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Photoremovable protecting groups: reaction mechanisms and applications Anna Paola Pelliccioli and Jakob Wirz * Institut für Physikalische Chemie, Universität Basel, Klingelbergstrasse 80, CH-4056 Basel, Switzerland. E-mail: [email protected]; Fax: 41 61 267 38 55; Tel: 41 61 267 38 42 Received 21st January 2002, Accepted 1st May 2002 First published as an Advance Article on the web 6th June 2002 Photolabile protecting groups enable biochemists to control the release of bioactive compounds in living tissue. ‘Caged compounds’ (photoactivatable bioagents) have become an important tool to study the events that follow chemical signalling in, e.g., cell biology and the neuro- sciences. The possibilities are by no means exhausted. Progress will depend on the development of photo- removable protecting groups that satisfy the diverse requirements of new applications—a challenging task for photochemists. Introduction Photolabile protecting groups have had a renaissance in recent years. They oer the means to deliver bioactive materials such as neurotransmitters, ATP or simply Ca 2 ions rapidly to small, addressable target sites and thereby enable biologists to follow the ensuing physiological events in real time. Amplied chemical eects may be achieved by controlling enzyme activity, gene expression or ion channel permeability with light (phototriggers). Recent developments include time-resolved X-ray crystallography, two-photon excitation using highly focussed, ultrashort infrared laser pulses, and kinetic studies on the early events in protein folding. The list of novel applications is growing rapidly. Consider the following criteria for the design of a good photoremovable protecting group. 1) In general, the photoreaction should be clean and occur with high quantum yield . 2) The chromophore should have high absorption coecients ε at wavelengths above 300 nm, where irradiation is less likely to be absorbed by (and possibly cause damage to) the biological environment. However, low absorption coecients may be required to achieve deep penetration when higher concen- trations are used. 3) The photochemical by-products accompanying the released bioactive reagent should not interfere with the photoreaction and ideally be transparent at the irradiation wavelength in order to avoid competitive absorption of the photolysing light. Moreover, they must be biocompatible, i.e., should not aect Jakob (“Joggi”) Wirz Jakob (“Joggi”) Wirz is Professor of Chemistry at the Institute of Physical Chemistry, University of Basel. He obtained his PhD in 1970 with Professor Edgar Heilbronner at the ETH Zürich. His thesis work on the photochemistry of triuoromethylnaphthols provided absolute rate constants dening excited state reactivity and quantifying substituent eects on excited states. He learnt all the secrets (like: beware of artefacts!), ups and downs of ash photolysis during his postdoctoral studies with Professor George Porter at the Royal Institution in London, where he elucidated the mechanisms of several preparative photoreactions developed by Derek Barton’s group at Imperial College. He takes continued pleasure in producing unusual molecules (cycloocta-1,5-diyne) and reactive intermediates (diradicals, ylides, carbenes, nitrenes, enols and ynols), and in exploring and exploiting their reactivity in solution. Throughout his career, he has been lucky to enjoy the collaborationwith creative and gifted co-workers as well as many colleagues and friends all over the world. Jakob Wirz is past-chairman of the European Photochemistry Association. Anna Paola Pelliccioli Anna Paola Pelliccioli received her degree in Chemistry from the University of Perugia, Italy, where she worked on the characterization of photochromic compounds in the laboratory of Professor G. Favaro. She then spent one year as a research assistant in the group of Dr M. A. J. Rodgers at the Center for Photochemical Sciences of Bowling Green State University, OH, studying the photo- physical properties of monomeric and dimeric metallophthalocyanines. At present she is completing her PhD studies in the Wirz group at the University of Basel, investigating the photochemistry of new photoremovable protecting groups. DOI: 10.1039/b200777k Photochem. Photobiol. Sci., 2002, 1, 441–458 441 This journal is © The Royal Society of Chemistry and Owner Societies 2002

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Page 1: Photoremovable protecting groups: reaction mechanisms and ... · protecting groups by structural variation. 2-Nitrobenzyl derivatives are by far the most common photolabile protecting

Photoremovable protecting groups: reaction mechanismsand applications

Anna Paola Pelliccioli and Jakob Wirz*

Institut für Physikalische Chemie, Universität Basel, Klingelbergstrasse 80, CH-4056 Basel,Switzerland. E-mail: [email protected]; Fax: �41 61 267 38 55; Tel: �41 61 267 38 42

Received 21st January 2002, Accepted 1st May 2002First published as an Advance Article on the web 6th June 2002

Photolabile protecting groups enable biochemists tocontrol the release of bioactive compounds in living tissue.‘Caged compounds’ (photoactivatable bioagents) havebecome an important tool to study the events that followchemical signalling in, e.g., cell biology and the neuro-sciences. The possibilities are by no means exhausted.Progress will depend on the development of photo-removable protecting groups that satisfy the diverserequirements of new applications—a challenging task forphotochemists.

IntroductionPhotolabile protecting groups have had a renaissance in recentyears. They offer the means to deliver bioactive materials suchas neurotransmitters, ATP or simply Ca2� ions rapidly to small,addressable target sites and thereby enable biologists to followthe ensuing physiological events in real time. Amplifiedchemical effects may be achieved by controlling enzymeactivity, gene expression or ion channel permeability with light

(phototriggers). Recent developments include time-resolvedX-ray crystallography, two-photon excitation using highlyfocussed, ultrashort infrared laser pulses, and kinetic studies onthe early events in protein folding. The list of novel applicationsis growing rapidly.

Consider the following criteria for the design of a goodphotoremovable protecting group.

1) In general, the photoreaction should be clean and occurwith high quantum yield �.

2) The chromophore should have high absorption coefficientsε at wavelengths above 300 nm, where irradiation is less likely tobe absorbed by (and possibly cause damage to) the biologicalenvironment. However, low absorption coefficients may berequired to achieve deep penetration when higher concen-trations are used.

3) The photochemical by-products accompanying the releasedbioactive reagent should not interfere with the photoreactionand ideally be transparent at the irradiation wavelength in orderto avoid competitive absorption of the photolysing light.Moreover, they must be biocompatible, i.e., should not affect

Jakob (“Joggi”) Wirz

Jakob (“Joggi”) Wirz is Professor of Chemistry at the Institute of Physical Chemistry, Universityof Basel. He obtained his PhD in 1970 with Professor Edgar Heilbronner at the ETH Zürich. Histhesis work on the photochemistry of trifluoromethylnaphthols provided absolute rate constantsdefining excited state reactivity and quantifying substituent effects on excited states. He learnt allthe secrets (like: beware of artefacts!), ups and downs of flash photolysis during his postdoctoralstudies with Professor George Porter at the Royal Institution in London, where he elucidated themechanisms of several preparative photoreactions developed by Derek Barton’s group at ImperialCollege. He takes continued pleasure in producing unusual molecules (cycloocta-1,5-diyne) andreactive intermediates (diradicals, ylides, carbenes, nitrenes, enols and ynols), and in exploringand exploiting their reactivity in solution. Throughout his career, he has been lucky to enjoy thecollaborationwith creative and gifted co-workers as well as many colleagues and friends all over theworld. Jakob Wirz is past-chairman of the European Photochemistry Association.

Anna Paola Pelliccioli

Anna Paola Pelliccioli received her degree in Chemistry from the University of Perugia, Italy,where she worked on the characterization of photochromic compounds in the laboratory of ProfessorG. Favaro. She then spent one year as a research assistant in the group of Dr M. A. J. Rodgers atthe Center for Photochemical Sciences of Bowling Green State University, OH, studying the photo-physical properties of monomeric and dimeric metallophthalocyanines. At present she is completingher PhD studies in the Wirz group at the University of Basel, investigating the photochemistry of newphotoremovable protecting groups.

DOI: 10.1039/b200777k Photochem. Photobiol. Sci., 2002, 1, 441–458 441

This journal is © The Royal Society of Chemistry and Owner Societies 2002

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the system investigated. Formation of free radicals should beavoided.

4) For time-resolved work the release rate of the bioagentmust exceed that of the response investigated.

5) The protected compounds should be soluble in the targetedbiological (mostly aqueous) media, and it may be required thatthey pass biological barriers such as cell membranes or showaffinity to specific sites such as the active site of enzymes.

6) Successful detection of the response under study oftendepends not only on the product ε�, which is proportional tothe amount of release for a given photon exposure at lowabsorbances, but also on the level of activity prior to irradiation.When dealing with molecules with several functional groups,finding the right substitution pattern that will render thecompound biologically inert may be a challenging task,such as in the case of ‘caged’ proteins. Background activity mayalso arise from contamination by deprotected material. Hence,the photosensitive compounds must be pure and stable inthe media of interest during the time required for samplepreparation.

No single system will excel in all of these requirements. Thevariety of applications is such that it is desirable to have a rangeof compounds with different characteristics to choose from.Efficient and fast systems for poor leaving groups such as RO�,RHN� or RS� are wanted in particular.

Two types of photorelease may be distinguished: a) directelimination of the substrate from a reactive (singlet or triplet)excited state, and b) the light-induced generation of reactiveground-state intermediates that have a strong driving force andlow energy barrier for release of the substrate. The firstapproach holds more promise for achieving rapid releaseof reluctant leaving groups. Another strategy for the releaseof poor leaving groups such as alcohols or amines is torelease carbonic or carbamic acid monoesters, which decar-boxylate when released. However, decarboxylation may thenbecome rate limiting for the release of the ultimate substrate.

Many photoreactions proceed via a cascade of reactiveintermediates and the release of the desired substrate may occurwith considerable delay after excitation by a short light pulse.As the release is in many cases a heterolytic elimination orrequires proton transfer, release rates and efficiencies maydepend strongly on solvent, pH and, in some cases, on bufferconcentration (general acid/base catalysis). Knowledge of theactual release rates under physiological conditions is a pre-requisite for studying biological response times, and knowledgeof the reaction mechanism allows the designed improvement ofprotecting groups by structural variation.

2-Nitrobenzyl derivatives are by far the most commonphotolabile protecting groups. They were originally developedfor use in organic synthesis.1 Their application to biochemistrywas pioneered 1978 in ground-breaking work of Kaplanet al.2 with the synthesis of ‘caged ATP’. Over 40 nitrobenzyl-protected biochemicals are now commercially available. Theirextensive application, often successful, has also broughtattention to the disadvantages of nitrobenzyl cages, such astoxic and strongly absorbing by-products, and rather slow ratesof release following excitation. A number of alternative activeprinciples have been developed in recent years. The first resultsfrom practical applications are promising and suggest thatsome of these protecting groups represent valid alternatives,with the potential of further extending the technique to moredemanding fields of application.

