Competing pathways in the photo-Favorskii rearrangement and release of esters: Studies on fluorinated p-hydroxyphenacyl-caged GABA and glutamate phototriggers

Article abstract of DOI:10.1021/jo900139h

(Chemical Equation Presented) Three new trifluoromethylated p-hydroxyphenacyl (pHP)-caged γ-aminobutyric acid (GABA) and glutamate (Glu) derivatives have been examined for their efficacy as photoremovable protecting groups in aqueous solution. Through the

Full text of DOI:10.1021/jo900139h

pubs.acs.org/joc
Competing Pathways in the Photo-Favorskii Rearrangement and Release
of Esters: Studies on Fluorinated p-Hydroxyphenacyl-Caged GABA and
Glutamate Phototriggers
Kenneth Stensrud,† Jihyun Noh,‡ Karl Kandler,‡ Jakob Wirz,§ Dominik Heger,^
and Richard S. Givens*,†
†Department of Chemistry, 1251 Wescoe Hall Drive, University of Kansas, Lawrence, Kansas 66045,
‡Department of Otolaryngology, 3500 Terrace Street, University of Pittsburgh, Pittsburgh, Pennsylvania
§
15208, Departement Chemie, Universitat Basel, Klingelbergstrasse 80, CH-4056 Basel, Switzerland, and
€
^Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
givensr@ku.edu
Received January 26, 2009
Three new trifluoromethylated p-hydroxyphenacyl (pHP)-caged γ-aminobutyric acid (GABA) and
glutamate (Glu) derivatives have been examined for their efficacy as photoremovable pro-
tecting groups in aqueous solution. Through the replacement of hydrogen with fluorine,
e.g., a m-trifluoromethyl or a m-trifluoromethoxy versus m-methoxy substituents on the pHP chromo-
phore, modest increases in the quantum yields for the release of amino acids GABA and glutamate as
well as improved lipophilicity were realized. The pHP triplet undergoes a photo-Favorskii rearrange-
ment with concomitant release of the amino acid substrate. Deprotonation competes with the
rearrangement from the triplet excited state and yields the pHP conjugate base that, upon
reprotonation, regenerates the starting ketoester, a chemically unproductive or “energy-wasting”
process. When picosecond pump-probe spectroscopy is employed, GABA derivatives 2-5 are
characterized by short triplet lifetimes, a manifestation of their rapid release of GABA. The
bioavailability of released GABA at the GABAA receptor improved when the release took
place from m-OCF3 (2) but decreased for m-CF3 (3) when compared with the parent pHP derivative.
These studies demonstrate that pKa and lipophilicity exert significant but sometimes opposing
influences on the photochemistry and biological activity of pHP phototriggers.
Introduction
and have become a subject of wide interest.1,2 They diminish
or block the normal reactivity of an active compound, which
can be restored upon exposure to light. When employed in
this capacity, light is regarded as a “traceless reagent” that
can be administered along specific spatial, temporal, and
concentration coordinates.1,3 These features are exploited,
for example, through the release of the carboxyl group of the
amino acids glutamate (Glu), γ-aminobutyric acid (GABA),
Photoremovable protecting groups or “phototriggers”
have found many applications in chemistry and biology
(1) (a) Dynamic Studies in Biology, Phototriggers, Photoswitches, and
Caged Compounds; Goeldner, M., Givens, R., Eds.; Wiley-VCH: Weinheim,
Germany, 2005. (b) Pelliccioli, A. P.; Wirz, J. Photochem. Photobiol. Sci. 2002,
1, 441–458. (c) Adams, S. R.; Tsien, R. Y. Annu. Rev. Physiol. 2000, 18, 755–
784.
(2) (a) Greene’s Protective Groups in Organic Synthesis, 4th ed.; Wutts,
P. G. M., Greene, T. W., Eds.; Wiley Interscience-John Wiley and Sons: New
York, 2007; pp 980-9832. (b) Protti, S.; Fagnoni, M. Chem. Commun. 2008,
3611–3621.
(3) Ni, J.; Auston, D. A.; Frelich, D. A.; Muralidharan, S.; Sobie, E. A.;
Kao, J. P. Y. J. Am. Chem. Soc. 2007, 129, 5316–5317.
DOI: 10.1021/jo900139h
r
Published on Web 07/02/2009
J. Org. Chem. 2009, 74, 5219–5227 5219
2009 American Chemical Society

Stensrud et al.
JOCArticle
or glycine in order to investigate neuronal processes in cell-
signaling, kinetic, and mechanistic studies of the central
nervous system.4
Only a very weak fluorescence is observed with this chromo-
phore.14d
Since our discovery that the p-hydroxyphenacyl (pHP)
chromophore will serve as a photoremovable protecting
group,5 several biologically relevant substrates such as phos-
phates (ATP5 and GTP6), thiols (Protein Kinase A7 and
glutathione8), and carboxylates (GABA9 and Glu10), includ-
ing the C terminus of the oligopeptide (bradykinin11,12
)
and examples of functional group protection in synthesis2,
have been reported. This has prompted us to investigate the
mechanistic features and substituent effects on the reaction.
Interestingly, the reaction results in a significant and useful13
blue shift because of the rearrangement of the chromo-
phore to (p-hydroxyphenyl)acetic acid (6) as a primary
product (eq 1). The reaction mechanism has been studied
in detail, predominantly by Phillips and co-workers14 and
by us.15 We have shown that the reaction of p-hydro-
xyphenacyl diethyl phosphate proceeds via a very short-lived
triplet state T1 (3τ = 60 ps in wholly aqueous solution)
and that the substrate is released concomitantly with the
decay of T1. Questions remain regarding the nature and
significance of competing pathways and the effect of sub-
stitution of the chromophore on the release of substrates.
In particular, the quantum yields for substrate release
are often substantially less than unity, suggesting that che-
mically nonproductive pathways may contribute to the
normal photophysical decay of the excited chromophore.
In previous studies, it was demonstrated that the intro-
duction of electron-donating groups such as OCH3 (4 and 5)
shifts the π,π* absorption of the chromophore to longer
wavelengths (λmax>300 nm), whereas electron-withdrawing
meta substituents (CO2Me or CONH2) have little influence
on λmax.
