A novel and unusually long-lived chemiluminophore based on the 7-hydroxycoumarin scaffold

Article abstract of DOI:10.1039/c1cc11919b

Synthesis and chemiluminescent properties of a new 1,2-dioxetane chemiluminophore bearing a 7-hydroxycoumarin moiety are presented. The 1,2-dioxetane decomposition ended up with strong and long-lived emission of light. This new structure opens way to the development of a new generation of bright chemiluminescent bio-probes.

Full text of DOI:10.1039/c1cc11919b

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COMMUNICATION
A novel and unusually long-lived chemiluminophore based on the
7-hydroxycoumarin scaffoldw
Jan Kocı ab Virgile Grandclaude,ab Marc Massonneau,b Jean-Alexandre Richard,ab
´
,
Anthony Romieu*ac and Pierre-Yves Renard*acd
Received 4th April 2011, Accepted 21st April 2011
DOI: 10.1039/c1cc11919b
Synthesis and chemiluminescent properties of a new 1,2-dioxetane
chemiluminophore bearing a 7-hydroxycoumarin moiety are
presented. The 1,2-dioxetane decomposition ended up with
strong and long-lived emission of light. This new structure opens
way to the development of a new generation of bright chemi-
luminescent bio-probes.
successful use in some bioanalyses and bioimaging applica-
tions due to the significant absorption of the emitted light by
some biomaterials.6 The use of relay fluorophores can be an
alternative to red-shift this short-wavelength emission. Thus,
two different strategies are generally adopted to transfer the
energy released by the chemiluminescent reaction to the
fluorophore moiety. The first strategy relies on the connection
of chemiluminophore and fluorescent dye by a linker which
allows an efficient chemiluminescence resonance energy
transfer (CRET)7 or through bond energy transfer (TBET).8,9
The second strategy is based on the concomitant encapsula-
tion of chemiluminophore and fluorescent dye within the same
confined environment of nanoparticles or micelles.10
Over the past decade, based on the strained and unstable
intermediate polyoxygenated rings formed in the bioluminescence
processes, many 1,2-dioxetane structures have been developed
as convenient tools for chemiluminescence (CL) studies1 and
to design chemoluminogenic probes suitable for sensitive
detection and quantitation of various bio-analytes in complex
biological mixtures.2 The origin of the CL for these
1,2-dioxetanes is a Chemically Initiated Electron Exchange
Luminescence phenomenon (CIEEL): the deprotonation of a
hydroxyaryl group bearing a 1,2-dioxetane unit (preferentially
at the meta position of the aromatic relative to the phenolate)
generates an unstable oxidoaryl-substituted 1,2-dioxetane
which undergoes intramolecular charge-transfer-induced
decomposition (CTID) to produce a singlet-excited carbonyl
fragment while effectively emitting light.3
To expand the scope of the first strategy to a wider range of
fluorescent reporter molecules, we wish to describe in this
communication the challenging synthesis and luminescent
properties of a new chemiluminophore 1 based on a dihydro-
furan ring directly bound at the 5-position to a pro-fluorescent
7-hydroxycoumarin unit (Fig. 1).
Indeed, the 7-hydroxycoumarin moiety is well-known for
its pro-fluorescent properties: substitution of its phenol by
any electron-withdrawing group completely abolishes its
fluorescence.11,12 By attaching a 1,2-dioxetane moiety at the
5-position of a 7-acetoxycoumarin unit (i.e., in the meta
position to the 1,2-dioxetane required for efficient CIEEL), it
will be possible to study the unveiling luminescence associated
to the phenol deprotection reaction which should end up with
a simultaneous release of the fluorescent properties of the
7-hydroxycoumarin and of the electron exchange based
luminescence.
Among the required stabilising moieties, the dihydrofuran
ring has been described in the late 90’s as a convenient scaffold
to obtain 1,2-dioxetanes exhibiting a remarkable thermal
stability.4 Thus, many examples of such oxy-substituted
dioxetanes have been synthesised during these last few years.5
The main drawback of these structures is their light emission
wavelengths, which are too close to the UV region for their
a Equipe de Chimie Bio-Organique, COBRA-CNRS UMR
7-Hydroxycoumarins are usually synthesised by the Pechmann
reaction, starting from the corresponding resorcinol and
b-keto acid (or ester) and involving acidic conditions.13
`
6014 & FR 3038, rue Lucien Tesniere, 76131 Mont-Saint-Aignan,
France. E-mail: anthony.romieu@univ-rouen.fr,
pierre-yves.renard@univ-rouen.fr; Web: http://ircof.crihan.fr;
Fax: +33 2-35-52-29-71; Tel: +33 2-35-52-24-27 or 24-76
b QUIDD, Pharmaparc II, baˆtiment D, Voie de l’innovation,
27100 Val-de-Reuil, France. E-mail: mcmm@quidd.com;
Web: http://www.quidd.com; Fax: +33 2-32-59-67-65;
Tel: +33 2-32-63-46-91
c Universite´ de Rouen, Place Emile Blondel,
76821 Mont-Saint-Aignan, France
d Institut Universitaire de France, 103 Boulevard Saint-Michel, 75005,
Paris, France
w Electronic supplementary information (ESI) available: Detailed
synthetic procedures and characterisation/spectral data for compounds
1–11. See DOI: 10.1039/c1cc11919b
Fig. 1 Chemiluminophore–fluorophore hybrid studied in this work,
whose CL reaction is based on the phenol deprotection.
c
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Chem. Commun., 2011, 47, 6713–6715 6713

