Chemistry on Boranils: An entry to functionalized fluorescent dyes

Article abstract of DOI:10.1021/ol3020573

A Boranil fluorophore bearing a nitro-phenyl group has been selectively reduced to its anilino form and then successfully converted to amide, imine, urea, and thiourea derivatives which are fluorescent dyes. Its isolated isothiocyanate intermediate deriva

Full text of DOI:10.1021/ol3020573

ORGANIC
LETTERS
2012
Vol. 14, No. 18
4774–4777
Chemistry on Boranils: An Entry to
Functionalized Fluorescent Dyes
ꢀ
Denis Frath, Sebastien Azizi, Gilles Ulrich,* and Raymond Ziessel*
ꢀ
Laboratoire de Chimie Organique et Spectroscopies Avancees (LCOSA),
UMR 7515 au CNRS, Ecole de Chimie, Polymeres, Materiaux de Strasbourg (ECPM),
ꢁ
ꢀ
25 rue Becquerel, 67087 Strasbourg, Cedex 02, France
gulrich@unistra.fr; ziessel@unistra.fr
Received July 25, 2012
ABSTRACT
A Boranil fluorophore bearing a nitro-phenyl group has been selectively reduced to its anilino form and then successfully converted to amide, imine,
urea, and thiourea derivatives which are fluorescent dyes. Its isolated isothiocyanate intermediate derivative was used ina model labeling experiment
with Bovine Serum Albumin (BSA). The purified labeled-BSA exhibits strong luminescence (Φf = 47%) in a phosphate buffer at pH = 7.4.
There is currently a renewed interest in the field of dyes
and fluorophores, propelled by a constant need for new
candidates for organic optoelectronic devices (OLED,
OPV),1 molecular sensors,2 and a parallel search for fluoro-
phore probes for biological labeling.3 There are currently
many leading families in the fluorophore research area such
as cyanines,4 rhodamines,5 squaraines,6 and BODIPYs.7
More recently, new fluorescent dyes based on borate com-
plexes have been developed by using the boron atom as a
structuring element to rigidify and flatten cyanine skeletons.
Some new structures constructed from a central NꢀBꢀO8 or
NꢀBꢀN9 pattern have generated optimism that a family of
value comparable to that of BODIPY may be envisaged. We
recently introduced a new family of fluorescent boron com-
plexes, so-called Boranils, built around a NꢀBꢀO pattern
and using salicylaldanilinimines.10 These compounds are
particularly appealing, due to their facile synthesis, poten-
tially on a large scale, and the ease of their postsynthetic
modifications. We describe here a particular procedure lead-
ing to a versatile amino-Boranil synthon suitable for further
substitution introducing a variety of functional sites.
€
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r
10.1021/ol3020573
Published on Web 09/07/2012
2012 American Chemical Society

