Development of Photolabile Protecting Groups and their Application to the Optochemical Control of Cell Signaling

Abstract

Many biological processes are naturally regulated with spatiotemporal control. In order to perturb and investigate them, optochemical tools have been developed that convey similar spatiotemporal precision during study of the system. Pivotal to optochemical probes are photolabile protecting groups, so called caging groups, and recent developments have enabled new applications to cellular processes, including cell signaling. This review focusses on the advances made in the field of caging groups and their application in cell signaling through caged molecules such as neurotransmitters, lipids, secondary messengers, and proteins.

Introduction

Application of light as a non-invasive external trigger allows spatiotemporal control in dynamic systems to study dynamic biological processes [1–5]. It started when Engels and Hoffman first reported photoactivation of cyclic adenosine monophosphate (cAMP) [6] and adenosine triphosphate (ATP) [7], using 2-nitrobenzyl and (2-nitrophenyl)ethyl photolabile groups, respectively. The photolabile protecting groups, more commonly referred to as “caging groups”, have since been a cornerstone in optochemical biology. They have been used for the light-regulation of a variety of biomolecules, ranging from oligonucleotides [8,9], carbohydrates [10], proteins [2], and peptides [11], to small molecules (such as, cell signaling molecules [12], fluorophores [13], and chemical inducers of dimerization (CIDs) [14]), thus providing precise spatiotemporal control over biological processes in cells and animals. Over the last five years, there has been a surge toward improving the photophysical properties of caging groups, by shifting their absorption maxima towards the use of long-wavelength light for photoactivation, which reduces the potential for phototoxicity and enhances tissue penetration, as well as enabling decaging via multi-photon excitation. Several excellent review articles on caging groups exist, including a very comprehensive one by Klan et al. [15], and others focusing on two-photon applications [16–18]. This review summarizes most recent caging group developments (predominantly within the last five years), as well as recent applications of caging methodologies to the optical control of cell signaling. Complementary to caging groups, synthetic photoswitchable molecules [19,20], as well as natural photoswitchable proteins have been reviewed elsewhere and in this issue by Leippe and Frank. [21,22].

Advances in caging group development

Recent advances in caging group design have focused on optimizing several desirable properties including [15]: 1) red-shifted absorption maxima (λ_max) towards far visible/NIR, 2) high molar extinction coefficient (ϵ) and quantum yield of decaging (φ_u) leading to higher decaging efficiency (ϵ x φ_u), 3) good aqueous solubility and stability, 4) non-toxic and low-absorbing photoreleased by-products, 5) large two-photon (2P) absorption (TPA) cross section (δ_a) which is used for quantifying the two-photon absorption of a chromophore, and 6) narrow absorption profile to enable multiplexing through orthogonal decaging experiments.

One challenge in caging group design is the difficulty in simultaneously optimizing both absorption maxima and quantum yield, where red-shifting the absorption by increasing conjugation sometimes leads to reduction in decaging efficiency. Additionally, introducing hydrophilic groups to achieve optimal solubility for in vivo applications often requires the presence of amine or hydroxy or alkyne handles on the caging group. The fine balance between background hydrolysis of caged compound and its rapid substrate release requires fine-tuning of pK_a of both caging group and substrate. Rapid kinetics will allow investigation of fast cellular processes like neuronal signal transduction. Moreover, lack of background activity of the caged compound indicating high light to dark activity switching is desirable.

Coumarin-based caging groups

Coumarin-based caging groups have been applied towards a variety of studies in recent years due to ease of synthesis and rapid release of substrate. Recently, structural modifications have been made towards improving the photophysical properties like quantum yield and aqueous solubility. Efforts have built onto the 7-(diethylamino)-4-(hydroxymethyl)coumarin (DEACM) scaffold (Figure 1b) [23] to red-shift the absorption maximum. The developments can be broadly classified based on their electronic structure: Donor-π system-Acceptor (D-π-A) and Donor-π system-Donor (D-π-D). The D-π-A category exhibits push-pull effect where the chromophore is end-capped with an electron donor and an electron acceptor [24]. Substrates caged by coumarins are typically connected to the caging group through a carbonate, carbamate, phosphate, or carboxy moiety due to the requirement of low pK_a in the leaving group [25]. Fournier et al. synthesized a series of such coumarin scaffolds where the structure bore an electron donating group (OMe/NEt₂) at the 7-position and different electron withdrawing groups at 2/3 position/s aimed at extending the π-conjugation system [26]. Benzoic acid was utilized as the substrate to cage, and extensive investigation of the photophysical properties yielded three best candidates 1a-1c (Figure 1a), selected based on red-shifted absorption maxima and good quantum yield (Table 1) [26]. The caged tamoxifen analog 2 was employed to photoregulate the activity of an engineered transcription factor En2 in En2-ERT2 mRNA injected zebrafish embryos. Photoactivation of 2 upon 470 nm irradiation for 10 minutes led to observing 50 % of the expected phenotype, a reduction in size/ total absence of eyes [27].

