Synthesis and photophysics of novel biocompatible fluorescent oxocines and azocines in aqueous solution

Article (PDF Available)inPhysical Chemistry Chemical Physics 15(39) · August 2013with 95 Reads
DOI: 10.1039/c3cp52228h · Source: PubMed
Abstract
The spectroscopic properties in water solution of the different prototropic forms of the strongly fluorescent hemiacetal 4,9-dihydroxy-1,2-dihydro-4,11a-methanooxocino[4,5-b]benzofuran-5(4H)-one (, monardine), the aza analogue 4,9-dihydroxy-3,4-dihydro-1H-4,11a-methanobenzofuro[2,3-d]azocin-5(2H)-one (, azamonardine) and the respective 2-carboxyl derivatives (, ) have been studied by experimental and quantum-chemical methods. Monardine and carboxymonardine are the major products of new fluorogenic, room-temperature reactions of hydroxytyrosol or salvianic acid in aqueous solution, respectively, and present unique photophysical properties. Near neutral pH (pKa = 7.2) monardine switches from a weakly emitting, UV-absorbing (382 nm) neutral species to a VIS-absorbing (426 nm), blue emitting (464 nm) anion form, with a fluorescence quantum yield ϕF = 1 and single-exponential decay τF = 2.74 ns. This binary-like spectroscopic change from the neutral to the anionic form was interpreted based on time-dependent density functional theory (TDDFT) calculations as due to (i) the reversal of (n,π*) and (π,π*) lowest-lying singlet excited states, and (ii) a change in the triplet-state distribution accompanying monardine ionization which may abolish de-excitation via intersystem crossing. A similar fluorogenic reaction takes place with catecholamines such as dopamine and DOPA, to yield fluorescent azocines and which, depending on pH, may be present as cationic, neutral or anionic species. TDDFT computations of these forms were also carried out to assign the corresponding excitation transitions and emission properties. Besides the analytical interest of the fluorogenic reactions, the photochemical stability and biocompatibility of the bright-dark pH-controlled molecular switches and may facilitate novel labels and probes to be developed for superresolution fluorescence microscopy.
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Cite this: DOI: 10.1039/c3cp52228h
Synthesis and photophysics of novel biocompatible
fluorescent oxocines and azocines in aqueous solution
A. Ulises Acun˜a,*
a
Mo
´
nica A
´
lvarez-Pe
´
rez,
a
Marta Liras,
b
Pedro B. Coto
c
and
Francisco Amat-Guerriz
b
The spectroscopic properties in water solution of the different prototropic forms of the strongly
fluorescent hemiacetal 4,9-dihydroxy-1,2-dihydro-4,11a-methanooxocino[4,5-b]benzofuran-5(4H)-one (1a,
monardine), the aza analogue 4,9-dihydroxy-3,4-dihydro-1H-4,11a-methanobenzofuro[2,3-d]azocin-5(2H)-
one (2a, azamonardine) and the respective 2-carboxyl derivatives (1b, 2b) have been studied by
experimental and quantum-chemical methods. Monardine and carboxymonardine are the major products
of new fluorogenic, room-temperature reactions of hydroxytyrosol or salvianic acid in aqueous solution,
respectively, and present unique photophysical properties. Near neutral pH (pK
a
= 7.2) monardine
switches from a weakly emitting, UV-absorbing (382 nm) neutral species to a VIS-absorbing (426 nm),
blue emitting (464 nm) anion form, with a fluorescence quantum yield f
F
= 1 and single-exponential
decay t
F
= 2.74 ns. This binary-like spectroscopic change from the neutral to the anionic form was
interpreted based on time-dependent density functional theory (TDDFT) calculations as due to (i) the
reversal of (n,p*) and (p,p*) lowest-lying singlet excited states, and (ii) a change in the triplet-state
distribution accompanying monardine ionization which may abolish de-excitation via intersystem crossing.
A similar fluorogenic reaction takes place with catecholamines such as dopamine and DOPA, to yield
fluorescent azocines 2a and 2b which, depending on pH, may be present as cationic, neutral or anionic
species. TDDFT computations of these forms were also carried out to assign the corresponding excitation
transitions and emission properties. Besides the analytical interest of the fluorogenic reactions, the
photochemical stability and biocompatibility of the bright–dark pH-controlled molecular switches 1a and
1b may facilitate novel labels and probes to be developed for superresolution fluorescence microscopy.
Introduction
The recent developments in fluorescence-based analytical and
imaging techniques resulted in unprecedented improvements
in detection sensitivity and spatial resolution, as well as in the
diversity of samples that may be quantitatively studied with
optical techniques, from a single molecule
1
to a living organism.
2
Successful application of these advances depends on the availability
of fluorescent labels and probes with specific properties,
3
and on
efficient methods of targeting fluorescent labeling.
4
Here we
report the synthesis and main photophysical properties of a
novel fluorescent molecular platform, which displays several
convenient features for these applications: (i) 100% emission
quantum yield in aqueous solution, (ii) pH-dependent switching
between bright ‘‘on’’ and dark ‘‘off’’ states and (iii) full compatibility
with biological media.
The origin of the new emitting materials is related to the
first reference to fluorescence (1565) credited to N. Monardes, a
physician from Seville (Spain) who noted the strange ‘‘color’’ of
the infusion of an American medicinal wood of uncertain
genealogy.
5
It has been shown recently that the wood was
obtained from a Mexican tree (Eysenhardtia polystachya (Ort.)
