A synthesized GFP analogue emits via a hydrogen-bonding system

Article abstract of DOI:10.1039/b927461h

Two GFP analogues o-HBDI and o-HBMO have been designed and synthesized. The former shows no fluorescence while the latter shows strong fluorescence due to the formation of an intramolecular hydrogen-bond. At low temperatures, both molecules have strong fl

Full text of DOI:10.1039/b927461h

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A synthesized GFP analogue emits via a hydrogen-bonding systemw
Juntao Kang, Guiyan Zhao, Jingwei Xu* and Wei Yang*
Received (in Cambridge, UK) 5th January 2010, Accepted 11th February 2010
First published as an Advance Article on the web 5th March 2010
DOI: 10.1039/b927461h
Two GFP analogues o-HBDI and o-HBMO have been designed
and synthesized. The former shows no fluorescence while the
latter shows strong fluorescence due to the formation of an
intramolecular hydrogen-bond. At low temperatures, both
molecules have strong fluorescence.
The synthesis of o-HBMO and o-HBDI was started from
2-hydroxybenzaldehyde (Scheme 1),3 which reacted with acetic
anhydride in dry diethyl ether to form 2-formylphenyl acetate.
Then a mixture of 2-formylphenyl acetate, 2-acetamidoacetic
acid and sodium acetate in acetic anhydride was stirred at
80 1C overnight to produce o-HBMO. o-HBDI was obtained
by a reflux of o-HBMO suspended in ethanol and aqueous
methylamine in the presence of K2CO3 at 80 1C for 5 h.3 The
product of each step was purified and then confirmed using
mass spectrometry and NMR (see ESIw).
o-HBMO analogues are notoriously unstable and tend to
rearrange to coumarin-like isomers.14 To reveal the structure
of o-HBMO under our experimental conditions, IR and NMR
investigations were carried out. The results indicated that
o-HBMO adopted the configuration as shown in Scheme 1
in water (see ESIw) instead of a coumarin-like structure.
Such a configuration has also been reported in dozens of
reactions.14
Green fluorescent protein (GFP) has been widely applied
on living cell labelling.1,2 Studies on the synthesized GFP
analogues are valuable not only in revealing the mechanism
of the protein fluorescence but also in developing fluorescent
probes.3–7 Despite great efforts devoted in the past decades,
the synthesis of a GFP-like probe with, for example, cation-
dependent fluorescence, has made less progress. In this study,
two GFP analogues, 4-(2-hydroxybenzylidene)-1,2-dimethyl-
1H-imidazol-5(4H)-one (o-HBDI) and 4-(2-hydroxybenzylidene)-
2-methyloxazol-5(4H)-one (o-HBMO), the latter of which
emits via a seven-membered-ring hydrogen-bonding system
in a pH-dependent manner, were synthesized.
o-HBMO showed a strong fluorescence emission at 390 nm
(Fig. 1) in aqueous solution. In DMSO, ethanol or
chloroform, the emission blue shifts to 389, 384 or 382 nm,
respectively. The maximum intensity in chloroform is B30%
greater than that in the other solutions. Further studies
confirmed that the fluorescence of o-HBMO is directly related
to the hydrogen-bond formation, which occurs simultaneously
with the phenol group protonation process, as expected.
First, the fluorescence of o-HBMO is pH-dependent (Fig. 1),
suggesting that the hydroxyl proton is directly involves in the
excitation-emission process. The quantum yield (f) of
o-HBMO was measured to be 0.09 at acidic pH by using
2-aminopyridine as standard,15 almost two orders higher than
that for the isolated GFP fluorophore. It dramatically
decreased at high pH values to o0.001.
The GFP chromophore is formed by the cyclization of a
Ser-Tyr-Gly tripeptide in a deeply buried environment.8 The
p-conjugated system of the fluorescent chromophore consists
of a phenol group and an imidazole ring that are linked with
an aryl–alkene bond (Scheme 1).9,10 Isolated chromophores as
well as most of the synthesized analogues possess only very
weak fluorescence, e.g. the quantum yields for p- and m-HBDI
are smaller than 0.001,11 which prevent their use as probes,
due to the free rotation about the aryl–alkene bond.12,13
In GFPs, this free rotation is inhibited by the tightly
packed protein structure. For isolated molecules, chemical
modification such as by complexation of a BF2 entity has
been used to fix the configuration of the molecules.5
Nevertheless, the boron-modified molecule is not an ideal
fluorescent probe due to its lability in solution.