1,9 None of these substituents significantly affected
the lifetime of the triplet state in contrast to their effects on
the quantum yields.1,16 Electron-withdrawing groups im-
prove the quantum yields for the photo-Favorskii rearrange-
ꢀ
ment, whereas electron donors are lower vis-a-vis the parent
pHP derivatives.
To combine the advantages of the red shift by m-methoxy
substitution and the improved quantum yields from elec-
tron-withdrawing groups, we introduced trifluoromethyl
and trifluoromethoxy groups and compared the photochem-
istry and biological efficacy with those of the methoxy and
the unsubstituted pHP GABA analogues. Among the factors
that are attendant with introducing a trifluoromethyl sub-
stituent are the significant increase in polarity due to the
pronounced electron-withdrawing inductive contribution
of F,17 the relatively modest increase in steric size, and the
potential improvement in biocompatibility.18 Although the
trifluoromethyl group has rarely been exploited as a sub-
stituent on phototriggers,19 we reasoned that it would
modify the reactivity without greatly disturbing the π system
of the chromophore. We report here the synthesis and photo-
chemistry of three trifluoromethyl modifications, CF3O-
pHP GABA (2), CF3-pHP GABA (3), and CF3-pHP Glu
(8), to test this hypothesis. We have also probed the differ-
ences between 2 and 3 upon GABA release at the GABAA
receptor using whole-cell patch clamp monitoring of neurons
in cortical slices and compared these derivatives with those of
our previous study employing unsubstituted pHP GABA (1)
and m-CH3O-pHP GABA (4).9
(4) (a) Hess, G. P. Reference 1, pp 205-231. (b) Callaway, E. M.; Yuste,
R. Curr. Opin. Neurobiol. 2001, 12, 587–592. (c) Pettit, D. L.; Augustine, G. J.
Ion Channel Localization 2001, 349–370.
(5) (a) Park, C.-H.; Givens, R. S. J. Am. Chem. Soc. 1997, 119, 2453–
2463. (b) Geibel, S.; Barth, A.; Amslinger, S.; Jung, A. H.; Burzik, C.; Clarke,
R. J.; Givens, R. S.; Fendler, K. Biophys. J. 2000, 79, 1346–1357.
€
(6) (a) Kotting, C.; Kallenbach, A.; Suveyzdis, Y.; Wittinghofer, A.;
Gerwert, K. Proc. Natl. Acad. Sci. 2008, 105, 6260–6265. (b) Warscheid, B.;
€
Brucker, S.; Kallenbach, A.; Meyer, H. M.; Gerwert, K.; Kotting, C. Vib.
Spectrosc. 2008, 48, 28–36. (c) Du, X.; Frei, H.; Kim, S.-H. J. Biol. Chem.
2000, 275, 8492–8500.
(7) Zou, K.; Cheley, S.; Givens, R. S.; Bayley, H. J. Am. Chem. Soc. 2002,
124, 8220–8229. Zou, K.; Miller, W. T.; Givens, R. S.; Bayley, H. Angew.
Chem., Int. Ed. 2001, 40, 3049–3051.
(8) Specht, A.; Loudwig, S.; Peng, L.; Goeldner, M. Tetrahedron Lett.
2002, 43, 8947–8950.
(9) Conrad, P. G. II; Givens, R. S.; Weber, J. F. W.; Kandler, K. Org.
Lett. 2000, 2, 1545–1547.
(10) Givens, R. S.; Jung, A. H.; Park, C.-H.; Weber, J. F. W.; Bartlett, W.
J. Am. Chem. Soc. 1997, 119, 8369–8370.
(11) Givens, R. S.; Weber, J. F. W.; Conrad, P. G.; Orosz, G.; Donahue,
S. L.; Thayer, S. A. J. Am. Chem. Soc. 2000, 122, 2687–2697.
(12) Sul, J.-Y.; Orosz, G.; Givens, R. S.; Haydon, P. G. Neuroglia Biol.
2004, 1, 3–11.
Results and Discussion
Synthetic Studies. The synthetic sequence for 3 shown
in Scheme 1 is the general route used for all three of the
new derivatives. Benzyl protection of commercially available
4-bromo-2-(trifluoromethyl)phenol (9) with BnBr/K2CO3/
CH3CN, followed by acetylation with a Stille protocol20
using Pd(PPh3)4/tributyl(1-ethoxyvinyl)stannane in toluene,
and hydrolysis of the resulting enol ether gave the fluorinated
(13) The blue shift of the chromophore of the product (p-hydroxyphenyl)-
acetic acid to below 300 nm avoids competitive absorption with pHP and
thus permits 100% conversion of pHP derivatives at incident wavelengths
>300 nm. The “photo-Favorskii rearrangement” was first reported by
Anderson and Reese: Anderson, J. C.; Reese, C. B. Tetrahedron Lett.
1962, 1–4.
(14) (a) Chen, X.; Ma, C.; Kwok, W. M.; Guan, X.; Du, V.; Phillips, D. L.
J. Phys. Chem. A 2006, 110, 12406–12413. (b) Chen, X.; Ma, C.; Kwok, W.
M.; Guan, X.; Du, V.; Phillips, D. L. J. Phys. Chem. B 2007, 111, 11832–
11842. (c) Ma, C.; Kwok, W. M.; Chan, W. S.; Du, Y.; Kan, J. T. W.; Toy, P.
H.; Phillips, D. L. J. Am. Chem. Soc. 2006, 128, 2558–2570. (d) Ma, C.;
Kwok, W. M.; Chan, W. S.; Zou, P.; Kan, J. T. W.; Toy, P. H.; Phillips, D. L.
J. Am. Chem. Soc. 2005, 127, 1463–1472. (e) Ma, C.; Zou, P.; Kwok, W. M.;
Chan, W. S.; Kan, J. T. W.; Toy, P. H.; Phillips, D. L. J. Org. Chem. 2004, 69,
6641–6657. (f) Ma, C.; Kwok, W. M.; Chan, W. S.; Zou, P.; Phillips, D. L. J.
