Fluorescence photoswitching based on a photochromic pKa change in an aqueous solution

Article abstract of DOI:10.1039/c2cc35889a

Reversible fluorescence photoswitching of RSA-AZO dyad 1 was clearly demonstrated in an acidic aqueous solution. The fluorescence photoswitching mechanism is based on the reversible ring opening/closing reactions of the RSA unit induced by a photochromic

Full text of DOI:10.1039/c2cc35889a

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Fluorescence photoswitching based on a photochromic pKa change in an
aqueous solutionw
Tuyoshi Fukaminato,* Emi Tateyama and Nobuyuki Tamaoki*
Received 15th August 2012, Accepted 19th September 2012
DOI: 10.1039/c2cc35889a
Reversible fluorescence photoswitching of RSA-AZO dyad 1 was
clearly demonstrated in an acidic aqueous solution. The fluorescence
photoswitching mechanism is based on the reversible ring opening/
closing reactions of the RSA unit induced by a photochromic pKa
change along with the photoisomerization of the AZO unit.
depend on the pH or the polarity of the surrounding environment,
that is, the open- and the closed-states of RSA exist in equilibrium
depending on the local pH or polarity.6b,d With decreasing pH of
the solvent, the ratio of the fluorescent open-state increases, and
vice versa. In this study, we focused on this pH dependence of ring-
opening/closing reactions of RSA. It is expected that ring opening/
closing reactions of RSA can be indirectly induced by connecting
a photochromic unit, which enables reversible control of the
local conditions such as polarity or acidity along with its photo-
isomerization. This approach may allow us to keep the photo-
induced fluorescent open-state of the RSA unit stable, which makes
it possible not only to increase the detectable photon number in
super-resolution microscopic applications but also to utilize the
derivative for other applications such as fluorescent photoswitch-
able bio-markers. Here we report on the molecular design and the
synthesis of a water-soluble fluorescent photoswitchable RSA
derivative based on photochromic pKa switching.
Fluorescent photochromic small molecules and proteins have
attracted increasing interest owing to their potential applications
in optical data storage and molecular switches.1,2 In addition,
biological applications such as bio-markers or super-resolution
fluorescence microscopes are becoming one of the trends in this
field.3 For the latter applications, the fluorescence photoswitching
property in aqueous conditions is indispensable. Although some
fluorescence photoswitching in aqueous conditions including intra-
cellular environments has been demonstrated by utilizing assembled
systems such as nanoparticles or vesicles containing photochromic
molecules,4 (non-assembled) water-soluble fluorescent photo-
chromic molecules are still rare5,6 and it is desirable to improve
their fluorescence properties and functionalities.
Rhodamine spiroamide (RSA) fluorophores6 are some of
the prominent functional groups of water-soluble fluorescent
photoswitchable molecules for super-resolution microscopic
applications because of their high fluorescence quantum yields
and robust photochemical stability in aqueous conditions. The
derivatives undergo photoinduced ring-opening and thermally
induced ring-closing reactions. The closed-state is transparent
in the visible region and is practically non-fluorescent. Upon
irradiation with UV light, the open-state, which absorbs in the
green region and emits at around 580 nm, is produced.
Although the open-state is highly fluorescent and enables the
detection of a fluorescence signal even at the single-molecule
level, the fluorescent state cannot be maintained for a long time due
to the rapid thermal back reaction (in a few milliseconds in polar
solvents).6a The fast thermal recovery restricts its photoswitching
application to only super-resolution fluorescence microscopy.
On the other hand, the ring opening/closing processes of RSA
In order to prepare our desired fluorescent photoswitchable
molecules, it was necessary to avoid spectral overlap between
the absorption bands of both isomers of a photochromic unit
and the fluorescence band of the RSA unit to deter fluorescence
quenching due to an intramolecular energy transfer. Azobenzene
(AZO) derivatives are one of the most popular photochromic
compounds and have been extensively used for the photocontrol
of biomolecular structures and biological functions.7 AZOs show
reversible cis-trans photoisomerization upon alternate irradiation
with appropriate UV and visible light even in aqueous conditions
and both isomers have an absorption band within relative short-
wavelength regions (l o 500 nm). In addition, it is well known
that AZOs reversibly change molecular polarity or basicity along
with the photoisomerization.8,9 Taking these advantages into
account, we selected AZOs as a suitable photoswitching unit in
our molecular design.
It has been challenging to develop a highly fluorescent
molecule to connect with the AZO unit, since the pico-second
photoisomerization dynamics of AZO can easily quench the
excited state of a fluorophore and therefore AZOs are commonly
utilized as efficient fluorescence quenchers.10 Eshow et al.11
recently reported that highly fluorescent properties can be
achieved for AZO-connecting fluorescent molecules by carefully
selecting a fluorescence unit, whose absorption and fluorescence
bands are far from the absorption band of the AZO unit.
According to their strategies, we designed and synthesized a
RSA-AZO dyad 1 (Scheme 1), in which an AZO derivative is
Research Institute for Electronic Science, Hokkaido University,
N20, W10, Kita-ku, Sapporo 001-0020, Japan.
E-mail: tuyoshi@es.hokudai.ac.jp, tamaoki@es.hokudai.ac.jp;
Fax: +81-11-706-9350; Tel: +81-11-706-9350
w Electronic supplementary information (ESI) available: Detailed
synthetic procedure of dyad 1, HPLC analyses of 1 upon irradiation
with 365 nm light and absorption spectra of dyad 1 in other pH
conditions. See DOI: 10.1039/c2cc35889a
c
10874 Chem. Commun., 2012, 48, 10874–10876
This journal is The Royal Society of Chemistry 2012