Sarg.) very rich in a rare glucosyl-dihydrochalcone,
6
which is
non-fluorescent. However, as soon as the non-emitting dihydro-
chalcone passes from the wood to water solution, a fast oxida-
tion reaction takes place to yield a strongly fluorescent glucoside
(matlaline, f
F
= 1.0), the source of the unusual ‘‘color’ of the old
a
Instituto de Quı
´
mica
´
sica ‘‘Rocasolano’’, C.S.I.C., Serrano 119, 28006 Madrid,
Spain. E-mail: roculises@iqfr.csic.es; Fax: +34 91 5642431; Tel: +34 91 7459501
b
Instituto de Quı
´
mica Orga
´
nica General, C.S.I.C., Juan de la Cierva 3,
28006 Madrid, Spain
c
Departamento de Quı
´
mica I, Universidad de Alcala
´
, E-28871,
Alcala
´
de Henares (Madrid), Spain
Electronic supplementary information (ESI) available: (1) General experimental
methods, (2) synthesis, purification and chemical characterization of oxocines
1a,b and azocines 2a,b, (3) reaction optimization and mechanism, (4) sample
titration plots of 1a and absorption and emission spectra of 1b, (5) NMR spectra
of products, (6) computational details: molecular orbitals, transition dipole
components, relative triplet energies and cartesian coordinates of oxo- and aza-
compounds. See DOI: 10.1039/c3cp52228h
Deceased on November 2011.
Received 27th May 2013,
Accepted 29th July 2013
DOI: 10.1039/c3cp52228h
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medicinal infusion.
7
The glucosilated fluorophore was found to
be preformed in another tree (Pterocarpus indicus Willd.) from
the Philippine Islands.
7
The unusual four-ring structure of the
natural dye was confirmed by reproducing the intramolecular
oxidation reaction with a synthetic dihydrochalcone analog,
7
which yielded the dye fluorescent core 1a (Fig. 1).
Recently, we discovered that 1a,aswellasanewfamilyof
strongly fluorescent oxocines, can also be prepared from catechols
(o-diphenol compounds) by a simple, one-step bimolecular reaction
in water solution at room temperature. This is an example of
a fluorogenic reaction
8
in which a non-emissive or weakly
UV-emitting compound (catechol) is quickly converted into a
bright VIS-emitting product. Moreover, monoamine o-diphenol
compounds (aminocatechols) of biological relevance, such as the
neurotransmitter dopamine and the amino acid DOPA (3-(3,4-
dihydroxyphenyl)-rac-alanine), also undergo the fluorogenic reac-
tion. Remarkably, the four-ring fluorescent azocine product of DOPA
reaction (2b) had been identified previously in a detailed investiga-
tion of oxidative transformation of o-quinones, a process related to
melanogenesis and insect cuticle sclerotization.
9
Here we describe in detail the fluorogenic reactions of pro-
totype catechols (hydroxytyrosol and salvianic acid) and amino-
catechols (dopamine and DOPA), as well as the spectroscopic
characterization of the corresponding fluorescent products
(Fig. 1): monardine
10
(1a), carboxymonardine (1b), azamonar-
dine (2a) and carboxyazamonardine (2b). The pH-dependent
spectral properties and fluorescence polarization data of these
compounds are reported, and an attempt is made to interpret
the absorption and emission experimental observations of the
different prototropic species in terms of the corresponding large
changes in singlet and triplet excited-state distribution, as
computed by TDDFT quantum chemical methods.
Experimental
Chemical methods
Oxocines 4,9-dihydroxy-1,2-dihydro-4,11a-methanooxocino[4,5-b]-
benzofuran-5(4H)-one (1a, monardine) and 4,9-dihydroxy-5-oxo-
1,2,4,5-tetrahydro-4,11a-methanooxocino[4,5-b]benzofuran-2-
carboxylic acid (2a, carboxymonardine)wereobtainedthrough
reaction of resorcinol (3) with hydroxytyrosol (4a) or salvianic
acid (4b), respectively, in alkaline water solution (Scheme 1).
A similar reaction with dopamine (5a)orDOPA(5b)yielded
the corresponding azocines 4,9-dihydroxy-3,4-dihydro-1H-4,11a-
methanobenzofuro[2,3-d]azocin-5(2H)-one (2a, azamonardine)or
4,9-dihydroxy-5-oxo-2,3,4,5-tetrahydro-1H-4,11a-methanobenzofuro-
[2,3-d]azocine-2-carboxylic acid (2b, carboxyazamonardine). Solvent
and reactant sources, reaction conditions, isolation and full
chemical characterization of the fluorescent reaction products
maybefoundintheESI.
Spectroscopic methods
Steady-state fluorescence excitation, emission and polarization
spectra were recorded using a photon-counting PC1 fluorometer
(ISS, US). All spectra and wavelengths shown here pertain to
corrected spectra. Instrument correction factors were determined
in-house using a Rhodamine B quantum counter
11
and a calibrated
tungsten-ribbon radiance source for the excitation and emission
channels, respectively. Emission anisotropy values were computed
from polarized intensities and corrected as described elsewhere.
12
Apparent ionization constants pK
a
were determined from absorp-
tion and/or fluorescence intensity titration
13
in the 2–12 pH range,
using dilute water solutions of the ‘‘universal’’ Britton–Robinson
buffer containing mixtures of sodium salts of acetic, boric and
phosphoric aci ds.
14
Time-resolved fluorescence data were acquired
by the time-correlated single-photon counting technique, exci ting
the samples at 375 or 407 nm with pulsed diode lasers (PicoQuant
GmbH, Germany) at an 8 MHz repetition rate. The instrument
response function of the detection channel, that included a 10 cm
monochromator and a R1564U microchannelplate photomultiplier
(Hamamatsu Photonics, Japan), was B70 ps. Decay traces were
analyzed as multiexponential functionsbyNLLSmethods,using
the Globals software package developed at the Fluorescence
Dynamics Laboratory, Univ. of Illinois, Urbana-Champaign (U.S.).
Fig. 1 Chemical structure of monardine (MON, 1a), carboxymonardine (CMON,
1b), azamonardine (AZMON, 2a) and carboxyazamonardine (CAZMON, 2b).
