Visible Light Mediated Bidirectional Control over Carbonic Anhydrase Activity in Cells and in Vivo Using Azobenzenesulfonamides

Article abstract of DOI:10.1021/jacs.0c05383

Two azobenzenesulfonamide molecules with thermally stable cis configurations resulting from fluorination of positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis configuratio

Full text of DOI:10.1021/jacs.0c05383

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Article
Visible light mediated bidirectional control over carbonic anhydrase
activity in cells and in vivo using azobenzene sulfonamides
Kanchan Aggarwal, Timothy P. Kuka, Mandira Banik, Brenda P. Medellin, Chinh
Q. Ngo, Da Xie, Yohaan Fernandes, Tyler L. Dangerfield, Elva Ye, Bailey Bouley,
Kenneth A Johnson, Yan Jessie Zhang, Johann K. Eberhart, and Emily L. Que
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c05383 • Publication Date (Web): 04 Jul 2020
Downloaded from pubs.acs.org on July 4, 2020
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Visible light mediated bidirectional control over carbonic anhydrase
activity in cells and in vivo using azobenzene sulfonamides
Kanchan Aggarwala, Timothy P. Kukab, Mandira Banika, Brenda P. Medellinb, Chinh Q. Ngoa, Da Xiea,
Yohaan Fernandesb,c, Tyler L. Dangerfieldb, Elva Yea, Bailey Bouleya, Kenneth A. Johnsonb, Yan Jessie
Zhangb, Johann K. Eberhartb,c, Emily L. Quea*
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[a] Department of Chemistry, University of Texas at Austin, 105 E 24th St Stop A5300, Austin, TX 78712
[b] Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, 100
E 24th St Stop A5000, Austin, TX 78712
[c] Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, 2500 Speedway, A4800,
Austin, TX 78712
KEYWORDS carbonic anhydrase, azobenzene, photoisomerization, visible light, zebrafish
ABSTRACT: Two azobenzene sulfonamide molecules with thermally stable cis configurations resulting from fluorination of
positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis
configurations. These fluorinated probes each use two distinct visible wavelengths (520 nm and 410 nm or 460 nm) for isomerization
with high photoconversion efficiency. Correspondingly, the cis isomer of these systems is highly stable and persistent (as evidenced
by structural studies in solid and solution state), permitting regulation of metalloenzyme activity without continuous irradiation.
Herein, we use these probes to demonstrate the visible light mediated bidirectional control over the activity of zinc-dependent carbonic
anhydrase in solution as an isolated protein, in intact live cells and in vivo in zebrafish during embryo development.
the fields of chemotherapeutics11-12 and neurobiology13-14
.
INTRODUCTION
Carbonic anhydrase (CA) is a promising target for
photoregulation due to its physiological and pathological
roles.15 The active site of most CAs contains a zinc ion bound
to three histidine residues and one water molecule. This protein
catalyzes a simple physiological reaction, the reversible
conversion of carbon dioxide and water to bicarbonate and
protons. CAs are involved in the maintenance of acid-base
equilibrium and CO2 homeostasis. However, they can be highly
overexpressed in diseases, including cancer, epilepsy, and
glaucoma, and hence, are important therapeutic targets.16
Effective photoswitchable inhibitors of CA have promise as
research tools as they can be activated in a spatially and
temporally specific manner. Therapeutic applications are also
possible, particularly for glaucoma due to its location in the eye
where light activation is very feasible.17
Photopharmacology, in which the activity of pharmacophores
can be controlled via application of specific wavelengths of
light, has emerged as an attractive method for controlling the
behavior of biological species on demand. Light serves as an
ideal external control element for in situ biochemical
manipulation because it offers a high level of spatiotemporal
resolution, it is minimally invasive, and its wavelength and
intensity can be readily tuned. One important strategy used in
the development of photoactivable probes is alteration of the
properties of a molecule (chemical structure, shape, polarity)
upon light irradiation resulting in different affinity towards the
desired target. Recent years have seen great advances in this
field where the control over biomacromolecular function has
been attained either by using a photoprotecting group1
(irreversible approach) or small photochromic moieties2
(reversible approach). The latter approach uses
a
Aryl sulfonamides show exquisite selectivity for CA
inhibition, combining metal coordination, hydrogen bonding,
and hydrophobic interactions to achieve this specificity.18
Others have reported azobenzene-based photoswitches for CA,
however studies have been limited to isolated protein or cell
lysates.19-21 Recently, we reported small molecule azobenzene
sulfonamide CAP1 that demonstrated differential regulation of
intracellular pH in the trans and cis isomeric forms in live
cancer cells.22 However, this probe had three major limitations:
use of UV light for photo-isomerization, poor photoconversion
photoswitchable bond that allows access to different molecular
structures induced by irradiation of light. Although stilbenes,
hemithioindigos and diarylethenes have been used as switches,
azobenzenes shows the most promise in achieving a larger
range of activation wavelengths and greater photoactivated
state stabilities.3 They have been readily used to manipulate a
wide variety of biological processes4 including regulation of
protein expression,5 enzyme folding6 and activity7-8, and DNA
conformation and transcription,9-10 and have been explored in
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display absorption bands corresponding to a -* transition (S2)
efficiency, and importantly, poor thermal stability of the cis
isomer, which limited long-term applications in live cells and
organisms.
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with a max of 312 nm for CAP-F3 and 306 nm for CAP-F5.
Furthermore, moderate absorbance bands at 436 nm for CAP-
F3 and 450 nm for CAP-F5 were observed, which correspond
to n-* transitions (S1). Irradiation of CAP-F3 and CAP-F5
with 520 nm light shifted their absorption profiles to ones
consistent with cis-isomer formation, including a significant
decrease in the intensity of the -* band. While the -* bands
underwent a slight hypsochromic shift in both cases, there was
a large hypsochromic shift in the n-* transition by 16 nm for
CAP-F3 and 37 nm for CAP-F5 upon photoiosmerization
(Table S1). The large separation of the n-* transition bands
between trans and cis isomers should result in high
photoisomerization conversions.23 Indeed, irradiation of
solutions with 520 nm light yielded high conversions to c-CAP-
F3 (71%) and c-CAP-F5 (87%) (19F NMR, CD3OD, Figure
S3). Isomerization from cis to trans can subsequently be
achieved by irradiating with 460 nm and 410 nm light for CAP-
F3 (58 %) and CAP-F5 (82 %) respectively. The high
conversion of CAP-F5 in aqueous buffer is better than our
previously reported probes (CAP1 and CAP2),22 and is on the
same order as other cis-trans isomerizing photoswitches studied
in buffer solution.2
To address these limitations, we identified ortho-fluorination
as a strategy to improve the performance of azobenzene-based
probes. Fluorination ortho to the azo moiety of the azobenzene
results in separation of the n-* absorption band in the trans
and cis isomers.23 This splitting of absorption bands can further
be increased by the incorporation of an electron withdrawing
group at the position para to the azo unit. This large separation
of n-* bands enables use of distinct visible wavelengths to
achieve higher isomer conversion in the photostationary state
when compared to other azobenzene derivatives. Remarkably,
the cis isomers of ortho-fluorinated azobenzenes are
exceptionally thermally stable with half-lives as long as several
days.24 Furthermore, unlike other visible light sensitive and
stable cis-azobenzene derivatives, ortho-fluorinated systems
are immune to the cytosolic reducing environment making them
more biocompatible.25 Ortho-fluorinated azobenzenes have
been extensively used for materials applications13, 26-29 and have
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gained interest recently in biological applications30-34
,
demonstrating the robustness of this system in the context of
biomolecules and cells. Herein, we report the first
demonstration of the ortho-fluorination strategy for controlling
the activity of a metalloenzyme. Importantly, we demonstrate
the efficacy of this approach for dynamic modulation of the
activity of Zn-dependent carbonic anhydrase in vitro with
isolated protein, in live cells, and for the first time, in vivo in
live zebrafish.