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Scheme 1 Photoisomerization of RSA-AZO dyad 1.
connected to the Rhodamine B fluorophore through an amino-
acid lysine spacer. In this molecular design, the lysine spacer
plays an important role in the separation of electronic states of
both RSA and AZO units and the high water-solubility. Dyad 1
was prepared according to Scheme S1 in the ESI.w Synthetic and
structural characterization details are provided in the ESI.w
The left sides of Fig. 1a and b show absorption and
fluorescence spectral changes of 1 upon alternate irradiation
with UV (365 nm) and visible (490 nm) light in an aqueous
solution (pH 3.8 Æ 0.1). The weak absorption band at 563 nm
and the pronounced absorption bands around 300–400 nm,
which correspond to the absorption band of the open-state of
the RSA unit and the p–p* transition band of the AZO unit,
are observed. The absorption spectrum suggests that the
equilibrium between the open- and the closed-state of the
RSA unit almost leans towards the closed-one before photo-
irradiation. Upon irradiation with 365 nm light to the 1Ct
(Closed-trans) solution, the absorption band at around
300–380 nm is gradually decreased and the characteristic
absorption band at 563 nm is dramatically increased and the
solution color changes from nearly colorless to pink, as shown
in the right side of Fig. 1a. These spectral and color changes
suggest that the ring-opening reaction of the RSA unit takes
place and the equilibrium between the open- and the closed-
state of the RSA unit leans towards the open one. From
HPLC analyses, the conversion ratio from the trans- to the
cis-isomer of the AZO unit of dyad 1 upon 365 nm light
irradiation at the photostationary state (PSS) was estimated to
be >95% (see Fig. S1 in ESIw). Upon irradiation with 490 nm
light, the absorption spectrum of the PSS solution nearly recovers
to the initial one (Fig. 1a). The rate constant of thermal back-
relaxation (k) for 1Oc (PSS under irradiation with 365 nm light)
was determined to be 2.2 Æ 0.1 Â 10À4 minÀ1 at 25 1C by plotting
the absorbance at 563 nm (Fig. 1c) (a detailed explanation is
described in ESIw). The small k value indicates that the photo-
induced open-state of RSA-AZO dyad 1 can be maintained for a
reasonably long time in comparison with the photoinduced open-
state of conventional RSA derivatives.6a
Fig. 1 (Left): (a) absorption and (b) fluorescence spectral changes in
an aqueous solution ([C] = 8.8 Â 10À6 M, pH 3.8 Æ 0.1) upon
alternate irradiation with UV (365 nm) and visible (490 nm) light;
before photoirradiation (1Ct, black-line), PSS under 365 nm light
irradiation (red-line), PSS under 490 nm light irradiation (green-line).
(Right): photographs of the color and fluorescence changes along with
photoisomerizations. (c) The change of absorbance at 563 nm of a PSS
solution (pH 3.8 Æ 0.1, 25 1C) under dark conditions; the dashed-line
corresponds to the absorption spectrum before photoirradiation and
each line is monitored at 1 hour intervals. Inset: the thermal fading of
1Oc in an aqueous solution (pH 3.8 Æ 0.1) at 25 1C under dark
