Tryptanthrin from indigo: Synthesis, excited state deactivation routes and efficient singlet oxygen sensitization

Article abstract of DOI:10.1016/j.dyepig.2019.108125

The microwave-assisted synthesis of tryptanthrin from indigo in mild oxidation conditions, and a comprehensive study of the excited state properties of this compound in a variety of solvents with different polarity and viscosity values at room and low temperatures are reported. In contrast with indigo, emission of the triplet state of tryptanthrin is observed with a very efficient singlet oxygen sensitization quantum yield, indicating that the triplet state is efficiently populated. From time-resolved fluorescence and femtosecond transient absorption data, further supported with time-dependent density functional theory (TDDFT) calculations, two species, with S₁ states with locally excited (LE) of π,π* nature and a charge transfer (CT) of n,π* characteristics, originated from an initially populated Frank-Condon S₂ state (π,π*), are observed. The two electronically independent species are energetically nearly degenerate and inter-conversion is predicted (and rate constants determined) to occur between LE (S₁) and CT (S₁) species. Due to the low value of the fluorescence quantum yield (~10^?3) and high triplet state yield (?_T≥?_Δ), the high stability of this compound is associated to the high efficiency of the radiationless deactivation processes which involve the formation of the CT state which efficiently converts, through S₁ ~~> T_n intersystem crossing, to the T₁ triplet state.

Full text of DOI:10.1016/j.dyepig.2019.108125

Journal Pre-proof
Tryptanthrin from indigo: Synthesis, excited state deactivation routes and efficient
singlet oxygen sensitization
Daniela Pinheiro, Marta Pineiro, João Pina, Pedro Brandão, Adelino M. Galvão,
J.Sérgio Seixas de Melo
PII:
S0143-7208(19)32032-7
DOI:
https://doi.org/10.1016/j.dyepig.2019.108125
DYPI 108125
Reference:
To appear in: Dyes and Pigments
Received Date: 28 August 2019
Revised Date: 24 October 2019
Accepted Date: 10 December 2019
Please cite this article as: Pinheiro D, Pineiro M, Pina Joã, Brandão P, Galvão AM, Seixas de Melo
JSé, Tryptanthrin from indigo: Synthesis, excited state deactivation routes and efficient singlet oxygen
sensitization, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2019.108125.
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Tryptanthrin from Indigo: Synthesis, Excited
State Deactivation Routes and Efficient Singlet
Oxygen Sensitization
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Daniela Pinheiroa, Marta Pineiroa, João Pinaa, Pedro Brandãoa,b,
Adelino M. Galvãoc, J. Sérgio Seixas de Meloa*
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aCQC, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535
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bCentro de Química de Évora, Institute for Research and Advanced Studies, University
of Évora, Évora, Portugal
cCentro de Química Estrutural, Instituto Superior Técnico (IST), Universidade de
Lisboa, Lisboa, Portugal
Keywords: Indigo; Tryptanthrin; Fluorescence; Singlet Oxygen Sensitization; TDDFT
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Email: sseixas@ci.uc.pt
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22 Abstract
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The microwave-assisted synthesis, of tryptanthrin from indigo in mild oxidation
conditions, together with a comprehensive study of the excited state properties of this
compound is made in a variety of solvents of different polarity and viscosity at room
and low temperatures. In contrast with indigo, emission of the triplet state of
tryptanthrin is observed with a very efficient singlet oxygen sensitization quantum
yield indicating that the triplet state is efficiently populated. From time-resolved
fluorescence and femtosecond transient absorption data, further supported with time-
depend density functional theory (TDDFT) calculations, two species, with S1 states
with locally excited (LE) of π,π* nature and a charge transfer (CT) of n,π*
characteristics, originated from an initially populated Frank-Condon S2 state (π,π*), are
observed. The two electronically independent species are energetically nearly
degenerate and inter-conversion is predicted (and rate constants determined) to occur
between LE (S1) and CT (S1) species. Due to the low value of the fluorescence quantum
yield (~10-3) and high triplet state yield (φT≥φ∆) the high stability of this compound is
associated to the high efficiency of the radiationless deactivation processes which
involve the formation of the CT state which efficiently converts, through S1 ~->Tn
intersystem crossing, to the T1 triplet state.
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2

