A protocol for the synthesis and measurement of the photochemical properties of modular caged compounds with clickable moieties is presented.
Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.
Caged compounds are designed synthetic molecules whose original functions are temporally masked by covalently attached photo-removable protecting groups. Interestingly, caged compounds of biologically relevant molecules provide an indispensable method for the spatiotemporal control of the cellular physiology1,2,3,4,5,6. In 1977, Engels and Schlaeger reported the 2-nitrobenzyl ester of cAMP as a membrane permeable and photolabile derivative of cAMP7. The following year, Kaplan reported the 1-(2-nitrophenyl)ethyl ester of ATP (NPE-ATP) and named this compound “caged” ATP8. Since then, a range of photochemically removable protecting groups such as 2-nitrobenzyls, p-hydroxyphenacyls9, 2-(2-nitrophenyl)ethyls10,11, 7-nitroindolin-1-yls12,13, and (coumarin-4-yl)methyls14,15,16 have been used for the preparation of caged compounds.
The synthesis of caged compounds with desirable additional properties such as membrane permeability, water solubility, and cellular targeting ability would be expected to facilitate cell biological applications. Since the physical and photochemical properties of these molecules depend primarily on the chemical structure of the photochemically removable protecting groups used to prepare them, a diverse repertoire of photo-caging groups is required. However, the structural diversity of currently available caging groups that exhibit high photolysis efficiencies is limited. This could be an obstacle to increasing the use of caged compounds.
To address this issue, the repertoire of photo-caging groups has been expanded by the chemical modification of existing photoremovable protecting groups or the design of new photolabile chromophores with superior photophysical and photochemical properties. Examples include nitrodibenzofuran (NDBF)17, [3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl] (DMNPB)18,19, a calcium-sensitive 2-nitrobenzyl photocage20, substituted coumarinylmethyls (DEAC45021, DEAdcCM22, 7-azetidinyl-4-methylcoumarin23, and styryl coumarins24), cyanine derivatives (CyEt-pan)25, and BODIPY derivatives26,27.
In addition, we previously developed the (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group and successfully synthesized various caged compounds of neurotransmitters28, second messengers29,30, and oligonucleotides31,32,33 exhibiting large one- and two-photon excitation cross-sections. If additional properties can be installed easily into the caging groups without compromising their photosensitivity, then the repertoire of caged compounds can be expanded34,35,36,37,38,39. We therefore designed modular caged compounds that comprise three parts, namely the Bhc group as a photo-responsive core, chemical handles for the installation of additional functionalities, and the molecules that are to be masked40,41.
Thus, this article provides a practical method for the preparation of caged compounds of biologically relevant molecules. The present protocol describes methods for the preparation of a clickable platform for photo-caging groups, the introduction of additional functionalities to expand the repertoire of caged compounds, the measurement of their physical and photochemical properties, and the cell-type selective targeting of a clickable caged compound for further cellular application.
1. Synthesis of the modular caging paBhc group for clickable caged compounds28,41
2. Preparation of clickable caged compounds
NOTE: The following procedures can be applied to the preparation of other clickable caged compounds containing hydroxyl, amino, and carboxylate functional groups.
3. Installation of a functional unit into the clickable caged compounds
4. Photolytic uncaging reaction of the caged compounds
5. Targeting of a clickable caged compound with a HaloTag ligand
NOTE: Prior to use, maintain the HeLa cells in Dulbecco’s modified Eagle medium (DMEM, low glucose, sodium pyruvate, l-glutamine) supplemented with 10% fetal bovine serum (FBS) containing 1% antibiotics (streptomycin sulfate, penicillin G, and amphotericin) at 37 °C and 5% CO2.
6. Photomediated modulation of a kinase localization using a clickable caged compound
NOTE: Prior to use, maintain the CHO-K1 cells in Ham’s F-12 medium supplemented with 10% FBS at 37 °C and 5% CO2.