Phys. Chem. B 2004, 108, 9264–9276.
(16) (a) Givens, R. S.; Conrad, P. G., II; Yousef, A. L.; Lee, J.-I.
Photoremovable protecting groups. CRC Handbook of Organic Photochem-
istry and Photobiology, 2nd ed.; CRC Press: Boca Raton, FL, 2004; pp 69/1-69/
46. (b) Givens, R. S.; Weber, J. F. W.; Jung, A. H.; Park, C.-H. New photo-
protecting groups: desyl and p-hydroxyphenacyl phosphate and carboxylate
esters. Methods Enzymol. 1998, 291 (Caged Compounds), 1-29.
(17) Pauling, L. The Nature of the Chemical Bond; Cornell University
Press: Ithaca, NY, 1960.
(15) Givens, R. S.; Heger, D.; Hellrung, B.; Kamdzhilov, Y.; Mac, M.;
Conrad, P. G.; Cope, E.; Lee, J. I.; Mata-Segreda, J. F.; Schowen, R. L.;
Wirz, J. J. Am. Chem. Soc. 2008, 130, 3307–3309.
(18) Ojima, I. ChemBiolChem 2004, 5, 628–635.
(19) Specht, A.; Goeldner, M. Angew. Chem., Int. Ed. 2004, 43, 2008–2012.
(20) Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508–524.
5220 J. Org. Chem. Vol. 74, No. 15, 2009

Stensrud et al.
JOCArticle
SCHEME 1. Synthetic Strategy for Trifluoromethyl-Substituted pHP Amino Acids
TABLE 1. UV Spectral Data, pKa, and Disappearance Quantum Yieldsa for pHP GABA Derivatives 1-5 and CF3-pHP Glu (8)
φdis(H2O)c
φdis(pH 7.3)d
φdis(pH 8.2)e
λmax (log ε), pH 9.0
φdis(pH 9.0)f
b
pHP
λmax (log ε)
pKa
1g
2
3
4
5
8
282 (4.16), 325 sh
274 (4.20), 331 (3.54)
328 (4.11) i
279 (3.97), 307 (3.90), 341 sh
303 (3.90), 355 (3.55)
328 (4.11) h
8.0
6.7
5.5
7.85
7.78
5.5
0.20
0.09h
0.17
0.06
0.03g
0.18
0.20
0.06
0.12
NA
NA
NA
0.09
0.02
0.08
0.02
NA
0.11
326 (4.06)
332 (4.18)
328 (4.14)
348 (4.05)
366 (4.30)
328 (4.11)
0.0041
0.0058
0.0081
NA
NA
0.0067
aQuantum yields for the appearance of GABA were within (10% of the disappearance quantum yields for pHP GABA, φdis. bpKa’s were determined
titrimetrically (see the Supporting Information). cDeionized water, pH=7.0. d0.01 M HEPES, adjusted to pH=7.3, e0.01 M HEPES adjusted to pH=8.2.
f0.01 M Tris adjusted to pH 9.0, 0.1 M LiClO4, irradiation λ=350 nm. gSee ref 1a, Chapters 1.3 and 2. hAn identical value was obtained with oxygen-
purged solutions (Ar, 30 min). iThe pKa (5.5) is below the pH for this pHP GABA derivative.
acetophenone 10. The Stille coupling of protected p-bromo-
phenols to stannyl vinyl ethers served as a high-yielding,
general route for us to synthesize a variety of p-hydroxy-
acetophenones (pHA)21 as opposed to traditional acylation
methodology. R-Bromination with dioxane dibromide in
dichloromethane (DDB/CH2Cl2), followed by esterification
with N-Boc-protected GABA and deprotection of the phenol
and amine groups with Pd/C (10% w/w)/H2 in EtOAc and 1:1
TFA/CH2Cl2, respectively, afforded 3 in 55% overall yield
from 9. Essentially the same route was followed for the
syntheses of 2 (57%) and 8 (38%).
Photochemical Studies. All three derivatives, 2, 3, and 8,
quantitatively released the amino acid upon irradiation
at λ > 300 nm in aqueous media under ambient conditi-
ons. The Favorskii rearrangement products 6 (R1=OCF3 or
CF3; R2 = H) along with small amounts of the minor
product, p-hydroxybenzyl alcohols 7, were characterized
by 1H, 13C, and 19F NMR and mass spectral analysis
and, for certain cases, by comparison with authentic
samples. In the case of the formation of p-hydroxy-m-
FIGURE 1. Species spectra of the singlet (absorption and stimu-
lated emission; red, dotted) and triplet (absorption; green, solid) of 1
(trifluoromethoxy)benzyl alcohol 7 from 2, for example,
the reaction products were also verified by NMR analysis
after spiking the photolysis mixture with an independen-
tly synthesized sample of 7. Another potential photohy-
drolysis byproduct, 2-hydroxy-1-[4-hydroxy-3-(trifluoro-
methoxy)phenyl]ethanone (12), which is structurally related
to products that had been reported by others,22 was
shown not to be present by 1H and 13C NMR analysis
by the addition of an authentic sample to the photolysis
mixture.
in water determined by global analysis of the spectra taken with
delays of 0.6-15 ps using a monoexponential rate law for fitting.
Quantitative analyses of the major products GABA and
the substituted (p-hydroxyphenyl)acetic acid, along with
unreacted caged GABA, were accomplished using LC/MS/
MS equipped with a C18 RP column (mobile phase gradient
1:1 MeOH/H2O with added NH4 HCO2 and acetamino-
3
phen as the internal standard). This highly sensitive LC/MS/
MS method for quantitative analysis coupled with rapid data
collection made possible the accurate analyses of the reaction
profiles for quantum yields, quenching experiments, and
media effects on the photochemistry at low conversions
and using less than a few milligrams of caged GABA. The
quantum yield data are given in Table 1.
ꢁ
(21) Stensrud, K. F.; Heger, D.; Sebej, P.; Wirz, J.; Givens, R. S.
Photochem. Photobiol. Sci. 2008, 7, 614–624.
(22) Zhang, K.; Corrie, J. E. T.; Munasinghe, R. N.; Wan, P. J. Am.