41 Introduction
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Indigo, an iconic molecule of colour [1-5] with a splendid blue color and many
natural and synthetic derivatives [6-10] is also the synthetic precursor of
trypthanthrin (denoted as TRYP). Although with a pale yellowish color, TRYP
(indolo[2,1-b]quinazoline-6,12-dione) (Scheme 1) has gained relevance in the past
decades. TRYP is a weakly basic indoloquinazoline alkaloid isolated from various
natural sources [11], including indigo plants, [12, 13] fruits [14] and mammals (e.g.
urine of Asian elephant) [15, 16].
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Scheme 1 –Structures of TRYP (left) and indigo (right).
TRYP is known for exhibiting several biological and pharmacological activities
including anti-pathogenic, antifungal, anti-parasitic, anti-tuberculosis, antimalarial,
anticancer, antioxidant and anti-inflammatory [17-23]. Due to the scarcity of the
natural existence of TRYP, together with its unique bioactive properties, the synthesis
of this compound has generated great interest with the development of several
synthetic strategies reported [14, 17, 24-28], such as the oxidation of indigo or the
cyclization of isatin and isatoic anhydride [16, 29]. Despite these studies, significant
synthetic challenges still remain, since many of these methods are burdensome and are
not environmentally friendly [13, 16, 26, 27, 29, 30]. Therefore the development of a
new efficient, low cost, one-pot protocol to access TRYP is both attractive and valuable.
In recent years, several tryptanthrin derivatives have been synthesized and
studied for their potential optical applications, such as their use as fluorescent
chemosensors for metal ions and their potential as dyes in fluorescent imaging.
Successful fluorescent imaging in cells and tissues requires the application of dyes
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which absorb and emit in the “optical window” (concerning the near-infrared (NIR)
region of the electromagnetic spectrum – 650-900 nm). This selectivity is required to
avoid the interference of endogenous substances during the fluorescent imaging
process, as well as to improve tissue penetration levels [30-35].
Recent studies of tryptanthrin derivatives with high fluorescence quantum
yields have been reported [21, 36, 37]. However, only a small number of works
describe optical (absorption and fluorescence) properties of TRYP itself, highlighting
some of its more emissive derivatives [38]. Indeed, Kawakami and co-workers
reported that 2-aminotryptanthrin, when compared with other tryptanthrin
derivatives, display interesting photophysical properties including broad wavelength
absorption and emission bands in the visible region, high fluorescence quantum yield
(φF) and large positive fluorescence solvatochromism. Noteworthy, this fluorescence
solvatochromism was attributed to the planar and polar structure of the 2-
aminotryptanthrin with an intramolecular charge transfer (ICT) involving the carbonyl
group of the five-membered ring and the amino group [38].
In this work, TRYP obtained via a recently described mild reaction condition
[39] was thoroughly studied concerning its photophysical properties. To achieve such
a goal, we have performed a comprehensive electronic spectral and photophysical
study in various solvents and temperatures investigating the properties of the singlet
and triplet excited states which are further rationalized with time-depend density
functional theory (TDDFT) calculations.
92 Experimental Section
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Materials
All the reagents for the synthesis, indigo, sodium hydride (60% dispersion in
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mineral oil stored in a dry box) and 1,2-diodoethane were obtained from commercial
sources and used without further purification. The solvents were used as commercial
pro analysis (P.A.) quality. For the spectral and photophysical determinations, the
solvents used were of spectroscopic or equivalent grade and were used as received.
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Equipment and Methods
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Microwave-assisted reactions were performed in a CEM Discover S-Class single-
mode microwave reactor, featuring continuous temperature, pressure and microwave
power monitoring.
Analytical thin-layer chromatography (TLC) was performed on Macherey-Nagel
ALUGRAM Xtra silica gel plates with UV254 indicator. Visualization was accomplished
by an ultraviolet lamp (254 nm). Silica gel column was carried out with silica gel (230-
400 mesh).
Nuclear magnetic resonance (NMR) spectra were recorded at room
temperature in CDCl3 solutions on a Bruker Avance III spectrometer and a Bruker DRX-
1
400 spectrometer, both operating at 400.13 MHz for H and 100.61 MHz for 13C.
Tetramethylsilane (TMS) was used as internal standard.
Gas chromatography-mass spectroscopy (GC-MS) analyses were performed on
a Hewlett-Packard 5973 MSD spectrometer, using electron ionization (EI) (70 eV),
coupled to a Hewlett-Packard Agilent 6890 chromatography system, equipped with a
HP-5 MS column (30 m x 0.25 mm x 0.25 μm) and high-purity helium as carrier gas.
The initial temperature of 70 ᴼC was increased to 250 ᴼC at a 15 ᴼC/min rate, and held
for 10 min. Then the temperature was increased to 290 ᴼC at a 5 ᴼC/min rate and held
for 2 min, giving a total run time of 32 min. The flow of the carrier gas was maintained
at 1.33 mL/min. The injector port was set at 250 ᴼC.
High resolution mass spectrometry (HRMS) was performed on a Bruker
microTOF-Focus mass spectrometer equipped with an electrospray ionization time-of-
flight (ESI-TOF) source, and is given in Fig. SI1.
Absorption and fluorescence spectra were recorded on a Shimadzu UV-2450
and Horiba-Jobin-Ivon SPEX Fluorolog 3-22 spectrometers respectively. Fluorescence
spectra were corrected for the wavelength response of the system.
The singlet extinction coefficients (εss) were obtained from the slope of the plot
of the absorption with (at least) five solutions of different concentrations versus the
concentration (correlation values >0.999).
The fluorescence quantum yields (ФF) at room temperature (T=293 K) were
measured using α-quaterthiophene, α4 (φF = 0.16) [40] in acetonitrile (MeCN) as
standard using the equation below (eq. 1):
5

