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Photoreleasable Protecting Groups Triggered by Sequential TwoPhoton Absorption of Visible Light: Release of Carboxylic Acids from a Linked Anthraquinone-N-Alkylpicolinium Ester Molecule Matthew D. Thum and Daniel E. Falvey* University of Maryland, College Park, Maryland 20742, United States ABSTRACT: A photoreleasable protecting group activated by sequential absorption of two visible photons is designed, synthesized and tested. Specifically, an anthraquinone-based chromophore is covalently attached to an N-alkylpicolinium ester. Photolysis of this linked system results in the clean release of a corresponding carboxylic acid. Through product analysis, laser flash photolysis and steady-state UV/vis spectroscopy it is demonstrated that carboxylate ion release is effected by sequential absorption of two photons. The initial photochemical step results in reduction of an anthraquinone chromophore to the corresponding hydroquinone. The latter either reacts with O2 to regenerate the starting material, or absorption of a second photon causes an electron transfer to the picolinium group triggering C–O bond scission and release of the carboxylate.
Scheme 1. A stepwise, two-photon process.
INTRODUCTION Photochemical reactions have long been valued for their ability to provide both temporal and spatial control of molecular processes. Recently attention has turned to photochemical processes that are initiated by multiple photons, as these reactions have the promise of providing more precise 3dimensional spatial control. One approach is non-resonant two-photon absorption.1–8 In this case, the targeted excited state is generated using two photons that, individually, would not possess sufficient energy to populate an excited state, but when absorbed simultaneously, can create one. Simultaneous absorption requires high photon densities and, in practice, is typically realized using focused, femtosecond laser pulses. An attractive feature of such systems is the ability to activate photochemical or photophysical responses in media that might absorb the lower wavelengths required for resonant excitation and/or with 3-dimensional control, as the desired two-photon processes only occur at the focal point of the laser beam. Applications of this phenomenon have been demonstrated in high resolution optical imaging,9–13 photolithography,10,14–19 3dimensional optical storage,20,21 and more.22–25 The present study is part of a program aimed at developing molecular systems that are activated through stepwise twophoton excitation.26–30 Scheme 1 illustrates the general design principles. Initial excitation of substrate A creates a short lived excited state or reversibly formed reactive intermediate B. The excited state of interest is created when B absorbs a second photon. In contrast to the high peak power requirements for non-resonant multiphoton absorption, stepwise, twophoton absorption can be achieved using low power, continuous wave (CW), light sources. Some examples of this approach have been successfully applied to fluorescence imaging, photochemical switches, photoacid generation, and synthesis.31–35 The experiments described below focus on the development of photoreleasable protecting groups (PRPGs) based on stepwise, two-photon excitation.
hν2
hν1
A
C
B
1/τ
PRPGs are molecular groups that can be covalently attached to a substrate molecule, masking its normal reactivity.36–45 Photolysis of the PRPG unit releases the original substrate. While most PRPGs are activated by single-photon absorption, some have shown useful behavior under non-resonant two-photon excitation.23,46 However, other than an interesting example described by Pirrung and coworkers using a modified onitrobenzyl group PRPG for alcohols, PRPGs activated by stepwise two-photon excitation remain underexplored.47 Herein we report the efficient photorelease of a carboxylate anion that is triggered by visible light, sequential, two-photon photoinduced electron transfer (PET) from an anthraquinone chromophore to a covalently attached N-alkyl picolinium (NAP) group by exploiting the photochemical reduction of anthraquinone to hydroquinone in the presence of alcohols. A previous report described single photon release of carboxylate anions (X-) using a N-alkylpicolinium (NAP) ion that was covalently attached to a light–absorbing mediator (M) via photoinduced electron transfer mechanism. The current report demonstrates that by tuning the redox potential of M it is possible to drive a similar release process through sequential release of two photons (Scheme 2). Scheme 2. Sequential, two-photon release of a compound, X, using two electron transfer steps. D M
M
D hν1
NAP
NAP
X
X
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M
NAP
X
kBET
kFRAG
hν2 kBET
M
NAP
+
X
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The Journal of Physical Chemistry
Scheme 5. Synthesis of a linked AQ-NAP complex.
