Site-selective hydrogen-bonding-induced fluorescence quenching of highly solvatofluorochromic GFP-like chromophores

Article abstract of DOI:10.1021/ol302237k

The unconstrained green fluorescence protein (GFP)-like chromophore m-DMABDI displays a high solvatofluorochromicity in aprotic solvents, but the fluorescence is quenched in protic solvents. According to the site-specific intramolecularly hydrogen-bonded analogs 1OH and 2OH, the hydrogen bonding to the carbonyl oxygen is more important than that to the imino nitrogen of the imidazolinone group in the fluorescence quenching.

Full text of DOI:10.1021/ol302237k

ORGANIC
LETTERS
2012
Vol. 14, No. 19
5034–5037
Site-Selective Hydrogen-Bonding-Induced
Fluorescence Quenching of Highly
Solvatofluorochromic GFP-like
Chromophores
Guan-Jhih Huang,† Jinn-Hsuan Ho,‡ Ch. Prabhakar,† Yi-Hung Liu,† Shie-Ming Peng,†
and Jye-Shane Yang*,†
Department of Chemistry, National Taiwan University, Taipei, Taiwan, 10617, and
Department of Chemical Engineering, National Taiwan University of Science and
Technology, Taipei, Taiwan, 10607
jsyang@ntu.edu.tw
Received August 12, 2012
ABSTRACT
The unconstrained green fluorescence protein (GFP)-like chromophore m-DMABDI displays a high solvatofluorochromicity in aprotic solvents,
but the fluorescence is quenched in protic solvents. According to the site-specific intramolecularly hydrogen-bonded analogs 1OH and 2OH, the
hydrogen bonding to the carbonyl oxygen is more important than that to the imino nitrogen of the imidazolinone group in the fluorescence
quenching.
Hydrogen bonding is a directional noncovalent interaction
that plays an important role in the structures and properties
of numerous natural and artificial molecules, supermolecules,
and polymers.1ꢀ7 Hydrogen-bonding interactions in the
excited state might lead to fluorescence quenching due to
rapid internal conversion or proton/electron transfer.2ꢀ7
However, the phenomenon of H-bonding-induced fluores-
cence quenching is barely predictable for a new chromo-
phore, as the fluorescence-quenching mode is sensitive to
substrate conformation4 and H-bonding location5 and/or
orientation.6 For a chromophore containing more than
one H-bonding site,7 experimental identification of the site
responsible for the fluorescence quenching is fundamentally
† National Taiwan University.
‡ National Taiwan University of Science and Technology.
(1) (a) Hu, W.; Zhu, N.; Tang, W.; Zhao, D. Org. Lett. 2008, 10,
2669–2672. (b) Hariharan, M.; Siegmund, K.; Lewis, F. D. J. Org. Chem.
2010, 75, 6236–6243. (c) Li, X.; Fang, Y.; Deng, P.; Hu, J.; Li, T.; Feng,
W.; Yuan, L. Org. Lett. 2011, 13, 4628–4631. (d) Gonzalez-Rodriguez,
D.; Schenning, A. P. H. J. Chem. Mater. 2011, 23, 310–325. (e) Such,
G. K.; Johnston, A. P. R.; Caruso, F. Chem. Soc. Rev. 2011, 40, 19–29.
(2) (a) Zimmer, M. Chem. Rev. 2002, 102, 759–781. (b) Waluk, J. Acc.
Chem. Res. 2003, 36, 832–838. (c) Zhao, G.-J.; Han, K.-L. Acc. Chem.
Res. 2012, 45, 404–413.
ꢀ
(5) Morimoito, A.; Yatsuhashi, T.; Shimada, T.; Biczok, L.; Tryk,
D. A.; Inoue, H. J. Phys. Chem. A 2001, 105, 10488–10496.
(6) (a) Sugitha, M.; Shimada, T.; Tachibana, H.; Inoue, H. Phys.
Chem. Chem. Phys. 2001, 3, 2012–2017. (b) Morimoto, A.; Yatsuhashi,
T.; Shimada, T.; Kumazaki, S.; Yoshihara, K.; Inoue, H. J. Phys. Chem.
A 2001, 105, 8840–8849.
(3) (a) Hermant, R. M.; Bakker, N. A. C.; Scherer, T.; Krijnen, B.;
Verhoeven, J. W. J. Am. Chem. Soc. 1990, 112, 1214–1221. (b) Lewis,
F. D.; Yoon, B. A.; Arai, T.; Iwasaki, T.; Tokumaru, K. J. Am. Chem.
Soc. 1995, 117, 3029–3036.
