uncaging for 3 upon irradiation in the enzymatic assay ( 3A). According to the production of short-lived radicals detected by ESR, we assume that the molecule decomposes, yielding a mixture of fragments instead of providing the actual CDK2 inhibitor AZD5438. Due to solubility issues of 3 the photochemical characterisation was performed in DMSO yielding uncaging of 3 without difficulty (3). However, biological evaluation takes place in aqueous solutions at µM compound concentrations. Thus, we performed respective experiments in aqueous solutions for 3 and 5. Indeed, under irradiation we observed a strong photodegradation of the compounds yielding non-defined product mixtures providing further evidence for radical reaction processes (5C, S16).
Photodegradation has already been reported for photosensitisers,
e.g. porphyrines and semi-porphyrines.63 Compared to features of these photosensitisers, solutions of compound 3 and 5 decoloured upon irradiation suggesting a type 2 photobleaching (true photobleaching) behaviour. Interestingly, not only photodegradation prevents a proper photochemical characterisation in aqueous solution, but also precipitation occurred in the samples upon irradiation probably as the result of radical polymerisation reactions. Similar to our observations, Descalzo et al. also reported about aggregation and dimerisation of BODIPY moieties64 in aqueous solutions, which are, however, indispensable for biological applications. We also observed precipitation of 3 and
5 when water was added to a DMSO stock solution (S14). Accordingly, aggregation of BODIPY moieties has been reported elsewhere to be responsible for precipitation when water was added.65,66 Furthermore, NMR experiments employing D2O to investigate these effects in more detail proved to be non-successful again due to precipitation.
Conclusions
Uncaging of BODIPY-caged compounds is well established in organic solvents and could be also achieved in this work.9,11 However, with regard to biological evaluation in aqueous solutions, the situation turned out to be different. In line with recent reports from Sitkowska et al. and Takeda et al., we observed unstable storage conditions for BODIPY derivatives.12,66 Although the BODIPY moiety is tempting for caging approaches based on its response to green light with wavelength of > 500 nm, we provide evidence for the BODIPY-caging concept being limited in an aqueous environment due to the production of singlet oxygen upon irradiation. Thus, the generatedThis journal is © The Royal Society of Chemistry 20xx J. Name., 2020, 00, 1-3 | 5
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radical species mediate non-selective degradation effects.
Journal of the American Chemical Society, 2017,Vi1e3w9A(r4tic2le),Online
Accordingly, biological activities of BODIPY containing compounds 3
15168.and 5 in a Panc89 cell proliferation assay turned out to be highly effective. In this assay, due to singlet oxygen generation, 3 as a caged compound is 30 times more active upon irradiation compared to the actually potent CDK2 inhibitor AZD5438. Hence, in order to avoid misinterpretation of the results for BODIPY caged compounds in biological applications we strongly recommend to investigate BODIPY moieties case-by-case towards their potential to produce ROS upon irradiation. Whether BODIPY derivatives will be useful in photodynamic approaches remains to be seen.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We would like to thank Martin Schütt and Dr. Ulrich Girreser from the Pharmaceutical Institute, Kiel, for excellent technical and analytical assistance. We also kindly acknowledge Till Priegann for technical support during fluorescence microscopy. At last, we gratefully acknowledge the Deutsche Forschungsgesellschaft for financial support (grant PE1605_2_2).
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This journal is © The Royal Society of Chemistry 20xx J. Name., 2020, 00, 1-3 | 7Photochemical & Photobiological Sciences Page 8 of 8Photochemical analysis and biological characterization in vitro revealed significant limitations of the BODIPY-caged inhibitor concept regarding uncaging in AZD5438 aqueous solution but also promising cancer cell treatment through photodynamic effects.