Chem. Soc. 1999, 121, 5625–5632.
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Stensrud et al.
JOCArticle
FIGURE 2. Pump-probe spectra of 2 in water, reconstructed after
factor analysis in the time range from 20 ps to 1.96 ns using the
species spectra shown in Figure 3.
FIGURE 3. Species spectra attributed to the triplet (solid, green)
and the conjugate base (anion) of pHP (see text) formed from the
triplet (dotted, blue) of 2 in water determined by global analysis of
the spectra using a biexponential fit. These spectra are equal to those
taken at delays of 4 ps and 1.96 ns, respectively.
The photorelease quantum yield of the trifluoromethoxy
derivative 2 in an unbuffered aqueous solution (φ=0.09) is
approximately twice that of the methoxy (4) or dimethoxy (5)
counterparts, whereas that of the trifluoromethyl analogue 3
(φ=0.17) is nearly the same as that of the parent pHP GABA
1. Stern-Volmer studies at high concentrations of sorbate
quenched these reactions, confirming a short-lived trip-
let reactive intermediate for pHP esters 2 and 3 (see the
Experimental Section) in accordance with our earlier results
with 1.5,9,23 In a HEPES buffer, pH 7.3 (1-9 mM pHP
GABA), the quantum yields were slightly lower, which we
attribute to pH and salt effects,24 a suggestion supported by
dramatic changes in the quantum yields when the pH is
varied. In the absence of buffers, the pH of the photolysis
solution decreases with increasing conversion because of the
production of two carboxylic acids of lower pKa’s than
the pHP phenol. Quantum yields are dependent on the pH
of the solution (vide infra).
TABLE 2. Singlet (1τ) and Triplet (3τ) Lifetimes and Rate Constants
Obtained from Pump-Probe and Stern-Volmer Measurements
a
pHP GABA
1τ/ps
3τ/ns
φdis
kpF/108 s-1 b
H2O
0.34
0.46
0.39
0.77
1
2
3
4
2.3
0.20
0.09
0.17
0.06
6.00
1.98
4.42
0.78
10% CH3CN/H2Oc
2
3
4
5
0.71
0.39
3.3
4.6
3.7
0.36
aSee Table 1. bCalculated as ki=φi/3τ, where φdis is the quantum yield
for the pHP GABA disappearance and kpF is the rate constant for the
photo-Favorskii process (see Scheme 2). cData were obtained by pump-
probe spectroscopy. Approximately 10% CH3CN was added to ensure
complete solubility of the substituted pHP GABA.
Increasing the pH to 8.2 or 9, values significantly above the
pKa’s of 2 or 3, lowered the quantum yields by half or more.
The observed strong red shift in the absorption spectra at the
higher pH is due to the formation of conjugate bases of these
phenolic chromophores, and photorelease from these is
decidedly less efficient, as was reported earlier.15,16
TABLE 3. Effect of H2O on the Rate of Disappearance of the 390-400-nm
Band
3kdis/s-1
mol % H2Oa
refsb
None of these photoreactions is appreciably quenched by
air, a manifestation of the very short lifetimes and high
reactivities of the pHP triplet excited states. The inefficiency
of O2 quenching was tested by purging the photolysis solu-
tion with argon (see 2, Table 1).
Time-Resolved Pump-Probe Studies. Picosecond transient
absorption spectra were obtained for compounds 1-5. The
samples were excited at 266 nm (pulse width 200 fs) and probed
by delayed supercontinuum pulses covering a range of 300-
650 nm. The main features in the transient spectra of 1up to 15 ps
are the absorptions by the excited singlet and triplet states of the
pHP chromophore, which are shown for 1 in Figure 1. Global
1.6 ꢀ 1010
9.95 ꢀ 109
3.45 ꢀ 109
3.30 ꢀ 109
1.89 ꢀ 109
1.25 ꢀ 109
7.81 ꢀ 108
4.17 ꢀ 108
1.11 ꢀ 108
3.84 ꢀ 107
1.15 ꢀ 107
6.56 ꢀ 106
2.72 ꢀ 106
2.62 ꢀ 105
100.00
95.14
89.77
74.53
66.11
55.64
49.38
34.05
24.54
15.03
10.00
7.51
this work
this work
Phillips14
this work
Phillips14
Phillips14
Phillips14
Phillips14
Phillips14
Hellrung
Hellrung
Hellrung
Hellrung
Hellrung
5.04
0.00
aCH3CN was the cosolvent. bData are combined from this work and
from previous studies (Hellrung, B.; Wirz, J., unpublished, and Phillips
and co-workers14 as indicated).
(23) (a) Conrad, P. G. II; Givens, R. S.; Hellrung, B.; Rajesh, C. S.;
Ramseier, M.; Wirz, J. J. Am. Chem. Soc. 2000, 122, 9346–9347. (b) See also:
Chan, W. S.; Ma, C.; Kwok, W. M.; Phillips, D. L. J. Phys. Chem. A 2005,
109, 3454–3469. (c) Chan, W. S.; Ma, C.; Kwok, W. M.; Phillips, D. L. J. Org.
Chem. 2005, 70, 8661–8675.
analysis of the spectral evolution with increasing delay up to 2 ns
of the probe pulse provided the singlet and triplet lifetimes, 1τ=
2.3 ps and 3τ=0.34 ns, respectively.
(24) We have found a salt effect on several of these photorelease reac-
tions, which we will report on more fully at a later time.
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Stensrud et al.
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The transient spectrum of the excited singlet state exhibits
an absorption maximum around 315 nm, but it is overlaid by
stimulated emission, which is responsible for the negative
peak observed at 420 nm (dotted red curve in Figure 1).
