Applications of p-hydroxyphenacyl (pHP) and coumarin-4-ylmethylphotoremovable protecting groups†
Richard S. Givens,*a Marina Rubinab and Jakob Wirzc
Received 7th December 2011, Accepted 20th January 2012DOI: 10.1039/c2pp05399c
Most applications of photoremovable protecting groups have used o-nitrobenzyl compounds and their(often commercially available) derivatives that, however, have several disadvantages. The focus of thisreview is on applications of the more recently developed title compounds, which are especially wellsuited for time-resolved biochemical and physiological investigations, because they release the cagedsubstrates in high yield within a few nanoseconds or less. Together, these two chromophores cover theaction spectrum for photorelease from >700 nm to 250 nm.
Introduction
o-Nitrobenzyl derivatives are by far the most commonly usedphotoremovable protecting groups (PPGs),1 despite their knowndisadvantages: following electronic excitation, the caged com-pounds are released on a time scale of microseconds at best,2
and o-nitroso aromatic ketones are produced as side products,which usually absorb more strongly than the parent PPG at the
irradiation wavelengths and which may interfere with the effectof the released bioactive material under study, because of theirtoxic effect on biological tissues.3 With the need for higher timeresolution in studies of the leading events in biological pro-cesses, there is a demand for clean, rapidly released triggers orinitiators of these processes.
The technology to monitor events on the nanosecond tofemtosecond time scales has become available and to exploitthese methods the initiating processes must match or exceed therates of detection. Recent advances in time-resolved Fouriertransform infrared (TR FTIR) and attenuated total reflectance(TR ATF), for example, permit investigations of protein bindingand enzyme catalysis at the nanosecond level.4 This reviewfocuses on relatively recent additions to the PPG variety,p-hydroxyphenacyl (pHP)5 and coumarylmethyl derivatives,6
which offer an alternative to o-nitrobenzyl.
Richard S. Givens
Richard S. Givens (1940) is anEmeritus Professor (2010) ofChemistry at the University ofKansas. He earned his BA(Chemistry, H-G. Gilde) fromMarietta College and PhD(H. E. Zimmerman) from theUniversity of Wisconsin in1966. He was an NIH postdoc-toral associate (G. A. Russell)at Iowa State. He has authoredor coauthored more than 120scientific publications and 5patents on physical organic
chemistry, photochemistry, and their applications in biology andis co-editor of Dynamic Studies in Biology: Phototriggers,Photoswitches and Caged Biomolecules (Wiley-VCH, 2005).
Marina Rubina
Marina Rubina received her BSdegree in chemistry and chemi-cal education from SyktyvkarState University (Russia) in1996. She was a researchassociate at the Moscow StateUniversity (Russia) beforeearning her PhD degree withVladimir Gevorgyan (2004) atthe University of Illinois atChicago. She was then a post-doctoral associate with MichaelRubin and Richard Givens atthe University of Kansas. Cur-
rently, she is Laboratory Director in the Department of Chem-istry and a Research Associate at the Center for EnvironmentallyBeneficial Catalysis at the University of Kansas.
†This paper is part of a themed issue on photoremovable protectinggroups: development and applications.
aDepartment of Chemistry, University of Kansas, Kansas, USA. E-mail:[email protected]; Tel: +1 785 864 3846bDepartment of Chemistry, University of Kansas, Kansas, USA. E-mail:[email protected]; Tel: +1 785 864 1574cDepartment of Chemistry, Klingelbergstrasse 80, CH-4056 Basel,Switzerland. E-mail: [email protected]; Tel: +41 76 413 47 48
Anderson and Reese discovered the molecular reorganizationthat occurs upon irradiation of pHP chloride.7 The mechanism ofthe photochemical release of the leaving group (LG) from pHPdiethyl phosphate (1) in aqueous solution is summarized inScheme 1.8 Release of a typical caged substrate upon irradiationin neutral aqueous solution at λ = 250–350 nm cleanly andefficiently proceeds through its triplet excited state forming aputative spirodiketone 2, termed the Favorskii intermediate. Thespirodiketone is subject to hydrolytic ring opening yielding p-hydroxyphenylacetic acid (3). The structural rearrangementclosely follows the classical ground state Favorskii rearrange-ment of α-haloketones, hence the origin of its moniker as the“photo Favorskii rearrangement”. In contrast to the startingmaterial, the photoproducts 3 (and traces of 4) are essentiallytransparent at wavelengths above 290 nm.
Leaving group effects
As a general rule, the most efficacious leaving groups are thosewith a pKa < 11. Alcohols and primary, secondary, and tertiaryamines with pKa’s above the threshold of 11, with a few excep-tions,9 have not been successfully released from pHP cagesalthough little has been reported on these functional groups(vide infra).
Several studies have shown that the pHP triplet lifetimes, 3τ,and the rate constants for release, krel = Φdis/
3τ, depend on theleaving group pKa.
10–13 As shown in Table 1, for pKa’s thatrange from the reactive mesylate (−1.54) and tosylate (−0.43) tothe less efficient phenols (8–11) and thiols (∼8) give a range ofrate constants krel that spans three orders of magnitude andquantum yields that vary by a factor of 20.
The dependence of the rate constants krel on the pKa’s of theLGs is demonstrated by a Brønsted linear free energy relation-ship, log(krel/s
−1) = (9.57 ± 0.14) − (0.24 ± 0.03)pKa, i.e., βLG =−0.24 (Fig. 1). This correlation implies significant heterolyticbond breaking at the transition state for release.
Laser flash transient absorption studies have detected theallyloxy–phenoxy biradical intermediate shown in Scheme 1,which intervenes between the excited triplet and the “Favorskii”intermediate.8 The biradical triplet results from the extrusion ofboth a proton and the leaving group anion from the triplet ketone31. This indicates that the leaving group departs in its groundstate since spin conservation requires only one triplet species beformed if the reaction is concerted. The simultaneous departureof both the proton and the leaving group is further supported bythe observation of a solvent kinetic isotope effect (SKIE kH2O/kD2O = 2.17).8 The Brønsted βLG = −0.24 for the photochemicalreaction finds precedence in gas-phase collision-induced dis-sociation (CID) fragmentation measurements of the conjugatebase of the pHP derivatives in Table 1.12 A correlation
Jakob Wirz
Jakob Wirz (1942) is EmeritusProfessor of Chemistry. Hestudied at the ETH Zürich withProf. E. Heilbronner and, afterpostdoctoral studies in Londonwith Professors G. Porter andD. Barton, habilitated at theUniversity of Basel, where heled a research group until2007. He co-authored the bookPhotochemistry of OrganicCompounds (Wiley, 2009) withProfessor P. Klán and pub-lished over 170 papers focusing
on photochemistry and spectroscopy, laser flash photolysis, reac-tion mechanisms, and photochemical protecting groups. He con-tinues to serve as Deputy editor-in-chief for Photochemical &Photobiological Sciences.