respectively. Thiourea derivatives 7 can be obtained in good
yield (89%) by the same procedure using thiophosgene.
Scheme 1. Reduction of the Nitro-Boranil
Significantly, the isothiocyanate intermediate 8 (νNCS
=
2102 cmꢀ1) can be isolated (77% yield), thus opening the
way to applications in biological materials labeling. The
isolated compound 8 was used (Scheme 3) to synthethize
thiourea 9 in good yield (73%), by reaction with hexyla-
mine. All the compounds were unambiguously character-
ized by their 1H and 13C NMR spectra, mass spectra, and
elemental analysis (see Supporting Information). Finally,
we used 8 to label lysine residues of BSA (Bovine Serum
Albumin) under conditions similar to those used with
commercial isothiocyanate-functionalized dyes (i.e., FITC:
fluorescein isothiocyanate).12 The BSA was labeled with an
excess of dye in basic aqueous DMSO and purified by use of
a Sephadex G25 gel column eluted with PBS.
Starting from the fluorescent Boranil compound A, we
investigated its conversion into more useful derivatives. In
contrast to known procedures where Boranil compounds
werereduced insitubyborane tothecorresponding amino-
phenol borate,11 we were pleased to find that, with soft
hydrogenation, we could obtain almost quantitatively the
amino derivative 1 (Scheme 1) without reducing the imino
fragment. This key synthon 1 was obtained by a standard
reduction of the nitro parent under H2 catalyzed by
palladium on carbon at rt. It should be stressed that the
Boranil core is stable enough to withstand these condi-
tions. Neither the boron center nor the imine group was
chemically reactive during the course of the reaction. This
was shown by the retention of the characteristic imine
proton multiplet at 8.36 ppm (reflecting 11B coupling),10 the
triplet in the 11B NMR spectrum at 0.84 ppm (J = 15.4 Hz),
the presence of the chelated imine vibrational absorption
at νNdC = 1620 cmꢀ1, and fluorescence properties in
accordance with the other members of the Boranil family.
Without formation of its boron complex, the nitrophenyl-
salicylaldimine present in A does not survive the reaction
conditions, giving an intractable product mixture, illus-
trating the valuable protective function of B(III) against
the imino and the phenol functions.
Table 1. Selected Optical Data Measured in Various Solvents
λabs
ε
λem
Δss
jfa
(%) (ns) solvent
τ
dye (nm) (Mꢀ1 cmꢀ1
)
(nm) (cmꢀ1
)
3
1
2
3
5
6
7
8
9
9
9
405
407
405
405
404
406
414
405
403
408
48000
56000
54000
46000
53000
58000
71000
56000
56000
57000
528
470
468
476
497
473
468
474
477
474
5800
3300
3300
3700
4600
3500
2800
3600
3900
3400
2
7
4
4
3
8
0.21 DCM
0.22 DCM
0.20 DCM
0.20 DCM
0.18 DCM
0.27 DCM
16 0.49 DCM
7
8
0.37 toluene
0.26 THF
10 0.35 DCM
a Using quinine sulfate as reference Φ=0.55inH2SO4 1N,λex =366nm
or Rhodamine 6G as reference, Φ = 0.88 in ethanol λex = 488 nm.
Using this versatile synthon, we were able to check the
reactivity and stability of the Boranil chromophore in
various types of reactions. Boranil 1 provides the amide
derivatives 2 and 3 in good yields (70ꢀ85%) through
peptidic coupling with both adipic acid and 3,4,5-tri-
methoxybenzoic acid, using DMAP and EDCI as coupling
agents (Scheme 2). A diagnostic νNHCO band was found at
1664 cmꢀ1 in the infrared spectra. Condensation with an
aldehyde, such as dimethylaminosalicaldehyde in EtOH,
afforded the corresponding salicylaldimine derivative 4 in
fair yields (51%). This compound has a borate complex
site and a free salicylaldimine chelating site, thus opening
the way to supplementary complexation. Urea Boranil
derivatives were prepared in a one-pot reaction. Initially,
the amino group was transformed into an isocyanate by
means of diphosgene in hot THF. No attack of the
coordinated phenol was observed under these conditions
providing again protection by borate complexation. Then,
nucleophilic attack of an aliphatic (hexylamine) or aro-
matic amine (anisidine) provided the corresponding ureas
5ꢀ6 in fair yields (37% to 68%). Characteristic stretching
All compounds exhibited absorption spectra typical
of Boranil derivatives (Table 1), with a λmax around
405ꢀ415 nm and a relatively high molar absorptivity
(50000ꢀ70000 Mꢀ1 cmꢀ1).10 The different substituents on
the phenyl groups have negligible influence on the absorption
spectra except in the case of the isothiocyanate (Figure 1)
where bathochromic and hyperchromic effects were appar-
ent, probably due to the electron-withdrawing character of
this group, which would induce a stronger dipole moment
and extended delocalization along the main molecular axis.
Figure 1. (a) Absorption, emission, and excitation spectra of
Boranil-ITC 8 in dichloromethane at rt. (b) Absorption, emis-
sion, and excitation spectra of labeled BSA-10 in PBS at pH =
7.4 and at rt. λexc = 380 nm.
vibrations were observed at νNHCONH = 1674 and 1705 cmꢀ1
,
(11) Barnes, S. S.; Vogels, C. M.; Decken, A.; Westcott, S. A. Dalton.
Trans. 2011, 40, 4707–4714.
Org. Lett., Vol. 14, No. 18, 2012
4775