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required lactone through a less commonly used Wittig reac-
tion, which involved the introduction of a carbonyl moiety at
the 1-position of the phenyl ring. Direct metallation of this
position gave low yields. However, an efficient and selective
bromination using N-bromosuccinimide in THF–water (95 : 5)
gave the product 5 with a high yield (91%). This bromination
reaction was best performed prior to the elimination process
leading to the dihydrofuran derivative 6. This latter reaction
involved standard chlorination of the tertiary alcohol followed
by spontaneous (and/or pyridine-mediated) elimination to
form 6 with a good yield (72%). Thereafter, bromine–lithium
exchange was performed using n-BuLi, and the aryllithium
intermediate was trapped by methyl chloroformate to give
methyl ester 7 in 83% yield. Further conversion into aldehyde
Scheme 1 Failure of the first synthetic route.
Thus, we first planned the preparation of 1 through the
introduction of the functionalised alkyl chain required for
the formation of the 1,2-dioxetane moiety, on the 5-position
of a 7-methoxycoumarin derivative readily obtained from
standard condensation (vide supra), and to build the tetra-
hydrofuran ring thereafter (Scheme 1).
Unfortunately and despite our efforts, the crucial synthetic
step which consists of the intramolecular cyclisation of the
benzylic anion to the ketone to form the tetrahydrofuran ring,
which requires strongly basic conditions, did not generate the
expected compound. Contrary to our expectations, the lactone
ring did not suffer much from these conditions (especially ring
opening), but we suspected that the electron delocalisation
within the coumarin ring dramatically lessens the nucleo-
philicity of the formed benzyl-type carbanion, thus preventing
the desired ring cyclisation. Using harsher reaction conditions
eventually destroyed the coumarin unit. To overcome this
failure, we turned our attention to an alternative and more
challenging approach which involves: (1) the synthesis of the
tetrahydrofuran ring, (2) the construction of the coumarin ring
under unusual conditions and finally (3) the formation of the
fragile 1,2-dioxetane moiety (Scheme 2).
was obtained by using
a two-step reduction–oxidation
sequence. Thus, methyl ester 7 was firstly treated with
DIBAL-H and the resulting primary alcohol was oxidised
with MnO2 in suspension in anhydrous toluene under reflux
to give the expected benzaldehyde in 53% overall yield. The
formation of the lactone ring has required the selective depro-
tection of one of the two methoxy groups. We were pleased to
observe that the presence of the ortho-aldehyde allowed the
selective removal of the desired methyl group with BCl3 to
afford the key benzaldehyde derivative 8. This product was
directly submitted to a Wittig reaction, involving the ylide
derived from (2-ethoxy-2-oxoethyl)triphenylphosphonium
salt, in anhydrous N,N-diethylaniline as solvent. The lactone
ring was readily formed to obtain 7-methoxycoumarin 9 in a
good yield (83%). As expected, since the first methoxy group
was very selectively removed, the second demethylation reac-
tion of the 7-position of the coumarin core was by far trickier.
We found that it could be achieved in quantitative yield by
treatment of 9 with a large excess of AlCl3 in dry CH2Cl2
under reflux. Subsequent protection of the released phenol
with an acetyl group under standard conditions afforded the
7-acetoxycoumarin 10. Finally, the 1,2-dioxetane moiety was
generated through a [2+2] cycloaddition reaction between
singlet oxygen and the enol ether of 10. 1O2 was generated
under mild conditions by standard type II photosensitisation
(i.e., oxygen, visible light and methylene blue as photosensitiser).
The 1,2-dioxetane 1 was found to be stable enough to be
purified by chromatography on a silica gel column and finally
recovered in 63% yield. All spectroscopic data, in particular
NMR and mass spectrometry, are in agreement with the struc-
ture assigned (see ESIw). Interestingly, this chemiluminophore
The coumarinic 1,2-dioxetane 1 has been synthesised from
commercially available (3,5-dimethoxyphenyl)methanol 2.
This latter primary alcohol was brominated with PBr3 to
obtain the corresponding benzyl bromide derivative 3 in 69%
yield. Substitution reaction with 2,2,4,4 tetramethylpentane-1,3-
diol provided quantitatively the intermediate hydroxyl ether
which was oxidised with pyridinium chlorochromate (PCC) to
afford the corresponding ketone in 92% yield. Use of this