Clickable caged compounds of some biologically interesting molecules, including arachidonic acid and paclitaxel, were successfully synthesized (Figure 1)28,41. Additional properties such as the water solubility and cellular targeting ability were introduced into paBhcmoc-PTX via the copper(I)-catalyzed Huisgen cyclization (“Click” reaction) (Figure 2). These clickable caged PTXs were then photolyzed to produce their parent PTXs upon irradiation at 350 nm (Figure 3), and the physical and photochemical properties of the clickable caged compounds are summarized in Table 1. The quantum yields of clickable caged compounds 2ʹ-glc-paBhcmoc-PTX (Φdis 0.14) and paBhc-AA (Φdis 0.083) were more than twice those of conventional Bhc caged compounds 2ʹ-Bhcmoc-PTX (Φdis 0.040) and Bhc-AA (Φdis 0.038)43. In addition, an improved water solubility was observed for 2ʹ-glc-paBhcmoc-PTX, which contains a glucose moiety.
In live cell experiments, the targeting of paBhc-hex-FITC/Halo to the cultured mammalian cells transiently expressing a fusion protein of a HaloTag protein and epidermal growth factor receptor (EGFR) was achieved successfully. Green fluorescence of the fluorescein moiety of paBhc-hex-FITC/Halo was observed on the cell membrane (Figure 4). Photo-mediated modulation of the subcellular localization of a kinase was achieved using a paBhc caged compound. The translocation of diacylglycerol kinase γ (DGKγ) has been reported to be activated in the presence of arachidonic acid (AA)44. CHO-K1 cells transiently expressing GFP-DGKγ were treated with either AA or paBhc-AA (5). Addition of AA caused the modulation of the subcellular localization of DGKγ (Figure 5A,B). Similar changes in the localization of DGKγ were observed for the paBhc-AA-treated cells after exposure to UV light (Figure 5C,D).
Figure 1: Preparation of the clickable caged compounds.
(A) Reagents and conditions: a. ethyl 4-chloroacetoacetate/conc. H2SO4/rt/7 days/91% yield, b. 1 M HCl/reflux/3 days/97% yield. c. N-methylpropargylamine /HCHO/EtOH, then add (1) and heat at reflux for 17 h/79% yield. (B) Syntheses of the clickable caged amine, PTX, and arachidonic acid. Please click here to view a larger version of this figure.
Figure 2: Installation of functional units into clickable caged compounds.
(A) Synthesis of a water-soluble caged PTX via the copper(I)-catalyzed Huisgen cyclization. (B) Structures of clickable caged compounds containing the HaloTag ligand for cellular targeting. Please click here to view a larger version of this figure.
Figure 3: Photolysis of 2ʹ-glc-paBhcmoc-PTX (6).
Samples (10 μM) in K-MOPS solution (pH 7.2) were irradiated at 350 nm. (A) Typical HPLC traces for the photolysis of 6 (measured at 254 nm). Samples were analyzed at the specified irradiation time. (B) Time course for the photolysis of 6. Blue circles show the consumption of 6. The solid line shows the least-squares curve fit for a simple decaying exponential for 6. Red squares show the yield of PTX. The error bars represent the standard deviation (±SD). Please click here to view a larger version of this figure.
Figure 4: Fluorescence images of cultured mammalian cells incubated with paBhc-hex-FITC/Halo (8).
Cells transfected with pcDNA3-Halo-EGFR were incubated with a 2 μM solution of compound 8 at 37 °C for 30 min. The images were obtained after repeated washing with PBS+. Mock-treated HEK293T cells (A: differential interference contrast (DIC) image and D: fluorescence image). HEK293T cells (B and E) and HeLa cells (C and F) transiently expressing Halo-EGFR (B and C: DIC images and E and F: fluorescence images). Please click here to view a larger version of this figure.
Figure 5: Fluorescence images after UV irradiation of the CHO-K1 cells incubated with Bhc caged arachidonic acid. CHO-K1 cells were transfected with a fusion protein DGKγ-EGFP.