Several excellent reviews have covered the synthetic andphysico-chemical 3,4 as well as the biochemical 5,6 aspects up tothe early nineties, and a book with 27 papers devoted to cagedcompounds appeared in 1998.7 The present review first sum-marises current knowledge about the reaction mechanismsof the more important photolabile protecting groups. Second,a selection of recent applications is given with a focus onapproaches that hold promise for further development.

Active principles: reaction mechanisms and releaserates

o-Alkylated nitrophenyl compounds

Electronic excitation of 2-nitrobenzyl compounds generatestheir aci-nitro tautomers very rapidly (<1 ns, Scheme 1). Never-

theless, quantum yields for this simple hydrogen shiftreaction are usually less than unity, only about 1% for parent2-nitrotoluene (1), and 0.3% for α,α,α-trideuterated 1,8 butup to about 0.6 for 1-(2-nitrophenyl)ethyl derivatives. TheE-stereoisomer of aci-1 is the major product when 1 isirradiated in Ar or N2 matrices at 12 K,9 suggesting that aci-1is formed adiabatically in an excited state, which yields bothstereoisomers upon internal conversion to the ground state.It is not clear whether the reaction proceeds from the excitedsinglet or triplet state or both.8,10

The phototautomerisation of 1 is largely reversible in aqueoussolution. The elementary steps promoting re-tautomerisationof aci-1 to 1 were recently identified.8 aci-1 is a moderatelystrong acid (Scheme 2), which ionizes to 1� with a rate constant

of about 2 × 107 s�1 in pure water. Thus, the equilibriumbetween the E- and Z-stereoisomers and their common anion isestablished within 100 ns (less in the presence of buffers orbase). Protonation of 1� at the methylene carbon is the rate-determining step regenerating 1. Hydronium ions protonate 1�

in the pH-range of 0–6, and solvent water is the proton sourceat pH >6. Acid catalysis saturates at 34 s�1, as the pH fallsbelow the pKa of aci-1, because [1�] becomes inversely pro-portional to [H�]. Carbon protonation of 1� by water is slow(k = 1.2 s�1) at physiological pH, and is unlikely to competeeffectively with the irreversible reactions of the nitrobenzylderivatives considered here.

Derivatives of 1 carrying a leaving group at the benzylic pos-ition release the protected substrate upon irradiation. The reac-tion proceeds via aci-nitro intermediates that are readilyobserved at λmax ≈ 400 nm by laser flash photolysis (LFP). Thedecay rates of these transients vary strongly with substitution,solvent, and with pH in aqueous solution. The pattern of thesechanges is not well understood. In spite of the long-standinginterest in the photochemistry of nitrobenzyl compounds,only fragmentary information is available about the elementarysteps that lead to deprotection. Several 2-nitrobenzyl esterswere studied by LFP.11 Time-resolved conductivity studiesindicated that the primary nitronic acids ionize in ethanol–water mixtures, but not in acetonitrile. Acceleration of the

Scheme 1 Phototautomerisation of 2-nitrotoluene.

Scheme 2 Tautomerisation and ionization constants of 2-nitro-toluene.8

442 Photochem. Photobiol. Sci., 2002, 1, 441–458

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decay rate by acid was observed, and attributed to protonationof the ester function. The most extensive mechanistic studieshave been done for ‘caged ATP’, the P3-1-(2-nitrophenyl)ethylester of adenosine triphosphate (2).

The appearance rates of the three products formed by LFPof 2, namely ATP (bioassay), 2-nitrosoacetophenone (absorp-tion at 740 nm), and H� (indicator dye), were each monitored,as well as the decay kinetics of the aci-nitro intermediate.12 Twosteps were kinetically resolved in aqueous solution at neutralpH: first, a proton is released by rapid ionization of aci-2 to2� (pKa ≈ 3.7). In the subsequent, slower reaction, the freenucleotide and the side product 2-nitrosoacetophenone areformed concomitantly with the decay of 2�. A reaction mech-anism consistent with these results was proposed (Scheme 3).

The acceleration of the second step with increasing protonconcentration in the pH range 6.3–7.9 was attributed toprotonation of the phosphate group, thereby acceleratingheterolytic elimination from a cyclic intermediate, whichwas assumed to coexist in rapid pre-equilibrium with 2�.Subsequent work using time-resolved infrared (TRIR) detec-tion and isotopic labelling established that the release of ATPoccurs in a first-order reaction that is synchronous with thedecay of 2� (k = 52 s�1 at pH 7, 10 �C).13 The TRIR technique 14

is essential to determine actual release rates, because most ofthe released groups do not give rise to significant changes inoptical absorption upon release.

Although the authors 12 carefully declared Scheme 3 as aminimal mechanism consistent with their kinetic results oncaged ATP, this mechanism has since frequently been assumedto hold for nitrobenzyl protecting groups in general. In par-ticular, aci-decay rates determined by LFP are usually taken toindicate substrate release rates. Recent work shows that thismechanism must be revised and that the aci-decay is not alwaysrate determining for the release. Scheme 3 does not accountfor the observation of acid catalysis with compounds carryingnon-basic leaving groups other than phosphate or carboxylicacid esters. ab initio and density functional theory calculationsfor the potential energy surface of 1 and its isomers predicted amuch higher activation barrier for cyclization of the aci-anion,1�, than for the neutral aci-form, aci-1.15 Moreover, these cal-culations predicted cyclization of aci-1 to be highly exothermic,whereas that of 1� was calculated to be endothermic. Similarconclusions were independently reached on the basis ofsemiempirical AM1 model calculations for derivatives of 1.16

Scheme 3 Reaction scheme proposed 12 for caged ATP.

In Scheme 4, cyclization is assumed to proceed irreversibly,and only from the neutral nitronic acid, which is formed bypre-equilibrium protonation of the aci-anion. A study of2-nitrobenzyl alcohol methyl ether (3) by LFP and TRIR innon-aqueous and aqueous solvents at various pH values gaveresults that were consistent with Scheme 4, but not with Scheme3.17 Both the cyclic benzisoxazoline intermediate 4 (Fig. 1) and

the subsequent hemiacetal 5 (not shown), have been identifiedby TRIR. The formation of 4 was observed by the resolvedgrowth of a band at 1090 cm�1. The N��O band due to 5(1500 cm�1) and the C��O band of the final product 2-nitroso-benzaldehyde (1700 cm�1) appear only at longer times. Furtherstrong evidence favouring the mechanism of Scheme 4 has beenreported recently (see Scheme 6 below).18

It may seem surprising that neutral nitronic acids cyclizemore readily than their conjugate anions. Cyclization ofaci-anions is unfavourable because the resulting NO� functionis a much stronger base than the nitronic acid anion. Themechanism shown in Scheme 4 implies specific acid catalysis forthe decay of the aci-anion of 2-nitrobenzyl derivatives, whichpredominates in a rapid pre-equilibrium with the neutralaci-form at near-neutral pH. On the other hand, the subsequentring opening and elimination reactions (Scheme 4) are catalysedby (general) base. It so happens that the aci-decay is therate-determining step for the release of ATP from caged ATP(2) at pH ≥ 6.5. On the other hand, with more reluctant leavinggroups, at lower pH or temperature, or in non-aqueous mediathe subsequent steps may be much slower than the aci-decay.This should be kept in mind when release rates are determinedfrom kinetic data of the aci-decay.

Several photoremovable nitrobenzyl compounds carry ahydroxy group in the α-position (e.g. ‘nitr-5’, see section on

Scheme 4 Revised 17 mechanism for 2-nitrobenzyl derivatives.

Fig. 1 TRIR spectra of the reaction aci-3 4 in CD3CN.17

Photochem. Photobiol. Sci., 2002, 1, 441–458 443

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Table 1 Common nitrobenzyl ‘cages’

R R� Acronym

H H o-Nitrobenzyl NBMe H o-Nitrophenethyl NPECO2H H α-Carboxy-2-nitrobenzyl CNBH OMe 4,5-Dimethoxy-2-nitrobenzyl DMNBMe OMe 4,5-Dimethoxy-2-nitrophenethyl DMNPE

2,2�-Dinitrobenzhydrol DNB

H OMe 6-Nitroveratryloxycarbonyl NVOCMe –OCH2O– α-Methyl-6-nitro-piperonyloxycarbonyl MeNPOC

Photorelease of Ca2�, below). 2-Nitrobenzyl alcohol (6, R = H)was thus investigated as a model compound.19 Irradiation of 6(R = H) and 1-(2-nitrophenyl)ethanol (6, R = Me) generates2-nitrosobenzaldehyde and 2-nitrosoacetophenone, respect-ively. The mechanism of this efficient reaction was studied byLFP, TRIR spectroscopy, and 18O-labeling experiments. Theprimary aci-nitro photoproducts react by two competingreaction paths (Scheme 5).

The balance between the two paths depends on the reactionmedium and on pH. pH–Rate profiles for the decay of theaci-intermediates and for the formation of the final productswere determined in aqueous solution. Reaction via a hydratednitroso compound formed by proton transfer from the enoloxygen to the nitronic acid function of aci-6 prevails in aproticsolvents and aqueous acid or base (Scheme 5, path a). Cycliz-ation to benzisoxazoline intermediates followed by ringopening to the carbonyl hydrate of the 2-nitroso productspredominates in neutral water (path b). Dehydration of thenitroso hydrate (about 1 × 103 s�1 at pH 7) is much slower thanthe decay of the aci-form at pH < 8. The dehydration of thecarbonyl hydrate is still slower. A proton transfer mechanism(Scheme 5, path a) was suggested by Tsien and co-workers toexplain the remarkably fast release rates of the widely usedphotosensitive ‘nitr-5’ Ca2� chelators (6, R = BAPTA).20

Pfleiderer and co-workers have developed yet another variantof the o-alkyl nitrophenyl theme for the release of nucleosides.21

Here, the leaving group is attached to the β-position of ano-ethyl substituent. The mechanism of this reaction wasrecently investigated by LFP (Scheme 6).18 β-Elimination isfavoured with good leaving groups –OR under conditionswhere deprotonation of the aci-nitro transient is efficient.Otherwise, formation of the nitroso product (without release ofROH) prevails. This work also indicates that the cyclizationreaction yielding the nitroso product proceeds from the neutralnitronic acid, not from the anion, as shown in the revisedmechanism of Scheme 4.