RESULTS AND DISCUSSION
We synthesized and characterized two fluorinated azobenzene
sulfonamide probes containing either three (CAP-F3) or five
(CAP-F5) fluorines. We anticipated that these probes would
achieve red-shifted wavelengths of isomerization relative to
non-fluorinated derivatives with improved switching fidelities
and increased thermal lifetime of the cis isomer (Scheme 1).
Full experimental details are provided in the Supporting
Information.
Scheme 1. Structures of CAP-F3 and CAP-F5, and
photoswitching in the presence of carbonic anhydrase
Figure 1. UV-vis spectra of CAP-F3 (A) and CAP-F5 (B) before
and after photoirradiation (cis 520 nm for 5 minutes; trans 460/410
nm for 2 min). The power densities for 410, 460 and 520 nm were
5.20 mW/cm2, 14.12 mW/cm2 and 9.18 mW/cm2, respectively. The
studies are performed in methanol at 298 K.
To further understand the spectroscopic properties, we
performed TD-DFT calculations to compute the energies of the
HOMO (n) and LUMO (π*) orbitals for both the trans and cis
isomers of CAP-F3 and CAP-F5 at the B3LYP/6-31G* level
and also included calculations for a CAP-F1 analogue, where
the ortho-fluorine atoms are all substituted by hydrogen atoms.
The n and π* orbitals of the cis isomers are all higher in energy
than the trans isomers. However, the relative energy of the n
orbital is also affected by the number of ortho-fluorine atoms
due to the -electron withdrawing effect of fluorine. As a result,
the calculations predicted a larger separation of n-π* transition
The photoisomerization properties of CAP-F3 and CAP-F5
were monitored in methanol (Figure 1A and 1B) and aqueous
buffer (Figure S1) using UV-vis absorption spectroscopy. All
relevant wavelengths are summarized in Table S1. The spectra
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bands between the trans and cis isomers for CAP-F5 compared
to CAP-F3 or CAP-F1 analogues (Table S1),
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Figure 2. (A) Thermal relaxation of c-CAP-F3 and c-CAP-F5 in aqueous buffer at 37 C. (B) 19F and 19F-{1H} spectra for CAP-F5 collected
in CD3OD. Single crystal structure of c-CAP-F5 (C) and t-CAP-F5 (D). Thermal ellipsoid plots at 50% probability are shown. Solvent
molecules and hydrogen atoms are omitted for clarity.
demonstrating the importance of ortho-fluorination in our
molecular design. A detailed explanation of calculations and
interpretation is provided in the Supporting Information.
in the cis isomer was further supported by correlation peaks
observed in its 19F-19F COSY spectrum (Figure S9). Finally, we
investigated how the rotation of the ring bearing Fb is affected
by the absence (CAP-F3) or presence (CAP-F5) of Fa on the
other phenyl ring using transverse relaxation time (T2)
measurements.39 The shorter T2 values of Fb in the cis isomer
(1.19 sec for c-CAP-F3; 0.73 sec for c-CAP-F5) indicate that
ring rotation is more constrained in c-CAP-F5; this constriction
likely stabilizes c-CAP-F5 in solution greater than c-CAP-F3.
The cis isomers of CAP-F3 and CAP-F5 both have increased
thermal stability compared to non-fluorinated analogues,22 with
exceptional thermal stability exhibited for cis-CAP-F5. The
thermal conversion from the cis to trans isomer of CAP-F3 and
CAP-F5 at 37 °C in aqueous buffer was studied under dark
conditions (Figure 2A). We observed a low ~15% conversion
of c-CAP-F5 to t-CAP-F5 over 48 hours, and a ~40%
conversion of c-CAP-F3 to t-CAP-F3. This difference is
unsurprising giving the n-* separation trend. The stability
driven by ortho-fluorination is better than what has been
observed for ortho-methyl substituted bistable azobenzenes.35
Previous reports have indicated that intramolecular interactions
may also contribute to the stability of the cis isomer of ortho-
fluorinated derivatives of azobenzene.36-37
The persistence of c-CAP-F5 was further demonstrated in
the solid state via single-crystal X-ray diffraction, a rare
example of a cis-azobenzene structure.36 In the crystal structure,
the distances between the ortho-fluorine atoms and ipso
carbons are 2.78 Å and 2.76 Å (Figure 2C). Both distances are
shorter than the C-F van der Waals distance (3.17 Å),
demonstrating the presence of CF interactions in the cis
isomer. Such favorable interactions were not observed in the
solid-state structure of t-CAP-F5 (Figure 2D). The increased
stabilization of these CF interactions should result in a
smaller Gibbs free energy difference between the trans and cis
isomers, and DFT calculated energies reflected this when CAP-
F5, CAP-F3, and CAP-F1 were compared (ΔGcis-trans, Table
S2).36
We examined potential interactions using NMR spectroscopy
in a polar protic solvent. In the 19F NMR spectrum of CAP-F5,
we observed that the peak splitting pattern for the cis isomer
was more complex than the trans isomer (Figure 2B), and
could not be accounted for solely by through-bond couplings.
