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DOI: 10.1039/D5OB00542F (Review Article) Org. Biomol. Chem., 2025, Advance Article

Shining light on photo-responsive molecular tools: advances in visible-to-NIR activatable photolabile protecting groups

Pin-Han Lin a, Megan R. Clotworthy b, Joseph J. M. Dawson a and Cassandra L. Fleming *ab
aSchool of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia. E-mail: cassandralee.fleming@sydney.edu.au
bCentre of Biomedical and Chemical Sciences, School of Science, Auckland University of Technology, Private Bag 92006, Auckland 1142, New Zealand

Received 1st April 2025 , Accepted 12th May 2025

First published on 19th May 2025

Abstract

The utilisation of light as an external stimulus to control the structure and function of bio-relevant molecules in healthy and diseased cells and tissues serves as a valuable means to probe fundamental molecular events of complex cellular processes. Indeed, the photocaging approach has proved highly popular, whereby bioactive molecules are modified with photolabile protecting groups, rendering them temporarily inactive. Biological activity is only restored upon irradiation with a defined wavelength of light, resulting in the irreversible cleavage of the caging group. While seminal works are reliant on the use of phototoxic UV light for photoactivation, the rapid evolution of the field of photopharmacology has facilitated the development of more sophisticated caging groups with physiochemical and photophysical properties better suited for in vivo applications. Herein, we highlight the recent progress made on the development of BODIPY-, cyanine- and xanthene-derived caging groups, which utilise visible-to-near-infrared light for photoactivation. The strategies employed to red-shift absorption spectra, as well as improve photolysis efficiencies and aqueous solubility have been of particular interest, and are therefore discussed in detail in this review.

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Pin-Han Lin

Pin-Han Lin received her B.S. degree in Chemistry in 2020 from Fu Jen Catholic University and her M.S. degree in Chemistry in 2022 from Fu Jen Catholic University. She is currently pursuing her PhD at the University of Sydney. Her research interests focus on the development of organic small-molecule fluorescent probes for biological applications and light-responsive drug delivery systems.

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Megan R. Clotworthy

Megan Clotworthy completed her Bachelor of Advance Science (Honours) in chemistry at Auckland University of Technology in 2023, after which she worked as a research assistant in the group of Dr Cassandra Fleming, focussing on the design and synthesis of photocaged bioactive molecules. Megan will commence her PhD at the University of Sydney, developing photocaged small molecule inhibitors as tools to study the effect of enzyme inhibition in live cells.

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Joseph J. M. Dawson

Joseph Dawson completed his Bachelor of Science at Auckland University of Technology in 2023. He is currently an Honours student at the University of Sydney, in the group of Dr Cassandra Fleming, focusing on the development of visible-light activated caging groups.

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Cassandra L. Fleming

Cassandra Fleming obtained her PhD in 2015 from Deakin University, Australia. She then joined the group of Professor Joakim Andréasson at Chalmers University of Technology. She spent a further two years in Sweden as a Marie Curie postdoctoral fellow in the group of Professor Morten Grøtli at the University of Gothenburg. In 2020, Cassandra moved to Auckland University of Technology as a Lecturer in Chemistry before moving to the University of Sydney in 2024. Her research focusses on the development of all-photonic molecular tools in which light can be used to both track and trigger the release of bio-relevant molecules.

1. Introduction

Optical control over biochemical processes presents a valuable means to interrogate underlying cellular and subcellular mechanisms with superior spatiotemporal control.1–4 Indeed, this has been achieved through the use of photoswitchable entities and/or photolabile protecting groups (PPGs).1–6 Reversible, photoswitchable drug-like molecules are typically realised through the inclusion of photoswitching moieties into the pharmacophore of the bioactive, giving rise to a photoswitchable variant that reversibly toggles between two (or more) distinct conformations, in which one form has been designed to have a higher affinity for the target than the other. To date, azobenzenes1 have been the dominant photochromic moiety of choice, with diarylethenes7–10 and spiropyrans11 also frequenting the literature in this context.12

Commonly referred to as caging groups, PPGs are introduced onto known bioactive molecules, rendering them inactive via impeding critical binding interactions between the bioactive and its corresponding target. Exposure to a defined wavelength of light results in the irreversible cleavage of the caging group and concurrent liberation of the bioactive in its now ‘active’ form. Since the seminal work by Barltrop et al. in the 1960s,13,14 which saw the development of photolabile ortho-nitrobenzyl-derivatives, a range of structurally diverse caging groups that largely cover the visible spectrum have populated the literature (Fig. 1). Most notably, those based on BODIPY,15,16 coumarin,17,18 cyanine19 and xanthene20 scaffolds have gained increasing interest of late. With the continual interest in using light as an external stimulus to control when and where small molecule bioactives exhibit their bio-relevant function, PPGs that display favourable physicochemical and photophysical properties for use in cellular and multicellular settings are highly sought after. High uncaging quantum yields and molar extinction coefficients at long wavelengths, the formation of non-toxic photo-products, and the use of visible-to-near-infrared (NIR) light to trigger photoactivation, are just some of the key factors that constitute an ideal PPG for more advanced biological applications. Up until recently, the vast majority of caging groups and photocaged substrates reported in the literature were reliant on the use of harmful UV light to achieve photoactivation.5,6,21 In addition to the phototoxicity towards cells and tissue that may arise upon prolonged exposure to UV light,22–24 the poor tissue penetration of UV light25 significantly prevents the utility of such light-responsive probes in more advanced multicellular applications.


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Fig. 1 Common classes of caging groups and their approximate absorption maxima.

In efforts to move away from this reliance on UV light and maximise the utility of caged substrates in more complex bio-relevant matrixes, academic groups have turned their attention to the development of new PPGs that employ visible-to-NIR light to trigger the light-induced uncaging event. While recent reviews in this space detail the design of BODIPY-26,27 and coumarin-derived28 caging groups, and the biomedical application of caging groups that employ >600 nm for photoactivation,29 this review serves to highlight the advancements made in the development of BODIPY-, cyanine- and xanthene-derived PPGs over the past 10 years, in which the strategies employed to tailor physiochemical and photophysical properties are of particular interest. While key photophysical properties are discussed and summarised (Tables 1–7), one should note that uncaging quantum yields are calculated for either the release of the payload or the consumption of the caged substrate. In theory, there should be little-to-no variation between the two approaches. However, in the case where competitive photolysis reactions occur, these values will differ. It is also important to note that the nature of the solvent used for the photolysis reaction influences uncaging efficiencies. For example, recent studies have shown that higher water content affords larger photolysis quantum yields for uncaging reactions that proceed via heterolytic cleavage.30–32 While in contrast, for PPGs that rely on a radical-type uncaging mechanism, higher quantum yields are reported in solvents with lower polarity.33 As such, one should take care when comparing photolysis quantum yields (Φu) and uncaging efficiencies (ε × ϕu) from different studies.

