Solid-phase combinatorial synthesis using microarrays of microcompartments with light-induced on-chip cell screening

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Abstract

The process of drug discovery includes individual synthesis and characterization of drug candidates, followed by a biological screening, which is separated from synthesis in space and time. This approach suffers from low throughput and associated high costs, which in turn lead to inefficiency in the field of drug discovery. Here, we present a miniaturized platform combining combinatorial solid-phase synthesis with high-throughput cell screenings. The method is based on the formation of nanoporous poly(2-hydroxyethyl methacrylate- co-ethylene dimethacrylate) layers patterned with hydrophilic spots separated from each other by superhydrophobic liquid-impermeable barriers. The porous polymer inside the hydrophilic spots is used as a support to conduct solid-phase synthesis. The hydrophilic spots can be then filled with droplets containing either reagents for synthesis or live cells. Upon irradiation with UV light, products of solid-phase synthesis are released from the porous polymer because of the photo-cleavable linkers used and diffuse into separate droplets. The light-induced release of the products allows the control of the release spatially, temporally, and quantitatively. To demonstrate the versatility and usability of the platform for various cell lines, we have successfully implemented peptide synthesis to create an exemplary chemical library and demonstrated high cell viability after the UV-triggered small-molecule release.

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Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

Petr Klán, Tomáš Šolomek, Christian G Bochet … (2012)