(4) Petkova, I.; Mudadu, M. S.; Singh, A.; Thummel, R. P.; van
Stokkum, I. H. M.; Buma, W. J.; Waluk, J. J. Phys. Chem. A 2007, 111,
11400–11409.
(7) Zhao, G.-J.; Liu, J.-Y.; Zhou, L.-C.; Han, K.-L. J. Phys. Chem. B
2007, 111, 8940–8945.
r
10.1021/ol302237k
Published on Web 09/17/2012
2012 American Chemical Society

important but is rarely demonstrated. We report herein one
such example with the modified green fluorescent protein
(GFP) chromophores meta-dimethylaminobenzylidenedi-
methylimidazolinone (m-DMABDI), 1OH, and 2OH (the
m-DMABDIs).
The chromophores m-DMABDI, 1OH, and 1OMe were
obtained from the common intermediate m-DMABO by
reacting with the corresponding aliphatic amines. The OH-
containing side chain in 2OH was introduced from the
reaction of m-DMABDI and acetaldehyde.11 Acetalde-
hyde instead of formaldehyde was used due to its synthetic
feasibility. The reference compounds 2OMe were obtained
by O-methylation of 2OH. Detailed synthetic procedures
and compound characterization data are supplied as
Supporting Information.
Scheme 1. Synthesis of the m-DMABDIs
Figure 1. m-DMABDIs and related chromophores.
Molecular design of the m-DMABDIs is based on our
previous observation of drastic fluorescence quenching of
m-ABDI on going from aprotic to protic solvents (e.g., fluo-
rescence quantum yield Φf = 0.34 in hexane and <0.001 in
MeOH), which indicates the presence of H-bonding-induced
excited-state deactivation.8 This differs from the extremely
weak (Φf < 0.001) and solvent-independent fluorescence
properties of the parent GFP chromophore, para-hydro-
xybenzylidenedimethylimidazolinone (p-HBDI), revealing
a significant meta-amino substituent effect.9 The H-bond-
ing modes associated with the imidazolinone group in
m-ABDI should be important, as the photoinduced internal
charge transfer increases the charge density of this group.
The presence of two H-bond acceptors, the carbonyl oxygen
and the imino nitrogen, in the imidazolinone group provides
a unique opportunity for investigating their relative con-
tribution to the observed fluorescence quenching. To avoid
the complication of H-bonding interactions between the
amino group (H-bond donor) in m-ABDI and polar aprotic
solvents (e.g., the nitrogen of MeCN), the dimethylamino
analog m-DMABDI and the site-specific intramolecularly
H-bonded derivatives 1OH and 2OH were designed. The
intramolecular H-bond is in a seven-membered ring in 1OH
but in a six-membered ring in 2OH. On the basis of DFT
(B3LYP/6-31G**) calculations, the optimized structures of
1OH and 2OH (Figure S1) exhibit the expected intramolec-
The X-ray crystal structures were determined for
m-DMABDI, 1OH, and2OH. The expected intramolecular
H-bond OH••••NdC is observed for 2OH, but the hydro-
xyl group in 1OH is H-bonded to the imino nitrogen of the
neighboring molecule (Figure S2). The benzylideneimida-
zolinone moiety is nearly coplanar and the dimethylamino
(DMA) group is in a syn orientation to the carbonyl group
(as shown in Figure 1) for all three cases. The discrepancy
in the DMA orientation and/or the H-bonding mode
between the DFT-optimized and X-ray crystal structures
could be attributed to the larger basicity12 of the imino
nitrogen vs the carbonyl oxygen and to the crystal packing
effect.
ular H-bond and are favorable by 2.49 and 5.72 kcal molꢀ1
,
The absorption spectra of the m-DMABDIs in hexane
are shown in Figure 2. The spectral maximum (λabs) is near
350 nm, and a long-wavelength shoulder with noticeable
absorbance up to 480 nm is present for all cases. The peak
maximum is of little dependence on the solvent polarity;
the magnitude of the solvatochromic shift from hexane to
MeOH is 1ꢀ3 nm (Table S3). The difference of λabs
between 1OH and 1OMe and between 2OH and 2OMe
respectively, relative to non-H-bonded conformations
(Tables S1 and S2). With the corresponding non-H-bonded
systems 1OMe and 2OMe as reference compounds, the
carbonyl vs imino H-bonding effect on the fluorescence
quenching can be addressed.