Table 1 Photophysical properties of 4-aryloxy BODIPY-derived caging groups
Compound λmax[thin space (1/6-em)]a (nm) ε (M−1 cm−1) λu[thin space (1/6-em)]b (nm) Φu[thin space (1/6-em)]c (%) ε × Φu (M−1 cm−1)
a Absorption maxima.b Irradiation wavelength for uncaging.c Uncaging quantum yields for consumption of caged substrate. Measurements performed ind 1% MeOH in CH2Cl2; ande MeOH.
1a[thin space (1/6-em)]d (ref. 34) 503 81[thin space (1/6-em)]000 500 7.40 × 10−5 6.0
1b[thin space (1/6-em)]d (ref. 34) 503 89[thin space (1/6-em)]000 500 7.10 × 10−4 64
1c[thin space (1/6-em)]d (ref. 34) 502 86[thin space (1/6-em)]000 500 1.30 × 10−3 111
1c[thin space (1/6-em)]e (ref. 33) 500 78[thin space (1/6-em)]000 500 2.56 × 10−3 199
1d[thin space (1/6-em)]e (ref. 33) 498 91[thin space (1/6-em)]000 500 5.38 × 10−3 490
4a[thin space (1/6-em)]e (ref. 36) 577 200[thin space (1/6-em)]000 580 4.90 × 10−4 98

Table 2 Photophysical properties of BODIPY oxime ester-derived caging groups
Compound λmax[thin space (1/6-em)]a (nm) ε (M−1 cm−1) λu[thin space (1/6-em)]b (nm) Φu[thin space (1/6-em)]c (%) ε × Φu (M−1 cm−1)
a Absorption maxima.b Irradiation wavelength for uncaging.c Uncaging quantum yields for consumption of caged substrate. Measurements performed ind MeOH[thin space (1/6-em)]:[thin space (1/6-em)]H2O (7[thin space (1/6-em)]:[thin space (1/6-em)]3); ande MeOH[thin space (1/6-em)]:[thin space (1/6-em)]H2O (4[thin space (1/6-em)]:[thin space (1/6-em)]1).
16[thin space (1/6-em)]d (ref. 35) 507 52[thin space (1/6-em)]800 503 7.20 × 10−4 38
19[thin space (1/6-em)]e (ref. 38) 626 56[thin space (1/6-em)]000 660 9.30 × 10−3 532

Table 3 Photophysical properties of meso-methyl BODIPY-derived caging groups
Compound λmax[thin space (1/6-em)]a (nm) ε (M−1 cm−1) λu[thin space (1/6-em)]b (nm) Φu[thin space (1/6-em)]c (%) ε × Φu (M−1 cm−1)
n.r. = not reported.a Absorption maxima.b Irradiation wavelength for uncaging.c Uncaging quantum yields for release of the payload.Measurements performed ind MeOH ande 5% CH3CN in 20 mM phosphate buffer (pH 7.4).f Uncaging quantum yields for consumption of caged substrate. Measurements performed ing 20% DMSO in 5 mM phosphate buffer (pH 7.5)h phosphate buffer (pH 7.4) andi aerated CH2Cl2 : MeOH (1[thin space (1/6-em)]:[thin space (1/6-em)]9).
25[thin space (1/6-em)]d (ref. 15) 519 57[thin space (1/6-em)]000 532 6.40 × 10−4 37
26[thin space (1/6-em)]d (ref. 15) 515 71[thin space (1/6-em)]000 532 9.90 × 10−4 70
27[thin space (1/6-em)]d (ref. 15) 544 62[thin space (1/6-em)]000 532 9.50 × 10−4 59
28[thin space (1/6-em)]d (ref. 15) 544 48[thin space (1/6-em)]000 532 4.00 × 10−4 19
29[thin space (1/6-em)]d (ref. 15) 553 49[thin space (1/6-em)]000 532 2.38 × 10−3 117
30[thin space (1/6-em)]e (ref. 16) 545 34[thin space (1/6-em)]500 540 6.00 × 10−4f 21
31[thin space (1/6-em)]e (ref. 16) 544 45[thin space (1/6-em)]000 540 1.50 × 10−4f 6.7
32[thin space (1/6-em)]e (ref. 16) 547 48[thin space (1/6-em)]400 540 1.60 × 10−3f 77
33[thin space (1/6-em)]e (ref. 16) 545 38[thin space (1/6-em)]600 540 1.80 × 10−4f 7
35[thin space (1/6-em)]g (ref. 39) 505 19[thin space (1/6-em)]000 530 4.20 × 10−5f 0.67
37[thin space (1/6-em)]g (ref. 39) 521 30[thin space (1/6-em)]000 530 3.80 × 10−5f 1.1
38[thin space (1/6-em)]h (ref. 40) 513 59[thin space (1/6-em)]200 507 0.084 n.r.
39[thin space (1/6-em)]h (ref. 40) 688 55[thin space (1/6-em)]400 700 0.035 n.r.
46[thin space (1/6-em)]i (ref. 41) 516 56[thin space (1/6-em)]000 525 0.165 n.r.

Table 4 Photophysical properties of meso-methyl BODIPY-derived caging groups with red-shifted absorption spectra
Compound λmax[thin space (1/6-em)]a (nm) ε (M−1 cm−1) λu[thin space (1/6-em)]b (nm) Φu[thin space (1/6-em)]c (%) ε × Φu (M−1 cm−1)
a Absorption maxima.b Irradiation wavelength for uncaging.c Uncaging quantum yields for release of payload. Measurements performed ind MeOH; ande 2% acetone, 10% CH2Cl2 and 88% MeOH.f Uncaging quantum yields for consumption of caged substrate.
26[thin space (1/6-em)]d (ref. 15) 515 71[thin space (1/6-em)]000 532 0.0999 70
59[thin space (1/6-em)]d (ref. 46) 586 61[thin space (1/6-em)]000 532 9.80 × 10−3 6.0
60[thin space (1/6-em)]d (ref. 46) 661 65[thin space (1/6-em)]000 532 4.10 × 10−3 2.7
61[thin space (1/6-em)]e (ref. 50) 549 89[thin space (1/6-em)]400 690 0.84f 223