Scheme 1 Mechanism for pHP photoisomerization and leaving group(LG) release.8
Table 1 Representative pKa’s, disappearance quantum yields of pHPderivatives (Φdis), triplet state lifetimes (3τ), and rate constants for therelease of the leaving groups (LG) calculated as krel = Φdis/
coefficient of βLG = −0.19 ± 0.02 for the appearance energies(AEs) vs. gas-phase acidities of the leaving groups (−ΔHacid)reveals a similar extent (19% vs. 24%) of bond breaking at thetransition state for the fragmentation process.
Chemical yield
An ideal PPG should give a quantitative yield of the unprotectedfunctional group. Moreover, the irradiation dose required shouldbe minimal to avoid unwanted photochemistry such as photo-reactions of the products. Meeting these objectives avoids dele-terious processes that compromise the efficacy of PPG-basedmethods. Poor chemical yields of released material, secondaryphotochemical reactions and over-irradiation leading to degra-dation of products and spectator components of the reactionmixture often result in complex mixtures of unwanted materials.For the p-hydroxyphenacyl derivatives, both criteria are metsince the conversions are spectacularly clean and the quantumefficiencies approach unity.
Stability to thermal degradation
Esters, phenyl ethers and thio ethers of pHP are very stable inorganic solvents. Most esters and all ethers are also stable inaqueous-based solvents at neutral pH and do not undergohydrolysis at ambient temperatures. However, as the leavinggroup reactivity increases, e.g., sulfonate leaving groups, thecaged compound’s stability decreases. Aqueous solutions of car-boxylate esters may undergo slow hydrolysis at room tempera-ture over a 24 h period forming the α-hydroxyketone. The rate ofhydrolysis increases at higher pH (>9). No rearrangement pro-ducts are detected, and thus no ground-state Favorskii rearrange-ment takes place under these basic conditions.14
Selected applications of pHP derivatives
The pHP PPG has been successfully deployed for time-resolvedbiochemical and physiological studies to delineate response kin-etics,15 or to define spatial resolution in tracking signal transduc-tion in neural networks.16 Several of these applications havebeen reviewed earlier.15 More recently, time-resolved absorptionspectroscopy,8,14,17–20 Fourier transform IR (TR-FTIR)3,4,21 andresonance Raman (TR3),10,22,23 techniques have become avail-able for better time-resolved applications in biological chemistry,such as examining substrate binding, enzyme conformationalchanges, dynamic inhibition and signal transduction kinetics.Several applications have incorporated an initiating pHP photo-activation step, again taking advantage of pHP’s rapid releaserate.
The pHP phosphate derivatives are most frequently deployedfor studies on enzyme catalysis, such as the photo-initiation stepfor kinetic studies,3,24 taking advantage of their very rapid andclean phosphate group release.3–5,10,18–24 For carboxylaterelease, such as the C-terminus of amino acids, peptides, and oli-gopeptides, the value has been in their ease of synthesis, clean,high yielding release, and biological compatibility. Bradykinin,L-glutamate, and GABA have been employed for several in vitroand in vivo studies, e.g., to activate the BK2 receptor, and as
agonists or antagonists in mouse neuroreceptor stimulationstudies of the auditory system.15,16 Assorted applications of pHPthio derivatives that take advantage of the high nucleophilicity ofthiol for direct incorporation of the protecting group using pHPBr have been reported.25,26
The first examples of pHP phosphate release came from theinvestigations by Fendler et al.24 on ATPase hydrolysis of Na+,K+-ATP and at about the same time by Du et al.3,27 on pHP GTPas an initiator of the catalytic hydrolysis of GTP by Ras protein–GTPase. These two hallmark applications demonstrated thepower of pHP as a phototrigger for the rapid release of thenucleotides ATP and GTP, in contrast to more commonlyemployed o-nitrobenzyl (oNB) and o-nitrophenylethyl (oNPE)PPGs. Fast cage release of the nucleotides was crucial for deter-mining rate constants of the earliest events in a catalytic hydroly-sis. In Du et al.’s work,27 kinetic isotope effects obtained fromthe rates of hydrolysis of 18O-labeled α-, β-, and γ-phosphates ofGTP were determined from time-resolved FTIR analyses of therates of bound and free phosphate. The changes in binding ofGTP in the catalytic pocket during its hydrolysis of GDP weredetermined using 18O-induced spectral shifts from photolysis ofthe three pHP 18O-labelled GTP isomers. An increase in β-phos-phate binding coupled with prior evidence of deprotonation ofthe γ-phosphate led the authors to conclude that hydrolysisoccurs through a dissociative mechanism forming meta-phos-phate intermediate and GDP. Rapid H2O addition to the meta-phosphate yields inorganic phosphate (H2PO4
2−, Pi), which isthen released from the catalytic site in the rate-determining step.These studies relied heavily on the rapid release rate of pHPGTP to monitor the initial rates for binding at the catalytic siteand to confirm its location (vide infra). Attempts to perform thesame experiments using oNPE GTP were unsatisfactory andresulted in the early onset of extraneous IR signals masking theslower evolution of signals associated with the changes inbinding from GTP to GDP.
In a follow-up communication,3 Du et al. documented thesource of the difficulties encountered with the oNPE GTP study.The oNPE photolysis byproduct results from a photoredox reac-tion of the chromophore forming an α-nitrosoacetophenone.Nitroso ketones and aldehydes are the common byproducts forall o-NB-based caged compounds and are very susceptible tonucleophilic attack by amines and thiols. To counteract thiseffect, a common practice has been to add dithiothreitol (DTT)to the photolysis mixture in order to trap or disable any aryl-nitroso byproducts. Spurious signals that appeared early in theoNPE GTP photolysis were shown to arise from reactions ofnitrosoacetophenone with the protein. In the absence of protein,the nitrosoacetophenone is readily detected.
The rate of oNPE cage release is also many orders of magni-tude slower than that of the pHP analogs: krel = 1.0–10 s−1
(oNPE)2 vs. 108–109 s−1 (pHP).8,17,22 The vital time framerequired for RasGTP catalysis by TR-FTIR was too fast foroNPE GTP initiation.3,4 The initial TR-FTIR fingerprints of thebinding changes of the α- and β-phosphate groups on GTP wereonly observed with labelled pHP GTP as phototriggers.3,21,28,29
A similar comparison of TR-FTIR analyses of pHP ATP andoNB ATP-catalyzed hydrolysis was reported by Fendler et al.24
Here, the authors reached the same conclusions regarding thequality of data using the two methods for triggering the
hydrolysis. Benzoin-protected ATP was also examined but didnot provide useful results, primarily due to fluorescence andcompetition for incident radiation by the phenylbenzofuranphotoproduct.
