A unique family of rigid analogues of the GFP chromophore with tunable two-photon action cross-sections for biological imaging

Article abstract of DOI:10.1002/anie.201303179

Conformationally restricted analogues of the GFP chromophore have been synthesized. The spectroscopic properties of GCTPOC, a unique family with tunable two-photon action cross-sections, were investigated. GCTPOC could be used as a robust two-photon platform for development of a two-photon fluorescent thiol sensor, which could stain endogenous thiols both in vitro and in vivo.

Full text of DOI:10.1002/anie.201303179

Angewandte
Chemie
DOI: 10.1002/anie.201303179
Fluorescent Probes
A Unique Family of Rigid Analogues of the GFP Chromophore with
Tunable Two-Photon Action Cross-Sections for Biological Imaging**
Lin Yuan, Weiying Lin,* Hua Chen, Sasa Zhu, and Longwei He
The green fluorescent protein (GFP) and related genetically
encoded fluorescent proteins have made a great impact on the
fundamental research of life science. As a fluorescent tag,
GFP is invaluable for studies of protein localization, dynam-
ics, and function. Furthermore, GFP has been engineered as
fluorescent sensors for a wide variety of targets including pH,
metal ions, and small biomolecules.[1–7] The chromophore in
GFP is p-hydroxybenzylideneimidazolone (p-HOBDI; Sche-
me 1A). However, p-HOBDI alone does not exhibit signifi-
employed by encapsulation,[10,13] reversible locking with
metal ions (for example p-HOPyDI:Zn)[8,14] and hydrogen
bonds,[15] or irreversible locking with a BF2 group (for
example p-HOBDI-BF2 and compound A).[9,14]
Two-photon dyes have attracted intense attention in the
recent years in light of their useful applications in diverse
fields such as two-photon fluorescence microscopy, localized
release of bioactive species, photodynamic therapy, optical
power limiting, three-dimensional (3D) optical data storage,
and microfabrication.[16–25] In particular, two-photon excita-
tion fluorescence (TPEF) microscopy, where the fluorescence
is triggered by spatially confined two-photon excitation, bears
several advantages over the conventional one-photon fluo-
rescence microscopy, including three-dimensional imaging of
living tissues, reduced photodamage to biosamples, increased
tissue penetration, and negligible background fluorescence.
To obtain two-photon fluorescence images with sufficient
brightness, two-photon dyes with efficient two-photon exci-
tation action cross-section are required (s’ is the two-photon
absorption cross-section times the fluorescence quantum
yield: s’ = s ꢀ F). Some p-HOBDI rigid analogues with
good one-photon properties have been reported;[8–10,13–15]
however, their two-photon properties are unknown. To
employ GFP chromophore analogues as two-photon fluores-
cent platforms for the development of two-photon fluorescent
sensors, it is necessary to develop GFP chromophore ana-
logues with tunable two-photon action cross-sections.
Herein, we describe the development of a unique family
of rigid analogues of the GFP chromophore, which we call
GCTPOC (Scheme 1B). Compared to the original GFP
chromophore and the reported rigid analogues of GFP
chromophore (Scheme 1A), GCTPOC compounds exhibit
several distinct features. First, from the structural point of
view, in GCTPOC, an oxygen-bridge is employed to lock the
chromophore system in a rigid conformation, which is similar
to the Z-isomer of GFP chromophore, to prevent the free
rotation of the aryl–alkene bond and the E/Z isomerization of
the double bond. Second, the two nitrogen atoms in the
imidazolinone ring of the GFP chromophore are replaced as
carbon atoms. Consequently, the amide function group in the
GFP chromophore is turned into a carbonyl group in
GCTPOC, which has better electron-withdrawing ability.
Thus, GCTPOC should be a more efficient push–pull system
than the GFP chromophore. This is desirable for dyes with
asymmetric structures to have good two-photon proper-
ties.[16,19] Third, from the optical point of view, the two-
photon properties of the above mentioned rigid analogues of
the GFP chromophore have not yet been reported. In
contrast, our GCTPOC display favorable two-photon proper-
ties. By combining these characteristics, we named the new
Scheme 1. A) Structures of the GFP chromophore and some represen-
tative rigid GFP chromophore analogues. B) Structures of GCTPOC
compounds. The novel two-photon rigid analogues of the GFP
chromophore feature an oxygen bridge and carbon substituents
compared to the classic GFP chromophore (p-HOBDI).