rather drastic oxidant was required, since all attempts using
milder conditions: Swern oxidation, IBX or TPAP/NMO
system gave poor yields. Next, we found that the best condi-
tions for the cyclisation involved the use of freshly prepared
dimethyl sulfoxide anion (potassium dimsyl) as a strong base
to form the corresponding tetrahydrofuran ring 4 in 70%
yield.14 In order to convert the resorcinol dimethyl ether 4
into the corresponding coumarin, we decided to generate the
Scheme 2 Synthesis of coumarinic chemiluminophore 1.
c
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red-shift emission of such chemiluminophores. Indeed, the
CL studies have revealed that the luminescence energy
produced at the 1,2-dioxetane moiety is efficiently transferred
to the coumarin unit to emit light at the maximum wavelength
of this fluorophore. Interestingly, functionalisation of its
4-position with a bio-orthogonal ‘‘handle’’ (e.g., an acetic acid
pendant arm) should make possible some applications in
bio-labelling. Further efforts are also devoted to the imple-
mentation of this promising strategy to red-emitting fluorescent
phenols such as conjugation-extended 7-hydroxycoumarin NIR
dyes recently reported by us.15
This work was supported by La Re
via the CRUNCh program (CPER 2007–2013) especially for a
post-doctoral fellowship to Jan Kocı, and Institut Universitaire
´
gion Haute-Normandie
´
de France (IUF). We thank Annick Leboisselier (INSA de
Rouen) for the determination of elemental analyses.
Fig. 2 CL spectra of 1 in DMSO (after adding 1.0 M TBAF in THF,
3% v/v) at 25 1C (concentration: 170 mM): (a) scan mode, (b) kinetic
mode at l = 470 nm.
Notes and references
1 For selected reviews, see: (a) M. Matsumoto, J. Photochem.
Photobiol., C, 2004, 5, 27; (b) W. J. Baader and E. L. Bastos, in
Science of Synthesis: Houben–Weyl Methods of Molecular Trans-
formations, ed. A. Berkessel, Thieme-Chemistry, Stuttgart, 2008,
vol. 38, p. 323.
was found to be full-stable for few days at room temperature
1
as inferred from H NMR analyses.
The photophysical properties of 7-acetoxycoumarin–dioxetane
hybrid 1 were evaluated in DMSO. The UV-vis absorption
spectrum displayed a broad band between 260 and 340 nm with
two maxima located at 278 (e 9700 dm3 molÀ1 cmÀ1) and 321 nm
(e 8200 dm3 molÀ1 cmÀ1) (see ESIw). The hypsochromic shift of
this absorption spectrum relative to the free fluorophore (60 nm)
is in agreement with the spectral behaviour currently observed
for phenol-based pro-fluorescent probes.12,15,16 Furthermore, as
expected, this compound was found to be virtually non-
fluorescent and its quantum yield was too low to be accurately
measured. The chemiluminescent characteristics of 1 were also
determined in DMSO after adding 1.0 M TBAF in THF
(basic fluoride ions) which acts as a CL triggering through the
removal of the acetyl group and subsequent generation of the
phenolate species.17 Fig. 2a shows the CL spectrum of 1. This
compound was confirmed to emit blue light with a single
maximum at 470 nm. This luminescence peak apparently
originated from the coumarin unit of 1, as inferred from the
similarity between Fig. 2a and the fluorescence emission
spectrum of the coumarin 11 released by the TBAF-induced
chemiluminescent reaction (see ESIw). Thus, the luminescence
energy produced at the 1,2-dioxetane moiety was completely
transferred to the energy acceptor coumarin.
2 For selected recent examples, see: (a) L. Liu and R. P. Mason,
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9 For the application of this concept to ROS-sensitive chemi-
luminescent probes, see: (a) R. Saito, A. Ohno and E. Ito,
Tetrahedron, 2010, 66, 583; (b) M. Sekiya, K. Umezawa, A. Sato,
D. Citterio and K. Suzuki, Chem. Commun., 2009, 3047.
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17 When aq. 1.0 M NaOH (3% v/v) was employed as CL trigger, two
emission maxima at 430 and 470 nm were observed, respectively,
assigned to cinnamate derivative (resulting from lactone
ring-opening) and compound 11 (see ESIw).
Furthermore, light emission was very strong once the trigger
was added, and its kinetic shape presents a long-lived decay,
which is a significant advantage in biological assays (Fig. 2b).
Indeed, significant light emission at 470 nm was still detected
even 10 hours after the initial TBAF-mediated phenolate release.
We have successfully synthesised 7-hydroxycoumarin-
attached 1,2-dioxetane 1 to develop a novel approach to
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 6713–6715 6715