Scheme 1 Synthesis of monardine (1a), azamonardine (2a) and the corre-
sponding carboxy-substituted derivatives. Reagents and conditions: equimolar
quantities of 1,3-dihydroxybenzene (resorcinol, 3) and the corresponding
1,2-dihydroxybenzene derivative (catechols 4 or aminocatechols 5) were dis-
solved in alkaline water (pH 8–11) in the presence of air at room tempe rature.
See ESI† for a detailed description of synthetic procedures.
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The fitting function was considered adequate if a minimum of
variable parameters yielded w
2
values in the 0.9–1.2 range and a
random distribution of weighted residuals. Nanosecond
fluorescence lifetimes are given with 0.05 ns uncertainty. See
ESI† for additional technical details.
Computational methods
The systems investigated in this work have been modeled using
density functional theory (DFT). Ground-state equilibrium
structures of the different molecules were computed using
the HSE06 functional
15
and Pople’s 6-31++G(d,p) basis set.
Solvation effects have been simulated by including the integral
equation formalism of the polarizable continuum model
(PCM),
16
using H
2
O as solvent. Vertical excitation energies of
the lowest-lying singlet and triplet states have been computed by
time dependent density functional theory (TDDFT), using the
HSE06 correlation–exchange functional and the 6-311++G(d,p)
basis set to get a balanced description of low-lying valence and
Rydberg states. Since this functional was not designed for the
description of charge transfer states, the possible impact of these
states in the above computations was investigated using specific
correlation–exchange functionals designed for this purpose (see
ESI†). The effect of solvent on excitation energies was incorpo-
rated by means of the PCM approach within the linear response
(LR) version of the TDDFT method.
17
All the calculations have
been carried out using Gaussian09 program package.
18
Results
Absorption and emission properties of monardine (1a) and
carboxymonardine (1b)
Monardine (1a) is virtually the sole product at short reaction
times of the fast oxidative coupling between resorcinol and
hydroxytyrosol (Scheme 1), using atmospheric oxygen as the
oxidant. The absorption spectrum of 1a in slightly alkaline
water solution displays an intense, structureless absorption
band at 425.5 nm (Fig. 2 and Table 1), that is blue-shifted to
382 nm in a reversible way in acidic solution, with a large
decrease in the absorption coefficient (Table 1). These changes
in absorption spectra are assigned to a prototropic equilibrium
between neutral (N) and anionic (A) forms, due to ionization of
the OH-group at position 9, as discussed below. The two species
are chemically and photochemically stable in the 3–12 pH range.
The anionic form shows a strong blue fluorescence, peaking
at 464 nm, with a Stokes shift of B2000 cm
1
and a quantum
yield f
F
= 1.0 0.1, while the neutral form is very weakly
emissive (Fig. 2 and Table 1). As expected, the shape of the
anion fluorescence band is a mirror image of the absorption
one. Absorption and fluorimetric titrations yielded the same
value for the prototropic equilibrium constant: pK
a
= 7.2 0.4,
20 1C (see Fig. S1 of the ESI† for sample titrations). The
fluorescence parameters of monardine listed in Table 1 were
obtained at pH values where the excitation and emission
spectra are independent of the emission–excitation wave-
lengths, indicating that a single molecular species populates
the ground state; the anion fluorescence decay is accurately
monoexponential, with a lifetime of 2.74 ns. Preliminary
experiments indicated that the monardine anion also presents
a large two-photon fluorescence excitation cross-section, with
l
max
B 400 nm. The two-exponential fit of the neutral form
weak emission yields a B70 ps major component (99.9%),
limited by the time resolution of our methods, and a residual
(o0.1%) slower decaying component (2.7 ns).
The reaction between resorcinol and salvianic acid
(Scheme 1) also proceeds quickly to yield carboxymonardine,
(1b), as a major reaction product. The spectroscopic properties
in acidic and alkaline solution, as well as the pK
a
value, iterate
those of the unsubstituted chromophore (Table 1 and Fig. S2 of
the ESI†). In alkaline solution (pH 9) carboxymonardine (1b)
should be in dianionic form, due to the ionization of both the
9-OH and 2-COOH groups. However, neither the carboxylic nor
the carboxylate groups affect the spectral properties of the
neutral and dianionic forms, which remain virtually identical
to those of the unsubstituted dye 1a.
Polarized fluorescence excitation and emission spectra of
the carboxymonardine dianion in alkaline glycerol at low
temperature (5 1C) are presented in Fig. 3 and 4. Compound
1b was selected for these experiments, instead of the parent dye
1a, to decrease further re-orientational motions within the
highly viscous glycerol matrix. The 380–460 nm range corresponds
to the S
0
–S
1
absorption band, approximately, with an average
anisotropy value r
ss
=0.383 0.006 very close to the one-photon
theoretical 2/5 value for coincident absorption and emission
transition moments. Instrument and sample-dependent factors
may account for this small discrepancy.
19
Absorption and emission properties of azamonardine (2a) and
carboxyazamonardine (2b)
The reaction of resorcinol with dopamine and DOPA yields 2a
and 2b, respectively, the aza-analogues of 1a,b, which are also
fluorescent. The spectroscopic properties of these compounds
depend on solution pH in a more complex way than in the
Fig. 2 Absorption and fluorescence spectra of neutral (N) and anionic (A) forms
of monardine, 1a, in aqueous solution. A
N
and F
N
: absorption and corrected
fluorescence spectra of the neutral species at pH 4. A
A
and F
A
: the corresponding
spectra of the monoanion species from the same solution at pH 9. [1a] E
2 10
6
M, l
exc
= 425 2 nm, T =201C.