Figure 1.

Structures of coumarin and BODIPY caged substrates; caging groups are shown in red. (a) Structures include D-π-A type (1-5) and D-π-D (6-7) coumarin caged substrates. The structures include caged benzoic acids (1, 4, 6–7), a caged tamoxifen analogue (2), a caged cyclic RGDfK peptide (3), caged glutamic acid (5a) and caged cAMP (5b). (b) BODIPY caged molecules include 4-methoxyphenol (8a), caged histamine (8b, 9), and caged 2,4-dinitrobenzoic acid (10-11).

Table 1.

List of the photochemical properties of some of the new caging groups mentioned in the development section. λ_max is the absorption maximum; ϵ is the molar extinction coefficient at the absorption maximum; Solvents used during photolysis: PBS (phosphate buffered saline pH=7.4), Tris is trisaminomethane buffer pH=7.5; φ_u is the quantum yield of decaging; δ_u = ϵ × φ_u is the decaging efficiency for single photon irradiation and is the product of the quantum yield and extinction coefficient; δu_2P = δ_a × φ_u is two photon decaging action cross section for two photon irradiation where δa is the two photon absorption cross section (1 GM = 10⁻⁵⁰ cm⁴ s photon⁻¹).

caged compound	λmax/nm	ϵ/mM−1cm−1	solvent	φu/10−2 (λ/nm)	δu/M−1cm−1	δu2P/GM (λ/nm)
1a	472	31	1:1 CH3CN:Tris	0.12 (365)	320	-
1c	487	33	1:1 CH3CN:Tris	0.07 (365)	2	-
2	492	30	1:1 CH3CN:Tris	0.24 (505)	58	-
4b	503	32	MeOH	<0.001 (505)	0.07	-
5a	450	43	PBS	39 (450)	16800	0.5 (900)
6	407	29	DMSO	16 (405)	4558	6 (760)
7a	430	30	9:1 MeOH:H2O	45 (450)	13500	26 (730)
7b	490	30	9:1 MeOH:H2O	40 (450)	12000	-
9	544	45	19:1 PBS:CH3CN	0.01 (540)	7	-
10a	538	61	MeOH	28 (507)	17056	-
11	661	65	MeOH	41 (532)	3	-
12a	668	40	DMSO	-	-	-
15a	397	8	PBS	15 (405)	1125	11 (800)
16	400	11	19:1 PBS:DMSO	22 (412)	2500	20 (800)
17a	443	30	MeOH	1 (355)	60
19	440	66	PBS	23 (410)	-	350 (810)
20	362	19	C6D6	30 (355)	5631	120 (680)

The Marchán group reported further improvement of green light activatable coumarin 1c and its application to a caged cRGDfK peptide 3 [28]. Presence of an α-methyl group speeds up decaging due to stabilization of the carbocation intermediate generated in the photolysis step [28]. Further red-shifted coumarin caged benzoic acids 4a and 4b (Figure 1a) were developed by replacing a cyano group in 1c with a nitrophenyl group [29]. However, the decaging quantum yield was significantly reduced (Table 1). The Ellis-Davies and Sabatini labs modified the DEACM scaffold with alkene and aspartate moieties yielding DEAC450 chromophore to shift the absorption maximum and enhance aqueous solubility, and applied it towards caging of glutamic acid (5a) [30]. Decaging of 5a with one-photon (473 nm) or two-photon (900 nm) irradiation at the spine heads on pyramidal neurons in isolated brain slices generated excitatory postsynaptic currents. Lack of response to 720 nm exposure enabled multiplexing of DEAC450 caging group with 4-carboxymethoxy-5,7-dinitroindoline (CDNI) and 4-methoxy-7-nitroindolinyl (MNI) scaffolds [15]. These results led to synthesis of caged cAMP 5b and its wavelength-selective decaging in presence of CDNI-caged γ-aminobutyric acid (GABA) [31].