1 Introduction Photoremovable (sometimes called photoreleasable, photocleavable or photoactivatable) protecting groups (PPGs) provide spatial and temporal control over the release of various chemicals such as bioagents (neurotransmitters and cell-signaling molecules), acids, bases, Ca2+ ions, oxidants, insecticides, pheromones, fragrances, etc. Following early reports on PPGs for use in organic synthesis by Barltrop, 1 Barton, 2 Woodward, 3 Sheehan 4 and their co-workers, applications to biology were sparked off by Engels and Schlaeger 5 and Kaplan and co-workers, 6 who first achieved the photorelease of cyclic adenosine monophosphate (cAMP) and ATP, respectively. The latter authors introduced the convenient, if somewhat misleading, term “caged” to designate a compound protected by a PPG. Two general perspectives 7 and many more specialized reviews covering applications of PPGs in synthesis, 8 biochemistry and neurobiology, 9 biomedicine, 10 volatiles release, 11 polymerization, 12 and fluorescence activation 13 have been published during the past decade, and a journal issue themed on these topics has recently been published. 14 The present review covers recent developments in the field, focusing on the scope, limitations, and applications of PPGs, which are used to release organic molecules. Photoactivation of small inorganic species and ions, such as NO, 15 CO, 16 Ca2+, 9h,17 Zn2+, 18 Cd2+, 19 or Cu+, 20 is not covered. Simplified basic structures of the photoremovable protecting groups discussed in this review are listed in Table 1 (the leaving groups are shown in red). Table 1 Photoremovable Protecting Groups The criteria for the design of a good PPG will depend on the application. No single system needs to fulfill all of the following requirements: (i) In general, the PPG should have strong absorption at wavelengths well above 300 nm, where irradiation is less likely to be absorbed by (and possibly cause damage to) the biological entity. 21 Moreover, the photoreaction should be clean and should occur with a high quantum yield or efficiency for release, Φ rel. The quantum yield Φ rel is equal to the amount of released substrate, n rel/mol, divided by the amount of photons at the irradiation wavelength λ, n p/mol = n p/einstein, that were absorbed by the caged compound: Φ rel = n rel/n p. An important measure for the efficacy of a PPG is the product of the quantum yield and the molar decadic absorption coefficient ε of the PPG, Φ relε(λirr), which is proportional to the amount of release at the given excitation wavelength. 22 (ii) Sensitive detection of the response under study often depends not only on the product Φ relε(λirr) but also on the background level of activity of the caged compound prior to irradiation. Hence, the PPGs must be pure, exhibit low intrinsic activity, and be stable in the media prior to and during photolysis. (iii) The PPGs should be soluble in the targeted media; they may further be required to pass through biological barriers such as cell membranes and show affinity to specific target components, for example, binding sites on cancer cells or the active site of an enzyme. (iv) The photochemical byproducts accompanying the released bioactive reagent should ideally be transparent at the irradiation wavelength to avoid competitive absorption of the excitation wavelengths. Moreover, they must be biocompatible, i.e., they should not react with the system investigated. (v) To study the kinetics of rapid responses to a released agent in samples such as brain tissue or single live cells, the PPG must be excited by a short light pulse and the appearance rate constant k app of the desired free substrate must exceed the rate constant of the response under investigation. Commonly, there are several reaction steps involving ground- and excited-state intermediates that precede the actual release of the free substrate. Therefore, detailed knowledge of the reaction mechanism is needed; in particular, the rate-determining step in the reaction path and its lifetime τrd or k app must be known, unless the appearance of the free substrate (k app) can be monitored directly by time-resolved techniques. Nitrobenzyl, nitrophenethyl compounds, and their dimethoxy derivatives (nitroveratryl) (section 3) are by far the most commonly used PPGs. The decay of their primary quinonoid intermediates on the microsecond time scale does not generally correspond to the rate-determining step of the overall reaction, and the release of the free substrate may be orders of magnitude slower. Moreover, photolysis of these compounds forms potentially toxic and strongly absorbing byproducts such as o-nitrosobenzaldehyde. Quite a number of alternative PPGs have been developed that do not suffer from these disadvantages. The appearance rate constant k app of the desired product is equal to the inverse of the rate-determining intermediate’s lifetime τrd, k app = 1/τrd, which often depends on the solvent as well as on the concentrations of acids and bases including those of the general acids and bases contained in buffers. Release rate constants, k r = ηr/τr, are sometimes quoted, where ηr is the efficiency of the releasing reaction step, ηr = k r /Σk, and τr = 1/Σk is the lifetime of the intermediate that is assumed to release the substrate; Σk includes k r and the rate constant of all competing reactions occurring from that intermediate. Note that the release rate constant k r may be higher or lower than the more relevant appearance rate constant k app of the desired substrate. Case (a): k r k r, i.e., if reactions other than substrate release contribute to the decay rate of the releasing intermediate. A trivial example of case (a) is shown in Scheme 1. Case (b): k r > k app if the actual release is preceded by a slower, rate-determining step of the reaction sequence. Scheme 1 Simple Case Where the Release Rate Constant of the Free Substrate (Leaving Group) X, k r, Is Smaller than Its Appearance Rate Constant, k app The speed of release is an ambivalent expression; it may refer to the efficiency of a PPG, Φ relε(λirr), the amount released by a given irradiation dose, or to the appearance rate constant in time-resolved work. The absorption spectra of a number of chromophores frequently encountered as PPGs are shown in Figure 1. Figure 1 UV spectra of selected chromophores. 23 (a) Benzophenone (ethanol; solid, red), 24 acetophenone (ethanol; dashed, blue); 24 (b) 1-acetyl-5-bromo-7-nitroindoline (acetonitrile; solid, red), 2-nitrotoluene (dashed, blue), (3,4-dimethoxy-6-nitrophenyl)methyl (nitroveratryl) derivative (acetonitrile, dashed, green); 25 (c) coumarin (acetonitrile, solid, red), p-hydroxyacetophenone (acetonitrile, dashed, blue), and benzoin (acetonitrile, dotted, black); (d) tris(bipyridyl)ruthenium(II) chloride (water). 26 2 Arylcarbonylmethyl Groups Aromatic ketones are thermally stable and synthetically readily accessible compounds; their photophysical and photochemical properties are well understood. The lowest energy transition of simple carbonyl compounds is typically a weak n,π* band (ε ≈ 10–100 M–1 cm–1). 27 The higher-energy π,π* absorption bands are strong, and internal conversion to the S1 state is very fast (e.g., ∼100–260 fs for acetophenone). 28 The electronic transitions of aromatic ketones are sensitive to solvent polarity and to substitution on the phenyl ring. Hydrogen bonding of protic solvents to the carbonyl oxygen stabilizes the oxygen nonbonding orbital, giving rise to a hypsochromic shift of the n,π* absorption band. Both electron-donating groups and polar solvents tend to stabilize the π,π* states. The strong bathochromic shift induced by para-amino substituents is attributed to a CT interaction 29 (for example, λmax for p-aminobenzaldehyde is ∼325 nm (π,π*) in cyclohexane). 24 Aromatic ketones are highly phosphorescent and only weakly fluorescent 30 due to their fast (>1010 s–1), very efficient intersystem crossing to the triplet state, for which two energetically close lying states (n,π* and π,π*) seem to play a crucial role, possibly due to a S1/T2/T1 intersection. 31 The singlet–triplet energy gap is much larger for π,π* than for n,π* states. The lowest π,π* and n,π* triplet states are nearly degenerate, and substitution on the phenyl ring as well as polar solvents may lead to triplet-state inversion. 27,32 Some of the important photophysical properties of acetophenone, the parent aryl ketone, are summarized in Table 2. Examples of the absorption spectra of other representative PPG aryl ketones are provided in Figure 1. Table 2 Photophysical Properties of Acetophenonea solvent E S/kJ mol–1 b τS/psc Φ f d Φ T e E T/kJ mol–1 f Φ p g nonpolar 330 30b 25 33 70 58 b methanol 0.09–0.20 58 b OC(=O)NR2 (amines) cyclohexane 0.054–0.089 59 b acetonitrile 0.035–0.070 59 97 59 b methanol 0.027–0.061 59 b a Appearance rate constant of the leaving group, calculated as k app = 1/τenol. b Slow, presumably 90% yield, free of any radical-derived decarboxylation products. 101 This confirmed the heterolytic pathway suggested by the groups of Givens, 85d,85e Falvey, 40 and Phillips. 99,100 The next layer of evidence on the photo-Favorskii mechanism arose from time-resolved transient absorption (TR-TA) studies, 85d which revealed two additional reactive intermediates: an early, very short-lived transient appearing on the tail of the triplet decay (Figure 5) and a later, long-lived species. The critical evidence for the first transient was obtained using pHP OTs (24, X = OTs), OMs, and DEP, all excellent leaving groups that depart efficiently and rapidly. For example, the transient formed from 3pHP DEP (lifetime, 3τ = 63 ps; in water) appears as a weak set of absorptions on the tail of the pHP triplet. These three maxima were assigned to an allyloxy–phenoxy triplet biradical (3 28, Scheme 19): the decay profile of 3 24 (3τ = 100 ps in 87% aqueous CH3CN) transforms into the profile of the slightly longer-lived transient 3 28 (τbirad = 500 ps). Scheme 19 Refined Mechanism Based on Time-Resolved Transient Absorption Analysis 85d The three weak absorptions of 28 were detected 85d only with the best leaving groups. The bands at 340, 430, and 440 nm were taken as evidence of a phenoxy radical intermediate and thus assigned to the biradical 28. As noted earlier, kinetic analysis of the ps-TR-RR spectra by Phillips and co-workers 85a,85b had suggested the intervention of an intermediate “M”, formed from the triplet state that proceeded to the final product 25. The intermediate “M” is now attributed 85d to the triplet biradical 3 28 that is assumed to be formed adiabatically. The formation of 3 28 can be viewed as being extruded from 3 24, leaving behind the leaving group X– and a proton in the ground state, thus obeying the Wigner spin rule. The fate of 3 28 is ISC and closure to an as yet undetected spirodienedione 26. The resulting traditional Favorskii-like intermediate very rapidly hydrolyzes to p-hydroxyphenylacetic acid, completing the formal ground state events normally proposed for the Favorskii rearrangement. 102 A further, long-lived intermediate was identified as the known p-quinone methide 103 29 (τ = 0.3 s) that hydrates yielding p-hydroxybenzyl alcohol (30). 85d The formation of small amounts of 30 is also a signature of the elusive spirodione intermediate 26, the lifetime of which appears to be shorter than its rate of formation under the reaction conditions. Thus, the validation of 26 is based solely on a requirement for the carbon skeleton reorganization and a very facile CO extrusion from 26 due to its strained bicyclic structure, and is complemented by DFT calculations. A summary of the photo-Favorskii mechanism, as currently understood, is outlined in Scheme 19. 104 The rearrangement proceeds from the chromophore’s triplet excited state (3 24) by a concerted departure of the leaving group and the phenolic proton generating the triplet biradical 3 28. Intersystem crossing of 3 28 gives an intermediate common to the ground state Favorskii rearrangement, 102a−102c the putative cyclopropanone, 86,98,102 that either hydrolyzes or decarbonylates on its pathway to the final products. The evidence provided, however, requires involvement of another intermediate, presumably the singlet allyloxy-phenoxy species 1 28, 98,99,104b to account for the complete racemization of a p-hydroxypropiophenone analogue where the leaving group is affixed to a stereogenic α-carbon. 104b This mechanism also provides a pathway for the minor photohydrolysis byproduct 24 (X = OH) that becomes predominant for the ring-contraction photoreactions of hydroxybenzocycloalkanonyl esters when ring strain discourages or prevents cyclopropanone formation. 98 Figure 5 Pump–probe spectra of pHP DEP (24, X = diethyl phosphate) in 87% aqueous CH3CN. The sample was excited with a pulse from a Ti/Sa–NOPA laser system (266 nm, 150 fs pulse width, pulse energy 1 μJ). The inset shows the species spectra of 3pHP DEP and biradical 3 28 that were determined by global analysis of the spectra taken with delays of 10–1800 ps using a biexponential fit. Reprinted with permission from ref (85d). Copyright 2008 American Chemical Society. Position and Requirement for a p-Hydroxy Group Unsubstituted phenacyl, which is also a PPG (section 2.1), does not undergo a photo-Favorskii rearrangement but rather reacts through a photoreduction mechanism. The p-OH modification of the phenacyl chromophore causes a profound change in the photochemical behavior. The search for alternative functional groups or other locations of the OH group on the phenacyl chromophore that would accommodate a Favorskii rearrangement pathway has met with very little success. Only 2-hydroxyphenacyl esters were shown to release carboxylic acids. 73 m-Hydroxyphenacyl acetate and a 5-hydroxy-1-naphthacyl analog were unreactive under photo-Favorskii conditions. 105 Electron donors such as p-methoxy and o-methoxyphenacyl have been tested as early as the seminal report of the photo-Favorskii rearrangement by Anderson and Reese. 87 For these examples, the photo-Favorskii rearrangement competes with photoreduction, forming mixtures of the corresponding methoxyacetophenones and phenyl acetates. In the early 1970s Sheehan and Umezawa developed the p-methoxyphenacyl derivatives as a PPG for photolysis in dioxane or ethanol, producing reduction products. 39,106 Givens and co-workers later showed that the reaction in methanol or t-butanol (hydroxylic solvents) did undergo the Favorskii rearrangement as the major pathway, yielding p-methoxyphenylacetates. The competing photoreduction pathway was also evident from the significant proportion of reduction to p-methoxyacetophenone. 107 Phillips and co-workers showed that p-methoxyphenacyl diethyl phosphate undergoes a rapid heterolytic cleavage that results in deprotection and formation of a solvolytic rearrangement product. 108 Other electron-donating substituents have met with even less success toward the rearrangement of the chromophore. 82 Although several p-methoxy and other p-alkoxy analogues have been successfully employed as PPGs for the release of carboxylates, 39,106,109 phosphates, 82,107 carbonates, and carbamates, 110 they do not lead to rearrangement of the chromophore. Nature of the Leaving Group The most efficacious leaving groups are conjugate bases of moderate to strong acids, e.g., sulfates, 85d,93 phosphates 7,9u,82−85,99 (thiophosphate), 100,111 carboxylates, 7b,9u,85a−85c,93−95,97b,97c,112 phenolates, 9u,93 and thiolates. 111,113 In general, quantum yields monotonically decrease with an increase in the acid leaving group’s pK a (Table 4), conforming to a Brønsted leaving group relationship (βLG), which correlates the pHP release rate constant with the K a of the leaving group. A correlation of the log of the rate constants (log k r), derived from the quantum yields and the triplet lifetimes as k r = Φ/3τ (see Table 4 footnote b for details), with the pK a of the leaving groups gave βLG = −0.24 ± 0.03. 9u,114 Table 4 Disappearance Quantum Yields, pK a’s, and Rates of Release for Different Leaving Groups (X) for pHP X (24) Arranged According to the pK a of HX X (released substrate) λmax (log ε)a (pHP X) pK a (HX) Φ –X b k r/108 s–1 c ref mesylate –1.54 0.93 50 (85d, 93, 114) tosylate –0.43 1.00 100 (85d, 93, 114) OPO3Et2 271 (4.18) 2.12 0.4 12. (82, 85a−85c, 96a, 99, 114) Glu 273 (3.94) 4.33 0.14 1.9 (83c, 97b, 97c) Ala·Ala 282 (4.12) 3.4 0.27 1.8 (83c, 112a) bradykinin 282 (4.07) 3.4 0.22 1.8e (83c, 112a) p-CF3C6H4CO2 – 3.69 0.2 3.2 (9u, 93, 114) formate 3.75 0.94 14 (9u) benzoate 4.21 0.32 2.8 (9u, 73, 85c, 93, 114) acetate 279 (4.09) 4.76 0.4 N/Af (85a−85c, 95, 96) GABA 282 (4.16); 325 4.76 0.2 6.2 (83c, 85e, 94, 97b, 97c) RPO3S– d 5.3 0.21 N/A (111) OPO3 –2 280 (4.48) 7.19 0.38 N/A (82) GTP 7.4 N/A N/A (91, 92) ATP 253 (4.3); 320 (2.70) 7.4 0.37 68 (82, 83c, 90) p-CNC6H4O– 7.8 0.11 0.76 (9u, 93) RS– d 8.4 0.085 N/A (111, 113, 115) C6H5O– 9.89 0.04 1.0 (9u, 93) HO– 15.7 315 nm). 179 Other interesting applications were developed in the preparation of otherwise inaccessible metal–organic frameworks, by inclusion of o-nitrobenzyl ethers and subsequent photolysis. 180 o-Nitrobenzylic PPGs have also been employed in natural product syntheses. One well-known and impressive application was reported by Nicolaou and co-workers in the total synthesis of the Calicheamicin γ1 where the high compatibility of 57 with other classical PGs and functions was demonstrated. 181 Another smart example is the use of 57 in the synthesis of an analogue of Leukotriene C4 (LTC4, Scheme 36). 182 In the final steps, deprotection by irradiation at 350 nm of the nitrobenzyl derivative affords the secondary amine in 74% yield. It is noteworthy that no isomerization of the triene part and no racemization were induced by photolysis. Scheme 36 Final Steps in the Total Synthesis of N-Methyl LTC4 182 Over the years, multiple modifications have been developed to tune the properties, mostly toward an increase in quantum yield, an increase in the rate of release, and increasing absorbance at longer wavelengths. Substitution at the benzylic site mainly affects quantum yields, whereas modification on the aromatic moiety affects the absorbance. Substitution at the Benzylic Position In addition to providing an electronic effect at the benzylic site, a second hydrogen-abstracting unit could, in principle, increase the efficiency. Already in the 1970s, Patchornik, Amit, and Woodward proposed this modification, which worked up to a certain point. 