Strong green chemiluminescence from naphthalene analogues of luminol

Article abstract of DOI:10.1039/c4nj00364k

Naphthalene analogues of luminol with several types of substituents were prepared. All molecules exhibit a strong green chemiluminescence in aqueous solution, which is catalysed by iron, pointing to the possibility of increasing the sensitivity of analytical methods currently based on luminol. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4nj00364k

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Strong green chemiluminescence from
naphthalene analogues of luminol†
Cite this:DOI: 10.1039/c4nj00364k
Govindasami Periyasami, Liliana Martelo, Carlos Baleizao* and
Mario N. Berberan-Santos*
´
˜
Received (in Montpellier, France)
12th March 2014,
Accepted 17th March 2014
DOI: 10.1039/c4nj00364k
www.rsc.org/njc
Naphthalene analogues of luminol with several types of substituents
were prepared. All molecules exhibit a strong green chemiluminescence
in aqueous solution, which is catalysed by iron, pointing to the possibility
of increasing the sensitivity of analytical methods currently based on
luminol.
The chemiluminescence of luminol, first reported by Albrecht in
1928,1 is extensively used in analytical and biochemical applications,
namely to monitor the production of reactive oxygen species in live
cells,2 and in forensics for the detection of bloodstains invisible to
the naked eye.3–5 In aqueous solution, and when mixed with an
oxidizing agent, luminol exhibits a striking blue chemiluminescence,
which is catalyzed by iron.3,4 In this way, a standard forensic test for
the visual detection of bloodstains consists of spraying a luminol
aqueous solution in a darkened environment. Selective chemilumi-
nescence from the sprayed bloodstains is then observed due to the
enhancing (catalytic) effect of the iron-containing heme group.5
However, the human eye has maximal sensitivity in the green
spectral region, a wavelength region where less interference from
background emission is also expected. For this reason, luminol
analogues with green chemiluminescence in aqueous solution are of
great interest.
Here we report the preparation of new luminol analogues with
green chemiluminescence in aqueous solution. The emission color
shifts from luminol’s standard blue and was obtained by both
extending the aromaticity, as a result of replacing the benzene
(luminol) with a naphthalene core, and by changing the position
of the amine group (position 5 or 6 in the naphthalene structure,
Chart 1).
Chart 1 Chemical structures of luminol and analogues 1–5, with
extended aromaticity and different substituents in position 5 or 6 of the
naphthalene ring.
The incorporation of benzyl groups as substituents in the
amine also tunes the chemiluminescence emission of the new
luminol analogues (Chart 1).
The synthetic strategy for the preparation of analogues 1–3
(amine substituents in position 5 of the naphthalene ring), is
presented in Scheme 1. For the synthesis of 1, we started with the
cyclization of 2,3-naphthalenedicarboxylic acid with hydrazine in
acetic acid, affording pyridazinedione 6.6 The subsequent step was
the nitration of 6, an electrophilic substitution which occurs at
electron rich positions. In the case of naphthalene and according to
the frontier orbital theory, the electron density is highest at position
1. In our case, because we have substitutions in the naphthalene
ring, positions 1 and 5 can be considered for an electrophilic attack.
However, because of the pyridazinedione in positions 2 and 3,
position 5 is more electron rich than position 1 and with less steric
hindrance. In our first attempts for the nitration of compound 6, we
used nitric acid but we could not control the selectivity. The multi-
nitration products obtained using nitric acid led us to use nitronium
tetrafluoroborate, a mild nitration agent.7 The nitration occurs with
high selectivity, but with low yields, as already reported for this type
of reaction.8,9 The selective nitration at position 5 was followed by an
efficient soft reduction with metallic iron and ammonium chloride10
yielding analogue 1. The direct preparation of analogues 2 and 3
from analogue 1 (mono- and di-benzyl amine protection) was not
possible (interference with the protons in the pyridazine group).
´
´
CQFM- Centro de Quımica-Fısica Molecular and IN- Institute of Nanoscience and
´
Nanotechnology, Instituto Superior Tecnico, Universidade de Lisboa, 1049-001
Lisboa, Portugal. E-mail: carlos.baleizao@tecnico.ulisboa.pt,
berberan@tecnico.ulisboa.pt
† Electronic supplementary information (ESI) available: Experimental proce-
dures, methods, and product characterization. See DOI: 10.1039/c4nj00364k
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Scheme 2 Synthesis of analogues 4 and 5.
groups of analogue 4 and 5, but without success. Other
attempts, namely the hydrolysis of the benzylamino groups
(H2 with Pd/C) of derivatives 19 and 20 or the ester hydrolysis of
compound 16 (and consequent hydrazine cyclization) under
basic and acidic conditions, proved to be unfruitful, as it was
not possible to isolate the products because of their zwitter-
ionic nature.