The decoupled 19F-{1H} spectrum was collected and revealed
coupling between Fa and Fb only in the cis isomer.37 As shown
in Figure 2B, the peaks corresponding to Fa and Fb in the trans
isomer appeared as singlet and doublet (due to the para-F),
respectively, and hence, there was no evidence of through-
space coupling between the ortho-fluorine moieties in this
isomeric form. The peaks corresponding to Fa and Fb in the cis
isomer appear as a triplet and quartet, respectively, as a result
Due to the increased thermal stability and higher
photoisomerization efficiency of c-CAP-F5 over c-CAP-F3,
we focused on CAP-F5 for more in-depth studies. The binding
interactions of t-CAP-F5 and c-CAP-F5 were studied with
bovine carbonic anhydrase (bCA) using a dansylamide (DNSA)
competition assay. DNSA, a sulfonamide based CA-inhibitor,
exhibits an enhanced fluorescence at 458 nm upon binding to
the active site of CA (apparent Kd = 1.6 M, under our
experimental conditions, Figure S10A), and a weakened
of through-space coupling between the ortho-fluorines (TSJFF
5.64 Hz for Fa, 5.67 Hz for Fb).38 Coupling between Fa and Fb
=
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’
fluorescence when an external ligand displaces it from the
active site (Figure S11). A solution of bCADNSA was titrated
with trans and cis isomers
isomers are drastically different, yielding Kd values of 9  1.3
nM for t-CAP-F5 and 106  16 nM for c-CAP-F5,
demonstrating an order higher binding affinity of the trans
isomer.
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A control molecule lacking the Zn2+-binding p-sulfonamide
group (CAP-con, Scheme S3) induces almost no change in the
fluorescence emission (Figure S12). We further employed
DNSA to investigate the isomerization of CAP-F5 in the active
site of CA, using CAP-F5, bCA, and an excess of DNSA.
Solutions of bCA, DNSA and CAP-F5 were irradiated with 520
nm (trans  cis) and 410 nm (cis  trans) alternately with
fluorescence measured after each irradiation (Figure 3B).
DNSA fluorescence intensity decreased after irradiation with
410 nm and increased after irradiation with 520 nm through
several cycles. These experiments demonstrate the reversible
and robust CAP-F5 photoisomerization even in the presence of
CA.
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To better understand the difference in binding preference
between the cis and trans isomers of CAP-F5 towards CA, we
analyzed the X-ray structure of hCAII with a related
azobenzene
sulfonamide
ligand,
(E)-4-(4-
aminophenyldiazenyl)benzenesulfonamide (L), bound to the
active site (Protein Data Bank: 5BYI).40 In this crystal
structure, L forms extensive hydrophilic and hydrophobic
interactions with hCAII at the Zn active site (Figure 4A). The
primary interaction between the protein and L is between the
deprotonated nitrogen of the -SO2NH2 and the Zn2+ center,
forming a tetrahedral coordination along with the three active
site histidines (H94, 96 and 119). The sulfonamide group of L
also hydrogen bonds to the backbone and side chain of Thr198
while the adjoining benzene ring sits in a hydrophobic pocket
formed by residues W208, L197 and L142. The other phenyl
ring hydrophobically stacks with P201 and forms a 6 Å T-shape
π-π stacking with F130 (Figure 4A).
Figure 3. (A) Change in fluorescence emission (λex = 280 nm; λem
= 458 nm) of bCA-DNSA mixture in the presence of different
concentrations of t-CAP-F5 and c-CAP-F5 to determine apparent
Kd values. (B) The binding of CAP-F5 is reversible in nature as
shown by irradiating the sample of CA, DNSA and probe with
alternate 520 nm and 410 nm for in situ isomerization and
monitoring DNSA fluorescence. Data represents mean  standard
deviation. Studies conducted in 50 mM HEPES, 0.1 M KNO3, pH
7.2, 298K, in the dark.
of CAP-F5 and the apparent Kd values were calculated. As
shown in Figure 3A, the binding profiles of the trans and cis
Figure 4. Docking of CAP-F5 in the active site of hCAII structure obtained with azobenzene ligand L bound to the active site (PDB 5BYI).
The secondary Protein (PDB 5BYI) is shown with side chains of active site residues in sticks. The favorable hydrogen bonds and -*
interactions are shown in yellow and orange dotted lines, respectively. (A) Docking of t-CAP-F5 into the hCAII active site after
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superimposing it onto the ligand L. (B) Alternative binding mode for t-CAP-F5 due to the presence of fluorine atoms. (C) Docking of c-
CAP-F5 when interactions between –SO2NH2 and Zn2+ are kept intact, steric clashes are shown as red disks. (D). Potential orientation of c-
CAP-F5 when steric clashes are removed, showing the probe retreats from the active site.
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The molecular structure of CAP-F5 highly resembles that of L
with the only alteration at the benzyl ring extending away from
the Zn2+ coordination site. We conducted molecular modeling
and docking using Maestro (Schrodinger Suite).41 Due to the
high structural similarity of t-CAP-F5 to L, the same
coordination to Zn2+ is expected to be conserved. The model is
then energy minimized in Maestro.42-43 Two configurations of
t-CAP-F5 are identified with favorable energy, both
maintaining similar binding mode to L without any steric
clashes (Figures 4A, 4B). The first configuration is identical to
the crystal structure of L binding with all favorable interaction
maintained (Figure 4A). The addition of fluorine atoms in
CAP-F5, however, decreases the electron-richness of the
benzene rings compared to L. Thus, an additional binding mode
is also possible for t-CAP-F5 when the azo bond flips by 180°
(Figure 4B). In this alternative configuration with similar
favorable binding energy, the fluoro-benzene ring rotates to a
configuration that forms a parallel π-π stacking of 3.7Å with
F130 while maintaining almost all other non-covalent
interactions. This orientation can be further stabilized by a
potential hydrogen bond between the azo group and Q92 with
no steric clashes with residues forming the active site pocket
(Figure 4B). Thus, t-CAP-F5 forms extensive favorable
contacts with the enzyme in two possible poses.
As CA can also catalyze bicarbonate dehydration, we also
investigated CAP-F5 in the context of this reaction, where
aqueous KHCO3 was used as the substrate. The initial velocity
of the CA-catalyzed reaction was 0.36 x 10-2 s-1 in the presence
of t-CAP-F5 and 1.0 x 10-2 s-1 in the presence of c-CAP-F5,
similar to the reaction rates in the presence (0.10 x 10-2 s-1) and
absence of acetazolamide (1.3 x 10-2 s-1) (Figure 5C). The
apparent Ki values of t-CAP-F5 and c-CAP-F5 were calculated
to be 30  3 nM and 205  14 nM respectively (Figure 5D).
These studies show that CAP-F5 isomers can be used to
modulate both the CO2 hydration and bicarbonate dehydration
activity of CA.
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When docking c-CAP-F5, the favorable interaction between
the Zn2+ and the sulfonamide nitrogen of the ligand cannot be
retained (Figure 4C). In order to maintain the same
coordination to Zn2+, the distal phenyl ring would be in close
contact with the active site pocket due to the cis-configuration
of the azo group, causing clashes with Q92, V121, V142 and
H64 (Figure 4C). To avoid steric clashes, c-CAP-F5 has to
adapt to a configuration retreating from the deep active site
pocket containing the Zn2+. As a result, the distance between the
sulfonamide nitrogen and the Zn2+ increase from 2.0 Å (as
found in L and more likely in t-CAP-F5) to 3.6 Å, which is no
longer optimal for Zn2+ coordination (Figure 4D). Furthermore,
the hydrogen bond between T198 and the sulfonamide group’s
nitrogen in the azobenzene ligand is unlikely to form in this
configuration (Figure 4D). Thus, c-CAP-F5 cannot show any
favorable interaction with Zn2+ in the active site, which makes
it more weakly binding than t-CAP-F5.