Scheme 5 Reaction paths determined for nitrobenzyl alcohols 6.

Important nitrobenzyl ‘cages’

2-Nitrobenzyl derivatives have found numerous applications asphotolabile protecting groups. Some of the most common areshown in Table 1, along with the associated acronyms.

o-Alkylated aryl ketones

The photoenolisation of o-alkylaryl ketones discovered byYang 22 is a well-documented reaction analogous to the intra-molecular hydrogen shift of o-nitrobenzyl compounds. Severalattempts have been made to exploit the high efficiency of thisreaction and the high reactivity of the primary o-quinonoidphotoenols for the release of protected compounds. Efficientphotorelease (� = 0.76) of HCl from 2,5-dimethylphenacylchloride in methanol was reported in 1978 by Bergmark.23 Thereaction proceeds from the major photoenol product, (Z )-xylylene (τ = 22 µs, Scheme 7) that is formed predominantly

from the singlet excited ketone.24 Release of poorer leavinggroups is, however, too slow to compete with intramolecularreketonisation of the (Z )-photoenol. Thus, the release ofcarboxylic acids upon photolysis of 2,5-dimethylphenacyl

Scheme 6 Reaction paths for β-substituted 2-nitrophenethyl deriv-atives.18

Scheme 7 Photorelease of HCl from 2,5-dimethylphenacyl chloride.24

444 Photochem. Photobiol. Sci., 2002, 1, 441–458

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esters 25 occurs only from the longer-lived (E )-stereoisomer (τ ≈2 ms in methanol) that is formed only by the triplet pathway.26

Release from the β-position of o-ethylbenzophenones byphotoenolisation (analogous to Scheme 6 with (C��O)Phreplacing NO2) was achieved by Tseng and Ullman.27 A similarsystem, o-(2-chloroethyl) acetophenone, is currently beinginvestigated.28 In acetonitrile solution the reaction quantumyield is � = 0.36 and formation of the photoproduct o-vinylacetophenone is complete after 0.5 µs, as shown by LFP. Themechanism probably proceeds by HCl elimination from the(Z )-isomer.

The benzoin group

Benzoin has interesting properties as a photolabile protectinggroup. The advantages include high quantum yields and ratesof release, and formation of an inert benzofuran by-product(Scheme 8). Its strong absorption centred at 300 nm 29–32 allows

convenient monitoring of the reaction progress by UVspectroscopy. On the other hand, this characteristic may be adrawback (light absorption by the product). The same can besaid for the strong fluorescence exhibited by the benzofuran.33

Since Sheehan and Wilson 34 reported that 3�,5�-dimethoxy-benzoin (3�,5�-DMB) acetate was cleaved efficiently and cleanlyto give the corresponding benzofuran with � = 0.64, researchershave experimented with other substitution patterns in orderto optimise quantum yield and product distribution, whichalso strongly depend on the leaving group.29–31,33–37 The mostwidespread caging moieties are parent benzoin 32,35,38–41 and3�,5�-DMB.29,31,33,34,42–45 Quenching experiments have shownthat photorelease from unsubstituted benzoin and 3�-meth-oxybenzoin derivatives proceeds through the triplet-excitedstate.34,38,40

Givens and co-workers 38 reported on the photolysis ofbenzoin phosphate triester as well as on the salts of water-soluble mono and diesters. Irradiation of the triester at 300 nmproduced the free phosphate diester in about 30% yield inmethanol, benzene and acetonitrile. The efficiency of photo-release of ionizable phosphate esters in aqueous acetonitrilewas strongly dependent on pH. Best results were obtainedin acidic media, presumably because protonation of thephosphate produces a better leaving group. All reactions werequenched upon addition of either piperylene or naphthalene,and the triplet lifetimes estimated from the quenching data werebetween 2 and 7 ns.

A study of benzoin diethyl phosphate by LFP gave directevidence that 2-phenylbenzofuran is formed within 25 ns fromthe triplet state.41 In water, trifluoroethanol and hexafluoro-propan-2-ol an additional transient, λmax= 570 nm, was detectedand assigned to a triplet cation that is formed adiabaticallyby heterolytic dissociation of the triplet state. Irradiation intrifluoroethanol produced benzoin trifluoroethyl ether as anadditional product (Scheme 9).

A different mechanism was proposed for 3�,5�-DMB deriv-atives. The reaction is not affected by addition of commontriplet quenchers,33,34 indicating either that the triplet state isvery short-lived, or that the reaction proceeds from the excitedsinglet state. Several hypotheses have been advanced. Sheehanet al.34 proposed a Paterno–Büchi reaction of the singlet-excitedstate to form a strained bicyclic intermediate, followed by ringopening and loss of acetate to give the benzofuran. Pirrung 33

and Cameron 31 suggested heterolytic cleavage and formation

Scheme 8 Photoreaction of the benzoin group.

of an ion pair directly from the singlet state, followed by ringclosure and elimination.

Shi et al.43 reported nanosecond LFP studies of several3�,5�-DMB esters. They proposed a singlet mechanisminvolving an intramolecular exciplex and observed a short-livedcationic intermediate as a precursor of the benzofuran product.According to this mechanism, activation of the 2�-position ofthe benzene ring by the two m-methoxy substituents facilitatesintramolecular cyclization. More recently, Rock and Chanstudied the photochemistry of the water-soluble 3�,5�-bis-(carboxymethoxy)benzoin (BCMB, Scheme 8, R = -OCH2-COOH) acetate.46 In this case the major photoproduct wasBCMB and the yield of benzofuran was only 30%. A biradicalintermediate that undergoes acetoxy migration to give a cyclicprecursor of both the benzoin and the benzofuran products wasproposed.

Several reports have described photoactivatable benzoinphosphate esters.29,33,37–39 Corrie and Trentham 29 studied therelease of phosphate from 3�,5�-DMB ester by flash photolysis.Benzoin-caged cAMP was also investigated.38 The nucleotidewas released in nearly quantitative yield, with a quantumefficiency of 0.33. The reaction was quenched by sodiumnaphthalene-2-sulfonate and Stern–Volmer analysis gave atriplet lifetime of approximately 0.5 ns which, when combinedwith the measured quantum yield, afforded a release rateconstant kr = �r

3τ = 7.1 × 108 s�1.Pirrung and Shuey 33 prepared 3�,5�- and 2�,3�-DMB phos-

phate triesters. They reported efficient release of phosphateand lack of triplet quenching, confirming Sheehan’s earlierobservations on the DMB derivatives. They also developed asynthetic procedure to obtain optically active benzoin, to beused as a cage for chiral alcohols. DMB-protected ATP wasalso synthesised and tested.47 It was found to have a much fasterrelease rate (>105 s�1) than nitrobenzyl-caged ATP 2.

Photogeneration of amines was achieved from 3�,5�-DMB-carbamates.31,45 Cameron et al.31 studied the photorelease ofcyclohexylamine from 3�,5�-DMB and 3,3�,5,5�-tetramethoxy-benzoin carbamate both in solution and in the solid phase,providing a photogenerator of organic bases. It should benoted, however, that slow decarboxylation of the carbamateanion is likely to limit the release rates in these reactions:Papageorgiou and Corrie determined a rate constant of 150 s�1

for amine liberation by decarboxylation of N-carboxymethyl-N-methylcarbamate dianion at 21 �C, pH 7 using BromothymolBlue as an indicator.48

Peach et al.32 used the benzoin moiety to cage the carboxylicesters of some oligopeptides and devised a synthetic methodto obtain optically active esters. Pirrung 44 used 3�,5�-DMBcarbonates to protect alcohols and nucleotides. As the devel-opment of this phototrigger is fairly recent, only a few practicalapplications have been described.

The p-hydroxyphenacyl group

Several features render the p-hydroxyphenacyl (pHP) cage oneof the most promising alternatives to the nitrobenzyl deriv-atives. A straightforward synthetic route from commerciallyavailable p-hydroxyacetophenone yields the protected com-pounds. The derivatives are soluble in aqueous media and stablein biological buffers for over 24 h. The main by-product,

Scheme 9 Phosphate photorelease from benzoin diethyl phosphate.

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Table 2 Quantum yields of release of p-hydroxyphenacyl (pHP) cages

Caged compound Substrate �dis �appa Solvent Reference

ATP 0.37 0.30 Tris Buffer 49,50

Phosphate 0.38 n.d. Aq. CH3CN 50

Ala–Ala 0.267 0.253 D2O 51,52

GABA 0.35 n.d. Buffer 51

Glutamate 0.12 0.08 Buffer 51

Bradykinin 0.205 0.219 D2O 52

a n.d. = not determined.

p-hydroxyphenylacetic acid, is water-soluble as well andnon-toxic, unlike the products formed by photolysis ofnitrobenzyl derivatives. Moreover, the UV absorption of p-hydroxyphenyl acetic acid is blue-shifted with respect to that ofits precursor. Therefore, if the irradiation wavelength is chosenproperly, the photoproduct does not interfere with light absorp-tion, allowing quantitative conversion. Unlike the benzoinderivatives, binding of the pHP group to the substrate doesnot introduce a chiral centre, avoiding the problem of mixturesof diastereomers when dealing with chiral compounds. Last,but not least, the release is remarkably fast (see below).