The m-DMABDIs were prepared according to a mod-
ified version of Niwa’s synthetic protocol (Scheme 1).10
is little or none, revealing a weak H-bonding effect on λabs
.
(8) Yang, J.-S.; Huang, G.-J.; Liu, Y.-H.; Peng, S.-M. Chem. Com-
mun. 2008, 1344–1346.
(9) Yang, J.-S.; Liau, K.-L.; Li, C.-Y.; Chen, M.-Y. J. Am. Chem.
Soc. 2007, 129, 13183–13192 and references cited therein.
(10) Kojima, S.; Ohkawa, H.; Hirano, T.; Maki, S.; Niwa, H.;
Ohashi, M.; Inouye, S.; Tsuji, F. I. Tetrahedron Lett. 1998, 39, 5239–
5242.
(11) Tanis, S. P.; Parker, T. T.; Colca, J. R.; Fisher, R. M.; Kletzein,
R. F. J. Med. Chem. 1996, 39, 5053–5063.
(12) Dong, J.; Solntsev, K. M.; Tolbert, L. M. J. Am. Chem. Soc.
2006, 128, 12038–12039.
Org. Lett., Vol. 14, No. 19, 2012
5035

A similar situation was also reported for an intramolecu-
larly H-bonded derivative of p-HBDI.13
model for the ZꢀE photoisomerization,16 the quantum
yield for the torsion of the exocyclic CdC bond is approxi-
mately equivalent to 2ΦZE, assuming that partitioning of
the perpendicularly twisted intermediate to the Z and the E
isomers is equal. That Φf þ 2ΦZE ≈ 1.0 for m-DMABDI,
1OMe, and 2OMe thus indicates that the excited-state
deactivation is mainly due to fluorescence and the Z f E
isomerization and other internal conversion channels are
unimportant. In contrast, it is Φf þ 2ΦZE , 1.0 for 1OH
and 2OH. The decrease in Φf and ΦZE for the H-bonded
and non-H-bonded couples (1OH vs 1OMe and 2OH vs
2OMe) are 4ꢀ50% and 38ꢀ64%, respectively, and the
difference is larger in more polar solvents. Evidently, the
intramolecular H-bonding induces a new nonradiative
decay channel that competes effectively with the fluores-
cence and Z,E-isomerization.
Unlike the case of λabs, the fluorescence maximum (λf)
exhibits a large solvatochromic effect (e.g., a shift of
4190ꢀ4770 cmꢀ1 from hexane to MeCN, Figure S3 and
Table 1), indicating the prominent charge-transfer char-
acter of the m-DMABDIs in the excited state. A spectrum
of color from blue to red is observed for the fluorescence of
m-DMABDI on going from hexane to MeCN (TOC
graphic). The prominent solvatofluorochromism of m-
DMABDI is reminiscent of the colorful mutants of
GFP.14 The solvatofluorochromic shift is slightly de-
creased for 1OMe and 2OMe. The λf values of 1OH and
The site-dependent H-bonding effect is even more ex-
plicit with the comparison of radiative (kr = Φf/τf) and
nonradiative (knr = (1 ꢀ Φf)/τf) decay rate constants
deduced from Φf and the fluorescence lifetimes (τf). These
data are shown in Table 1, and the uncertainty is 10% of
the values. All the fluorescence decay profiles can be well fit
Table 1. Photophysical and Photochemical Data for the
m-DMABDIs in Hexane (Hex), THF, and MeCN
a
λf
Φf ΦZE
τfb
kf
knr
(108 sꢀ1
)
compd
solvent (nm) (%) (%) (ns) (108 sꢀ1
)
Figure 2. Absorption and fluorescence spectra of the m-DMAB-
DIs in hexane.
m-DMABDI Hex
492 46 21
584 14 38
22.5
15.3
8.6
0.20
0.09
0.06
0.21
0.10
0.07
0.21
0.09
0.05
0.20
0.08
0.06
0.19
0.09
0.08
0.24
0.56
1.11
0.27
0.73
3.50
0.26
0.59
1.22
0.33
0.67
1.87
0.25
0.56
1.22
THF
MeCN 643
5
53
1OH
Hex
506 43 n.d. 20.8
2OH are at longer wavelengths relative to those of 1OMe
and 2OMe, which is consistent with H-bonding interac-
tions at the acceptor moiety of a donorꢀacceptor chro-
mophore. The slightly larger difference in λf for 1OH and
1OMe (15ꢀ24 nm) compared to 2OH and 2OMe (10ꢀ16
nm) shows a larger electronic perturbation by H-bonding
to the carbonyl oxygen than to the imino nitrogen.