Gerwert et al.21 extended the TR-FTIR technique by develop-ing it into a rapid high-throughput screening method as a com-petitive drug–protein binding assay. Using TR-FTIR differencespectra to tease out the changes in GTP–GDP binding in thepresence and absence of small molecular weight pharmaceuticalcandidates, the degree of inhibition at key functional groups onthe protein and/or induced changes in the protein’s conformationare measurable. The “on” and “off” signalling can be automatedfor high-throughput screening, a novel application of caged com-pounds with even broader applications for pharmaceutical drugdevelopment.30 This approach may be useful in assaying infor-mation about the active binding site of potential GTPaseinhibitors.
Kötting et al.4,28 also determined the kinetic parameters andactivation energies for GTP hydrolysis by GTPase in water andcatalyzed by the Ras protein-GTPase in order to evaluate theextent of entropic vs. enthalpic influence of the protein on thecatalytic cycle. Ras increased the rate of hydrolysis by 105 rela-tive to H2O and 107 when Mg2+ was present in the aqueous sol-ution. Temperature dependent rates showed that the ΔΔH0* of5.2 kcal mol−1 for water vs. RasGTP was essentially the totalsource of the diminished activation barrier to the hydrolysis. TheTΔΔS0* value was less than 0.5 kcal mol−1, essentially demon-strating that the protein was not providing enhanced ordering atthe transition state in the hydrolysis process.
These values were determined using 18O frequency shifts at1143 cm−1 for RasGTP and 1114 cm−1 for RasGDP to registerthe rate of formation of the RasGTP complex and its hydrolysisupon photolysis of pHP GTP. The frequency shifts for the α andγ labelled phosphates showed an upshift in comparing theGTPase activity in H2O vs. with protein-bound RasGTPase. Theβ-phosphate caused a downshift indicating a build-up of negativecharge density at that position in accord with the earlier findingsof Du et al.3 This was accounted for by the proximity of the
Mg2+ ion and Lys16 near the β phosphate to encourage negativecharge migration to the phosphate oxygen through electrostaticattraction (Fig. 2).
It should be pointed out that the synthesis of 18O-labeledphosphates of nucleotides like ATP and GTP is readily accom-plished since the synthetic strategy for pHP caged phosphatessimply involves the condensation of labelled pHP OPO(OH)2with the mono- or diphosphorylated nucleoside bases(Scheme 2).3–5,15
Wittinghofer, Gerwert, Kötting, and coworkers have madeimportant contributions to unveiling the specificity of Ras andRap, two GTPase activating proteins (GAPs) that assist in cataly-tic hydrolysis of GTP.4,29,31,32 Subtle changes in the amino acidsequence and mutations can greatly affect the GTP and GDPbinding and hydrolysis of GTP by RasGTPase altering the“on”–“off” signalling by the enzyme, which is considered one ofthe major determinants in uncontrolled cell growth.4
Regulation of phosphotyrosines, important in transmembranesignalling, is governed by the competitive rates of formation andhydrolysis of tyrosine phosphates. The analysis of the rates ofphosphotyrosine formation by phosphotyrosine phosphatase(PTP) has been the target of several mechanistic investigationsincluding small molecule inhibition. The hydrolysis of tyrosinephosphate involves the intervention of a neighbouring cysteine atthe active site which attacks the phosphate forming a cysteinethiophosphate intermediate that is hydrolyzed to Pi. α-Halo-ketone derivatives including phenacyl halides act as small mol-ecule inhibitors that block this pathway by reacting covalentlywith the cysteine thiol as “suicide inhibitors”.
Pei and coworkers33 showed that pHP Br inhibited PTPhydrolysis by reaction with the key cysteine (Cys 453) located atthe enzyme active site. For PTP SHP1(ΔSHP2) the “suicide inhi-bition” was complete in a few minutes (kinact = 0.40 min−1)which could be replicated in human B cells. In this case, inhi-bition was shown to cause protein hyperphosphorylation. Unliketraditional “suicide inhibitors,” Pei’s work demonstrated that thepHP thio ether linkage could be severed from the enzyme photo-chemically at 350 nm. Up to 80% of the PTP SHP1(ΔSHP2)activity was restored with a 15 min exposure, providing a modelfor mechanistic investigations of “on”–“off” inhibition ofenzyme catalysis and other fast kinetic studies at cysteine activesites.
Goeldner25 has further demonstrated that pHP thioethersundergo the same rearrangement as the ester counterparts to give90% of the rearranged p-hydroxyphenylacetate products alongwith ∼10% of p-hydroxyacetophenone, suggesting a smallradical component for this reaction. In fact, the release of thethiol leads to disulfide dimers in ∼70% yields with a quantum yieldof 0.085 for 3′-thio-dTMP in Tris–HCl buffer (pH 7.2). No attemptswere made in this study to suppress radical coupling reactions
Fig. 2 Mg2+ and Lys16 from Ras and the arginine finger from GAPdraw negative charge towards the β-phosphate of GTP. The main cataly-tic effect of Ras and GAP is enthalpic and seems to originate from theseelectrostatic interactions. (Reprinted with permission by ChemicalPhysics (2004).28)
with citrate or other moderators. An interesting variant in theproduct mixture was 30% formation of p-hydroxyphenylacetatethioester 6. Its formation may be the result of nucleophilicaddition by the released thiol (pKa = 8.4) to the spirodiketone 5,which then opens to 6 (Scheme 3).
Three thiol derivatives (N-benzylcysteine, 3′-thio-deoxythymi-dine phosphate (3′-thio-dTMP, a substrate analog for thymidilatemonophosphate kinase), and glutathione) were pHP “protected”by direct displacement on pHP Br with the thiols in 80–90%yields. The ease of thiol protection–deprotection by addition ofpHP Br followed by photochemical deprotection has served as abasis for several studies on cysteine and thiophosphatesubstrates.
Bayley et al.26 compared the protection–deprotection effec-tiveness of oNB and pHP for thiophosphorylated tyrosine. Bothprotection sequences were accomplished in high yield (75% and90%, respectively) by treatment of the thiophosphorylatedtyrosyl peptide EPOYEEIPILG with oNB Br and pHP Br. Bothwere equally stable to storage as well as to reaction conditionswithout light (Scheme 4).
Irradiation at 312 nm released the thiophosphated peptide in50–70% yields with quantum yields of 0.25–0.37 (oNB) and0.56–0.65 (pHP). The chemical and quantum yields were pHdependent between pH 5.8 to 7.3, but at pH 8.2 the reactantsand/or the products were not stable. Irradiation conditions at312 nm resulted in deprotection from pHP ten times faster thanfrom oNB due primarily to a combination of higher efficiencyand larger absorptivity for pHP at this excitation wavelength.Both deprotection reactions provided the oligopeptide regaining50–70% of its original binding capacity as determined by radio-labelled binding studies with recombinant SH2 domain.
