Ortho Green Fluorescence Protein Synthetic Chromophore; Excited-State
Intramolecular Proton Transfer via a Seven-Membered-Ring
Kew-Yu Chen, Yi-Ming Cheng, Cheng-Hsuan Lai, Cheng-Chih Hsu, Mei-Lin Ho,
Gene-Hsiang Lee, and Pi-Tai Chou*
Department of Chemistry, National Taiwan UniVersity, Taipei, 106, Taiwan, R.O.C.
Received February 7, 2007; E-mail: firstname.lastname@example.org
Green fluorescence protein (GFP), which serves as an energy
acceptor and emitter for bioluminescence in the sea pansy Renilla
reniformis and the jellyfish Aequorea Victoria, has drawn much
attention because of its applications in molecular biology and
biochemistry.1GFP takes advantage of the presence of a chro-
mophore that is anchored both covalently and via a hydrogen-bond
5(4H)-one (p-HBDI, see Scheme 1), which undergoes excited-state
proton transfer (ESPT)2via the proton relay of water molecules
and a remote residue such as E222,3resulting in a very effective
and intense anion fluorescence.
Nevertheless, studies reveal a strong cutoff between the properties
of wild type GFP (or certain GFP mutants) and the synthetic
analogue chromophores of p-HBDI.4In view of photophysics, the
fluorescence yield of the protein-free chromophore in fluid solvents
is much weaker and strongly temperature dependent. The results
suggest an efficient radiationless transition operating in p-HBDI,
most probably induced by conformational relaxation along torsional
deformation of the two exocyclic C-C bonds to a nonfluorescent
twisted intermediate.5More recently, it has been proposed that the
shallow potential energy surface of the intermediates may conically
intersect with that of the ground state, inducing the dominant
radiationless deactivation.4c-d,6Such a conformational relaxation
is greatly suppressed in GFP by its proton relay, rigid environment.
In view of chemistry, most of the research has been focused on
the chemical modification of p-HBDI analogues at the C(1)
position.4c,7Conversely, in this study, we are interested in the
derivatization on the phenyl ring. As an ingenious approach,
switching the hydroxyl group from the C(8) position to the C(6)
position (see Scheme 1), forming 4-(2-hydroxybenzylidene)-1,2-
dimethyl-1H-imidazol-5(4H)-one (o-HBDI), a structural isomer of
p-HBDI may reveal several novel features with respect to p-HBDI.
The geometry optimization (B3LYP/cc-pVDZ and aug-cc-pVDZ,
see ESI) of o-HBDI unveils the existence of a seven-membered-
ring intramolecular hydrogen bond between -OH and the N(2)
atom. This intramolecular hydrogen-bonding configuration should,
in part, hinder the exocyclic torsional deformation such that the
radiationless deactivation may be reduced. More importantly,
theoretical approaches also predict that excited-state intramolecular
proton transfer (ESIPT) from the OH proton to the N(2) atom is
thermally favorable (vide infra), forming a zwitterionic tautomer
species (see Table of Content, TOC).
In light of these perspectives, we have thus expended great effort
to make a facile synthesis of o-HBDI. Briefly, the o-methoxybenz-
aldehyde was used as a starting reactant (see Scheme 1). Because
of the lack of the o-hydroxyl group and hence the intramolecular
lactonation, 3 was obtained with a good yield (70%). Subsequent
reaction of 3 with methylamine, followed by deprotection of the
methyl group of o-MBDI by BBr3, afforded o-HBDI with an overall
product yield of 43%. Detailed synthetic procedures and product
characterization are provided in ESI.
The structure of o-HBDI was further confirmed by single-crystal
X-ray diffraction analysis. As depicted in Figure 1, the nearly planar
configuration between phenol and imidazolidinone rings was
established by∠N(2)-C(3)-C(5)-C(6) of -1.05°. This, together
with 2.63 Å of O(2)-N(2) distance and 176° of ∠N(2)-H-O(2),
strongly supports the seven-membered-ring intramolecular hydrogen-
bonding formation. In good agreement with this observation, the
1H NMR spectrum (in CDCl3) revealed a significantly downfield
signal at δ13.7, giving a clear indication of the strong hydrogen-
Figure 2 shows the absorption and emission spectra of o-HBDI
in cyclohexane. The absorption is characterized by a lowest-energy
absorption band maximized at 385 nm, for which the ? value of
(2.0 ( 0.3) × 104M-1cm-1makes its assignment to the π f π*
transition unambiguous. The steady-state emission consists soley
of one band maximized at as long as 605 nm with a moderate
quantum yield of (3.1 ( 0.2) × 10-3. To further verify the origin
of the emission, o-MBDI, (see Scheme 1), was also investigated.