nC2P
nr2ef
I(
λ
)CP d
λ
∫
∫
φFCP
=
φFRe f
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(1)
Re f
I(
λ
)
dλ
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I(λ λ
)CPd
)
where (
is the integrated area under the emission spectra of the
∫
of the reference (ref) solution, ꢀꢁꢂ and ꢀꢄꢃꢅꢆ are the
I(
λ
)
Re f d
λ
ꢃ
136
compound (cp) and
∫
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refractive index of the solvents in which the compounds and the reference were
Re f
respectively dissolved and
is the fluorescence quantum yield of the standard.
φF
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Phosphorescence measurements were recorded with a Horiba-Jobin-Ivon SPEX
Fluorolog 3-22 spectrometer using a 150 W pulsed Xenon lamp. The phosphorescence
spectra were corrected for the wavelength response of the system. Phosphorescence
experiments were performed with a quartz NMR-like (EPR) tube. Due to the smaller
optical pathway, l, an absorbance of ~0.4 (checked with a 1 cm length cuvette) was
used in contrast to the ~0.2 used in fluorescence experiments. This tube was put inside
a dewar (fixed to the equipment with an appropriate holder) filled up with liquid
nitrogen.
The phosphorescence quantum yields were determined using biacetyl
(φPh=0.23) [41] in a mixed solvent containing diethyl ether, isopentane and ethanol in a
5:5:2 (v/v/v) ratio as standard [42].
Singlet oxygen formation quantum yields (φ∆) were obtained by direct
measurements of the phosphorescence emission spectra of singlet oxygen, 1O2,
centered at 1276 nm with a Horiba-Jobin-Ivon SPEX Fluorolog 3-22 spectrometer
equipped with a near-infrared (NIR) detector Hamamatsu R5509-42 photomultipler
cooled to 193 K in a liquid nitrogen chamber. To avoid the overlap of the fluorescence
emission second harmonic signal with the sensitized singlet oxygen phosphorescence
emission at 1275 nm a Newport longpass dielectric filter with 1000 nm cut-on
(reference 10LWF-1000-B) was used. The sensitized phosphorescence emission
spectra of 1O2 from optically matched solutions of the samples and that of the reference
compound were obtained in identical experimental conditions. The singlet oxygen
formation quantum yield was then determined by comparing the integrated area under
I(λ λ
)CP d
)and that of the reference
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the emission spectra of the sample solutions (
∫
I(λ)Re f dλ
solution (
) and applying the equation (eq. 2)[43],
∫
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I(
λ
)CP d
λ
∫
∫
φ∆CP
=
φ∆Re f
(2)
Re f
I(
λ
)
dλ
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with ∅ꢄ∆ꢅꢆ the singlet oxygen formation quantum yield of the reference compound. 1H-
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phenalen-1-one (phenalenone) in acetonitrile (φ∆ = 0.98) as used as standard [44].
Fluorescence decays were measured using a home-built picosecond time-
correlated single photon counting (ps-TCSPC) apparatus described elsewhere [45]. The
excitation source consists of a tunable picosecond Spectra-Physics mode-lock Tsunami
laser (Ti:Sapphire) model 3950 (80 MHz repetition rate, tuning range 700−1000 nm),
pumped by a 532 nm continuous wave Spectra-Physics Millennia Pro-10s laser. The
excitation wavelengths used in this work (409 nm, 411 nm and 422 nm) were obtained
with a Spectra-Physics harmonic generator, model GWU-23PS. The fluorescence decay
curves were deconvoluted using the experimental instrument response function signal
collected with a scattering solution (aqueous Ludox solution). The deconvolution
procedure was performed using the modulation function method, as implemented by
G. Striker in the SAND program, and previously reported in the literature [46].
The experimental setup for the ultrafast spectroscopic and kinetics
measurements consisted of a broadband (350–1600 nm) HELIOS pump-probe
femtosecond transient absorption spectrometer from Ultrafast Systems, pumped by an
amplified femtosecond Spectra-Physics Solstice-100F laser (800 nm central
wavelength, 128 fs pulse width and 1 kHz repetition rate) and coupled to a Spectra-
Physics TOPAS Prime F optical parametric amplifier (195–22000 nm tuning range).
The probe light in the UV range was generated by passing a small portion of the 800
nm light from the Solstice-100F laser through a computerized optical delay (with a
time window of up to 8 ns) and then focusing in a vertical translating CaF2 crystal to
generate a white-light continuum (350–750 nm). All the measurements were obtained
in a 2 mm quartz cuvette, with absorptions of 0.3 at the pump excitation wavelength.
To avoid photodegradation, the solutions were stirred during the experiments or kept
in movement with a frequency lower than the laser using a motorized translating
sample holder. Background signals from impurities or unwanted coherent effects were
ruled out by scans of the neat solvent. The spectral chirp of the data was corrected
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using Surface Xplorer PRO program from Ultrafast Systems. Global analysis of the data
(using a sequential model) was performed using Glotaran software [47].
The experimental setup (applied photophysics laser flash photolysis apparatus
pumped by a Spectra-Physics Nd:YAG laser) used to obtain the nanosecond-
microsecond transient absorption spectra has been described elsewhere [48, 49].
All theoretical calculations were of the density functional theory (DFT) type,
carried out using GAMESS-US [50] version R3. A range corrected CAMB3LYP [51]
functional, with 65% HF exact exchange at long range and 19% at short range, was
used in both ground- and excited-state calculations. TDDFT calculations, with similar
functionals, were used to probe the excited-state potential energy surface (PES). The
solvent was included using the polarizable continuum model with the solvation model
density to add corrections for cavitation, dispersion, and solvent structure. In TDDFT
calculation of FC (Franck-Condon) excitations the dielectric constant of the solvent was
split into a “bulk” component and a fast component, which is essentially the square of
the refractive index.[52] In “adiabatic” conditions only the static dielectric constant is
used. A 6-31G** basis set was used in either DFT or TDDFT calculations. CAMB3LYP
slightly overestimates excitations with a scaling correction applied to the reported
values (Ereported = ETDDFT × 0.92 - 0.19) [6]. Excitations were corrected for zero point
vibrational energy by using, essentially, the frequency of the relaxing vibrational mode
(±0.06 eV). Triplet energetics was estimated from UHF CAMB3LYP calculations with
multiplicities of 1 and 3.
215
Synthesis of Tryptanthrin
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In a glass microwave reactor charged with a magnetic stirring bar, indigo
(100mg, 1 eq.) and sodium hydride (2 equiv) were dissolved in 1 mL of DMF. The 1,2-
diodoethane was added (2 equiv) and solvent was added to a total volume of 2 mL. The
o
resulting mixture was heated under microwave irradiation at 50 C for 15 minutes.
After cooling down to room temperature, a sample was collected, dissolved in 1 mL of
dichloromethane and washed with 1 mL of water. The organic layer was analyzed by
GC-MS and the analysis of the reaction media showed the presence of isatin, isatoic
anhydride, tryptanthrin and unreacted indigo [39]. The crude was extracted with
dichloromethane and isolated by column chromatography (SiO2) using hexane:ethyl
acetate (8:2) as eluent to yield pure TRYP (yellow solid; 25 mg; yield: 26%). The
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chemical structure of tryptanthrin was confirmed by GC-MS and HRMS analysis as well
1H and 13C spectroscopy.
Tryptanthrin:
1H NMR (CDCl3, 400 MHz), δ (ppm): 8.61 (d, J=8.1Hz, 1H, ArH), 8.42 (dd, J=7.9Hz,
J=1.3Hz, 1H, ArH), 8.02 (d, J=7.8Hz, 1H, ArH), 7.90 (d, J=7.6Hz, 1H, ArH), 7.82-7.87 (m,
1H, ArH), 7.76-7.80 (m, 1H, ArH), 7.64-7.68 (m, 1H, ArH), 7.40-7.44 (m, J=7.5Hz,
J=0.4Hz, 1H, ArH); 13C NMR (CDCl3, 101 MHz) δ (ppm): 182.6 (C=O), 158.1 (C=O),
146.6, 146.4, 144.4, 138.3, 135.1, 130.8, 130.3, 127.6, 127.2, 125.4, 123.8, 122.0, 118.0;
GC-MS: m/z [M+] = 248.0; HRMS (ESI-TOF-MS): m/z [M+1]+ =249.0659 obtained for
C15H9N2O2 (calc. 249.0659), see SI for further details.
238 Results and Discussion
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Synthesis
TRYP was obtained from indigo using a trio oxidant system - NaH, an iodine
source and N,N’-dimethylformamide (DMF) - under microwave (MW) irradiation, as
recently described by our group (Scheme 2). Briefly, we verified that under these
reaction conditions, indigo is converted to isatin and isatoic anhydride, which further
condensate to afford tryptanthrin [39]. This approach is a faster and greener
methodology to achieve this compound from indigo, and requires milder conditions
when compared with previously described methods.
247
O
O
O
H
MW (50oC)
15 min
N
O
1,2-diiodoethane
O
NaH
DMF
N
N
N
H
O
H
H
O
Indigo
O
N
N
O
TRYP
248
249
Scheme 2 – Schematic synthetic pathway for the synthesis of TRYP from indigo.
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Excited State Characterization of Tryptanthrin
Absorption and Steady State Fluorescence
The absorption, fluorescence emission and excitation spectra of TRYP were
obtained at T= 293 K, in six organic solvents with different dielectric constant (ε) and
in a viscous mixture of methanol:glycerol (70:30, v/v). The solvents range from
polarities of ε=1.84 (n-pentane) to ε= 36.71 (DMF) see Table 1. The absorption,
excitation and fluorescence emission spectra of TRYP in dichloromethane (DCM)
(moderately polar aprotic solvent), MeCN (polar aprotic solvent) and methanol
(MeOH) (polar protic solvent) at T= 293 K are depicted in Figure 1. Spectral data is
summarized in Table 1.
10