Because both of these transitions arise from the same state,
they exhibit the same kinetic behavior. Singlet-triplet inter-
system crossing (ISC) results in a rise of the strong triplet-
triplet absorption band of 31 at 400 nm (Figure 1). A similar
400-nm absorption band was observed under conditions
similar to those of pHA, which is quenchable with potassium
sorbate and has previously been assigned as being due to
the triplet-triplet absorption for pHA.23 Similar triplet-
triplet absorption spectra were observed for the other four
pHP derivatives, i.e., a maximum at 400-420 nm with
a shoulder at ∼520 nm. Sorbate quenching of the 400-nm
bands of the pHP derivatives likewise established them as
triplet-triplet absorptions in agreement with our previous
studies15,23a and those by the Phillips group14,23b,23c derived
from time-resolved transient absorption and resonance
Raman spectral analyses with pHP phosphate and carboxy-
late esters.
The pump-probe time scan from 20 ps to 1.96 ns for 2 in
water is shown in Figure 2, and the species spectrum attrib-
uted to the triplet of 2 is given in Figure 3 (solid green line).
Also shown in Figure 3 is the spectrum of a species that
persists after the decay of the triplet, λmax ≈ 340 nm (dotted
blue line). A decay rate constant of kdec ∼ 6.5ꢀ106 s-1 was
determined for the 340-nm transient by nanosecond flash
photolysis of 2 in water containing ca. 10% acetonitrile,
similar to the results obtained with pHP phosphates.15,23
In order to identify the 340-nm transient, we examined the
transient spectra of pHA in aqueous solution. Pump-probe
spectra showed the formation of both the 400-nm absorption
band due to the triplet and a 340-nm transient lifetime ex-
ceeding that accessible by the delay line (e2 ns). Nanosecond
LFP of pHA showed that neither the lifetime nor the
amplitude of the 340-nm transient were affected by the
addition of up to 10 mM sorbate, whereas the lifetime of
the 400-nm triplet transient was reduced by more than 10-
fold (kq ≈ 2 ꢀ 109 M-1 s-1). Thus, the 340-nm transient is not a
triplet. It showed first-order decay kinetics (1τ ∼ 10-5 s1), and
with the addition of 10-4
M HCl in a solution
containing 10 mM sorbate, the lifetime of the 340-nm
transient was quenched (kHsorb=4.6 ꢀ 109 M-1 s-1), suggest-
ing that it is the ground-state anion of pHA. Indeed, the
onset of the absorption by pHA- follows the shape of the
340-nm transient down to 350 nm, but it continues to rise to
its maximum at 325 nm. The shape of the transient absorp-
tion (Figure 3, dotted blue line) with an apparent maximum
FIGURE 4. Effect of H2O on the rate of disappearance of the 390-
400-nm band (the blue squares are from Hellrung and Wirz, the
green circles are from Phillips et al.,14 and the red triangles are this
work).
SCHEME 2. Competing Photo-Favorskii and Deprotonation of pHP Esters
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TABLE 4. C log P and EC50 Values for Fluoro-pHP GABA 2 and 3
pHP
φdis(1-octanol)
C log P
EC50 (μM)a
1
2
3
4
0.12
0.14
0.10
0.042c
1.19
1.93, -1.59b
2.02, -1.90a
-3.38
49.2
119.8
93.4
aEffective concentration for 50% activity. See Figure 7. bThis value is
for the conjugate base of 2, which predominates at pH 7.3. cThe solvent
was 1-pentanol.
at 340 nm is, however, distorted by the onset of absorption
by ground-state pHA below 350 nm.
25
€
On the basis of Forster’s cycle, triplet excited pHA is
predicted to be a moderately strong acid, pKa ≈ 3.6.23a
Ionization of triplet pHA in aqueous solution occurs on a
time scale of 10ns.23c This explainswhy quenchingofthe pHA
triplet with up to 10 mM sorbate does not reduce the yield of
the ground-state anion absorbing at 340 nm. Acceptor-
substituted pHP derivatives such as the pHP GABAs are
likely to have a somewhat lower pKa. Thus, we attribute the
pathway competing with Favorskii rearrangement to triplet-
state deprotonation of the phenol,15,23,25 forming the triplet
conjugate base of pHP GABA. Subsequent ISC of the anion
triplet and neutralization regenerate the starting pHP ester.
The overall process must be viewed as a competing “energy-
wasting” sequence responsible for the diminished release
quantum yields. This and other competing pathways from
-
the triplet lower the release yield in the order R2CO2
<
R2O2PO2-<RSO3-. Together, these resultssuggestthatbetter
leaving groups influence the partitioning of the triplet more
favorably toward the photo-Favorskii pathway.14d,15,23a,26
Thus, the short triplet lifetimes of less than a nanosecond
in wholly aqueous solution observed for the pHP esters can
be attributed to a facile heterolysis of the leaving group. As
reported earlier, water has a significant effect on the rate and
quantum yield.21 For example, the triplet lifetime of pHP
diethyl phosphate with its better leaving group decreases
dramatically as the proportion of water in acetonitrile is
increased (Table 3 and Figure 4).14,15,23a Nevertheless, the
quantum yields for the disappearance of the pHP phosphate
and the appearance of the rearranged phenylacetic acid
remain significantly below unity. These observations con-
firm that an additional process is competing with the re-
arrangement/release process in aqueous environments.
For the rearrangement pathway, as we had previously
proposed, an adiabatic C-O bond heterolysis is accompa-
nied by deprotonation of the phenol15,21,23 to generate the
oxyallyl-phenoxy triplet biradical 314 (λmax=445 and 420 nm;
τ ∼ 0.6 ns). This biradical was also observed, but barely
detectable, in the pump-probe absorption spectra of the
present series (see the Supporting Information for the bir-
adical triplet-triplet transient absorption spectrum for 314).
