Novel Arylindigoids by Late-Stage Derivatization of Biocatalytically Synthesized Dibromoindigo

Article abstract of DOI:10.1002/chem.202005191

Indigoids represent natural product-based compounds applicable as organic semiconductors and photoresponsive materials. Yet modified indigo derivatives are difficult to access by chemical synthesis. A biocatalytic approach applying several consecutive selective C?H functionalizations was developed that selectively provides access to various indigoids: Enzymatic halogenation of l-tryptophan followed by indole generation with tryptophanase yields 5-, 6- and 7-bromoindoles. Subsequent hydroxylation using a flavin monooxygenase furnishes dibromoindigo that is derivatized by acylation. This four-step one-pot cascade gives dibromoindigo in good isolated yields. Moreover, the halogen substituent allows for late-stage diversification by cross-coupling directly performed in the crude mixture, thus enabling synthesis of a small set of 6,6’-diarylindigo derivatives. This chemoenzymatic approach provides a modular platform towards novel indigoids with attractive spectral properties.

Full text of DOI:10.1002/chem.202005191

Communication
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&
CÀH Functionalization |Hot Paper|
Novel Arylindigoids by Late-Stage Derivatization of
Biocatalytically Synthesized Dibromoindigo
Christian Schnepel,[a, b] Veronica I. Dodero,[a] and Norbert Sewald*[a]
abilities of N,N’-aryl-substituted indigos pointed to a useful
Abstract: Indigoids represent natural product-based com-
pounds applicable as organic semiconductors and photo-
responsive materials. Yet modified indigo derivatives are
difficult to access by chemical synthesis. A biocatalytic ap-
proach applying several consecutive selective CÀH func-
tionalizations was developed that selectively provides
access to various indigoids: Enzymatic halogenation of l-
tryptophan followed by indole generation with
tryptophanase yields 5-, 6- and 7-bromoindoles. Subse-
quent hydroxylation using a flavin monooxygenase fur-
nishes dibromoindigo that is derivatized by acylation. This
four-step one-pot cascade gives dibromoindigo in good
isolated yields. Moreover, the halogen substituent allows
for late-stage diversification by cross-coupling directly per-
formed in the crude mixture, thus enabling synthesis of a
small set of 6,6’-diarylindigo derivatives. This chemoenzy-
matic approach provides a modular platform towards
novel indigoids with attractive spectral properties.
strategy to tailor their photochemical properties.[7–9] Synthetic
routes towards 1 were developed by Baeyer and by Heumann
and paved the way to multi-ton production, yet its dibrominat-
ed counterpart 2 has never entered an industrial scale produc-
tion.[10]
Today biocatalysis offers a versatile methodology to address
selectivity issues, e.g., arising from similarly reactive CÀH moi-
eties.[11] Recent advancements on enzyme discovery, engineer-
ing as well as tremendous efforts on process development
open up elaborate transformations that can be carried out
under mild conditions, often with excellent selectivities.[12–14]
Immense progress has been achieved in enzyme-catalyzed CÀ
H functionalization.[15] Especially oxyfunctionalization is a para-
mount approach to activate CÀH bonds by using biocatalysts
that are capable of utilizing molecular oxygen. Manifold
biocatalytic approaches in this rapidly evolving field, especially
on the use of P450 enzymes, were extensively reviewed, giving
a wide overview on the current state of the art.[16–19] The first
fermentative synthesis of indigo was reported by Ensley and
later by Lee et al. using a dioxygenase for indole hydroxylation.