Figure 5. Change in absorbance of phenol red (=557nm) due to
CO2 hydration activity (A) and HCO3- dehydration activity (C) of
CA in the absence and presence of 1eq of trans and cis CAP-F5;
Change in CO2 hydration (B) and HCO3- dehydration (D) catalytic
velocity as a function of trans and cis isomer concentration. Studies
are conducted in 50 mM HEPES, 0.1M KNO3 pH 7.2 at 25 C using
stop flow apparatus. Data represents mean  the standard deviation.
-
CA catalyzes the hydration of CO2 and dehydration of HCO3
reversibly. The inhibitory effect of the trans and cis isomers of
CAP-F5 on CO2 hydration activity of bCA was measured by
tracking changes in solution pH using phenol red as a pH
indicator, and a solution of saturated CO2 as a substrate (Figure
5A).44 The observed rates for CO2 conversion by bCA in the
absence and presence of known CA inhibitor acetazolamide
were 0.113 s-1 and 0.027 s-1 respectively. Correspondingly, the
initial velocities of the catalytic reaction in the presence of trans
and cis isomers of CAP-F5 were found to be 0.025 s-1 and 0.082
s-1 respectively, demonstrating the superior ability of the trans
isomer to inhibit CA enzymatic activity. Using this same
stopped flow method, the apparent Ki values for t-CAP-F5 and
c-CAP-F5 were calculated to be 36  2 nM and 164  8 nM
respectively (Figure 5B), indicating ~5 times greater potency
of t-CAP-F5.
Having demonstrated the efficacy of CAP-F5 to
differentially interact with isolated CA protein in the trans and
cis isomeric forms, we next applied our photoswitch to control
the activity of CA in cell culture. We note that CAP-F5
isomerization is reversible over several cycles in cell culture
media (Figure S16). CAII is the cytosolic isoform of CA
responsible for maintaining intracellular pH, and is known to be
overexpressed in certain cancer cells.16 It has been reported that
the resting intracellular pH of cells is affected by CA inhibitors,
and this change highly depends on the cell type and
environment.45-47 Previously, we showed differential effects of
trans and cis isomers of CAP1 on intracellular pH, however
thermal instability of cis isomer prevented applications over
longer time periods.22 With our improved probe design with
highly stable cis isomer, we compared the ability of trans and
cis isomers of CAP-F5 to modulate intracellular pH in HeLa
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cells at both short (30 min) and long (24h) incubation times. We both short and long incubation times, whereas cells incubated
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performed a cellular cytotoxicity assay to determine probe
concentrations at which minimal toxicity would be observed for
the cis isomer (Figure S17). We observed that cells incubated
with the cis isomer maintained a similar pH to control cells after
with the trans isomer displayed decreased intracellular pH in
both scenarios
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Figure 6. (A) The intracellular pH of HeLa cells as a result of incubation of t-CAP-F5 and c-CAP-F5 for 30 minutes, as analyzed by flow
cytometry. The results are shown as an average of six independent experiments. Box plot indicates the range and average values for each
group. Asterisks denote statistically significant differences (p < 0.05; one-way analysis of variance). The change in intracellular pH with
respect to time as a result of addition of CO2 in the presence of c-CAP-F5(B) and t-CAP-F5 (C) due to the mechanism shown in (D). The
change in intracellular pH of cells incubated with c-CAP-F5 before and after irradiating with 410 nm to isomerize cis isomer to trans isomer.
These studies performed in Live Cell Imaging Solution at 4C.
(Figure 6A and S20). We repeated these experiments with
isolated cis and trans species (via preparative HPLC), and
similar results were obtained, indicating the PSS isomers are
sufficient to obtain differential cellular response (Figure S20).
Intracellular c-CAP-F5 can further be photostimulated in situ
to activate CA inhibition. Cells were treated with pHrodo dye,
c-CAP-F5 or DMSO in the similar way as described earlier.
The c-CAP-F5 treated cells were divided into two groups,
where only one group was irradiated with 410 nm light for 3
minutes (no phototoxicity observed) to isomerize the cis form
to trans form in situ right before the addition of CO2, and were
compared with non-irradiated cells. As shown in Figure 6E, the
kinetics of intracellular pH change in cis-treated cells was faster
than cells irradiated with 410 nm (converting cis to trans) prior
to the addition of CO2.This experiment demonstrates that probe
isomerization can be achieved within the complex cellular
environment, and indeed could be used to inhibit the CA
activity upon light irradiation.
We next explored the effect of the isomers of CAP-F5 on the
response of the cytoplasmic CA machinery to exogenous
perturbations of CO2. When the level of extracellular CO2
increases, it passively diffuses into the cytoplasm through the
cell membrane where it is converted to carbonic acid by
cytoplasmic CAs, resulting in decrease in intracellular pH
(Figure 6D).48 Inhibition of cytoplasmic CA reduces the rate of
intracellular acidification following increases in extracellular
48-49
CO2 concentration.45-46,
We used this rate of change of
intracellular pH to demonstrate the real-time inhibition of
cytosolic CA by t-CAP-F5. In our experiment, HeLa cells were
loaded with pHrodo and incubated with 25 M t-CAP-F5, c-
CAP-F5, or a DMSO control for 30 minutes. A bolus of
saturated aqueous CO2 was added to the cell suspension and
intracellular pH changes were monitored using flow cytometry.
As shown in Figure 6B, c-CAP-F5 treated cells had similar
kinetic changes in intracellular pH over time as compared to
DMSO-treated control cells, signifying that cytosolic CA
remains active in both groups. However, t-CAP-F5 treated cells
displayed a slower rate of change in pH upon CO2 addition,
consistent with inhibition of the cytosolic CA machinery of CO2
hydration (Figure 6C).