RESULTS AND DISCUSSION
NH2 Cl
Previous work demonstrated that the NAP (Scheme 3) group can be used as a conventional, one-photon PRPG for carboxylic acids and amines.48–52 One-electron reduction of the NAP group triggers a rapid and efficient C–O bond scission releasing a carboxylate or carbamate ion. The reduction step can occur either directly from an excited-state electron donor (e.g. N-methylcarbazole), or though mediated electron transfer. In the latter case, the mediator (M) is an excited state oxidant (e.g benzophenone) that abstracts an electron (or H atom) from a sacrificial donor (Scheme 3) and subsequently reduces the NAP group in a spontaneous, ground state process. Adapting this reaction to a stepwise two-photon scheme requires that the first photon generate an intermediate that will not spontaneously reduce the NAP group, but will only do so after absorbing a second photon. Scheme 3. Photorelease via electron transfer to the NAP group. D+• D
X
M* M-•
X H
X
hν
X = Amines Carboxylates
M N
N
N
Scheme 4. Photochemical reduction of AQ 3* X
O (1) hν
DH
D•
O2
O
O
O O O
O N
4 eq.
O AQH•
N
H N
Cl O
THF reflux 18 hours
O PAQ-1
2-Aminoanthraquinone was covalently liked to a picolyl ester according to the route described in Scheme 5. In brief, 2aminoanthraquinone and 2-chloroacetylchloride were combined in dichloromethane at 0 o C for one hour followed by stirring at room temperature overnight yielding 2-chloro-N(9,10-anthraquinone-2-)acetaniline. The final linked chromophore-acceptor, PAQ-1, was prepared by heating 2-chloro-N(9,10-anthraquinone-2-)acetaniline and picolyl phenyl acetate in THF at reflux for 18 hours.
Photolysis of PAQ-1 in isopropyl alcohol or methanol, either with high intensity visible light (447 nm, ca. 1 W) or with sequential application of 350 nm (broadband) light followed by 447 nm light causes clean release of phenylacetic acid. These solvents were chosen because they are expected to serve as H atom donors to the triplet state of AQ and thus produce the intermediates that could be subsequently photolyzed to initiate release of from NAP. The AQ chromophore has an absorbance maximum at 337 nm, as well as a long wavelength tail that extends out beyond 450 nm. Thus, both light sources are expected to excite AQ and the subsequently formed intermediates (see below). A specific example of the photolysis illustrated in Figure 1. In this case, a 0.24 mM solution of PAQ-1 in isopropyl alcohol was irradiated for one hour at 447 nm. After removal of the solvent only phenyl acetic acid and PAQ-0 were detected by 1H NMR (nuclear magnetic resonance) and ESI+ (electro-spray ionization) mass spectrometry. Following the irradiation, 1H NMR shows clear change as
PAQ-1 is converted into PAQ-0 and phenyl acetic acid (Figure 2). The formation of PAQ-0 was further confirmed by comparing photolyzed samples to that of an independently synthesized standard. Similar photolyses carried out on air-equilibrated samples also produced some phenylacetic acid, but with a more complex mixture of byproducts.
O AQ3*
OH X
X k [AQH•] d
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Ph
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OH
Cl O
AQ (X=NH2)
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1.4 eq.
CH2Cl2 18 hours
X
AQ
O Cl
(b) Photochemical deprotection
Anthraquinone (AQ, Scheme 4) derivatives were chosen as mediators for sequential two photon excitation purposes. With appropriate substitution they absorb in the visible region of the spectrum and, in most cases, excitation results in efficient formation of excited triplet states, AQ3*. Under reducing conditions (i.e. in the presence of electron donors, or in solvents that donate H atoms), AQ3* is known to generate three potential intermediates that could serve as B in Scheme 1.53–59 One – electron reduction forms the anion radical (AQ• ) and/or its conjugate acid, the semiquinone radical (AQH•). The latter radicals can either undergo a disproportionation reaction, or a sequential H atom abstraction, to form hydroquinones (AQH2). The reduction potential of the anthraquinone chromophore (-0.62 V vs. SCE), is less negative than that of the NAP group (-1.1 vs SCE), therefore rapid and spontaneous – electron transfer from AQ• to the NAP group should be disfavored. Likewise, the protonated AQH• and the hydroquinone, AQH2, are expected to be even less labile toward the subsequent electron transfer to NAP. Therefore any of these reduced intermediates could serve as the initially prepared state in the two-photon scheme.