Tryptophanase that originated from endogenous tryptophan
catabolism was exploited to obtain indole.[20,21] Heme-depen-
dent monooxygenases (MOs) were later evolved towards C3-
hydroxylation of indole.[22–24] Flitsch et al. used formation of
indigo-derived pigments for detecting activity of MO mu-
tants.[25,26] Biocatalytic functionalization of unprotected indole
was achieved using engineered myoglobin variants which cata-
lyze non-native carbene transfer in whole cells.[27] Besides
using heme-dependent enzymes, also flavin-dependent MOs
play a key role in oxyfunctionalization. Accordingly a flavin
monooxygenase from Methylophaga sp. (mFMO) was estab-
lished for the biotechnological production of indigo and indi-
rubin.[28,29] Nevertheless, the synthesis of valuable halogenated
indigos has remained on analytical or small preparative scale,
particularly due to the low efficiency of halogenases as a
severe bottleneck. In more recent studies, Tischler and co-au-
thors reported on the conversion of haloindoles using a small
array of styrene MOs.[30] One-pot synthesis of indigoids either
in bacteria or plant as the hosts was recently achieved: By in-
troducing a tryptophan halogenase into the host strain along
with a hydroxylase, production of indigoids from tryptophan
was feasible, omitting the need for costly substituted indole
substrates through exploiting the cellular metabolism.[31,32]
However, product titers remained low and the isolation of the
pigment from the cultivation broth can become a tedious pro-
cedure. Moreover, structural modifications that can be, for ex-
Indole is a widespread heterocycle found in many natural
products.[1] For instance, indigo dyes derived from indole have
been applied in textile dyeing for thousands of years due to
their outstanding spectral properties.[2,3] Indigo (1) developed
to a bulk chemical in the last century whereas its C6-brominat-
ed analogue, 6,6’-dibromoindigo (6,6’-2), the major component
of the high-value pigment Tyrian purple, still remains a rarity
(Scheme 1A). Thanks to tremendous efforts by several research
groups traditional indigoids recently turned into focus as natu-
ral product-based, non-toxic materials for sustainable organic
electronics.[4–6] Topical studies examining the photoswitching
[a] Dr. C. Schnepel, Dr. V. I. Dodero, Prof. Dr. N. Sewald
Organische und Bioorganische Chemie, Fakultät für Chemie
Universität Bielefeld, Universitätsstraße 25, 33615 Bielefeld (Germany)
E-mail: norbert.sewald@uni-bielefeld.de
[b] Dr. C. Schnepel
Present address: School of Chemistry
Manchester Institute of Biotechnology, The University of Manchester
131 Princess Street, Manchester, M1 7DN (UK)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/chem.202005191.
ꢀ 2021 The Authors. Published by Wiley-VCH GmbH. This is an open access
article under the terms of the Creative Commons Attribution Non-Commer-
cial NoDerivs License, which permits use and distribution in any medium,
provided the original work is properly cited, the use is non-commercial and
no modifications or adaptations are made.
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Scheme 1. A. Structure of natural indigo dyes and diversification strategies. B. Biocatalytic retrosynthesis of haloindigo via multiple steps of CÀH functionaliza-
tion.
ample, introduced by chemocatalysis, prove challenging when
applying whole cell catalysts. As an alternative, in vitro enzyme
cascades offer a more modular approach for scaffold diversifi-
cation.
cally address the C5-, C6-, or C7-position of the indole moiety
(Scheme 2).
Formation of crosslinked enzyme aggregates (CLEAs) ena-
bles preparative-scale bromination of 3 resulting in product
titers of 280 mgLÀ1. As demonstrated in earlier studies, CLEAs
permit facile upscaling of the halogenation reaction. The bro-
minated amino acid is selectively obtained whereas protein
contaminants are simply filtered, therefore providing a good
starting point for cascade reactions.[38] In the next step, trypto-
phanase catalyzes the cleavage of the amino acid backbone to
release bromoindole (5): In a pyridoxal phosphate-dependent
reaction, a 1,4-elimination takes place leading to 5 and pyru-
vate. The gene encoding the tryptophanase TnaA (EC 4.1.99.1)
from E. coli was subcloned, and the resulting enzyme was uti-
lized in biotransformations (Figure S1, S2).[45,46] Initially, the ac-
tivity of TnaA towards l-6-bromotryptophan (6-4) was con-
firmed in analytical-scale reactions. Upon incubation of 6-4
with purified TnaA, 6-bromoindole (6-5) was successfully
formed (Scheme S3). This motivated performing TnaA-cata-
lyzed elimination preceded by enzymatic halogenation in a
one-pot fashion on a milligram scale (Figure 1). After quantita-
tive halogenation of 3 (0.1 mmol) using the C6-halogenase
Thal along with auxiliary enzymes (combiCLEAs) a cell-free ex-
tract containing overexpressed TnaA was simply added to the
We embarked on the development of a modular biocatalytic
platform to afford the synthesis of haloindigo that can be
easily isolated and/or diversified by addressing the halogen
substituents using Pd-catalyzed cross-coupling. Assembly of
the indigoid scaffold is achieved by stepwise CÀH functionali-
zation utilizing selective halogenation and hydroxylation start-
ing from l-tryptophan (3, Scheme 1B).[15] Various recent
achievements underscore that flavin-dependent tryptophan
halogenases are capable of regioselective aryl halogenation
using O2, FADH2 and a halide salt.[33,34] Current efforts on
random and targeted engineering, immobilization and combi-
nation with chemocatalysis considerably extended the synthet-
ic utility of these enzymes.[35–40] Recent reports also showcase
applications of halogenases for the synthesis of valuable chem-
icals using cascades in vivo and in vitro.[41À43] However, reports
on enzymatic halogenation of free indole indicate that the
substitution usually occurs in the electronically most favored
C3 position.[44] To retain regioselectivity at the benzene ring, it
is necessary to halogenate l-tryptophan (3) instead, since the
toolkit of regiocomplementary halogenases allows to specifi-
Scheme 2. General overview of the enzyme cascade for the synthesis of brominated indigo dyes starting from l-tryptophan (3). Sequential CÀH activation
was initiated by enzymatic halogenation of the indole side chain occurring selectively at C5, C6, or C7, respectively. Elimination of the amino acid backbone
and final hydroxylation at C3 position that spontaneously dimerizes gives target compound 2.