Next, we applied CAP-F5 in an in vivo system. Embryonic
zebrafish (Danio rerio) are an excellent animal model for
studying drug induced toxicity due to their high fecundity,
external fertilization, rapid development, and the ease of
maintaining large numbers in a small space.50 Substances of
interest can be administered in the media and then are rapidly
up taken into the embryo. Importantly, the embryos are also
optically transparent, which allows for study of photo
responsive materials. CA is a vital component in acid-base
regulation as well as ionic and osmotic balance in vertebrates.51
Just like mammals, fish possess an abundance of CA isoforms
that vary in molecular sequence, tissue distribution and
subcellular localization.52 They also have different kinetic
properties and active site structure and thus different
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susceptibility to inhibitors. They are necessary during the early
with CA inhibitor can display small otoliths, an irregular jaw,
enlarged heart and yolk sac, and impaired locomotion.54-55
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stages of development in zebrafish for effective CO2
excretion.53 Inhibition of CA by inhibitors like acetazolamide
has a great effect on zebrafish embryogenesis; embryos treated
We used phenotypic analysis to differentiate the behavior of
cis and trans CAP-F5 in zebrafish. Wild-type (AB strain)
embryos were collected and transferred into standard embryo
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Figure 7. (A) The timeline representing probe treatment and fish analysis. The morphological appearance (B), swimming behavior (C) and
otolith development (D) in fish in the absence (vehicle) and presence of t-CAP-F5 and c-CAP-F5. The data represents mean values  the
standard deviation. Asterisks denote statistically significant differences (p < 0.0001; one-way analysis of variance). Scale bars represents
500 m and 50 m for B and D respectively. The morphological (E) and otolith (F) development as a result of in situ activation of probe
from cis to trans isomer by irradiating the fish with 410 nm at different time point during embryo development. These developments are
compared with ex situ generated trans isomer treatment at the respective time points. The representative images for normal, broken and
absent otoliths can be found in supplementary. The results are represented as mean of 30 fish in three independent experiments. Data
represents mean values  SEM. Asterisks denote statistically significant differences (**p < 0.005, ***p < 0.0001; one-way analysis of
variance).
media56 and sorted by developmental stage.57 At six hours post
fertilization (6hpf), zebrafish were exposed to media containing
1% DMSO and varying concentrations of CAP-F5 in both trans
and cis forms. Embryos were incubated in a light-proof
container at 28.5 C until 48hpf. They were then transferred to
fresh media and raised to 5 days post fertilization (5dpf), when
they
treated with 2.5 µM of trans and cis isomers of CAP-F5, and
were raised to 5dpf as described above. As shown in Figure 7B,
zebrafish treated with t-CAP-F5 showed multiple
morphological abnormalities, including failure to form a swim
bladder, pectoral fin defects, and cardiac edema, consistent with
previous reported effects of CA inhibitors.54-55 100% of
embryos (n=30) treated with the same concentration of c-CAP-
F5 developed normally, signifying the normal function of CA
in cis-treated fish. Furthermore, the zebrafish were fixed and
processed for otolith analysis. The t-CAP-F5 treated zebrafish
had hollow and underdeveloped otoliths as opposed to c-CAP-
F5 treated fish, where 100% (n=30) had normally developed
were assessed for viability (Figure 7A). As expected, t-CAP-
F5 induces higher toxicity than c-CAP-F5 (Table S5). Further,
the distinct effect of the isomers on embryogenesis was
evaluated. For morphological endpoints, the zebrafish were
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Journal of the American Chemical Society
otoliths (Figure 7D). This is likely caused by the inhibition of
Page 8 of 17
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General materials and synthetic methods, experimental protocols,
CA isoforms present in inner ear hair cells by t-CAP-F5 only,
−
tables and figures, sample characterization data, and supporting
explanation of TD-DFT calculation (PDF)
disrupting the supply of HCO3 to the endolymph for otolith
calcification.56 Potentially as
a result of poor otolith
development or other developmental defects, fish treated with
t-CAP-F5 showed poor locomotion, while c-CAP-F5 treated
fish exhibited normal locomotive behavior (Figure 7C). Our
results signify that the isomers of CAP-F5 are capable of
differential effects in this complex multicellular system that are
associated with CA inhibition.
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Crystal data of t-CAP-F5 and c-CAP-F5 (cif)
AUTHOR INFORMATION
Corresponding Author
After establishing that different isomers of CAP-F5 can have
different effects on the developmental and behavioral properties
of fish, we investigated if the probe could be activated in situ
within the embryo for CA inhibition during development.
Embryos were treated with c-CAP-F5 as described above, but
the probe was activated to the trans isomer in situ (within the
fish) through photoirradiation with 410 nm (0.50 mW/cm2;
3min) at different time points during embryogenesis (12 hpf, 24
hpf, 30hpf), delineated in Figure 7A. These irradiation
conditions do not affect the development of embryos at any
given irradiation time point. As CA isoforms play an important
role in meeting the requirements for CO2 excretion between
24hpf and 48hpf in developing fish, these time points were
chosen to assess the effect of inhibition before and during that
window.53 The results were compared to fish that were treated
with ex situ generated trans isomer (positive control) and
vehicle (negative control) at all the respective time of cis
activation. The degree of edema observed in the photoactivated
fish are similar to the t-CAP-F5 treated fish at all points of
photoirradiation, signifying the capability of activating the
probe within the embryos (Figure 7E). Moreover, as we
progress further along the post-fertilization timeline for probe
activation, the inhibitor efficacy appears to be decreasing.
These results are consistent with the known crucial role of CA
role in the initial phases of fish development.53 Otolith
development at various stages of probe activation shows a
similar trend (Figure 7F), further corroborating the relation of
phenotypic changes with in situ CA inhibition in this live
organism model system. These experiments represent a key
demonstration of using cis stabilized ortho-fluorinated
azobenzenes in an in vivo system and achieving in situ
photoactivation over extended time periods (up to 48 h).
Corresponding Author
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*E-mail: emilyque@cm.utexas.edu.
ORCID
Emily L. Que: 0000-0001-6604-3052
Author Contributions
All of the authors reviewed the results and approved the final
version of the manuscript.
Funding Sources
This project was supported by grants from NIH (R35 GM133612
ELQ, RO1 GM125882 YJZ, R01 DE020884 and R35 DE029086
JKE, f31 AA026781 TPK, K99 AA027567 YF), Brain &
Behavior Research Foundation (BBRF) 27321 YF, the Welch
Foundation (F-1883 ELQ and 1778 YJZ), and the University of
Texas at Austin (start-up funds ELQ, undergraduate research
fellowship MB). Some NMR spectra were acquired on a Bruker
AVIII HD 500 instrument acquired through a National Institutes
of Health equipment grant (J. Sessler, 1 S10 OD021508-01).
Notes
The author declare no competing financial interest.
ACKNOWLEDGMENT
Flow cytometry experiments were performed at the Microscopy
and Imaging Facility of the Center for Biomedical Research
Support at UT Austin. We are thankful to Gregory Thiabaud and
Dr. Jonathan Sessler for training and use of their HPLC setup. We
would like to acknowledge Steve Sorey and Angela W.
Spangenberg from the UT Austin Chemistry NMR facility for
guidance on 2-D NMR experiments.
