Boron dipyrromethene as a fluorescent caging group for single-photon uncaging with long-wavelength visible light

Article abstract of DOI:10.1021/cb500525p

Caged compounds are useful tools for precise spatiotemporal modulation of cell functions, but in most cases uncaging requires ultraviolet (UV) light, which is cytotoxic and has limited tissue penetration. Therefore, caged compounds that can be activated by longer-wavelength light are required. Here we describe a novel photoelimination reaction of 4-aryloxy boron dipyrromethene (BODIPY) derivatives and show that BODIPY can function as a caging group for phenol groups. We developed a novel BODIPY-caged histamine compound, which is photoactivatable with blue-green visible light to stimulate cultured HeLa cells in a spatiotemporally well-controlled manner. This caging strategy is expected to be widely applicable to develop tools for probing various cellular functions. (Chemical Equation Presented).

Full text of DOI:10.1021/cb500525p

Letters
pubs.acs.org/acschemicalbiology
Boron Dipyrromethene As a Fluorescent Caging Group for Single-
Photon Uncaging with Long-Wavelength Visible Light
†
,⊥
‡,⊥
‡
†
,‡,∥
†,§
Nobuhiro Umeda, Hironori Takahashi, Mako Kamiya, Tasuku Ueno, Toru Komatsu,
†
†
†
Takuya Terai, Kenjiro Hanaoka, Tetsuo Nagano, and Yasuteru Urano*
†
‡
Graduate School of Pharmaceutical Sciences and Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
§
Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8
Honcho, Kawaguchi, Saitama 332-0012, Japan
∥
Basic Research Program, Japan Science and Technology Agency, 3-5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
*
S Supporting Information
ABSTRACT: Caged compounds are useful tools for precise
spatiotemporal modulation of cell functions, but in most cases
uncaging requires ultraviolet (UV) light, which is cytotoxic and
has limited tissue penetration. Therefore, caged compounds
that can be activated by longer-wavelength light are required.
Here we describe a novel photoelimination reaction of 4-
aryloxy boron dipyrromethene (BODIPY) derivatives and
show that BODIPY can function as a caging group for phenol
groups. We developed a novel BODIPY-caged histamine
compound, which is photoactivatable with blue-green visible
light to stimulate cultured HeLa cells in a spatiotemporally
well-controlled manner. This caging strategy is expected to be
widely applicable to develop tools for probing various cellular functions.
aged compounds have been widely used as a biological
tool for investigating or perturbing cellular dynamics/
with a blue-green visible light source (∼500 nm), we developed
C
photoactivatable caged histamine, which can be used to
stimulate living cells in a precisely controlled manner.
functions of living cells in a spatiotemporally controlled
manner. However, uncaging of conventional caged compounds
by single photon excitation generally requires ultraviolet (UV)
We first synthesized 1,3,5,7-tetramethyl-BODIPY derivatives
bearing aryl groups at the boron position according to the
1
,2
15
to violet light (250−400 nm), which is cytotoxic to living
literature (1−7, Figure 1a and Supporting Information). We
3
cells and has low tissue penetration. Therefore, caged
found that the fluorescence quantum efficiency (ϕ ) of these 4-
fl
compounds that can be activated by longer-wavelength light
have been desired. Despite massive efforts to develop such
caged compounds, most suffer from drawbacks, such as a
aryloxy BODIPY derivatives decreased as the HOMO energy
level of the aryloxy groups increased, while the absorption/
emission wavelengths and the molar absorption coefficient (ε)
varied only slightly (Figure 1c, Supplementary Table S1). Since
it has been reported that BODIPY fluorescence can be
quenched by photoinduced electron transfer (PeT) from
4
requirement of additional reagents or lack of evidence for
5,6
practicality as caged compounds in living systems. Amine-
ruthenium complexes can be photolyzed by visible wavelengths
7
−9
up to 532 nm to induce cellular response, but their relatively
broad absorption spectrum hampers fluorescence observation
using fluorescent indicators such as Rhod-2, and there remain
concerns about potential photodamage, since they are originally
16
adjacent aryl groups, the quenching of these 4-aryloxy
BODIPY derivatives is also considered to be due to PeT
from the 4-aryloxy groups. During these spectroscopic
investigations, we serendipitously discovered the very intriguing
phenomenon that some of these 4-aryloxy BODIPY derivatives
were photocleavable at the 4-position by visible light
irradiation. Indeed, we confirmed by HPLC analysis and
NMR characterization that solvolyzed BODIPY and a hydroxy
aryl compound were produced after irradiation with visible light
10
based on photosensitizers. Recently, blue-cyan light-absorbing
caging groups based on coumarin derivatives have also been
1
1,12
reported.