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Table 1. Results of enzymatic synthesis of bromoindole applying a se-
quential cascade of halogenation and TnaA-catalyzed elimination. Differ-
ent halogenated regioisomers were obtained by choosing regiocomple-
mentary halogenases (5-Br: PyrH; 6-Br: Thal; 7-Br: RebH) in the first step
(Figure 1A).
#
Product
isomer (5)
TnaA formulation
Scale
[mmol] total yield[b]
Conversion,[a]
1
2
3
4
5
6-Br
6-Br
6-Br
5-Br
7-Br
crude lysate
(0.15 L E. coli culture)
CLEA
(1.5 L E. coli culture)
freeze-dried cells (1 mgmLÀ1
0.10
1.0
98%,
not isolated
>99%,
60%
>99%,
68%
96%,
60%
91%,
63%
)
0.25
À0.50
0.50
freeze-dried cells
(1 mgmLÀ1
)
freeze-dried cells
(3 mgmLÀ1
0.25
)
[a] Conversion of l-bromotryptophan by TnaA was determined by RP-
HPLC. [b] Final yield refers to purified product obtained from the two-
step cascade.
for biocatalysis.[28] Enzyme assays carried out with 1 mm 6-5
and ADH/iso-propanol for concomitant NADPH regeneration
initially gave a purplish suspension at 258C. Whilst the sub-
strate was fully consumed, LC-MS indicated 6-bromo-3-
hydroxyindole (6) as a minor species present (Figure S6A). Not
surprisingly, under aerobic conditions, the hydroxylation of 6-5
directly initiates transformation of the hydroxylated intermedi-
ate into the insoluble pigment 6,6’-2. Despite low solubility in
DMSO and poor ionization abilities, ESI-MS analysis of the col-
ored sediment suggests the presence of 6,6’-dibromoindigo
(6,6’-2) as the biotransformation product (Figure S6B).
Figure 1. A. One-pot synthesis of 5-, 6-, and 7-bromoindole, respectively, by
sequential combination of halogenase and tryptophanase. B. Cutout of RP-
HPLC traces of two-step biocatalysis highlighting the selective synthesis of
6-bromoindole (6-5) starting from Trp by sequential halogenation and elimi-
nation.
Encouraged by these results, the hydroxylation of 5 was fur-
ther optimized to obtain an efficient process for the synthesis
and isolation of halogenated indigo. Seminal achievements by
Fraaije et al. inspired to generate a bifunctional biocatalyst by
fusion of mFMO with phosphite dehydrogenase (PTDH).[48–52]
N-terminal PTDH genetically fused to mFMO is capable of
NADPH regeneration by oxidation of phosphite while the
monooxygenase catalyzes the hydroxylation. In case of sub-
strate 6-5, the self-sufficient enzyme reached a total conversion
of approx. 96% (HPLC), whereas other regeneration systems
were less productive (Table S1). Comparing the substrate con-
version under optimized conditions revealed that unsubstitut-
ed indole causes a 2.5-fold higher TTN than its brominated de-
rivatives (Scheme S1). However, both 5- and 6-bromoindole
(5-/6-5) are being reasonably converted by PTDH-mFMO with a
slight preference for the C6-isomer. Only 7-5 could not be con-
verted into 7,7’-dibromoindigo.
biotransformation mixture. Moreover K2HPO4 was added to a
final concentration of 50 mm because potassium cations were
reported to have a positive impact on TnaA,[47] whilst K+ ions
were omitted during halogenation to suppress undesired activ-
ity of endogenous tryptophanase. By incubation for 24 h at
258C, >98% of substrate was selectively converted into 6-bro-
moindole (6-5), thus avoiding intermediary isolation of 6-4
(Figure 1B). Notable side products did not occur, which indi-
cates the high selectivity of the consecutive biotransformations
to isolate haloindoles in two steps that can be hardly synthe-
sized otherwise.