Table 5 Photophysical properties of meso-methyl BODIPY-derived caging groups with improved uncaging efficiencies
Compound λmax[thin space (1/6-em)]a (nm) ε (M−1 cm−1) λu[thin space (1/6-em)]b (nm) Φu[thin space (1/6-em)]c (%) ε × Φu (M−1 cm−1)
n.r. = not reported.a Absorption maxima.b Irradiation wavelength for uncaging.c Uncaging quantum yields for release of the payload. Measurements performed ind aerated MeOH ande CDCl3[thin space (1/6-em)]:[thin space (1/6-em)]MeOD (1[thin space (1/6-em)]:[thin space (1/6-em)]1).
26[thin space (1/6-em)]d (ref. 51) 517 71[thin space (1/6-em)]000 365 0.14 n.r.
28[thin space (1/6-em)]d (ref. 51) 544 48[thin space (1/6-em)]000 365 0.29 n.r.
62[thin space (1/6-em)]d (ref. 51) 545 56[thin space (1/6-em)]300 507 0.70 n.r.
29[thin space (1/6-em)]d (ref. 51) 553 49[thin space (1/6-em)]000 507 0.99 n.r.
63[thin space (1/6-em)]d (ref. 51) 514 104[thin space (1/6-em)]400 507 0.15 n.r.
64[thin space (1/6-em)]d (ref. 51) 512 68[thin space (1/6-em)]500 507 5.5 n.r.
65[thin space (1/6-em)]d (ref. 51) 513 55[thin space (1/6-em)]300 507 4.4 n.r.
66[thin space (1/6-em)]d (ref. 51) 517 58[thin space (1/6-em)]300 507 0.57 n.r.
67[thin space (1/6-em)]d (ref. 51) 538 60[thin space (1/6-em)]700 507 15.6 n.r.
68[thin space (1/6-em)]e (ref. 55) 514 69[thin space (1/6-em)]000 532 0.10 69
69[thin space (1/6-em)]e (ref. 55) 510 52[thin space (1/6-em)]000 532 1.0 340
70[thin space (1/6-em)]e (ref. 55) 535 57[thin space (1/6-em)]000 532 0.15 86
76[thin space (1/6-em)]d (ref. 46) 647 49[thin space (1/6-em)]000 532 0.084 41
77[thin space (1/6-em)]e (ref. 53) 708 108[thin space (1/6-em)]000 532 0.05 53
78[thin space (1/6-em)]e (ref. 53) 681 124[thin space (1/6-em)]000 532 3.75 4650

Table 6 Photophysical properties of cyanine-derived caging groups
Compound λmax[thin space (1/6-em)]a (nm) ε (M−1 cm−1) λu[thin space (1/6-em)]b (nm) Φu[thin space (1/6-em)]c (%) ε × Φu (M−1 cm−1) Yieldaer[thin space (1/6-em)]d (%) Yielddeg[thin space (1/6-em)]e (%)
n.r. = not reported.a Absorption maxima.b Irradiation wavelength for uncaging.c Uncaging quantum yields for release of the payload.d Chemical yield of the payload under ambient conditions determined in CD3OD.e Chemical yield of the payload under oxygen-free conditions determined in CD3OD. Measurements performed inf 1% DMSO in H2O (degassed) andg phosphate buffer.h Uncaging quantum yield for consumption of caged substrate. Measurements performed ini MeOH.j 20% DMSO in phosphate buffer (pH 7.4).k Uncaging quantum yield for photooxidative decomposition.
105[thin space (1/6-em)]f (ref. 82) 746 276[thin space (1/6-em)]000 760 0.33 921 n.r. n.r.
111[thin space (1/6-em)]g (ref. 83) 786 155[thin space (1/6-em)]400 810 2.20 × 10−5h n.r. n.r. n.r.
112[thin space (1/6-em)]i (ref. 84) 845 71[thin space (1/6-em)]430 820 <2 n.r. 58 96
113a[thin space (1/6-em)]j (ref. 78) 809 120[thin space (1/6-em)]900 820 3.90 × 10−5k n.r. 44 92
113b[thin space (1/6-em)]j (ref. 78) 811 94[thin space (1/6-em)]200 820 5.80 × 10−5k n.r. 63 103
113c[thin space (1/6-em)]j (ref. 78) 809 117[thin space (1/6-em)]300 820 4.50 × 10−5k n.r. 47 88
113d[thin space (1/6-em)]j (ref. 78) 808 115[thin space (1/6-em)]300 820 6.00 × 10−5k n.r. 41 n.r.

Table 7 Photophysical properties of xanthene-derived caging groups 121–123
Compound λmax[thin space (1/6-em)]a (nm) ε (M−1 cm−1) λu[thin space (1/6-em)]b (nm) Φu[thin space (1/6-em)]c (%) ε × Φu (M−1 cm−1)
a Absorption maxima.b Irradiation wavelength for uncaging.c Uncaging quantum yields for consumption of caged substrate.d Measurements performed in H2O[thin space (1/6-em)]:[thin space (1/6-em)]CH3CN (9[thin space (1/6-em)]:[thin space (1/6-em)]1, pH 7.4).
121[thin space (1/6-em)]d (ref. 86) 538 22[thin space (1/6-em)]700 549 6.2 1070
122[thin space (1/6-em)]d (ref. 86) 570 70[thin space (1/6-em)]000 605 1.9 1020
123[thin space (1/6-em)]d (ref. 86) 641 86[thin space (1/6-em)]000 685 2.2 1400

2. BODIPY-derived caging groups

Due to their sharp absorption spectra, high molar extinction coefficients (ε ≈ 40[thin space (1/6-em)]000–150[thin space (1/6-em)]000 M−1 cm−1), long excitation wavelengths, resistance to photobleaching and good biocompatibility, an array of structurally diverse BODIPY-derived caging groups have proven an exciting class of caging groups that are readily cleaved at wavelengths >500 nm.27 The payload can be introduced at either the boron atom (i.e. the 4-position),34 the 2,6-position via an oxime functionality,35 or more commonly, the meso-position (i.e. the 8-position, Fig. 2).15,16
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Fig. 2 The inclusion of the payload (=LG) at different positions of the BODIPY core.

2.1 4-Aryloxy BODIPY caging groups

In 2014, Urano and co-workers reported on the serendipitous finding that 4-aryloxy BODIPY derivatives (compounds 1a–c) underwent a photoelimination reaction at the boron–aryloxy bond when irradiated with 500 nm to give the corresponding phenol 3 and solvated BODIPY photoproduct 2 (Fig. 3).34 Photo-induced electron transfer (PeT) between the BODIPY core and the adjacent aryl group was found to be the driving force behind the uncaging reaction, in which authors propose that photoexcitation results in the formation of a charge-separated intermediate, consisting of a cation radical of the aryl group and an anion radical on the BODIPY, and subsequent solvolysis of the B–O bond.
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Fig. 3 BODIPY-derived caging groups with the payload introduced at the boron atom.