conditions. (d) Photoswitching cycles of 1 in an aqueous solution
(pH 3.8 Æ 0.1) upon alternate irradiation with UV (365 nm) and visible
(490 nm) light. The changes of absorbance at 563 nm (open-circle) and
fluorescence intensity at 580 nm (solid-circle) were monitored in an
aqueous solution (pH 3.8 Æ 0.1).
under 365 nm light irradiation was estimated to be 0.37 Æ 0.05
in an aqueous solution (pH 3.8) (see ‘‘General’’ in ESIw),
which is almost similar to that of typical RSA derivatives.6d
Therefore, this result indicates that the quenching effect of the
AZO unit on the fluorescence of the RSA unit was negligible.
These reversible absorption and fluorescence spectral
changes in dyad 1 may be attributed to the change of the
equilibrium constant between the open- and the closed-states
of the RSA unit along with the photoisomerization of the
AZO unit. In order to confirm the origin of the photoinduced
ring-opening/closing reaction of the RSA unit, pH depen-
dences of absorption spectra of RSA were measured for both
cis-AZO and trans-AZO isomers. In an aqueous solution of
pH 5.0, the absorption band at 563 nm was not clearly
observed (see Fig. S2a in ESIw), while a characteristic strong
absorption band at 563 nm was observed in a solution of
pH 3.0 (see Fig. S2b in ESIw). In both conditions, however, the
As shown in the left side of Fig. 1b, reversible fluorescence
intensity changes were also observed along with the photo-
isomerization. Before photoirradiation, very weak orange
fluorescence centred at 580 nm was observed under excitation
with 520 nm light. When the solution reached the PSS under
irradiation with 365 nm light, the fluorescence intensity
increased to around 15 times the initial intensity and a strong
orange emission was readily visible (right side of Fig. 1b). The
fluorescence intensity decreased again upon visible (490 nm)
light irradiation. These absorption and fluorescence spectral
changes were repeatable for at least more than five-cycles
(Fig. 1d). Fluorescence quantum yield (Ff) for the PSS state
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10874–10876 10875

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Fig. 2 Titration curves of the trans-AZO isomer (black-square) and
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In conclusion, a water-soluble fluorescent photoswitchable
molecule, RSA-AZO dyad 1, was designed and synthesized, and
the reversible fluorescence photoswitching was successfully
demonstrated in aqueous solution. The mechanism in the
fluorescence photoswitching of dyad 1 is based on the reversible
change of molecular pKa induced by the photoisomerization of
the AZO unit. It is clearly confirmed that the fluorescence
switching mechanism based on a photochromic pKa change is
useful for the design of water-soluble fluorescent photoswitch-
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useful pH range in this system are under progress.
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This work was supported by JSPS KAKENHI; Grant-in-
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10876 Chem. Commun., 2012, 48, 10874–10876
This journal is The Royal Society of Chemistry 2012