By applying TnaA crude lysate, freeze-dried cells or CLEAs,
haloindoles were afforded in quantities sufficient for multistep
synthesis and characterization (Table 1; Figure S4). 5-, 6-, and 7-
bromo isomers of 5 hence are obtained with >90% conver-
sion on larger scale. Solely, the TnaA loading had to be in-
creased in case of 7-5, presumably due to a lower activity.
Final isolation of 5 merely requires desalting over a plug of re-
versed-phase silica leading to a satisfying product purity of
>95% (Figure S5). Application of this cascade provides 5-, 6-
and 7-bromoindole (5) in good yields ranging from 60–70%
for two synthetic steps, starting from 3.
The optimized biotransformation steps were combined to
provide a one-pot cascade allowing stepwise transformation of
3 into dibromoindigo (2). In a sequential fashion, halogenation
of 3 was followed by elimination and final hydroxylation
simply by adding the enzymes and cofactors required for each
step without isolation of reaction intermediates (Figure 2A). Fi-
nally, the hydroxylation step reached full conversion after 96 h
on 0.1 mmol scale (Figure 2B). Initial C6-halogenation using
the halogenase Thal thus furnishes a purplish-colored solid
Inspired by these results, 6-5 was subjected to enzyme-cata-
lyzed C3-hydroxylation. The flavin-dependent monooxygenase
(mFMO) from Methylophaga spp. was cloned and established
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Figure 2. A. Cascade synthesis of Boc-protected dibromoindigo using multiple biocatalysis and Boc protection. The latter step facilitates solubilization and
final purification of the protected indigoid. Yields mentioned for 7 refer to the total 4-step cascade; calculations were based on the quantity of substrate 3.
B. HPLC diagram of PTDH-mFMO-catalyzed hydroxylation of 6-5. The substrate is continuously converted into insoluble pigment. The intermediary brominat-
ed indoxyl is only present in minor quantities while other side products are negligible. C. Identification of N,N’-Di-Boc-6,6’-dibromoindigo (6,6’-7) as the major
product of the final acylation step. ESI-ToF MS analysis confirms the identity of the dibrominated compound from the isotope pattern.
pointing to the successful formation of 6,6’-2 (Figure S8). Like-
wise, the halogenase PyrH (C5 halogenation) results in 5,5’-2 as
a blue-colored pigment.
average, based on three independent replicates. Furthermore,
a biotransformation on 0.50 mmol scale gave a comparable
yield (52%). Likewise, the cascade afforded 5,5’-7 in a similar
yield of 46%. Taking into account that the final product 7 is fi-
nally isolated after four sequential reactions, these results indi-
cate a straightforward method towards haloindigos.
As a consequence of its insolubility in aqueous medium, pig-
ment 2 precipitates quantitatively and can be simply harvested
by centrifugation. However, its isolation and purification from
the biotransformation extract, e.g., from protein precipitate,
proves challenging due to its low solubility. In light of a previ-
ous study by Sariciftci et al., acylation of the indigo nitrogen
enhances solubility by suppression of the intramolecular hy-
drogen bonds.[53] Hence, subsequent to biocatalysis the crude
solid was subjected to N,N’-Boc protection. As a test system to
obtain a suitable procedure applicable to cascade conditions, a
crude extract containing 1 was first employed (cf. Supp.