The utility of this new class of caging group was further demonstrated when a BODIPY-derived caging group was introduced onto the terminal amine of histamine via a benzyloxycarbonyl self-immolating linker (compound 5, Fig. 3), in which, upon irradiation with visible light, phenolate 7 was released and further decomposes to give histamine 9, quinone methide 8 and carbon dioxide (Fig. 3). The caged substrate 5 was used to stimulate cultured HeLa cells in a light-dependent manner when exposed to blue-green visible light.34

Despite the benefits obtained from using visible light to trigger the release of the payload, these caging groups suffer from poor uncaging quantum yields, especially when these are compared to those caging groups that employ shorter wavelengths of light for the photolysis reaction.34 In a follow-up study, the same authors looked at the effect of varying the electron-donating substituent at the para-position of the aryl group on uncaging quantum yields.33 While a handful of electron-donating groups were introduced at the 4-position of the aryl moiety, the inclusion of a methyl substituent saw a 2.1-fold increase in uncaging quantum yield over that of the previously reported methoxy variant 1c (Φu of 53.8 × 10−4 and 25.6 × 10−4 for 1d and 1c, respectively when uncaging was performed in MeOH, Table 1). The effect of solvent polarity on uncaging efficiencies was also of interest, in which authors noted higher uncaging quantum yields in solvents of lower polarity.

More recently, the same group extended the π-conjugation of the BODIPY core through the addition of furo[b]fused ring system (compound 4, Fig. 3), resulting in a red-shifted absorption maximum from 500 nm to 577 nm (when compared to 1c).36 Indeed, irradiation with 580 nm afforded the release of the 4-methoxy phenol payload with an uncaging efficiency of 98 M−1 cm−1 (Table 1). The inclusion of the furo[b]fused BODIPY caging group onto the transient receptor potential vanilloid 1 agonist, N-vanillylnonanamide, afforded optical control over neuronal activity and behaviour in Caenorhabditis elegans (compound 4b, Fig. 3).

2.2 Inclusion of the payload at the 2,6-position

In 2016, Urano and co-workers unexpectedly discovered that 2,6-disulfonamide BODIPY derivatives (compound 10, Fig. 4) were photolabile when exposed to visible light (λirr = 490 nm).37 Upon further investigation and the identification of 13 and 14 photoproducts, the authors proposed that photoexcitation results in the formation of a charge-separation state between the carboxylate group and BODIPY core (compound 11), followed by decarboxylation. A subsequent radical reaction proceeds via one of two pathways: (A) cleavage of the sulfonamide and release of the unsubstituted BODIPY 13; or (B) deboronation of the BODIPY core to give 14 (Fig. 4). Recognising the potential as new BODIPY-derived caging groups, the amino moiety of the neurotransmitter, γ-aminobutyric acid (GABA) was caged with a BODIPY PPG (compound 15, Fig. 4), masking GABA's cellular activity. Exposure to visible light released the active form of GABA to stimulate neuronal cells.
image file: d5ob00542f-f4.tif
Fig. 4 BODIPY-derived caging groups with the payload introduced at the 2,6-position.

In 2019, Sambath et al. reported the BODIPY oxime ester 16 as a new uncaging platform for the photo-release of aliphatic and aromatic carboxylic acids (Fig. 5).35 Using the known histone deacetylase inhibitor, valproic acid 18, as the model payload, exposure to green light (λirr = 503 nm) cleaves the N–O bond to release the active form of valproic acid and the 2-cyano-BODIPY 17 uncaged product with an uncaging efficiency of 38 M−1 cm−1 (Table 2). The light-dependent anticancer properties of compound 16 was demonstrated in HeLa cells, in which cell death was only observed when cells, treated with 16, were exposed to 503 nm. The same authors installed a diphenyl-sulfide styryl group at the 3,5-position of the BODIPY core (compound 19, Fig. 5), resulting in a 119 nm red-shift of the absorption maximum of the BODIPY oxime ester 16 (λmax = 507 and 626 nm for compounds 16 and 19, respectively).38 Irradiation with 626 nm resulted in the photo-induced release of valproic acid payload with a 14-fold increase in uncaging efficiency (532 M−1 cm−1 for compound 19, Table 2).


image file: d5ob00542f-f5.tif
Fig. 5 BODIPY-derived caging groups with the payload introduced at the 2,6-position.

2.3 meso-Methyl BODIPY caging groups

The emergence of the meso-methyl BODIPY-derived caging groups in 2015 presented a versatile caging group scaffold in which the payload is readily released upon irradiation with visible-to-NIR light.15,16 Analogous to the design of coumarin- and xanthene-derived caging groups, Weinstain and Winter simultaneously reported the installation of a methylhydroxy moiety at the meso-position of the BODIPY scaffold, affording visible light absorbing caging groups, in which the hydroxy group can be readily coupled to amino, hydroxy and carboxylic acid functionalities through the formation of carbamate, carbonate and ester groups, respectively (see Fig. 7).

While the exact uncaging mechanism is yet to be fully elucidated, it is proposed that the uncaging reaction proceeds via a SN1-type mechanism.15 Observations from initial mechanistic studies suggest that photo-release occurs from either the singlet or triplet excited state, in which heterolytic cleavage of the C–O bond affords the corresponding contact ion pair (21 and 22, Fig. 6). The cation 21 directly reacts with the solvent, forming the corresponding solvated product 23.


image file: d5ob00542f-f6.tif
Fig. 6 Proposed uncaging mechanism for the photolytic reaction of meso-methyl BODIPY-derived caging groups.

Utilising a 3,5-dimethyl and 1,3,5,7-tetramethyl BODIPY core, Smith and Winter reported the photolysis of meso-methyl BODIPY-derived caging groups with varying substituents at the 2,6-position (compounds 25–29, Fig. 7).15 The inclusion of iodo-substituents at the 2,6-position resulted in a pronounced red-shift in absorption maxima as well as a 6-fold increase in uncaging efficiency when compared to the 2,6-dichloro BODIPY 28 (19 M−1 cm−1 and 117 M−1 cm−1 for compounds 28 and 29, respectively, Table 3). The authors propose that this increase in uncaging efficiency is attributed to the iodo groups promoting intersystem crossing to the triplet excited state, which in turn, affords more time for the photolysis reaction to occur.


image file: d5ob00542f-f7.tif
Fig. 7 meso-Methyl BODIPY-derived caging groups.

In the study described by Weinstain, the meso-methyl BODIPY caging group was introduced onto amino (primary and aromatic amines), phenol and carboxylic acid model payloads via carbamic, carbonic and ester linkages, respectively (compounds 30–31, Fig. 7).16 Caged substrates absorbed strongly around 545 nm, exhibited good stability in the absence of light, and upon exposure to 540 nm, readily released the corresponding payload in aqueous media. The nature of the payload appeared to influence the rate of the uncaging reaction as well as uncaging efficiencies. More specifically, the photo-release of the aromatic amine payload from compound 30 occurred approximately 5-times faster than that of the primary amine from compound 31 (t½ = 120 s and 600 s for 30 and 31, respectively), with a 3.1-fold increase in uncaging efficiency (Table 3). While photolysis of carbonate 32 rapidly afforded the corresponding para-nitrophenol (t½ = 140 s) with an uncaging efficiency of 77 M−1 cm−1. The authors also demonstrated good cell compatibility through the caging of biogenic amines, histamine and dopamine, in which their bioactivity was readily photoregulated when exposed to visible light in cultured cells and neurons, respectively.