3 It should be kept in mind that most substitutions at this site create a chiral center, which can become a drawback if chiral molecules have to be protected (and that is the case for most of the relevant applications, such as amino acids, carbohydrates, and oligonucleotides), unless the added subsituent is identical to the aromatic core. A second o-nitrophenyl would fit in this category. However, an increase in photolysis quantum yields was not explicitly mentioned, and the increase in chemical yield might just be the consequence of the formation of a ketone instead of an aldehyde side-product, which is less prone to the parasitic imine formation. Thus, 59 was able to release carboxylic acids quantitatively and inorganic phosphate with 85% yield, 106a whereas 60 released amino acids in yields between 70% and 95% in the absence of a carbonyl scavenger. Various derivatives have been prepared since, among them 61 and 62, which liberated acetic acid in good quantum yields (Φ = 0.2 for 61), 168b or 63. 183 Despite the problem of chirality, a phenyl group can have a beneficial effect, as had been shown much earlier by Barltrop and co-workers with esters of 64 and carbamates of 65, 151 and was later used in linkers for solid-phase synthesis. 184 Simpler substituents, such as a methyl group, have a beneficial impact on the quantum yield. Thus, pivalate esters of 57 have release quantum yields in a polymeric matrix of 0.04 and 0.13 in acetonitrile solution, whereas they are much higher for pivalate ester 66 (Φ = 0.09 in polymer and 0.64 in acetonitrile). 185 Solution-phase quantum yields cannot be directly extrapolated to those of the solid phase, as the available conformations can be quite different. 186 Derivatives of 66 have many other uses, such as the release of biologically active carboxylic acids in plant cells, 187 or on solid support. 188 Specht and Goeldner proposed the introduction of a strongly electron-withdrawing group at the benzylic carbon, which had a massive effect on the quantum efficiency on choline and arseniocholine ether derivatives (Φ = 0.7 for 67 and Φ = 0.43 for 68) and sugar ethers (Φ = 0.62 for 67 and Φ = 0.52 for 68). Such derivatives were, however, not widely used in organic synthesis, as they were assembled by Mistunobu reactions, which were sluggish for secondary alcohols. Nevertheless, interesting applications, such as the release of choline or arsenocholine, were used as examples. 189 Jullien and co-workers prepared and evaluated the 6-nitroveratryl (NV, or 4,5-dimethoxy-2-nitrobenzyl, DMNB) derivatives 69, 70, 71, and 72 with electron-withdrawing substituents at the benzylic site; despite earlier claims, it was found that the substituents at the benzylic site, while having an unambiguously observable effect (up to a factor 3 between the most and least efficient), did not increase the quantum efficiency by orders of magnitude (Φ = 0.013 for −Br and Φ = 0.003 for −CN). 190 Interestingly, the cyano compound had been independently identified by a clever combinatorial technique by Pirrung and co-workers as being quite efficient. 191 Such apparent contradictions have been reported in the field of PPGs for decades and could potentially slow down future development of new or modified PPGs, as no clear trend emerges from the huge amount of sometimes unreliable data accumulated. The determination of quantum yields remains a delicate operation subject to significant experimental error; furthermore, all these different groups are frequently tested for the release of different leaving groups (phenols, carboxylic acids, amine, aliphatic alcohols, and carbamates) under different irradiation wavelengths, in different solvents and buffers, and each with a different photochemical apparatus. We have seen above the enormous importance that the effects of solvation and proton transfer play on the reaction mechanism. As mentioned for the benzoin PPG (see section 2.4), water-solubility is a crucial point for biological applications, and this property can be achieved by the introduction of a CO2H substituent. Hess and co-workers designed the α-carboxynitrobenzyl (α-CNB) derivative 73 for the release of phenolic OH groups; there was a significant increase in the release rate, but this was observed only at shorter wavelengths. 169,192 Bassani and co-workers recently reported that the α-carboxy-6-nitroveratryl (α-CNV) analogue 74 released carboxylic acids, in quantitative chemical yields with Φ = 0.17 and a rate of release 2–3 orders of magnitude larger than the parent compound, 193 and capsaicin through an ether bond. 194 A related strategy (based on 75) had been devised by Schaper and co-workers. 195 Thiols could also be released with the derivative 76. 196 A time-resolved infrared study of the photoreactivity of α-CNB 74 was performed by Corrie and co-workers. 197 Related groups were studied computationally by Schaper and co-workers. 198 Pirrung and co-workers designed a clever way of dealing with the nitroso side-product, responsible for the internal filter effect. It was trapped in situ by a Diels–Alder reaction with a diene function included at the benzylic site (Scheme 37). 199 These pentadienylnitrobenzyl PPG esters, ethers, and carbamates 77 were used to release carboxylic acids, alcohols, and amines (Φ = 0.22, 0.41, and 0.38, respectively); an analogue with a methylenedioxy group on the aromatic ring was also discussed, with improved properties (in this case, the increase in absorbance more than compensated for the usual decrease in quantum yields). Scheme 37 Trapping of the Nitroso Byproduct in a Diels–Alder Cycloaddition 199 Substituents on the Aromatic Ring of the o-Nitrobenzyl Chromophore Because of the problems of chirality, the synthetic issues, and the limited impact on the absorbance, considerably less work was devoted on substitution at the benzylic site than the very numerous modifications on the aromatic ring. Such modifications bring three features: (a) the tuning of the absorbance with concomitant effects on the quantum yield, (b) the possibility to anchor the group on a solid support or on a linker (this will not be discussed here, as a book chapter reviewed the field recently), 200 and (c) the possibility to modulate the solubility properties of the group. A second electron-withdrawing nitro group was added to increase the hydrogen-abstraction capacity (reminiscent of 59 and 60), and the release of amines from carbamates 78 was much more efficient than for 58 (Φ = 0.62 versus Φ = 0.13 in the solid state). 186 Interestingly, in the same study, the carbamate derivative of 66 showed a quantum yield of release of 0.11, which differs from earlier data. This was attributed to the influence of the matrix. The analogue 79 was also exploited a few years later. 187 Various groups have been added to increase the hydrophilicity of the cage again to address the water-solubility issue. As an example, the carboxylic acid 80 with a one-carbon spacer was reported by Allan et al. 187 Similarly, 81 and 82 were also exploited. 169b,201 Early on, two methoxy groups were added to increase the absorbance at longer wavelengths (λ > 350 nm, and Rayonet lamps at 420 nm still provide photolysis at reasonable rates), leading to the 6-nitroveratryl (NV, 83) and 6-nitroveratryloxycarbonyl (NVOC, 84) moieties. These are undoubtedly the most frequently used groups to date both in solution 3 and in the solid phase (for two recent, among many other, examples, see ref (202)). Multiple variants have been evaluated, such as the introduction of an α-methyl group at the benzylic site: the methyl-6-nitroveratryl (MeNV, 85) and methyl-6-nitroveratryloxycarbonyl (MeNVOC, 86) groups. Very close analogues, but with slightly altered properties, presumably due to a restriction in the freedom in aligning the nonbonding orbitals of the oxygen with the aromatic π system, are the methylenedioxy derivatives 87 and 88. Further addition of an α-methyl group led to α-methyl-(6-nitropiperonyloxymethyl) (MeNPOM, 89) and the now famous analogous 3,4-(methylenedioxy)-6-nitrophenylethoxycarbonyl (MeNPOC 90) groups. The latter moiety was used in the automated synthesis of DNA chips. 203 A solution-phase comparison of the quantum yield of release of thymidine derivatives of NVOC (Φ = 0.0013) and MeNPOC (Φ = 0.0075) was performed. 204 Caged nucleobases were used for the transient disruption of DNA hybridization to photochemically controlled DNAzyme activity. 205 This strategy is illustrated in Scheme 38 where the 89 (MeNPOM)-protected thymidine phosphoramidite is incorporated into DNA using classical synthetic procedures. The caged-DNAzyme thus obtained can be quantitatively reactivated by irradiation at higher wavelengths. MeNPOM presents several advantages compared to the parent compounds 73, 87, and 90: the very efficient conversion upon irradiation at 365 nm, the high stability of the caged molecules in aqueous media, the less toxic acetophenone byproduct, and its easy incorporation via the chloromethyl ether derivative. 206 Scheme 38 Preparation of Caged-DNAzyme and Activation by Nonphoto-Damaging UV Light 205 Inactivation of fluorophores (see section 11) was effected by similar caging, for example, by binding nitroveratryl groups to rhodamines and fluoresceins. 207 A recent computational study 198 was complemented by an experimental systematic evaluation of substituents on the aromatic ring and at the benzylic site 168b and showed surprisingly low influence of these groups (except for the strongly electron-releasing amine of 91, an effect that was reported earlier). Indeed, one of us reported a very significant lowering of the deprotection efficiency by having electron-releasing groups para to the nitro group, a manifestation of the CT-character of the excitation, as mentioned earlier. 208 This was exploited in a so-called safety-catch approach, where esters of 92, 93, 94, and 95 were protected against photolysis in normal conditions. Upon protonation with a strong acid, the reactivity was restored, thus allowing normal photolysis. 209 Similarly, the reactions of the compounds 96, 97, and 98, evaluated by Jullien and co-workers, were found to be less efficient than those of the nonsubstituted congeners (in fact, they were too slow to be measured in the case of 96 and 97). 190 The same authors also examined the effect of other groups, in para-position to the nitro group, with or without additional conjugation (99, 100, and 101). 190 As was the case for the benzylic substituent, these extra groups had a limited impact on the quantum yield, although the highly conjugated group in 101 decreased the reaction efficiency. The authors also explicitly pointed out an inverse correlation between the quantum yield and longer absorption wavelengths. Various other o-nitrobenzyl alcohol (e.g., 102 and 103) derivatives substituted in the ortho-position to the benzylic group, or meta-position to the nitro group, have been reported. 12b,185,208 The reaction efficiency of their ethers was tested in terms of kinetics of release, quantum yields in solution, or quantum yields in the solid state as a function of the irradiation wavelength for some of them. In an effort to increase the absorbance at longer wavelength, Singh and Khade extended the aromatic core as a naphthalene 210 or 7-methoxynaphthalene. 211 The corresponding esters 104 were able to release aromatic rings containing carboxylic acids at 380 nm, with Φ ranging between 0.08 and 0.16 (in acetonitrile–water). 210 The nitrodibenzofuran 105 also had very interesting properties (such as efficient deprotection), which were applied in recent examples. 164,165 The carbonyl group is among the groups most frequently in need of facile protection–deprotection protocols, so it is surprising that very few PPGs have been developed for it. Gravel and co-workers were among the first to address this issue, by first introducing the ketal 106 in 1974. 212 One issue was the generation of a mixture of diastereoisomers if the ketone to be protected was nonsymmetrical. The same group found later that the even simpler ketal 107 could circumvent the problem. 213 A solid-support version of 106 was developed. 214 Another way of dealing with the issue of chirality was proposed by one of us with the ketal 108, derived from the very easily prepared enantiopure (1R,2R)-bis(o-nitrophenyl)ethanediol, 215 which was used by Walsh and co-workers for the preparation of the otherwise too sensitive intermediate. 216 The substituted version 109 with a red-shifted absorption was proposed later. 217 On the other hand, for the release of simple aldehydes, it is not necessary to consume two aromatic-releasing units, and α-acetyl acetals have been shown to release aldehydes efficiently upon irradiation (Scheme 39). 218 Scheme 39 Photolysis of α-Acetyl Acetals 218 In a similar strategy, the o-nitrobenzaldehyde derived acetals 110 or 111 were used to protect 1,2- or 1,3-diols, principally for protection of saccharides. 219 However, in this case, only one alcohol can be deprotected, leading to photostable 2-nitrosobenzoic acid derivatives. Moderate to high regioselectivities were obtained during the deprotection, mostly determined by steric and electronic effects of the nonsymmetrical substrates (Scheme 40). 219a The final deprotection of diols could be achieved under basic conditions or by intracellular hydrolysis. A reverse strategy was developed by Iwamura et al. with the treatment of 111 by boron trifluoride in the first step to release an ether of 57, which was subsequently cleaved photochemically. Scheme 40 Selectivity in the Deprotection of 110-Protected 1,2-Diol 219a Two-Photon Absorption The two-photon irradiation technique (see also section 9) enhances the efficient temporal and spatial control of biomolecule delivery. Taking advantage of this for a biological application, Kao and co-workers reported that vanilloid derivatives of oNB (57) and NV (83) can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons with one-photon quantum efficiencies of 0.13 and 0.041 (Scheme 41). 220 In another example, retinoic acid derivatives of 72 and 83 were prepared and photolyzed, first at 365 nm. 221 The presence of a long-wavelength tail in the absorption spectrum does not prevent liberation, although isomerization of double bonds did occur. In the two-photon mode (excitation at 750 nm; 25 mGM) retinoic acid was also liberated from cage 57, and dynamic studies on zebrafish embryogenesis were carried out (vide infra). Scheme 41 Two-Photon Activation of Vanilloid Derivatives 220 An interesting alternative to the simultaneous absorption of two photons is the use of lanthanide-containing upconverting nanoparticles (UCNPs; see section 8.3). In this case, two near-infrared (NIR) photons are absorbed sequentially, and the nanoparticle re-emits one higher-energy photon, in a spectral range that can be exploited by PPGs. Such an example was recently shown by Branda, Zhao, and co-workers, where nitroveratrylester-containing micelles were dissociated upon irradiation with a continuous-wave NIR laser. 222 A similar strategy was also used to photolyze benzoin esters. 223 Isotopic Substitution As the transfer of hydrogen from the benzylic site to the nitro group is involved in the primary photochemical step, it was found that the rupture of the stronger C–D bond was less efficient. 224 The photolysis of the esters 57 bearing a perdeuterated benzylic position was 3.8–8.3-times less efficient (depending on the irradiation wavelength). The substituents on the aromatic ring in the analogous esters 83 diminished this isotope effect. The origin of these observations has not yet been fully elucidated; so far the phenomenon was utilized in the selective removal of two photolabile (oNB and NV) groups (Scheme 42 a and b depicts two different isotopically labeled derivatives). 225 The photochemistry of the oNB derivatives is summarized in Table 8. Scheme 42 Impact of Isotopic Substitution on the Photoreactivity 225 Table 8 Photolysis Quantum Yields for Nitrobenzylic Derivatives PPG X (leaving group) solvent (λirr/nm) Φ ref 57 (oNB) thymidine-OCO2 MeOH/H2O, 1:1 (365) 0.033 (226) 57 (oNB) t-BuCO2 MeCN (254) 0.13 (185) 61 CH3CO2 MeCN (254) 0.2 (168b) 66 t-BuCO2 polymer (254) 0.09 (185) 66 t-BuCO2 MeCN (254) 0.64 (185) 66 c-Hex-NHCO2 polymer (254) 0.11 (186) 67 choline-O buffer, pH 7 (364) 0.7 (189) 67 sugar-O buffer, pH 7 (364) 0.62 (189) 69 arsenocholine-O buffer, pH 7 (364) 0.43 (189) 69 sugar-O buffer, pH 7 (364) 0.52 (189) 70 coumarin-CO2 MeCN/buffer, pH 7, 1:1 (325) 0.013 (190) 71 coumarin-CO2 MeCN/buffer, pH 7, 1:1 (325) 0.011 (190) 72 coumarin-CO2 MeCN/buffer, pH 7, 1:1 (325) 0.003 (190) 73 PhCO2 EtOH/H2O, 1:1 (355) 0.17 (193) 77 PhCO2 CDCl3 (350) 0.22 (199) 77 BnO CDCl3 (350) 0.41 (199) 77 c-Hex-NHCO2 CDCl3 (350) 0.38 (199) 79 c-Hex-NHCO2 THF (254) 0.62 (186) 79 c-Hex-NHCO2 polymer (254) 0.13 (186) 83 (NV) thymidine-OCO2 MeOH/H2O, 1:1 (365) 0.0013 (204) 83 coumarin-CO2 MeCN/buffer, pH 7, 1:1 (325) 0.006 (190) 90 (MeNPOC) thymidine-OCO2 MeOH/H2O, 1:1 (365) 0.0075 (204) 104 RCO2 MeCN/H2O, 3:2 (>370) 0.08–0.16 (210) 3.2 o-Nitro-2-phenethyloxycarbonyl Groups In the late 1990s, Hasan and co-workers designed the oNB one-carbon homologue 112 (1-(2-nitrophenyl)ethyloxycarbonyl, NPEOC; “OC” stands for the −OC(=O)– group) and its α-methylated analogue 113 (NPPOC). 226 Interestingly, despite the structural similarity between 112/113 and 57, the release mechanism differs markedly, and the observation of a nitrostyrene side product is compatible with a photoinduced elimination (Scheme 43), 226 reminiscent to the photoelimination from ketones by photoenolization mentioned earlier (Scheme 11). It turned out that 112 eliminated a 5′-O-nucleoside carbonate (Φ = 0.042) with a higher quantum yield than that of its oNB analogue 57 (Φ = 0.033). The substitution of the benzylic center with a methyl considerably added to the efficiency (Φ = 0.35), which made 113 a candidate of choice for oligonucleotide synthesis. 227 This group was further modified to increase the absorbance, such as in the 2-(3,4-methylenedioxy-6-nitrophenyl)propoxycarbonyl (MNPPOC; 114) 204,228 or analogous 3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl (DMNPB, section 9) 229 derivatives, but 113 was applied in automated light-directed olgonucleotide synthesis (DNA-chips). 230 Amino acids 231 and carbohydrates were protected with NPPOC at the 6-position, including thiophenyl glycoside donors. 232 NPPOC derivatives are themselves relatively inefficient in two-photon deprotections; however, in the presence of sensitizers (section 8) (it has been shown by Wöll and co-workers that NPPOC can be sensitized), 166,233 the cross section can reach useful levels for several applications. 234 This is discussed in more detail in section 9. A very recent example of sensitization was shown by Winssinger and co-workers, where a thioxanthenone sensitizer is bound to a nucleic acid sequence, which is able to promote the photolysis only when hybridized with a NPPOC-containing sequence with the matching complementary bases. 