1.5
1.0
0.5
1.5
1.0
0.5
A
0.0
1.5
0.0
1.5
B
C
1.0
0.5
1.0
0.5
0.0
1.5
0.0
1.5
1.0
0.5
0.0
1.0
0.5
0.0
300
400
500
600
700
λ
(nm)
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Figure 1 - Normalized absorption (black line), excitation (blue line) and fluorescence
emission (red line) spectra in dichloromethane (DCM) (A), acetonitrile (MeCN) (B) and
methanol (MeOH) (C) solutions at T=293 K. Fluorescence emission spectra obtained
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with excitation wavelength λexc =340 nm and the excitation spectra collected with λem
=
505 nm and λem =550 nm. The dashed vertical line is just meant to be a guideline for
the eye.
Figure 1 shows that the emission band is strongly red-shifted relative to the
longest wavelength absorption band. The emission is also solvent dependent as seen in
Figure 1 by the red-shifting with the solvent polarity. It can also be observed a good
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overlap between the absorption and the excitation spectra, thus attesting the purity of
TRYP.
Table 1- Spectral data including absorption (λabs), fluorescence (λFluo) and
phosphorescence (λPh) wavelength maxima, Stokes shifts (rSS), molar extinction
coefficient (εSS) and singlet (ES) and triplet (ET) state energies for tryptanthrin in
different solvents at room temperature. For indigo the same parameters are also
presented in DMF for comparison.
c
d
ηb
λabs
λfluo
∆
εSS
ES
λph
ET
∆ES-T
(eV)
SS
Compound
Indigoa
Solvent
ε
(cP)
(nm) (nm) (cm-1) (M-1 cm-1) (eV)
(nm)
(eV)
DMF
n-pentane
DCM
0.924 36.71 610
653
506
508
513
514
539
552
551
1080
5878
5378
5888
5925
7154
7591
7427
22140
1553
6623
3545
3576
4169
3736
3527
1.97
2.90
2.81
2.79
2.78
-
-
0.91e
N.D.f
N.D.f
N.D.f
N.D.f
0.45
N.D.f
N.D.f
0.214
0.410
1.84
8.93
390
399
N.D.f
N.D.f
N.D.f
N.D.f
N.D.f
N.D.f
N.D.f
N.D.f
MeCN
DMF
0.345 35.94 394
0.924 36.71 394
1.200 24.55 389
0.593 32.66 389
7.700g 36.70g 391
TRYP
EtOH
2.84 567, 615 2.39
MeOH
2.78
2.76
N.D.f
N.D.f
N.D.f
N.D.f
MeOH:Glycerol
(70:30)
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283
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aFor indigo in DMF data from ref. [4]. bη= Viscosity. c∆SS= Stokes shifts. dεSS= Molar extinction coefficient.
f
g
eData taken from ref. [53]. N.D.=Not determined. For MeOH:Glycerol 70:30 volume ratio data from ref.
[54].
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293
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Table 1 summarizes the spectral (absorption and emission) data for TRYP in
the investigated solvents and that of indigo in DMF for comparison. Observation of data
in Table 1 shows several interesting features: (i) the absorption spectra are relatively
unaffected by changes in solvent polarity and by differences in the hydrogen bonding
ability of the alcoholic solvents; (ii) the red-shift of the emission wavelength band; (iii)
the high dependence of the emission band with the solvent polarity; (iv) the high
Stokes shift (∆SS) in protic alcohols, thus supporting the occurrence of an excited state
charge transfer state; (v) the significant blue shift of TRYP, both in the absorption and
fluorescence emission maxima, relative to indigo and (vi) the much lower molar
extinction coefficient values of TRYP relative to indigo. The molar absorption
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coefficients (εSS) are, however, of the same order of magnitude (εSS ≈ 103 M-1 cm-1) of
previously reported values for TRYP derivatives [32, 36].
To further evaluate the effect of the solvent on the fluorescence emission
(excited state interactions) of TRYP, the Stokes shift has been plotted against the
Dimroth-Reichardt ꢇꢈꢉ polarity parameter [55]. This plot is shown in Figure SI2 and
found linear, thus indicating that solute-solvent dipole–dipole interactions are mainly
responsible for the solvent-dependent observed fluorescence shift [56, 57]. The nature
of the charge transfer singlet excited state will be further discussed at the light of the
TDDFT calculations.
Photophysical Data
A summary of the photophysical parameters, obtained in different solvents,
including fluorescence, phosphorescence, intersystem crossing triplet and internal
conversion quantum yields, lifetimes and rate constants for the radiative and
radiationless processes, is given in Table 2.
Table 2- Photophysical data including fluorescence (ϕF), phosphorescence (ϕPh),
intersystem crossing triplet (ϕT) and internal conversion (ϕIC) quantum yields,
fluorescence (τF) and phosphorescence lifetimes (τPh) and rate constants (fluorescence
(kF), nonradiative (kNR), internal conversion (kIC) and intersystem crossing (kISC)) for
Tryptanthrin in various solvents at 293 K and 77 K. For indigo the same parameters
are also presented in DMF for comparison.
τPh
kF
kNR
kIC
kISC
φF
τF
φPh
φ∆≈φΤ
φIC
ηb
(cP)
Compound
Solvent
ε
(ps)c
(ms)
(ns-1)d (ns-1)d (ns-1)d (ns-1)d
(293 K) (293 K) (77 K) (77 K)
(293 K) (293 K) (293 K)
(293 K)
(293 K)
(293 K)
0.0066f
0.77
0.991
0.22
0.017
0.017
0.020
0.011
0.013
0.015
7.390
7.341
0.049
Indigoa
TRYP
DMF
MeCN
DMF
0.924 36.71 0.002
0.345 35.94 0.008
0.924 36.71 0.007
1.200 24.55 0.003
0.593 32.66 0.002
7.700e 36.70e 0.002
135
499
368
255
146
127
-
-
1.987
2.698
3.911
6.836
7.859
0.440
1.547
EtOH
MeOH
0.0027 10.3
MeOH:Glycerol
(70:30)
319
13