These first two reports on meso-methyl BODIPY caging groups spurred a significant increase in the utility of BODIPY-derived caging groups. Sitkowska et al. investigated the caging of primary and secondary amines with meso-methyl BODIPY PPGs via a carbamate linker (compounds 34–37, Fig. 7).39 Irradiation with 530 nm resulted in the rapid release of the 4-fluorobenzylamino moieties, albeit with poor uncaging quantum yields (Table 3).

Štacko and Klán extended the utility of meso-methyl BODIPY caging groups to the photoactivation of hydrogen sulfide.40 To aid water solubility, polyethylene glycol chains were either introduced onto the boron atom, replacing the fluorine atoms on the BF2 group (compound 38, Fig. 8), or appended to styryl units introduced at the 3,5-positions of the BODIPY core (compound 39). The latter also extended the π-conjugation of the BODIPY core, red-shifting the absorption maxima as a result (Table 3, see section 2.3.1 for further discussion on red-shifting of the absorption profile of meso-methyl BODIPY cages). Employing a thiocarbamate as the payload, irradiation with visible-to-NIR light resulted in the release of thiocarbamate 42 and subsequent fragmentation to Ph2NH 44 and COS 45, after which, the release of H2S was facilitated upon the addition of carbonic anhydrase (Fig. 8).


image file: d5ob00542f-f8.tif
Fig. 8 Thiocarbamate meso-methyl BODIPYs 38 and 39 and their proposed uncaging mechanism.

More recently, Wohlrábová et al. reported the sulfonothioated meso-methyl BODIPY 46, which, upon irradiation with 525 nm, gave the corresponding dansyl sulfonate 58 and reactive sulfur species (H2Sn, Fig. 9).41 Mechanistic studies suggest a dual uncaging mechanism, whereby the weak sulfonothioate bond undergoes both homolytic (pathways A and B, Fig. 9) and heterolytic cleavage (pathway C), releasing H2S2 and a mixture of BODIPY-derived photoproducts (compounds 51–54 and 57).


image file: d5ob00542f-f9.tif
Fig. 9 Proposed uncaging mechanism for the photolytic reaction of meso-methyl BODIPY 46.

Given the versatility of the functional groups/payloads that can be caged with meso-methyl BODIPY PPGs, meso-methyl BODIPY-derived caging groups have found use in the caging of various bio-relevant molecules, including small molecule enzyme inhibitors,18 receptor agonists,42 nucleotides43 and large bio-molecules under high-vacuum.44 Despite their popularity, this class of caging group often suffers from poor solubility in aqueous milieu and sub-optimal uncaging efficiencies. Addressing these shortcomings, together with further tuning their absorption properties to longer wavelengths, has been of interest. As such, the remainder of section 2 will focus on the recent advancements made to further improve the physiochemical and photophysical properties of meso-methyl BODIPY caging groups.

2.3.1 Red-shifting absorption spectra. One of the appeals of the BODIPY platform is the relative ease of tuning its absorption properties to longer wavelengths, which has mostly been achieved by extending the conjugation at the 3,5-positions of the BODIPY core.45 In 2018, Smith and Winter showed that the extension of the π-conjugation at the 3,5-positions of the BODIPY core through the installation of styryl groups, resulted in a significant bathochromic shift to 586 nm and 661 nm for monostyryl 59 and distyryl 60, respectively (Fig. 10 and Table 4).46 Unfortunately these modifications have come at a cost of uncaging efficiencies, with the uncaging quantum yields for 59 and 60 an order of magnitude lower than the unconjugated variant 26 (Table 4). However, due to their favourable red-shifted absorption profile, styryl BODIPY-derived caging groups have found utility in the photoactivation of dopamine,47 receptor agonists48 and immunomodulatory drugs.49
image file: d5ob00542f-f10.tif
Fig. 10 meso-Methyl BODIPY-derived caging groups with red-shifted absorption.

More recently, Wang and co-workers report the use of a photosensitiser as a means to initiate the uncaging of BODIPY-derived caged substrates employing longer wavelengths.50 In short, this upconversion-like process entails the excitation of the photosensitiser via singlet–triplet absorption, followed by triplet–triplet energy transfer to the caging group, resulting in photolysis. The inclusion of the iodo atoms at the 2,6-position of the BODIPY core is critical for promoting intersystem crossing to the triplet excited state (compound 61, Fig. 10). Employing the anticancer agent, chlorambucil, as a model substrate, the photo-induced release of chlorambucil was achieved upon irradiation with 690 nm, with an uncaging quantum yield of 0.84% in the absence of oxygen (Table 4). This mechanism relies on an oxygen-sensitive energy transfer process, making its utility in conventional biological milieu challenging, however, the authors showed that by encapsulating the photosensitiser and caged anticancer agent within a nanoparticle, the photo-induced activation of chlorambucil inhibited the growth of cancer cells.

2.3.2 Increasing uncaging efficiencies. While the overall uncaging quantum yields observed in the first-generation meso-methyl BODIPY PPGs are significantly lower when compared to the ortho-nitrobenzyl- and coumarin-derived caging groups, the higher extinction coefficients of the BODIPY core partially compensate for this.21 Nevertheless, a number of structural modifications have been pursued in efforts to accelerate intersystem crossing to the triplet excited state,51 stabilise the carbocation intermediate to impede ion pair recombination51,52 or prevent unproductive non-radiative decay processes,53 thus improving uncaging quantum yields, and in turn, uncaging efficiencies as a result. Further to these efforts, electronic structure-photoreactive studies, aided by DFT-calculations, have highlighted the complexity of the photophysical and photochemical channels that are competing with the photo-induced uncaging pathway.54