O
O
O
(a) Design and Synthesis
X=H, NH2, NHAc OH AQH2
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hν, 447 nm
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This observation, as well as experiments on unlinked model systems described below, lead us to conclude that the triplet AQ either abstracts a H atom from the solvent, or relaxes to ground state. Any direct interaction with the NAP group would be negligible. Following the decay triplet, a new, longer lived, species with an apparent lmax at 410 nm is seen. Again, comparison with previously reported spectra allows the peak at 410 nm to be assigned to the semiquinone.55 The latter is observed to decay but is too persistent (t1/2 ca. 300 us, See SI Figures S-15). for accurate kinetic analysis given the limits of the LFP set up The long lifetime of the AQH• argues against a direct, exergonic H atom or electron transfer to the NAP group.
Figure 1. 1H NMR spectra from the photolysis of PAQ-1 in isopropyl alcohol. A stock solution of PAQ-1 (0.24 mM in isopropyl alcohol) was prepared and filtered through a 0.45 µm cellulose acetate syringe filter prior to photolysis. To a 1 cm quartz cuvette with a stirbar was added 1.5 mL of PAQ-1 stock. The cuvette was purged with N2 for 10 minutes in the solution and an additional 5 minutes in the headspace. The solution was photolyzed at 447 nm for the desired time before the solvent was removed under reduced pressure and CD3OD was added. Samples were photolyzed for 0, 10 and 50 min and then analyzed by mass spectrometry (ESI+, see SI Figures S4-S6). PAQ-0 appears after 10 minutes of photolysis, and after 50 minutes, it becomes the only major product. Free phenylacetic acid was also identified by 1H NMR in the photolysis solution by the addition of standard phenyl acetic acid after the solvent was removed and CD3OD was added. By monitoring changes in their relative peak intensities it was seen that the ester peak at 7.4 ppm is replaced by a new signal at 7.2 ppm upon irradiation corresponds with formation of free phenylacetic acid. However, free phenyl acetic acid could not be explicitly quantified due to the loss of product during the removal of solvent. The formation of phenylacetic acid, along with the presence of PAQ-0 as the only major photoproduct, suggests a mechanism involving PET to the attached NAP group and release of substrate while NAP group remains covalently linked to the chromophore.
Figure 2. Transient absorption spectrum of PAQ-1 in 90:10
isopropyl alcohol/methanol.
(b) Mechanism of photochemical deprotection Laser flash photolysis (LFP) with UV/vis detection was used to identify and characterize the relevant short-lived intermediates in the photorelease process. The transient absorption spectrum from pulsed photolysis of PAQ-1 (354.7 nm, 10 mJ, 5-7 ns) is shown in Figure 2. Immediately following excitation there appears a sharp absorption feature at 490 nm which is assigned to the triplet state localized on the anthraquinone. This assignment is made on the basis of triplet state spectra for structurally similar anthraquinone derivatives and oxygen quenching experiments (See SI Figure S-14).55 Its lifetime of 833 ns in an H-atom donating solvent (isopropyl alcohol) is largely consistent with similar AQ derivatives measured in previous work.55 Donor substitution at the 2-position attenuates, but does not prevent H atom transfer. The detection of the triplet on a ns timescale also suggests that the triplet does not directly transfer energy or electrons to the NAP group. In a linked system such as PAQ-1, exergonic electron and energy transfer process generally occur on a ps or shorter timescale.