Bayley et al.26 extended this approach to the protection–depro-tection of thiophosphorylated threonine, Thr-197, in the catalyticsite of a cell-signalling protein kinase A found in the catalyticCα subunit by phosphorylation. The phosphothreonine unit playsa key role in stabilizing the active conformation of the Cα cataly-tic subunit. The thiophosphorylated threonine was effectivelyprotected by direct reaction with pHP Br to yield pHP-PST
197
Cα. It is noteworthy that the thiophosphorylated threonine (pKa
= 5.8) was selectively protected with pHP Br in the presence of
potentially competing cysteine residues. Both the binding capa-bility and the specific activity of the caged enzyme were dramati-cally reduced, to 2.4% (binding assay measured on TNB-thioagarose beads) and by a 17-fold decrease in 32P kinase activity.Photolysis at 312 nm at pH 7.3 restored 85–90% of the bindingactivity and the specific activity was nearly recovered (15-foldincrease) with a quantum yield of 0.21. The role of the pHPgroup in this instance is to block or diminish H-bonding andelectrostatic attractions between the threonine-197 and Arg-165with Lys-189, the amino acids that stabilize the closed form ofthe Cα subunit.
Protecting groups have a long history of application, whichtakes advantage of the ability to control the exposure of reactivefunctional groups to reactive environments during multistepsyntheses of peptides, nucleotides and polymers.34 While photo-removable caged compounds have long been considered forapplications in these areas, related applications on solid sur-faces35 are receiving more attention. Three attractive character-istics for photochemical approaches toward systematic exposureof functional groups are the location and the density of theexposed functionalities. Added to this is the temporal controlwhich can be important for investigations of signal processing.These attributes have encouraged an explosive array of appli-cations36 and surface-bound caged compounds have beenexploited for controlled exposure of groups such as carboxylicacids, amines, thiols, and many other reactive functional groupsbound to glass surfaces, beads, polymeric supports, and othersolid surfaces.
Photoremovable protecting groups applied to regulation ofPCR and gene expression have been explored with mixedresults. Many attempts to control polynucleotide selectivity andactivity using PPGs have also been attempted.37 However, arecent study by Pickens and Gee9 has opened a potentiallyuseful new application using pHP protection of the 3′-hydroxygroup on thymidine that, as its corresponding phosphoramidite,could be employed in PCR. In this study, the 3′-hydroxy wasprotected as its pHP ether. Photolysis of pHP 7 released thymi-dine quantitatively in 15 s (Scheme 5). The authors suggestedthat the protected phosphoramidite might serve as a photoreversi-ble blocker for selective PCR modification of the nucleotidearray (Fig. 3).
(Coumarin-4-yl)methyl [coumarylmethyl]photoremovable protecting groups
The development of coumarylmethyl cages as a new class ofphotoremovable groups commenced with the discovery by
Scheme 3
Scheme 4
Scheme 5 Synthesis and release of pHP caged thymidine 7.
Givens and Matuszewski of the photoreactivity of a coumarinyl-methyl group in releasing phosphate ester 8 (Scheme 6).6
Schmidt and coworkers38 have probed the mechanism ofrelease from several coumarylmethyl cages CM–LG using quan-titative quantum yield and time-resolved measurements of thephotochemistry and fluorescence of coumarylmethyl esters andthe corresponding alcohols (vide infra, Scheme 14 and Table 3).Heterolytic bond cleavage, kcl, proceeds from the relaxed excitedsinglet state 1CM–LG* in competition with fluorescence andnonradiative decay processes, kf and knr, forming a singlet ionpair 1[CM+⋯LG−] in the initial reaction step (Fig. 4). Escapefrom the solvent cage first affords the solvent-separated ionsCM+ and LG−. The coumarylmethyl cation CM+ reacts withwater to yield the product alcohol CM–OH and H+ in addition tothe leaving group anion LG−.
The cleavage rate constants, kcl, were estimated from measure-ments of the fluorescence quantum yield, Φfl, and the fluor-escence lifetime, τfl, of the alcohols CM–OH and thefluorescence and photochemical quantum yields, Φfl and Φr, ofthe coumarylmethyl esters CM–LG. The resulting rate constantskcl range from 0.1 to 50 × 109 s−1 and obey a linear free energyrelationship with the acidity constants pKa of the LG acids, i.e.,those releasing the strongest acid are fastest. A similar relation-ship was found for the heterolytic cleavage from the triplet state
of pHP esters (Fig. 1). The ratio of escape to recombination fromthe ion pair could not be determined directly, but the measure-ments indicated that the factors that accelerate the heterolyticbond cleavage retard the ion recombination reaction.
Coumarin-based PPGs have gained considerable attention,especially for applications in biological chemistry,39 primarilydue to its longer-wavelength absorption, extending farther intothe visible region (400–500 nm) and the fast release rate from itsexcited singlet. These advantages have been offset by coumarin’smarginal quantum yields, low aqueous solubility and theoccasional inconvenience of a strong fluorescence and competi-tive absorptivity from the coumarin photolysis byproducts.However, many successful efforts have been reported whereclever modifications to the coumarin group have resulted insomewhat higher quantum yields and better aqueous solubility.These, together with adaptations that expand the leaving grouprange (including alcohols and amines), have raised researchers’awareness to this relatively new class of PPGs.
The first generation of coumarylmethyl cages possessing an–OH or –OMe substituent at C7 were characterized by very fastrelease rates and great hydrolytic stability. However, they had adrawback of poor solubility and relatively low quantum yieldsfor all leaving groups except phosphates. 6-Bromo-derivativeswhere designed to lower the pKa of the 7-hydroxy group by twounits to effect a complete deprotonation at physiological pH,thereby enhancing water solubility and causing a bathochromicshift of 60 nm (Table 2). The downside of this modification wasthe increased susceptibility of some of the bromo-substitutedcaged compounds to hydrolyze in the dark. Further developmentin this area resulted in emergence of a second-generation of cou-marylmethyl PPGs bearing an amino group at C7, which signifi-cantly improved the spectroscopic and photochemical propertiesof the cage, moving the absorption maxima to 350–400 nm andrecording the highest quantum yields among the analogues.Solubility of the aminocoumarylmethyl cages could be improvedby appending polar groups such as carboxylates to the anilinemoiety. Strong fluorophoric properties, which are even moreenhanced in the polyaromatic analogs, lend a convenient tool formonitoring of the reaction course.
Scheme 6
Fig. 3 Caging methodologies to regulate oligo synthesis. (With per-mission of Tetrahedron Lett.)
Fig. 4 Mechanism of the photocleavage of coumarylmethyl cages.Adapted from ref. 38.
Phosphates. Photolysis of 4-methoxycoumarylmethyl phos-phate was the first example that employed a coumarylmethylPPG6 and current literature reveals that coumarinylmethyl phos-phates continue to be the most frequently encountered appli-cation of the coumarin cage. Most caged phosphates have goodstability to hydrolysis (although some analogues may havelimited water solubility), adequate quantum efficiencies (Φ =0.03–0.2), and are easily synthesized, which, taken together withvery fast release kinetics, make coumarin-caged phosphates very
attractive candidates for biological applications. Depending onspecific needs, properties of caged phosphate can be tuned bymodifying substituents on the coumarin core. Studies by Limaet al. also emphasize the importance of pH control in photolysisof ionizable 4-methylcoumarin-caged phosphates, which showsignificant dependence of quantum yields on pH variations.64 Areview by Furuta provides useful guidelines for choosing a suit-able cage.65 The most recent applications of caged phosphates inbiological studies are described below.