Givens and co-workers reported photorelease of a variety ofbioactive molecules (including ATP,49 phosphate,50 glutamate,51

GABA,51 the dipeptide Ala–Ala,51,52 and the nonapeptideBradykinin 52) from the corresponding pHP derivatives. Thequantum yields, ranging from 0.1 to 0.4, are appealing (Table2). The product distribution depends on the leaving groupand on the solvent. While irradiation of pHP phosphatein aqueous Tris buffer yields free phosphate and p-hydroxy-phenylacetic acid, photolysis of pHP phosphate diethyl esterin either MeOH or t-BuOH yields p-hydroxyacetophenone asa main product.50

In all cases investigated by Givens’ group the reaction wasquenched upon addition of sodium naphthalene-2-sulfonate,indicating a reaction pathway through the triplet-excited state.Stern–Volmer quenching data yielded estimated triplet lifetimesof 0.5 ns for the ATP 50 and 6 ns for the Ala–Ala 52 derivatives.Later, Zhang et al.53 concluded that photorelease from pHPcarboxylic esters occurs directly from the singlet excited state.However, in a subsequent study of pHP diethyl phosphate usingnanosecond flash photolysis and picosecond pump–probespectroscopy, reaction via the triplet pathway was unequiv-ocally established.54 The triplet excited state was detected inacetonitrile (λmax = 380 nm), and was quenched upon admissionof oxygen and upon addition of piperylene, kq = 4.5 × 109 M�1

s�1. Its decay rate increased upon addition of water to the

solution, and the transient was no longer detectable bynanosecond LFP when the water content was above 10%.Consistently, pump–probe spectroscopy indicated a triplet life-time of 0.4 ns in a 1 : 1 mixture of acetonitrile and water.Quantitative agreement between the LFP and steady-statequenching data was obtained, which strongly supports amechanism where the triplet is the reactive state. The effect ofwater on the triplet lifetime is attributed to acceleration of theheterolytic phosphate release. It remains to be shown whetherthe apparent difference in the behaviour of pHP carboxylate 53

and phosphate 54 esters must be attributed to the leaving group.Apart from the multiplicity of the reactive state, the

mechanism of photorelease has not yet been established. Aworking hypothesis is shown in Scheme 10. According to this

mechanism the release of the protected ligand L is synchronouswith the decay of the exited triplet state (108–109 s�1). However,attempts to identify the spirodienedione intermediate by TRIRfailed.55

Scheme 10 Photocleavage of p-hydroxyphenacyl (pHP) derivatives.

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A disadvantage of the pHP protecting group is its lowabsorption coefficient at wavelengths above 320 nm. In anattempt to shift the π,π*-absorption to longer wavelengths,Conrad et al.56 synthesised 3,5-dimethoxy-pHP, which absorbsup to 400 nm. However, the quantum yield for release ofglutamate and GABA is reduced to 0.03–0.04 and the reactionfollows a different path. Along with the free substrates amixture is formed where the p-hydroxyphenylacetic acid eitheris a minor product or is not formed at all. The reactions werequenched by potassium sorbate, indicating a triplet pathway.The rate of release was assumed to be equal to the inverse of thetriplet lifetime, kr = 2.2 × 107 s�1. The biological utility of the3,5-dimethoxy-pHP photoprotecting group for GABA wastested measuring whole cell patch clamp recordings on CA1neurons in acute hippocampal brain slices.

Ras is a small protein that plays an essential role in cellularsignal transduction pathways. Its ability to transmit a signal canbe switched on upon binding with a GTP molecule, and turnedoff by hydrolysis of GTP to GDP. Du et al.57 applied the pHPand the 1-(2-nitrophenyl)ethyl (NPE) cages to the study of thishydrolysis reaction. The caged GTP–Ras complexes wereirradiated in an IR cell and their photolysis was followed byTRIR. In both cases the difference spectra showed positivebands ascribed to free phosphate and to GDP–Ras complexformation and negative bands due to the depletion of theGTP–Ras complex. In the experiment with the NPE groupseveral peaks were detected during the first minutes ofphotolysis, which were absent in the spectra obtained with thepHP derivatives. The former were attributed to a reactionproduct between Ras and nitrosoacetophenone. The observ-ation may serve as a caveat emptor for the use of nitrobenzylcompounds as photolabile protecting groups in biologicalsystems.

The coumarinyl group

Although a report by Givens and Matuszewski on the photo-activity of 7-methoxycoumarin-4-ylmethyl (MCM) estersdates back to 1984,39 the use of coumarin derivatives asphotolabile protecting groups is fairly recent.58–63 Irradiationof MCM esters in aqueous media leads to release of the pro-tected carboxylic acid and formation of the correspondinghydroxymethyl coumarin (MCM–OH, Scheme 11).60

The spectral range of irradiation extends to the visibleregion, and can be shifted further by addition of suitablesubstituents.62 Substituents are also important to regulate thehydrophilicity of the compound and to improve the quantumyield of the reaction.64 This is not very high,60,62,63,65 up to 0.25at most.64 Nevertheless, the high absorption coefficient, and thefast rate of photorelease make this system suitable for a numberof applications, such as photorelease of carboxylic acids,65

phosphates,39,65 cyclic nucleotides,58,60,61,63–65 and amino acids.62

Furuta et al. reported that the coumarinyl group is suitable fortwo-photon excitation experiments (vide infra), thanks to itsrelatively high two-photon excitation cross section.62 Theseauthors also reported that their MCM esters hydrolyzed in thedark with half-lives on the order of 50–200 h, faster than thecorresponding carbamates.

Schade et al.65 investigated the photolysis of several MCMesters and noticed a correlation between the reaction quantumyield and the leaving group ability of the protected substrate.

Scheme 11 Photocleavage of MCM esters.

Experiments in solvent mixtures showed that the efficiency ofthe photoreaction increased with the amount of polar proticsolvent in the mixture. These data suggested an ionic mech-anism. Photolysis of a phosphate ester in a 3 : 7 mixture ofacetonitrile and 18O-labeled water showed no isotope incorpor-ation in the free phosphate. The authors therefore proposed anSN1 mechanism involving heterolysis from the singlet-excitedstate to form an ion pair, implying that photorelease occurs onthe nanosecond time scale.

Direct evidence for fast formation of the alcohol MCM–OHfrom MCM phosphate esters comes from fluorescence studies.63

Irradiation of a solution of esters of 8-bromoadenosinecyclic 3�,5�-monophosphate and 8-bromoguanosine cyclic3�,5�-monophosphate yielded MCM–OH and was accom-panied by a considerable fluorescence enhancement. Thefluorescence kinetics gave evidence for re-excitation of thephotoproduct within the laser pulse, indicating that MCM–OHis formed within a few nanoseconds. Evidence put forth fora triplet state reaction is less convincing.62 The strongfluorescence exhibited by MCM–OH has been suggested as atool for monitoring the progress of the photoreaction.63 How-ever, in an experiment on HEK 293 cells, the fluorescence ofMCM–OH was almost completely quenched.

The synthesis and photochemical properties of a cagedcytidine 5�-diphosphate (caged CDP), which contains thephotolabile (7-diethylaminocoumarin-4-yl)methyl moiety, havebeen reported very recently. The caged CDP triggers the releaseof CDP when irradiated with wavelengths up to 436 nm. Therelease rate of CDP is 2 × 108 s�1 and the quantum efficiency forthe disappearance of caged CDP is 2.9%.66

�-Substituted methylphenols

Quinomethane intermediates are formed by water eliminationupon irradiation of α-hydroxymethylphenols.67 Eliminationof p-cyanophenol and of a tertiary amine was also shownto be efficient.68 Application of this reaction to the release ofcarboxylic acids is under study (Scheme 12).69

1-Acyl-7-nitroindolines

Corrie 70 and co-workers have revived a photoactive systemoriginally developed by Patchornik, 1-acyl-7-nitroindolines(NI). The proposed primary reaction is transfer of an acylgroup to the neighbouring nitro substituent (Scheme 13).

Scheme 12 Photorelease of acetic acid from 8-acyloxymethyl-1-naphthol.

Scheme 13 Proposed photolysis mechanism for 1-acyl-7-nitro-indolines.

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Experiments with 18O-labeled water showed that the oxygenatom introduced to form the free carboxylate is not from thesolvent. The rate constant for release of the phosphate ester of5-hydroxypentanoic acid [R = (CH2)4OPO3

2�] was determinedby LFP and TRIR techniques to be at least 2700 s�1, but morerecent work in progress 71 shows that the true half-time forphotorelease is in the sub-microsecond time domain. Thequantum yield of release is rather low, � ≈0.04, but suffices forpractical applications, because these compounds exhibitvery low physiological background activity.71 Furthermore, thequantum yield can be improved upon introduction of suitablesubstituents. A 4-methoxy-7-nitroindoline (MNI, R� = H)derivative of glutamate was photolyzed with an efficiency 2.2times greater than that of the analogous NI caged compound,with � = 0.085. Further mechanistic studies of this reaction arein progress.

A rather miraculous phototransformation of N-methyl-N-(o-nitrophenyl)carbamates to N-methyl-2-nitrosoaniline wasrecently observed by Loudwig and Goeldner.72 Alcohols areeasily protected and efficiently released on subsequent irradi-ation. It remains to be established whether the mechanismof this photoreaction bears any relation to that of the NIprotecting group.

Release initiated by electron transfer

Banerjee and Falvey designed a photoremovable protectinggroup based on photoinduced electron transfer.73 The systemconsists of a protecting group P attached to the substrateM and of a sensitizer S, which can either be bound to theprotecting group or just added to the solution (Scheme 14).

Upon irradiation S absorbs the light and transfers an electronto the protected molecule, giving the radical anion P–M��,which dissociates yielding the free substrate in its anionic form.To avoid the formation of free radicals, the sensitizer must becovalently linked to the protecting group. The main advantageof this strategy is that the irradiation wavelength can bechanged and shifted up to the visible by varying the sensitizerwithout affecting the release mechanism and yield. The protect-ing group of choice was the phenacyl group and it was used incombination with several sensitizers, among which N,N-dimethylaniline and 9,10-dimethylanthracene. Quantitativedeprotection was observed for many carboxylic acids botharomatic and aliphatic. Satisfactory results were also obtainedwith esters of t-Boc derivatives of glycine, phenylalanine,4-chlorophenylalanine and of the dipeptide isoleucine–glycine.The phenacyl ester of diethyl phosphate was converted quanti-tatively as well.

Deprotection of alcohols was achieved by protecting themas phenacyl carbonates.74 In this case a quantum yield ofdeprotection 5–6 times lower than for the carboxylic acid wasobserved. In all the above cases, the phenacyl-protected sub-strates were photolyzed in solution (typically acetonitrile) in thepresence of the sensitizers. The protected compounds quenchedthe fluorescence of either N,N,N�,N�-tetramethylphenylene-diamine or 9,10-dimethylanthracene with nearly diffusioncontrolled rate constants, indicating that the electron transferoccurs from the singlet excited state of the sensitizers. Lee andFalvey also described a system where sensitizer and protectinggroup are linked covalently.75 In the case of N,N-dimethyl-aniline they obtained efficient release of carboxylic acids,whereas no release was observed using an anthracene chromo-phore. A drawback of the method is its limitations regardingthe substrate. When the molecule to be protected is reduced

Scheme 14

more easily than the phenacyl group, undesired side reactionstake place.