Second, the hydroxyl proton was observed for the
fluorescent o-HBMO in aqueous solution by NMR (Fig. 2).
Correspondingly, the pKa of o-HBMO is 10.6, significantly
higher than that for o-HBDI (9.1) or phenol group (B10),
suggesting the proton is well protected from solvent exchange.
Third, the pKa obtained from the fluorescence study
We hypothesized that the free rotation about the aryl–
alkene bond could be inhibited by single hydrogen-bond,
allowing the molecule to function as a pH probe. o-HBDI
consists of a phenol group and an imidazole ring, similarly to
GFP, with the only modification of moving the hydroxyl
group from the p- to o-position. One nitrogen atom in the
imidazole ring of o-HBDI was replaced by an oxygen atom
in o-HBMO. The structure characteristics of these two
compounds make them suitable for testing this hypothesis.
(10.7
Æ
0.1, Fig. 1, inset) and the absorption study
The State Key laboratory of Electroanalytic Chemistry, Changchun
Institute of Applied Chemistry, Changchun, 130022, Jilin, China.
E-mail: yangwei@ciac.jl.cn, jwxu@ciac.jl.cn; Fax: 86-431-8526-2349;
Tel: 86-431-8526-2354
w Electronic supplementary information (ESI) available: Detailed
synthesis procedures, mass spectra, NMR, IR, and UV-vis results.
See DOI: 10.1039/b927461h
Scheme 1 Synthesis of o-HBMO and o-HBDI. The structure of the
GFP chromophore (p-HBDI) is also shown at the bottom.
ꢀc
This journal is The Royal Society of Chemistry 2010
2868 | Chem. Commun., 2010, 46, 2868–2870

View Article Online
make o-HBMO, to our knowledge, the first synthesized GFP
analogue that can function as a fluorescent probe for protons
and provides a step forward for fluorescent probe develop-
ment. On the contrary, o-HBDI has a Hill constant of 0.7 from
the UV-vis study, suggesting only a process of protonation.
o-HBDI has a similar p-conjugated system as natural GFP,
suggested by the UV-vis spectra. The absorption maximum of
o-HBDI is at 375 nm at pH 4 and 444 nm at pH 13, while the
enzyme-digested GFP has absorptions at 380 and 445 nm at
acidic and basic pH, respectively.12 However, the fluorescence
intensity of o-HBDI is weak (f o 0.001) as for other GFP
analogues. In solvents including ethanol, DMSO and chloro-
form, the fluorescence was not significantly improved at room
temperature.
To further show the fluorescent differences between o-HBDI
and o-HBMO, we carried out low-temperature studies. The
molecule temperatures were lowered to below À100 1C to
reduce the vibration and rotation of molecules. At such low
temperatures, the hydroxyl proton no longer determined the
fluorescence intensity. Indeed, strong fluorescence signals were
observed for both o-HBDI and o-HBMO both in the presence
and absence of NaOH.
Fig. 1 The emission spectra of o-HBMO (black) and o-HBDI
(red/gray) with excitation at 320 nm. The spectra of o-HBDI at
different pH almost overlap and that at pH 2.0 is shown here. The
pH for o-HBMO (top to bottom) are 9.0, 10.2, 10.6, 10.8, 11.0, 11.2,
11.6, 12.0 and 13.0, respectively. (Inset) The fluorescent intensities at
390 nm of o-HBMO as a function of pH are shown.
Different from GFP proteins that have the same emission
wavelength at neutral and ionic states, o-HBMO and o-HBDI
emit at different wavelengths with or without the hydroxyl
proton. At temperatures below À100 1C, the quantum yield of
o-HBMO was enhanced more than 2-fold to 0.24 (Fig. 3) while
the addition of solid NaOH shifted the emission wavelength
from 384 to 450 nm with an enhanced intensity (f = 0.51).
For o-HBDI, fluorescent signals were observed with
emission maxima at around 440 and 570 nm in the absence
and presence of NaOH at below À100 1C (Fig. 3). Corres-
ponding quantum yields were estimated to be 0.11 and 0.14,
Fig. 2 1H NMR spectra of o-HBMO in H2O at pH 4 (a), in D2O at
pH 4 (b), and in H2O at pH 13 (c), and o-HBDI in H2O at pH 3 (d).