Caged peptide 9 was used as a modular domain-bindingpeptide in the investigation of PI3K kinase-mediated phos-phorylation of proteins. Irradiation of 9 in K-MOPS buffer sol-ution quantitatively produced phosphopeptide 10 andhydroxymethylcoumarin 11 with a quantum yield of 0.12(Scheme 7).66 Peptide 10 was also successfully released in livingcells upon exposure at 365 nm without causing any toxicity.
Schultz and coworkers demonstrated that 7-diethylaminocou-marin-4-ylmethyl-caged bioinactive phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) can be effectively transported throughthe plasma membrane and then uncaged to release PI(3,4,5)P3,
Table 2 Typical coumarin chromophores
λmax Structure Abbreviation Solventa Ref.
320–330 nm HCM (R = H) C 40D 41
7-MCM, Bpm (R = Me) C 40,42F 43A,D 38,41,44
CMCM (R = CH2CO2H) D 45
330–380 nm DMCM (X = Y = OMe, Z = H) F 43A 38,46
BCMCM (X = Y = OCH2CO2H, Z = H) D 45B 47
7,8-BCMCM (X = H, Y = Z = OCH2CO2H) G 48BHCM, Bhc (X = Br, Y = OH, Z = H) E 49,50
G 51F 43
BMCM (X = Br, Y = OMe, Z = H) E 49BBHCM (X = Br, Y = OH, Z = N(CH2CO2Bu
t)2) G 51(X = Br, Y = n-C17H35CO2, Z = H) B 52
350–400 nm ACM (R = H) D 41DMACM (R = Me) B 53
A 38,44DEACM (R = Et) H,B 54–57
D 58F 43A 38,46
BCMACM (R = CH2CO2H) B 59,60D
BBCMACM (R = CH2CO2But) G 61
340–360 nm Obb (X = H) C 42,62Bbl (X = MeO)
Obc (X = OH, Y = H) C 42,62,63Obm (X = OMe, Y = H)Bba (X = H, Y = OMe)
inducing membrane ruffling and pH-domain translocation in thepresence of the PI3-kinase inhibitor wortmannin (Fig. 5).67 Thecell entry by PI(3,4,5)P3 was conveniently monitored by fluor-escence microscopy. Biological response was observed within1 min following irradiation by a short pulse of a 375 nm laser,and was superior to that of the membrane-permeable uncagedanalogue. It should be mentioned that synthesis of PI(3,4,5)P3suffered from low yield (2.5% overall) due to poor stability ofthe coumarylmethyl group under alkylation conditions.
Due to very fast release rates (2 × 108 s−1), high photoefficien-cies and hydrolytic stability, the coumarin-4-ylmethyl cage isideal for studies of biological processes involving release ofnucleotides and nucleosides. Furthermore, the intense absorptiv-ity of 7-aminocoumarins at longer wavelengths (350–400 nm),far beyond that of other photoremovable groups, permitsefficient photorelease nicely avoiding biologically lethal wave-lengths. Thus, Bendig and Giese suggested 7-diethylaminocou-marin-4-ylmethyl cytidine 5′-diphosphate (CDP) 12 (λmax =392 nm; ε = 16 200 M−1 cm−1) as a suitable model for studiesof a long-range radical transfer mechanism in E. coli ribonucleo-tide reductase (RNR) (Scheme 8).57 An important advantage ofthis model over o-nitrobenzyl and other analogues is that photo-activation of CDP occurs extremely fast, which permits analysisof conformational changes preceding the radical transfer, and atwavelengths where the molar absorptivity of RNR is very low.57
The release of ATP in cell cultures and in acutely isolatedbrain slices was tested to probe the ability to evoke Ca2+ ionwaves53 (Scheme 9). The release of nucleoside triphosphate
from caged precursor 13 in HEPES solution was twice asefficient as that of the nitrobenzyl-caged analog at 365 nm andten times more efficient at 405 nm. To measure the response incells, cytosolic Ca2+ concentrations were recorded fromconfluent astroglial cultures stained with the Ca2+ indicator fluo-3. The experiments confirmed biological inertness of the cagedATP 13. In contrast, irradiation of the latter with an argon laser at364 nm caused a notable increase of fluorescence in cells closeto the UV spot. The response was successfully elicited threetimes, provided the cells were allowed to recover after each doseof radiation. The Ca2+ response showed the typical time courseof an ATP-triggered signal: a fast increase in cytosolic Ca2+ con-centration and a subsequent slow decay back to the initialconcentration.53
Light-controlled, in vitro enzymatic polymerization of nucleicacids through the release of ATP from a coumarin-4-ylmethyl-caged precursor was demonstrated by Baptista (Scheme 9)54 byirradiating a ribonucleotide mixture (CTP, UTP, and GTP) andcaged ATP 13. A control sample (no light) showed no reaction,whereas formation of the transcription product was formed onirradiation, reaching a maximum at 25 μM of ATP followed by adecrease in RNA formation. The decrease in RNAwas explainedby inhibition of transcription due to accumulation of byproduct15.54
The release rate of the cyclic nucleotide depends on its struc-ture and the solvent polarity.68 High electron donor ability of thepurine base compared with diethyl phosphate leads to morestable intermediate tight ion pairs resulting in greater quantumyields. Furthermore, the higher quantum yields for the axial vs.equatorial isomers of caged cyclic phosphates (0.13–0.21 for 16vs. 0.07–0.09 for 17, Scheme 10) were attributed to increasedsteric interaction and larger partial charge transfer between thepurine base and coumarylmethyl cation for the axial vs. equator-ial isomer.