D-π-D coumarin chromophores, generated through attachment of electron donating group at the 3-position – see 6 and 7, exhibit impressive two-photon properties (Table 1) [24,32,33]. Chitose et al. synthesized 6 which has a TPA cross section of 5.6 GM at 760 nm [33], 8-fold higher than the commonly used MNI chromophore [34], where GM (Goppert Mayer) is an unit of TPA cross section [35]. The Zhu group synthesized the coumarin chromophores 7a and 7b with electron rich styryl appendages at the 3-position [32]. This led to higher absorption maximum but more importantly produced an unconjugated by-product through intramolecular sequestration of the generated carbocation after photolysis. This prevents competitive absorption of the by-product at the absorption wavelength of the caged substrate.

Borondipyrromethene (BODIPY)-based caging groups

The BODIPY chromophore was serendipitously discovered as a caging group by the Urano group when they observed the release of an aryloxy group upon irradiation of 8a (Figure 1b) with 500 nm light [36]. An optimization study showed inverse correlation showed between fluorescence (φ_fl) and decaging quantum yields (φ_u), suggesting a photoinduced electron transfer (PeT)-based decaging process [15,25]. A major factor behind its excellent decaging efficiency is the large extinction coefficient (Table 1). Through a clever relay mechanism involving aryloxy decaging and subsequent 1,6-elimination and decarboxylation of a carbamate, the BODIPY group was applied to the caging of histamine (8b) [37].

The groups of Weinstain and Winter used a different connectivity, the meso-methylhydroxy position of BODIPY for caging [38,39]. Based on DFT calculations by the Klan group, BODIPY has a similar excited state structure as coumarins, xanthenes, and methine cyanines [40]. Decaging of 9 in HeLa cells using 500 nm light led to histamine-induced release of Ca²⁺ [38]. Systematic SAR studies on 32 meso-BODIPY scaffolds optimized their photophysical properties, leading to some candidates that showed exceptional promise in terms of absorption maxima and quantum yield (10a and 10b) [41]. The increased quantum yield for halogenated caged compounds provide evidence that decaging proceeds through a triplet state, as heavy atoms facilitate intersystem crossing (ISC) from the singlet to the triplet state [42]. The decaging efficiency which measures how efficiently a caged substrate is released and is the product of the extinction coefficient (ϵ) and the quantum yield (φ_u), surpassed 10,000 M⁻¹ cm⁻¹ for a few BODIPY chromophores. This can complicate handling of the caged compounds under ambient light. Winter recently reported a family of one-photon excitable BODIPY caging groups bearing styryl moieties that exhibit the most red-shifted absorption maxima for a BODIPY dye thus far (11) [43]. As a proof of principle, fluorescence of 11 was measured upon 635 nm light exposure and subsequent release of a quencher in HeLa cells [43].

Cyanine-based caging groups

Heptamethine cyanines commonly used for fluorescence applications [44] were employed by the Schnermann group as innovative near-infrared caging groups by converting the liability of cyanine photobleaching into a decaging strategy via localized photooxidation and cleavage of C-C double bonds upon irradiation with 690 nm light [45,46]. Caging and precise spatiotemporal release of 4-hydroxycyclofen 12a (Figure 2a) was demonstrated in MCF-7 cells [45]. The same chromophore was employed to cage combretastatin A4 as part of an antibody-drug conjugate (ADC) [47]. The antibody panitumumab ensured the localization of 13 to cells expressing EGFR while 690 nm irradiation controlled the release of the drug in a temporal fashion in a mouse xenograft model [47]. While the use of antibodies provides precise control over the delivery of the drug, the inherent fluorescence of cyanine enables tracking of the ADC and drug release can be monitored through decrease in fluorescence. The use of near-IR light further ensures good tissue penetration [48].

Figure 2.

Structures of heptamethine cyanine and new nitrobenzyl (NB) caged substrates; caging groups are shown in red. (a) Caged substrates include 4-hydroxycycofen (12), combretastatin A4 (13), and duocarmycin (14), the last two substrates were conjugated to the antibody panitumumab. (b) Structures include NPE-type (17-18 and 21) and NPP-type (15-16) caged molecules. Caged substrates include GABA (15), gibberellic acid (16), glutamic acid (17), deoxyadenosine (18), calcium chelators (19-20), and 3,4-dimethoxyphenylacetic acid (21);

Optimization of 12a via addition of lipophilic esters to both the caging group and substrate (12b, Figure 2a) enhances cellular uptake and localization [49]. Rational design of the cyanine structure led to development of a red-shifted and more hydrolytically stable dye [50]. Altering the heterocycle of the core and the linker domain yielded 14 which was used to spatiotemporally regulate release of duocarmycin from a cyanine-duocarmycin-panitumumab conjugate both in vitro and in vivo [50].