235 Scheme 43 Photolysis of 2-Nitro-2-Phenethyl Derivatives 226 Further modification of the backbone may increase the two-photon absorption cross section (section 9), such as in the biphenyl derivative 115(236) or highly conjugated systems 116 and 117. 237 This has also been recently demonstrated by Goeldner and co-workers, who studied the photochemical properties of biphenyl-containing photoremovable protecting groups possessing the o-nitrobenzyl, phenacyl, and 2-(o-nitrophenyl)propyl functionalities. 238 Replacement of the methoxy substituent of 115 by an amino group increased significantly the two-photon cross section (section 9). 239 The high efficiency of NPPOC was exploited in many applications. 166,204,228,230−234,236,240 3.3 o-Nitroanilides Nitroanilide derivatives, such as 118, were shown in the 1970s to be photolabile and to release carboxylic acids (Scheme 44) and various byproducts depending on the conditions. 241 The early works concluded that the reaction is a rearrangement and not a solvolysis, as no isotopic incorporation was observed when the photolysis was carried out in H2 18O. Likewise, derivatives of 119 are also photolabile, but their cyclic nature prevents a direct H-abstraction on the amide alkyl substituent. 242 Failure to provide esters when the reaction is carried out in alcoholic solvents was an additional argument against a simple solvolysis. Scheme 44 Photoactive o-Nitroanilide Derivatives 241 Neither 118 nor 119 were widely exploited in synthesis, to the exception of the carbamate derivatives of 118 (R = O–alkyl, R1 = Me, R2 = H) that were used as a PPG for alcohols (Scheme 45). 243 Scheme 45 Use of o-Nitroanilide Carbamates as PPGs for Alcohols 243 The size of the fused nitrogen heterocycle proved to be crucial for the reactivity, and the 5-membered analogues of 119 (Scheme 44), such as 120 and 121 (R = alkyl), released carboxylic acids very efficiently upon photolysis. 244 Most remarkably, when the reaction was carried out in nucleophilic solvents, such as methanol or ammonia-containing dichloromethane, the primary amide was obtained. Corrie, Bochet, Toscano, and co-workers succeeded in providing a comprehensive picture of the reaction mechanism. 245 The mechanistic studies concluded that a photoinduced acyl migration from the indoline nitrogen atom of 122 to one of the nitro group oxygens occurs, leading to a highly electrophilic N–O–acyl intermediate 123 (Scheme 46). 245a,245b Scheme 46 Diverging Reaction Pathways in Organic Solvents and in Water 245a,245b Interestingly, this intermediate further reacts via two distinct pathways depending on the solvent. In water, deprotonation releases a carboxylic acid, with the concomitant formation of a nitrosoindole 124 (pathway b, Scheme 46), whereas in moist organic solvents, the acyl moiety undergoes a nucleophilic attack either by the solvent (water or alcohol) or by an external nucleophile (pathway a). Variation in the substitution pattern on the indoline aromatic ring can to a certain extent influence the ratio of the side-products. 246 These 7-nitroindolines release their protected function efficiently, 247 and tuning of the aromatic substituents at the C-4 and C-5 position has an impact on their reactivity. Thus, the acetamide 120 (R = Me) releases acetic acid with quantum yields of 0.06 and 0.12 in water and MeCN/H2O 99:1, respectively. 245a On the other hand, the reaction of 125 was about 2.5 times more efficient than that of 121 or 126, and 5 times more efficient than that of 127; 128 and 129 were totally photochemically inert, possibly due to a low-lying charge transfer excited state. 246,248 Corrie and co-workers also designed a clever combination of a nitroindoline core and an absorbing “antenna”, which will be discussed later (section 8.1). 249 The compounds 130(250) and 131(251) have recently been proposed as PPGs. The bromonitroindoline 120 was used in an early example of peptide synthesis as a protecting group for the C-terminus. 252 The solvolysis pathway was used to give the carboxamide by using an ammonia-containing solvent, a feature frequently sought in peptide synthesis. A few years later, a spectacular exploitation of this nucleophilic attack of the reactive intermediate was published by Pass, Amit, and Patchornik, with the use of a N-nucleophilic peptide fragment (Scheme 47). 253 Otherwise sensitive glycopeptides were assembled using this strategy. 254 Scheme 47 Peptide Coupling Using Acylated 5-Bromo-7-Nitroindolines (Bni) 253 More recently, the derivatives 121 were used in regular organic synthesis for preparing amides from amines, 255 but also by using weaker nucleophiles in the formation of thioesters from thiols 256 and esters from alcohols. 257 The preparation of more complex derivatives of 121 can be difficult, as the nucleophilicity of the indoline nitrogen is greatly reduced by delocalization through the two strong electron-withdrawing groups, but indirect routes were designed and the synthesis of a series of amino acids was recently published. 258 The availability of such photoactivable amino acids allowed the all-photochemical synthesis of peptides, by exploiting the different-wavelengths sensitivity of the indoline protecting group on the N-terminus. 259 For other application of this chromatic orthogonality, see section 10. Carbamates can also be prepared by photoinduced transfer of fluorenylmethyloxycarbonyl (Fmoc) 132 or benzyloxycarbonyl (Cbz) 133 derivatives. So far, the Boc analogues were significantly inferior (Scheme 48). 260 Scheme 48 Photochemical Introduction of the Fmoc and Cbz Groups 260 Other functional groups were released from nitroindoline derivatives; Hassner et al. published the release of alcohols from carbamates of 120 (R = OR), or amines from ureas of 120 (R = NR1R2). 261 The 7-nitroindolines found the applications in neuronal studies 262 and other physiological sciences. For example, derivatives of 125 were found to be active in releasing glutamate or GABA in two-photon absorption mode. 249e,263 In some instances, 7-nitroindolines proved to be inferior compared to nitrobenzyl cages. 264 In general, photoinduced acyl transfers are relatively rare, 255c and recent attempts to design groups with similar properties were only moderately successful. 265 4 Coumarin-4-ylmethyl Groups Photolysis of 7-methoxycoumarinyl-4-methyl derivatives, which established coumarins as a photoactivatable phosphate-releasing group, was first demonstrated by Givens with diethyl phosphate ester 134 (Scheme 49). 266 Scheme 49 Release of Phosphates from 7-Methoxycoumarin Derivatives 266 This discovery set the stage for the development of a new class of (coumarin-4-yl)methyl cages (Table 9). The attractive features of the coumarin phototriggers, such as large molar absorption coefficients at longer wavelength, and fast release rates, taken together with improved stability and fluorescent properties that provide a tag for convenient monitoring of the reaction course, have raised researchers’ awareness to this relatively new class of PPGs. 9f,9u,267 Clever modifications of the substituents at C6 and C7 in the coumaryl group addressed low water solubility, typical of the first-generation 7-alkoxycoumaryl PPG (types 135 and 136; Table 9), and also helped extend the absorption maximum of the group into the biologically benign range (≥350 nm). Thus, installation of an additional alkoxy group at C6 in the structure of parent 7-alkoxycoumarinylmethanol produced a shift of the absorption maximum by 30 nm. The 6-bromo-derivative was designed to lower the pK a of the 7-hydroxy group by two units to effect a complete deprotonation at physiological pH, thereby enhancing water solubility and causing a λmax shift of 60 nm. Introduction of the second-generation of coumarinylmethyl PPGs with 7-amino substituents (type 137; Table 9) further improved the spectroscopic and photochemical properties of the cage, moving the absorption maxima to 350–400 nm and recording the highest quantum yields (0.21–0.28) among the analogues. The problem of water solubility, which can be an obstacle to biological application, has been addressed by appending polar groups such as carboxylates to the aniline moiety. Polyaromatic analogues were also synthesized to improve fluorophoric properties of the cage for use both as fluorescent tags and as PPGs (type 136; Table 9). Comprehensive reviews by Furuta, 268 Dore, 269 and Loudwig and Bayley, 270 and more recently one by Givens, Rubina, and Wirz, 9u encompass much of the groundwork on the photochemistry of coumarylmethyl PPGs with an emphasis on biological applications. Important mechanistic aspects, synthesis, and several selected recent applications are given below. Table 9 Typical Coumarin Chromophores Applied as PPGs a Leaving groups are in red. b Solvent: HEPES/MeOH (i); HEPES buffer (ii); EtOH (iii); PB or PBS buffer (iv); KMOPS buffer (v); KMOPS/MeOH (vi); HEPES/MeCN (vii); and water (viii). Synthetic Approaches to Coumarin-Caged Compounds Coumarin-4-ylmethanol 138, easily available from the corresponding halides, is the common precursor of coumarin-caged esters, including phosphates, carboxylates, and sulfonates as well as carbonate, carbamate, and anhydride derivatives (Scheme 50). 9a Carboxylic acids, including amino acids, can also be efficiently caged via SN2 substitution of coumarinylmethyl bromide 139. Likewise, coumarin-4-methylamines and -thiols can be directly obtained from bromides 139 by standard alkylation. Coumarin-caged diols, e.g., acetals 140, were prepared in two steps from aldehyde 141, which in turn is available from the corresponding alcohol 138 by oxidation with manganese dioxide (Scheme 50). 295 Aldehyde 141 is also a convenient precursor to 4-diazomethylcoumarin 142, which has proven to be effective in protecting complex phosphates, particularly cyclic adenosine nucleotides, when standard approaches employing coumarin-4-ylmethanol 138 or coumarin-4-yl bromide 139 fail to provide the desired product. 271a,279a Scheme 50 Synthetic Approaches to Coumarin-Caged Compounds Mechanism of Photorelease and Selected Applications A general mechanism of photorelease of phosphates, sulfonates, and carboxylic acids, the most successfully applied leaving groups, is summarized in Scheme 51. 276,296 After initial light absorption, relaxation takes place to the lowest 1(π,π*) excited singlet state, which partitions between unproductive radiationless decay and fluorescence, and heterolytic C–X bond cleavage. The initially formed tight ion pair (coumarinylmethyl cation and leaving group conjugate base) is the key intermediate. The coumarinylmethyl cation either reacts directly with adventitious nucleophiles or solvent to generate a new stable coumarylmethyl product. Alternatively, the tight ion pair escapes the solvent cage and reacts with available nucleophiles. Although there is evidence of intersystem crossing occurring with 7-aminocoumarins, 284c there are no indications of triplet reactivity. Recombination of the tight ion pair regenerates the ground-state caged derivative, an additional nonproductive pathway. Time-resolved absorption studies have demonstrated that the heterolytic bond cleavage is very fast with rate constants reaching 2 × 1010 s–1 (measured for phosphate esters), among the most rapid photorelease rates for any caged compounds. 297 However, ion pair recombination dominates the subsequent reactions and is about 10 times faster than nucleophilic trapping of the coumarinylmethyl cation by solvent. 276,296 Evidence in support of SN1 cleavage at carbon, vis-à-vis a photosolvolysis pathway, was obtained using 18O-labeled water, which afforded labeled coumarinylmethanol and label-free phosphoric acid (Scheme 51). 297 Installation of electron-donating substituents on the coumaryl moiety and choice of a leaving group with low pK a values facilitate the reaction and, at the same time, impede ion pair recombination. 276 Scheme 51 Mechanism of Photorelease of Coumarin-Caged Compounds 297 Poor leaving groups such as alcohols, phenols, and thiols render (coumarin-4-yl)methyl derivatives resistant to heterolysis. Such groups can be more efficiently released when caged through a carbonate linkage with the (6-bromo-7-hydroxy/alkoxycoumarin-4-yl)methyl moiety 143 (X = O, S, Scheme 52). The initially liberated carbonic or thiocarbonic acid is unstable and undergoes decarboxylation to give free alcohol or thiol. 196,272,275,289 The rates of these decarboxylation reactions are usually quite slow, k –CO2 = 10–3 s–1, 298 and subject to both acid and base catalysis. Scheme 52 Decarboxylative Photorelease of Alcohols, Thiols, and Amines Analogous to alcohols, release of amines from coumarin-4-ylmethyl carbamates 143 (X = NR′′) proceeds uneventfully, although at a slower rate (Scheme 52). 272,288,290,299 As in the case of carbonates, the rate-limiting step is decarboxylation of the released carbamate anion, which is more strongly dependent on the pH and on the nature of the released amine or amino acid. 62,300 This could become a limitation for applications requiring a very rapid substrate release. Carbamate-linked coumaryl PPG was used by Hayashi, Kiso, and co-workers to mask the benzylamine moiety and suppress the pharmacophore activity in isotaxel (Scheme 53). 283 The resulting photoresponsive prodrug phototaxel (144) was selectively activated using visible light (430 nm) to release isotaxel (145), which converted into paclitaxel (146) by a spontaneous intramolecular O- to N-acyl migration. 283 To improve water solubility of the phototaxel prodrug, the N-ethyl groups of the coumarin cage were replaced with N-(2-(dimethylamino)ethyl)acetamide groups. 301 It should be mentioned that an alternative O-protected paclitaxel prodrug with a carbonate-linked coumarin was unstable in aqueous solutions. Scheme 53 Activation of Paclitaxel by Visible Light 283 Among the most significant advances in the photochemistry of coumarin-based protecting groups has been the discovery and development of two-photon activation (see section 9). 9h,302 Initially reported for the two-photon release from coumarin-caged substrates by Furuta, Dore, Tsien, and co-workers 303 this approach for decaging has developed into a major area of research and discovery. 9a,269 Block methylmethacrylate copolymer 147 esterified with the (7-diethylaminocoumarin-4-yl)methyl group exhibiting a large two-photon absorption cross section was used by Morris, Zhao, and co-workers to study light-responsive micelle disruption (Scheme 54). 304 UV irradiation of the micelles loaded with the hydrophobic Nile red dye revealed nonradiative energy transfer between the coumarin chromophore and the dye. Micelles without dye showed significant fluorescence self-quenching due to tight packing of the coumarin groups within a confined space; however, photorelease of the chromophore was accompanied by micelle disruption, which caused a dramatic increase in its fluorescence. Experiments with dye-loaded micelles placed in a dialysis cap immersed in water showed efficient diffusion of photoreleased coumarin into water (measured by fluorescence emission), while hydrophobic guest dye molecules remained within the cap. Multiphoton irradiation using near-infrared light was less disruptive for micelles yet sufficient to release loaded dye. 304 Scheme 54 Light-Responsive Micelle Disruption 304 An efficient multiphoton uncaging permitted exposure of the functional groups (primary amines and sulfides) buried within modified aragose gel 148, creating complex 3D sites ready for selective immobilization of biologically relevant entities without affecting the mechanical properties of the patterned material (Scheme 55). 299,305 Scheme 55 Selective Photoactivation of Functional Groups within Aragose Gel 299,305 A single example of photodeprotection of a coumarinylmethylamine through a C–N bond cleavage has been reported. This reaction proceeded efficiently only in the presence of an excess of a hydrogen-atom donor, such as n-decanethiol or 1,4-cyclohexadiene (Scheme 56). 306 A radical mechanism has been proposed that involved electron transfer between the amine and coumaryl moieties forming the intramolecular radical ion pair 149. Subsequent cleavage of the C–N bond generated an aminyl radical and the resonance-stabilized coumarinylmethyl radical 150, both of which were trapped by hydrogen-atom donors. Scheme 56 Uncaging of Amines via Direct C–N Bond Cleavage 306 When the released group is the conjugate base of a very strong acid, the caged substrate becomes a “caged proton source” that permits the spatial and temporal control of rapid change in pH. Thus, the release of phosphoric acid, methanesulfonic acid, or sulfuric acid derivatives from their coumarin-caged precursors 151 has been employed as a proton trigger in studies of proton-dependent cellular signal transduction (Scheme 57). 307 The release occurred within 2 ns, giving a significant drop in the pH of up to three units. It was also noted that hydrophobic phosphate derivatives penetrate cell membranes whereas charged sulfate was membrane-impermeant. Scheme 57 Caged Proton Source: Photorelease of Strong Acids 307 Photorelease of diols from cyclic (coumarin-4-yl)methyl acetals 152 proceeds via an ion-pair intermediate 153, which then reacts with water to produce the hemiacetal 154. The latter decomposes to give free diol and coumaryl aldehyde 155 (Scheme 58). 308 Interestingly, the photodegradation of coumaryl-caged diols was found to be dependent on the size of the cyclic acetal. Thus, 1,2- and 1,4-diols were released equally efficiently (Φ = 0.005–0.03), whereas the corresponding 1,3-diols appeared to be completely inert to photolysis. Although different stereoelectronic properties of the three acetals precluded direct comparison of the propensity toward ring-opening, 308 it is probable that the divergent quantum yields result from lack of recyclization because of the increasing ring strain for 5- and 7-membered acetals (by ca. 6 kcal mol–1) compared with recyclization to the “strainless” 6-membered analogue. 309 Scheme 58 Photorelease of Coumarin-Caged Diols 308 A complementary approach for protecting carbonyl compounds in the form of a photolabile cyclic coumaryl acetal has also been demonstrated (Scheme 59). 287,291 Remarkably, both single- and two-photon uncaging (section 9) of 4-coumarin-4-yl-1,3-dioxolanes 156 were successfully achieved under physiological conditions. In line with the above example, the proposed mechanism involved photoinitiated heterolysis and subsequent solvolysis of the zwitterionic species 157. Scheme 59 Photorelease of Coumarin-Caged Carbonyl Compounds 287,291 Caged carbonyl compounds 158 were obtained from (coumarin-4-yl)methyl chloride (159) by Wittig olefination followed by dihydroxylation of the resulting alkene with OsO4 and subsequent acetalization or ketalization under standard conditions (Scheme 60). 