Inform. for further details, Table S2). According to these opti-
mized conditions, Boc2O (15 equiv) and DMAP (3 equiv) were
added to a freeze-dried crude extract containing 6,6’-2 re-sus-
pended in DMF, which led to formation of Boc-protected di-
bromoindigo (7) after stirring for 3 d at r.t. as proven by LC-MS
(Figure 2C). The soluble 6,6’-7 was purified by reversed-phase
silica resulting in good purity and an overall yield of 24% after
four synthetic steps referring to starting compound 3 (Fig-
ure 2A; S9). The final Boc protection step could be optimized
by portion-wise addition of Boc2O/DMAP and the use of CH2Cl2
as an alternative solvent. In total, the four-step cascade starting
from 0.25 mmol of 3 resulted in an increased yield of 50% on
We further embarked on investigations to diversify 2 by Pd-
catalyzed cross-couplings. Aryl substitution of indigoids is a
hitherto unexplored research area. Initially cross-coupling of
Boc-protected 6,6’-7 to the electron-rich 3-aminophenylboronic
acid was attempted in order to enhance the polarity of the
target compound and to facilitate detection by ESI-MS used as
a simple reaction screening. However, as the brominated start-
ing material was prone to Boc cleavage as well as decomposi-
tion in basic medium hindering formation of the desired aryl
species, we concentrated on arylation of 2 instead.
Even though the cross-coupling of the insoluble unprotect-
ed dibromoindigo (2) in the crude extract proved more chal-
lenging, this approach would provide convenient diversifica-
tion of the natural dye and potentially enhance solubility. 6-5
(1.0 mmol) was subjected to hydroxylation and the resulting
crude precipitate containing 6,6’-2 was incubated with 3-ami-
nophenylboronic acid, Pd(sSPhos) and K3PO4 at 1308C in a
DMF suspension.
Unexpectedly, LC-MS analysis indicated formation of a
mono-methylated diarylindigo whereas dimethylation was not
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Figure 3. A. Analytical-scale Suzuki–Miyaura cross-coupling of crude 6,6’-dibromoindigo (6,6’-2) affording diarylindigo derivatives. B. HPLC (280 nm) of the
cross-coupling reaction identifies diarylindigo 6,6’-8 in the soluble fraction and in the precipitate (re-dissolved in DMF prior to analysis). C. ESI-MS data of the
reaction of 2 and 3-aminophenylboronic acid prove the successful synthesis of aniline derivative 9 evident from [M+2H]2+ (m/z=223.09 calc.) and [M+H]+
(m/z=445.17 calc.).
observed (Figure S10). Pd-catalyzed aniline N-methylation has
been previously described.[54] Cross-coupling at lower tempera-
ture (908C) in iPrOH under microwave irradiation in the pres-
ence of 5 equiv of arylboronic acid, 0.1 equiv Na2PdCl4,
0.25 equiv sSPhos, and 6 equiv K3PO4 gave the unmethylated
target compound, accompanied only by minor side products
(Figure 3A/B). Formation of the diarylindigo 6,6’-8 succeeded
when using 4-hydroxyphenylboronic acid: The target diarylin-
digo among other cross-coupling components and homo-
coupled product were detectable in the reaction solution as
well as in the precipitate (Figure 3B). Furthermore, MS analysis
of the coupling reaction of 3-aminophenylboronic acid perfect-
ly correlates with the twofold coupled aniline derivative 6,6’-9
whilst not resulting in the methylated side product (Figures
3C, S11, S12).
porting Information, Analytical Data). Notably, distinctly sepa-
rated elution peaks occurred in RP-HPLC for all the diarylindi-
gos under dilute acidic conditions giving different UV/Vis-spec-
tra, but identical mass spectra. This observation indicates the
To implement indigoid arylation within the cascade, 6,6’-di-
bromoindigo (6,6’-2) was synthesized starting from 3. The
crude extract was subjected to arylation under reflux and Ar
atmosphere for 4 h to form diarylindigo. In addition to 6,6’-8
and 6,6’-9, the phenylacetic acid-substituted derivative 6,6’-10
was obtained by using the corresponding boronic acid
(Scheme 3). Its carboxylate salt is also soluble in aqueous basic
medium leading to a notable improvement for handling and
application of indigoids.
Based on this procedure a small set of 6,6’-diarylindigos was
isolated on a low milligram scale upon purification by column
chromatography. Product identities were unambiguously con-
firmed by high-resolution mass spectrometry (cf. Supporting
Information, Analytical Data). The HPLC data, instantly record-
ed after isolation, corroborate sufficient product purity (cf. Sup-
Scheme 3. Overall chemoenzymatic cascade for the synthesis of indigo de-
rivatives. The initial substrate l-tryptophan (3) is successively converted into
insoluble dibromoindigo (2) that is subsequently either N-acylated or em-
ployed for cross-coupling using a suspension of the crude extract. Particular-
ly, coupling of more polar residues improves compound solubility. Results of
5,5’- and 6,6’-disubstituted indigoids 8–10 as well as final product yields re-
ferring to the starting material 3 are mentioned.