cant fluorescence owing to the free rotation of the aryl–alkene
bond and the E/Z isomerization of the double bond in
solution.[8–11] We performed time-dependent density func-
tional theory (TD-DFT) calculations on the E/Z isomers of p-
HOBDI, and the data indicate that it could show strong
fluorescence in the Z-isomer (a large oscillator strength: f =
0.9321) and relatively weak fluorescence in the E-isomer (f =
0.002; Supporting Information, Figure S1). These results are
in good agreement with the previous reports.[9,12] To improve
the fluorescence of p-HOBDI, it is necessary to put it in
a rigid environment, such as in the barrel structure of GFP.
Toward this end, several elegant approaches have been
[*] L. Yuan, Prof. W. Lin, H. Chen, S. Zhu, L. He
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering, Hunan University
Changsha, Hunan 410082 (China)
E-mail: weiyinglin@hnu.edu.cn
[**] Funding was partially provided by NSFC (20872032, 20972044,
21172063), NCET (08-0175), the Doctoral Fund of Chinese Ministry
of Education (20100161110008), and the Fundamental Research
Funds for the Central Universities, Hunan university.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201303179.
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compounds as GCTPOC, which refers to GFP chromophore
two-photon analogues with an oxygen bridge and carbon
substituents. Although the synthesis of GCTPOC analogues
has been reported,[26] to the best of our knowledge, their
optical properties were completely unknown. Herein, we
reveal the optical properties of GCTPOC for the first time.
Thus, from the optical point of view, GCTPOC is a unique
family of conformationally restricted analogues of the GFP
chromophore with tunable two-photon action cross-sections.
TD-DFT calculations were conducted to examine the
potential effect of the position of the hydroxy group on the
fluorescence properties. When the hydroxy group is at the 2-
position (para-position), the oscillator strength is maximal
(Supporting Information, Table S1), indicating that the hy-
droxy group at the 2-position may be optimal for the
fluorescence, consistent with the position of the hydroxy
group at the GFP chromophore (Scheme 1A). To investigate
the structure two-photon properties, we further designed
a series of GCTPOC derivatives (1a–2d, Scheme 1B). The
novel type of compounds have different electron-donating
groups at the para-position, which may have an impact on the
nature of the push–pull system. Thus, we envisioned that these
new compounds should have distinct optical properties.
GCTPOC derivatives, 1a–d and 2a–d, possess a five- or six-
membered carbon ring, respectively.
tion and emission peaks at 395/445 (extinction coefficient e >
20000 LmolÀ1 cmÀ1) and 513 nm, respectively, in PBS
(Figure 1). Interestingly, these properties resemble those of
the classic green fluorescence protein (labs = 395/475 nm,
lems = 509 nm, e = 21000 LmolÀ1 cmÀ1).[27] The large Stokes
shift of compound 1a, just like that of fluorescent proteins,
may be attributed to the excited-state proton transfer
(ESPT).[28] Furthermore, like GFP chromophore, the absorp-
tion profiles of the dye 1a is pH-dependent. With the
enhancement of pH from 3 to 11, the absorption band at
around 392 nm, ascribed to the phenolic form of 1a, under-
goes a red-shift to a peak at around 445 nm, attributed to the
phenolate form of 1a (Supporting Information, Figure S2A).
There is a well-defined isosbestic point at 403 nm in the
absorption spectra, indicating the equilibrium of the phenolic
and phenolate forms of 1a (Supporting Information, Fig-
ure S2B). Excitation of dye 1a at 445 nm affords one-photon
fluorescence spectrum with a maximal emission at 513 nm
(F = 0.29). The TPEF of compound 1a displays a maximal
emission peak at 512 nm (Figure 1C), and the shape of TPEF
spectrum well resembles that of the one-photon fluorescence
spectrum (Supporting Information, Figure S3). Significantly,
compound 1a in the phenolate form exhibits excellent two-
photon properties with a two-photon cross-section (s) above
800 GM and a two-photon excitation action cross-section
(brightness, s’) above 230 GM (Supporting Information,
Table S2). The two-photon cross-section (s) per molecular
weight (s/MW) of dye 1a is 3.96, indicating that the dye is
potentially useful for bioimaging applications.[19] Compound
2a, the six-membered ring analogue of dye 1a, shows similar
excellent two-photon properties (Supporting Information,
The dyes 1a–d and 2a–d were prepared by reaction of the
substituted salicylaldehyde with 2-cyclopenten-1-one/2-cyclo-
hexen-1-one by a Baylis–Hillman reaction[26] (Supporting
Information, Scheme S1). The structures of all the com-
1
pounds were characterized by H NMR and 13C NMR spec-
troscopy and HRMS.