The effect of substituents on coumarin PPGs was systemati-cally investigated for a series of caged cAMPs (Scheme 11).44 Inaddition to influencing the absorption maxima (Table 2), chan-ging donor substituents at the 6- and 7-position of coumarin alsosignificantly affects the quantum yields. The highest quantumyields are obtained for 7-dialkylamino derivatives (0.21–0.28),which were rationalized by more efficient stabilization of thecoumarylmethyl carbocation through the electron-donatingamino substituents and, as a result, more efficient ion pair escape(Scheme 11).44,68
Iwamura and coworkers were the first to probe photorelease ofcAMP from a coumarin cage in studies of biological responseswithin living cells.69,70 A test experiment using zebra fish mela-nophores demonstrated that inactive, caged cAMP (18, X = Br,
Fig. 5 Mechanism of action of PI(3,4,5)P3: (a) cell entry; (b) enzy-matic removal of Bt and AM protecting groups produces cgPI(3,4,5)P3;(c) light-induced removal of coumarin protecting group; (d) PI(3,4,5)P3induces translocation of EGFP-Grp1-PH domains to the plasmamembrane.67
Y = AcO, Scheme 11) successfully penetrated through theplasma membrane into the melanophore and released a sufficientamount of cAMP upon irradiation at 340–365 nm to inducemotile response of melanin granules. This has been revisited for
coumaryl PPGs possessing a polar aminobis(methylcarboxylate)group (19, Scheme 12), which significantly improved water solu-bility of the caged nucleoside monophosphates.47,60 Further-more, the fluorescence quantum yields of 19a,b were an order ofmagnitude lower than that of the corresponding coumarin-4-ylmethanol 20, which permitted convenient monitoring of thereaction by fluorescence spectroscopy. Upon irradiation ofHEK293 cells transfected with DNA encoding CNGA2 chan-nels, loaded with 19b, an increase in both the intracellular fluor-escence and the cAMP-induced current resulting from activationof olfactory neurons (CNGA2) was observed.47 From thesestudies, coumarylmethyl PPGs can be used as an analyticalmethod for quantitative dose–response relationships for cellularreactions triggered by cAMP.
In addition to significant advances achieved in caging of DNAand RNA through the phosphate backbone using methodsdescribed above, complementary approaches for base-cagednucleosides have also been developed to enable control ofWatson–Crick pairing and double strand formation. Bromocou-marylmethyl-caged deoxycytidine 21 and deoxyadenosines 22used by Furuta et al. proved most efficient compared to o-nitro-benzyl caged analogues in the release of nucleosides uponirradiation at 350 nm (Scheme 13).49
The coumarylmethyl chromophore has been increasinglyexamined for 2-photon excitation efficacy using near IR(>700 nm) lasers.71 Thus, it was also shown that the two-photonuncaging action cross-section of 22a (0.35 GM at 740 nm) is inthe practical range for biological applications. The O-cageddeoxyguanosines 23 reported by Heckel showed significantlylower quantum yields at 365 nm as compared to P-caged cyclicnucleotides or the 2-(o-nitrophenyl)propyl analogue 24;however, the high extinction coefficient of 23 makes photoche-mical effectiveness for release nearly two-fold better than thatfor 24.72 Furthermore, uncaging of nucleobase in 23 at 405 nmproceeded 80 times faster than the corresponding oNE PPG in24, permitting wavelength-selective photorelease.
Release of carboxylate: carboxylic acids and amino acids. Thephotoefficiency of the 7-alkoxycoumarin-caged carboxylates,especially aliphatic acids, is generally rather poor,65 primarilyattributed to the higher pKa of a carboxylic acid as comparedwith a phosphate leaving group. Thus, Schmidt et al. demon-strated a strong decrease of the rate constants of heterolytic clea-vage, kcl, with increasing pKa of the released acid (Fig. 4,Scheme 14, Table 3).38
The release of 2,4-D represents a practical, agricultural appli-cation of coumarins as PPGs. The UV-Vis photochemistry of2,4-D caged by 7-substituted coumarylmethyl protecting groupswas measured in several aqueous organic solvents to explore the
Scheme 12
Scheme 13
Table 3 Effect of pKa on quantum yields and cleavage rate constantskcl
effects of substituents and the pH on the solubility and the con-version at different wavelengths (Scheme 15).73 All compoundsin the tested series showed rather low efficiencies; the highestquantum yield was observed for 7-hydroxy-derivative 26 (0.018and 0.012 at 310 and 350 nm, respectively).
Similarly, a comparison of quantum yields of three GABAconjugates 25a–c at different wavelengths performed by Costaet al. revealed rather low photoefficiency in the UV rangeoptimal for most biological applications (≥350 nm)(Scheme 16).42,68
In contrast to poor quantum yields observed in the previousexamples employing 7-alkoxycoumaryl group, photorelease ofamino acids linked through the carboxylate moiety to 7-amino-coumarylmethyl chromophore was much more efficient(Scheme 17).46,55,56,74–76 Quantum yields for caged GABA,glycine and glutamate derivatives range from 10 to 20%(Table 4).46,56 It was proposed that the photocleavage productyield can be attenuated by ion-pair internal return of the releasedgroup in competition with escape from the solvent cage.46 Stab-ility of the carboxylate-caged amino acids toward hydrolysismay become an issue and thus should always be examined prior
to photolysis to ensure stability of the caged compound inaqueous buffer solution. In fact, it was found that phenylalaninecaged with the water-soluble bis(carboxymethyl)amino deriva-tive (26c) was unstable in HEPES buffer at pH 7.2.74 Spon-taneous hydrolysis was also observed for glutamate when cagedwith 6-bromo-7-hydroxycoumarin-4-ylmethyl.66
Coumarin-caged neurotransmitters are ideal models becausethey can be efficiently photolyzed by visible and near UV lightthat is much less harmful to cells, thereby opening biologicalstudies to both in vivo and in vitro activation of agonists andantagonists at various neurotransmitter receptors. On the otherhand, the photolysis byproducts do not appear to inhibit or acti-vate these same receptors. The experimental design in thesestudies vary from more sophisticated laser-flash, time-resolvedprocedures to the more readily available, inexpensive lightsources such as Rapp flash lamps.
Hess and coworkers employed photorelease of caged GABA,glycine, and glutamate as a tool for studying the mechanism ofaction of neurotransmitter receptors using HEK 293 cells trans-fected with cDNA encoding the corresponding receptors. Flashlamp photolysis (385–450 nm) uncaged glycine56 and gluta-mate55 from precursors 26d,e in HEK extracellular buffer. Theefficiency of photorelease was assessed by the whole-cell currentevoked by the released amino acid.55,56 Both caged glycine(26d) and glutamate (26e) were inert toward the correspondingreceptors. The analogous GABA derivative 26f, however, inhib-ited GABAA receptors.75,76 This problem was addressed throughthe installation of an additional substituent at 4-methylene group(26g, X = CONHCH2CO2Et, Scheme 17) of the coumarinphototrigger which helped eliminate unwanted activity of thecaged precursor.76
Carbamate-caged amino acids. To address the hydrolysisissue, amino acids caged at the N-terminus via a carbamatelinkage were employed. Thus, carbamate-caged blockers for glu-tamate transporter, L-threo-β-benzyloxyaspartate (L-TBOA) andits more potent m-(trifluoromethyl)benzamide analog (L-TFB–TBOA) were found to be more stable in aqueous solutions thanthe corresponding carboxylates (Scheme 18).45 Furthermore,hydrophilic coumarin-4-ylmethylcarbamate cage 27b,c (R2 =OCH2COOH) photolyzed efficiently with quantum yields 0.03and 0.02 (for 27b and 27c, respectively), which is comparable tothat of the complementary carboxylate-caged analogs. Tests wereconducted using 27a–c on biological activity by the glutamateuptake inhibition assay using excitatory amino acid transporter(EAAT2) stably expressed on MDCK cells. It was shown that
Scheme 15
Scheme 16
Scheme 14
Scheme 17
Table 4 Photorelease of coumarylmethyl-caged neurotransmitters
Y X LG λ/nm (pH) Φ Ref.
