A Reversible Photoacid Functioning in PBS Buffer under Visible Light

Article abstract of DOI:10.1021/jacs.5b06218

A metastable-state photoacid that can reversibly release a proton in PBS buffer (pH = 7.4) under visible light is reported. The design is based on the dual acid-base property and tautomerization of indazole. The quantum yield was as high as 0.73, and moderate light intensity (10² μmol·m²·s^-1) is sufficient for the photoreaction. Reversible pH change of 1.7 units was demonstrated using a 0.1 mM aqueous solution. This type of photoacid is promising for control of proton-transfer processes in physiological conditions and may find applications in biomedical areas.

Full text of DOI:10.1021/jacs.5b06218

Communication
pubs.acs.org/JACS
A Reversible Photoacid Functioning in PBS Buffer under Visible Light
Nawodi Abeyrathna and Yi Liao*
Department of Chemistry, Florida Institute of Technology, Melbourne, Florida 32901, United States
S
* Supporting Information
highly acidic metastable form.13 For example, compound 1,
which is the first mPAH reported and has been used in several
applications, is shown in Scheme 1. This type of mPAH is
ABSTRACT: A metastable-state photoacid that can
reversibly release a proton in PBS buffer (pH = 7.4)
under visible light is reported. The design is based on the
dual acid−base property and tautomerization of indazole.
The quantum yield was as high as 0.73, and moderate light
intensity (102 μmol·m2·s−1) is sufficient for the photo-
reaction. Reversible pH change of 1.7 units was
demonstrated using a 0.1 mM aqueous solution. This
type of photoacid is promising for control of proton-
transfer processes in physiological conditions and may find
applications in biomedical areas.
Scheme 1. (a) Structures of the mPAH 1 and 2, (b)
Proposed Mechanism of the Photoreaction of 2, and (c)
Synthesis of 2
hotoacids are molecules that transform into strong acids
P
upon irradiation. They can be utilized for remote, spatial,
and temporal control of proton concentration and proton-
transfer processes. The irreversible photoacid generators
(PAGs) have been extensively studied as photo initiators for
cationic polymerization and applied to photolithography.1−5
Excited-state photoacids have been studied since the 1970s and
have been utilized to investigate fast proton-transfer pro-
cesses.6−11 Hydroxyazobenzenes have also been utilized for
photogeneration of pH jump.12 The recently discovered
metastable-state photoacids (mPAH) can produce a large
proton concentration with high efficiency and good reversi-
bility.13−15 In addition, visible light with moderate intensity,
e.g., LED and sunlight, can be used to activate mPAHs.
Therefore, mPAHs can be conveniently incorporated in
different systems to control various proton-transfer processes.
Several applications of mPAHs have been demonstrated
recently including control of acid-catalyzed reactions, volume
change of hydrogels, polymer conductivity, bacteria killing,
odorant release, and color change of materials.13,16−19 They
have also been utilized to control supramolecular assemblies,20
molecular switches,21 microbial fuel cells,22 and cationic
sensors.23 Given that proton transfer is involved in the
mechanisms of many enzymes and abnormal cellular pH is
related to many diseases including cancer, cardiovascular
diseases, and Alzheimer’s disease, etc., mPAHs have a lot of
potential in the biomedical area. However, the bioapplications
of mPAH are limited by the fact that none of the previously
reported mPAH works at a pH of 7.4, which is the common
physiological pH. Herein, we report a novel mPAH, which
functions in 1× PBS buffer (pH = 7.4) under visible light.