Figure 3. Steady state photolysis of PAQ-1 at 447 nm in isopropyl alcohol. To characterize the intermediates present over longer timescales, steady state photolysis of PAQ-1 with a 447 nm, 1W CW diode laser in isopropyl alcohol was carried out and the long–lived products were detected by UV/vis spectroscopy. Figure 3 shows that this photolysis generates a new absorption band at 390 nm, which is confidently attributed to the hydroquinone.55,59 The latter, which is formed with a quantum yield of 0.2 (See SI Figures S-12 and S-13), persists indefinitely (>1 day) and upon bubbling with molecular oxygen, reverts back to its original spectrum of the starting quinone. However, it should be noted that the hydroquinones derived from PAQ-1 and PAQ-0 are not readily distinguished by UV/vis absorption. Thus, from this experiment alone, it is not clear if the observed AQH2 species is an intermediate on the pathway to
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The Journal of Physical Chemistry
release, or if it merely accumulates from subsequent photoreduction of PAQ-0. O
O H N
O O
N
O
1
ClO4
2
Figure 4. Model compounds used in the un-linked experiment performed in this study. For synthesis and characterization, please see SI. Unlinked model compounds 1, 2-acetylamino anthraquinone and 2 (a NAP ester) were prepared and used to test the possible mechanisms. Figure 5 shows the transient absorption spectrum following excitation of 1 in isopropyl alcohol. As with the linked PAQ system, a short-lived feature at 490 nm due to the triplet decays to leave a long-lived signal at 410 nm, attributed to the semiquinone. In this case the ratio of triplet to semiquinone formation is somewhat larger than it is for PAQ1. The reasons for this were not examined in detail. In any case, the semiquinone from 1, does not react at a significant rate with NAP derivative 2. Quenching experiments where the semiquinone signal was monitored at 410 nm as the concentration of 2 was increased up to its solubility limit in isopropyl alcohol, (Figure 6) demonstrate the that lifetime of the semiquinone is insensitive to the presence of 2. On this basis, we exclude ground state electron transfer from the singly-reduced aminoanthraquinone to the NAP group as a significant pathway for release of carboxylic acid in the PAQ-1 photolysis.
Two experiments show that photolysis of AQH2 results in NAP reduction and carboxylate release. First, the unlinked AQH2 was generated independently (i.e. in a nonphotochemical manner) and its photolysis in the presence of NAP derivative 2 results in carboxylate release. As illustrated in Figure 7, a solution of 1 and 2 in CD3OD was treated with the reducing agent, sodium dithionite, in D2O under an inert atmosphere. As expected, UV/vis spectroscopy showed that this results in the formation of the corresponding AQH2 species. When photolysis of the latter is carried out in the presence of NAP ester 2, clean release of the corresponding acid is seen by 1H NMR. Control experiments showed that the release requires the presence of reducing agent (to form AQH2) and light. Omitting either leaves ester 2 unchanged. * O O O O* H H N N * O *O * * HO O * + O O* O
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Figure 7. 1H NMR spectra of the control experiments performed with compounds 1 and 2. Blue: A solution of 1 and 2 were kept in the dark for 60 minutes in a solution of 2:1 CD3OD/D2O. Red: A solution of 1 and 2 were photolyzed in a solution of 2:1 CD3OD/D2O at 447 nm for 60 minutes. Green: A 1 mL stock solution of 1 in CD3OD was purged with nitrogen for 5 minutes. After purging, 0.5 mL of 39.4 mM sodium dithionite was added via cannula. The solution was then kept in the dark for 60 minutes. Pink: A 1 mL stock solution of 1 in CD3OD was purged with nitrogen for 5 minutes. After purging, 0.5 mL of 39.4 mM sodium dithionite was added via cannula. The solution was then photolyzed at 447 nm for 60 minutes. Figure 5. Transient absorption spectrum of 1 in 90:10 isopropyl alcohol/methanol.
Figure 6. Waveforms of 1 at 410 nm with increasing concentrations of 2.
It is also possible to prepare the linked hydroquinone in a similar way. In this second experiment, PAQ-1 was reduced using sodium dithionite in a mixture of D2O/CD3OD (Figure 8). UV/vis spectroscopy showed that, under an inert atmosphere, PAQ-1 was quantitatively converted into the doublyreduced hydroquinone (PAQ-1H2 Scheme 6). Two identical samples were prepared, the first of which was photolyzed at 447 nm. After 60 min, clean conversion to PAQ-0 resulting from release of the carboxylic acid was observed. The dark control, however, showed no reaction. (see SI Figure S-10). These observations further establish that the PAQ-1H2 is capable of acting as an excited state donor to NAP. Significantly, the PAQ-1H2 does not trigger release of the carboxylate at a significant rate in the absence of light.