The resonance at 8.28 ppm in H2O (a, arrow) is from the hydroxyl
group, which was missing in D2O at pH 4 (b) and not observed at pH
13 (c). This proton was also not observed for o-HBDI (d). The
numbers at the top of signals represent the relative area (the most left
peak in (a)–(c) and the singlet peak at 7.35 ppm in (d) were assumed to
be 1 for each spectrum). The peaks with relative areas o0.4 in (c) are
unlabelled. A single set of five protons other than the hydroxyl in this
region were observed in (a), (b) and (d) as expected. In (c), more than
five peaks with irregular area ratio were observed, suggesting at least
double sets of configurations for o-HBMO, likely the E-and Z-forms
of o-HBMO since generally the molecule has lower energy when the
two rings are in the same plane.
Fig. 3 o-HBMO and o-HBDI in ethanol (melting point À114 1C)
were frozen by liquid nitrogen. Then the fluorescence spectra were
collected when the ethanol ice was just melted and the temperature was
kept below À100 1C before the scan was completed (in 1 min). o-HBDI
showed significantly enhanced signals with (.) and without (K) the
addition of NaOH compared to that at room temperature (Fig. 1). An
emission was retained at 381 nm and an additional emission at 364 nm
were observed for o-HBMO without NaOH (m). The emission shifts to
450 nm for o-HBMO with NaOH (’). A fluorescence spectrum of
o-HBMO at room temperature collected at the same time is also
shown (J).
(10.6 Æ 0.1, ESIw) is almost the same, suggesting that the
fluorescence quenching accompanies the deprotonation
process. In addition, a Hill coefficient constant of about 2
(1.7 Æ 0.3 and 2.4 Æ 0.4, respectively, obtained from two
studies) implies that two processes—protonation of the
hydroxyl group and formation of the hydrogen-bond—
cooperatively take place.16 The pH-dependent properties
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 2868–2870 | 2869

View Article Online
more than two orders higher than those at room temperature.
However, we have not observed emissions at 495 and 600 nm
for o-HBDI as reported in ref. 17 under our examined
conditions (it is noteworthy that the emission in nonpolar
solvents such as C6H12 usually blue-shifts compared to that in
polar solutions such as water). The results clearly showed that
it is the thermal motions that lead to the non-radiative
quenching of o-HBDI at room temperature. This further
supports the existence of an oxazolone-like structure of
o-HBMO because, in the coumarin-like isomer, the labile
proton is not located at a key position that would influence
the thermal motions of the rings. In such a case, an emission
wavelength shift, as observed at À100 1C for o-HBMO,
instead of fluorescence quenching is more reasonable.
transitions among multiple configurations (less likely). A high
E–Z transition energy barrier has been reported in a GFP
analogue (compound 4c in ref. 5).
On the contrary, o-HBMO provided only one set of signals
at low pH and two sets at high pH (Fig. 2). The two sets of
NMR signals are possibly from (1) E- and Z-forms of
o-HBMO or (2) o-HBMO and its coumarin-like isomer. Since
a coumarin-like structure lacks the aryl-alkene bond, its
fluorescence is less likely to be so sensitive to temperatures.
Therefore, the loss of fluorescence at room temperature
implies that the two sets of NMR signals are from E- and
Z-forms of the molecule.
In conclusion, two GFP chromophore analogues o-HBDI
and o-HBMO have been designed and synthesized. A single
atom substitution in the imidazole ring converts non-
fluorescent o-HBDI (f o 0.001) to fluorescent o-HBMO
(f = 0.09) at room temperature. The former lacks a hydrogen-
bond (pKa = 9.1) while the latter emits in a pH-dependent
manner (pKa = 10.7). At À100 1C, both o-HBDI (f = 0.11
and 0.14) and o-HBMO (f = 0.24 and 0.51) possess strong
fluorescence with and without the hydroxyl proton. Although
further investigations are necessary on the detailed mechanism
leading to the difference and the biological implications of the
molecules, the results provide clues to design probes with less
non-radiation quenching.
The studies in different solvents excluded viscosity as the
determining factor for the fluorescence behaviours of the two
molecules. The absence of the intramolecular hydrogen-bond
or only a very weak hydrogen-bond, at least in aqueous
solutions, plays the key role for the quenching of o-HBDI at
room temperature. Correspondingly, the pKa of o-HBDI is 9.1
from UV-vis absorption study, lower than the pKa of about 10
for phenol. At the same time, the hydrogen from the hydroxyl
group could not been detected by NMR in aqueous solution
and can only be detected in organic solvents (see ESIw). These
results suggest that the hydroxyl proton in o-HBDI undergoes
fast exchange with solvent protons.