(A) A fluorescence image of the transfected cells. (B) 100 s after the addition of a 10 μM solution of arachidonic acid. (C) Cells were incubated with a 10 μM solution of paBhc-AA (5) at 37 °C for 5 min. (D) 100 s after 20-s UV irradiation (330–385 nm). Please click here to view a larger version of this figure.
compounds | λmax (nm)a | εmax (M-1 cm-1)b | ϕdisc | εϕdisd | Solubility (μM)e |
PTX | 1.0 | ||||
2'-Bhcmoc-PTX | 340 | 10500 | 0.040 | 400 | 55 |
2'-paBhcmoc-PTX | 359 | 9300 | 0.059 | 670 | 8.3 |
2'-glc-Bhcmoc-PTX | 373 | 12300 | 0.14 | 1280 | 650 |
Bhc-AA | 341 | 10800 | 0.038 | 390 | |
paBhc-AA | 366 | 10300 | 0.083 | 750 |
Table 1: Physical and photochemical properties of the clickable caged compounds.
a. Absorption maximum (nm), b. Molar absorptivity at λmax (M−1 cm−1), c. Quantum yield of the disappearance of the starting materials at 350 nm, d. The product of molar absorptivity and the quantum yield of disappearance at 350 nm, e. The concentration of the saturated solution in K-MOPS (pH 7.2) (μg mL−1).
We previously developed Bhc caged compounds of various biologically active molecules that exhibit high photolytic efficiencies28,45,46,47. With the aim of expanding the repertoire of Bhc caging groups, we also reported platforms of modular caged compounds that can be modified easily by the introduction of various functional units32,40,41. The present protocol therefore represents a method for the synthesis of a clickable precursor of Bhc caging groups that can be modified via the copper(I)-catalyzed Huisgen cyclization. The synthesis of the clickable precursor, paBhcCH2OH (2), was achieved via a four-step reaction sequence starting from the commercially available 4-bromoresorcinol (Figure 1A). The advantage of the present protocol is that no laborious purification steps (e.g., column chromatographic separations) are required.
As clickable precursor paBhcCH2OH (2) can be used to mask various functional groups, clickable caged compounds of amines, alcohols, and carboxylic acids were synthesized using 2 as the precursor (Figure 1B). Amines were modified as their carbamates while alcohols were modified as their carbonates. In general procedures 1 and 2, CDI was used for the preparation of clickable carbamates, while 4-nitrophenyl chloroformate was used for the preparation of carbonates. As indicated by the reaction mechanism, both reagents can be used for the preparation of carbamates and carbonates. It should also be noted that the yield of the desired caged compound depends on the chemical structure of the molecule to be caged. Other examples can be seen in our previous reports28,30,33,48.
Click modification was then performed using a slight modification of the reported procedure49. The addition of tris(triazolylmethyl)amine-based ligands is necessary to obtain the desired products in good to high yields. Since a variety of azides are readily available both from commercial sources and from literature procedures, we can prepare various modular caged compounds with additional properties such as water solubility and cellular targeting ability (Figure 2).
The quantum yield of photolysis was then measured according to a reported procedure28,50. Figure 3 shows that the photolytic consumption of 2ʹ-glc-paBhcmoc-PTX and the release of PTX were approximated by single-exponential decay and rise, respectively, suggesting no inner filtering of the radiation or undesired secondary effects. Improved photolysis quantum yields (Φ) and photolysis efficiencies (εΦ) were observed for the clickable paBhc caged compounds compared to those of the previously reported Bhc caged compounds (Table 1)41,43. Since the photolysis efficiencies (εΦ) of Bhc caged compounds are more than one hundred times higher than those of 2-nitrobenzyl-type caged compounds48, the marked improvement due to the presence of paBhc caging groups is clearly an advantage for this system.
As a proof-of-concept experiment, a hydrophilic moiety was introduced into 2ʹ-paBhcmoc-PTX (4) and a cellular targeting ligand was introduced into compound 3 (Figure 2). The water solubility of 2ʹ-glc-paBhcmoc-PTX was 650 times higher than that of the parent PTX (Table 1). Selective cellular targeting was achieved using a tag-probe system, and paBhcmoc-hex-FITC/Halo (8) bearing the HaloTag ligand was successfully targeted to the cell membrane of cultured mammalian cells expressing the HaloTag/EGFR fusion protein (Figure 4). Photo-mediated modulation of the subcellular localization of a kinase was also achieved using a clickable caged compound 5 (Figure 5).