The new dyes were readily soluble in PBS at micromolar
concentrations suitable for optical measurement and imaging
applications. The absorption, one-photon, and two-photon
fluorescence spectra of 1a–d and 2a–d in PBS are shown in
Figure 1 (for photophysical data, see the Supporting Infor-
mation, Table S2). Compound 1a displays maximal absorp-
Figures S3–S5) with a two-photon cross-section above
810 GM and a two-photon excitation action cross-section
above 270 GM (Supporting Information, Table S2). Notably,
these data are significantly larger than those of the fluorescent
proteins.[29] A brief comparison of some representative
conformationally locked GFP-chromophore analogues
reported in the literature[8–10,13–15] and in this work is given
in the Supporting Information, Table S3. However, the
comparison of the two-photon properties of dyes 1a, 2a
with those of the rigid GFP chromophore analogues such as p-
HOPyDI:Zn, p-HOBDI-BF2, and compound A is not possi-
ble, as surprisingly the two-photon properties of them were
not revealed in previous reports. Thus, our dyes 1a and 2a are
a unique type of GFP chromophore rigid analogue with
excellent two-photon properties.
Dyes 1d and 2d, which bear no electron-donating group
at the para-position, display weak fluorescence and negligible
two-photon excitation action cross-section (Figure 1; Sup-
porting Information, Table S2). In contrast, dyes 1c and 2c,
which contain a strong electron-donating group, diethylamino
at the para-position, exhibit strong fluorescence and large
two-photon excitation action cross-section, highlighting the
significance of the push–pull character for two-photon
properties.
We further examined the photophysical properties of
compound 1b, the methoxy analogue of dye 1a. The maximal
absorption peak of 1b is at 386 nm, which is blue-shifted when
compared to that of 1a. However, the fluorescence quantum
Figure 1. The absorption (A), one-photon fluorescence (B), and two-
~
*
photon fluorescence (C) spectra of compounds 1a ( ), 1b ( ), 1c
&
^
( ), and 1d ( ) (5 mm) in PBS containing 1% EtOH). D) Two-photon
~
*
excitation action cross-section of 1a ( ) and 1b ( ) in PBS containing
1% EtOH.
2
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yield of 1b is 0.008, significantly less than that (0.29) of 1a.
Notably, the two-photon excitation action cross-section (s’) of
1b is only less than 1 GM, which is drastically reduced when
compared to that (232 GM) of compound 1a (Figure 1D;
Supporting Information, Table S2). It is known that the
phenolate form of fluorescent proteins is fluorescent, whereas
the phenolic form of fluorescent proteins is essentially non-
fluorescent.[28,30] Thus, analogously, the extremely low two-
photon excitation action cross-section of 1b may be attributed
to the alkyloxy substituent of 1b, which precludes the
formation of the fluorescent phenolate form upon excitation
by the ESPT mechanism.[28,30]
ure 2A; Supporting Information, Figure S7). In good agree-
ment, a large (up to 30-fold) fluorescence increase in the two-
photon fluorescence profile is observed (Supporting Infor-
mation, Figure S8). The pseudo first-order rate constant of
There is a striking distinction in TPEF (more than 300-
fold) between the compounds 1a and 1b (Supporting
Information, Figure S5A). Thus, importantly, these data
suggest that the two-photon properties of the new dye 1a
are tunable by modifications on the hydroxy group. The
similar relationship between compounds 2a/2b is also noted
(Supporting Information, Table S2, Figure S5B).
Figure 2. A) The one-photon fluorescence spectra of the sensor 3
(5 mm) incubated with GSH (5 mm) for 0–60 min in PBS containing
1% EtOH. B) The fluorescent intensity of the sensor 3 (5 mm) at
516 nm in the presence of various biological relevant analytes (1 mm)
for 1 h in PBS containing 10% EtOH. 1) free, 2) Ala, 3) Arg, 4) Gly,
5) GSH, 6) Leu, 7) Phe, 8) Ser, 9) Tyr, 10) Val, 11) Glu, 12) Asn, 13) Lys,
14) H2O2, 15) glucose, 16) Ca2+, 17) Zn2+
.