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oxo-chromophore. In acidic solution (pH 4) the shape of the
absorption spectrum of azamonardine (2a) is very similar to
that of monardine neutral form (Table 1 and Fig. 5) but slightly
red-shifted. Approaching neutral solution (BpH 6) the absorption
spectrum shifts to the blue, with small changes in the band shape
and the absorption coefficient (Fig. 5), indicating the presence of a
new species in the ground state. Finally, at pH 9–10 an intense
absorption band at 419 nm dominates the spectrum, with the
shape and the absorption coefficient similar to those of monardine
anionic form. This anion form is strongly fluorescent, with a
quantum yield of B50% (Table 1). These spectral changes are
fully reversible and were assigned to different prototropic forms
of the aza-compound as discussed below, i.e., to cationic (C),
neutral (N) and monoanion (A) forms of 2a. The spectroscopic
parameters of the neutral form cannot be determined with
accuracy due to the large overlap with the corresponding
spectra of the cationic and anionic species. Nevertheless, it
was found that both cationic and neutral forms are fluorescent
(Fig. 5), with f
F
(N) E 3 f
F
(C). Two macroscopic pK
a
values
Table 1 Absorption and fluorescence properties of monardine (1a, MON), carboxymonardine (1b, CMON), azamonardine (2a, AZMON) and carboxyazamonardine
(2b, CAZMON) in water solution as a function of pH
a
Compound pH l
max
/nm (e/M
1
cm
1
) l
F
/nm f
F
c
t
F
/ns pK
a
1a, MON 4 260 (n.d.)
b
, 305 (8680 100), 382 (22 930 600) 453 0.018 0.07
d
7.2 0.4
9 275 (5070 200), 324 (2830 200), 425.5 (48 600 1100) 464 1.0 2.74
1b, CMON 4 260 (n.d.), 305 (8160 100), 382 (21 200 600) 453 0.02 0.09
d
7.4 0.4
9 275 (4660 200), 324 (2810 200), 425.5 (44 200 300) 464 1.0 2.74
2a, AZMON 3 258 (n.d.), 308 (8100 400), 389 (25 000 100) 460 0.16
e
5.0 0.5
6 B301 (9000)
f
, B377 (22 600)
f
(n.d.) (n.d.) (n.d.) 7.6 0.4
10 275 (5000 200), 320 (2895 200), 419 (47 250 1300) 462 0.47 h1.23i
g
2b, CAZMON 3 258 (n.d), 308 (7820 400), 389 (24 700 600) 460 0.25
e
5.0 0.5
6 B301 (8400)
f
, B377 (21 500)
f
(n.d.) (n.d.) (n.d.) 7.6 0.4
10 275 (5700 200), 320 (3015 200), 419 (44 680 1300) 462 0.45 h1.14i
g
a
pK
a
: apparent ioniz. constant.
b
Not determined.
c
10%.
d
Major component (99.9%).
e
See Table 2.
f
Estimated values of the (N) form.
g
hti =
P
a
i
t
i
.
Fig. 3 Corrected fluorescence excitation intensity (TT ) and anisotropy ()
spectra of carboxymonardine (1b) dianion in 99.5% alkaline glycerol solution
at 2 10
6
M, pH E 10, l
em
= 470 4 nm, T =51C.
Fig. 4 Corrected fluorescence emission intensity (TT ) and anisotropy () spectra
of carboxymonardine (1b) dianion in 99.5% alkaline glycerol solution at 2
10
6
M, pH E 10, l
exc
= 420 1 nm, T =51C.
Fig. 5 Absorption and fluorescence spectra of different prototropic forms of
azamonardine (2a) in aqueous solution. A
C
and F
C
: absorption and fluorescence
spectra of the cationic species at pH 4. A6: absorption spectrum at pH 6. A
A
and
F
A
: absorption and fluorescence spectra of the anionic species at pH 9.[2a] E
10
6
M, l
exc
= 389 (cation), 419 nm (anion) 2 nm, T =201C.
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could be estimated from absorption and/or fluorescence titra-
tions, with different levels of uncertainty, as shown in Table 1.
The time-resolved analysis of the emission of the protonated
and anionic forms of 2a indicates a complex excited-state
behavior (Table 2). Fitting the emission decay in acidic solution
at pH 3 (cationic species) required a numerical model with a
rise-time component and two decay times (Table 2); in addition,
the decay of monoanion fluorescenceinalkalinesolution(pH10)
is clearly biexponential. The analysis of the emission at near-
neutral pH is further complicated by the simultaneous presence
of all prototropic forms, and was not investigated further.
Carboxyazamonardine (2b) spectral properties depend on
pH in the same way as those of the unsubstituted dye, and three
prototropic absorbing species can also be postulated. These
properties, listed in Tables 1 and 2, are also virtually identical to
those of the unsubstituted dye 2a, apart from some differences
in the fluorescence lifetime of the dianion species.
Discussion
Monardine prototropic species
The fluorogenic reaction between hydroxytyrosol and resorcinol
yields 1a, a very stable aromatic hemiacetal characterized by
large changes in absorption and emission properties as a
function of solution pH. The highly fluorescent species giving
rise to the intense absorption at 425.5 nm is most likely the
phenolate anion (A) formed by ionization of the 9-OH group of
the neutral species N (Fig. 6) as in the related compound
matlaline,
7
since compound 1a does not contain alternative
ionizable groups in the near-neutral pH range. The experimental
pK
a
value (7.2) of the neutral species (Table 1) is comparable to
that of substituted 7-OH coumarins,
20
and the electronic factors
giving rise to the increased acidity of the 9-OH phenol group, as
compared to that of simpler monophenol compounds, are also
expected to be similar.
21
Interestingly, monardine ionization
equilibrium provides a fluorescence equivalent to a binary
switch.
22
Monardine electronic transitions and photophysics
Theoretical values of singlet and triplet relative energies and
vertical electronic transitions for the neutral and anionic forms
of the dye were estimated by quantum-chemical computations at
the TDDFT level (HESE06/6-311++G(d,p)//HSE06/6-31++G(d,p)).
As indicated above, the aqueous environment was simulated
using the PCM approximation. In this way, specific dye–water
interactions (which should be more important for the anionic
species) have not been considered. The calculated geometrical
parameters of ground-state monardine (1a,4S,11aS)(Fig.7and
ESI†) agree quite well with the corresponding experimental bond
distances and angles determined from the single-crystal X-ray
structure of 1b methyl ester.