In conclusion, we successfully demonstrated a method for the preparation of clickable platforms for photo-caged compounds of biologically interesting molecules that can be modified easily with additional properties, such as water solubility and a cellular targeting ability. Since the paBhc caging group can be used to prepare any molecules with modifiable functional groups, the application of the present protocol is not limited to the molecules described herein. Using a modular platform, namely the paBhc caging group, the desired caged compounds can be easily prepared, and their physical and chemical properties can be modulated via click modification.
This work was supported by JSPS KAKENHI grant number JP16H01282 (TF), a Grant-in-Aid for Scientific Research on Innovative Areas "Memory Dynamism," and JP19H05778 (TF), "MolMovies."
Name | Company | Catalog Number | Comments |
acetonitrile, EP | Nacalai | 00404-75 | |
acetonitrile, super dehydrated | FUJIFILM Wako | 010-22905 | |
Antibiotic-Antimycotic, 100X | Thermo Fisher | 15240062 | |
4-bromoresorcinol | TCI Chemicals | B0654 | |
N,N’-carbonyldiimidazole | FUJIFILM Wako | 034-10491 | |
chloroform | Kanto | 07278-71 | |
Copper (II) Sulfate Pentahydrate, 99.9% | FUJIFILM Wako | 032-12511 | |
dichloromethane, dehydrated | Kanto | 11338-05 | |
N,N'-Diisopropylcarbodiimide (DIPC) | TCI Chemicals | D0254 | |
4-dimethylaminopyridine | TCI Chemicals | D1450 | |
dimethylsulfoxide, dehydrated -super- | Kanto | 10380-05 | |
DMEM - Dulbecco's Modified Eagle Medium | Sigma | D6046-500ML | |
dual light source fluorescence illuminator, IX2-RFAW | Olympus | ||
Ethanol (99.5) | FUJIFILM Wako | 054-07225 | |
Ethyl 4-Chloroacetoacetate | TCI Chemicals | C0911 | |
Ham's F-12 with L-Glutamine and Phenol Red | FUJIFILM Wako | 087-08335 | |
hydrochloric acid | FUJIFILM Wako | 087-01076 | |
inverted fluorescent microscope IX-71 | Olympus | ||
ISOLUTE Phase Separator, 15 mL | Biotage | 120-1906-D | |
L-(+)-Ascorbic Acid Sodium Salt | FUJIFILM Wako | 196-01252 | |
laser scanning fluorescence confocal microscopy, FLUOVIEW FV1200/IX-81 | Olympus | ||
Lipofectamine 2000 Transfection Reagent | Thermo Fisher | 11668027 | lipofection reagent |
3-(N-morpholino)propanesulfonic acid | Dojindo | 345-01804 | MOPS |
4-nitrophenylchloroformate (4-NPC) | TCI Chemicals | C1400 | |
Opti-MEM I Reduced Serum Medium, no phenol red | Thermo Fisher | 11058021 | reduced serum medium contains no phenol red |
1,10‐Phenanthroline Monohydrate | Nacalai | 26707-02 | |
Photochemical reactor with RPR 350 nm lamps | Rayonet | ||
Potassium Trioxalatoferrate (III) trihydrate | FUJIFILM Wako | W01SRM19-5000 | |
Sodium Acetate Trihydrate | Nacalai | 31115-05 | |
Sodium Bicarbonate | FUJIFILM Wako | 199-05985 | |
Sulfuric Acid, 96-98% | FUJIFILM Wako | 190-04675 | |
Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) | ALDRICH | 762342-100MG | |
tri‐Sodium Citrate Dihydrate | Nacalai | 31404-15 | |
Xenon light source, MAX-303 | Asahi Spectra |
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