Photoacids and bases

Numerous photoreactions produce acids or bases and can beused for pH-jump experiments.76 We note only two recentadditions to the variety of photoacids 77 and photobases 78 anda review on applications of photoacids in photoimaging.79

Applications to protein-folding studies are mentioned below.

Recent applications

Photorelease of neurotransmitters

One of the fields in which photoactivatable molecules have beenmost successful is that of the neural sciences. This broad area ofresearch has been covered in several reviews,3,5,80 so we willmention only a few examples of recent developments. Manyphotoactivatable derivatives of neurotransmitters and neuro-transmitter agonists are available. Among these are glutamate,γ-aminobutyric acid (GABA), glycine, carbamoylcholine,aspartate, phenylephrine, and kainic acid. These compoundshave proved useful in investigations on the mechanism of chem-ical synaptic transmission. When neurotransmitters bind to areceptor they cause either directly or indirectly the opening ofion channels. By combining the use of caged neurotransmitterswith the patch-clamp technique to measure the resulting cur-rent it is possible to monitor the kinetics of the process (Fig. 2).

Grewer et al.81 reported the development of a nitrobenzylcage with higher quantum yield of release and greater thermalstability at physiological pH. The α-carboxy-2-nitrobenzyl ester(CNB, Table 1) of glycine reacted with a quantum yield of0.38. The decay of the aci-nitro intermediate occurred withbiexponential kinetics with lifetimes of 7 and 170 µs, whichwere about 200 times faster than those of the previouslyreported N-protected CNB glycine. However, coincidenceof the release rate with the aci-decay rate was not established.The compound was biologically inert in the dark and uponphotolysis was able to induce whole cell currents in HEK 293cells. The same protecting group was used to obtain cagedserotonin,82 which was applied in studies on the serotonin5-HT3 receptors. Both O- and N-protected serotonin weresynthesised, but only the first showed the desired character-istics. Irradiation of the compound yielded an aci-nitro inter-mediate that decayed with a pH-independent lifetime of 16 µs.The quantum yield was only 0.03, but it was enough to activatethe receptors in NIE-115 mouse neuroblastoma cells. Theauthors pointed out that the pH-independent rate of thetransient decay and the yield of reaction were not in agreementwith results obtained for other caged phenols. They suggestedthat a different pathway of cleavage of the aci-nitro inter-mediate could be the reason, although no alternative reactionscheme was proposed.

Fig. 2 Activation of an ionotropic receptor by photorelease of aneurotransmitter.

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Gee et al.83 developed a new photoactivatable derivative ofN-methyl--aspartic acid (NMDA), a molecule that specificallybinds to NMDA-type glutamate receptors. A NMDA moleculeprotected with the CNB group had been described previously,but was not practically useful because it was biologicallyactive prior to photolysis. The use of a bulkier protectinggroup, 2,2�-dinitrobenzyl (DNB), successfully took care of theproblem. In rat hippocampal neurons (the cerebral area calledhippocampus is essential for the formation of memory) thecompound was inert in the dark and upon photolysis eliciteda current that rose with a rate constant of 100 s�1. The aci-nitrointermediate decayed with single exponential kinetics with apH-dependent lifetime, ranging from 3.8 µs at pH 3.8 to 13.8 µsat pH 10.6. The quantum yield of release was 0.18. A drawbackof this caging group is that it renders the compound lesswater-soluble, so DMSO has to be used as a co-solvent if highconcentrations are needed.

The same group investigated the benzoin esters of GABAand glutamate.40 The authors prepared O-desyl GABA, γ-O-desyl glutamate, α-O-desyl glutamate and N-desyl glutamate(desyl = 2-oxo-1,2-diphenylethyl). Irradiation of γ-O-desylglutamate and O-desyl GABA gave benzofuran with aquantum yield of 0.14, along with the released amino acids.The reactions proceeded through the triplet pathway, as provedby quenching experiments. On the other hand, photolysisof α-O-desyl glutamate and N-desyl glutamate gave benzil as amajor product, and in the case of N-desyl glutamate nobenzofuran was produced.

Bradykinin is one of the most active pain-producing agents.It is known to excite sensory neurons to produce an increase ofCa2� concentration. p-Hydroxyphenacyl (pHP) Bradykinin wasapplied to rat dorsal root ganglions 52 and photolyzed at 337 nmwith light from a nitrogen laser that was directed to the targetby fibre optics. The intracellular Ca2�-level was then measuredon single neurons using a calcium-sensitive fluorescent dye.A single laser pulse sufficed to produce a detectable transientsignal. When the Bradykinin receptor antagonist HOE 140 wasadded to the bath in a control experiment, irradiation failed toincrease the Ca2� concentration.

Kandler et al.84 used pHP glutamate in the study of long termpotentiation (LTP) and long term depression (LTD), two neuralprocesses that are thought to be involved in the mechanismof memory and learning. Thanks to the photoactivatableglutamate approach, the role of postsynaptic cellular changesin CA1 hippocampal pyramidal cells could be studiedindependently from presynaptic stimulation. The synaptictransmission was blocked and irradiation of the protectedamino acid induced a glutamate concentration jump. This,paired with depolarisation, produced LTD of glutamatereceptors, which proved that alteration of the postsynapticglutamate receptors occurs independently from presynaptic

neurotransmitter release. In the same study the use of com-mercially available forms of photoprotected glutamate (CNBand bis-CNB glutamate) were unsuccessful, because bothcompounds reacted in the dark inducing synaptic activity priorto irradiation.

Application of NI derivatives (Scheme 13) of glutamate,glycine, GABA and of a MNI caged glutamate have alsobeen reported.71 Irradiation of NI-glutamate in hippocampalneurons activated both AMPA- and NMDA-receptor mediatedcurrents. Monitoring of the kinetics yielded rise times of τ ≈ 0.6and τ ≈ 12 ms, respectively. Similar results were obtained withthe MNI derivative, which was also photolyzed more efficiently.In both cases the caged compound did not have any effect onthe receptors prior to irradiation. On the other hand, the NIderivatives of GABA and glycine were shown to act asantagonists at the target receptors, producing inhibition andslowing down the rate of activation by the uncaged neuro-transmitters. Finally, photolysis of a control compound thatreleased an inert phosphorylated carboxylate did not affect theAMPA and NMDA receptor responses, proving that thenitrosoindoline produced does not interfere with the systeminvestigated.

Photorelease of calcium

Binding of a hormone or a neurotransmitter, the ‘first messen-ger’, to the external domain of a receptor causes a change inthe level of a ‘second messenger’—an intracellular regulatorymolecule that triggers cell responses. The rapid increase inintracellular Ca2� concentration through the opening of Ca2�

channels in the plasma membrane or sarco/endoplasmicreticulum is the signalling event that is responsible forthe initiation of many physiological processes. Photolabilechelators that release Ca2� upon irradiation are used to definethe role of this important second messenger in cellularprocesses such as muscle contraction and synaptictransmission.

The ‘caging’ strategy for Ca2� ions is based on chelatormolecules, which change their affinity for calcium uponirradiation. The first to be developed were the ‘nitr’ series ofcompounds 20 and DM-nitrophen,85 based, respectively, on theknown BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N�,N�-tetraacetic acid] and EDTA (ethylenediamine tetraacetic acid)chelators. The two systems work on different principles: in thenitr series formation of a carbonyl group upon irradiationcauses a decrease of the electron donating ability of the chelate,lowering its ability to complex Ca2�. Irradiation of DM-nitrophen leads to cleavage of the chelator.

The main advantage of the nitr compounds over DM-nitrophen is their selectivity for calcium ions. On the otherhand, DM-nitrophen exhibits a much greater affinity change

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Table 3 Dissociation constants for several caged Ca2� chelators

Kd(Ca2�) a Kd-ratio Kd(Mg2�) a �Before After Before

Nitr-5 20 1.45 × 10�7 6.3 × 10�6 43 8.5 × 10�3 0.035DM-Nitrophen 85 5.0 × 10�9 3.0 × 10�3 600000 2.5 × 10�6 0.18NP-EGTA 86 8.0 × 10�8 1.0 × 10�3 12500 9.0 × 10�3 0.23DMNPE-4 87 4.8 × 10�8 ∼1 × 10�3 ∼21000 ∼7 × 10�3 0.20Azid-1 88 2.3 × 10�7 1.2 × 10�4 521 7.6 × 10�3 1

a Dissociation constants expressed in M. Values determined at pH 7.2 and 0.1–0.15 M ionic strength.

towards Ca2� upon photolysis, and it reacts with a higherquantum yield. Its affinity towards Mg2�, while useful for thephotorelease of magnesium ions, is a complicating feature forsome applications. Nitrophenyl-EGTA,86 and DMNPE-4,87 themost recent member in the family of nitrobenzyl derived caginggroups for calcium, work similarly to DM-nitrophen but exhibita higher affinity for calcium over magnesium. The data on thefour calcium photoprotective chelators are summarised inTable 3 and compared to those for azid-1, a novel systemdeveloped by Adams et al.88

Azid-1 was obtained from fura-2, a fluorescent calciumindicator, by adding an azide substituent on the benzofuran 3position. Upon irradiation the compound does not fluoresce,but undergoes an irreversible photochemical reaction yieldingfree calcium with unit quantum yield. This, together with a veryhigh absorption coefficient (33000 M�1 cm�1 at 342 nm) makesit a promising candidate for two-photon photolysis applications(vide infra).