7
The molecular plane defined by the
phenyl and furanyl rings deviates very little (B101)fromthat
defined by the C6aQC6 double bond and the carbonyl group.
The deviation from coplanarity is even lower for the anion
species (B81), facilitating the formation of a p-electron conjugated
system extending over the whole molecule. Bond distances C7–C8
and C10a–C10 are shorter than other C–C bond lengths in the
phenyl ring, indicating a quinoid character of this cycle
23
which
increased in the anion form (Fig. 6). The molecular framework is
very rigid, due to the locking effect of the tetrahedral carbon atom
C12, among other factors.
The relative energy and electronic character of the five
lowest lying singlet and triplet states of neutral monardine,
computed as detailed above, are shown in Table 3. The maximum
of the first experimental absorption band (382 nm, 3.25 eV)
compares reasonably well with the optically allowed vertical excita-
tion to the S
1
state, represented mostly by a HOMO - LUMO (p,p*)
transition. Excitation to the S
2
state, nearly degenerated with S
1
within the accuracy of the method, is optically forbidden due to the
dominant HOMO 1 - LUMO (n,p*) transition (see ESI†). The
remaining absorption bands, observed at 305 nm (4.07 eV) and
260 nm (4.77), correspond nicely with the excitation energy and
character of S
3
and S
5
(Table 3). In the triplet manifold two different
states T
2
(p,p*) and T
3
(n,p*) are located very close in energy to the
lowest S
1
and S
2
states. According to the experimental observations,
the lowest-lying excited singlet of the neutral species in aqueous
solution is very efficiently deactivat ed. Vibronic interactions
between close-lying S
1
(p,p*) and S
2
(n,p*) states would enhance
Table 2 Time-resolved fluorescence decay of azamonardine (2a) and carboxy-
azamonardine (2b) in acidic and alkaline aqueous solution
a
Compound pH l
exc
l
em
a
1
a
2
a
3
t
1
t
2
t
3
2a, AZMON 3 375 460 0.2 0.78 0.02 0.22 0.5 2.5
10 407 470 0.77 0.23 1.04 1.86
2b, CAZMON 3 375 460 0.2 0.77 0.03 0.24 0.67 2.1
10 407 470 0.52 0.48 0.8 1.5
a
Uncertainty of pre-exponential factors and lifetimes (ns) B10%.
Fig. 6 Neutral (N) and anionic (A) species of monardine (1a) in aqueous
solution.
Fig. 7 Optimized geometry of ground-state neutral and anionic forms of
monardine (1a,4S, 11aS).
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the rate of intersystem crossing to nearby triplet states,
24
further
favored by spin–orbit coupling between singlet and triplet states of
different electronic symmetry,
25
as well as other radiationless
processes. As a result of that the fluo rescence of neutral monardine
would be largely suppressed.
In the anion species, the large changes in the absorption
spectrum are also well reproduced by the computed data
(Table 3). The intense absorption at 426 nm (2.9 eV) can be
assigned to excitation to the optically allowed S
1
(p,p*) state
(3.3 eV), which in the anion is stabilized relative to the S
2
(n,p*)
state preventing interstate coupling. Note that the calculated S
1
energy is overestimated due to the known limitations of the
PCM approach in accounting for specific solvation effects of the
charged phenolate species. The weak absorption bands at
320 nm (3.8 eV) and 275 nm (4.5 eV) are assigned to excitation
to S
3
(p,p*) and S
5
(p,p*) states, respectively, which also show a
reduced oscillator strength. This assignment is also consistent
with the anion fluorescence excitation anisotropy spectrum
depicted in Fig. 3. According to the computed data (Table S1,
ESI†), the S
0
–S
1
transition moment is essentially contained
within the molecular plane, as defined above, and aligned with
the long molecular axis, while transition moments S
0
–S
3
and
S
0
–S
5
are oriented +611 and 601 relative to S
1
, and also mostly
included in that plane, as illustrated in Fig. 8. The spectrum of
Fig. 3 shows an important decrease of anisotropy by excitation
in the 320 nm range (S
3
), not as large as expected due probably
to vibrational coupling of this weakly allowed state or to overlap
with high vibrational states of S
1
. The high fluorescence efficiency
of the anion is associated to the S
1
(p,p*) excited state, with large
oscillator strength and well separated from the nearest (n,p*)
state, and to a very rigid molecular framework. In addition, the
unusual distribution of triplet states (Table 3) is likely the crucial
factor behind the 100% emission efficiency. There is a large
energy gap between S
1
and T
1
,whichareofthesameelectronic
configuration, while the closely grouped T
2
–T
5
states are expected
to be located at higher energy than the solvent-relaxed emitting
state.Asaresultofthattheintersystemcrossingratebecomes
negligible.
It was shown above that the carboxy-substituted compound
1b displays identical spectral properties and pH-dependent
changes as 1a. The covalent binding of dyes to biomolecules
is frequently achieved by the reaction of amino or sulfhydryl
groups of the target compound with functionalized dye deriva-
tives. Therefore, the carboxylic group at position 2 in 1b
appears as an ideal point to attach a reacting handle while
preserving the favorable spectral properties of the dye. The
saturated structure of this part of the molecule very effectively
Table 3 Monardine (1a): relative energy (D E, eV), oscillator strength (f), weight of the most significant singlet excitation contribution (MSE) and character of the five lowest-lying singlet and triplet excited states of the
neutral and anionic species in aqueous solution, computed at the LR-TDDFT-PCM level of theory
a
(see ESI)
Neutral monardine Monardine anion
Singlets Triplets Singlets Triplets
State DE
f
MSE Character State D E Character State DE
f
MSE Character State DE Character
S1 3.4 0.396 HOMO - LUMO (0.60) (p,p*) T1 2.2 (p,p*) S1 3.3 0.973 HOMO - LUMO (0.71) (p,p*) T1 1.9 (p,p*)
S2 3.5 0.148 HOMO 1 - LUMO (0.59) (n,p*) T2 3.2 (n,p*) S2 3.6 0.000 HOMO 2 - LUMO (0.70) (n,p*) T2 3.3 (p,p*)
S3 4.1 0.245 HOMO 2 - LUMO (0.62) (p,p*) T3 3.4 (p,p*) S3 3.8 0.015 HOMO 1 - LUMO (0.62) (p,p*) T3 3.4 (n,p*)
S4 4.7 0.001 HOMO 4 - LUMO (0.51) (n,p*) T4 3.9 (n,p*) S4 3.9 0.007 HOMO 3 - LUMO (0.61) (n,p*) T4 3.5 (n,p*)
S5 5.0 0.038 HOMO - LUMO + 1 (0.66) (
p,p*) T5 4.0 (p,p*) S5 4.5 0.048 HOMO - LUMO + 1 (0.69) (p,p*) T5 3.6 (n,p*)
a
HSE06/6-311++G(d,p)//HSE06/6-31++G(d,p).