Significantly, the above findings that there is a drastic
distinction between the two-photon properties of compounds
1a/1b and 2a/2b, which implies that the new family of
conformationally locked GFP chromophore two-photon
analogues represented by 1a and 2a may be exploited as
novel two-photon based platforms for the design of two-
photon fluorescent sensors for biological imaging applications
in living tissues by easy modifications on the hydroxy
group.[31] This is further encouraged by the observation that
cells stained with 2a displayed strong fluorescence and those
stained with 2b showed almost no fluorescence (Supporting
Information, Figure S6). To demonstrate the use of our two-
photon dyes as effective platforms, for proof-of-concept, we
further engineered compound 3 (Scheme 2) as a novel
sensor 3 for GSH, cysteine, and homocysteine (100 equiv)
were determined to be k’ = 0.05828, 0.06082, and
0.04421 minÀ1, respectively (Supporting Information, Fig-
ures S9–S11). It seems that the sensor reacted almost equally
well with GSH, cysteine, and homocysteine. The second-order
rate constant of sensor 3 for GSH was determined to be k =
0.04843 LmolÀ1 minÀ1 (Supporting Information, Figure S12).
Titration experiments indicated that the emission intensity of
sensor 3 at 516 nm is linearly proportional to the amount of
GSH (2–200 mm) and cysteine (2–250 mm; Supporting Infor-
mation, Figure S13) with a detection limit (S/N = 3) of 0.80
and 0.75 mm, respectively, in pH 7.4 PBS containing 1%
EtOH as a cosolvent, suggesting that the probe is potentially
useful for quantitative determination of thiol concentrations
over a large dynamic range.
The reaction product between compound 3 and thiols was
isolated and confirmed to be compound 2a by mass spec-
trometry (Supporting Information, Figure S14). Furthermore,
as shown in Figure 2B and the Supporting Information,
Figure S15, the sensor 3 is highly selective to typical thiols (for
example GSH, cysteine, and homocysteine) over other
biorelevant species, such as Ala, Arg, Leu, Phe, Ser, Tyr,
Scheme 2. The design and synthesis of two-photon fluorescence turn-
on thiol sensor 3: a) 4-Nitrophenyl chloroformate, then PhSH; b) RSH.
candidate of two-photon fluorescence turn-on sensor by
simple modifications on the hydroxy group of platform 2a
for fluorescence imaging of thiols in living tissues. Small-
molecular-weight biological thiols play an important role in
many biological processes. However, abnormal levels of thiols
are associated with various diseases including liver damage,
skin lesions, and slowed growth.[32] Thus, it is of great interest
to monitor biological thiols in living systems by fluorescent
sensors.[33]
The compound 3 is stable in the solid form, and it can be
stored for more than one year in a freezer without any
hydrolysis. As designed, the free sensor 3 is essentially
nonfluorescent in PBS (25 mm, pH 7.4, 1% EtOH), and its
fluorescence intensity did not display observable changes for
5 days (Supporting Information, Figure S7). In contrast, the
addition of a representative thiol, glutathione (GSH), elicits
a significant fluorescence enhancement at 516 nm (Fig-
Val, Glu, Asn, Lys, H2O2, glucose, Ca2+, Zn2+, Mg2+, Cd2+,
À
Fe2+, Cu2+, Co2+, NO, FÀ, ClÀ, BrÀ, IÀ, AcOÀ, N3À, NO2
,
NO3À, SCNÀ, SO3 SO4 CNÀ, and CO3 in PBS contain-
ing 10% EtOH, suggesting that the sensor is promising for
applications in biological systems.