In efforts to elucidate what structural modifications could be made to the BODIPY core that could be further translated into a rational design approach for improving uncaging efficiencies, Slanina et al. conducted a systematic structure-activity-relationship study, in which the effects of halogens at the 2,6-position and alkyl substituents on the boron atom on uncaging quantum yields were of particular interest.51 As anticipated, the inclusion of heavy atoms at the 2,6-position saw a decrease in fluorescence quantum yields in the order of H > Cl > Br > I (due to increased intersystem crossing efficiency), while the uncaging quantum yields were inversely proportional to the fluorescence quantum yields with the 2,6-diiodo variant 29 (Fig. 11) exhibiting a 7-fold increase in uncaging quantum yield when compared to the non-halogenated BODIPY 26 (Φu of 0.14% and 0.99% for 26 and 29, respectively, Table 5). On the basis of further mechanistic studies, the authors also proposed that increasing the electron density of the BODIPY core should stabilise the carbocation intermediate following heterolytic cleavage, and in turn, improve the uncaging efficiency. Indeed, the replacement of the two fluoride groups on the boron atom with methyl groups on the 1,3,5,7-tetramethyl BODIPY 64, in which phenylacetic acid was employed as the model payload, resulted in a 36-fold improvement in uncaging quantum yields (Φu of 0.15% and 5.5% for 63 and 64, respectively, Table 5 and Fig. 11). Diethyl- and diphenyl boron derivatives were also synthesised (compounds 65 and 66, respectively), however, the increased steric bulk introduced by these substituents resulted in a decrease in uncaging quantum yield (Table 5). Having identified two structural motifs that lead to increased uncaging quantum yields in meso-methyl BODIPY PPGs, the combination of 2,6-diiodo substituents with boron methylalkylation was investigated (compound 67, Fig. 11), and afforded the uncaging of phenylacetic acid with a further improved uncaging quantum of 15.6% (Table 5).


image file: d5ob00542f-f11.tif
Fig. 11 meso-Methyl BODIPY-derived caging groups 26, 28–29 and 62–70.

Winter and co-workers further extended this study to investigate the direct photo-induced release of alcohols (via an ether linkage) using boron-alkylated BODIPY PPGs.55 Again, the alkylation of the boron atom with two methyl groups resulted in a 10-fold increase in uncaging quantum yield and a 5-fold increase in overall uncaging efficiency (69 M−1 cm−1 and 340 M−1 cm−1 for compounds 68 and 69, respectively, Table 5 and Fig. 11). However, in contrast to the earlier report, the inclusion of the 2,6-diiodo substituent on the BODIPY core (compound 70) afforded a significant drop in uncaging efficiency (340 M−1 cm−1 and 86 M−1 cm−1 for compounds 69 and 70, respectively, Table 5).

More recently, Ljubić et al. identified competing photolysis pathways when studying the uncaging of phenols employing boron-alkylated BODIPY 71 (Fig. 12).56 Using MeOH as the solvent, the authors showed that compound 71 was prone to photocleavage of the B–C bond and subsequent solvolysis with MeOH to yield methane and the corresponding photoproduct 73. Interestingly, the inclusion of a methoxy group at the ortho-position of the caged phenol (compound 72, Fig. 12) did not result in the photolysis of the B–C bond, but instead the heterolytic cleavage of the bond between the O and C atom of the phenyl ring, affording the BODIPY photoproduct 74 and methoxy uncaged product 75.


image file: d5ob00542f-f12.tif
Fig. 12 Proposed uncaging pathways for meso-methyl BODIPY-derived caging groups 71 and 72.

Given the improved uncaging efficiencies afforded by boron-alkylated BODIPYs, it is no surprise that they have proven valuable in a number of bio-relevant settings, including the photoactivation of DNA binders,57 anticancer agents,58 neurotransmitters59 and receptor agonists and antagonists.60

As highlighted in section 2.3.1, styryl-substituted BODIPY PPGs suffer from poor uncaging efficiencies. Smith and Winter proposed that the flexibility around the C–C single bonds at the 3,5-positions of the BODIPY core, as well as cis–trans isomerisation of the stilbene moieties, contributed to unproductive non-radiative decay processes.53 Therefore, in efforts to increase the uncaging efficiencies of the styryl-substituted BODIPYs, whilst maintaining the favourable red-shifted absorption spectra, the same authors synthesised conformationally restrained variants consisting of a fused polycyclic scaffold (compounds 77 and 78, Fig. 13).53 The additional rigidity proved highly successful, with BODIPY 77 showing significantly improved uncaging efficiencies ca. 100-fold greater than the stilbene derivatives (41 M−1 cm−1 and 4650 M−1 cm−1 for compounds 76 and 78, respectively, Table 5). However, it should be noted that acetic acid was employed as the payload in compounds 77 and 78 in place of phenylacetic acid used for the stilbene variant 76. Interestingly, exchanging the boron-fluorinated functionality for a boron-methylated group saw a slight hypsochromic shift in absorbance maxima, but a 93-fold increase in uncaging efficiencies (53 M−1 cm−1 and 4650 M−1 cm−1 for compounds 77 and 78, respectively, Table 5). In 2022, Winter and co-workers reported on a systematic study looking at the scope of the functional groups that can be caged using 79 (i.e. carboxylic acid, amine, alcohol, phenol, phosphate and halide) and their uncaging efficiencies (Fig. 13).61 Interestingly, there is little correlation between the pKa of the leaving group and uncaging quantum yields, suggesting that factors other than the quality of the leaving group influences uncaging quantum yields. The utility of fused-ring BODIPY PPGs have been extended to the photoactivation of antibiotics,49 antitumour immunosuppressant62 and enzyme inhibitors.63


image file: d5ob00542f-f13.tif
Fig. 13 meso-Methyl BODIPY-derived caging groups 76–80.

In 2022, Szymański and Feringa reported on a general strategy for the improvement of uncaging quantum yields for PPGs that undergo a SN1-type uncaging mechanism upon irradiation with light.52 More specifically, the cation on the alpha-carbon that forms following heterolytic cleavage is stabilised by a prenyl substituent at the BODIPY alpha-carbon, preventing contact ion pair recombination as a result (compound 80, Fig. 13). The photolysis reaction depicted in Fig. 13 follows a SN1′-type uncaging pathway, in which irradiation with visible light (530 nm) resulted in the rapid and clean release of the acetic acid payload, and formation of alcohol 81 as the photoproduct (formed as a result of water trapping of the cationic intermediate). While no uncaging quantum yields are reported for BODIPY 80, the authors do report a 7-fold increase in the rate of uncaging when compared to the parent compound 26 (4.41 × 10−4 s−1 and 3.00 × 10−3 s−1 for compounds 26 and 80, respectively).

2.3.3 Improving water solubility. BODIPY-derived caging groups are inherently hydrophobic, resulting in poor aqueous solubility. In efforts to improve water solubility, the inclusion of sulfonic acid side chains at the 3- and/or 5-positions of the BODIPY core has proven effective, with little effect on uncaging efficiencies (compounds 82 and 83, Fig. 14).64 Interestingly, the authors showed that the degree of sulfonation (i.e. mono vs. di) influences cell permeability, with the mono-sulfonated BODIPY 82 readily crossing the cell membrane into the intracellular environment, while the di-sulfonated BODIPY 83 is cell impermeable. The latter proving useful for the photo-induced release of bio-relevant molecules that target cell-surface receptors.
image file: d5ob00542f-f14.tif
Fig. 14 BODIPY-derived caging groups with improved water solubility.