We are grateful for the financial support of this work by the
NSFC grant 30870491 to W. Y.
Such a weak hydrogen-bond in o-HBDI is unexpected as an
intramolecular hydrogen-bond has been observed in o-HBDI
crystals.17 In addition, compared to o-HBMO, the proton
donor and acceptor for the hydrogen-bond as well as the
allowed configurations are all maintained in o-HBDI. A
TD-DFT calculation has suggested that excited-state intra-
molecular proton transfer via the hydrogen-bond is thermally
favourable with an energy difference of B7.8 kcal molÀ1 for
o-HBDI.17 Clearly, the model did not fully present the real
state of o-HBDI in solution.
The crystal structure of o-HBDI17 and the NOE study on
o-HBMO show that the hydrogen bond connects the hydroxyl
group and the nitrogen atom. To satisfy the O–N distance
requirement for the hydrogen-bond,18 the molecules need to
adopt several irregular bond lengths and angles as found in the
crystal structure of o-HBDI,17 or allow two rings to be twisted
out of the same plane.
Notes and references
1 N. C. Shaner, G. H. Patterson and M. W. Davidson, J. Cell Sci.,
2007, 120, 4247–4260.
2 R. Y. Tsien, Annu. Rev. Biochem., 1998, 67, 509–544.
3 X. He, A. F. Bell and P. J. Tonge, Org. Lett., 2002, 4, 1523–1526.
4 S. Kojima, H. Ohkawa, T. Hirano, S. Maki, H. Niwa, M. Ohashi,
S. Inouye and F. I. Tsuji, Tetrahedron Lett., 1998, 39, 5239–5242.
5 L. Wu and K. Burgess, J. Am. Chem. Soc., 2008, 130, 4089–4096.
6 I. V. Yampolsky, A. A. Kislukhin, T. T. Amatov, D. Shcherbo,
V. K. Potapov, S. Lukyanov and K. A. Lukyanov, Bioorg. Chem.,
2008, 36, 96–104.
7 I. V. Yampolsky, S. J. Remington, V. I. Martynov, V. K. Potapov,
S. Lukyanov and K. A. Lukyanov, Biochemistry, 2005, 44,
5788–5793.
8 M. Zimmer, Chem. Rev., 2002, 102, 759–781.
9 M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien and
S. J. Remington, Science, 1996, 273, 1392–1395.
10 F. Yang, L. G. Moss and G. N. Phillips Jr., Nat. Biotechnol., 1996,
14, 1246–1251.
11 J. Dong, K. M. Solntsev and L. M. Tolbert, J. Am. Chem. Soc.,
2006, 128, 12038–12039.
12 C. W. Cody, D. C. Prasher, W. M. Westler, F. G. Prendergast and
W. W. Ward, Biochemistry, 1993, 32, 1212–1218.
13 A. Follenius-Wund, M. Bourotte, M. Schmitt, F. Iyice, H. Lami,
J. J. Bourguignon, J. Haiech and C. Pigault, Biophys. J., 2003, 85,
1839–1850.
Either of these two configurations enhances the energy level
of the molecules, which needs to be compensated by the
formation of the hydrogen-bond. In solution and at room
temperatures, the strength of the hydrogen-bond in o-HBDI
hardly stabilizes the configuration while that in o-HBMO
does. Currently, we assign this bond energy difference as
resulting from the local electron density difference between
two molecules. The substitution of a nitrogen atom with an
oxygen atom in the imidazole ring provides an additional lone
pair of electrons, which should facilitate the proton relocation.
14 J. Cornforth and M. H. Du, J. Chem. Soc., Perkin Trans. 1, 1991,
2183–2187.
15 D. F. Eaton, J. Photochem. Photobiol., B, 1988, 2, 523–531.
16 S. Forsen and S. Linse, Trends Biochem. Sci., 1995, 20, 495–497.
17 K. Y. Chen, Y. M. Cheng, C. H. Lai, C. C. Hsu, M. L. Ho,
G. H. Lee and P. T. Chou, J. Am. Chem. Soc., 2007, 129,
4534–4535.
1
In H NMR spectra, o-HBDI provided one set of signals
at all pHs (Fig. 2), possibly due to the molecule either
having only one major state (more likely) or undergoing fast
18 W. W. Cleland, Arch. Biochem. Biophys., 2000, 382, 1–5.
ꢀc
This journal is The Royal Society of Chemistry 2010
2870 | Chem. Commun., 2010, 46, 2868–2870