2À,
2À,
2À
Encouraged by the above prominent features of the
sensor 3 and the advantages of two-photon fluorescence
microscopy, we decided to examine the feasibility of the
sensor to detect endogenous thiols in living tissues by two-
photon fluorescence microscopy. After incubation with 5.0 mm
sensor 3 for 1 hour, a fresh mouse liver slice was washed with
PBS. The dye-stained liver slice displays bright two-photon
fluorescence (Figure 3a; Supporting Information, Fig-
ure S16c), consistent with the two-photon fluorescence turn-
on response as observed in solution (Supporting Information,
Figure S8). Moreover, the two-photon fluorescence images at
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with an oxygen-bridge and carbon atoms. Significantly, the
two-photon action cross-sections of GCTPOC is tunable by
easy modifications on the hydroxy group of GCTPOC
compounds, such as 1a and 2a, indicating that 1a and 2a
could be exploited as novel platforms to design two-photon
fluorescent sensors. Based on the platform 2a, the novel two-
photon fluorescent thiol sensor 3 was constructed. We have
further demonstrated that the two-photon fluorescence turn-
on sensor 3 could stain endogenous thiols both in vitro and
in vivo. The design strategy will lead to the development of
many GFP chromophore rigid analogues, and the robust two-
photon fluorescent platforms GCTPOC will open new
avenues for the development of two-photon fluorescent
sensors as molecular tools for studies in living systems.
Figure 3. Two-photon fluorescence images (with a magnification at
20ꢀ) of a mouse liver slice that was a) not treated or b) pretreated
with NEM (5 mm) for 1 hour before being stained with 5 mm sensor 3
for 1 h. The two-photon fluorescence emission was collected at
between 470 and 570 nm upon excitation at 780 nm with a femto-
second pulse. Scale bar=20 mm. (For a color version, see the Support-
ing Information, Figure S22.)
Received: April 16, 2013
Revised: June 23, 2013
Published online: && &&, &&&&
the different depths (0–180 mm) show that the sensor can
image thiols distribution in each xy plane along the z direction
(Supporting Information, Figure S17). The observation that
the sensor can endogenously image thiols in living tissues at
a depth of 180 mm is in good agreement with the large two-
photon excitation action cross-section of the dye 2a (the
reaction product of the sensor with thiols). Control experi-
ments indicated that the compound 2a-stained liver slice
(positive control) displayed strong fluorescence (Supporting
Information, Figure S16a) and compound 2b-stained liver
slice (negative control) exhibited almost no fluorescence
(Supporting Information, Figure S16b). Furthermore, in
another control experiment, a fresh mouse liver slice was
pre-treated with N-ethylmaleimide (NEM, a thiol-reactive
agent), and further incubated with the sensor 3, and the two-
photon fluorescence decreased significantly (Figure 3b; Sup-
porting Information, Figure S16d), suggesting the selective
reaction of the sensor with thiols.
After we had demonstrated that the sensor could monitor
endogenous thiols in the isolated tissues (in vitro), we then
proceeded to investigate whether our sensor could stain
endogenous thiols in intact tissues in living mice (in vivo).
200 nmol sensor 3 was injected into the living mice via a tail
vein, and the mice were anesthetized and sacrificed by
cervical dislocation at 1.5 h post-injection. The organs such as
liver, lung, heart, and kidney were then quickly removed and
washed with cold PBS buffer and imaged with a one-photon
in vivo imaging system. The results indicate that the liver and
kidneys have stronger fluorescence than other organs such as
lung and heart (Supporting Information, Figure S18). The
tissue slices (thickness 400 mm) of the liver and kidney were
then prepared and subjected to two-photon fluorescence
microscopic analysis. The sensor could stain endogenous
thiols in the kidney and liver sections with two-photon
fluorescence at a depth up to 190 mm (Supporting Informa-
tion, Figures S19–S22). Furthermore, in general, the two-
photon fluorescence in the kidney slices is brighter than that
in the liver slices (Supporting Information, Figure S19–20),
which is consistent with the one-photon in vivo fluorescence
images (Supporting Information, Figure S18).
Keywords: fluorescence imaging · fluorescent dyes ·
.
fluorescent probes · GFP chromophore analogues ·
two-photon dyes
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Angew. Chem. Int. Ed. 2013, 52, 1 – 6
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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.
Angewandte
Communications
Communications
Fluorescent Probes
Conformationally restricted analogues of
the GFP chromophore have been synthe-
sized. The spectroscopic properties of
GCTPOC, a unique family with tunable
two-photon action cross-sections, were
investigated. GCTPOC could be used as
a robust two-photon platform for devel-
opment of a two-photon fluorescent thiol
sensor, which could stain endogenous
thiols both in vitro and in vivo.
L. Yuan, W. Lin,* H. Chen, S. Zhu,
L. He
&&&& — &&&&
A Unique Family of Rigid Analogues of the
GFP Chromophore with Tunable Two-
Photon Action Cross-Sections for
Biological Imaging
6
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1 – 6
These are not the final page numbers!