Here, we focused on the widely known boron
13,14
dipyrromethene (BODIPY) fluorophore,
since it has a
sharp and intense absorption profile that might minimally
occupy the wavelengths needed for fluorescence observation
and shows negligible triplet-state formation, which should
guarantee low phototoxicity and high biocompatibility. On the
basis of our serendipitous finding that some 4-aryloxy BODIPY
derivatives undergo photoelimination reaction under irradiation
Received: July 1, 2014
Accepted: August 20, 2014
©
XXXX American Chemical Society
A
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ACS Chemical Biology
Letters
Figure 1. Photoelimination reaction of 4-aryloxy BODIPY derivatives. (a) General scheme of the photoreaction. (b) Time course of BDP-7HC (4)
−2
consumption and photoproducts production upon irradiation. Irradiation: 475−490 nm, 15 mW cm . (c and d) Fluorescence quantum efficiency
(
ϕ ) and uncaging quantum efficiency (ϕ ) of 4-aryloxy BODIPY derivatives are plotted against HOMO energy level of the corresponding phenols.
fl
u
Figure 2. Development of BODIPY caged histamine. (a) Scheme of uncaging reaction of BcHA-1 (13) and -2 (18). (b) Cellular distribution of 1
μM BcHA-1 and 5 μM BcHA-2 in HeLa cells. Scale bars, 100 μm. (c) Absorption (black solid line) and fluorescence (red solid line) spectra of
−2
BcHA-2. (d) Time course of BcHA-2 consumption and histamine production. Irradiation: 460−500 nm, 29 mW cm . Data are presented as mean
SEM from three independent experiments. (e) Dose−response curves of BcHA-2 and histamine. Data are presented as mean ± SEM, n > 40 cells
for each condition from two independent experiments.
±
(
475−490 nm) (Figure 1a,b and Supplementary Figures S1 and
O bond of the positively charged aryl group. As the previous
S2). More precisely, the efficiency of photocleavage (ϕ ) of 4-
report suggested that cationization can greatly reduce the pK
u
a
17
aryloxy BODIPY derivatives was greatly dependent on the
HOMO energy level of the aryl group; the derivatives whose ϕfl
was low due to the quenching via PeT were efficiently
decomposed with high ϕ upon irradiation with blue-green
light (Figure 1d, Supplementary Table S1). This inverse
of the hydroxy aryl groups, we speculated that production of
the CS state would be an important determinant of
photocleavage of the BODIPY caging group. We also found
that the PeT-based quenching of fluorescence is a necessary but
not sufficient condition for efficient uncaging because a
u
correlation between ϕ and ϕ suggests that the uncaging
decrease of ϕ above −0.2 hartree was observed, which might
fl
u
u
reaction of the BODIPY caging group proceeds via a PeT
process in which a charge-separation intermediate (CS state)
involving the cation radical of the aryl group and anion radical
of the fluorophore is produced, followed by solvolysis of the B−
result from a faster back electron transfer (BeT) process,
18
shortening the lifetime of the CS state. Concerning the values
of uncaging quantum efficiency (ϕ ), the values of our
u
BODIPY derivatives are lower than those of known UV-
B
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ACS Chemical Biology
Letters
2
24
excitable caging groups (Supplementary Table S1). However,
this can be compensated by the higher molar absorption
coefficient (ε) of BODIPY derivatives, since the product of ε
derivatization using o-phthalaldehyde and thiol (Figure 2d).
While BcHA-2 was consumed completely after sufficient light
irradiation, histamine was released in a maximum yield of 40%,
probably owing to a photoinduced side reaction, though this
has not yet been clarified. We also confirmed that the agonist
activity of BcHA-2 for histamine H1 receptor was greatly
suppressed compared to that of histamine by measuring the
fluorescence change of Rhod 2-AM, a Ca indicator, which was
preloaded into HeLa cells (Figure 2e). Further, BcHA-2
showed no antagonist activity toward H1 receptor at the
maximum experimental concentration of 5 μM (Supplementary
Figure S5).
Next, we tested whether single-photon uncaging of BcHA-2
can induce an increase of intracellular Ca2+ in HeLa cells. Light
irradiation via a confocal laser scanning microscope equipped
with argon lasers successfully evoked a Ca2+ response (Figure
3a, top row). Cells showed no response in the control
and ϕ is as high as 100. Indeed, as described later, we
u
demonstrated that these values are sufficient for practical usage
to uncage a physiological concentration of bioactive molecules
with a conventional laser setup for fluorescence microscopy.
Further, uncaging with even longer-wavelength light was
accomplished based on another π-extended BODIPY derivative,
dihydronaphtho BODIPY (DHNB), which shows absorption at
over 600 nm (8 and 9, Supplementary Figure S3 and
Supplementary Table S2), by introducing an appropriate
aryloxy group on the boron atom to quench the fluorescence of
DHNB. To our knowledge, this is the first report of a caging
group activated by single-photon excitation at a wavelength
over 600 nm.