Its weak signals were overlaid by the strong triplet absorp-
tions, which are longer-lived than those for 1 (Table 2). The
two competing pathways for pHP GABA are summarized in
Scheme 2.15,21
FIGURE 5. Dose-response of 2 photolysis. (A) Sample traces of
whole-cell currents elicited by the photolysis of 2 with 50-ms UV
light pulses (horizontal bar; UV). (B) Specific GABAA receptor
antagonist Gabazine (10 μM) completely blocked currents evoked
by the photolysis of 2 (100 μM, flash duration 10 ms). Washout of
Gabazine partly restored the initial response. (C) Current-voltage
curve: Peak amplitudes of responses are plotted versus the holding
membrane potential (-70 to þ20 mV; n = 3 neurons). The inset
shows sample traces at the various holding potential (100 μM 2,
flash duration 50 ms). (D) 2 having no effect on the holding currents
or membrane input resistance. Perfusion with ACSF containing
100 μM 2 for 2 min (horizontal bar) did not change the membrane
input resistance (1), whereas bath application of 100 μM GABA for
2 min decreased the membrane input resistance because of the
activation of GABAA-activated chloride channels.
the conjugate base of the trifluoromethyl pHP GABA
derivatives demonstrated a decided diminution in the quan-
tum yields at pHs above the pKa of the phenolic group. The
lower photoreactivity of the phenolates may follow a com-
pletely different mechanistic pathway, although the photo-
products remain the same.
The replacement of methyl with trifluoromethyl is accom-
panied by a significant increase in the lipophilicity of the two
GABA derivatives for 2 and 3, nearly doubling C log P, as
determined by partitioning between 1-octanol and water,
compared with the unsubstituted pHP GABA (Table 4). The
effects of increased lipophilicity on the photoreactions of 2
and 3 were reflected by the quantum yields in 1-octanol and
in mixtures of CH3CN/H2O or dimethyl sulfoxide (DMSO)/
H2O. In 1-octanol, 2 and 3 had quantum yields for the
Finally, the effects of the pH on the quantum yields
prompted further investigations of 2 and 3. Photolysis of
ꢂ
(25) Klan, P.; Wirz, J. Photochemistry of Organic Compounds; Wiley:
Chichester, U.K., 2009.
(26) Cope, E. Ph.D. Thesis, The University of Kansas, Lawrence, KS,
2008, unpublished results .
5224 J. Org. Chem. Vol. 74, No. 15, 2009

Stensrud et al.
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FIGURE 6. Dose-response of 3 photolysis. (A) Examples of membrane current responses of a neuron upon photolysis of 3 at concentrations
of 50, 100, and 500 μM. (B) Responses elicited by the photolysis of 3 (200 μM) blocked by the specific GABAA receptor antagonist Gabazine
(10 μM). Upon 15 min washout of Gabazine, the response partially recovered. These results indicate that photochemically released GABA
from 3 activated GABAA receptors. (C) 3 (200 μM, application during the black bar) itself having no effect on the holding currents or the
membrane input resistance, indicating that 3 itself did not activate GABAA receptors. In contrast, in the same neuron, 200 μM GABA elicited a
strong inward current and decreased the input resistance, as indicated by an increase in the amplitude of the injected current necessary to
produce a 5 mV membrane potential depolarization (example traces to the right).
disappearance of 0.14 and 0.10, respectively. In aqueous-
organic mixed solvents, the quantum yields increased with
an increase in the water content in accordance with ear-
lier observations with mixed acetonitrile/H2O solvent mix-
tures.14,15,21,23 Typically, for biologically benign solvents like
CH3CN or DMSO, the quantum yield for 2 increased by a
factor of 4 upon the addition of 10% (v/v) H2O, i.e.,
increasing from 0.03 in dry CH3CN to 0.12 in 10% H2O/
CH3CN. Above 25% H2O, the quantum yields remained
relatively constant.
Results of the CF3 Substituent Effect on the Photorelease of
FIGURE 7. Comparison of GABAA receptor activation by rapid
GABA in Biological Studies. The trifluoromethoxy- and
trifluoromethyl-caged pHP GABAs 2 and 3 were tested for
photolysis of pHP GABA. Dose-response curves for 2 (blue, n = 7
neurons), 3 (red, n = 6 neurons), and 4 (black, n = 6 neurons) with 4
their efficacy in whole-cell patch clamp studies in neurons in
cortical slices of mice27 and compared with earlier results for
the methoxy (3) and dimethoxy (4) GABA derivatives.9
Local photolysis with short UV light pulses (10-50 ms)
delivered through a small-diameter optical fiber produced
transient whole-cell inward currents. As shown in Figures 5
and 6, increasing the concentration of the photolyzed deri-
vative generated currents of larger amplitude and longer
duration.
To elucidate whether photolysis of these new derivatives
was evoking activation of the GABAA receptor, the effect of
the specific GABAA receptor antagonist SR 95531 (Gaba-
zine; Tocris, Ellisville, MI) on the photolysis responses and
the reversal potential of 2 and 3 was determined. SR 95531
completely abolished photolysis-induced membrane cur-
rents (10 μM; Figures 5B and 6B), indicating that the
population data of the peak currents normalized to the maximum
peak response. Data were fitted by Hill’s equation to yield EC50
.
EC50 and Hill’s coefficient values were as follows: 2, 49.2 μM, 1.8, n = 7
neurons; 4, 93.4 μM, 1.9, n = 6; 3, 119.8 μM, 2.73, n = 6.
response is mediated by activation of the GABAA receptor.
Current-voltage relationships of responses revealed a re-
versal potential of -14.2 ( 2.9 mV (n=3; Figure 5C) for 2.
This value is close to the theoretical value of -20 mV as
calculated by the Nernst equation for the chloride concentra-
tion between internal (60 mM) and external (133 mM) solu-
tions. Taken together, these results demonstrate that 2 and 3
photolysis-evoked membrane currents are mediated by speci-
fically activating the GABAA chloride receptor channel.
To determine whether nonphotolyzed 2 could act as an
agonist for GABAA receptors, the effect of 2 on the mem-
brane input resistance was measured by monitoring current
responses to short 5 mV depolarizations from a holding
(27) Kim, G.; Kandler, K. Nat. Neurosci. 2003, 6, 282–290.
J. Org. Chem. Vol. 74, No. 15, 2009 5225

Stensrud et al.
JOCArticle
potential of -70 mV in the presence and absence of GABA
(100 μM) or 2 (100 μM). As expected, GABA (100 μM)
reduced the membrane input resistance because of its activa-
tion of GABAA chloride channels. In contrast, 2 had no
effect on the membrane input resistance (Figure 5 D),
indicating that only photolyzed 2, but not 2 itself, activates
GABAA chloride receptor channels. A parallel study on 3
gave the same result (Figure 6C).