Owing to its lack of a hydroxyl proton, o-MBDI serves as a model
to represent the prohibition of the proton-transfer reaction. As
depicted in Figure 2, the S0f S1absorption (λmax≈ 370 nm) and
the corresponding emission peak wavelength (λmax≈ 425 nm) for
o-MBDI reveal a mirror image with a normal Stokes shift.
Scheme 1. The Synthesis of o-HBDI and the Structure of p-HBDIa
aThe Non-IUPAC atom label is for the convenience of discussion.
Figure 1. The molecular structure of o-HBDI, thermal ellipsoids drawn at
the 50% probability level.
Published on Web 03/27/2007
4534 9 J. AM. CHEM. SOC. 2007, 129, 4534-4535
10.1021/ja070880i CCC: $37.00 © 2007 American Chemical Society
Accordingly, the ∼605 nm emission in o-HBDI with an anoma- Download full-text
lously large Stokes shift (peak-to-peak) of ∼10000 cm-1relative
to the S0f S1absorption, is unambiguously ascribed to a tautomer
emission resulting from the ESIPT reaction, most probably via the
phenolic proton to the N(2) nitrogen, forming a zwitterionic species
(see TOC for structure). With a femtosecond fluorescence up-
conversion technique, the population decay of the 605-nm emission
band was measured to be 32 ( 0.2 ps, while the corresponding
rise time was beyond the system response of 150 fs, which consists
with the system response limited decay time monitored at 450 nm,
presumed to be the origin of normal emission.
To further gain insights into the ESIPT kinetics, the H-deuterated
O-D compound of o-HBDI, o-dBDI, was also prepared (see
Supporting Information) and investigated. Under the same experi-
mental condition as that performed for o-HBDI, the up-converted
tautomer emission in o-dBDI also revealed a system response
limited rise time and a population decay of 33 ( 0.3 ps. The results
demonstrate that the rate of ESIPT is quite insensitive to an H/D
exchange and point to an essentially barrierless potential energy
surface along the ESIPT reaction. Further support was also given
by the theoretical approach. Based on the time dependent DFT
method (TDDFT/B3LYP/cc-pVDZ and aug-cc-pVDZ) implemented
in the TURBOMOLE 5.8 software package9(see SI), the ESIPT
process was calculated to be thermally favorable by ∼7.8 kcal/
mol (see TOC). Moreover, upon Franck-Condon excitation and
execution of the geometry relaxation, the TDDFT method could
not locate the energy minimum of the excited normal species, a
result which is consistent with a barrierless ESIPT process
As compared to those generally observed weak emissions for
p-HBDI (Φf≈10-4and τf≈ 1.7 ps in toluene) at room temperature,4d
the tautomer emission yield of 3.1 × 10-3with τf ≈ 32 ps in
cyclohexane implies that the hydrogen-bonding strength may, in
part, hinder the exocyclic C-C bonds rotation. Further support is
rendered by the much weaker normal emission (Φf≈ 5 × 10-4
and τf≈ 4 ps in cyclohexane, see Table 1) in o-MBDI that lacks
the seven-hydrogen bond formation. Nevertheless, the somewhat
weak proton-transfer tautomer emission may indicate the active
operation of the conformational relaxation owing to the loose
rigidity of the seven-membered-ring hydrogen bond. Note that
ESIPT still takes place in the solid film (vapor deposition onto a
quartz plate) of o-HBDI (Figure 2), resulting in a ∼595 nm tautomer
emission with Φfas high as 0.4 (τf≈ 1.7 ns).
As listed in Table 1, similar ultrasfast ESIPT was also observed,
giving a unique tautomer emission, in all aprotic solvents. However,
in protic solvent such as water (pH ) 7), dual emission, consisting
of normal (∼495 nm) and tautomer (∼600 nm) emission, was
resolved (see SI). The result in water can plausibly be rationalized
by the partial rupture of the intramolecular hydrogen bond,10
resulting in the prohibition of ESIPT. This viewpoint can be
supported by the lack of correlation between the finite decay of
normal emission (0.9 ps, Table 1) and system response limited-
rise dynamics of the tautomer (<150 fs). In pH ) 12, the o-HBDI
anion exhibits absorption and emission at 445 and 580 nm,
respectively. Comparing the anionic species, the red shift of the
zwitterionic emission can be rationalized by the reduction of
electron donating strength at the protonated N(2) nitrogen, resulting
in a decrease of the LUMO energy.