b
320
321
aFor indigo in DMF data from ref.[3]. η= Viscosity. cThe decay time considered here is the slower
φF
τF
(1−φF
)
1−φF
−
φT
φT
τF
component in the fluorescence decays. d
;
;
;
kF =
kNR =
kIC =
kISC =
τF
τ F
322
323
324
325
326
327
∅
= 1 − ∅ꢈ − ∅ꢌ . eFor MeOH:Glycerol (70/30) volume ratio data from ref. [54]. fData from ref. [53].
ꢊꢋ
Observation of Table 2 shows that the fluorescence quantum yield is higher in
MeCN and DMF than in the polar protic solvents, MeOH and ethanol (EtOH) and in the
methanol:glycerol (70:30, v/v) mixture studied. The fluorescence lifetimes also show a
gradual decrease going from polar aprotic to protic solvents. Noteworthy, the highly
efficient singlet oxygen sensitization of TRYP in aerated MeCN solution, φ∆= 0.77 (see
Figure SI3 and Table 2), which establishes the S1~~>T1 as the dominant deactivation
channel, clearly contrasting with indigo where the S1~~>S0 (internal conversion) is the
dominant deactivation channel with more than 99.99% of the quanta loss occurring
from this channel [53, 58, 59].
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
Also in contrast to indigo [53, 59] and derivatives [4, 60], where no
phosphorescence emission was observed, in the case of TRYP phosphorescence in
ethanol glass at 77 K could be observed (see Figure 2). A broad phosphorescence
emission band is observed with two vibronic bands (567 and 615 nm) which partially
overlaps the fluorescence emission (also collected at low temperature, 77 K), as
illustrated in Figure SI4. A phosphorescence lifetime of 10.3 ms was obtained (see
Figure SI5 for the mono-exponential decay profile). This indicates the nature of the
triplet state to be of n,π* origin [61]. The very close proximity with the S1 (CT), i.e., the
small singlet-triplet energy gap of 0.45 eV (see below) further supports the very
efficient S1~~>T1 intersystem crossing deactivation channel [61, 62]. In addition,
strong triplet-triplet absorption bands were found for TRYP (see below), thus giving
support for the close proximity of the S1 and T1 states.
14

1.2
1.0
0.8
0.6
0.4
0.2
0.0
500
600
700
λ
(nm)
346
347
Figure 2 - Phosphorescence spectra for TRYP in ethanol at T= 77 K.
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
From the photophysical data in Table 2, it can also be seen that (and
considering that φT≥φ ) the internal conversion deactivation channel (φ ) represents,
IC
∆
ca. 22% of the quanta loss (φ =1- φT - φF).
IC
Due to Franck-Condon forbidden nature of the absorption spectra of TRYP the
singlet energy values were obtained from the interception of the normalized
absorption (lowest energy band) and the fluorescence emission spectra. The triplet
energy value was taken from the energy of the 0-0 vibronic band of the
phosphorescence spectra. The obtained singlet-triplet energy gap, ∆ES-T = 0.45 eV
(3629 cm-1), is lower than the value found for indigo ∆ES-T = 0.91 eV (7340cm-1) [2, 53].
The small ∆ES-T value found for TRYP together with the observed properties of the
time-resolved data will be discussed in detail in the DFT/TDDFT calculations section.
TDDFT calculations
TDDFT calculations show that the observed lowest absorption band
corresponds to an S0→S2 (π→π*) transition (392 nm). The S2 state is predicted to have
a strong S2→S14 transient absorption at 478 nm (f=0.38), in agreement with the fs-TA
spectroscopic data (see below). Table 3 summarizes the data for the calculated
transitions, namely wavelength maxima, λ (nm), oscillator strength (f) and the major
15

367
368
369
370
371
contributions of the Molecular Orbitals (MO) of the transitions involved. The
calculated UV-VIS and fluorescence spectra are given in Fig. SI6.
Table 3- Predicted singlet and triplet transitions for Tryp in the gas phase and in MeCN
including the expected wavelength maxima and the orbitals involved in the transition.
Solvent/state
λ (nm)
f
Major orbitals involved in
the transition *
HOMO-2 → LUMO (-0.89)
Gas phase
S0 → S1
Gas phase
S0 → S2
Gas phase
S2 → S14
Gas phase
T1 → S0
Gas phase
S1 → S0
450
392
478
591
489
508
0
HOMO → LUMO (-0.92)
0.17
0.38
0
(S2) HOMO-2 → LUMO (-0.89)
(S14) HOMO-7 → LUMO (-0.77)
**
HOMO → LUMO (0.95)
HOMO → LUMO (0.97)
0.08
0.17
Acetonitrile
S1 → S0
372
373
374
* In brackets the coefficient contribution of this transition.
** T1 calculated by DFT as the ground state triplet
375
376
377
378
379
380
The frontier orbitals representing the main contributions CT and LE states
involving respectively major contributions of n→π* and π→π* transitions are
presented in Figure 3. The CT state mainly results from the transition from the I
(HOMO-2) to the III (LUMO) orbitals (n→π* character) whereas the LE mainly results
from the transition from II (HOMO) to III (LUMO) orbitals (π →π* nature).
Ι
ΙΙ
ΙΙΙ
(LUMO )
(HOMO-2)
(HOMO)
381
382
383
Figure 3 – Topological plots of n (I), π (II) and π* (III) orbitals of TRYP.
16

384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
In low polar solvents the calculations always end-up with degenerate S1 and S2
states. Which suggests that a Hybrid LE/CT can be proposed for this system. In gas
phase, the calculations show a decrease in the energetic separation between S1 and S2
making hybrid LE/CT states more favorable but more difficult to analyze by TDDFT
since they may have different sensitivities to the amount of HF exact exchange used in
calculations and its ordering reversed.
Nevertheless in MeCN the LE and CT are clearly distinct states and probing the
PES of both the S1 and S2 states (in MeCN and gas phase) allowed us to find that the
excited state can decay according to two different pathways: it relaxes to a CT state of
n,π* nature (which can convert to a T1 state predicted in gas phase at 591 nm, which is
in agreement with the phosphorescence first vibronic band at ~ 567 nm, see Figure 2)
and to a LE π,π emissive state. The later shows a strong emissive dependence with
the solvent polarity red-shifting by 19 nm from gas phase (489 nm) to MeCN (508 nm),
in agreement with the data in Table 1 and Figure SI2. This effect brings the energy of
the emissive state closer to that of the triplet state, therefore increasing the efficiency
of the S~~~>T intersystem crossing (ISC) in polar solvents making it the dominant
deactivation channel.
Scheme 3 summarizes, conceptually, the results of DFT/TDDFT calculations,
used to provide insight on the photophysics of TRYP. It is worth noting that in Scheme
3, the xx’ and yy’ axes are neither to scale nor represent a physical relaxation
coordinate. The vibronic levels are not associated to calculated vibrations, with the
exception of the lowest vibronic which correspond to the energy minimum corrected
to zero point vibrational energy (see Experimental Section).
As explained below, due to the energetic proximity of the states obtained by
either pathway (CT and LE) they can interconvert originating double exponential
decays in time-resolved fluorescence.
17