Ellis-Davies and co-workers 89 investigated the rate of Ca2�

release from NP-EGTA and DM-nitrophen. The main questionwas whether the fragmentation rate of the chelate was equal tothe rate of calcium release. The possibility that the dissociationmight occur from a fragment of the photolyzed chelate (with arate dictated by its own dissociation constant) could not beexcluded a priori. Monitoring of the emission kinetics of afluorescent Ca2� indicator, Ca-orange-5N, yielded rate con-stants of 6.8 × 104 and 3.8 × 104 s�1 for Ca2�-release fromNP-EGTA and DM-nitrophen, respectively. Flash photolysisof the chelate yielded biexponential kinetics that were attrib-uted to the decay of an s-cis and an s-trans form of the aci-nitroanion intermediates with rate constants of 5 × 105 and1 × 105s�1 for NP-EGTA, and 3.8 × 104 and 1.1 × 104 s�1 forDM-nitrophen. Based on their kinetic model the authorsconcluded that, in the case of NP-EGTA, the rate-limiting stepis the release from the post-photolysis complex, whereas it isthe chelator fragmentation in the case of DM-nitrophen.Surprisingly, the 458 nm transient decays, which were attributedto the aci-nitro intermediates, hardly depended on pH. Tsienand co-workers 20 monitored formation of the unchelatednitrosobenzophenone chromophore at 366 nm by flashphotolysis of nitr-5 in the presence of a weak Ca2� chelator.The major fraction appeared with a rate constant of k = 3000s�1, followed by about one third of the total absorbance changewith k = 0.17 s�1. These rates were taken to be a lower limit forthe release of Ca2� from nitr-5.

The ability to produce a jump of the otherwise very low (10�7

M) Ca2� concentration within the cytoplasm has been usedextensively in the investigation of a number of cellular events,including muscle contraction and neurotransmitter release. Theinterested reader is referred to the copious literature on thesubject.5,90

Photorelease of other second messengers

A different approach to induce a calcium concentration jump incells is the use of photoactivatable inositol 1,4,5-triphosphate 91

(InsP3). When InsP3 binds to receptors located on theendoplasmic reticulum, it causes the opening of calciumchannels and the release of Ca2� from internal stores into the

cytoplasm. Li et al.92 synthesised a DMNB ether of an InsP3

analogue and esterified the three phosphate groups to renderthe molecule cell-permeable. Once inside the cell the esterswere cleaved by intracellular esterase. The compound wasphotolyzed at 365 nm with a quantum yield of 0.09. The releaseof the InsP3 analogue elicited Ca2� spikes that stimulated geneexpression by activating the nuclear factor of activated T cells(NF-AT). Interestingly, using a given amount of caged InsP3,the amplitude of the response was dependent on the irradiationprotocol. The most effective consisted of a sequence of lightflashes separated by 1 min intervals. Either more frequentpulses or continuous irradiation resulted in a poorer NF-ATactivation. The results point to the importance of concen-tration oscillations of Ca2� (or possibly of InsP3 itself ) withinthe cell.

Nicotinic acid adenine dinucleotide phosphate (NAADP) isa potent calcium mobilising agent discovered in sea urchineggs. Nanomolar concentrations suffice to trigger Ca2� releasefrom intracellular stores by stimulating its receptors. NAADPwas photoprotected through a reaction with 1-(2-nitrophenyl)-diazoethane.93 In an experiment on live eggs, photolysis ofcaged NAADP occurred with only 1% efficiency. Nevertheless,due to the high sensitivity of the system, it induced Ca2� oscil-lations, monitored by a fluorescence indicator, that lasted formore than 30 minutes. Having previously eliminated externalcalcium, this proved unequivocally that Ca2� release occurredfrom intracellular stores. Similar results were obtained withanother newly discovered second messenger, cyclic adenosinediphosphate ribose (cADPR).94 In both cases photolysis couldbe induced by the light of a spectrofluorimeter, which indicatesthat the two caged compounds can be conveniently used forexperiments in any laboratory.

The role of nitric oxide in cellular signalling has become oneof the most rapidly growing research fields in biology.95 Notonly does NO stimulate formation of cGMP, a key player in theregulation of various physiological processes, it also functionsas a retrograde messenger. Being able to cross the plasmamembrane, this small molecule can carry information fromthe postsynaptic neuron back to the presynaptic cell and isprobably involved in LTP. Makings and Tsien described cagingof nitric oxide 96 with a system based on the known decom-position of diazeniumdiolates (Scheme 15, Table 4).97

The photosensitive diazeniumdiolate derivatives 9–13 wereirradiated in aqueous solution and surprisingly produced freeNO within the 5 ms resolution of the LFP set-up. In spite of thelow quantum yields (Table 4), the compounds were able toinhibit thrombin stimulated platelet aggregation. Moreover,depending on the substituents R, it was possible to achieveextracellular-only (with compound 12), or intracellular-only(with compound 13) NO release. In the latter case, the esterifiedcarboxylic groups allowed the molecule to permeate through

Scheme 15 Decomposition of diazeniumdiolates.

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the plasma membrane into the cell, where the esterases restoredthe carboxylic groups, trapping it inside.

A recent study on the photochemistry of some diazenium-diolate derivatives in acetonitrile, among which compound 10,revealed that NO is probably not produced by the expectedphotodeprotection of the diazeniumdiolate anion, but ratherby secondary photolysis of the main reaction product N-nitrosodiethylamine. The latter has been shown to be carcino-genic in a number of species and this might render practicalusage problematic. Studies with similar systems producingnon-toxic nitrosamines are in progress.98

Some alternative compounds that photorelease NO havebeen described. Irradiation of ruthenium nitrosyl chlorides,in particular the compounds RuNOCl3 and K2RuNOCl5 at300–350 nm led to production of free NO with Φ = 0.012 and0.06, respectively. LFP showed that the release occurred within1 µs. The efficacy of the compounds was shown by their abilityto induce relaxation of contracted rabbit aortic rings.99

Very promising results for the photorelease of NO wereobtained with derivatives of N,N�-dimethyl-N,N�-dinitroso-p-phenylenediamine (14) developed by Fujimori and co-workers(Scheme 16).100 The release of two NO radicals from a single

molecule ensures formation of a closed-shell (nonradical)product along with free NO. Substituents are used to tunetheir solubility, so that 15 is soluble in water, 14 in lipids, and16 in various organic solvents. The compounds exhibit strong

Scheme 16 Photorelease of NO from 14–16.

Table 4 Quantum yields of NO formation 96

R �NO

HOCH3

OCH2CO2EtOCH2CO2

�K�

OCH2CO2CH2O2CCH3

0.050.010.020.010.02

910111312

absorption extending to 425 nm. The chemical yields of NO arequantitative and �NO ≈ 2. Thus, the product ε� is much higherthan those reported for other NO cages, 6 × 103 M�1 cm�1 for15 against 15–70 M�1 cm�1 of compounds 9–13. LFP showedthat NO release occurs in two steps, with τUC1 < 10 ns and τUC2

between 5 and 19 µs. The efficacy of 14–16 in biological studieswas proved by the photoinduced relaxation of rat aortic strips.

Adenosine 3�,5�-cyclic monophosphate (cAMP) is an activ-ator of protein kinases. Furuta et al. described the photo-chemistry of an MCM ester of cAMP.58,60 Irradiation at340 nm in either dioxane–water (1 : 1) or 1% DMSO in Ringer’ssolution produced free cAMP and (4-hydroxymethyl)-7-meth-oxycoumarin with � = 0.07 and 0.012, respectively. Comparedto desyl-protected cAMP the compound was stable muchlonger in Ringer’s solution. In experiments on fish melano-phores, the MCM-cAMP permeated into the cells was able,upon UV irradiation, to stimulate the dispersion of pigmentgranules, which is known to be regulated by intracellularcAMP. Even higher biological responses were obtained using7-acyloxycoumarin-4-ylmethyl esters.61 These were synthesisedwith the purpose of tuning the lipophilicity of the system, inorder to favour penetration into cells. Once inside the cell,the ester group was cleaved by intracellular esterases to givea hydroxycoumarin (HCM–OH). The higher hydrophiliccharacter of the HCM-esters prevented the system from leakingout of the melanophores, so higher intracellular concentrationswere achieved.

Photorelease of peptides and proteins

Peptides exhibit a wide range of biological activities andfunction, for example, as hormones or neurotransmitters.Synthetic peptides can be used as selective inhibitors of proteinactivity. Therefore, photoactivatable peptides have the potentialfor extensive application. The design involves identification ofamino acid substitution patterns that inhibit peptide activity,for example preventing the peptide from binding to a targetprotein.

One approach is to introduce an amino acid previouslyprotected with a photolabile protecting group at the desiredposition using automated solid-phase peptide synthesis. Walkeret al. used this method to insert nitrobenzyltyrosine into RS-20,a target peptide for calmodulin, which binds calcium and isinvolved in a number of Ca2� mediated events.101 The affinity ofthe caged RS-20 for calcium–calmodulin was 50 times lowerthan that of RS-20. The system was used to study the role ofthe calcium–calmodulin complex and myosin II in the rapidlocomotion of newt eosinophils (leukocytes). Similarly, Tatsu etal.102 synthesised caged neuropeptide Y (NPY), a 36-amino acidpeptide that contains 5 tyrosine residues, two of which are atthe N- and C-termini. The authors introduced 2-nitrobenzyl-tyrosine at either one or both termini, and assessed that the

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biological activity was reduced by about one order of mag-nitude in the mono-protected peptides, and two orders ofmagnitude upon introduction of nitrobenzyl groups at bothtermini. The activity was promptly restored upon UV irrad-iation. An attempt to obtain caged Angiotensin II with thesame approach was unsuccessful, because the nitrobenzyl groupdid not inhibit the activity of this peptide.

The main drawback of this method to obtain photoactiv-atable peptides is that the current synthetic technology islimited to about 70 amino acids. Another possibility is tochemically modify targeted amino acids of an already existingpeptide. Kemptide is a peptide used as a substrate for cAMP-dependent protein kinase (PKA). Pan and Bayley used 2-nitrobenzyl derivatives to protect the thiol group of cysteinein C-kemptide (Leu-Arg-Arg-Ala-Cys-Leu-Gly), and of thio-phosphoryl serine in kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly).103 In the case of a kemptide CS-dimer (a 14-mercontaining one unit of C-kemptide and one unit of kemptide),they were able to selectively protect thiophosphoryl serine inthe presence of a cysteine residue. The caged peptides weredeprotected in aqueous solution with quantum yields rangingfrom � = 0.06 to 0.62.

Phosphorylation of proteins on tyrosine residues is one ofthe most important post-translational modifications, playingcentral roles both in physiological processes such as transmem-brane signalling and in pathological processes such as cancerand immune dysfunction. The levels of tyrosine phosphoryl-ation are regulated by the opposing actions of protein tyrosinekinases (PTKs), which catalyse formation of phosphotyrosine(pY) in proteins, and protein tyrosine phosphatases (PTPs),which hydrolyse pY residues. Pei and co-workers 104 found thatα-haloacetophenone derivatives such as pHP are potent,photoreversible, and membrane-permeable inhibitors of PTPs.Bayley and co-workers 105 reported that tyrosine-containingpeptides can be thiophosphorylated with cognate kinases, andsubsequently caged by reaction with electrophilic reagents suchas 2-nitrobenzyl bromide or pHP bromide. The caged peptidesare no longer able to bind to an SH2 domain, but this activity isrestored upon photolysis.