291 Alternatively, caged progesterone 160 was synthesized by Hagen and co-workers via ketalization of the commercially available pregnenolone 161, followed by Oppenauer oxidation of intermediate 162 (Scheme 61). 310 Photoactivation of coumaryl-caged progesterone produced 163 in only 30% yield, which, however, was sufficient to initiate progesterone-mediated response in sperm. Two-photon excitation of 160 (755 nm) also caused release of progesterone, albeit with ca. 5 times lower efficiency compared with photolysis of the analogous glutamate derivative. 301 Scheme 60 Synthesis of Coumarin-Caged Carbonyl Compounds 291 Scheme 61 Photoactivation of Coumaryl-Caged Progesterone 310 5 Arylmethyl Groups 5.1 Simple Arylmethyl Groups A large family of photolabile protecting groups is based on photochemically induced hydrolysis (or less commonly alcoholysis) of benzyl or heterobenzyl esters or ethers 164 (Scheme 62). Scheme 62 Photochemical Cleavage of Benzyl Protection In fact, photochemical release of glycine from its benzyloxycarbonyl derivative was the first application of a “photosensitive protecting group” in organic chemistry. 1 Barltrop and Schofield reported that irradiation of N-benzyloxycarbonylglycine 165 with 254 nm light resulted in the release of glycine in 75% yield and a quantum yield of 0.15 (Scheme 63). Photolabile protection of amino acids and dipeptides was further demonstrated by Chamberlin, who used the 3,5-dimethoxybenzyloxycarbonyl protecting group (166). 311 Substrates were released in up to 85% yield upon illumination with a high-pressure mercury lamp for 1.5 h. Scheme 63 Uncaging of Glycine 1,311 From a mechanistic point of view, this photosolvolysis can proceed via initial light-induced homolysis of the C–O bond, followed by rapid electron transfer to give an ion pair (Scheme 64). The latter is then trapped by solvent to give the final products. Pincock and co-workers have shown that this mechanism is predominant in the photolysis of simple benzyl derivatives. 101 In fact, photosolvolysis of benzyl esters is often accompanied by the formation of radical products. 312 Scheme 64 Photochemistry of Arylmethyl Ethers Calculations by Zimmerman and co-workers 312a,313 suggested that heterolysis of the C–O bond is energetically preferred over the homolysis, especially in the presence of ortho and meta substituents stabilizing the cation. While studying the photochemical solvolysis of benzyl acetates, Zimmerman observed the now famous “meta-effect”: a higher efficiency of photocleavage for meta-methoxy-substituted benzyl esters. Structural features that help to delocalize positive charge in the ground state enhance the efficiency of the electron-transfer step (Scheme 64), whereas excited-state cation-stabilizing moieties promote the direct photoheterolysis of the C–O bond. The efficiency of the uncaging reaction depicted in Scheme 64 is defined by the competition between solvent capture of the ion pair to give the liberated hydroxyl compound ROH and recombination leading to the starting material (polar protic solvents enhance both the heterolytic cleavage and electron transfer). Therefore, photodeprotection of carboxylates, carbonates, carbamates, phosphates, and other good leaving groups is usually quite efficient, whereas uncaging of alcohols is more challenging. Enhanced stability of the benzyl cation and increased steric hindrance around the cationic center also help to slow down the ion recombination. Thus, the design of new PPGs of this class was mostly focused on the incorporation of features helping to delocalize positive charge. However, there is a pitfall associated with this approach: a very stable cation makes the PPG susceptible to acid-catalyzed hydrolysis. In fact, the conventional acid-labile trityl protecting group can be removed from caged nucleosides (167) in 71–99% yield upon 254 nm irradiation with ca. 10% quantum efficiency (Scheme 65). 314 Scheme 65 Photocleavage of the Trityl PPG 314 Wang and co-workers systematically explored various trityl derivatives and found that the 3-(dimethylamino)trityl group (168, DMATr, Scheme 65) is the most efficient alcohol-caging group in the trityl series (Φ = 0.2). 315 Introduction of a strong electron-donating meta-substituent in one of the trityl rings improves the photochemical reactivity due to the meta-effect, stabilizing the PPG derivative to hydrolysis in the dark. This PPG has a relatively long absorption wavelength (λmax = 309 nm), is stable in acidic media, and releases alcohols with 0.12–0.20 quantum yield efficiency. DMATr can be easily installed by simple solvent-free heating of the DMATr acetate with the alcohol. Several alcohols, including sugars and thymidine, have been protected and released upon irradiation with a medium-pressure mercury lamp in methanol with 80–90% yields. DMATr is also orthogonal to conventional methoxytrityl protecting groups, as it can be removed by irradiation in methanol, whereas the methoxytrityl protection is cleaved upon treatment with 80% acetic acid. A similar α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl PPG (169, Ddz, Scheme 66) was used by Cameron and Frechet for caging of amines. 316 Irradiation of these thermally stable carbamates with a medium-pressure mercury lamp results in liberation of free amines both in THF solution and in the solid state. Scheme 66 Uncaging of Amines 316 The stability of 9-phenylxanthylium cation prompted the development of the 9-phenylxanthyl (170, pixyl, Px) PPG for primary alcohols (Scheme 67). 317 Scheme 67 Protection and Photochemical Release of Primary Alcohols 317 The pixyl PPG is readily introduced in 65–74% yield by treating alcohols with commercially available pixyl chloride in the presence of pyridine. Irradiation of the pixyl ethers at 254 or 300 nm results in the release of the alcohol in 78–97% yield. On the basis of deprotection of the nucleoside thymidine, the authors suggested that Px could be an effective protecting group for the combinatorial synthesis of oligoribonucleotides. The 9-phenylthioxanthyl group (171, S-pixyl, S-Px, Scheme 68) was found to be an even more efficient cage for primary alcohols, as it absorbs at longer wavelengths and has a higher quantum yield of deprotection. 318 It was also shown that i-butyryl and benzoyl protection of amino groups in nucleobases is not affected by the photolysis. The chemical yield of deprotection varied from 75% for cytidine to 97% for thymidine. Scheme 68 Use of the S-Pixyl PPG for Caging and Release of Nucleosides 318 Further studies of the substituent effects on the photochemical release of thymidine from the S-Px cage showed that an electron-donating group in the 3-position enhances the efficiency of the photolysis by a factor of 3. 319 Moreover, some of these compounds proved to be suitable for deprotection under irradiation with 350 nm light. For example, 5′-O-(3-methoxy-9-phenylthioxanthyl)-2′-deoxythymidine (172, Scheme 68) quantitatively releases thymidine by irradiation at 300 or 350 nm. Direct photosolvolysis of ethers, such as 167, 168, and 170–172, has great advantage for alcohol caging over the use of photolabile carbonates. Substrates are released directly from the excited state of the photocage within a few microseconds in the former case, whereas decarboxylation of photochemically generated monocarbonate in the latter case is a much slower process. Photosolvolysis of arylmethanol derivatives has also been used for the deprotection of carbonyl compound components of photolabile acetals. Wang and co-workers developed a photoremovable protecting group for carbonyl compounds based on trityl photochemistry. 320 Ketones and aldehydes are converted into cyclic acetals (173) by treatment with 5-methoxysalicylic alcohol derivatives (Scheme 69). Upon irradiation, the carbonyl compounds are released in 75–90% yield and good quantum efficiency (Φ = 0.11). The authors proposed that this reaction proceeds via a two-step mechanism involving a zwitterionic intermediate. However, it is also possible that initial photocleavage of the benzylic C–O bond produces a hemiacetal, which undergoes further hydrolysis. This cage is remarkably stable to a variety of reagents including metalloorganic (PhLi, LiAlH4, and t-BuOK), strong acids (AcOH, TFA, and concentrated HCl), and mild oxidants (DDQ). Besides, Thevenet and Neier have recently studied photorelease of alcohols from the corresponding naphthalen-2-ylmethylidene and arylethynylmethylidene acetals. 321 Scheme 69 Photolabile Acetals for the Protection of Ketones and Aldehydes 320 The methoxy substituent in 173 was strategically positioned to exploit Zimmerman’s meta-effect. 312a,313 The second methoxy group in 174 makes photoheterolysis of benzylic C–O even more efficient, as noted earlier. A series of carbonyl compounds were protected in the presence of p-toluenesulfonic acid 322 or in the neat state and with no added catalysts at 140 °C 323 and then released (Φ = 0.17–0.23) 320c,320e from the photocage 174 upon irradiation with a 450 W medium-pressure mercury lamp in aqueous acetonitrile. Both protection and uncaging proceeded in 74–99% yields. Introduction of electron-donating or -withdrawing substituents in the para-position of the phenyl substituents in 174 allows for the adjustment of λmax of the chromophore. 320c The majority of trityl- and benzyl-based PPGs require irradiation with light of rather short wavelength to achieve efficient substrate release. To shift the caging chromophore absorbance to longer wavelengths, the photocleavage of several polyaromatic analogues of the benzyl protecting group, including (2-naphthyl)methyl, 324 (anthracen-9-yl)methyl, 325 (pyren-1-yl)methyl (175, Scheme 70), 326 (perylen-3-yl)methyl, 327 and (phenanthren-9-yl)methyl, 326a−326c was explored. Scheme 70 Photocleavage of the Fluorescent (Pyren-1-yl)methyl Protecting Group 326 These cages have significant absorbance at 350 nm and show weak to moderate fluorescence (Φ fl = 0.01–0.2). Polyaromatic PPGs are well-suited for release of very good leaving groups, such as phosphates (175a). Uncaging of carboxylic acids, on the other hand, is less reliable, and alcohols are released with 280 92 0.17 (315) 176 (Aqm) carboxylate, alcohol (as a carbonate), carbonyl 330 350 ∼80 ∼0.1 (324,326d) 181 (BHQ) carboxylate, phosphate, carbonyl 369 365 ∼90 0.04 (330a, 331, 335) 184 (NQMP) alcohol, phenol, carboxylate 307 300 or 350 72–100 0.1–0.3 (338) 186 (DHB) alcohol, phenol, carboxylate, carbonyl, glycol 297 300 60–100 0.1–0.3 (339, 340) 194 carboxylate, alcohol 438 >410 94–97 0.072–0.093 (327) 6 Metal-Containing Groups Metal-containing photoremovable protecting groups upon irradiation have been shown to release coordinated metal ions, gaseous inorganic molecules, such as NO or CO, or organic molecules, often of biological interest. This innovative research topic has recently been reviewed by Haas and Franz, 347 Schatzschneider, 9o Ciesienski and Franz, 9t and Schiller and his co-workers; 10b thus, it is not covered comprehensively here. These cages are metal complexes that undergo a change in the metal coordination environment upon single-photon or multiphoton excitation. Several types of organic ligands, such as amines, nitriles, azides, or thioethers, have been shown to be released. Upon irradiation with visible light above 480 nm, the most common [Ru2+(bpy)2]2+ core (195; an amine is a leaving group in this case) liberates various nitrogen atom-containing ligands (Scheme 82), such as 4-aminopyridine, serotonin, butylamine, tryptamine, tyramine, 348 γ-amino butyric acid, 349 glutamate, 350 or azide. 351 One of the amino group-containing ligands has also been replaced by triphenylphosphine to increase the release quantum yield. 349b Etchenique and collaborators have reported that ruthenium–bipyridine-based caged nicotine ([Ru(bpy)2(nic)2]2+ where nic = nicotine) is released with the quantum yield Φ = 0.23 upon irradiation with blue (473 nm) or green (532 nm) light. 352 Gobetto, Sadler, and co-workers have used spectroscopic and DFT computational methods to study the release mechanism. 353 They showed that the photochemical activity is related to the presence of singlet and triplet transitions involving σ-antibonding orbitals and identified and characterized the triplet state responsible for the photodissociation process. 353a Excitation of the metal–ligand-to-ligand charge-transfer (MLLCT) band of the [Ru(bpy)(4AP)4]2+ complex (4AP = 4-aminopyridine) was found to provide selective photodissociation of two 4AP moieties in two consecutive steps with quantum yields of approximately 6 × 10–3 and 2 × 10–4, respectively. 353c The photorelease of an aliphatic amine from a ruthenium–bipyridine-based PPG attached to silica surfaces has recently been shown to occur upon single-photon (460 nm) and two-photon (900 nm) excitation (section 9). 354 A caged glutamate can also be liberated upon single- and two-photon activation from a Ru complex. 355 In addition, a photoactivatable fluorescent probe 356 as well as caging peptidomimetic inhibitors 357 are recent examples in which a ruthenium–bipyridine-based PPG releases a nitrile ligand. Thioether ligands, such as thioether-cholestanol hybrid, 358 N-acetylmethionine, 359 or biotin, 359 can also be liberated from the [Ru2+(bpy)2]2+ core upon irradiation with visible light. Scheme 82 Photorelease from the [Ru2+(bpy)2]2+ Cage 7 Miscellaneous Groups 7.1 Pivaloyl Group Initially used as part of a photolabile linker for solid-phase synthesis, pivaloyl derivatives such as 196 fragment by a Norrish-type I mechanism equally well in solution, releasing carboxylic acids or alcohols (Scheme 83). 360 The carbonyl group might seem to be a nuisance due to cross-reactivity, but these pivaloyl esters show remarkable stability under typical synthetic conditions (such as acids, bases, or transition-metal catalysts). The side-products are all volatile: carbon monoxide, isobutene, and acetone, and the quantum yield of release is quite high (Φ = 0.56). They require, however, the use of a relatively short-wavelength source (280–340 nm), which can be problematic when dealing with biomolecules. Scheme 83 Photolysis of Pivaloylglycol Derivatives 360 7.2 Esters of Carboxylic Acids The 2-benzoylbenzoic acid moiety (197) has been utilized as a PPG for primary and secondary alcohols, or thiols (Scheme 84). 361 This chromophore exhibits photochemical reactivity typical for benzophenone, such as photoinitiated H-atom or electron transfer to the excited carbonyl group. Alcohol release is accompanied by the formation of a dimeric side-product 198 in the presence of an H-atom donor, whereas the isobenzofuranone derivative 199 is obtained by photoreduction with, for example, amines. Scheme 84 Photochemistry of 2-Benzoylbenzoic Acid Esters 361 2-Benzoylbenzoic acid derivatives were utilized as photoreversible inhibitors of serine proteases by Jones and Porter. 362 Various esters 200 served as efficient chymotrypsin (a digestive enzyme) inhibitors (Scheme 85). The synthesized acyl-chymotrypsins were found to be stable to hydrolysis from hours to months; a sharp increase of enzyme activity was then observed upon irradiation at 366 nm: up to 80% of the preinhibition activity was recovered. The corresponding 2-aroylbenzoic acid was formed as a side-product; Scheme 85 shows one of the proposed reaction mechanisms. Scheme 85 Chymotrypsin Photorelease 362 A photofragmentation reaction of xanthenoic esters has been shown to give lactones of various ring sizes. 363 The initial homolytic O–C bond cleavage of the esters 201 leads to xanthene and formyl radicals, which rearrange to lactones 202 and 203 in moderate to low chemical yields (10–55%; Scheme 86). The corresponding alcohols 204 and formates 205 were isolated as side-products. Scheme 86 Photofragmentation of Xanthenoic Esters 363 A water-soluble visible-light absorbing (ε400 > 9000 M–1 cm–1) PPG for carboxylic acids based on the aminonitrophenyl chromophore 206 has been demonstrated to photorelease β-alanine (207), which then activates the inhibitory glycine receptor in the mammalian central nervous system (Scheme 87). 364 β-Alanine was found to be released in 5 μs with rather low quantum yields of Φ = 0.03 and 0.002, when irradiated at λ = 360 or 450 nm, respectively. Scheme 87 Liberation of β-Alanine from the Aminonitrophenyl Chromophore 364 7.3 Arylsulfonyl Group The photochemical generation of acids (photoacids) is an important strategy in the development of coating and imaging technologies as well as the synthesis of polymers. 365 In such a process, the acids are typically photochemically produced from relatively small amounts of an excited initiator to start a chain reaction. However, this topic is beyond the scope of this review; here we only cite a few recent reports 366 as examples. Arylsulfonyl ester (Scheme 88) or amide photoacids are also frequently used as PPGs (e.g., ref (367)). For example, this strategy was utilized for dopamine release from a photocleavable biotin linker 368 or for reactivation of a benzenesulfonyl-caged Zn2+ tetraazacyclododecane complex. 369 Acid (proton) photorelease has been used to study proton binding in the sarcoplasmic reticulum. 370 Scheme 88 Photochemistry of Arylsulfonyl Esters 7.4 Ketones: 1,5- and 1,6-Hydrogen Abstraction A considerable focus has been devoted to the development of photochemically releasable volatile odoriferous compounds—fragrances. Such applications have been the subject of several recent reviews. 11,371 Aldehydes and ketones are important classes of fragrance molecules. Herrmann and co-workers have shown that protected carbonyl compounds can be photoreleased from α-keto esters (208) via the Norrish Type II photofragmentation under mild conditions in the presence of air (Scheme 89). 372 The initially formed 1,4-biradical reacts with air oxygen followed by the release of the aldehyde or ketone 209, carbon dioxide, and the corresponding carboxylic acid. In the absence of oxygen, 208 can also undergo β-cleavage to give 209 and a ketene intermediate. Scheme 89 Photochemistry of α-Keto Esters 372 Photochemically triggered hydrolysis of protected volatile aldehydes or ketones has also been demonstrated using 1,5-diketones with abstractable γ-hydrogen 373 or 1-alkoxy-9,10-anthraquinones, e.g., 210 (Scheme 90). 