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toward NAP in the along with the demonstrated PET reaction between PAQ-1H2 and NAP. Scheme 7. Proposed Photorelease Mechanism.
Scheme 6. Photolysis of Thermally Generated PAQ-1H2. O O
O O
O
N
H N
O
OH
Cl Sodium Dithionite
O
H N
OH
rk
Da
O O
N
H N
O
Cl
H2O2
N
O2
Cl
PAQ-13* λmax = 490 nm
Ph
Cl
O
N
H N
Cl
OH
N
H N
Disproportionation
hν2, 447 nm PET -H+
Cl
O
O
OH
Ph O
O OH
OH
OH H
O
O
+
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Cl
O
PAQ-1
H N
N
H N
hν1, 447 nm
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O
O O
N
H N
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in
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D2O/CD3OD
O
N
nm 7 44 1) O2 2)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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PAQ-1
PAQH•-1
O
OH PAQH2-1 λmax = 390 nm
PAQH-1 λmax = 410 nm O
Ph O
OH
N
H N
Rearrange Re-Oxidation
O O
O O O H
+
N
H N
Cl
O O PAQ-0
CONCLUSION
Figure 8. PAQ-1 was converted to PAQ-1H2 by reduction with sodium dithionite in CD3OD/D2O under an inert atmosphere. To 1.5 mL of a 0.893 mM solution of PAQ-1 in CD3OD under N2, 0.5 mL of 0.1 M sodium dithionite in D2O was added via cannula. The UV/vis spectrum shows the chemical conversion to hydroquinone using sodium dithionite. The dark control showed no change by UV/vis. Given these observations, along with the transient spectroscopy and photoproduct analysis demonstrated earlier, a mechanism for the stepwise, two-photon release of carboxylate is described in Scheme 7. The first photon excites the AQ chromophore, forming the corresponding triplet state, PAQ-13*. The latter abstracts a H atom from the solvent, to form the PAQ-1H•. A relatively slow disproportionation reaction of PAQ-1H• provides the hydroquinone PAQ-1H2. The second photon drives a PET from PAQ-1H2 to the NAP, resulting in release of the substrate. A relatively slow reaction of PAQ1H2 with O2 resets the chromophore back to its original state (PAQ-1). In the present system, the overlap of PAQ-1H2 absorption with that of PAQ-1, along with a fast intramolecular photoreduction of the NAP group, make accumulation of the intermediate complex infeasible. Moreover, given the stability of PAQ-1H2 along with its slow rate of formation via disproportionation, it is not expected that the release process will exhibit the sort of non-linear intensity dependence that is characteristic of reaction driven by non-resonant two-photon absoprtion. However, the requirement for sequential absorption of two photons is demonstrated through the lack of ground-state reactivity of the key intermediates (PAQ-1H•, PAQ-1H2)
The experiments described herein demonstrate a linked chromophore-protecting group system, PAQ-1, which can release a model carboxylate upon sequential absorption of two visible light photons in the presence of a hydrogen atom donor. The mechanism involves photochemically generating hydroquinone in solution and eventual photolysis of the hydroquinone triggering PET to the NAP group and release of free carboxylate. Further work is currently in progress to increase the substrate scope to include amines, and other biologically relevant molecules such as amino acids and phosphates. Furthermore, it would be ideal if the absorption spectrum of groundstate and intermediate or transient species (in this case the groundstate anthraquinone and the groundstate hydroquinone) were shifted relative to one another to allow for selective irradiation of the intermediate. To address this issue and improve upon the current system, additional chromophores are being investigated. The development of sequential two-photon PRPGs will allow for continued improvement in the spatial control over photochemical process.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author *
[email protected] Author Contributions All authors have given approval to the final version of the manuscript.
Funding Sources
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The Journal of Physical Chemistry
This work was supported by the National Science Foundation (CHE-1112018)
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ABBREVIATIONS CW, continuous wave; PRPG, photoreleasable protecting group; PET, photoinduced electron transfer; NAP, N-alkyl picolinium; NMR, nuclear magnetic resonance; ESI, electrospray ionization; LFP, laser flash photolysis.
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