26a N(CH2CO2H)2 H GABA 380 (7.2) 0.2 226b N(CH2CO2H)2 H Glu 380 (7.2) 0.1 226c N(CH2CO2H)2 H Phe — (7.2) — 7126d NEt2 H Gly 400 (7.4) 0.12 5526e NEt2 H Glu 400 (7.4) 0.11 2826f NEt2 H GABA 400 (7.4) 0.14 2926g NEt2
the activity of carbamate- and carboxylate-protected blockerswas efficiently masked by the coumarin cage. The release of themore potent L-TFB-TBOA upon photolysis provided a suffi-ciently effective concentration of the blocker for applications inbiological preparations. It was also confirmed by uptake assaythat the blocker activity of the released L-TFB-TBOA was notimpeded by the photolysis byproducts.45
Comparison of irradiation doses required to release aminoacids caged through different linkages revealed the followingrelative photosensitivity order: anhydride > ester > carbamate >carbonate, with carbamates being slightly more resistant toirradiation than carboxylates (Scheme 19).40 It should be men-tioned that the rate-limiting step in all these cases except carbox-ylate 31 is decarboxylation of the released carbamic andcarbonic acid, which depends on the nature of the releasedgroup,77,78 and varies with pH. The rates of decarboxylationreactions are usually quite slow, k−CO2
= 10−3 s−1,79–81 andsubject to both acid and base catalysis.
Schmidt’s calculated shifts in the λmax of 7-amino-substitutedcoumarin-caged GABA 32 suggested a notable bathochromicshift as compared with the 7-hydroxy- and 7-methoxy
derivatives.41 Indeed, the absorption maximum of caged GABA32 was shifted by 22–24 nm and readily released amino acid at300–400 nm with a quantum yield of 0.04 (Scheme 20). Pho-tolysis of caged GABA 32 on auditory neurons in the lateralsuperior olive in brainstem slices of mice triggered membranecurrent mediated by specific activation of the GABAA receptorby whole-cell patch clamp recordings of individual neurons.41 Itwas shown that GABA derivative 32 had no intrinsic biologicalactivity and good stability in dark solutions, while another cagedneurotransmitter, 6-bromo-7-hydroxycoumarin-4-ylmethoxycar-bonyl dopamine, was found to be more susceptible to hydrolysisin aqueous solutions, which, however, was slow enough on thetimescale of the physiological experiments to allow usefulstudies.51
Applications of photoremovable groups in protein chemistryface a number of limitations, including synthetic challengesassociated with chemo- and site-specific installation of PPGs,difficulty in transporting caged proteins into a cell, poor tissuepenetration and damaging effects by UV light causing unwantedside reactions, such as photooxidation, and incomplete uncagingin biological environment. Often, small PPG chromophores failto efficiently suppress the activity of large biological entitiesresulting in notable residual activity.82 Described below areseveral recent reports of the successful use of coumarin PPGs incaging strategies for peptides and other large biomolecules.
The coumarylmethyl cage was successfully used to mask theactivity of oligopeptides Aβ1–24 and Aβ27–42 (Scheme 21).61
An elegant approach termed “click peptide” involved an O–Nintramolecular acyl migration triggered by cleavage of the PPG.Screening of different photoremovable groups, including 6-nitro-veratryl, p-dimethylaminophenacyl and various 7-aminocoumar-ylmethyl analogs revealed that only the bis(carboxymethyl)aminocoumarylmethyl derivative possessed the necessarilyimportant features of sufficient hydrophilicity and stability tohydrolysis. Importantly, the protected oligopeptide precursor 33was significantly more water soluble than the target peptideAβ1–42 (35), and did not self-assemble, because the O-acyl(instead of N-acyl) fragments in 33 suppressed the conformation-al transition into a β-sheet analogous to 35. Irradiation of 33 at355 nm quantitatively removed the protecting group within
2 min; subsequent incubation of 34 for 30 min at 37 °C enabledsmooth acyl migration to produce 35 (Scheme 21).61
Another example showcasing the application of a photoremo-vable coumarin group in synthesis of peptidoconjugates wasdemonstrated by Nagamune et al. (Scheme 22).83 Temporal andspatial control of peptide release was achieved upon irradiationof a peptide conjugate possessing a photocleavable backbone.Solutions of conjugates 36 and 38 possessing a bifunctional bro-mocoumarin carbamate linker were photolyzed at 350 nm,releasing peptides 37 and 39 with quantum yields of 0.16 and0.26, respectively. A similar approach was employed for site-specific caging and photorelease of plasmid DNA.84
Coumarin-containing lipids are another example for successfuluse of photocleavable fragments implanted in the backbone of acomplex biological molecule (Scheme 23).52 Light-mediatedrelease of the liposomal content via lipid cleavage represents anattractive alternative for controlled drug release. A hydrophilichead of the lipid (an amino acid) was separated from the hydro-phobic tail by a bifunctional coumarin phototrigger carbamatelink at one end and a carboxylate group at C7 as the tether (40).The hydrophobic head was cleaved upon irradiation, whichreleased the amino acid from the coumarylmethyl stearate 41.Liposomes that incorporated 20% of 40 were compared in sizebefore and after irradiation and found to decrease in diameter by50% as a result of the liposome reorganization and leakage.52
A caging strategy for masking the biological activity of acomplex, heavily functionalized molecule such as Isotaxel wasdemonstrated by Kiso et al. (Scheme 24).85 Isotaxel, an O-acylisoform of the anticancer agent Paclitaxel, was modified to sup-press the pharmacophore activity through caging the benzyl-amine moiety with a coumaryl PPG. The resultingphotoresponsive prodrug, called Phototaxel (42), was selectivelyactivated by visible light (430 nm) releasing Isotaxel (43), whichin turn was converted into Paclitaxel (44) by a spontaneous intra-molecular O–N-acyl migration (Scheme 24).85 The yield ofreleased Paclitaxel was found to be 69%; partial loss of materialwas attributed to formation of unidentified side photoproducts.