Fig. 8 Orientation of the transition dipole moment of the lowest-lying (p,p*)
excitations in monardine anion.
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isolates the p conjugated system from intramolecular electronic
interactions.
Azamonardine prototropic species
Dopamine and DOPA yield the aza-analogues of monardine 2a
and 2b (Fig. 1). The carboxy-substituted compound 2b was first
isolated and identified by Crescenci et al.
9
in a study of the
mechanism of the melanogenesis reaction in vitro,asmentioned
above. These authors also noted the fluorescence of the novel
compound which, presumably, was a mixture of 2b prototropic
species.
The overall features of the absorption and emission spectra
of azamonardine in the 4–9 pH range are similar to those of
monardine (Fig. 5 and Table 1); there is also a strongly absorb-
ing species under near-neutral conditions (pK
a
= 7.6) with high
fluorescence quantum yield B0.5 (Table 1). On the other side,
the detailed emission properties of azamonardine are much
more complex than those of the oxo-compound. Thus, in the
pH range 4–6 a protolytic equilibrium between two species with
a similar absorption spectrum can be detected (Fig. 5). The
species appearing near pH 4 is very likely the cationic form of
2a (C in Fig. 9) with a protonated amino group. This species is
fluorescent, with a quantum yield of 0.16 and an emission
spectrum similar to that of the anionic form (Table 1 and Fig. 5).
Increasing solution pH yields a new blue-shifted absorbing
species which is also fluorescent, tentatively assigned to the
neutral form N(1) (pK
a
E 5) shown in Fig. 9, in which the 9-OH
group is not ionized. This assignment is based on the absence of
the intense red-shifted absorption band characteristic of the
phenolate group, although a zwitterionic form N(2) cannot be
ruled out. In fact, the emission spectrum (not shown) and
quantum yield (E0.4) of the neutral form are very similar to
those of the anionic species. These observations may be
conciliated if the dominant structure in the emitting singlet-
state is the zwitterion N(2), as a result of proton dissociation due
to the increase in acidity of the 9-OH group in the excited
electronic-state.
26
Finally, the strong absorption band and
intense fluorescence appearing in alkaline solution (Fig. 5) are
likely due to the anionic species (A) characterized by a pK
a
value
similar to that of 1a. The anion fluorescence decay is clearly
biexponential (Table 2), with lifetimes E1and2ns,presumably
due to some excited-state process, because under the conditions
of the lifetime experiments (pH = 10) only one prototropic form is
to be found in the ground-state.
Azamonardine electronic transitions and photophysics
TDDFT methods, as detailed above, were also applied to the
neutral, cationic and anionic species of azamonardine (Fig. 9)
in water solution, and the most relevant data are listed in
Table 4 and in Section S6 of the ESI.† The computed bond
distances and angles of the neutral N(1) and anion species of
the aza-compound 2a are very similar to those of the oxo-
compound (see Fig. S5 of the ESI†); in fact, the geometry of
the phenol and furanyl rings is virtually identical to that of 1a.
The experimental absorption maxima of the cationic C and
neutral N(1) forms compare well with the computed vertical
excitation energies and oscillator strengths. The observed
bands can be assigned to the corresponding three optically-
allowed lowest (p,p*) transitions listed in Table 4. The intense
absorption of the anion form at 419 nm (2.96 eV) should be
assigned to the S
1
(p,p*) transition in Table 4; as noted above
the computed energy of this transition is overestimated (3.3 eV)
due to the limitations of the PCM model. The cationic species
of azamonardine displays a modest fluorescence yield (0.16)
consistent with the (p,p*) character of the computed lowest
excited-state S
1
(Table 4). The time-resolved emission of this
form required a three-exponential function with one negative
pre-exponential coefficient (Table 2), indicating that at least a
fraction of the emitting species is being produced in the excited
state. Solvent-mediated proton-transfer or intramolecular
photo-tautomerization reactions in the electronically excited-
state may be responsible for the observed rise-time in the
200 ps time-range.
The high fluorescence yield of the anion form (0.46) is
probably related to a distribution of singlet and triplet states
similar to that of monardine. The energy separation between
S
1
(p,p*) and S
2
(n,p*) excited-states is relatively large (0.3 eV)
preventing interstate coupling, and T
2
–T
5
states are located at
higher energy than S
1
(see Table S4 in the ESI†). In fact, the S–T
energy separation would be even higher for the solvent-relaxed
S
1
state, reducing intersystem crossing de-activation.
The emission properties of the species appearing at pH 6
could only be determined with a large uncertainty, as noted
above. Nevertheless, it may be shown that both fluorescence
band shape and quantum yield are similar to those of the anion
species. These properties are not consistent with the computed
singlet-state distribution of the neutral N(1) species presented
in Table 4, in which the lowest excited state is an optically-
forbidden (n,p*) state. In fact, these properties might be better
Fig. 9 Cationic C, neutral N(1), zwitterionic N(2) and anionic A forms of
azamonardine (2a) in aqueous solution.