S14
S14-n
π,π
S2 ( *)
470 nm (TA)
(478 nm, S2-> S14 )
π,π
S1/LE ( *)
S2~~>S1
< 250 ps
π
S1/CT (n, *)
8.9 ps
π
T1 (n, *)
513 nm
(508 nm)
394 nm
(392 nm)
τT 1.95
s
τPh
10.3 ms
567 nm
(615 nm)
466 ps
S0
411
412
Scheme 3 – Schematic diagram with potential energy curves for predicted states and
transitions resulting from the experimental and TDDFT data for TRYP in acetonitrile
(MeCN), in gas phase the absorption band is predicted at 392 nm. The values in
brackets represent the TDDFT predicted transitions. A S1-S14 transition is predicted
from TDDFT and corroborated by fs-TA experiments. The S2 state rapidly deactivates
to the S1 state. The S1 (LE) interconverts in 8.9 ps into S1 (CT) (dashed line), which in
466 ps decays to ground state. The triplet state formed, T1, during the deactivation of
S1, has a phosphorescence lifetime of 10.3 ms.
413
414
415
416
417
418
419
420
421
422
423
424
With irradiation of the electronic ground state of TRYP, with 392 nm (λpump),
the Frank-Condon state S2, π,π* is populated (vertical transition) which, within the IRF
of the pump-pulse rapidly decays (<250 ps) to S1 LE (π,π*) that can interconvert to S1
CT (n,π*) (Scheme 3).
18

425
426
427
428
429
430
431
432
433
434
435
436
437
438
From the fs-TA data analysis in MeCN using a sequential model it was found that
the CT state can be formed at the expenses of LE state ( 8.9 ps rise-time) and
depopulates (through ISC to form the triplet state, by fluorescence or by internal
conversion to the ground-state) in 466 ps. This is further corroborated by time-
resolved fluorescence. This will be discussed in detailed in the next two sections.
Time-resolved fluorescence
Upon excitation to the S2 state of TRYP and rapid vibrational relaxation to S1
(LE) and S1 (CT) species whose fluorescence decays (according to Scheme 4) are
obtained in a ps time scale (with 4 ps time resolution). These were obtained in
different solvents at room temperature, with excitation at 409 nm, 411 nm and 422 nm
and collected at different emission wavelengths (Table 4).
Global analysis of the data revealed that the fluorescence decays are fitted with
sums of two exponentials according to eq. 3:
2
I(t) = a e−t/
τi
∑
ij
i, j=1
439
440
441
442
443
444
445
446
447
448
(3)
with identical decays times (τi) but different pre-exponential factors (aij) all over the
emission spectra (see Figure SI7). The data reveal that, with exception of MeCN and
DMF, at higher energies the shorter decay component dominates, whereas at the tail of
the emission band the longer decay components dominates with the shorter
component now displaying a negative associated pre-exponential factor, thus
corroborating the presence of an excited state dynamic process (LE state giving rise to
the CT state).
449
450
Scheme 4 - Kinetic Scheme for the excited behavior of TRYP in S1.
19

451
452
453
454
455
456
457
Table 4 - Time resolved fluorescence data (lifetimes, τi, pre-exponential factors, ai, and
chi-squared values, χ2) obtained with Picosecond Time-Correlated Single Photon
Counting (ps-TCSPC) technique for tryptanthrin, collected at different emission
wavelengths obtained with λexc = 409 nm, λexc = 411 nm and 422 nm at T= 293 K. For
indigo the same parameters are also presented in DMF for comparison.
λexc
λem
τ1
τ2
χ2
Compound
Indigoa
Solvent
DMF
a1
a2
(nm)
(nm) (ps)
(ps)
630
10
660
0,61 0.39 1.10
-0.51 1.00 1.03
0.15 0.85 1.06
0.11 0.89 1.16
0.05 0.95 1.12
0.12 0.88 1.11
0.03 0.97 1.08
-0.03 1.00 1.15
0.78 0.22 1.07
0.42 0.59 0.92
-0.27 1.00 0.91
0.73 0.27 1.23
0.25 0.75 1.13
-0.48 1.00 1.34
0.73 0.27 1.06
0.23 0.77 1.11
-0.35 1.00 1.30
378
135
480
411
MeCN
DMF
65
29
23
12
17
499
510
630
480
510
630
490
540
620
500
550
600
500
550
600
409
422
368
255
146
127
TRYP
EtOH
MeOH
409
MeOH:Glycerol
(70:30)
458
459
460
461
462
463
464
465
466
467
468
In the case of indigo the bi-exponential nature of the decay was found to be
consistent with a keto-excited form, giving rise to the enol form by single fast proton
transfer [3, 4] Still for indigo, the presence of the rising component at longer emission
wavelengths is particularly relevant, and demonstrates that the second species (the
longer-lived enol) is formed at the expense of the shorter-lived keto species [2, 3].
From Table 3 it can be seen that for TRYP with the exception of MeCN, where no rising
component is observed, in all the other solvents including the methanol:glycerol
mixture a rising component (indicated by the negative pre-exponential value) at the
longer wavelength emission is present (Figure 4).
20

469
470
471
472
473
474
Therefore, the solution of this 2-state kinetic scheme (see Scheme 4) allows one
to obtain the kinetic constants for the formation (ka) and deactivation (kd) of the CT
and LE species.[43] It is worth mentioning that in Scheme 4, the dashed arrow
associated with the decay of the CT state/species reflects the fact that this is a dark
state and the decay is essentially non-radiative.
475
476
477
478
479
480
481
482
483
484
Figure 4 - Fluorescence decays of TRYP obtained in methanol (MeOH) at T= 293 K.
The fluorescence decay times (τi), in ps, and the associated pre-exponential factors (aij)
are presented in the inset. For a better judgment of the quality of the fits, the weighted
residuals, autocorrelation functions (ACs) and chi-squared (χ2) values are also shown.
The dashed lines in the decays are the pulse instrumental response.
The differential equations ruling out the time dependence concentration of the
two excited species are, instantaneous formation of the excited species), according to
Scheme 4, given by [63-65]:
















−kX kd
ka −kY
d
LE *
CT *
LE *
CT *
485
486
(4)
=


dt


where LE* and CT* are the concentrations of S1(LE) and S1(CT) in the excited state and
21

487
488
kX = ka + kLE
kY = kd + kCT
(5)
(6)
489
490
491
where kLE is the reciprocal of the lifetime of the LE species and kCT represents the
deactivation of the CT species (since this is a dark state this deactivation essentially
mirrors the radiationless process). Integration of eq. 4 leads to
492
(7)
493
494
495
where the eigen values λi are the reciprocal decay times of the shorter (λ2=1/τ2) and of
the longer (λ1=1/τ1) species and are related to the rate constants in Scheme 4 by the
characteristic equation [66]:
496
(8)
497
498
the solutions of which are given by eq.9.