374 The reaction starts with 1,6-hydrogen atom abstraction followed by electron transfer to form the zwitterion 211, which is trapped by an alcohol. The final fast hydrolytic step liberates the protected compound 212. Scheme 90 Photochemistry of 1-Alkoxy-9,10-anthraquinones 374 7.5 Carbanion-Mediated Groups Another type of PPG relies on the photochemical formation of a carbon-centered anion, which drives the subsequent release of a leaving group via β-elimination (Scheme 91). This strategy, developed by Scaiano and co-workers, 375 is often based on the photochemical decarboxylation in the side-chain of a chromophore and is compatible with an aqueous environment. Benzophenone (LG = carboxylate; Φ 500 nm) were shown to mediate electron transfer between dithiothreitol, a good electron donor, and an N-methylpicolinium ester in aqueous solution with quantum yields in the range between 0.5 and 4.5, suggesting involvement of a radical chain mechanism. 434 Ketocoumarin dyes were also used in a similar application. 435 Tris(bipyridyl)ruthenium(II) as both a photosensitizer and a mediator for electron transfer between a good electron donor and the newly synthesized water-soluble 2-cyanopicolinium protecting group was recently demonstrated to result in the release of various carboxylic acids. 436 Scheme 118 Photorelease via Mediated Electron Transfer Falvey and co-workers then exercised the same principle to control the viscosity of aqueous solutions of 251 and cetyl trimethylammonium bromide by using visible light to initiate sensitized photorelease from the picolinium group and UV light to control photoisomerization of the attached cinnamic acid (Rubpy = tris(bipyridyl)ruthenium(II) as a sensitizer; Scheme 119). 437 The irradiation protocols are thus conveniently orthogonal to one another. Scheme 119 Orthogonal Photorelease and Photoisomerization 437 Visible-light-absorbing tris(bipyridyl)ruthenium(II) has also been recently used to mediate electron transfer to N-methylpicolinium carbamates (252) to give free amines in several steps (Scheme 120). 438 Another application of a visible-light photoredox catalyst, such as Ir[dF(CF3)ppy]2(dtbbpy)PF6, in the oxidation of electron-rich arenes resulting in the selective deprotection of p-methoxybenzyl ethers in 69–91% yields has been demonstrated by Stephenson and collaborators. 344a 2,3-Dichloro-5,6-dicyano-p-benzoquinone as an electron acceptor has been used for deprotection of various benzyl ethers by Toshima and collaborators. 344b In addition, Lechner and König have shown that flavin photocatalysis can be used as visible-light-absorbing sensitizers in the deprotection of benzyl amines. 345 Scheme 120 Visible-Light Deprotection of N-Methylpicolinium Carbamates 438 Benzyl ethers can release alcohols through oxidatively sensitized irradiation; 439 however, no additional reports have been published in the past decade. In a different strategy, Tu and Floreancig demonstrated the protection of carbonyl and carboxyl compounds, which can be released by intramolecular photoinduced electron transfer from a phenethyl alcohol/ether group to covalently tethered anthraquinone (as a sensitizer) in 253 according to Scheme 121. 440 The reported chemical yields of the deprotection were between 6 and 97%. Scheme 121 Intramolecular Sensitization of the Phenethyl Alcohol/Ether Group 440 Cossy and Rakotoarisoa have discovered that the 2-acetoxyethyl group can be used to protect secondary amines. 441 The deprotection is facilitated by photoinduced electron transfer from the acetoxyethyl derivative 254 to the dimethoxybenzophenone sensitizer 255 via a cation radical intermediate and the iminium salt 256, which subsequently hydrolyzes to release the secondary amine 257 in 60–80% chemical yields (Scheme 122). Scheme 122 Sensitization of Acetoxyethyl Derivatives 441 A blue-light triggered photorelease system based on the riboflavin-binding protein dodecin (a dodecameric hollow-spherical flavoprotein with six flavin binding sites) has recently been developed by Noll and co-workers. 442 The release of the reduced flavin derivatives is reported to be triggered by irradiation with blue light of the dodecin-bound flavin anchor. Kutateladze and co-workers have developed a novel strategy of aldehyde and ketone (258) protection using the dithiane moiety, which can be efficiently removed via oxidative photoinduced electron transfer in the presence of a sensitizer (sens), such as benzophenone (Scheme 123). 443 The starting compounds 259 are easily synthesized from the lithium salts of 1,3-dithianes and the corresponding carbonyl compounds. Upon irradiation, the carbonyl compounds are released in excellent to mediocre chemical yields (e.g., 35–97%) 443 and with reasonable efficiency (Φ = 0.06–0.17). 444 Scheme 123 Photochemistry of Dithiane Moiety 443 A simplified mechanism of the oxidative deprotection is shown in Scheme 124. The reaction of 260 is facilitated by electron transfer to the excited state of benzophenone, resulting in formation of the cation radical 261 and the benzophenone radical anion 262. Depending on the solvent polarity, 262 deprotonates 261 in the solvent cage, or both species escape the solvent cage and form the same compounds through the reaction with water. It was evident from LFP studies that in-cage deprotonation occurs in dry acetonitrile whereas the latter process is dominant in more polar acetonitrile/water mixtures. 445 Deprotonation of the radical cation of an adduct of dithiane and, for example, a t-butyl-substituted carbonyl compound gives the zwitterionic intermediate 263, which may, following intramolecular electron transfer, lead to an alternative C–C bond scission to give a stable t-butyl radical. 446 However, the detailed mechanism is still not completely clear. 447 Adducts of trithianes and trithiabicyclo[2.2.2]octanes, analogous to those of 1,3-dithianes, have also been studied as protection moieties. 448 In addition, the dithiane moiety was successfully coupled with amino acids to form novel dithiazane photocleavable linkers. 449 9H-Thioxanthen-9-one has also been introduced as a sensitizer in the dithiane-, trithiane-, and dithiazane-based photolabile scaffolds. 450 Scheme 124 Mechanism of Oxidative Deprotection of Dithiane Moiety 445,446 The dithiane-based linkers were explored for various applications, such as photolabile phospholipids and amphiphiles, 451 calixarene-based rosette, 452 or barbiturate receptors. 453 Dithiane-spiro-crown ethers (e.g., 264; Scheme 125) were used as photolabile moieties, which allowed photochemical interruption of transport through liquid membranes, 448a or as photolabile linkers. 454 Scheme 125 Photochemistry of Dithiane-spiro-crown Ethers 448a Triggering of two-photon fluorescence (see also section 9) as a reporting function has been developed. 455 Kutateladze and co-workers used dithianes to mask the sensitizer molecules immobilized on beads, dendrimers, or peptides to demonstrate that excitation of a single free sensitizer leads to liberation of its own copy, thus leading to signal amplification. 456 Events, which occur on an attomolar concentration scale, can now be detected via molecular recognition-triggered photoamplified fluorescence quenching. 457 Dithiane-based photolabile tags have also been used for encoding and direct screening of solution-phase combinatorial libraries. 458 Dalko and co-workers have recently reported on a very interesting bichromophoric system composed from hydroxymethyl quinolene-derived probes, which are activated through interactions of X-ray or γ-irradiation with gadolinium(III) complexes (Scheme 126). 459 These metal complexes act as intramolecular antennae and convert part of the energy resulting in electron transfer and subsequent fragmentation of the quinolines. Scheme 126 Photo- and Radiolysis of Caged Hydroxymethyl Quinolone Derivatives 459 8.3 Sensitized Release: Light Upconverting Nanoparticles In contrast to multiphoton excitation, which comprises the simultaneous absorption of two or more photons using pulses with femtosecond duration (section 9), photon upconversion is a sequential absorption of two or more photons. 460 Light upconverting nanoparticles (UCNPs) have attracted considerable attention as visible or near-IR (NIR) harvesting light antennae that, upon energy or heat transfer (photothermal effect), can trigger a release of species, such as drugs. 461 Branda and co-workers used a dimethoxybenzoin PPG coupled to monodispersed core–shell nanoparticles (NPs) composed of NaYF4 nanocrystals doped with lanthanides to release carboxylates upon NIR light activation (980 nm) followed by NP fluorescence, exciting the benzoin chromophore. 223 The wavelength of emission may be controlled by the power density of lasers used for NPs excitation. 462 The same principle has also been demonstrated on photoswitches. 463 Remotely triggered release was achieved by heat transfer from gold nanoparticles, which can be tethered to, encapsulated within, or suspended freely outside liposomes or micelles that can serve as drug carriers. 222,464 In a similar study, gold NPs were enclosed in biodegradable and biocompatible microspheres (1–15 μm) containing the antitumor drug paclitaxel. 465 The NIR-activated photothermal effect leads to efficient paclitaxel release. A variety of target compounds, such as oligonucleotides or si-RNA, can also be adsorbed on the modified surface of a gold NP 466 or coupled to the NP surface via gold–thiol conjugation. 467 For example, a high payload of doxorubicin was coated and successfully released from both the outer and inner shells of polyethylene glycol (PEG) hollow gold nanoparticles. 468 Chromatic orthogonality (see also section 10) to release two different DNA oligonucleotides has been demostrated on gold nanorods differing in their aspect ratio, 469 which is promising for future applications in cancer theranostics. 470 9 Two-Photon Excitation-Induced Photorelease The removal of the photolabile protecting groups discussed in this review usually requires UV irradiation. These conditions, however, are not compatible with many biomedical applications, as UV–vis light is efficiently absorbed by the tissue. 471 The major absorbing species in vertebrate tissues is oxyhemoglobin, which filters practically all the light of wavelengths shorter than 650 nm. 471 On the other hand, water becomes increasingly absorbant at wavelengths longer than 950 nm. These two factors define the “phototherapeutic window”, the region of relative tissue transparency between 650 and 950 nm. 472 The increased depth of tissue penetration of radiation in this region is accompanied by additional advantages: lower scattering and reduced phototoxic effects. However, red and NIR photons have relatively low energy, limiting the range of processes they can initiate. In addition, the majority of chromophores with useful single-photon absorbance in the “phototherapeutic window” are also sensitive to visible light, complicating the handling of the caged substrates. One of the approaches that overcomes most of these problems is to use nonresonant two-photon excitation (2PE). At high light intensity, chromophores may simultaneously absorb two red/NIR photons, producing higher-energy excited states, the same as or similar to those accessible by direct excitation with UV photons of about twice the frequency. 473 Additionally, focusing the irradiation of 2PE on UV chromophores within UV-absorbing materials provides an opportunity to control substrate release in three dimensions. The probability of 2PE is proportional to radiant intensity squared, which is many orders of magnitude higher in the λ3 volume around the focal point than in other areas of the laser beam. 474 This phenomenon has made possible the development of 3-D applications in fluorescent imaging, 473a,474b,475 microscopy, 474b,476 photonics, 477 3-D fabrication, 478 and potentially even drug delivery 304,479 and photodynamic therapy. 480 Substantially less data are available on two-photon induced photochemical reactions 473c,481 and their use in biochemistry. 164,476b,482 The 3-D capabilities of two-photon-induced uncaging were successfully used in neuron mapping, 10d,483 investigation of intracellular processes, 484 and regulation of protein activity in vitro 221,485 and in vivo, 221,485b as well as in the development of 2PE-uncaging microscopy. 486 2PE requires very high light intensities, which can be achieved only using ultrafast pulsed lasers. Thus, a commercially available mode-locked Ti:sapphire laser can provide a photon irradiance (E p) of 1025 photons cm–2 s–1 (3 mm beam at 100 fs pulse duration, and repetition frequency ν = 90 MHz) or more. The principal emission of the most common Ti:sapphire lasers is at 800 nm, which almost perfectly corresponds to the minimum of mammalian tissues absorbance. Lasers that can be tuned to shorter wavelengths are usually more expensive. The differential form of the Beer’s law for two-photon excitation 487 is shown below (eq 2), where E p is the photon irradiance (photons cm–2 s–1), δ is the two-photon cross section (cm4 s photon–1 molecule–1), N is the concentration (molecules cm–3), and x is the sample thickness (cm). 2 3 As light intensity virtually does not change when passing through the sample due to low two-photon absorption, we can write eq 2 in a simplified form (eq 3). The rate of photoreaction with quantum yield Φ (molecule photon–1) is proportional to photon irradiance absorbed by the sample, which is in turn proportional to squared photon irradiance (eq 4). 481f The coefficient 1/2 in eq 4 reflects the two-photon nature of the process. 4 5 Integration of eq 4 and taking into account the pulsed nature of the irradiation gives eq 5. 481f N 0 and N t represent starting and current concentration of a substrate; E p represents the averaged photon irradiance during the pulse, t pulse is pulse duration, ν is laser repetition rate, and t irr is the time of exposure. Strictly speaking, E p 2 should be integrated for the duration of the pulse, but this approximation introduces insignificant error. For low-conversion experiments that use the Ti:sapphire laser discussed above, we can write a simplified eq 6, where δ is expressed in Goeppert-Mayer units (1 GM = 10–50 cm4 s photon–1 molecule–1). 6 Thus, to achieve 1% release of a substrate within the focal volume of a laser beam using a PPG with a quantum yield of uncaging Φ = 0.5 and a 2PE cross-section of δ = 1 GM, the sample has to be irradiated for about 30 min. One should also take into account the background one-photon absorbance of tissue materials, which is about 10–3 per 1 cm at 750 nm, 476b,488 and the broadening of ultrashort pulses in dense media. For an efficient two-photon photochemistry in tissues, the product δE p should be at least 10–25 cm2 molecule–1. 487 This means that, at light intensities produced by a Ti:sapphire laser, the two-photon absorption cross section δ of the PPG should be around 1 GM or higher. The cross section of two-photon absorption for most organic chromophores is rather low (δ 365 0.01 800 (496) 0.23c 740 (221, 485b) 0.004c–0.025 750 (221, 485b) 57 (o-nitrobenzyl, oNB; R = Cl, OH, MeO, NH2, p-MeOC6H4, etc.) 0.001–0.1 325 0.015–0.065 750 (190) 265 (3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl, DMNPB) 0.26 365 0.17 720 (492) 115 ((4′-methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl, PMNB) 0.1 315 0.45 800 (236) 3.1 740 (237) 266 ((4′-tris-ethoxymethoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl, PENB) 0.1 315 3.7 740 (493) 117 (2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene, BNSF) 0.25 354 5.0 800 (237) 267 ((7-hydroxycoumarin-4-yl)methyl) 0.025 365 1.07 740 (331) 0.13 800 (303) 268 ((6-chloro-7-hydroxycoumarin-4-yl)methyl) 0.01 365 1.07 740 (303) 0.34 800 (303) 143 ((6-bromo-7-hydroxycoumarin-4-yl)methyl, BHCM) 0.084 350 0.35 740 (288) 0.019–0.037 365 0.51–1.99 740 (294, 303) 0.21 780 (330a) 0.37–0.42 800 (303) 269 ({7-[bis(carboxymethyl)-amino]coumarin-4-yl}methyl, BCMACM) 0.29 330–430 ca. 1c 800 (286) 270 ({7-[4-(dimethylamino)styryl]coumarin-4-yl}methoxycarbonyl) 0.83 × 10–3 407 0.26 800 (497) 271 ((3,6,8-tribromo-7-hydroxycoumarin-4-yl)methyl) 0.065 365 0.96 740 (303) 3.1 800 (303) 181 ((8-bromo-7-hydroxyquinoline-2-yl)methyl, BHQ) 0.29–0.39 365 0.59–0.90 740 (331) 0.087 780 (330a) 272 ((8-cyano-7-hydroxyquinoline-2-yl)methyl, CyHQ) 0.31 365 0.32 740 (335a) 273 ((8-chloro-7-hydroxyquinoline-2-yl)methyl, CHQ) 0.10 365 0.12 740 (335a) 274 ([7-(dimethylamino)quinoline-2-yl]methyl, DMAQ) 0.046 365 0.13 740 (335a) 275 ([7-(dimethylamino)-4-chloroquinoline-2-yl]methyl, DMAQ-Cl) 0.09 365 0.47 740 (335a) 276 ((7-mercaptoquinoline-2-yl)methyl, TQ) 0.063 365 0.42 740 (335a) 277 ((Z)-butyl 3-(4-(diethylamino)-2-hydroxyphenyl)-2-acetamidoacrylate) 0.05 300–400 0.3 750 (393b) 278 (3,5-dibromo-2,4-dihydroxycinnamate) 0.05 369 1.6 750 (390) 0.05 300–400 0.6 750 (393a) 279 ((E)-3-(6-hydroxy-benzo(1,3)dioxo-5-yl)acrylate) 0.03 300–400 3.8 750 (393a) 280 ((E)-3-(4-diethylamino-2-hydroxy-phenyl)acrylate) 0.02 300–400 2.0 750 (393a) 281 ((Z)-butyl 2-acetamido-3-(5-hydroxybenzo[d][1,3]dioxol-6-yl)acrylate) 0.07 300–400 2.0 750 (393b) 282 ((E)-3-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido(3,2,1-ij)quinolin-9-yl)acrylate) 0.03 300–400 4.7 750 (393a) 125 (4-methoxy-7-nitroindolinyl, MNI) 0.085 350 0.006 730 (10d, 483) 127 (4-methoxy-5,7-dinitroindolinyl, MDNI) 0.47 350 0.06c 730 (494) 283 (4-carboxymethoxy-5,7-dinitroindolinyl, CDNI) 0.5c 334–364 0.06 720 (483a) 130 (5-methoxy-8-nitro-1,2-dihydroquinolinyl, MNDQ) 0.05 365 <0.06c 720 (250) a The single-photon quantum yield of substrate release. b The two-photon cross section of uncaging. c By comparison with the literature data. 10 Chromatic Orthogonality The diversity of different PPGs, operating by different mechanisms and bearing different types of chromophores, opens the door for wavelength-selective deprotection. Indeed, the possibility of releasing on demand various types of molecules (e.g., in a cell, or wherever access by conventional injection techniques is not possible) is a very appealing prospect. A first-order approximation would suggest that absorption maxima sufficiently distant could work; however, fast energy transfer may thwart such a strategy, in particular for cases where both chromophores are part of the same molecular entity. Careful choice of PPG pairs made such a wavelength discrimination possible, allowing for the selective release of two different carboxylic acids, in what was an early example of chromatic orthogonality (Scheme 127). 208,498 Scheme 127 Example of Intermolecular Chromatic Orthogonality 498 The same benzoin (section 2.4)/nitroveratryl (NV, section 3.3) pair was then shown to be equally orthogonal in the intramolecular case, where spatial proximity is more critical than in separate molecules (Scheme 128). 