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matched by the excited-state distribution of zwitterionic aza-
monardine, in which the lowest singlet excited-state is of (p,p*)
character with large oscillator strength. However, it should be
recalled that HSE06 functional was not specifically designed for
charge transfer states (see ESI† for further details of this assign-
ment). As noted above, the absorption and emission properties of
the species appearing at pH 6 might indicate that an excited-state
proton-transfer reaction of the absorbing neutral form N(1) is
taking place.
The spectral properties and complex excited-state kinetics of
the carboxy-substituted aza compound 2b are similar to those
of the parent 2a, apart from small differences in quantum yield
and emission decay. This again reflects the absence of large
perturbing effects of the carboxylic group at position 2. The
structure of the 2b cationic species would be similar to that of
2a shown in Fig. 9, apart from the carboxylic group. In alkaline
solution, pH > 8, a dianionic species would be present, due
to ionization of the 9-OH and 2-COOH groups, as in the oxo-
compound. Near neutral pH the absorption spectrum of 2b is
consistent with a non-ionized 9-OH group, while the emission
approaches that of 2b phenolate, as in the unsubstituted com-
pound. However, the assignment of ground-state species is much
more uncertain in the present case, due to additional zwitterion
formsasCOO
/NH
+
, and was not investigated further.
Conclusions
A simple, one-step fluorogenic reaction between hydroxytyrosol
and resorcinol in aqueous solution yields the novel oxocine dye
4,9-dihydroxy-1,2-dihydro-4,11a-methanooxocino[4,5-b]benzofuran-
5(4H)-one (1a, monardine) essentially as a single reaction product.
The neutral form of 1a is weakly fluorescent while the anion
species, generated by 9-OH group ionization (pK
a
=7.2),shows
strongly polarized fluorescence in the blue range with unity quan-
tum yield and single-exponential decay. Based on high-level quan-
tum chemical calculations this switching of the fluorescence yield
was interpreted as due to the reversal of (p,p*) and (n,p*) lowest
singlet excited-states and a crucial change in the triplet-state
distribution that accompanies monardine ionization. The
convenient spectral properties of the dye are not affected by
the presence of an additional reacting group, as shown here for
the 2-carboxy compound 1b that results from the fluorogenic
reaction of salvianic acid.
The fluorogenic reaction also takes place with monoamino
catechols, as illustrated here with the reaction of dopamine
and DOPA to yield the strongly fluorescent aza-analogues of
monardine 2a and 2b, with emission efficiency as high as E0.5.
The presence of the amino group gives rise to three prototropic
species of azamonardine (2a) in the pH range 3–8, with differ-
ent emission efficiency and complex excited-state properties.
The fluorogenic reactions of aminocatechols may have useful
applications for detecting very low amounts of these important
neurochemicals. Overall, th e interesting spectroscopic properties of
the novel blue fluorophores and the relative simplicity of the
fluorogenic reactions in water solution may be of utility in a variety
of applications.
Table 4 Azamonardine (2a): relative energy (D E, eV), oscillator strength (f), weight of the most significant singlet excitation contribution (MSE) and character of the five lowest-lying singlet states of neutral, cationic and
anionic species in aqueous solution, computed at the LR-TDDFT-PCM level of theory
a
(see also ESI)
Neutral azamonardine Azamonardine cation Azamonardine anion
State D E
f
MSE Character State D E
f
MSE Character State D E
f
MSE Character
S1 3.2 0.007 HOMO 1 - LUMO (0.69) (n,p*) S1 3.4 0.549 HOMO - LUMO (0.68) (p,p*) S1 3.3 0.971 HOMO - LUMO (0.70) (p,p*)
S2 3.5 0.539 HOMO - LUMO (0.68) (p,p*) S2 3.9 0.171 HOMO 1 - LUMO (0.61) (p,p*) S2 3.6 0.003 HOMO 1 - LUMO (0.69) (n,p*)
S3 4.2 0.223 HOMO 2 - LUMO (0.67) (p,p*) S3 4.0 0.099 HOMO 2 - LUMO (0.61) (n,p*) S3 3.7 o0.000 HOMO 3 - LUMO (0.70) (n,p*)
S4 4.5 0.012 HOMO 3 - LUMO (0.66) (n,p*) S4 5.0 0.016 HOMO - LUMO + 1 (0.60) (p,p*) S4 3.9 0.014 HOMO 2 - LUMO (0.70) (p,p*)
S5 5.0 0.045 HOMO - LUMO + 1 (0.66) (
p,p*) S5 5.2 0.047 HOMO 3 - LUMO (0.58) (p,p*) S5 4.5 0.054 HOMO - LUMO + 1 (0.69) (p,p*)
a
HSE06/6-311++G(d,p)//HSE06/6-31++G(d,p).
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Phys. Chem. Chem. Phys.
Acknowledgements
We thank Drs B. Rodriguez and O. Castan
˜
o for helpful discussions,
and Dr P. Lillo for experimental assistance. PBC thanks Prof. L. M.
Frutos for his hospitality during his stay in the RESMOL group at
the University of Alcala
´
. M.L. acknowledges a JAE-Doc Grant from
Consejo Superior de Investigaciones Cientı
´
ficas (CSIC). This work
was financed by Grants CTQ-2010-16457 (AUA) and CTQ2012-36966
(PBC) of the Min. Ciencia Innov. Spain.