ABBREVIATIONS
CAP1, carbonic anhydrase azobenzene photoswitch 1; CAP2,
carbonic anhydrase azobenzene photoswitch 2; CAP-F3, carbonic
anhydrase azobenzene photoswitch- with three fluorines;
CAP-F5, carbonic anhydrase azobenzene photoswitch- with five
fluorines; CAP-con, carbonic anhydrase azobenzene photoswith-
control with no sulfonamide; CA, carbonic anhydrase; DFT,
Differential Functional Theory; DNSA, Dansylamide; t, solution
after irradiation with 410 nm (CAP-F5) or 460 nm (CAP-F3) light;
c, solution after irradiation with 520 nm light; PSS, photostationary
CONCLUSIONS
In conclusion, we have demonstrated the in vitro, cellular, and
in vivo application of an azobenzene photopharmacophore that
targets the active site of the metalloenzyme carbonic anhydrase.
Ortho-fluorination serves to red-shift photoisomerization
wavelengths, improve photoconversion efficiency, and
importantly, stabilize the cis isomer. These properties enable
longitudinal studies in cells and, for the first time, in vivo,
allowing in situ temporal control of CA activity and
demonstrating the potential use of this strategy for spatially and
temporally regulated metalloenzyme activity. These strategies
should not be limited to carbonic anhydrase and current efforts
are focused on targeting metalloenzyme classes with relevance
to cancer and other diseases.
state; NMR, nuclear magnetic resonance; XRD, X-Ray Diffraction;
TS
J , Through space coupling constant between Fluorine atoms.
FF
REFERENCES
1.
Klan, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.;
Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J., Photoremovable protecting
groups in chemistry and biology: reaction mechanisms and efficacy. Chem.
Rev. 2013, 113 (1), 119-91.
2.
Szymanski, W.; Beierle, J. M.; Kistemaker, H. A.; Velema, W.
A.; Feringa, B. L., Reversible photocontrol of biological systems by the
incorporation of molecular photoswitches. Chem. Rev. 2013, 113 (8), 6114-
78.
ASSOCIATED CONTENT
Supporting Information
3.
Mahimwalla, Z.; Yager, K. G.; Mamiya, J.; Shishido, A.;
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
Priimagi, A.; Barrett, C. J., Azobenzene photomechanics: prospects and
potential applications. Polym. Bull. 2012, 69 (8), 967-1006.
ACS Paragon Plus Environment

Page 9 of 17
Journal of the American Chemical Society
4.
biomolecules. Chem. Soc. Rev. 2011, 40 (8), 4422-37.
5. Ogasawara, S., Duration Control of Protein Expression in Vivo
by Light-Mediated Reversible Activation of Translation. ACS Chem. Biol.
2017, 12 (2), 351-356.
Beharry, A. A.; Woolley, G. A., Azobenzene photoswitches for
Crystal Polymer Networks. Angew. Chem., Int. Ed. Engl. 2016, 55 (34),
9908-12.
27.
1
2
3
4
5
6
7
8
Bushuyev, O. S.; Tomberg, A.; Friscic, T.; Barrett, C. J.,
Shaping crystals with light: crystal-to-crystal isomerization and
photomechanical effect in fluorinated azobenzenes. J. Am. Chem. Soc.
2013, 135 (34), 12556-9.
6.
Martin, N.; Ruchmann, J.; Tribet, C., Prevention of aggregation
and renaturation of carbonic anhydrase via weak association with
octadecyl- or azobenzene-modified poly(acrylate) derivatives. Langmuir
2015, 31 (1), 338-49.
28.
Castellanos, S.; Goulet-Hanssens, A.; Zhao, F.; Dikhtiarenko,
A.; Pustovarenko, A.; Hecht, S.; Gascon, J.; Kapteijn, F.; Bleger, D.,
Structural Effects in Visible-Light-Responsive Metal-Organic Frameworks
Incorporating ortho-Fluoroazobenzenes. Chemistry 2016, 22 (2), 746-52.
7.
Weston, C. E.; Kramer, A.; Colin, F.; Yildiz, O.; Baud, M. G.;
Meyer-Almes, F. J.; Fuchter, M. J., Toward Photopharmacological
Antimicrobial Chemotherapy Using Photoswitchable Amidohydrolase
Inhibitors. ACS Infect. Dis. 2017, 3 (2), 152-161.
29.
Accardo, J. V.; Kalow, J. A., Reversibly tuning hydrogel
stiffness through photocontrolled dynamic covalent crosslinks. Chem. Sci.
2018, 9 (27), 5987-5993.
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
8.
Szymanski, W.; Ourailidou, M. E.; Velema, W. A.; Dekker, F.
30.
Trads, J. B.; Burgstaller, J.; Laprell, L.; Konrad, D. B.; de la Osa
J.; Feringa, B. L., Light-Controlled Histone Deacetylase (HDAC)
Inhibitors: Towards Photopharmacological Chemotherapy. Chemistry
2015, 21 (46), 16517-24.
de la Rosa, L.; Weaver, C. D.; Baier, H.; Trauner, D.; Barber, D. M., Optical
control of GIRK channels using visible light. Org. Biomol. Chem. 2016, 15
(1), 76-81.
9.
Wang, X.; Huang, J.; Zhou, Y.; Yan, S.; Weng, X.; Wu, X.;
31.
Wegener, M.; Hansen, M. J.; Driessen, A. J. M.; Szymanski, W.;
Deng, M.; Zhou, X., Conformational switching of G-quadruplex DNA by
photoregulation. Angew. Chem., Int. Ed. Engl. 2010, 49 (31), 5305-09.
10.
Zhou, X.; Wang, S.; Zhou, X., Small-Molecule-Triggered and Light-
Controlled Reversible Regulation of Enzymatic Activity. J. Am. Chem. Soc.
2016, 138 (3), 955-61.
Feringa, B. L., Photocontrol of Antibacterial Activity: Shifting from UV to
Red Light Activation. J. Am. Chem. Soc. 2017, 139 (49), 17979-86.
32.
Reversible Spatiotemporal Control of Induced Protein Degradation by
Bistable PhotoPROTACs. ACS Cent. Sci. 2019, 5 (10), 1682-90.
33.
Messerer, R.; Littmann, T.; Wolber, G.; Holzgrabe, U.; Decker, M.,
Fluorination of Photoswitchable Muscarinic Agonists Tunes Receptor
Pharmacology and Photochromic Properties. J. Med. Chem. 2019, 62 (6),
3009-3020.
Tian, T.; Song, Y.; Wang, J.; Fu, B.; He, Z.; Xu, X.; Li, A.;
Pfaff, P.; Samarasinghe, K. T. G.; Crews, C. M.; Carreira, E. M.,
Agnetta, L.; Bermudez, M.; Riefolo, F.; Matera, C.; Claro, E.;
11.