1/2
λ2,1 = (k + k )± (k − k )+4k k
X Y X Y a
(9)
2

d 
499
500
The sum and product of the lambda values (λ1 and λ2) resulting from eq. 9 are
respectively given by:
501
502
503
(10)
k X + kY = λ1
+ λ 2
and
(11)
504
505
The pre-exponential factors aij are the linear combinations of the eigenvector
basis set which obey the following initial conditions:
506
507
a11 +a12 =1
a21 +a22 = 0
(12)
(13)
22

508
509
510
511
512
513
It is worth noting that application of these initial conditions to the investigated
systems is dependent on the excitation wavelength. This means that by using different
excitation wavelengths one should expect to obtain different α values. Simple
manipulation of eq. 4 and 7, together with eq. 11 and considering the initial conditions
given by eqs 12 and 13, leads to the following relations for the pre-exponential
amplitudes (aij):
kX − λ2
514
515
516
517
(14)
(15)
(16)
(17)
a11 =
a12 =
a21 =
a22 =
λ1 − λ2
λ
1 − kX + kd
λ1 λ2
−
kX − λ1
λ2 λ1
−
kX − λ2
λ1 λ2
−
518
519
The system is therefore reduced to 3 unknowns (ka, kd, and kCT), to be evaluated
from λ2
,
λ1, A and B (equations 18 and 19).
a12 kX − λ1
520
521
(18)
(19)
A =
B =
=
a11
λ2 − kX
a22
a21
= −1
522
523
which are obtained from the pre-exponential ratio at the emission of LE (A) and CT (B)
species/states.
524
525
Rearrangement of eq. 18 leads to:
Aλ2
+
λ1
(20)
kX =
A+1
526
Further manipulation yields:
23

A
λ2 + λ
1 − kLE
527
528
529
(21)
(22)
ka =
A+1
and from equation 11, the following equation is obtained
kX kY −λ1λ2
kd =
ka
530
and from finally from eq. 6 one obtains the value of kCT=kY-kd.
531
532
533
534
535
536
537
538
539
540
541
542
543
544
From the decay parameters, aij and τi and kLE which is the reciprocal of the
lifetime of the model compound, here taken as the same of that for indigo (see ref. [3]
with a value of 152 ps) we obtained kCT values in the 3.10-8.30 ns-1 for TRYP in the
protic polar solvents methanol, ethanol and methanol:glycerol (see Table 5).
Noteworthy, is the insignificant change in the decay times going from MeOH to
the viscous methanol:glycerol (70:30, v/v) mixture thus excluding the formation of
different TRYP conformers in the excited state. Indeed, it has been reported that TRYP
can adopt two conformations when adsorbed in highly oriented pyrolytic graphite, i.e.,
a planar structure where the nitrogen on the amide moiety is sp2 hybridized and a non-
planar form in which this same nitrogen is sp3 hybridized [19]. However, and in
agreement with our work, in solution it was shown that TRYP assumes a planar
conformation [19]. Indeed, as suggested by crystallographic studies [67], TRYP was
computationally modeled in a completely planar conformation, which maximizes the
aromaticity and represents the lowest energy conformer.
545
546
547
Table 5- Time-resolved data (λ1, λ2 and A) and rate constants (ka, kd and kCT)
recovered the kinetic analysis resulting from Scheme 4.
Solvent
A
ka (ns-1)
kd (ns-1)
kCT (ns-1)
λ1
λ2
EtOH
3.92
6.85
43.14
80.44
3.55
2.70
27.93
53.99
9.45
3.10
6.94
MeOH
19.77
MeOH:Glycerol
(70:30)
7.85
59.27
2.70
38.81
13.43
8.30
548
549
24

550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
The data in Table 4 indicates that the rate constant for the formation of the
S1(CT) from the S1(LE) state, ka, is higher in methanol but essentially of the same order
of magnitude in all solvents. Very interesting is to note the close proximity between the
ka and kd values for the investigated solvents, which indicates a highly reversible
system and a very efficient deactivation of the excited state of TRYP as predicted from
TDDFT.
Time-resolved femtosecond transient difference absorption spectra (fs-TA)
Additional information on the excited-state deactivation process in TRYP was
obtained from femtosecond (fs)-transient absorption (TA) data (Table 6).
Table 6- Results of the global fit analysis (lifetimes, τi,) obtained from the time-
resolved transient absorption data collected with excitation at 300 nm together (for
comparison) with the fluorescence decay times obtained from Picosecond Time-
Correlated Single Photon Counting technique for Tryptanthrin in solvents of different
viscosity (η) and dielectric Constant (ε) at 293 K.
[τ3]a,b
(µs)
[τ1]a
(ps)
[τ2]a
(ps)
τ1
τ2
a
a
Solvent
η (cP)
ε
(ps)
(ps)
MeCN
MeOH
0.345 35.94
0.593 32.66
8.9
3.1
466
165
1.95
51
65
499
12
17
146
127
MeOH:Glycerol
(70:30)
7.700c 36.70c
2.2
156
51
b
567
568
569
aDecay time values, τi, from fs-TA in brackets and the three last values from ps-TCSPC. This transient
lifetime was fixed in the global analysis to the triplet lifetime obtained by ns-TA. cFor MeOH:Glycerol
(70/30) volume ratio data from ref. [54].
570
571
572
573
574
575
The time-resolved femtosecond transient difference absorption spectra (fs-TA)
for TRYP was recorded in aerated MeOH, methanol:glycerol (70:30, v/v) and MeCN
solutions in the 340-750 nm range with excitation at 300 nm and 430 nm (see Figure 5
for MeCN and Figure SI8 for MeOH and methanol:glycerol mixtures). The fs-TA spectra
are dominated by strongly overlapped positive broad transient absorption bands in the
25