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Supplementary resources

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    As a well-known copper-containing oxidase, tyrosinase has been anticipated to serve as the biomarker of skin diseases. We describe here an exquisite label-free fluorescent and colorimetric dual-readout assay of its activity, inspired by the specific oxidation ability of monophenolamine substrates to catecholamines and a unique fluorogenic reaction between resorcinol and catecholamines. By employing commercially available tyramine as the model substrate (dopamine as the product), it is found that the tyrosinase-incubated tyramine solution exhibits obvious pale yellow with intense blue fluorescence in the presence of resorcinol and O2, where the absorbance and fluorescence intensity are directly related to the concentration of added tyrosinase (i.e., the amount of conversion of tyramine to dopamine). The overall process of sensing tyrosinase activity takes less than 100 minutes at ambient temperature and pressure conditions with exceedingly simple operation procedure, explicit response mechanism, and formation of fluorophore with high quantum yield from scratch. Furthermore, such a convenient, rapid, cost-effective, and highly sensitive dual-readout assay exhibits promising prospect for the tyrosinase activity in extensive bioassays and clinic researches, as well as in screening potential tyrosinase inhibitors.
  • ... After reaction, the characteristic adsorption peak of DA is disappeared which indicates the DA has reacted completely. Additionally, a new strong adsorption peak at 420 nm demonstrates the generation of aza- monardine [30]. The fluorescence emission spectrum displays a single emission peak at 475 nm when excited at 420 nm, and the fluorescence intensity as a function of reaction time with different alkali concentrations. ...
    ... At optimized pH, the 11- OH group in azamonardine becomes anionic form resulting in strong fluorescence. This result is similar to reference [30]. ...
    ... It is found that the reaction with Na 2 CO 3 needs shorter time to equilibrate than that with NaOH at any concentrations (Fig. 3b). The possible reason is that the reaction is associated with oxygen concentration in solution [30]. NaOH tends to absorb CO 2 from atmosphere, which may cause the decrease of dissolved oxygen in solution, and slows down the reaction . ...
    Article
    A simple, fast and low-cost method for dopamine (DA) detection based on turn-on fluorescence using resorcinol is developed. The rapid reaction between resorcinol and DA allows the detection to be performed within 5 min, and the reaction product (azamonardine) with high quantum yield generates strong fluorescence signal for sensitive optical detection. The detection exhibits a high sensitivity to DA with a wide linear range of 10 nM–20 μM and the limit of detection is estimated to be 1.8 nM (S/N = 3). This approach has been successfully applied to determine DA concentrations in human urine samples with satisfactory quantitative recovery of 97.84%–103.50%, which shows great potential in clinical diagnosis.
  • Chapter
    The determination of ionization constants by ultraviolet or visible spectrophotometry is more time-consuming than by potentiometry. However, spectrometry is an ideal method when a substance is too insoluble for potentiometry or when its pK a value is particularly low or high (e.g. less than 2 or more than 11). The method depends upon the direct determination of the ratio of molecular species (neutral molecule) to ionized species in a series of non-absorbing buffer solutions (whose pH values are either known or measured). For this purpose, the spectrum of the molecular species must first be obtained in a buffer solution whose pH is so chosen that the compound to be measured is present wholly as this species. This spectrum is compared with that of the pure ionized species similarly isolated at another suitable pH. A wavelength is chosen at which the greatest difference between the absorbances of the two species is observed. This is termed the ‘analytical wavelength’. Fig. 4.1, in which the base 2-amino- pyridine is used as an example, shows how these two pH values can be found.
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    After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measurements are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature.
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    Under biomimetic conditions, both phloroglucinol and resorcinol cause significant inhibition of the oxidative conversion of dopa to dopachrome, a key step in the biosynthesis of melanin pigments. The major product (λmax = 377 nm) from oxidation of dopa in the presence of phloroglucinol was characterized as 2, containing the unusual tetrahydromethanobenzofuroazocine ring system. Another product, formed in somewhat smaller amount, was formulated as 3, arising from coupling of phloroglucinol with 2 dopaquinone derived units. Analogous cross coupling adducts 4 and 5 were obtained by reaction of dopa or dopa methylester with resorcinol, respectively. These results suggest the trapping of labile dopaquinone, generated in situ from oxidation of dopa, by the reactive phenolic compounds, leading to the novel adducts 2, 4 and 5.
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    The first two examples of naturally occurring C-glucosyl-α-hydroxydihydrochalcones have been isolated from Eysenhardtia polystachya, a Mexica
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    The kinetics of enol—keto (lactim—lactam) photoautomerism of 7-quinolinol in methanol have been studied by picosecond fluorescence spcctroscopy using a laser—streak camera system. On excitation of the enol ground slate, approximately half of the excited enol tautomcrizes to the keto form. An upper limit to the tautomerization rate constant of 4.8 × 109 s−1 has been determined.
  • Article
    The quantum statistical approach to relaxation phenomena was employed in a numerical investigation of the effect of vibronic interaction between the first and second excited states on the radiationless decay rate of the first excited state. It is shown that the vibronically active out‐of‐plane modes may be the dominant accepting modes for the radiationless transitions of N‐heterocyclics with close‐lying nπ∗ and ππ∗ excited states. The decay rate was found to vary with the strength of nπ∗–ππ∗ vibronic interaction, the nπ∗ and ππ∗ separation, the energy gap between the initial and final electronic states of the radiationless process, and isotopic substitution, in a manner which is consistent with experimental observation.
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    Design and synthesis of various types of photoswitchable fluorescent molecules, which are applicable to “single-molecule optical memory” and “super-resolution fluorescence microscopy”, have been reviewed.
  • Article
    The kinetics and mechanism of the reaction in the photo-excited state of 7-hydroxycoumarin and its derivative have been studied in aqueous solutions by means of measuring the fluorescence spectra and the fluorescence lifetime. The four fluorescence bands have been identified as emissions from the neutral, anionic, tautomeric, and cationic forms of 7-hydroxycoumarins. The quantitative analysis was performed by using the rate equation which included the term for the direct conversion of the excited neutral molecule to its tautomer in addition to the usual reaction term of dissociation. The rate constants of the tautomerization and dissociation were discussed in detail, and it was revealed that the tautomerization in aqueous solutions was mainly caused by the transfer of a proton between two active sites in the molecule. Such an intramolecular proton-transfer of the coumarins, found first in this paper, is considered to be promoted by water molecules with a hydrogen bond surrounding the excited molecule.