499 Reactivity tuning by way of a kinetic isotope effect was also exploited to make two derivatives of o-nitrobenzylic esters (section 3.1) chromatically orthogonal. 225 Scheme 128 Example of Intramolecular Chromatic Orthogonality 499 Examples of this principle were shown soon after in the solid-phase synthesis of peptides 360c and in the selective release from resins by using the nitroveratry/pivaloylglycol pair. 500 del Campo and co-workers investigated a more complex version of the wavelength-selective cleavage by putting seven photolabile protecting groups on various functionalities immobilized through an organosilane tether on a glass surface against each other. 240e,501 Systematic analysis of photolytic characteristics of common photolabile groups helped establish several protecting strategies that permit the simultaneous use of up to four orthogonal photoactivated groups, such as benzoin (λex = 255 nm, section 2.4), p-hydroxyphenacyl (pHP, λex = 275 nm, section 2.3), 5,7-dinitroindolinyl (DNI, λex = 360 nm, section 3.3), and [7-(diethylamino)coumarin-4-yl]methyl (DEACM, λex = 435 nm, section 4), that can be cleaved sequentially from the same solid support under different irradiation conditions. 240e It was also noted that two-photon excitation can potentially expand the spectral window and increase the number of possible functional levels for selective spatiotemporal activation. Applications in biochemistry are appearing more and more frequently, such as the protection of cysteines (using variously substituted coumarins), 196 nucleotides (using a coumarin/nitrophenethyl pair), 502 or both, 503 and the release of both substrates and inhibitors (also using a coumarin/nitrophenethyl pair), 504 or the selective release of glutamate or GABA by two-colors/two-photon excitation. 263b The latter strategy was also applied very recently with a NV/p-MeO-phenacyl pair by Emptage, Conway, and co-workers. 106c The same concept was shown to be valid in the solid phase, where the wavelength-selective modification of surfaces was performed with orthogonal pairs 135a,501 or quartets. 240e 11 Photoactivatable Fluorescent Dyes Photoactivatable (caged) fluorophores 13b are fluorogenic and release a fluorescent molecule upon irradiation. They are obtained by coupling a fluorescent dye to a PPG that prevents it from displaying fluorescence. Caged fluorescent dyes provide a highly sensitive tool to monitor the flow of liquids (rheology) and to follow the movements and the distribution of particular species of interest at the single molecule level and with a spatial resolution at the nanometer scale, i.e., beyond the diffraction limit of optical microscopy (∼250 nm). Fluorescence imaging microscopy is among the most powerful techniques for observing dynamic processes in living cells. To monitor the movement of target molecules or species in real time, it is necessary to create a local region within the cell where the fluorescence intensity is higher than in the bulk. Imaging with high target-to-background contrast requires a high activation ratio, i.e., a high ratio of postactivation to preactivation signal intensity. Thus, the signal intensity arising from the caged fluorophore should be close to zero. The classic method for creating a differentially labeled region has been fluorescence photobleaching, i.e, reducing fluorescence intensity by irradiation, usually by reaction of a fluorescent dye with singlet oxygen. Alternatively, photochemically labile fluorophores such as the aminophthalimide–serine shown in Scheme 129 can be used. 378 Disadvantages of photobleaching through singlet oxygen are that it may cause local damage to proteins and membranes and that it is difficult to track a region of reduced fluorescence within a background of higher fluorescence. The use of a nonbleachable reference fluorophore has been proposed to track the distribution of the bleached molecules by image differencing. 505 Scheme 129 “Armed” Phthalimide (λmax = 340 nm, Exhibits Strong Fluorescence, λmax = 513 nm, in Aqueous Media at pH = 7; Irradiation Liberates Acetate and CO2, and the Fluorescence Decreases) 378 To overcome these limitations, numerous photoactivatable fluorophores have been developed. The design and use of caged fluorophores have been reviewed. 9n,13a,13b,506 Desiderata for an effective caged fluorescent probe are biostability and -compatibility; a high affinity to or, preferentially, specificity for the target (e.g., cancer cells); 507 rapid and efficient photoactivation providing a high ratio of pre- and postirradiation fluorescence; a high fluorescence quantum yield; photostability of the uncaged fluorophore; and practicable synthesis. Up to 1998, 506 caged fluorophores have all used variants of the oNB (section 3) caging group (e.g., 284). The full activation of such bis-caged lactone (leuco) forms of rhodamine or fluorescein requires the removal of both PPGs. Removal of one PPG is sufficient to restore the chromophore, but the remaining nitroaromatic group often largely quenches the fluorescence emission of the monocaged dye. Full deprotection therefore requires high doses of UV irradiation. On the other hand, the non-negligible fluorescence quantum yield of monoprotected fluorescein hampers its use as a caged fluorophore, because the activation ratio upon deprotection is usually insufficient. A monoprotected variant of fluorescein (TokyoGreen, 285, Scheme 130) has, however, been developed that exhibits very little fluorescence. 508 The fluorescence quantum yield of singly 2-(2-nitrophenyl)prop-1-yl (NPP) (section 3.1) protected TokyoGreen (286) is less than 1/100th that of protected fluorescein. It liberates the highly fluorescent free form (Φ fl = 0.96) by removal of the PPG with a quantum yield of 0.03. 509 Scheme 130 Deprotection of Mono-Caged Tokyo Green (Its Fluorescence Is Quenched by Intramolecular Electron Transfer in the Excited Singlet State) 508 Several other examples of fluorescence activation by photochemical removal of a quencher moiety attached near the fluorophore have been reported. Other than by electron transfer, fluorescent singlet states can be quenched by Förster resonance energy transfer (FRET, somewhat inadequately called “fluorescence resonance energy transfer” by most biochemists) 510 to an adjacent, nonfluorescent, but photoremovable chromophore. For example, a synthetic route to incorporate a photocleavable 4-dimethylaminoazobenzene-4′-sulfonyl (287, dabsyl) 511 moiety and fluorescein at adjacent cytidines in the middle of a 25-mer oligodeoxynucleotide has been reported. 512 UV irradiation removed the dabsyl moiety, which increased the fluorescence intensity 51-fold and restored the melting temperature of the nucleotide. Such caged fluorescent oligodeoxynucleotides will allow many DNA processes to be controlled with light. The same principle was used to regulate the DNA polymerase reaction by the Klenow fragment of polymerase I with UV light. A 25-mer caged fluorescent oligodeoxynucleotide as the template was functionalized with a fluorescein reporter and the photoremovable dabsyl quencher moiety (287). With this template, the Klenow fragment was blocked from extending a complementary 12-mer primer. Removal of the quencher by short UV photolysis partly restored the activity of the Klenow fragment, and the reactivation could be monitored by fluorescence. 511 A related approach was used to provide for real-time monitoring of Smad2, a key protein involved in the transforming growth factor β (TGF-β) signaling pathway, spatial and temporal control of its activity, and differentiation between its active and inactive state. 513 The protein was labeled at neighboring sites with a fluorescein chromophore and with a 4-dimethylaminoazobenzene-4′-carboxylic (dabcyl) acid quencher through a photocleavable 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy] butanoic acid linker. This both suppressed the protein activity and quenched its fluorescence. Photocleavage of the caging group resulted in the simultaneous restoration of protein activity and luminescence of the fluorescent tag. Numerous photocleavable (coumarin-4-yl)methyl esters with fast release have been described (section 4). 276,289,296 NPP-caged coumarins that display more than 200-fold fluorescence enhancement upon UV irradiation were developed (Scheme 131). 491 The combined advantages of robust fluorescence contrast enhancement, high uncaging efficiency, noninvasive cellular delivery, and flexible chemistry for bioconjugations are promising for the use of these caged coumarins in biochemical and biological research. The high uncaging efficiency is attributed to the antenna function of the coumarin, i.e., light absorbed by the coumarin chromophore is utilized to cleave the NPP cage. It should, however, be noted that NPE-caged fluorophores are subject to the known disadvantages of o-nitrobenzyl photochemistry (see section 3.1). Scheme 131 Caged Coumarin 491 To prepare a caged coumarin emitting a different color, the o-nitrobenzyl-caged coumarin was linked to a water-soluble fluorescein dye emitting at 520 nm. 514 The caged dye can be localized in the sample prior to photolysis by directly exciting the fluorescein moiety at 490 nm. After uncaging the coumarin chromophore, the green emission can be excited at 410 nm due to efficient FRET from the coumarin moiety to the fluorescein dye. This is especially desirable for experiments demanding highly localized photoactivation by two-photon uncaging, which requires knowing the distribution of the label in three dimensions. These probes thus offer new opportunities to image molecular and cellular dynamics. A light-activated fluorescent reporter of intracellular protein kinase activity has been designed (Scheme 132) that furnishes a fluorescent readout. 515 The photolytically labile appendage affords control over both the timing and the amount of active sensor release. The quantum yield for photolytic conversion is 0.06. The caged fluorescent substrate was introduced into HeLa cells via microinjection. Following in situ illumination of the caged peptide, time-dependent changes of the fluorescence intensity due to phosphorylation of the released hydroxyl function provided a measure of protein kinase activity in the cells. Scheme 132 Fluorescent Reporter of Protein Kinase Activity 515 Azidomethyl-caged fluorescein derivatives (288 and 289) were utilized to monitor the dynamics of oligonucleotides in living human cells. Both compounds were rapidly activated upon brief irradiation and showed a strong increase in fluorescence intensity. 516 The addition of lithiated dithianes to 2-amidothioxanthones disrupts conjugation, resulting in a blue-shift of the absorption and a dramatic decrease in fluorescence intensity. The product represents a new caging system capable of fast quantification of released payloads such as TentaGel beads by single- or two-photon fluorescence. 455 Irradiation induces homolytic C–C fragmentation followed by disproportionation of the two radicals (Scheme 133). The half-life of the radical intermediates was 1.7 μs. Scheme 133 Caged Thioxanthone 455 Rhodamine dyes having a 2-diazo caging group incorporated into a spiro-9H-xanthene fragment have been synthesized by reaction of diazomethane with the acid chloride of rhodamine B. 517 The yellow crystalline diazoketone 290 was obtained in high yield (Scheme 134). Caged rhodamines with an additional carboxy group in the benzoyl fragment of the diazoketone that may be linked to an amino or a thiol reactive site were also made. Application of a related system featuring a high contrast ratio (1:140) and activation by visible light (λ < 420 nm) to high-resolution microscopy was recently reported. 518 Scheme 134 Photochemistry of Caged Rhodamine (a Wolff Rearrangement) 517 A ruthenium-rhodamine complex 291 (see Section 6) has been evaluated as an activatable fluorescent probe (Scheme 135). 356 Upon irradiation, it releases rhodamine in a fast and clean reaction, increasing its fluorescence nearly 6-fold. Scheme 135 Photochemistry of Caged Rhodamine 356 A photoactivatable nitrobenzyl-caged (section 3.1) green fluorescent protein (292, GFP) that is practically nonfluorescent prior to irradiation was synthesized recently (Scheme 136). 519 The relative brightness at the wavelength of maximum emission (486 nm) increased by at least 4 orders of magnitude during photoactivation at 365 nm. Scheme 136 o-Nitrobenzyl-Caged GFP 519 Photoactivatable fluorescent proteins have become an important addition to the set of molecular probes used to understand cellular function. Wild-type GFP 520 is in fact already a caged fluorophore of sorts. The protein can adopt two conformations with different absorption maxima, and irradiation shifts the molecules toward the longer-wavelength absorbing form. Photoconversion in wild-type GFP is thought to involve rotation of threonine 203 and decarboxylation of glutamic acid 222. A mutant of wild-type GFP, in which the threonine 203 position is replaced by histidine, was shown to increase its fluorescence intensity (λex = 488 nm, λem = 520 nm) 100 times upon intense irradiation at 413 nm. 521 The activated protein remains stable for days under aerobic conditions. This photoactivatable variant of the Aequorea victoria GFP was used both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange. Optical microscopy provides spatial resolution down to the diffraction limit of ∼250 nm, but single fluorescent particles can now be localized down to ∼1 nm precision, and spectroscopic techniques, such as FRET, can offer yet smaller distance changes. Super-resolution (subdiffraction) imaging techniques based on sequential imaging of sparse subsets of single molecules have used fluorophores whose emission can be photoactivated or photoswitched. 522 The common principle is that only one pointlike light source is active in a diffraction-limited area at any time. Imaging with a sensitive camera localizes this fluorophore. Its exact location can then be determined to well below the diffraction limit by fitting a point-spread function. In the beginning of data acquisition, all except a few fluorophores are prepared in the off-state and the number of active fluorophores is kept constant by applying an activating light source that compensates for the loss of fluorophores by photobleaching or switching off. A super-resolution image of a labeled complex structure can then be reconstructed from many successive rounds of weak photoactivation and fitting. This method has been used to image intracellular proteins at nanometer spatial resolution. As an alternative to caged fluorophores for super-resolution microscopy, on- and off-states have been engineered by controlling the photophysics of fluorophores by electron transfer reactions. 523 Because good organic fluorophores are more photochemically stable than most fluorescent proteins, organic fluorophores have a potential benefit in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. The design and application of target-specific an azido derivative of 2-dicyanomethylene-3-cyano-2,5-dihydrofuran, a photoactivatable push–pull fluorogen that produces bright fluorescent labels suitable for single-molecule super-resolution imaging in live bacterial and fixed mammalian cells, were reported (293). 524 The same authors subsequently demonstrated that the azide-to-amine photoactivation process is generally applicable to a variety of push–pull chromophores, which provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy. 525 Moreover, these photoactivated push–pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling). The design, synthesis, and photophysical characterization of a class of ligand-directed, photoactivatable, turn-on fluorescent probes for the spatially controlled imaging of microtubules in live mammalian cells was reported. 526 A series of taxoid–tetrazoles was reported in which 7-β-alanyltaxol L was attached to a water-soluble tetrazole bearing an o-allyloxy group at the N-phenyl ring via a flexible linker. Upon irradiation at 302, 365, or even 405 nm, the tetrazoles 294 undergo deazotization generating reactive nitrile imine dipoles that spontaneously react with the prealigned allyl group to form pyrazoline fluorophores (Scheme 137). Whereas none of the taxoid–tetrazoles was fluorescent, the taxoid–pyrazolines exhibited strong fluorescence emission in the range of 500–800 nm. With this photoactivatable fluorescent probe, it should be possible to label microtubules asymmetrically within a single cell and identify factors that break cellular symmetry during cell division. Scheme 137 Intramolecular Photoclick Reaction 526 12 Conclusion Photoremovable protecting groups (PPGs) release molecules such as enzymes, neurotransmitters, signaling molecules, fluorophores, insecticides, pheromones, and fragrances that thereby exhibit desirable physical, chemical, or biological qualities upon photoactivation. They are synthetically malleable and accessible, offering a wide range of structures for designed applications. They offer excellent spatial and temporal control for the substrate release. Their applications span many scientific fields, from DNA chip technology, drug delivery, and photoregulation of proteins, to rheology, solid-phase synthesis, surface chemistry, and nanotechnology. PPGs are excellent, versatile tools for time-resolved studies of chemical processes in living cells. Multiphoton excitation can provide superior spatial resolution, and the new chromophores included herein offer more precise temporal control for addressing the dynamics of in vivo events in living organisms. There has been an upsurge in interest in PPGs over the past decade that has resulted in a dramatic increase in the number of new designs and the development of both new and known PPGs to fulfill the demands for better sensitivity, faster kinetics, and more demanding bioanalytical applications. The many new discoveries have brought substantial improvements and versatility to the synthesis, thermal stability, solubility, and absorption properties of PPGs, as well as improved efficiencies and rates of release. Nonetheless, there still remain many important research goals, such as extending the wavelength coverage for activation of PPGs with visible and infrared light and improving the absorption properties for wavelength-selective, orthogonal activation, and melding these features with the inherent advantages of photoactivation that PPG possess as outlined at the beginning of this review. Our goal has been to aid and assist researchers in applying these tools and to attract and encourage their participation in this rapidly developing field.