Scheme 1 Synthesis of analogues 1–3.
The chemiluminescence of the different analogues was
A different pathway was delineated for analogues 2 and 3, which studied in aqueous solutions (5% DMSO, due to the insolubility
started with the quantitative esterification of 2,3-naphthalenedi- of analogues 3 and 5 in pure water), using potassium persulfate
carboxylic acid (compound 8),11 followed by nitration at position as an oxidant.14 This procedure also slows down the reaction
5 (compound 9, confirmed by 1H–1H and 1H–13C COSY NMR rates, allowing the facile recording of the spectra, when com-
correlation spectra-ESI†) and consequent quantitative reduction pared with the Fe–NaOH system, which has faster kinetics. The
with iron (compound 10), as used in analogue 1 synthesis. The chemiluminescence spectra of luminol and those of analogues
mono- and di-benzylation of the amine group was selectively 1–5 in H2O/DMSO (5%) after addition of K2S2O8 are presented
achieved using potassium carbonate (compound 11) and sodium in Fig. 1. Although the intensities are comparable, the analogues
hydride (compound 12), respectively.12 The ester hydrolysis of the exhibit a very large bathochromic shift of the chemiluminescence
5-((mono or di)benzylamino) diethyl naphthalene-2,3-dicarboxylate maxima with respect to that of luminol: the standard luminol
(11 and 12) was performed under basic conditions,13 and the chemiluminescence has a maximum at ca. 420 nm, whereas the
cyclization of 5-((mono or di)benzylamino) naphthalene-2,3- maxima of analogues 1–5 vary between 490 nm and 590 nm,
dicarboxylic acid (13 and 14) with hydrazine in acetic acid afforded depending on the position of the amine and substituents.
analogues 2 and 3 in high yields. We also attempted the hydrolysis Analogues 4 and 5, with the amine group in position 6 of the
(using H2 with Pd/C) of the benzylamino groups of analogue 2 and 3 naphthalene ring, both have a maximum at 490 nm. When the
to obtain analogue 1, but without success.
amine group (either, mono or di-benzyl substituents) is in position
The preparation of analogues 4 and 5 (amine substituents in 5 of the naphthalene ring (analogues 2 and 3), the chemi-
position 6 of the naphthalene ring) is presented in Scheme 2. luminescence maximum further shifts to 540–550 nm. This can
The synthetic strategy followed for these compounds was based be explained by an increase of the electron density in position
on ref. 12, aiming at using 2,3-naphthalimide derivatives as 5, which is already high, with a consequent change in the
probes for monitoring protein–protein interactions. Compound intramolecular charge distribution of the analogue. In Fig. 1A,
15, diethyl 6-nitro-2,3-naphthalenedicarboxylate, was an inter- as inset, we show color pictures of chemiluminescence of
mediate of the final products in the study, and we use it as the luminol and analogue 2, with the change from dark blue to
starting point for the synthesis of analogues 4 and 5. Com- green being apparent. The highest shift is obtained with
pound 15 is in fact a structural isomer of compound 9 analogue 1 (primary amine in position 5 of the naphthalene
(Scheme 1), and the synthetic strategy employed was the same: ring) whose chemiluminescence maximum occurs at 590 nm. It
reduction of the nitro group to an amine (compound 16), is clear that the presence of the benzyl substituents in the
followed by mono- and di-benzylation of the amine (com- amine, affects the chemiluminescence, most probably because
pounds 17 and 18), basic hydrolysis of the esters groups of the reduction of the electron donor behavior of the amine.
(compounds 19 and 20) and finally the cyclization with hydra- The synthesis and chemiluminescence of analogue 1 are
zine in acetic acid, obtaining analogues 4 and 5 in good yields. described in the literature,15,16 however a chemiluminescence
We also tried to obtain an unsubstituted amine analogue maximum similar to that of luminol (ca. 420 nm) was reported
through the hydrolysis (H2 with Pd/C) of the benzylamino (for the corresponding secondary amine, used for amino acid
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Fig. 2 Chemiluminescence spectra (solid lines, using Fe/NaOH) of luminol
and analogue 2, and fluorescence spectra (dashed lines) of 3-APA and
oxidation product 13 in H2O/NaOH/H2O2. The inset shows color pictures of
chemiluminescence from luminol and analogue 2.