The appendage of sulfonic groups on the π-extended BODIPY 84 also afforded a water-soluble variant (Fig. 14).65 Using acetic acid as the model payload, mechanistic studies showed that upon irradiation with 633 nm an oxidative cleavage of one of the styryl double bonds occurs prior to the release of acetic acid (compound 85). It is also interesting to note that in addition to the formation of the corresponding alcohol uncaged product 86, the formation of a photoproduct bearing a methyl group at the meso-position was also observed (compound 87).

2.3.4 Organelle-targeting caging groups. The additional functionalisation of photolabile caging groups with organelle-targeting ligands has proven valuable in further enhancing spatial resolution. While a handful of organelle-targeting coumarin-derived caging groups have dotted the literature,66–70 to date, there is only one report of this approach being applied to BODIPY-derived caging groups.71

Kand et al. report on the late-stage functionalisation of a meso-BODIPY caging group with phenyl sulfonamide, morpholine and triphenylphosphonium functional groups for the targeting of the endoplasmic reticulum (ER), lysosome and mitochondria, respectively.71 In efforts to demonstrate their utility, the protonophore, 2,4-dinitrophenol was caged with the mitochondrial-targeting caging group (compound 88, Fig. 15) and selectively released in the mitochondria of HeLa cells upon irradiation with 545 nm. The protein synthesis inhibitor, puromycin, was also caged with the ER-targeting caging group, in which light-induced release of puromycin in the ER of HeLa cells was observed (compound 89, Fig. 15).


image file: d5ob00542f-f15.tif
Fig. 15 BODIPY-derived organelle-targeting caging groups.

3. Cyanine-derived caging groups

Cyanine dyes are well-known NIR chromophores, valued for their high molar extinction coefficients (ε ≈ 100[thin space (1/6-em)]000–250[thin space (1/6-em)]000 M−1 cm−1) and excellent biocompatibility.72,73 Among them, heptamethine cyanines have been widely used in in vivo imaging studies due to their good tolerance and minimal toxicity in living systems.72,73 These favourable properties have also made cyanine an attractive platform for NIR caging groups, and to date, have found use in the NIR-photoactivation of anticancer agents,19,74,75 antibody–drug conjugates enabling the targeted release of potent antitumor agents in vivo,76,77 antihypotensive agents78 and nano gels.79

In 2014, Gorka et al. reported the first example of a cyanine-derived caging group, in which irradiation of the C4′-N,N’-dialkyldiamine-substituted heptamethine cyanine 90 resulted in the photooxidative cleavage via a self-sensitised 1O2-mediated mechanism (Fig. 16).19 The C–C cleavage of compound 90 at C1–C1′ and C2′–C3′ bonds yield the intermediate products 91 and 92, which subsequently undergo spontaneous C–N bond hydrolysis, liberating a secondary amine 95, which then cyclises onto a pendant carbamate 96 to release the corresponding phenol-containing payload 97. The utility of this new class of caging group in live cells was demonstrated with the caged estrogen receptor antagonist 98, in which the NIR light-triggered release of the estrogen receptor antagonist, 4-hydroxycyclofen, inhibiting cell viability and regulating gene expression in a ligand-dependent CreERT/LoxP-reporter cell line derived from transgenic mice (Fig. 17).


image file: d5ob00542f-f16.tif
Fig. 16 Proposed uncaging mechanism for cyanine-derived caging group 90.

image file: d5ob00542f-f17.tif
Fig. 17 Cyanine-derived caging groups 98–101.

In a follow-up study by Gorka et al., cellular imaging studies showed that the caged cyclofen 98 predominately localised in the lysosomes, resulting in poor cellular retention of the caged substrate.75 In efforts to improve cellular uptake and intracellular retention, the authors exchanged the dimethylamino moiety on the cyclofen payload and the sulfonate groups on the cyanine scaffold for lipophilic n-butyl esters (compound 99, Fig. 17). These modifications promoted intracellular ester cleavage, enhancing uptake and spatial regulation over Cre-mediated recombination.

Given the biological significance of nucleic acids and other amine-containing molecules, the development of NIR-responsive photocages for amine release has attracted considerable interest. Yamamoto et al. designed heptamethine cyanines bearing aryl amine substituents at the C4′ position (compound 100, Fig. 17), which release amines upon cyanine photooxidation and subsequent C–N hydrolysis (analogous to the first two steps of the mechanism in Fig. 16).80 While irradiation of compound 100 with 780 nm resulted in rapid photooxidation (t½ = 5.7 min), <1% of the fluorescent payload (7-amino-4(trifluoromethyl)coumarin) was detected after 1 hour due to slow C–N hydrolysis. In efforts to improve the release of the coumarin-derived payload, the unsymmetrical merocyanine-flavylium conjugate 101 was developed, consisting of an electron-donating indolenine and an electron-deficient flavylium heterocycle. Indeed, irradiation with 690 nm enabled rapid photooxidation and subsequent β-elimination afforded 33% of the desired payload within 1 hour.

These early examples of cyanine-derived caging groups are reliant on the presence of oxygen for photoactivation. To address this, Guo and co-workers reported on a radical-mediated photoactivation strategy that is independent of oxygen consumption, enabling photoactivation under hypoxic conditions.81 Under normoxic conditions, the cyanine photocage 102 follows the traditional 1O2-mediated photocleavage pathway, affording a non-fluorescent uncaged product 104 (Fig. 18). However, in the absence of oxygen (i.e. hypoxia), photoactivation of 102 generates free radicals, which, in the presence of water, further reacts to yield a fluorescent uncaged product 103. The formation of fluorescent and non-fluorescent photoproducts 103 and 104 under hypoxia and normoxia, respectively, affords a valuable real-time reporting mechanism, which serves as a powerful tool for deep-tissue penetration or phototherapy.


image file: d5ob00542f-f18.tif
Fig. 18 Proposed uncaging pathways for cyanine-derived caging groups 102 and 105.

More recently, computational approaches have been employed to design oxygen-independent NIR photocages. In 2022, Feringa and co-workers employed DFT-calculations to screen potential NIR dyes for a Node-to-Lobe Shift in their frontier molecular orbital configurations, which is thought to drive photo-release in caging groups that proceed via heterolytic cleavage.82 This led to the rational design of the cyanine-derived PPG 105 that is not reliant on the presence of oxygen for photoactivation or the inclusion of an additional linker between the payload and cyanine scaffold (Fig. 18). Irradiation of 105 with 760 nm saw the photo-release of the model payload, acetic acid, and the subsequent formation of photoproducts 109 and 110, with an uncaging quantum yield of 0.33% (Table 6).