Metastable-state photoacids are generally designed by linking
an electron-accepting moiety and a weakly acidic nucleophilic
moiety with a double bond. Photoinduced trans−cis isomer-
ization of the double bond allows a nucleophilic cyclization
reaction to occur between the two moieties, which generates a
related to the photoreaction of spiropyrans, which has been
utilized to control proton transfer.24 All the previously reported
mPAHs have phenol derivatives as the nucleophilic moiety.13,15
Plain phenol has a pKa of 10. The pKa is lowered when it is
linked to a strong electron-accepting moiety. Due to the acidity
of the phenol moiety of the mPAHs, proton dissociation occurs
at a pH of 7.4 in the dark. The deprotonation also shifts the
thermal equilibrium to the cyclized acidic form. Consequently,
a large portion of the mPAHs release protons in the dark, which
is the reason that none of the previously reported mPAH can
function at a pH of 7.4. Figure 1 (left) shows the UV−vis
spectrum of 1 in the PBS buffer. Approximately 83% of 1
changed to the deprotonated ring-opening form (PA−) or
cyclized form (PA′−) in the dark. The dark acidity of mPAH
may be lowered by using a nucleophilic moiety with a weaker
acidity than that of phenol. However, in the mechanism of
mPAH, the nucleophilic cyclization involves a proton transfer
or a proton release.13 A low acidity of the nucleophilic moiety
may retard the cyclization reaction.
In this work, a novel mPAH (2 in Scheme 1) using indazole
as the nucleophilic moiety was designed and synthesized.
Received: June 15, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b06218
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
addition of HCl solution), indicating that the protonated form
is predominant. Therefore, 2 does not release its proton at a pH
of 7.4 in the dark. The stability of 2 in PBS buffer was also
studied by monitoring the decrease of the UV−vis absorption
with time. These data were fitted to a first-order equation, and
the half-life was calculated to be ∼48 h, which is long enough
for some bioapplications. The details are described in the SI.
The photoactivity of 2 was studied by irradiating a PBS
solution (1% DMSO) of 2 using a 470 nm LED. The UV−vis
spectrum obtained after 3 min irradiation is shown in Figure 1.
The peak at 422 nm disappeared after irradiation, and multiple
peaks between 350 and 250 nm appeared, which are assigned to
the cyclized acidic form. Besides the disappearance of the peak
at 422 nm, the most pronounced change is that the dip at 284
nm in the spectrum before irradiation changed to a strong peak
after irradiation. After the light was turned off, the reformation
of PAH was monitored using UV−vis spectroscopy (Figure
S5). Since the reverse reaction in the PBS buffer is slow, we
studied whether irradiation with UV light (254 and 365 nm)
could accelerate the reverse reaction. We found that irradiating
a solution of PA′H with 365 nm light slowed down the reverse
reaction because PAH also substantially absorbs at this
wavelength (Figure 1). When 254 nm light was used, the
UV−vis spectrum was different from that of PAH indicating a
different photoreaction occurred.
The photoreaction was also studied by NMR analysis. A
deuterated DMSO solution of 2 (∼3 mM) was irradiated for 10
min and then scanned by NMR. The obtained spectrum
showed the expected photoproduct (PA′−) together with
substantial amount of cis-PAH and small amount of trans-PAH
(Figures S2 and S3). Increasing the irradiation time did not
change the spectrum, which indicates that the mixture was due
to the reverse reaction that occurred during the NMR
measurement (∼8 min including time for setup and scan)
rather than an uncompleted photo reaction. In fact, when the
sample was kept in the dark for 30 min after irradiation, peaks
for trans-PAH became predominant and very little cis-PAH and
PA′− can be observed. After 1 h, the spectrum became the same
as that of the unirradiated sample except that the NH peak was
broader than before.
To confirm that 2 indeed releases its proton upon irradiation,
photoinduced pH change of a solution of 2 in deionized water
containing 10% DMSO was studied. The concentration was
about 0.1 mM, which is nearly the maximum concentration.
The pH change was measured using both a pH meter and pH
indicators. The pH of the solution was 6.0 in the dark. This pH
is due to the acidity of deionized water but not the photoacid.
To confirm this, the ground-state pKa was experimentally
determined to be 10.1. (The details are described in the SI.) A
0.1 mM solution of 2 in pure water should result in a pH close
to 7 instead of 6. Deionized water often has a pH ∼ 6 due to
absorption of CO2. The solution of 2 was irradiated using a 470
nm LED light for 5 min. The pH after irradiation was 4.3,
which was 1.7 units lower than the initial pH. After the
irradiated solution was kept in the dark for 48 h, the pH
returned to the original level.