In summary, we successfully prepared new luminol analogues
with extended aromaticity and different substituents in the aromatic
ring, leading to green chemiluminescence. The new luminol
analogues have a naphthalene core (whereas luminol has a benzene
core), with the amine group in position 5 or 6 of the polycyclic ring.
The increase of aromaticity leads to a bathochromic shift of the
chemiluminescence maximum by 70 nm when compared with
luminol, if the amine is in position 6 of the naphthalene ring.
However, for the analogues with the amine in position 5, the
chemiluminescence maximum red shifts by 120–130 nm, when
compared with luminol. This is due to the changes in the electron
density on the aromatic rings. The presence of substituents in the
amine group also influences the chemiluminescence performance.
The highest increase in the chemiluminescence maximum was
obtained for an analogue without substituents in the amine group
(in position 5), with a shift of 170 nm when compared with luminol.
Chemiluminescence bathochromic shifts are also observed upon
using iron as a catalyst, and this new evidence can open new doors
to develop more sensitive chemiluminescence detection methods in
blood analysis or for new sensing systems, taking into account
possible toxicity issues of the reactants, when relevant.
Fig. 1 (A) Normalized chemiluminescence emission spectra of luminol
and analogues 1–5 in H2O/DMSO (5%) with K2S2O8. The inset shows color
pictures of chemiluminescence from luminol and analogue 2. (B) Color
pictures of the fluorescence of oxidation products in H2O/DMSO (5%)
under excitation at 365 nm.
labeling, a chemiluminescence maximum wavelength of 480–
510 nm is indicated).15,16
As mentioned earlier, and as is well known, the chemi-
luminescence is the result of an oxidation reaction, and the
oxidation products are the emitting species.2,5,17 The oxidation
products of our analogues were also synthesized, namely:
analogue 5 vs. compound 20; analogue 4 vs. compound 19;
analogue 3 vs. compound 14; analogue 2 vs. compound 13;
analogue 1 vs. compound 10 (nonhydrolyzed ester), and luminol vs.
3-aminophthalic acid (3-APA). In Fig. 1, we present color pictures of
the fluorescence of oxidation products in H2O/DMSO (5%) under
excitation at 365 nm. It is possible to see that the chemilumines-
cence of the new analogues cover a wide range of the visible
spectrum. The chemiluminescence colors of compounds 13 and
14 (Fig. 1B) are similar to that of analogue 2 (Fig. 1A, inset).
One of the main applications of the chemiluminescence of
Experimental
luminol is analytical, especially in blood analysis and detection, Chemiluminescence procedure: the chemiluminescence experi-
where iron-containing heme is the catalyst, as mentioned above. In ments were performed in a quartz cuvette (1 Â 1 cm). Typically,
Fig. 2 we present the chemiluminescence spectra of luminol to a solution (2.5 mL) of luminol or analogues (5 Â 10À5 M) in
and analogue 2, using iron as a catalyst. Although the chemi- H2O (with 5% DMSO), a solution of K2S2O8 (6 Â 10À2 M) in H2O
luminescence maximum of analogue 2 experiences a significant was added (100 mL). In the case of iron catalyzed chemi-
hypsochromic shift (from 550 nm to 490 nm, compare Fig. 1), a luminescence, to a solution (2.5 mL) of luminol or analogues
green chemiluminescence is still observed (Fig. 2, inset). The (5 Â 10À5 M) in H2O, a solution of H2O2 (1 Â 10À2 M) and
chemiluminescence decay time measured for analogue 2 (260 s), K3Fe(CN)6 (2 Â 10À3 M) in alkaline H2O (NaOH, 2.5 Â 10À2 M)
is even higher that that obtained for luminol (170 s) under the was added (200 mL). The chemiluminescence spectra were
same conditions. The similarity of the fluorescence spectra of the recorded immediately after mixing. The fluorescence spectra
oxidation products, 3-APA and compound 13, with the respective of 3-APA and analogue 2 (5 Â 10À5 M) in alkaline H2O (NaOH,
chemiluminescence, can also be observed.
2.5 Â 10À2 M) were recorded at room temperature.
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Acknowledgements
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ˆ
This work was partially supported by Fundaçao para a Ciencia e a
Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects
RECI/QEQ-QIN/0189/2012 and PTDC/QUI-QUI/123162/2010. P.G.
and L.M. thanks FCT for Post-Doctoral (SFRH/BPD/66457/2009)
and PhD (SFRH/BD/47660/2008) grants, respectively.
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