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 (Scheme 24).86 N- andO-protected Paclitaxel prodrugs 45 and 46 with carbamate andcarbonate-linked coumarins were synthesized. Both caged com-pounds showed excellent water solubility; however, carbonate 46was not stable to aqueous solutions. Notably, direct continuousirradiation (365 nm) by a UV ray lamp released Paclitaxel from45 very effectively, whereas pulsed laser activation (355 nm)resulted in substantial decomposition.86
Effective release by irradiation at long wavelengths and excel-lent quantum yields for the 7-aminocoumarin derivatives makethem the protecting group of choice vis-à-vis other PPGs forwavelength-selective uncaging.87,88 Thus, del Campo et al. useda pair of wavelength-based nearly orthogonal photoactivated pro-tecting groups, a 7-diethylaminocoumarin (λmax = 390–400 nm)and a nitroveratryloxycarbonyl (λmax = 356 nm), each individu-ally attached through an amino group tethered to a silica surface(Scheme 25).87 Sequential spatially controlled wavelengthexposures, first with 412 nm and then 345 nm light, selectivelydeprotected coumarin and nitroveratryloxycarbonyl groups creat-ing patterns which were visualized by in situ subsequent coup-ling of the uncaged amino groups with different fluorescentdyes. Reversing the exposure sequence to first 345 nm light fol-lowed by 412 nm resulted in simultaneous cleavage of both
chromophores at 345 nm due to the overlap and similar extinc-tion coefficients of both PPGs at this wavelength.
The study further expanded the controllable variables fromspatial and time-resolution to include wavelength selectivity byemploying a variety of caging groups that are activated over arange of excitation wavelengths. A clear objective of this studywas to identify truly orthogonal chromophores for activation offunctional groups, an objective of many synthetic applications inprotecting group chemistry. Seven caging chromophores, rangingfrom pHP (λmax 270 nm) to coumaryl (λmax 350 nm), wereattached to a carboxylate group poised on an alkyl tether boundto a glass surface by a Si(OMe)3 linkage. Systematic variation ofthe exposure wavelength to the coated silica plate released thefunctional carboxylate groups. The chromophores gave an effec-tive wavelength range between 250 and 450 nm. Analysis of theseven chromophores further demonstrated that pairwise, tripletand quartet sets of orthogonal protecting groups from the varietyof caging chromophore combinations could be generated on asingle platform. This approach holds promise for new appli-cations of sensors that offer more precise control for moleculardetection and for communication purposes.
A photoremovable coumarin protection was employed byWylie and Shoichet in a clever design of three-dimensionalpattern-writing medium (Scheme 26).89 The matrix, composedof modified aragose 47 possessing an exposed amino group pro-tected with 6-bromo-7-hydroxycoumarin, was chosen for itshigh efficiency in two-photon activation. Confocal microscopyusing a Ti-sapphire laser for 3D multiphoton uncaging cleavedthe coumarin protecting group liberating the primary amine moi-eties in the gel.
Thiols. Analogously, unmasking sulfide groups upon UVlight exposure in thiolated aragose gels 48 provided a con-venient, chemospecific 3D pattern at the reaction site in prep-aration for selective immobilization of biologically relevantsubstrates (Scheme 26).90 This approach preserves the mechan-ical properties of the patterned material, enabling the creation ofcomplex 3D biochemical sites buried within hydrogels.89,90
Photolabile coumarin thiocarbonate protecting groups forthiols were developed by Hagen et al.48,74 Two complementaryPPGs, 7-aminocoumarin 49 (λmax 450 nm) and 7,8-biscarboxy-methoxycoumarin 50 (λmax 324 nm), were employed to mask
two different cysteine residues in resact, a sea urchin peptidehormone guanylate cyclase plasma membrane (Scheme 27).48
Notably, both PPGs were stable to TFA, which makes themorthogonal to many conventional peptide protecting groups.Irradiation of the protected resact with 402 nm light led to selec-tive cleavage of 49, releasing 50-OH and two resact isomers, theresult of an S-to-S acyl shift of the remaining 50 group, therebyfreeing a Cys1 side chain. Subsequent irradiation of the productmixture at 325 nm quantitatively removed 50 from the peptide.Although careful tuning of substituents on the PPG allowed
clean wavelength-selective photochemistry, the observed acylmigration within resact rendered thiocarbonate PPGs inappropri-ate for selective protection of cysteine residues in a peptide.48
Alcohols and phenols. Furuta et al. compared several carbon-ate-tethered coumarin phototriggers for their efficient release ofalcohols (Scheme 28).43 All coumarin PPGs tested were foundto be at least an order of magnitude more efficient than o-nitro-benzyl. Within the coumarins, the 7-methoxy- and 6-bromo-7-hydroxy-analogs 51a and 51b provided the highest quantumyields, and 51b was the most efficient among all the testedcages. A few biologically relevant alcohols caged with 51bshowed moderate to good resistance to hydrolysis, and all cagedcompounds efficiently released substrates upon photolysis at350 nm.43
Alkylation of the phenolic moiety of rosamine (52) with cou-marin-4-ylmethyl chloride followed by base-promoted spiro-cyclization destroyed conjugation in the xanthene ring andcompletely shut off the fluorescence of 53 (Scheme 29).91 Briefexposure of 53 to visible light induced a large fluorescenceenhancement typical for rosamine dye (577 nm), indicatingefficient probe release and restored conjugation in xanthene.Notably, the fluorescence spike in this case was much more pro-nounced indicating very efficient photorelease, when comparedwith release of rhodamine from the o-nitrobenzyl cage.92
Caged capsaicin 54, possessing the {7-[bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl PPG, was employed tostudy capsaicin’s effect on HEK 293 cells transfected withTRPV1 channels – cation channels that mediate pain perceptionin nociceptive somatosensory neurons (Scheme 30).93 Prelimi-nary tests showed that carbonate 54 had no residual activity atthe micromolar level, was sufficiently resistant to hydrolysis inthe dark, and efficiently released capsaicin 55 at 365 or405 nm.51,93 Isothermal titration calorimetry showed that themodified capsaicin 54 did not measurably translocate across aphospholipid membrane and thus can be selectively applied to
one side of the plasma membrane. The effect of intracellular andextracellular photorelease of capsaicin from membrane-imper-meant 54 was investigated. It was found that extracellular releaseof 55 triggered large whole-cell current and strong desensitiza-tion, whereas the analogous response was rather modest fromintracellular release of capsaicin.93
Carbonyl compounds. The first example of utilizing photoactivation of coumarylmethyl-caged carbonyl compounds in abiological system was reported by Hagen et al. (Scheme 31).94
The rate of the primary reaction (56 → 57) was found to be(1.2 ± 0.5) × 108 s−1 and the yield of progesterone (57) releasewas about 30%. The quantum yield was low but acceptable forthe application, due to high absorptivities that ensure high photo-sensitivity. Caged 56 was also sensitive to 2PE; exposure of 56to femtosecond pulses at 755 nm caused significant release ofprogesterone. However, the efficiency was ca. 5 times lower thanthat observed for an analogous glutamate derivative.66
Conclusions
The coumarylmethyl and pHP photoremovable protectinggroups are being developed as photoinitiators for mechanisticstudies in biological transformations that require nanosecondrelease rates. They are synthetically accessible and can beinstalled readily on most substrates. Recent advances in timeresolved spectroscopy encourage yet further development ofthese new PPGs.
Acknowledgements
We thank the NIH (RO1GM72910) for support.
Notes and references
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