For photoactivatable proteins the synthetic task becomes evenmore challenging.106 Reversible control (switching) of proteinactivity with photoisomerizable molecules has been achieved.107

Here, we consider irreversible photoactivation (triggering) ofinactivated proteins. One way to do it is by coupling the proteinwith a reagent aimed at one particular kind of amino acid. Itmay, however, be difficult to identify exactly where along thepeptide chain the modification has taken place. Techniquessuch as UV spectroscopy can help to estimate the number ofprotecting groups present. With such an approach Marriottobtained caged G-actin using NVOC-Cl, a well-known photo-sensitive reactant for amines.108 The protein contained betweenthree and five protected lysine residues. These were sufficient tohamper its polymerisation to F-actin, which was triggered withmore than 90% efficiency upon irradiation.

Optimisation of the protecting ratio plays an essential rolein the synthesis of caged proteins. An excessive number ofprotecting groups may denature the protein or render itinsoluble, and it may make photoactivation less efficient. On theother hand, too small a labelling ratio does not guarantee thatthe essential amino acid that needs to be modified will beprotected, because this will not necessarily be the most reactive.The procedure developed by Bayley mentioned above forpeptides is suitable to be used in the synthesis of photoactivat-able proteins as well, where cysteine residues that are eithernaturally occurring within a protein or that have been intro-duced by mutagenesis at desired positions can be protected. Anexample is the protection of a C subunit of cAMP-dependentprotein kinase, where derivatisation of the Cys-199 residueinhibited the enzymic activity, which increased 20–30 fold uponirradiation (Fig. 3).109 This caged kinase provides an important

new reagent to investigate cell signalling. With a similarapproach, using 4,5-dimethoxy-2-nitrobenzyl bromide as aphotolabile reagent to protect Cys-707, Marriott and Heideckerobtained caged Heavy Meromyosin.110

Photoactivatable fluorophores

Photoactivatable fluorophores are molecules that yield a fluor-escent species upon irradiation. They have been obtained bycoupling a fluorescent dye to a photoremovable protectinggroup which prevents it from displaying its usual emission.UV-irradiation frees the fluorescent species and restores itsabsorption and emission properties in the visible region. Atypical example is photoactivatable fluorescein.111,112 The freespecies exists as two tautomeric forms, a fluorescent carboxylicacid and a non-fluorescent lactone. Caging is achieved by intro-ducing two nitrobenzyl groups at the phenolic oxygens, whichconstrain the molecule in its lactone form, and which can beremoved upon irradiation, yielding the fluorescent carboxylicacid tautomer. By an analogous strategy caged rhodamineshave been developed.111 Other successful examples of photo-activatable fluorophores are caged resorufin 111,112 and caged8-hydroxypyrene-1,3,6-trisulfonic acid (caged HPTS).113

Successful application of such compounds has been reportedin various fields. The ability to easily monitor the formationof the deprotected substrate makes them useful calibrationsystems. Kiskin et al. used NPE-HPTS in an assay of synapticphotodamage upon two-photon excitation.114 The samemolecule was used to estimate the extent of photolysis ofanother caged compounds.113 Jasuja investigated the chemo-tactic responses of Escherichia coli to small aspartate con-centration changes generated by the photolysis of caged-aspartate. Comparison with the fluorescence of HPTSobtained upon irradiation of its non-emitting NPE-ether wasused to assess the amount of free aspartate produced in thebiological assays. Since the protected substrates do not displaya particular biological activity, practical application ofthese compounds often involves covalent linking to some

Fig. 3 Ribbon diagram of the ‘closed’ form of the wild-type C subunitof protein kinase complexed with an inhibitor peptide (red) and ATP.Thr-197 and Cys-199 are in the ‘activation loop’ of the kinase (yellow).Reprinted with permission from ref. 109 © 1998 American ChemicalSociety.

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macromolecule of interest. This way they function as photo-activatable fluorescent tracers.

Theriot and co-workers used caged resorufin coupled toG-actin to investigate the intracellular motility of Listeriamonocytogenes.115 This bacterium, a common food pathogen,once inside the infected cell rapidly induces polymerization ofG-actin to filaments forming ‘comet tails’, which quickly propelit through the cytoplasm. Irradiation of the tails allowedmonitoring of the movement and turnover of the labelledfilaments by fluorescence videomicroscopy, giving useful insightinto the general actin-based motility of pathogens within cells.

Vincent and O’Farrell applied a photoactivatable fluorescenttracer to the study of cell lineage during Drosophila embryosdevelopment.116 An NB-caged fluorescein (where NB wasmodified to increase water solubility) was connected to adextran backbone and the latter to a nuclear localizationpeptide. The dextran molecule served the purpose of preventingintercellular diffusion, whereas the peptide induced localizationof the tracer at the nuclei, improving the physical separationamong the targets and making it easier to distinguish the singlecells. The compound was injected into the synctyal blastoderm,i.e., at the stage when the nuclei derived from the early mitoticdivisions still reside in a common cytoplasm, allowing the tracerto diffuse and localize at the nuclei. After the subsequent cellu-larization phase, when membranes form around each nucleusand the embryos acquire a cellular organization, single cells werefluorescence-labelled upon irradiation. This way the develop-ment during the subsequent steps could be followed. The methodis suitable for application on live specimens, so that with the aidof a CCD camera “movies” of gastrulating embryos (gastrul-ation is the step following cellularization, where the cells arereshuffled to form three germinal layers) can be recorded.

Along with the successful application some limitations ofthe photoactivatable fluorophores developed so far have beenpointed out.111 Caged fluorescein, for instance, is highlyhydrophobic, and most proteins tend to aggregate if they arelabelled with it. Both caged fluorescein and resorufin are subjectto photobleaching after activation, which causes problems intheir imaging in cells.

Two-photon excitation

One of the advantages of using photoactivatable compounds inthe investigation of biological systems is the improved temporaland spatial resolution of the experiments compared to othermore conventional techniques. A further step in this direction isgiven by two-photon excitation of photoactivatable com-pounds, which is able to give three-dimensional control over thelocalisation of substrate release.117

In two-photon photolysis UV excitation is replaced by thesimultaneous absorption of two IR photons of equivalent totalenergy. The probability of excitation is proportional to I2 and toδ, where I is the light intensity and δ the two-photon absorptioncross section measured in GM (1 Göppert-Mayer = 10�50 cm4 sphoton�1). Therefore, the event is confined to a small regionnear the laser focus and is negligible in the surrounding area, sothat the release can be restricted to volumes smaller than afemtolitre.118,119 A further advantage of this technique is that IRphotons are less likely to cause photodamage to biologicalmolecules.

However, a word of caution comes from some recent studiesthat show how the toxicity of the irradiation is related to thelaser power.114,120 In the systems investigated, an upper limit of5–10 mW was established, above which photodamage due tomultiphoton absorption set in. The photosensitivity of amolecule to two-photon excitation is expressed by its two-photon action cross section δu, which is given by �δ, where � isthe reaction quantum yield. The application of the techniquehas been hitherto hindered by too low δu values of the availablephotoactivatable groups, such as those of nitrobenzyl deriv-

atives. It was estimated that the δu needed for practicalapplication at safe laser power values is higher than that of anyknown photoremovable protecting group (3–30 GM).114,120

Nevertheless, successful two-photon uncaging of calciumfrom azid-1 and glutamate from a coumarinyl derivative hasbeen achieved. Brown et al.119 compared the two-photon actioncross sections of DM-nitrophen, NP-EGTA and azid-1. Azid-1and DM-nitrophen had a maximum cross section of ∼1.4 GM at700 nm and ∼0.013 GM at 730 nm, respectively. NP-EGTA didnot show any detectable uncaging signal. The authors calculatedthat a 10 µs pulse train of ∼7 mW at 700 nm would photolyze allazid-1 within the focal volume, whereas DM-nitrophen wouldneed about 74 mW at 730 nm in the same conditions, a valueprone to cause damage in the biological environment. Yet,DM-nitrophen has been used to generate spatially-confinedartificial Ca2� sparks inside cardiac myocytes.121

In another two-photon excitation experiment 62 the 6-bromo-7-hydroxycoumarin-4-ylmethyl group was linked to -glutamatevia a carbamate bond. The system had a two-photon uncagingcross section of ∼ 1 GM at 740 nm. It was applied to brain slicesof rat cortex and hippocampus. Monitoring of the currentgenerated by two-photon scanning photochemical microscopyprovided three-dimensional maps of the glutamate sensitivityof neurons.

Time-resolved X-ray

Because of the long time required for crystallization (days orweeks) it is not possible to co-crystallize an enzyme–substratecomplex; the reaction would take place long before the crystalswere ready for data collection.122 Therefore one of the bigchallenges in time-resolved X-ray measurements is finding asuitable way to trigger the reaction within the crystal.

Photolabile protecting groups may be used in combinationwith Laue crystallography for mechanistic studies of enzymaticactivity. A short laser pulse initiates the reaction and a delayedsynchrotron X-ray pulse is used to monitor reaction progressby time-resolved structure analysis. Irradiation of a photo-activatable substrate or coenzyme within the crystal can rapidlytrigger the enzyme turnover, allowing crystallographic detec-tion of reactive intermediates. The intrinsic time resolution ofsuch experiments depends on the duration of the two pulsesand on the jitter in the time delay between them. At present it ispossible to reach the subnanosecond domain.123 A spectacularexample is provided by the nanosecond crystallographicsnapshots taken of protein structural changes following thephotodissociation of CO from the heme of myoglobin.124

Nitrobenzyl derivatives were used in the few examples withartificial photolabile groups reported so far, but protectinggroups with faster release rates will be required to make themost of this powerful technique. Alternatively, cryophotolysisfollowed by appropriate temperature profiles might be used totrap reaction intermediates in protein crystals for study bymonochromatic X-ray crystallography.125

Schlichting and co-workers were the first to use photoactiv-atable GTP in a time-resolved Laue study.126 A crystal of theprotein with NPE-GTP at its active site was used to obtain thestructures of the Ras p21-GTP and Ras p21-GDP complexesand to investigate the conformational changes accompanyingGTP hydrolysis.