2+
19
Next, in order to investigate biocompatibility, we aimed at
developing biofunctional caged compounds based on our
BODIPY derivatives. Due to the lower ϕ of DHNB derivatives
u
compared to that of 1,3,5,7-tetramethyl-BODIPY derivatives,
we decided to design caged compounds based on the latter. As
a target molecule, we chose histamine, which is a biogenic
amine that plays an important role in various physiological and
pathophysiological processes, including sleep-wakefulness,
20,21
allergy, and inflammation.
In the molecular design of
caged histamines (BcHAs), the terminal amine group of
histamine was caged with BODIPY through a benzyloxycar-
bonyl linker, so that the phenolate group released upon light
irradiation further decomposes to produce free histamine
together with CO and quinone methide, which is immediately
2
22
hydrolyzed to generate 4-hydroxybenzyl alcohol (Figure 2a).
We first developed BcHA-1 (13) based on conventional
1,3,5,7-tetramethyl BODIPY (Table 1). Although it exhibited
Table 1. Spectroscopic and Photochemical Properties of
BcHA-1 and BcHA-2
a
a
a
λabs (nm)
λem (nm)
ϕfl
ϕu
3.0 × 10−
3.9 × 10−
4
a
b
BcHA-1 (13)
BcHA-2 (18)
498
510
511
524
0.13
0.11
4
a
b
Measured in 0.1 M sodium phosphate buffer pH 7.4. Measured in
.2 M sodium phosphate buffer pH 7.4.
0
good photocleavability in vitro, it failed to stimulate cultured
HeLa cells (Supplementary Figure S4). There are two possible
reasons for this failure: the low release of histamine or
mismatched localization of the caged compound and target
receptor. Since even prolonged photoirradiation did not induce
a cellular response, we considered that the latter was more
likely. Therefore, we examined the cellular localization of
BcHA-1 by means of fluorescence microscopy, utilizing the
suppressed BODIPY-derived fluorescence. As a result, we
found that BcHA-1 tends to localize in membranous structures
in cells, presumably due to its hydrophobic nature (Figure 2b,
left). Considering that the histamine H1 receptor exists on the
cell surface, we considered that we needed a more hydrophilic
derivative, which should be localized in the extracellular space.
Therefore, we newly designed BcHA-2 (18) (Table 1), based
on more hydrophilic 2,6-dicarboxy-1,3,5,7-tetramethyl BODI-
Figure 3. Application of BcHA-2 to HeLa cells. (a) Uncaging
experiments of BcHA-2. BcHA-2 and pyrilamine were used at 5 and 1
μM, respectively. Scale bar, 100 μm. (b) Statistical analysis of data in
panel a. Mean ± SEM, *** p < 0.001, Games-Howell method, n = 30
cells from two independent experiments. (c) Representative traces of
intracellular Ca2 dynamics in Supplementary Figure S7b.
+
conditions, and the Ca2+ response was inhibited in the presence
of pyrilamine, an H1-antagonist (Figure 3a,b and Supple-
25
mentary Figure S6), indicating that HeLa cells respond
specifically to histamine produced by uncaging of BcHA-2. We
also attempted uncaging of BcHA-2 to specifically stimulate
cells in a small or broad region of interest. In the presence of
BcHA-2, light irradiation evoked a cellular response in a small
square region located in the center of the field of view of the
confocal laser scanning microscope (Supplementary Figure S7a
and Movie S1). The response subsequently extended outside
the irradiated area, presumably due to the diffusion of released
23
PY (dicarboxy BODIPY), which was indeed localized in the
extracellular medium (Figure 2b, right). We also confirmed that
BcHA-2 is photodecomposed to produce histamine in a light-
dependent manner by HPLC analysis after fluorescent
C
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ACS Chemical Biology
Letters
histamine. Then, we also tried to uncage BcHA-2 over a
broader area (a quarter of the field of view). As a result, we
succeeded in inducing a cellular response in a broad area,
demonstrating that BcHA-2 would serve as a tool for
modulating cellular functions even over broad areas (Supple-
mentary Figure S7b and Movie S2). Further, the fluorescence
traces of Rhod 2-AM exhibited characteristic intracellular Ca2+
increase and oscillation in several cells (Figure 3c), in
accordance with the previous report that histamine stimulation
JSPS Core-to-Core Program, A. Advanced Research Networks,
as well as The Daiichi-Sankyo Foundation of Life Science and
The Naito Foundation Natural Science Scholarship (grant to
Y.U.), The Mochida Memorial Foundation for Medical and
Pharmaceutical Research (grant to M.K.), The Tokyo Society
of Medical Sciences (grant to M.K.), and a JSPS stipend to
N.U.
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