The Φf and the Z f E isomerization quantum yield
(ΦZE) of the m-DMABDIs in hexane, THF, and MeCN
are reported in Table 1. The Φf of the m-DMABDIs in
hexane is in the range 0.37ꢀ0.46, which is larger than that
of m-ABDI (Φf = 0.34 in hexane)8 and the other uncon-
strained GFP-like chromophores in solutions.15 As in the
case of m-ABDI, the Φf decreases as the solvent polarity
increases, and the fluorescence is nearly quenched in
MeOH (Φf < 10ꢀ3). According to the one-bond-flip
THF
585 12 26
17
12.1
2.8
MeCN 656
2
1OMe
2OH
Hex
491 45 n.d. 21.2
THF
563 13 42
40
14.7
7.9
MeCN 632
4
Hex
506 37 n.d. 18.9
THF
585 11 27
16
13.2
5.2
MeCN 642
3
2OMe
Hex
495 43 n.d. 22.4
THF
569 14 47
44
15.3
7.7
MeCN 632
6
a For the purpose of solubility (10ꢀ3 M), Hex and MeCN solutions
contain 20% THF for the measurement of ΦZE. Data are not deter-
mined (n.d.) in hexane because of poor solubility, even containing 20%
THF. Excitation wavelength is 350 nm. b The τf was determined with
excitation and emission around the spectral maxima.
with a single-exponential function. Unlike the subpicosecond
fluorescence lifetime of p-HBDI,16 the τf of 2OMe is as long
as 22.4 ns in hexane, which is to our knowledge unprece-
dented for an unconstrained GFP-like chromophore.15,17
The τf and kf decrease with increasing solvent polarity.
This might indicate intensity borrowing18 from the higher
˚
(13) (a) Petersen, M. A.; Riber, P.; Andersen, L. H.; Nielsen, M. B.
Synthesis 2007, 23, 3635–3638. (b) Lincke, K.; Solling, T.; Andersen,
L. H.; Klaerke, B.; Rahbek, D. B.; Rajput, J.; Oehlenschlaeger, C. B.;
Petersen, M. A.; Nielsen, M. B. Chem. Commun. 2010, 46, 734–736.
(14) (a) Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509–544. (b)
Shaner, N. C.; Patterson, G. H.; Davidson, M. W. J. Cell. Sci. 2007, 120,
4247–4260.
(15) (a) Ivashkin, P. E.; Yampolsky, I. V.; Lukyanov, K. A. Russ. J.
Bioorg. Chem. 2009, 35, 652–669. (b) Huang, G.-J.; Yang, J.-S. Chem.;
Asian J. 2010, 5, 2075–2085. (c) Chuang, W.-T.; Hsieh, C.-C.; Lai, C.-H.;
Lai, C.-H.; Shih, C.-W.; Chen, K.-Y.; Hung, W.-Y.; Hsu, Y.-H.; Chou,
P.-T. J. Org. Chem. 2011, 76, 8189–8202. (d) Lee, J.-S.; Baldridge, A.;
Feng, S.; SiQiang, Y.; Kim, Y. K.; Tolbert, L. M.; Chang, Y.-T. ACS
Comb. Sci. 2011, 13, 32–38.
(16) Zimmer, M. Cis-trans Isomerization in Biochemistry; Dugave, C.,
Ed.; Wiley-VCH: Weinheim, Germany, 2006; p 77.
(17) Tolbert, L. M.; Baldridge, A.; Kowalik, J.; Solntsev, K. M. Acc.
Chem. Res. 2012, 45, 171–181 and references cited therein.
(18) Bixon, M.; Jortner, J.; Verhoeven, J. W. J. Am. Chem. Soc. 1994,
116, 7349–7355.
5036
Org. Lett., Vol. 14, No. 19, 2012

excited state of the more allowed transition (Figure 2). In
more polar solvents, the energetic separation between the
fluorescing and the higher excited state is larger, and thus the
intensity borrowing becomes lower. The natural fluorescence
rate constant (kf) of all five compounds in the same solvent is
equivalent within experimental uncertainty, and thus the
difference in kf between 1OH and 1OMe and that between
2OH and 2OMe is negligible. In contrast, the difference in
knr (Δknr) for the H-bonded vs non-H-bonded couple is
substantial and solvent-dependent (Figure 3a). The Δknr is
small((1ꢀ8) ꢁ 106 sꢀ1) in hexane but significant ((0.7ꢀ2.3) ꢁ
108 sꢀ1) in MeCN for both H-bonding modes. The Δknr can
be attributed to the rate constant of nonradiative decay
induced by the intramolecular H-bond (kHB = Δknr).