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presence of cis/trans isomers (Figures S13–S15).[9] In general, N-
unsubstituted indigos do not photoisomerize because of domi-
nating and fast deactivation mechanisms.[55] On the other
hand, under acidic conditions and in the gas phase a trans/cis-
protoisomerization has been detected for indigo and its bis-
imine derivatives.[55] The presence of the trans/cis-isomers in
the diarylindigoids suggests that aryl substitutions at the
indigo core reduce the double bond character of the central
C=C bond, thus facilitating isomerization in the protonated
state (Scheme S2).[55] NMR characterization of the aryl products
proved challenging, probably as a consequence of the resul-
tant low quantities along with isomerism and a potential light
sensitivity. For example, 1H- and 13C-NMR signals could be
clearly assigned for 6,6’-8 and 6,6’-10. Additional peaks detect-
ed in the spectra originate from isomerization as well as minor
impurities that were taken into account when calculating the
yields (Table 1, Scheme 3). Cross-couplings using 5,5’-2 were
less efficient and potential products could not be isolated in
satisfactory purities.
UV/Vis and fluorescence spectra were recorded to study the
spectroscopic properties of the novel diarylindigo derivatives.
Substituents at the benzene ring of the indigo moiety do not
have a pronounced effect on the UV/Vis absorption. +M Sub-
stituents like halogens or methoxy groups at the 5,5’- or 7,7’-
positions of indigo are supposed to cause a bathochromic
shift, while at the 6,6’-positions a hypsochromic shift is expect-
ed. This characteristic is mainly known for Tyrian purple (6,6’-
2), which has its visible band at lower wavelengths than indigo
(1).[56] In case of diarylindigoids, the aromatic rings mainly sta-
bilize the coplanar conformation of the molecule with a mar-
ginal mesomeric effect; however a fine-tuning of the maximal
wavelength might be possible by changing the peripheral sub-
stituents.[57]
In DMSO, only diarylindigo 6,6’-10 with the para-carboxy-
methylbenzoic acid moiety shows an intense band in the deep
red region at 626 nm similar to the band of 1 at 619 nm (Fig-
ure 4A, dashed line). Besides, a second intense band at 384 nm
is different to the shoulder observed for indigo (1) at 333 nm.
Another band appeared at 318 nm, while 1 shows a signal
below 300 nm. Notably 6,6’-10 is soluble in water, in which a
strong hypsochromic effect was observed. The change of sol-
vent from DMSO to water did not change the absorption maxi-
mum at 624 nm.
Figure 4. Solvent-dependent UV/Vis absorbance (b) and fluorescence
emission (c) spectra of 6,6’-10 and indigo (1). A. in DMSO. B. in water;
only 6,6’-10 is soluble (lex =320 nm). C. A qualitative comparison of the fluo-
rescence intensity of the 6,6’-derivatives at around 660 nm in DMSO is
shown (25 mm, 208C), spectra were acquired at optimum excitation wave-
lengths of each compound. Further spectra and experimental details are
presented in the Supporting Information (S16, S17). Identical experimental
conditions were applied for all compounds analyzed (25 mm, 208C; cf. Sup-
porting Information).
For the other arylindigo derivatives, a hypsochromic effect in
comparison to indigo (1) was obvious. Trace amounts of water
might be the reason, as 8 and 9 turned out to be rather hygro-
scopic. The aminophenyl-substituted indigoid (6,6’-9) shows
strong hypsochromic and bathochromic shifts to 597 nm and
650 nm, respectively (Figure S16). On the contrary, the
hydroxyaryl derivative (6,6’-8) shows a different tendency with
a maximum at 630 nm for 6,6’-8.
Generally, indigo and its derivatives often suffer from low
fluorescence and low quantum yields due to a dominant radia-
tionless decay (e.g. Figure S17).[55] Interestingly, 6,6’-10 shows
intense fluorescence emission at 664 nm that is even three
times higher than that of indigo (1) under identical experimen-
tal conditions (Figure 4A, full line, Figure 4C). However, in
water this fluorescence band does not occur, so that 6,6’-10
merely shows a significant emission at 425 nm pointing to an
influence of the solvent on the fluorescence properties of this
compound (Figure 4B, full line). A qualitative comparison of
the fluorescence intensity of the other 6,6’-derivatives in DMSO
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Manuscript received: December 3, 2020
Accepted manuscript online: January 26, 2021
Version of record online: February 26, 2021
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