Janeková et al. reported a class of heptamethine cyanine-derived caging groups that enable the direct attachment of carboxylic acids to the cyanine scaffold without an additional linker (Fig. 19).83 Using para-fluorobenzoic acid as a model payload, irradiation of 111 at 780 nm resulted in the rapid depletion of the caged substrate with an uncaging quantum yield comparable to the first generation of meso-methyl BODIPY PPGs (compound 111 Φu = 2.2 × 10−5%, Table 6). Mechanistic studies showed that the uncaging reaction proceeds via the photooxygenation pathway opposed to the photoheterolytic pathway previously reported by Feringa and co-workers.


image file: d5ob00542f-f19.tif
Fig. 19 Cyanine-derived caging groups 111–115.

In light of the competing photooxidation and photoheterolytic uncaging pathways, a follow-up study by the same authors demonstrated that the inclusion of a phenyl substituent at the meso-position of the cyanine PPG stabilised the contact ion pair that forms upon photo-induced heterolytic cleavage between the payload and the cyanine scaffold.84 Indeed, irradiation of compound 112 at 820 nm in deoxygenated MeOH afforded the efficient release of the para-fluorobenzoic acid payload with a chemical yield of 96%, a 1.7-fold improvement over that observed in the presence of oxygen (chemical yield of 58%, Fig. 19 and Table 6).

More recently, Štacko and co-workers demonstrated the versatility of heptamethine cyanine-derived caging groups, extending their utility to the photo-release of alcohol, phenol, amine and thiol payloads (compounds 113a–d, Fig. 19).78 Upon irradiation with NIR light (λirr = 820 nm), the uncaging reaction proceeds via two distinct pathways (photooxidation and photoheterolysis). Again, the chemical yields for the release of the payload under oxygen-free conditions were higher than when uncaging occurred in the presence of oxygen (Table 6). To demonstrate the utility of these caging groups in a bio-relevant setting, the cardiac stimulant, etilefrine, was caged and incubated in iPSC-derived cardiomyocytes, in which NIR light could be used to modulate the beating rates of the cardiomyocytes (compound 114, Fig. 19).

Employing the aforementioned heptamethine cyanine scaffold, the same authors reported the concurrent release of H2S and a fluorescent coumarin payload upon irradiation with 820 nm (compound 115, Fig. 19).85 In contrast to previous examples, the uncaging reaction was not dependent on the presence/absence of oxygen, rendering compound 115 suitable for both normoxic and hypoxic bio-relevant applications.

4. Xanthene-derived caging groups

Recognising the structural similarities with coumarin-derived caging groups, in 2013, Klán and Wirz reported on the development of xanthene-derived caging groups 116a–c, which were isolated as DDQ complexes (Fig. 20).20 Indeed, irradiation of caged substrates with visible light (λ = 538–546 nm) in aqueous phosphate buffer (at pH 7.0) afforded the payload (R–H), as well as photoproducts 118 and 119, whereby it is proposed that the hydrolysis of DDQ to give 117, oxidised the expected uncaging product 120 to 118. Interestingly, the ratio of the uncaged products 118 and 119 was dependent on the solvent used for photolysis. While 118 was obtained as the major uncaged product in phosphate buffer, irradiation of 116a in 1[thin space (1/6-em)]:[thin space (1/6-em)]1 H2O/MeOH resulted in only the formation of photoproduct 119.
image file: d5ob00542f-f20.tif
Fig. 20 Proposed uncaging pathway for xanthene-derived caging group 116.

Despite being one of the first class of organic caging groups reported to absorb >500 nm, surprisingly, xanthenes are yet to find significant use as PPGs – presumably due to the fact that they were not obtained as pure substances. It wasn't until 10 years later when Kele and Bojtár described the development of water soluble xanthenium-derived caging groups (compounds 121–123, Fig. 21), which unlike the earlier report, were isolated as pure substances.86 Using phenylacetic acid as the model payload, photo-release was achieved upon irradiation with 549 nm, 605 nm and 685 nm for rhodol 121, xanthenium 122 and carboxanthenium 123, respectively, with uncaging efficiencies ca. 1020 M−1 cm−1 (Table 7). The carboxanthenium-derived PPG was used to mask the inhibitory properties of the topoisomerase I inhibitor, SN38, via a self-immolative linker. Irradiation of the caged inhibitor 124 with red light (λirr = 650 nm) resulted in the decrease of the caging group and subsequent self-immolation to afford the active form of SN38.


image file: d5ob00542f-f21.tif
Fig. 21 Xanthene-derived caging groups 121–124 and their proposed uncaging mechanism.

While the uncaging mechanism of xanthenium-derived caging groups is yet to be fully elucidated, TD-DFT calculations, together with preliminary mechanistic studies suggests either a photo-induced SN1-type mechanism, whereby photoexcitation results in the formation of an ion-pair complex 127, or the formation of a radical pair 126 following irradiation and subsequent electron transfer to yield 128 is plausible (Fig. 21).

5. Summary and outlook

Since the early reports of their use in the 1960′s, photolabile caging groups have evolved into versatile light-responsive molecular tools for superior spatiotemporal control of biomolecular activity. The rapid growth and advancements in the field of photopharmacology over the past decade, has no doubt facilitated the discovery and, in turn, the development of new classes of caging groups with physiochemical and photophysical properties more amenable to in vivo applications. This includes the development of long-wavelength-activated caging groups, particularly those triggered with visible-to-NIR light, which has further enhanced their utility in biological systems by enabling deeper tissue penetration while reducing phototoxicity.

The BODIPY scaffold has proven to be a versatile platform for visible-light-activated PPGs, with the tuning of their absorption spectra to longer wavelengths and improvements to their uncaging efficiencies readily achieved through modest structural modification. Unfortunately, BODIPYs are inherently hydrophobic, posing challenges around solubility in aqueous media. While the inclusion of sulfonic acid has resulted in improved aqueous solubility, the degree of sulfonation has also shown to influence cell permeability. As such, further advancements in this space are warranted.

Although remarkable advancements have been made with the development of cyanine-derived caging groups as NIR-activated PPGs, many examples are either dependent on the presence of oxygen for photoactivation, or have competitive photooxidation/photoheterolytic cleavage uncaging pathways, restricting their utility in bio-relevant applications as a result. Furthermore, the lower energy excitation wavelengths result in short-lived excitation states, affording poor uncaging quantum yields. Detailed studies dedicated to increasing photolysis quantum yields are needed for this class of PPG.

Surprisingly, the versatility of xanthene-derived caging groups is yet to be fully exploited and holds much promise as red-light-activatable PPGs. Despite the substantial contributions made in the literature over the past 10 years, photocaging remains an active and evolving field of research, in which we can expect to see exciting advancements in the discovery of novel photocaging architectures, alternative uncaging mechanisms and new strategies for orthogonal activation to further improve biocompatibility and functionality in complex biological environments.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

PHL acknowledges funding from The Department of International and Cross-Strait Education of the Ministry of the Republic of China (Taiwan) and the University of Sydney. MRC and CLF acknowledges funding from the Health Research Council of New Zealand.

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