The activity of the proton released from the mPAH is
demonstrated by protonation of an acridine dye (Figure 2).
This acridine dye changes from orange to blue upon
protonation, and its λmax changes from 440 to 604 nm.19 The
protonated form has a pKa of 4.7. A solution of 2 (0.25 mM),
and the dye (0.25 mM) in DMSO was irradiated for 5 min. The
color of the solution changed from orange to dark green
Figure 1. UV−vis spectrum of mPAH 1 in PBS buffer in the dark
(left) and that of 2 before and after irradiation. (PAH is the protonated
form, PA− is the deprotonated form of PAH, and PA′− is the
deprotonated cyclized acidic form.)
Indazole is both a weak acid and a weak base. As described
above, its acidity determines the dark acidity of the
corresponding mPAH. The pKa of its NH acidity is 13.86,25
which is nearly 4 units higher than that of phenol. The
photoinduced acidity of the corresponding mPAH is limited by
its basicity since the proton released from the photogenerated
acid can bind to the indazole moiety. Therefore, the pKa of the
photogenerated acid is close to that of the conjugate acid of
indazole, which is 1.25. This pKa is more than 6 units lower
than 7.4 and thus is enough for releasing essentially all the
protons at this pH. Indazole has two tautomers, i.e., 1H-
indazole and 2H-indazole. Although 1H tautomer is more
stable than 2H tautomer, the energy difference is very small (15
kJ/mol).26 The photoinduced cyclization of 2 may occur
without releasing a proton before the nucleophilic reaction
since the 2H tautomer can act as a nucleophile (Scheme 1).
The synthetic route of 2 is shown in Scheme 1. Heating a
mixture of 1,3-propane sultone and 2-methyl-benzothiazole
yielded 2-methyl-1-(3-sulfonatepropyl)-benzothiazolium, which
was reacted with 1H-indazole-7-carbaldehyde in ethanol to
yield 2 as an orange precipitate. A small amount of ammonium
acetate was used as a catalyst in the second step to increase the
yield. Given the photosensitivity, workup needs to be done in
the dark. The procedures are given in the Supporting
Information (SI). The photoacid 2 has a low solubility in
water and alcohols but can be dissolved in DMSO.
To study the behavior of 2 at a pH of 7.4, 2 was dissolved in
a 1× PBS buffer containing 1% of DMSO. The UV−vis
spectrum of the solution is shown in Figure 1. A strong
absorption band with a λmax at 422 nm (molar absorptivity 2.9
× 104 L mol−1cm−1) was observed, which is assigned to the
protonated photoacid (PAH). The λmax is close to that of 1
(424 nm). The absorption of the deprotonated form (PA−) was
studied using a pH jump experiment, which is described in the
SI. The λmax was determined to be 498 nm. Unlike the UV−vis
spectrum of 1 in PBS buffer, no absorption peak was observed
at the λmax of the deprotonated form in the spectrum of 2
(Figure 1). To confirm that the protonated form is
predominant, a small volume of concentrated HCl was added
to break the buffer and change the solution to be strongly acidic
(pH ∼ 1). A previous study showed that the protonated form
of mPAH is predominant in strongly acidic conditions.14
Therefore, comparing the amount of the protonated form in
PBS buffer with that in the acidic solution allows us to estimate
the portion of the protonated form in PBS buffer. After
acidification, no significant change of the absorbance was
observed (after considering the volume change due to the
B
DOI: 10.1021/jacs.5b06218
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
ACKNOWLEDGMENTS
■
Support from the Air Force Office of Scientific Research
(FP048956) and the National Science Foundation (DMR
1342940) are gratefully acknowledged.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b06218.
■
S
Details of synthesis, NMR spectra, stability measurement,
ground-state pKa measurement, determination of the
UV−vis absorption of the deprotonated form (PDF)
AUTHOR INFORMATION
Corresponding Author
*yliao@fit.edu
■
Notes
The authors declare no competing financial interest.
C
DOI: 10.1021/jacs.5b06218
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX