Photochemical generation of a new, highly fluorescent compound from non-fluorescent resveratrol

Article abstract of DOI:10.1039/c2cc30940h

UV irradiation of trans-resveratrol leads to its photochemical transformation to a new, highly fluorescent compound, whose chemical structure was unambiguously identified. The new compound has large values of fluorescence quantum yield, Stokes' shift, and two-photon absorption cross section, which make it suitable for bio-imaging, multi-color labeling, and two-photon microscopy. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc30940h

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COMMUNICATION
Photochemical generation of a new, highly fluorescent compound from
non-fluorescent resveratrol
Ilseung Yang,wa Eunha Kim,wa Junhee Kang,b Hyouksoo Han,c Soohwan Sul,c
Seung Bum Park*d and Seong Keun Kim*d
Received 9th February 2012, Accepted 24th February 2012
DOI: 10.1039/c2cc30940h
UV irradiation of trans-resveratrol leads to its photochemical
transformation to a new, highly fluorescent compound, whose
chemical structure was unambiguously identified. The new compound
has large values of fluorescence quantum yield, Stokes’ shift,
and two-photon absorption cross section, which make it suitable
for bio-imaging, multi-color labeling, and two-photon microscopy.
the entire photochemical reaction pathway have been left
virtually uninvestigated so far.
Several research groups have sporadically reported observing
fluorescence from the solution of UV-irradiated trans-resveratrol.7,8
Roggero and Garciaparrilla found that intense UV irradiation of
an alcoholic solution of trans-resveratrol first induces isomerization
to the cis-form followed by formation of an unidentified, highly
fluorescent compound.8a Using a fluorometric technique to assess
the local stilbene content in grapevine leaf, Poutaraud et al.
reported a broad emission band of unknown origin from a
UV-irradiated resveratrol solution.8b In addition, Martinez-Ortega
et al. confirmed the presence of a highly fluorescent compound in
their quantitative analyses of resveratrol in red wine.7a In all these
previous studies, however, no structural information was given
about the highly fluorescent species and its chemical identification
has remained elusive to date.
Luminescence is a broad term used to describe the conversion of
energy into light in matter. Different sources of energy such as
chemical reactions, electrical potential, or light irradiation produce
different types of light emission such as chemi-, electro-, or photo-
luminescence, respectively.1 In this communication, we report a
novel luminescence phenomenon resulting from the combination
of photo- and chemi-luminescence of a non-emitting molecule
(trans-resveratrol in this study) that is transformed into a highly
fluorescent species by a photochemical reaction triggered by light.
This is a distinctly different phenomenon from photocaging of
fluorophores2 or photoactivated switching3,4 as it involves only a
single-moiety compound, with no release of a component species,
no protected (‘‘caged’’) molecules involved, no need for an
activator or foreign agent such as thiol,3 or no shelving–deshelving
process to achieve reversibility.4
Resveratrol (3,5,40-trihydroxy-trans-stilbene), 1, is a naturally
occurring phytoalexin compound found mostly in berries and
nuts, whose main function is protection against infection and other
external stresses.5 Its lifespan-extending effect for simple forms of
life such as nematodes and fruit flies has drawn considerable
interest in recent years, along with its significant medicinal effects
as an anti-oxidant, anti-carcinogenic, and cardio-protective agent.6
Chemically, the mechanism for photoisomerization from trans- to
cis-resveratrol has been studied in view of its application in
chemical content analysis,7 but chemical transformations along
In our experiment, we irradiated 125 mM methanol solution of
trans-resveratrol (R5010, Sigma) with UV light for 90 seconds,
which is much shorter than in earlier studies (Bhours),8 using a
6 watt UV lamp (lmax = 305 nm) with a photon intensity of
0.6 mW cmꢀ2 at 295 K. The photoproducts were analyzed by high-
performance liquid chromatography (HPLC). The wavelength of
the UV lamp was chosen to match that of the maximum absorp-
tion for trans-resveratrol, ensuring the highest yield in the shortest
time. In agreement with the earlier studies,8 we confirmed the
existence of three major species using HPLC (middle panel of
Fig. 1(a)): trans-resveratrol, cis-resveratrol, and an unknown
fluorescent compound (‘‘X’’ hereafter). Once photochemically
produced, this compound remains quite stable in aqueous solution.
Under prolonged exposure, however, it undergoes photodegrada-
tion (bottom panel of Fig. 1(a)). Fig. 1(b) shows that the absorp-
tion spectrum of X is rather different from that of trans- or
cis-resveratrol, while its emission in the visible range is quite a
distinct feature from the two resveratrol compounds, which are
non-fluorescent. Fig. 1(c) compares the actual photographs of
luminescence from the solution before and after UV irradiation.
In order to identify X chemically, we first isolated it from the
other resveratrol compounds by using preparatory thin-layer
chromatography where the mobile phase was composed of
a Department of Chemistry, Seoul National University,
Seoul 151-747, Korea
b WCU Department of Biophysics and Chemical Biology,
Seoul National University, Seoul 151-747, Korea
c Analytical Science Group Samsung Advanced Institute of Technology,
Yongin-si, Gyeonggi-do 446-712, Korea
d Department of Chemistry and WCU Department of Biophysics and
Chemical Biology, Seoul National University, Seoul 151-747, Korea.
E-mail: sbpark@snu.ac.kr, seongkim@snu.ac.kr;
Fax: +82-2-884-4025, +82-2-889-5719; Tel: +82-2-880-9090,
+82-2-880-6659
w These authors contributed equally.
c
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Chem. Commun., 2012, 48, 3839–3841 3839

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and 13C NMR with DEPT (distortionless enhanced polarization
transfer), HSQC (heteronuclear single quantum correlation), and
HMBC (heteronuclear multiple bond correlation) analysis along
with FT-IR spectroscopy. The coupling constant between the
1
two peaks of H NMR (Fig. 2(b)) at d 7.56 and at d 6.71 was
13.5 Hz, indicating the presence of trans-olefin, whereas that
between the two peaks at d 7.56 and at d 7.50 was 7.25 Hz,
indicating the presence of two aromatic protons. The 13C
chemical shift of X at d 201 (Fig. 2(c)) indicates the presence of
an aldehyde or ketone carbonyl group (CQO). DEPT analysis
(Fig. 2(d)) of the 1H NMR peak at d 2.3 suggests a methyl
group adjacent to the carbonyl (–CO–CH3). HSQC and HMBC
analyses (Fig. 2(e)) suggest that the carbons of the trans-olefin
are connected directly to the carbon of the acetyl group and
indirectly to the carbons of the two coupled aromatic protons
via one quaternary carbon. The presence of an acetyl group and
an olefin moiety in X deduced above is also consistent with the
FT-IR spectrum (Fig. 2(f)).
Considering all the results above, we propose that the structure
of the unknown fluorescent compound X should be that of (E)-4-
(6,8-dihydroxynaphthalen-2-yl)but-3-en-2-one (Fig. 3), which
has never been registered in the CAS. It will be interesting to
see if this relatively simple molecule can be synthesized without
resorting to a photochemical reaction. For this structure of X,
which for now we would like to name ‘‘resveratrone’’ for short,
we performed density functional theory calculations with the
Gaussian03 program package using the B3LYP/6–31G* basis set
and found that its simulated IR spectrum (Fig. 2(g)) is in good
agreement with the experimental FT-IR spectrum.
Fig. 1 (a) HPLC data for the reaction products obtained after
different durations of exposure to UV irradiation. The unknown,
highly fluorescent species is denoted by X. (b) Absorption (open
circles) and emission (solid circles) spectra of trans-resveratrol,
cis-resveratrol, and X. No emission is detected from trans- and
cis-resveratrol whereas X gives a strong emission in the visible range
when excited at 390 nm. (c) Photographs showing photoluminescence
(or lack thereof) of the resveratrol solution before (left) and after
(right) UV irradiation at 305 nm.
methanol and methylene chloride (1 : 20). A high-resolution
mass spectrometer (LTQ Orbitrap, Thermo Scientific) showed
that the molecular mass of HPLC-separated X is the same as
that of resveratrol itself (Fig. 2(a)), suggesting an isomerization
reaction as the main pathway for the photochemical generation
of X. For structural identification of X, we employed 1H NMR
Our proposed structure and reaction pathway for X are
somewhat similar to those suggested by Ho et al.,9 who found
a series of photocyclization, hydrogen shift, and ring opening
in their study of acid-assisted switching of the reaction pathway
for stilbene analogues upon UV irradiation. However, as shown
1
Fig. 2 (a) High-resolution mass, (b) H NMR, (c) 13C NMR, (d) DEPT NMR, (e) 2D NMR, (f) FT-IR, and (g) simulated IR spectrum of X.
c
3840 Chem. Commun., 2012, 48, 3839–3841
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be B250 ps in methanol by time-correlated single photon counting
(TCSPC) (Fig. 5(a)), which is somewhat shorter than those of
other dyes with similar quantum yields. In addition to the
single-photon emission (Fig. 5(b)), we observed a two-photon
fluorescence emission from resveratrone (Fig. 5(c) and (d)). The
two-photon absorption cross section of resveratrone measured
by the two-photon induced fluorescence method10 is B132 GM
(1 GM = 10ꢀ50 cm4 s) in ethanol, which is of comparable
magnitude to other commonly used two-photon dyes.
Fig. 3 Photochemical reaction pathway for the generation of the
highly fluorescent compound X (‘‘resveratrone’’) by UV light.
In conclusion, we identified the highly fluorescent yet elusive
compound that results from UV irradiation of trans-resveratrol.
The reaction pathway toward the observed luminescence starts
with photoisomerization of trans-resveratrol to cis-resveratrol,
which then undergoes another isomerization reaction that leads
to the product (named ‘‘resveratrone’’), (E)-4-(6,8-dihydroxy-
naphthalen-2-yl)but-3-en-2-one, presumably through photoin-
duced ring opening and closing. This new molecule is thermally
stable and highly fluorescent with a large quantum yield. It also
has a large Stokes’ shift appropriate for multicolor labeling and
in vivo imaging, and is even suited for two-photon microscopy
with its high two-photon absorption cross section.
This work was supported by the National Research Foundation
of Korea through grants to S. K. K. (Star Faculty grant,
NRF-2005-084-C00017) and S. B. P. (NRF-2009-0078236). Both
S. K. K. and S. B. P. also acknowledge the support of this work by
the Chemical Genomics grant (M10526020002-08N2602-00210)
and the World Class University grant (R31-2010-100320). S. K. K.
also acknowledges the support of this work by the Global Frontier
R&D Program on Center for Multiscale Energy System funded by
the National Research Foundation. Both I. Y. and E. K. are
grateful for their graduate fellowships awarded by the BK21
Program.
Fig. 4 Hypothetical photochemical reaction mechanism for the
generation of resveratrone.
in the hypothetical mechanism we propose in Fig. 4, the photo-
chemical transformation of resveratrol was achieved in the absence
of acidic conditions. It is also essential to have the 40-hydroxyl
moiety, which was confirmed by the failure of 40-methylated
resveratrol to undergo the desired photochemical transformation.
To characterize some crucial optical properties of this newly
identified compound, we measured its fluorescence quantum
yield, excited-state lifetime, and two-photon absorption cross
section. The quantum yield of resveratrone as measured by an
absolute quantum yield measurement system (QE-1000, Otsuka
Electronics) is 0.49 in DMSO and 0.31 in iso-propanol, which are
comparable to those of many commonly-used Alexa family dyes.
The Stokes’ shift of resveratrone is very large (B150 nm, Fig. 1(b)),
which makes it an ideal fluorophore in multicolor labeling and
in vivo imaging. The excited-state lifetime was measured to
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Fig. 5 (a) A single-exponential temporal decay in TCSPC measure-
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c
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Chem. Commun., 2012, 48, 3839–3841 3841