576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
350-650 nm range with maxima at 460 nm in MeOH and in the methanol:glycerol
mixture while in MeCN the maxima is at 470 nm.
The fs-TA data in the 340-750 range show that the decays are well fitted with a
sum of three exponential (see Table 6 and Table SI1). The fast decay transients (τ1)
with values in the 2.2-8.9 ps range, which are associated with a negative pre-
exponential values when the decays are collected at 465 nm (Table SI1), are not
consistent with the solvation dynamics times previously reported for these solvents
[68] and are therefore associated to the S1(LE) to S1(CT) state inter-conversion
previously described. Indeed, from Table 6 it can be seen that, in general, the
intermediate decay components, τ2, are in good agreement with the fluorescence
lifetimes assigned to the decay of the CT state. The longer component (τ3) although
needed to properly fit the decays, comes undefined in fs-TA measurements and is
defined from ns-TA experiments (see below).
The nanosecond-microsecond transient absorption spectra, ns-TA, for TRYP
were also obtained by laser flash photolysis with excitation at 355 nm in degassed
MeOH and MeCN solutions (Figure 6). The observed transient lifetimes were quenched
by oxygen and thus are attributed to the triplet excited state absorption. Worth
mention that, with the exception of the fs-TA data collected in MeOH and
methanol:glycerol mixture with excitation at 430 nm (which decayed completely
within the fs-TA observation time-window), in general, the positive TA bands at higher
delay times are in good agreement with the ns-TA bands (see Figures 5 and SI8-9) and
thus are attributed to the excited state triplet-triplet transient absorption.
26

0.18 ps
0.43 ps
1.3 ps
3.1 ps
349 ps
7.0 ns
0.4 µs (ns-TA)
A
1.0
0.5
0.0
1.0
0.35 ps
0.97 ps
5.1 ps
144 ps
634 ps
6.9 ns
B
0.5
0.0
360
420
480
540
600
λ (nm)
599
600
Figure 5 - Time-resolved transient absorption data for TRYP obtained with excitation
at 300 nm (A) and 430 nm (B) in aerated acetonitrile (MeCN) solutions at T= 293 K.
For the data collected with excitation at 430 nm the spectral range 419-436 nm is not
shown because it is disturbed by the scattered pump beam. The dashed vertical lines
are just meant to be a guideline for the eye. The normalized triplet-triplet absorption
spectra collected by ns-TA is presented in (A) to show the good agreement with the TA
band collected at higher delay times in the fs-TA time-resolved spectra and assign the
later to the characteristic triplet-triplet absorption.
601
602
603
604
605
606
607
608
609
610
Noteworthy is the appearance of a new band in the ns-TA spectra in MeCN
solution with maxima at 550 nm (see the spectra obtained at 0.4 µs and 0.6 µs delay
times, Figure 6B) and at 370 nm in MeOH solution (data collected at 4 µs and 10 µs,
Figure 6A).
611
612
613
614
615
27

0.1
0.0
4
6
8
10
16
24
µ
µ
µ
s
s
s
µ
µ
µ
A
B
s
s
s
370 nm
475 nm
550 nm
0.4
0.6
0.8
1.0
1.6
2.4
µs
µs
µs
µs
µs
µs
0.2
0.1
0.0
300
400
500
600
λ
(nm)
616
617
618
619
620
621
622
Figure 6 - Room-temperature transient singlet–triplet difference absorption spectra
for TRYP collected with excitation at 355 nm in degassed methanol (A) and
acetonitrile (B) solutions at T= 293 K.
623 Conclusions
624
625
626
627
628
629
630
631
632
633
634
635
The synthesis of TRYP from indigo, through MW irradiation, and a detailed
investigation of the excited state deactivation pathways has been undertaken. The
results showed that the excited state deactivation pathways are remarkably different
to those of indigo. Moreover, in contrast to indigo where the S1 ~~> S0 internal
conversion dominates the excited state deactivation (with more than 99.99 % of the
quanta loss) in TRYP the highly efficient radiationless deactivation channel has a
different nature with the formation (detected from longer decay components in fs-TA
and ns-TA absorption) of a poorly emissive triplet state (with a phosphorescence
quantum yield of 2.7x10-3) and an efficient singlet oxygen sensitization, φ∆=0.77. From
time-resolved fluorescence and fs-TA absorption two clear dominant decay
components, τ1 and τ2 are observed which is further interpreted on the basis of the
existence of LE and CT species from TDDFT. These two species are found with S1 states
28

636
637
638
639
640
of n,π* (CT) character coexisting with a LE state with dominant π,π* character. The
inter-conversion, in the excited state, between the CT and LE species was further
investigated and the obtained rate constants found to be on the order of 1010 s-1 with a
reversible process of the same order of magnitude.
641 Acknowledgments
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
This work was supported by Project “Hylight” (no. 031625) 02/SAICT/2017
which is funded by the Portuguese Science Foundation and Compete Centro 2020 and
project “SunStorage - Harvesting and storage of solar energy” for financial support,
reference POCI-01-0145-FEDER-016387, funded by European Regional Development
Fund (ERDF), through COMPETE 2020- Operational Programme for Competitiveness
and Internationalization (OPCI), and by national funds, through FCT. We acknowledge
funding by Fundo Europeu de Desenvolvimento Regional (FEDER) through Programa
Operacional Factores de Competitividade (COMPETE). The Coimbra Chemistry Centre
is supported by the Fundação para a Ciência e a Tecnologia (FCT), Portuguese Agency
for Scientific Research, through the Project PEst-OE/QUI/UI0313/2014. The FCT is
also acknowledged for a PhD grant to D. Pinheiro (ref SFRH/BD/74351/2010) and a
PhD grant to P. Brandão (ref PD/BD/128490/2017 - CATSUS FCT-PhD Program). D.
Pinheiro also acknowledges the project “SunStorage - Harvesting and storage of solar
energy” for a research Grant.
Supplementary information
Figures SI1 to SI7 and Table SI1.
660 References
661
662
663
664
665
666
667
668
669
670
[1] Seixas de Melo JS. The Molecules of Colour and Art. Molecules With History and
Modern Applications. In: Albini A, Prodi S, editors. Photochemistry. London: RSC; 2020. p.
196–216.
[2] Seixas de Melo JS. The Molecules of Colour. Photochemistry: Volume 45: The Royal
Society of Chemistry; 2018. p. 68-100.
[3] Pina J, Sarmento D, Accoto M, Gentili PL, Vaccaro L, Galvão A, et al. Excited-State
Proton Transfer in Indigo. J Phys Chem B. 2017;121(10):2308-18.
[4] Seixas de Melo JS, Rondão R, Burrows HD, Melo MJ, Navaratnam S, Edge R, et al.
Spectral and Photophysical Studies of Substituted Indigo Derivatives in Their Keto Forms.
ChemPhysChem. 2006;7(11):2303-11.
29