Photoactivatable NADP was developed and used to invest-igate the activity of isocitrate dehydrogenase.127 This enzymecatalyses the oxidation–decarboxylation of isocitrate to α-ketoglutarate in a reaction that requires formation of a ternaryenzyme–isocitrate–NADP complex. Introduction of the CNBgroup on the nicotinamide moiety produced a ‘catalytically-caged’ form of NADP (17), which could bind to the enzyme butwas not able to exert its oxidising function.

Incorporation of the DMNPE group on the 2�-phosphate ledto formation of an ‘affinity-caged’ NADP (18), which was

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unable to interact with the enzyme prior to photolysis. Thequantum yields of NADP release from the two compoundswere assumed to be equal to those of formation of theiraci-nitro intermediates that were 0.09 and 0.19, respectively. Inthe case of DMNPE-NADP (18) the intermediate decay in thepresence of dithioerythritol (DTT) was fitted with a doubleexponential and the fast component, 1.3 × 104 s�1, was assumedto represent the rate of product formation. The single expon-ential decay of the transient of CNB-NADP (17) exhibited amuch slower decay rate, 30 s�1. The two systems were testedalong with NPE-isocitrate 128 in time-resolved crystallographicexperiments, whereby the effects of different triggeringstrategies could be observed. The best results were obtainedusing DMNPE-NADP (18). The compound allowed pre-binding of isocitrate in the dark and upon irradiation releasedNADP, which could bind with full affinity to the enzyme andundergo the redox reaction. The α-ketoglutarate–enzyme com-plex, which has a half-life of about 10 ms, could be visualisedand its structure determined.

Goeldner and co-workers reported preliminary experimentson caged choline and carbamylcholine, with a view to applyingsuch systems to study the enzyme cholinesterase by time-resolved crystallography.129

The speed limit of protein folding

Kinetic studies of protein folding were hitherto largely limitedto stopped-flow methods with a time resolution of 100 µs atbest. The formation of local structures is thought to take placeon time scales of τ ≈ 1 µs (β-hairpins) and τ ≈ 0.1 µs(α-helices).130 Laser-induced temperature or pH 131 jumps, aswell as energy transfer methods 132 are increasingly applied tostudy these processes. Hochstrasser and co-workers 133 reporteda clever attempt to develop an optical trigger for proteinfolding. Aryl disulfides embedded in a polypeptide, such thatthe disulfide bridge constrains the peptide in a nonhelicalconformation, were cleaved within 1 ps of excitation andformed arylthiyl radicals with strong absorption in the visibleregion. However, the envisaged time-resolved studies of foldinginto a α-helical conformation were hampered by rapid geminaterecombination of the thiyl radicals.

More recently, the benzoin group was used to investigate thekinetics of α-helix formation.36 A subdomain (35 residues) ofthe protein villin, which autonomously folds to an α-helicalstructure in aqueous solution, was used. A small loop wasformed by binding the N-terminus of the peptide with a cyst-eine residue on the side chain of one of the internal amino acids(Scheme 17) using the benzoin as a linker. The cross-link, whichprohibits folding, was cleaved by steady-state irradiation, and

Scheme 17 Determination of protein folding kinetics.

α-helix formation of the resulting linear peptide was establishedby observing the changes in circular dichroism. Time-resolvedphotoacoustic calorimetry, which detects volume changes,revealed two kinetic phases with time constants of about 100and 400 ns. It was deemed likely that helix formation wasresponsible for at least one of the phases. More informativemethods such as time-resolved IR spectroscopy will be requiredto establish this point. The approach holds promise for furtherdevelopments.

Photoactivation of gene expression

A report appeared describing the first application of a photo-activatable protecting group to targeted expression of geneticmaterials.134 Plasmids coding for luciferase were photoprotectedwith the DMNPE group and introduced into rat skin cells.Luciferase expression was suppressed by the presence of theprotecting group and was triggered by irradiating the cells with355 nm laser light. Similar results were obtained in vitro usingprotected Green Fluorescent Protein (GFP) plasmid in HeLacells. Transcription assays showed that the blocking occurredat the level of mRNA production. In both cases it was notpossible to obtain complete restoration of biological activity. Itseems unlikely that a reaction of the nitrosoketone inhibitsplasmid expression. In fact, it was estimated that less than 3 %of the phosphate groups in the plasmid were photoprotected,so the concentration of nitrosoketone produced should bemuch lower than 0.001 M (∼10�6 M in the in vitro experiment),a typical value in caged compound experiments.

Targeted genetic expression has been recently achieved usingcaged mRNA in zebrafish embryos.135 Reaction of 6-bromo-4-diazomethyl-7-hydroxycoumarin with mRNA encoding GreenFluorescent Protein (GFP) produced caged Gfp mRNA,probably by formation of ester bonds with the phosphates, withan average ratio of approximately 1 coumarinyl group per 35bases. Experiments in vitro and in vivo showed that cagingcaused almost complete loss of translational activity of themRNA. Injection of embryos with photoactivatable mRNAresulted in severely reduced or no GFP fluorescence when com-pared to those with intact Gfp mRNA. Irradiation with350–365 nm laser light led to substantial recovery of the GFPsignal. Positive results were also obtained with photoactivatableβ-galactosidase (lacZ) mRNA and eng2a mRNA. Finally, thesame coumarinyl group was used to prepare photoactivatableplasmid DNA encoding GFP and proved able to inhibittranscription in vivo, which could be restored by uncaging.

Synthesis of molecular arrays and other applications

Nitrobenzyl derivatives have been applied to light-directedsynthesis of high-density arrays of peptides and oligonucleo-tides (biochips). In this technique the protected ‘buildingblocks’ are attached to a solid support and irradiated through amask. Deprotection occurs selectively on the molecules exposedto the light, leaving the others unchanged. The free functionalgroups are then ready for coupling with a second ‘caged’molecule. Irradiation with a different mask leads to photo-activation of another set of molecules, which are now free toreact. Repetition of these irradiation and coupling steps, usingmasks with varying patterns and different compounds, leads tothe synthesis of the desired set of products.

Fodor and co-workers, who were the first to report thisapplication,136 succeeded in obtaining an array of 1024 peptidesusing the NVOC protecting group. The ability to producehigh-density oligonucleotide arrays, the so-called DNA chips(GenChip® probe arrays), is utilised by Affymetrix in DNAsequencing technology, using the Sequencing By Hybridisation(SBH) method to supplant the current laborious electro-phoretic techniques. SBH involves the use of short oligonucleo-tide probes to search for complementary sequences in a longerDNA strand. Pease et al.137 showed the utility of this technique

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by synthesising an array of 256 octanucleotides. Fluorescein-labelled octanucleotides of known sequence were used to provethe specificity of the binding. Epifluorescence measurementsshowed that complementary probes yielded signals 5–35 timesstronger than those with single or double base-pair mismatches.

With a similar approach a method for immobilisation ofmacromolecules at precise locations on solid supports wasdeveloped, based on the high-affinity biotin–streptadivin-binding interaction.138 Photoprotected biotin is covalentlyattached to a solid support. Irradiation of the surface through aphotolithographic mask yields free biotin in the selectedregions, which is now available for coupling with streptadivin.Finally a solution of a biotinylated macromolecule is applied tothe system, and the desired compound bound to the surface.The process can be repeated to attach other molecules ontoother locations. The authors used the system to immobilisetwo antibodies, mouse and rabbit IgG. The localisation of thetwo molecules was evident upon addition of fluoresceinatedsecondary antibodies.

Vossmeyer and co-workers have reported an interestingextension of Fodor’s technique, where the NVOC group wasapplied in the light-directed assembly of nanoparticles ontoa solid substrate.139 NVOC-Protected glycine was attached to asilicon or glass plate. Irradiation through a microchip maskproduced a pattern of free amino groups on the surface. Theplate was treated with a solution of 12-aminododecane-cappedgold particles, which underwent ligand exchange with the freeamino groups, yielding selective binding of the gold particlesonto the irradiated portions of the plate. Subsequently, in orderto amplify the effect, a solution of octane-1,8-dithiol was usedto generate free surface-bound thiol groups in the area wheregold particles were already attached to the surface, and thesystem was treated again with the nanoparticle solution.Repetition of the dithiol amplification process led to formationof well-defined patterns. With an analogous approach, usingyellow and red luminescent CdSe/CdS-core/shell structurednanocrystals, repeating the deprotection and cappingsteps, polychromatic patterns were obtained. The techniquecould find application in the preparation of multicolourednanocrystal-based photonics devices.

Other interesting applications in various scientific fields havebeen reported. In searching for a new way to obtain patternedfunctional images on polymer films, Kim and co-workers 140,141

synthesised a methacrylate copolymer with pendant aminogroups protected by the α-MNVOC (α-methylnitroveratryl-oxycarbonyl) group. The polymer was spin-coated on a plateand irradiated through a mask. It was then immersed into a dyesolution and the chemisorption of the dye resulted in clearcoloured patterns.

In an application to analytical chemistry, 4,5-dimethoxy-nitrobenzyl was used to photodeprotect 3-(4-hydroxyphenyl)-propionic acid (HPPA), a reagent used in the assay ofthe enzyme peroxidase. The uncaging of HPPA in situ uponirradiation at 350 nm circumvents the problem of the poorstability of the compound in solution.141

Photosensitive dendrimers were obtained by incorporatingphotolabile groups near the core unit of benzyl aryl etherdendrimers. Irradiation led to fragmentation into dendrimerfragments.142 Watanabe and co-workers 143 synthesised dendriticcaged compounds, consisting of a dendritic core and α-carboxy-2-nitrobenzylleucine–leucine methyl ester attached at itsextremities. Photolysis of the dendrimer yielded freeLeuLeuOMe. The efficiency improved as the generation of thedendritic core increased.

This review covers an arbitrary selection of the abundantrecent developments, which have been published up to the endof 2001, in the area of photoremovable protecting groups. Wehope to have whetted the appetite of readers not previouslyworking in the field, so that they will be stimulated to contributefrom their own area of expertise.

Acknowledgements

This work was supported by the Swiss National ScienceFoundation, Project No. 20-61746.00. We thank ProfessorJ. E. T. Corrie for valuable comments on the manuscript.

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