In order to compare the relative sensitivity of photolyzed
2 and 3 with the other caged GABAs, peak amplitudes of the
membrane currents elicited by the photolysis of 2 and 3 at
increasing concentrations were plotted as concentration-
response curves, which were fitted by Hill’s equation. The
best-fit curves showed EC50 values of 49.2, 119.8, and
93.4 μM for 2-4, respectively (Figure 7 and Table 4),
demonstrating that photorelease from 2 is more effective in
eliciting GABA responses than either 3 or 4.
agonist at the GABAA receptor in whole-cell, patch clamp
studies with neurons in cortical slices. Photoreleasing
GABA from 2 was >50% more effective as an agonist
than from its nonfluoro analogue. This was not true for the
CF3 derivative, which was less effective by ca. 30% rela-
tive to the m-CH3O-pHP GABA in spite of its greater
quantum yield.
Further studies on the complex effects that substitutents
have on the photochemical and photophysical properties of
the pHP chromophore, including ionic strength and leaving
group effects on the partitioning of the two dominant triplet
pathways, are needed to more fully understand the efficacy
of the pHP phototrigger reaction and are currently being
pursued by our groups.
Experimental Section
Quantitative photolysis conditions for the determination of
)
quantum yields and Stern-Volmer quenching constants (KSV
were as follows: The lamp light output (in millieinsteins
per minute) was established using the potassium ferrioxalate
method.28 Milligram quantities of caged compounds and caf-
feine or acetamidophenol were weighed out on a Fisher brand
microbalance and dissolved in 4 mL of 18 MΩ ultrapure water;
salt solutions of various concentrations, buffers with or without
adjusted ionic strengths, or purified organic solvents were then
added to a 10 mm ꢀ 75 mm quartz tube and vortexed, resulting
in a homogeneous solution of the caged compound and internal
standard. The same tubes and mixing procedures were em-
ployed for actinometry (vide infra). Concentrations of the caged
compounds ranged from 1 to 9 mM. At these concentrations,
the absorbance was greater than 4 through the excitation range
of the 300-nm lamps, assuring the complete absorption by the
sample at low conversion over the irradiation wavelength range
employed. These tubes were then placed in a Rayonet MGR
100 carousel within a Rayonet photochemical reactor equipped
with two 3000-A, 15-W lamps. Samples of 100 μL were removed
at 30-s intervals up to 5 min using a 250-μL microsyringe after
vortexing of the reaction tube and the samples diluted to 1 mL
with water using 1-mL volumetric flasks. The samples were
thoroughly mixed before LC/MS/MS analysis. Each run gave
linear time-dependent conversions up to <20% conversion of
the pHP ester.
Conclusions
We have shown that the competing “energy-wasting”
triplet pathway is the deprotonation of the phenol group in
the photolysis of pHP GABA derivatives. In aqueous phase
photolyses at low to moderate pH, it is the triplet state that
undergoes the photo-Favorskii rearrangement, concomi-
tantly releasing the substrate. The efficiency of the triplet
excited-state rearrangement pathway depends on several
factors, the most important of which is the nucleofugality
of the caged substrate and the pKa of the chromophore. The
carboxylate leaving group, e.g., GABA release, is less effec-
tive than phosphates,5,14-16,21-23 for example, as expressed
by the higher quantum yields for phosphate release. In fact,
carboxylates are among the least efficient substrates (φd =
0.04-0.20) that we have reported. Additional factors also
influence the partitioning of the pathways, including the
water content of the solvent, pH, pKa of the pHP, and media
effects.
Evidence that the conjugate bases are much less efficient
chromophores for photorelease was reinforced with both
trifluoromethyl derivatives through studies at pH 9.0, well
above the pKa’s of the pHP derivatives, and at 350 nm, where
only the conjugate base absorbs. Thus, substituents on the
chromophore can attenuate the effective pH range and
wavelength region available for photorelease by their influ-
ence on the pKa of the p-hydroxy group. In the present case,
electron-withdrawing substituents on the chromophore suf-
ficiently lower the pKa to a point where the conjugate base is
the only species present at pH 9. Consequently, the quantum
yields are depressed to<0.02 because of the poor reactivity
of the conjugate base. In a similar manner, photoinduced
deprotonation to the conjugate base is an unproductive
pathway.
ꢂ
˚
The same procedures were used for actinometry using the
same volumes in the sample tubes. Complete light absorption
by the ferrioxalate solutions was accomplished by following the
procedure of Hatchard and Parker.28 A linear response for the
light output was also obtained in each series.
Quantitative analysis was achieved by LC/UV or HPLC/MS/
MS. The LC/MS/MS instrument was equipped with a triple-
quadrupole, electrospray ionization mass spectrometer, out-
fitted with an autosampler. UV-vis detection consisted of a
dual-wavelength detector set at 220 and 240 nm. The reservoirs
used were as follows: (A) 99% water, 1% methanol, 10 mM
ammonium formate, and 0.06% formic acid; (B) 99% methanol,
1% water, 10 mM ammonium formate, and 0.06% formic acid.
The column was of reversed-phase (C18), 4 μm mesh, and
50 mm. Injections of 100 μL were made with an automated
sampler for each run for a total of three injections per vial. A
mobile phase gradient was utilized to optimize compound
separation. The flow rate was set at 300 μL/min. Data analysis
was performed using MassLynx software. Smoothing functions
were used for peak analysis of the chromatographic peaks.
Calibration curves to obtain R values from linear least-squares
The fluoro substitution did improve the lipophilicity of the
chromophore, a feature that may have future implications
regarding the transport of caged compounds across mem-
branes in biological studies. A test on the photochemical
efficacy in more lipophilic environments such as octanol,
acetonitrile, and DMSO showed that photorelease occurs,
but the quantum yields improved with added H2O as a
cosolvent, as was the case for acetonitrile/H2O mixtures.14c
As a further test, the efficacy of fluorinated pHP GABA
derivatives were tested for controlled release of GABA as an
(28) Hatchard, C. G.; Parker, C. A. Proc. R. Soc. London, Ser. A 1956,
235, 518–536.
5226 J. Org. Chem. Vol. 74, No. 15, 2009

Stensrud et al.
JOCArticle
regression were determined at concentrations of the reactants
and products in photolyses by systematic increases of pHP-
caged GABA, free GABA, and p-hydroxyphenylacetic acid
concentrations to determine correlations with internal stan-
dards caffeine or 4-acetamidophenol. The quantum efficiencies
were then calculated from the ratio of the reactant or product
concentrations to the photons absorbed using the actinometer
values obtained as indicated above. A minimum of three in-
dependent, quantitative iterations were conducted for each
compound to underpin the veracity of reported quantum effi-
ciencies.
of Pittsburgh. Mice (aged postnatal days 10-11) were anesthe-
tized with isoflurane and decapitated. The brain was quickly
removed from the mouse skull and immersed into an ice-cold
artificial cerebrospinal fluid (ACSF), which contained 1 mM
kynurenic acid and was composed of the following (in mM): 124
NaCl, 26 NaHCO3, 10 glucose, 5 KCl, 1.25 KH2PO4, 1.3
MgSO4, and 2 CaCl2 (pH 7.4 when aerated with 95% O2/5%
CO2). The brain was blocked, and coronal slices (300 μm thick)
were obtained from the cortex using a vibrating microtome.
Slices were incubated in an interface-type chamber in a 95%
O2/5% CO2 atmosphere for 1 h at room temperature (20-25 ꢀC)
before commencing electrophysiological recordings.
For electrophysiological recordings, slices were transferred to
a chamber mounted to a fixed-stage microscope where they were
superfused with ACSF solutions at a rate of 2-3 mL/min using a
gravity-driven perfusion system. Whole-cell patch clamp re-
cordings were made in voltage-clamp mode using an Axo-
clamp-1D amplifier with a Digidata-1440A A/D converter
under the control of pClamp10. The recording pipettes (resis-
tance of 2-3 MΩ) were constructed from borosilicate glass
capillaries using a P-97 puller. Recording pipettes were filled
with a pipet solution containing (in mM): 54 D-gluconic acid,
54 CsOH, 56 CsCl, 1 MgCl2, 1 CaCl2, 10 Hepes, 11 EGTA, 0.3
Na-GTP, 2 Mg-ATP, and 5 QX-314 (pH 7.2, 280 mOsm/L).
Recordings were taken at a holding potential of -70 mV, unless
otherwise specified. Caged compounds and pharmaceutical
drugs were dissolved in ACSF immediately before application.
The area around the recorded neuron was illuminated with
UV light using an optical-fiber-based system consisting of a
fused-silica fiber (inner diameter 20 μm) coupled to a 100-W
mercury arc lamp. The duration of the light pulses was regulated
by an electronic shutter located between the lamp and the optical
fiber. The location of the light spot on the slice was monitored
with a CCD camera. Data are presented as mean ( SEM. The
results are shown in Figures 4-6.
The Stern-Volmer quenching method29 was employed to
determine the triplet lifetimes of pHP derivatives. Potassium
sorbate served as the quenching agent. Solutions of pHP GABA
(0.001-0.01 M) were diluted with sorbate solutions of increas-
ing concentration (0-0.1 M) and photolyzed under the afore-
mentioned conditions to ascertain the changes in quantum
efficiencies of GABA release. Stern-Volmer constants, KSV
,
were determined from the slope of φ0/φ vs [Q]. To determine the
triplet lifetime, τ3, the rate of quenching, kq, was assumed to be
the rate of bimolecular diffusion, kdiff =7.2 ꢀ109 s-1 (water).
The KSV values in H2O were 20 M-1 for 2 and 33 M-1 for 3.
Femtosecond transient absorption spectra were measured
with the pump-supercontinuum probe technique.30 The sample
was excited with a frequency-quadrupled pulse from a Ti/Sa
laser system [775 nm, pulse energy 0.8 mJ, full width at half-
maximum (fwhm) 150 fs, operating frequency 426 Hz] described
previously.31 The output at 540 nm was frequency-doubled to
266 nm and after compression provided pump pulses with an
energy of 1 μJ and a <100 fs pulse width. A probe beam
continuum was produced by focusing the 775 nm, 1 mm pulse
in front of a CaF2 of 2 mm path length. The second harmonic
(400 nm) of the fundamental generated a supercontinuum probe
in the range 270-690 nm. The pump and probe were focused in a
0.2-mm spot on the sample flowing in an optical cell of 0.4-mm
thickness. The probe signal was spectrally dispersed and regis-
tered with a photodiode array (512 pixels). The pump-probe
cross-correlation was well below 100 fs over the whole spectrum.
The experimental transient spectra ΔA(λ,t) were corrected
for the chirp of the supercontinuum and for the solvent con-
tribution.
Acknowledgment. This work was supported by NIH
Grant GM069663 (to R.S.G.), Grant R01 GM72910 (to R.
S.G.), Grant DC-04199 (to K.K.), the Czech Ministry of
Education, Youth and Sport MSM0021622413 and Grant
ME090217 (to DH), and the Swiss National Science Founda-
tion (J.W.). We thank Dr. Hellrung for experimental assis-
tance on the effects of H2O on the pHP GABA rate constants.
We thank the reviewers for very constructive critical com-
ments and valuable suggestions on the original manuscript.
Biological Activity. Experimental Determinations. Experi-
mental procedures were in accordance with the U.S. National
Institutes of Health guidelines and were approved by the
Institutional Animal Care and Use Committee at the University
Supporting Information Available: Synthetic procedures, 1H,
13C, and 19F NMR spectra, and HRMS data for 2, 3, and
6-12. This material is available free of charge via the Internet
at http://pubs.acs.org.
(29) Desilets, D. J.; Kissinger, P. T.; Lytle, F. E. Anal. Chem. 1987, 59,
1244–1246.
(30) Details for the pump-probe experiments can be found in ref 21.
€
(31) Muller, M. A.; Gaplovsky, M.; Wirz, J.; Woggon, W.-D. Helv.
Chim. Acta 2006, 89, 2987–3001.
J. Org. Chem. Vol. 74, No. 15, 2009 5227