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DNA microarray technology: devices, systems, and applications.

Michael Heller (2002)

In this review, recent advances in DNA microarray technology and their applications are examined. The many varieties of DNA microarray or DNA chip devices and systems are described along with their methods for fabrication and their use. This includes both high-density microarrays for high-throughput screening applications and lower-density microarrays for various diagnostic applications. The methods for microarray fabrication that are reviewed include various inkjet and microjet deposition or spotting technologies and processes, in situ or on-chip photolithographic oligonucleotide synthesis processes, and electronic DNA probe addressing processes. The DNA microarray hybridization applications reviewed include the important areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). In addition to the many molecular biological and genomic research uses, this review covers applications of microarray devices and systems for pharmacogenomic research and drug discovery, infectious and genetic disease and cancer diagnostics, and forensic and genetic identification purposes. Additionally, microarray technology being developed and applied to new areas of proteomic and cellular analysis are reviewed.

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Printing proteins as microarrays for high-throughput function determination.

S L Schreiber, G MacBeath (2000)

Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

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P.A. Levkin

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Journal ID (nlm-ta): Mater Today Bio

Journal ID (iso-abbrev): Mater Today Bio

Title: Materials Today Bio

Publisher: Elsevier

ISSN (Electronic): 2590-0064

Publication date PMC-release: 20 August 2019

Publication date Collection: June 2019

Publication date (Electronic): 20 August 2019

Volume: 3

Electronic Location Identifier: 100022

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[a ]Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Hermann-von Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany

[b ]Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, 76131, Karlsruhe, Germany

[c ]Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, Hermann-von Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany

[d ]Karlsruhe Institute of Technology (KIT), Karlsruhe Nano Micro Facility, Hermann-von Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany

[e ]Karlsruhe Institute of Technology (KIT), Institute for Applied Materials, Hermann-von Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany

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[∗ ]Corresponding author. Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Hermann-von Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany. levkin@ 123456kit.edu

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Publisher ID: S2590-0064(19)30048-1 Publisher ID: 100022

DOI: 10.1016/j.mtbio.2019.100022

PMC ID: 7061619

PubMed ID: 32159150

SO-VID: b0e1e552-e1ca-4e6b-a711-e949b87c00a6

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Date received : 9 May 2019

Date revision received : 19 July 2019

Date accepted : 25 July 2019

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DNA microarray technology: devices, systems, and applications.

Printing proteins as microarrays for high-throughput function determination.

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