To sum up, we have synthesized a structural isomer of the core
chromophore (p-HBDI) in GFP. o-HBDI possesses a seven-
membered-ring hydrogen bond, from which ultrafast ESIPT takes
place, resulting in a proton-transfer tautomer emission of ∼605 nm
in nonpolar solvents. Although the bioactivity of o-HBDI is pending
further exploration, its future chemical derivation is versatile. It is
believed that fine tuning the proton-transfer emission can be
achieved via the derivation at the C(1) position, while the
radiationless quenching process may be further reduced by anchor-
ing bulky groups at the C(4) position, generating a new series of
isomers of p-HBDI with remarkable ESIPT properties.
Supporting Information Available:
procedures, spectroscopic data, and X-ray studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
Details for experimental
(1) (a) Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C.
Science 1994, 263, 802. (b) Lippincott-Schwartz. J.; Patterson, G. H.
Science 2003, 300, 87. (c) Ormo, M.; Cubitt, A. B.; Kallio, K.; Gross, L.
A.; Tsien, R. Y.; Remington, S. J. Science 1996, 273, 1392. (d) Zimmer,
M. Chem. ReV. 2002, 102, 759. (e) Tsien, R. Y. Annu. ReV. Biochem.
1998, 67, 509. (f) Sullivan, K. F.; Kay, S. A. Green Fluorescent Proteins;
Academic Press: San Diego, CA, 1999.
(2) (a) Hosoi, H.; Mizuno, H.; Miyawaki, A.; Tahara, T. J. Phys. Chem. B
2006, 110, 22853. (b) Agmon, N. Biophys. J. 2005, 88, 2452. (c) Stoner-
Ma, D.; Jaye, A. A.; Matousek, P.; Towrie, M.; Meech, S. R.; Tonge, P.
J. J. Am. Chem. Soc. 2005, 127, 2864.
(3) Stoner-Ma, D.; Melief, E. H.; Nappa, J.; Ronayne, K. L.; Tonge, P. J.;
Meech, S. R. J. Phys. Chem. B 2006, 110, 22009.
(4) (a) Dong, J.; Solntsev, K. M.; Tolbert, L. M. J. Am. Chem. Soc. 2006,
128, 12038. (b) Brejc, K.; Sixma, T. K.; Kitts, P. A.; Kain, S. R.; Tsien,
R. Y.; Orm, M.; Remington, S. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
2306. (c) Litvinenko, K. L.; Webber, N. M.; Meech, S. R. J. Phys. Chem.
A 2003, 107, 2616. (d) Mandal, D.; Tahara, T.; Meech, S. R. J. Phys.
Chem. B 2004, 108, 1102.
(5) (a) Stavrov, S. S.; Solntsev, K. M.; Tolbert, L. M.; Huppert, D. J. Am.
Chem. Soc. 2006, 128, 1540. (b) Gepshtein, R.; Huppert, D.; Agmon, N.
J. Phys. Chem. B 2006, 110, 4434. (c) Usman, A.; Mohammed, O. F.;
Nibbering, E. T. J.; Dong, J.; Solntsev, K. M.; Tolbert, L. M. J. Am. Chem.
Soc. 2005, 127, 11214.
(6) (a) Webber, N. M.; Litvinenko, K. L.; Meech, S. R. J. Phys. Chem. B
2001, 105, 8036.
(7) He, X.; Bell, A. F.; Tonge, P. J. Org. Lett. 2002, 4, 1523.
(8) He, X.; Bell, A. F.; Tonge, P. J. J. Phys. Chem. B 2002, 106, 6056.
(9) Ahlrichs, R.; Bar, M.; Haser, M.; Hom, H.; Kolmel, C. Chem. Phys. Lett.
1989, 162, 165.
(10) McMorrow, D.; Kasha, M. J. Phys. Chem. 1984, 88, 2235.
Figure 2. The absorption and emission spectra of o-HBDI in cyclohexane
(black solid line) and solid film (red solid line, emission only) and o-MBDI
(blue solid line) in cyclohexane.
Table 1. The Photophysical Properties of o-HBDI and o-MBDI in
Various Solvents at Room Temperature
H2O (pH ) 7)
H2O (pH ) 12)4451.0
aUnit: nm.bUnit: ps.cSum of the dual emission.
C O M M U N I C A T I O N S
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