Although the DFT calculations suggest a more stable
H-bonded state for 2OH vs 1OH, the kHB is 3.4-fold larger
for 1OH vs 2OH in MeCN. Evidently, the H-bonding to the
carbonyl oxygen is more impactful than that to the imino
nitrogen on the excited-state quenching. As indicated by the
λf of 1OH vs 2OH, which is the same in hexane but larger in
MeCN, the larger H-bonding effect in 1OH results from
stronger H-bonding interactions associated with the carbonyl
vs the imino group in the electronically excited state.
Figure 3. (a) Comparison of kHB in different solvents and
H-bonding modes; (b) MeOH-induced fluorescence quenching
for m-DMABDI in MeCN ([MeOH] = 0.0ꢀ1.0 M), and the
corresponding (c) plot of log(Φf0/Φf ꢀ 1) against log[MeOH]
and (d) SternꢀVolmer plot.
indicates that a significant barrier exists in the quenching
process or it is static quenching due to ground state
complexation. ThelowΦf (<10ꢀ3) inneatMeOH suggests
a kHB value larger than 1 ꢁ 109 sꢀ1, provided that all of the
substrate forms the complex and the kf is on the same order
as that in MeCN. A smaller kHB for 1OH in MeCN (2.3 ꢁ
108 sꢀ1) vs m-DMABDI in MeOH (>1 ꢁ 109 sꢀ1) reflects
the low population of the optimal H-bonded form of 1OH.
In summary, the H-bonding to the carbonyl oxygen of
the m-DMABDIs plays a more important role than that to
the imino nitrogen in deactivating the excited state. The
intriguing dependence of fluorescence properties on sol-
vent polarity and proticity for the m-DMABDIs and their
analogs might prove of value as fluorescent probes for site-
selective fluorescence imaging of biological systems.
The solvent effect on kHB provides insight to the nature
of the H-bond-induced decay channel. In principle, the
intramolecular H-bond is more favorable in less polar
solvents.19 A larger H-bonded population is thus expected
for 1OH and 2OH in hexane than in MeCN. However, the
kHB is much smaller in hexane vs MeCN, which indicates
that the quenching of the excited state is not simply due to
internal conversion. An occurrence of excited-state proton
transfer (ESPT) from MeOH tothe carbonyl oxygen or the
imino nitrogen that results in a polar zwitterion might
account for the larger kHB in more polar solvents due to
better solvation.
The effect of intermolecular H-bonding on fluorescence
quenching was also investigated with MeOH titration of
m-DMABDI in MeCN (Figure 3b). Figure 3c shows the
log(Φf0/Φf ꢀ 1) vs log[MeOH] plot, where Φf0 and Φf
correspond to the fluorescence quantum yield in the
absence and presence of MeOH. The slope 1.02 ( 0.03
indicates that the fluorescence quenching results from a 1:1
complex of m-DMABDI and MeOH.4 According to the
slope (3.35 ( 0.07 Mꢀ1) of the SternꢀVolmer plot
(Figure 3d) and fluorescent lifetime (8.6 ns) in MeCN,
Acknowledgment. We thank the National Science Coun-
cil of Taiwan and National Taiwan University for financial
support. The computing time granted by the Computing
Center of NTU is acknowledged.
Supporting Information Available. Detailed experimen-
tal methods and synthesis, fluorescence spectra, X-ray
crystal and DFT-optimized structures, table of maxima
of UV/vis absorption (λabs) and fluorescence (λf), and
CIF file for the m-DMABDIs. This material is available
free of charge via the Internet at http://pubs.acs.org.
the calculated quenching constant is 3.6 ꢁ 108 Mꢀ1 sꢀ1
.
This value is nearly 100 times smaller than the diffusional
rate constant in MeCN (2.3 ꢁ 1010 Mꢀ1
s
ꢀ1),3a which
(19) Kohmoto, S.; Iwasaki, N.; Fukui, D.; Nishio, T.; Iida, I.;
Kishikawa, K.; Yamamoto, M.; Yamada, K. J. Chem. Soc., Perkin
Trans. 2 1996, 985–988.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 19, 2012
5037