Sheldon, J. E.; Dcona, M. M.; Lyons, C. E.; Hackett, J. C.;
Hartman, M. C., Photoswitchable anticancer activity via trans-cis
isomerization of a combretastatin A-4 analog. Org. Biomol. Chem. 2016,
14 (1), 40-49.
12.
Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.;
34.
Heinrich, B.; Bouazoune, K.; Wojcik, M.; Bakowsky, U.;
Jalinot, P.; Hasserodt, J.; Rehberg, M.; Delattre, M.; Zahler, S.; Vollmar,
A.; Trauner, D.; Thorn-Seshold, O., Photoswitchable Inhibitors of
Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell
2015, 162 (2), 403-411.
Vazquez, O., ortho-Fluoroazobenzene derivatives as DNA intercalators for
photocontrol of DNA and nucleosome binding by visible light. Org.
Biomol. Chem. 2019, 17 (7), 1827-1833.
35.
Lin, W. C.; Tsai, M. C.; Rajappa, R.; Kramer, R. H., Design of
13.
Tochitsky, I.; Banghart, M. R.; Mourot, A.; Yao, J. Z.; Gaub, B.;
a Highly Bistable Photoswitchable Tethered Ligand for Rapid and
Sustained Manipulation of Neurotransmission. J Am Chem Soc 2018, 140
(24), 7445-7448.
Kramer, R. H.; Trauner, D., Optochemical control of genetically engineered
neuronal nicotinic acetylcholine receptors. Nat. Chem. 2012, 4 (2), 105-11.
14.
Nagpal, J.; Rutter, G. A.; Rhee, J.-S.; Gottschalk, A.; Brose, N.; Schultz,
C.; Trauner, D., Photoswitchable diacylglycerols enable optical control of
protein kinase C. Nat. Chem. Biol. 2016, 12 (9), 755-762.
Frank, J. A.; Yushchenko, D. A.; Hodson, D. J.; Lipstein, N.;
36.
Saccone, M.; Siiskonen, A.; Fernandez-Palacio, F.; Priimagi,
A.; Terraneo, G.; Resnati, G.; Metrangolo, P., Halogen bonding stabilizes a
cis-azobenzene derivative in the solid state: a crystallographic study. Acta
Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater. 2017, 73 (2), 227-
233.
15.
Supuran, C. T., Structure and function of carbonic anhydrases.
Biochem. J. 2016, 473 (14), 2023-32.
16.
Carbonic Anhydrases: Role in pH Control and Cancer. Metabolites 2018, 8
(1), 19.
17.
of carbonic anhydrase inhibitors. Subcell Biochem 2014, 75, 349-59.
18. Kiefer, L. L.; Paterno, S. A.; Fierke, C. A., Hydrogen Bond
Network in the Metal Binding Site of Carbonic Anhydrase Enhances Zinc
Affinity and Catalytic Efficiency. J. Am. Chem. Soc. 1995, 117, 6831-6837.
37.
Rastogi, S. K.; Rogers, R. A.; Shi, J.; Brown, C. T.; Salinas, C.;
Mboge, M. Y.; Mahon, B. P.; McKenna, R.; Frost, S. C.,
Martin, K. M.; Armitage, J.; Dorsey, C.; Chun, G.; Rinaldi, P.; Brittain, W.
J., Through-space 19F-19F spin-spin coupling in ortho-fluoro Z-azobenzene.
Magn. Reson. Chem. 2016, 54 (2), 126-31.
Scozzafava, A.; Supuran, C. T., Glaucoma and the applications
38.
Rastogi, S. K.; Rogers, R. A.; Shi, J.; Gao, C.; Rinaldi, P. L.;
Brittain, W. J., Conformational Dynamics of o-Fluoro-Substituted Z-
Azobenzene. J. Org. Chem. 2015, 80 (22), 11485-90.
39.
magnetic relaxation. Nature 1947, 160 (4066), 475.
40. Runtsch, L. S.; Barber, D. M.; Mayer, P.; Groll, M.; Trauner,
D.; Broichhagen, J., Azobenzene-based inhibitors of human carbonic
anhydrase II. Beilstein J. Org. Chem. 2015, 11, 1129–35.
41.
Bloembergen, N.; Purcell, E. M.; Pound, R. V., Nuclear
19.
Rina Mogaki, K. O., and Takuzo Aida, Adhesive Photoswitch:
Selective Photochemical Modulation of Enzymes under Physiological
Conditions. J. Am. Chem. Soc. 2017, 139 (29), 10072-78.
20.
DuBay, K. H.; Iwan, K.; Osorio-Planes, L.; Geissler, P. L.;
Schrödinger Release 2020-1: Maestro, Schrödinger, LLC, New
Groll, M.; Trauner, D.; Broichhagen, J., A Predictive Approach for the
Optical Control of Carbonic Anhydrase II Activity. ACS Chem. Biol. 2018,
13 (3), 793-800.
21.
a photoswitchable affinity label. Chembiochem 2008, 9 (2), 191-3.
22. Aggarwal, K.; Banik, M.; Medellin, B.; Que, E. L., In Situ
Photoregulation of Carbonic Anhydrase Activity Using
Azobenzenesulfonamides. Biochemistry 2019, 58 (1), 48-53.
23. Bleger, D.; Schwarz, J.; Brouwer, A. M.; Hecht, S., o-
York, NY, 2020.
42.
Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen,
W. L., Evaluation and Reparametrization of the OPLS-AA Force Field for
Proteins via Comparison with Accurate Quantum Chemical Calculations on
Peptides†. J. Phys. Chem. B 2001, 105 (28), 6474-6487.
Harvey, J. H.; Trauner, D., Regulating enzymatic activity with
43.
Shivakumar, D.; Williams, J.; Wu, Y.; Damm, W.; Shelley, J.;
Sherman, W., Prediction of Absolute Solvation Free Energies using
Molecular Dynamics Free Energy Perturbation and the OPLS Force Field.
J. Chem. Theory Comput. 2010, 6 (5), 1509-1519.
Fluoroazobenzenes as readily synthesized photoswitches offering nearly
quantitative two-way isomerization with visible light. J. Am. Chem. Soc.
2012, 134 (51), 20597-600.
44.
Khalifah, R. G., The carbon dioxide hydration activity of
carbonic anhydrase. I. Stop-flow kinetic studies on the native human
isoenzymes B and C. J. Biol. Chem. 1971, 246 (8), 2561-73.
45.
Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit
Nonpigmented Ciliary Epithelium. J. Membr. Biol. 1998, 162, 31-38.
46.
Acetazolamide on Intracellular pH and Bicarbonate Transport in Bovine
Corneal Endothelium. Exp. Eye Res. 1995, 60, 425-434.
47.
24.
Knie, C.; Utecht, M.; Zhao, F.; Kulla, H.; Kovalenko, S.;
Wu, Q.; W.M. Pierce, J.; Delamere, N. A., Cytoplasmic pH
Brouwer, A. M.; Saalfrank, P.; Hecht, S.; Bleger, D., ortho-
Fluoroazobenzenes: visible light switches with very long-Lived Z isomers.