Ortho-nitrobenzyl (NB)-based caging groups

Despite being the most widely used caging group in biological applications, development of NB caging groups with more desirable properties has been challenging due to difficulties in optimizing the absorption and decaging properties simultaneously [51]. Increased conjugation has enhanced two-photon sensitivity but resulted in decrease in one-photon decaging efficiency in some cases [51–53]. Thus, recent efforts have focused on developing two photon sensitive chromophores.

Dialkylamino-biphenyl caging groups were introduced by Specht and Goeldner based on 2-(2-nitrophenyl) prop-1-yl (NPP) scaffold [54]. They synthesized caged GABA 15a and 15b (Figure 2b) which exhibited extraordinary TPA cross section of 11 GM at 800 nm and exhibited improved decaging efficiency over previously used coumarin caged GABA (0.37 GM at 800 nm) [53]. Irradiation of 15a or 15b with 800 nm light induced inhibitory postsynaptic GABAergic currents in rat cortical brain slices [54]. The dialkylamino group enables functionalization with PEG/2-carbonylmethyl moieties to improve aqueous solubility of the caged compound. Wombacher introduced a new caging group based on 15 where introduction of an alkyne led to a two-fold improvement in two-photon decaging efficiency by extending the π-conjugation, but there was no significant shift in the absorption maximum compared to 15. The caging group was installed on a plant-based CID, gibberellic acid (GA₃) 16, to trigger protein dimerization and mitochondrial translocation of EGFP in COS-7 cells after 412 nm irradiation [55]. Supported by DFT calculations, Kobayashi developed a dialkyl-dihydronaphthalene and applied it to the caging of the glutamate derivatives 17a and 17b [56], which exhibited improved two-photon sensitivity compared to 15. Becker et al. recently reported a new red-shifted caged oligonucleotide 18, having excellent two-photon decaging properties at 840 nm but inert to one-photon irradiation [52]. Due to the exclusive response to two-photon photolysis, this caging group could be utilized in an orthogonal decaging approach with one-photon labile red-shifted caging groups.

Ellis-Davies and coworkers developed a bis-styrylthiophene (BIST) caging group with improved two-photon sensitivity and an absorption maximum at 440 nm [57]. The calcium chelator 19 generated by appending EGTA to the BIST caging group produced rapid “Ca²⁺ waves” in cardiac myocytes upon irradiation with both 405 nm and 810 nm [57]. This caging group can be multiplexed with other caged metal chelators to study the effect of controlled release of select ions in different signal transduction pathways [58]. Jakkampudi et al. synthesized the Ca²⁺ chelator 20 as a cell permeable acetoxymethyl (AM) ester which showed impressive two-photon properties (Table 1) [59]. Cultured neurons containing 20 were irradiated with 720 nm light leading to induction of calcium response as monitored through inhibitory post synaptic currents (IPSCs) at the dendritic edges [59]. Analysis of the photolysis products reveals that 19 and 20 selectively undergo release of the α-substituent, instead of fragmenting through an also conceivable β-elimination pathway [15]. Nakad et al. recently reported a series of optically activated reporter molecules 21a-c which upon decaging yields a fluorescent by-product [60], which can be used to monitor the amount of decaging.

Applications of caging groups in cell signaling studies

Some of the new, recently reported caging groups have found application in the context of cell signaling through caging of small molecule ligands [37,38], calcium ions [57,59], and secondary messengers [30,31,54]. Upon photoactivation, caged small molecule ligands bind to cell surface receptor and promote downstream signaling events through secondary messengers. Alternatively, caged secondary messengers, when photoreleased, activate intracellular targets to induce signal transduction and subsequently elicit a cellular response (Figure 3a).

Figure 3.

Examples of applications of coumarin and BODIPY caging groups in cell-signaling. (a) The general approach includes a caged inactive biomolecule that is rendered functional upon light irradiation. The active biomolecule can act as a receptor ligand or a secondary messenger, both of which induce downstream signaling events and lead to a cellular response. (b) Whole cell current clamp recordings demonstrate the wavelength-selective, bi-directional modulation of neuronal firing in striatal cholinergic neurons through decaging of DEAC450-cAMP (5b) and CDNI-GABA within the same cell using one-photon irradiation. While decaging of 5b causes increase in the firing rates, decaging of CDNI-GABA transiently inhibits it. (c) Dendrimer-cloaked DEAC450-GABA (22) shields the substrate from interacting with the GABA-A receptors thereby reducing background activity. (d) Upon irradiation of BODIPY-caged histamine 8b, histamine binds to the H₁ receptor and induces the G-protein mediated activation of phospholipase C, leading to calcium release. Adapted with permission from ref. [31], Copyright 2013 American Chemical Society.

Orthogonal decaging of neurotransmitters

The Ellis-Davies group synthesized DEAC-450 caged-cAMP (5b, Figure 1a) and used it in combination with CDNI-caged GABA. Using one-photon (473 nm and 365 nm) irradiation in a sequential fashion, they interrogated the bi-directional neuronal firing rates in single striatal cholinergic neurons [31]. Patch clamped single neurons from brain slices were incubated with 5b and CDNI-GABA, and whole-cell current clamp recordings were used to monitor spontaneous action potential firings. Irradiation with 473 nm enhanced the action potential firing rate while irradiation with 355 nm transiently inhibited it (Figure 3b). The wavelength orthogonality was demonstrated through induction of stimulatory and inhibitory response patterns by varying the order of the wavelength applied (Figure 3b). The orthogonality was extended to two-photon uncaging using DEAC450-Glu and CDNI-GABA, where irradiation with 900 nm along a basal dendrite elicited an action potential only to be reversed by the inhibitory currents induced through decaging of CDNI-GABA at 720 nm [12]. The neurotransmitter receptors being densely clustered, two-photon decaging offers a higher level of spatial control due to the irradiation of single spines and synapses. Additionally, orthogonally decaging excitatory and inhibitory compounds in one system allows precise study of the neurons in their natural environment where they are subjected to a complex mix of rapid excitatory and inhibitory stimuli to produce output signals.

Reduction in background activity of caged neurotransmitters

A major limitation of the caged compounds (Glu and GABA) is that they exhibit significant “GABA-A antagonism”, where the caged compound interacts with GABA-A receptors and dampens the spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in patch clamped single neurons. This effect is pronounced for commonly used (MNI/CDNI) caged neurotransmitters employed at higher concentrations (1–12 mM), required for two-photon photolysis experiments, applied either through bath application or local perfusion [34,61]. To alleviate this issue, a “cloaked cage” strategy was developed where a dendrimer is attached to the caging group through “click” chemistry [62]. The bulky dendrimer envelopes the caged compound and prevents interaction with the receptors until photolyzed, similar to previous applications of PEG-modified caging groups blocking biological interactions until light-induced cleavage [63–65]. The application of the dendrimer-caged GABA 22 (Figure 3c) to patch-clamped prefrontal cortical neurons showed reduced binding affinity to receptors (IC₅₀ = 0.9 mM) when compared to antagonists like bicuculline (IC₅₀ = 0.5 μM) and caged GABA probes like DEAC-450-GABA (IC₅₀ = 0.5 μM), as determined through blockade of electrically evoked inhibitory currents. Additionally, the higher two-photon cross-section of 22 enabled use of it at lower concentrations (66 μM) for two-photon decaging [30,34].

Decaging of small molecule ligands

The Urano group utilized 8b (Figure 1b) in HeLa cells to optically regulate histamine release as indicated by changes in intracellular Ca²⁺ concentration [37]. Irradiation of 8b with 500 nm light released histamine which targets membrane localized histamine H₁ receptors to activate phospholipase-C (PLC) through the binding of G_α subunit of H₁-GPCR. PLC further activates downstream targets like IP₃ to eventually release calcium from the endoplasmic reticulum (Figure 3d). The di-carboxy group facilitates membrane localization of 8b near H₁ receptors [66]. Treatment with pyrilamine, an H₁ antagonist suppressed the Ca²⁺ response, indicating cells specifically responded to histamine produced through decaging of 8b [37].

Decaging of secondary messengers

In addition to applications of newly developed caging groups, the well-established nitrobenzyl and coumarin caging groups have found further use in recent biological applications, including control of cell signaling, due to their robust photophysical properties, ease of synthesis, and stability under physiological conditions [15]. The concentration gradients of distinct lipid species and their localization within the cell regulates signaling events [67]. Different potency due to subtle structural differences among lipids have prompted development of chemical tools to spatiotemporally investigate their function [67]. Wagner et al. reported a “click-cage” approach to synthesize organelle-specific DEACM caged lipid secondary messengers - arachidonic acid and sphingosine derivatives (Figure 4a) [68]. The click-cage is comprised of a DEACM caging group with a clickable alkyne handle enabling a modular design for attachment of established organelle targeting scaffolds [69]. Mitochondria, endoplasmic reticulum (ER), lysosome, and plasma membrane targeted sphingosine and arachidonic acid caged derivatives were synthesized and, along with corresponding organelle markers, applied to HeLa cells containing a fluorescent Ca²⁺ reporter. Localization of the caged compounds and markers was as expected (Figure 4a). Irradiation of defined regions in the cell with 385 nm light led to observation of vastly different signaling patterns in distinct cell organelles [68]. Robust calcium transients were observed in lysosomes, endoplasmic reticulum, and mitochondria but not at the plasma membrane through decaging of caged sphingosine derivatives, while stronger calcium responses were seen at the plasma membrane and mitochondria through decaging of caged arachidonic acid derivatives, compared to lysosomes. The observed results supports findings that TPC1, the main intracellular target of sphingosine is localized in lysosomes, while GPR40, a known target of arachidonic acid, is mainly localized at the plasma membrane [70,71]. This study shows that induction of lipid messengers at precise intracellular locations is critical for signaling outcome.

Figure 4.

Use of nitrobenzyl and coumarin caging groups in the control of cell signaling. (a) Organelle targeting DEACM-caged arachidonic acid and sphingosine derivatives are localized as expected: mitochondria (upper left), lysosome (upper right), plasma membrane (lower left), and endoplasmic reticulum (lower right). (b) A kinase is rendered inactive through caging of a catalytic site lysine with 7-hydroxycoumarin, blocking ATP binding until exposure to 365 nm irradiation. The active kinase then phosphorylates its downstream substrate. This was applied to MEK1 within the Ras/MAPK signaling pathway in developing zebrafish embryos. Hyperactivation of MEK1 induces embryo dorsalization leading to an elongation phenotype at 8 hours post fertilization. (c) LCK is rendered inactive through caging of its catalytic site until light irradiation with 375 nm activates the enzyme, which in turn activates ITAMs through phosphorylation. ZAP70 gets recruited by ITAMs and phosphorylated by LCK to activate downstream effector molecules and induce multiple signaling pathways. Adapted with permission from ref. [72] and [73], Copyright 2018, John Wiley and Sons and Copyright 2017, American Chemical Society, respectively.

Genetic code expansion using caged amino acids

Genetic code expansion, through expression of an engineered orthogonal tRNA/aminoacyl-tRNA synthetase pair, enables incorporation of unnatural amino acids into proteins in response to amber stop codons in cells and animals [74,75]. Among various chemically modified amino acids that were genetically encoded, caged analogs of tyrosine, cysteine, and lysine have found numerous applications in the control of protein function [2]. The Deiters lab utilized a genetically encoded 7-hydroxycoumarin lysine (HCK) replacing a critical lysine residue in the active site of MEK1 to extend applications of the optical control of MEK/ERK signaling from mammalian cells [76] to zebrafish embryos [72]. The caging group renders the MEK1 protein catalytically inactive by blocking the binding pocket of ATP until irradiation with 365 nm light makes the protein functional and allows phosphorylation of downstream targets (Figure 4b). Light activation of caged-MEK1 led to dorsalized embryos which was comparable to embryos expressing constitutively active MEK1 (caMEK1 in Figure 4b). The phenotypic results upon optical activation of caged-MEK1 at different time points revealed an essential time window (until 8 hours post-fertilization) for the MEK/ERK pathway to affect dorsal patterning. This critical information may be translated to treat human developmental defects due to MEK hyperactivation at an early developmental stage with pharmacological inhibitors. An earlier genetically encoded nitrobenzyl-caged lysine [77], was applied in triggering the TCR signaling pathway by James and Chin via optical regulation of lymphocyte tyrosine kinase (LCK) [73]. LCK was rendered inactive by caging the critical lysine residue K273 in the ATP binding pocket until 375 nm illumination led to phosphorylation of its downstream target ITAM. The kinase ZAP70 was then recruited and phosphorylated to initiate further downstream target phosphorylation and docking of binding proteins (Figure 4c). The kinetics of ZAP70 phosphorylation were measured by phospho-western blot analysis at different time points after decaging. Additionally, the authors revealed that LCK activity is mediated by autophosphorylation of its critical Y394 and a stimulatory role of the TCR coreceptor CD8 was observed through increased membrane recruitment of ZAP70 [73]. This work provides valuable mechanistic insights on the TCR signaling pathway and can be extended to other members of the SRC family of kinases.

Conclusions

Newer caging groups have improved several photophysical properties, especially shifting the absorbance maxima further into the visible/NIR region and enhancing the decaging efficiency. Increased π-conjugation has improved the decaging efficiency and has further sensitized nitrobenzyl and coumarin chromophores towards two-photon excitation which provides improved three-dimensional spatial control and opportunities for orthogonal decaging. The obtained wavelength orthogonality is an improvement over sequential activation studies which requires initial decaging of the red-shifted chromophore with longer wavelength, followed by activation of a second chromophore with shorter wavelength, typically UV light. Development of green light activatable BODIPY and near-IR activatable cyanine caging groups have further expanded the scope of one-photon irradiation, obviating the need to for a two-photon setup. The new caging groups have a broad substrate scope and have been applied to light-triggered release of alcohols, amines, thiols, carboxylates, and phosphates. The coumarin and BODIPY chromophores, however, generally require a carbonate or carbamate linkage, owing to the lower pKa requirement in the photolysis step compared to nitrobenzyl-based chromophores. In addition to choosing a caging group with red-shifted absorption maxima and good quantum yield for in vivo applications, fluorescent reporters with minimal spectral overlap with the caging group should be considered. The hydrophilicity/hydrophobicity of the caging group should be carefully considered based on solubility requirements and intracellular or membrane localization of the target. Moreover, decaging kinetics of caging groups should be considered for investigating fast cellular processes.

In the context of cell signaling, coumarin caging groups have been used to cage neurotransmitters like GABA and glutamate, secondary messengers like cAMP and lipid molecules like DAG, arachidonic acid, and sphingosine. Wavelength-selective decaging studies using DEAC450-cAMP and CDNI-GABA have enabled mimicking the complex signaling dynamics occurring at synapses and wavelength orthogonality may further be used to study highly dynamic protein kinase/phosphatase signaling pathways and their crosstalk. The use of PEGs and dendrimers to alleviate background activity highlights the need for optimal sterics in the design of a caged compound. Induction of local concentration bursts through decaging of DEACM-caged sphingosine and arachidonic acid has been applied in conjunction with organelle targeting motifs to study lipid-mediated calcium signaling events at pre-defined intracellular locations with improved spatial resolution. The use of modular ‘click’ approach overcomes the limitation of synthesizing specific sets of caged compounds for each organelle where the caging group is directly attached to the targeting scaffold. While BODIPY-caged histamines have enabled investigation of the histamine-mediated calcium signaling pathway using green light, near-IR activatable cyanine dyes has so far been applied exclusively to targeted drug delivery. The cyanine dyes can be applied to orthogonal decaging experiments in combination with nitrobenzyl or coumarin caging groups. Caged amino acids have expanded the substrate scope of unnatural amino acid mutagenesis and provide blocking of protein function through caging of specific amino acid residues, which can be selected based on structural and/or mechanistic data. The loss of the caging group upon photolysis prevents potential perturbation of native protein function. 7-Hydroxycoumarin lysine was used to investigate the MAPK/ERK signaling pathway in zebrafish embryos and the study revealed an essential time window of MEK1 hyperactivation for potential therapeutic intervention in congenital diseases. Furthermore, a nitrobenzyl-caged lysine was used to investigate the TCR signaling pathway and quantify LCK catalytic activity in cells through direct activation of LCK thereby overcoming the limitation of steady-state measurements. Taken together, optochemical tools employing highly modular and synthetically readily accessible caging groups are highly tunable in their photophysical properties and have found applications in cell signaling, ranging from the optical control of metal ions and small molecule messengers to lipids and protein kinases. The developed probes have been applied in single cells, tissues, and whole animals, laying the foundation for a wide range of future studies.