Chemistry 2014, 20 (50), 16492-501.
Bonannpo, J. A.; Srinivas, S. P.; Brown, M., Effect of
25.
Ahmed, Z.; Siiskonen, A.; Virkki, M.; Priimagi, A., Controlling
azobenzene photoswitching through combined ortho-fluorination and -
amination. ChemComm 2017, 53 (93), 12520-12523.
26.
Puech, C.; Chatard, M.; Felder-Flesch, D.; Prevot, N.; Perek, N.,
Iamsaard, S.; Anger, E.; Asshoff, S. J.; Depauw, A.; Fletcher, S.
Umbelliferone Decreases Intracellular pH and Sensitizes Melanoma Cell
P.; Katsonis, N., Fluorinated Azobenzenes for Shape-Persistent Liquid
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 10 of 17
Line A375 to Dacarbazin. Comparison with Acetazolamide. Curr. Mol.
Pharmacol. 2018, 11 (2), 133-139.
54.
Matsumoto, H.; Fujiwara, S.; Miyagi, H.; Nakamura, N.; Shiga,
Y.; Ohta, T.; Tsuzuki, M., Carbonic Anhydrase Inhibitors Induce
Developmental Toxicity During Zebrafish Embryogenesis, Especially in
the Inner Ear. Mar. Biotechnol. 2017, 19 (5), 430-440.
1
2
3
4
5
6
7
8
48.
Mizumori, M.; Meyerowitz, J.; Takeuchi, T.; Lim, S.; Lee, P.;
Supuran, C. T.; Guth, P. H.; Engel, E.; Kaunitz, J. D.; Akiba, Y., Epithelial
carbonic anhydrases facilitate PCO2 and pH regulation in rat duodenal
mucosa. J. Physiol. 2006, 573 (Pt 3), 827-42.
55.
Aspatwar, A.; Becker, H. M.; Parvathaneni, N. K.; Hammaren,
M.; Svorjova, A.; Barker, H.; Supuran, C. T.; Dubois, L.; Lambin, P.;
Parikka, M.; Parkkila, S.; Winum, J. Y., Nitroimidazole-based inhibitors
DTP338 and DTP348 are safe for zebrafish embryos and efficiently inhibit
the activity of human CA IX in Xenopus oocytes. J. Enzyme Inhib. Med.
Chem. 2018, 33 (1), 1064-1073.
49.
Rasmussen, J. K.; Boedtkjer, E., Carbonic anhydrase inhibitors
modify intracellular pH transients and contractions of rat middle cerebral
arteries during CO2/HCO3- fluctuations. J. Cereb. Blood Flow Metab. 2018,
38 (3), 492-505.
50.
Truong, L.; Harper, S. L.; Tanguay, R. L., Evaluation of
56.
use
Westerfield, M. The zebrafish book : a guide for the laboratory
of zebrafish Danio (Brachydanio) rerio.
embryotoxicity using the zebrafish model. Methods Mol. Biol. 2011, 691,
271-79.
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https://zfin.org/zf_info/zfbook/cont.html.
57. Kimmel, C. B.; Ballard, W. W.; Kimmel, S. R.; Ullmann, B.;
Schilling, T. F., Stages of embryonic development of the zebrafish. Dev.
Dyn. 1995, 203 (3), 253-310.
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acid-base homeostasis in zebrafish. PLoS One 2012, 7 (6), 39881.
52. Gilmour, K. M.; Perry, S. F., Carbonic anhydrase and acid-base
regulation in fish. J. Exp. Biol. 2009, 212 (Pt 11), 1647-61.
53. Gilmour, K. M.; Thomas, K.; Esbaugh, A. J.; Perry, S. F.,
Postel, R.; Sonnenberg, A., Carbonic anhydrase 5 regulates
Carbonic anhydrase expression and CO2 excretion during early
development in zebrafish Danio rerio. J. Exp. Biol. 2009, 212 (Pt 23), 3837-
45.
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UV-vis spectra of CAP-F3 (A) and CAP-F5 (B) before and after photoirradiation (cis 520 nm for 5 minutes;
2
trans 460/410 nm for 2 min). The power densities for 410, 460 and 520 nm were 5.20 mW/cm , 14.12
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mW/cm and 9.18 mW/cm , respectively. The studies are performed in methanol at 298 K.
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Structures of CAP-F3 and CAP-F5, and photoswitching in the presence of carbonic anhydrase
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(A) Change in fluorescence emission (λ = 280 nm; λ
= 458 nm) of bCA-DNSA mixture in the presence
em
ex
of different concentrations of t-CAP-F5 and c-CAP-F5 to determine apparent K values. (B) The binding of
CAP-F5 is reversible in nature as shown by irradiating the sample of CA, DNSA and probe with alternate
520 nm and 410 nm for in situ isomerization and monitoring DNSA fluorescence. Data represents mean ±
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standard deviation. Studies conducted in 50 mM HEPES, 0.1 M KNO , pH 7.2, 298K, in the dark.
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Docking of CAP-F5 in the active site of hCAII structure obtained with azobenzene ligand L bound to the
active site (PDB 5BYI). The secondary Protein (PDB 5BYI) is shown with side chains of active site residues in
sticks. The favorable hydrogen bonds and π-π* interactions are shown in yellow and orange dotted lines,
respectively. (A) Docking of t-CAP-F5 into the hCAII active site after superimposing it onto the ligand L.
(B) Alternative binding mode for t-CAP-F5 due to the presence of fluorine atoms. (C) Docking of c-CAP-F5
2+
when interactions between –SO NH and Zn
are kept intact, steric clashes are shown as red disks. (D).
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Potential orientation of c-CAP-F5 when steric clashes are removed, showing the probe retreats from the
active site.
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(A) The timeline representing probe treatment and fish analysis. The morphological appearance (B),
swimming behavior (C) and otolith development (D) in fish in the absence (vehicle) and presence of t-CAP-
F5 and c-CAP-F5. The data represents mean values ± the standard deviation. Asterisks denote statistically
significant differences (p < 0.0001; one-way analysis of variance). Scale bars represents 500 μm and 50 μm
for B and D respectively. The morphological (E) and otolith (F) development as a result of in situ activation
of probe from cis to trans isomer by irradiating the fish with 410 nm at different time point during embryo
development. These developments are compared with ex situ generated trans isomer treatment at the
respective time points. The representative images for normal, broken and absent otoliths can be found in
supplementary. The results are represented as mean of 30 fish in three independent experiments. Data
represents mean values ± SEM. Asterisks denote statistically significant differences (**p < 0.005, ***p <
0.0001; one-way analysis of variance).
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Table of Content graphics for paper
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