Scheme 2. Synthetic Transformations of Amino-Boranil 1
of an intramolecular charge transfer (ICT) emissive state.
However, the emission profile of Boranil 9 does not exhibit
a structured emission and the emissive properties are
not solvent dependent (Table 1). This suggests, as in the
case for 10, a weakly polarized excited state sensitive to
intermolecular interactions. The emission lifetimes are
short (ca. 200ꢀ500 ps), which confirms the high rate of
nonradiative deactivation. To demonstrate the ability of
boranil-isothiocyanate 8 to label biomolecules, we reacted
it with BSA in aqueous DMSO containing NaHCO3
(see Supporting Information). To compare the optical
properties and estimate the ratio of labeling, we prepared
several solutions by mixing BSA with compound 9. Ab-
sorption measurements on these solutions provided molar
absorption coefficients of 11000 Mꢀ1 cmꢀ1 at 277 nm and
36000 Mꢀ1 cmꢀ1 at 399 nm for 9 inPBS (Table2, Figure2).
By subtracting the electronic absorption spectrum of BSA
from that of 10, and by comparing the ratio between the
two bands, we estimated an average labeling ratio of 17
boranil cores per BSA protein. Unlabeled BSA protein has
ca. 30 lysine residues.13 An interesting comparison may be
Table 2. Optical Data of Solution of 9 with Genuine BSA and of
Labelled BSA-10 in PBS Buffer at RT
λabs
ε
λem
Δss
jfa
(%)
τ
(ns)
b
dye
neq (nm) (Mꢀ1 cmꢀ1
)
(nm) (cmꢀ1
)
3
BSA
ꢀ
277
277
399
43000
52000
33000
ꢀ
ꢀ
ꢀ
ꢀ
4
ꢀ
BSAþ 0.8
ꢀ
ꢀ
0.91(59%)
3.35(41%)
9 (1
0.9
508
5400
equiv)
BSAþ 4.9
277
397
97000
ꢀ
ꢀ
ꢀ
0.74(75%)
3.12(25%)
9 (5
5.0
181000
535
6500
2
equiv)
BSAþ 11.5 277
169000
384000
ꢀ
ꢀ
ꢀ
0.68(79%)
3.06(21%)
9 (10
equiv)
10
10.7 397
539
6600
2
17.0 277
17.1 399
230000
615000
ꢀ
ꢀ
ꢀ
0.92(66%)
513
5600
47 3.14(34%)
a Using Rhodamine 6G as reference, Φ= 0.88 in ethanol λex = 488 nm.
b Calculated number of Boranil dyes considering an average absorp-
tion coefficient for each Boranil of 11000 Mꢀ1 cmꢀ1 at 277 nm and 36000
M
ꢀ1 cmꢀ1 at 397/399 nm.
(12) (a) Hungerford, G.; Benesch, J.; Mano, J. F.; Reis, R. L.
Photochem. Photobiol. Sci. 2007, 6, 152–158. (b) Cherukuri, A.; Durack,
G.; Voss, E. W., Jr. Mol. Immunol. 1997, 34, 21–32. (c) The, T. H.;
Feltkamp, T. E. W. Immunology 1970, 18, 865–873.
All the dyes are fluorescent in dichloromethane with
an emission at 470ꢀ500 nm and a large Stokes shift (ca.
3000ꢀ4000 cmꢀ1). The shape of the emission band with
several shoulders on the low energy side and the nonmirror
symmetry with the large absorption bands are suggestive
ꢀ
(13) van Regenmortel, M. H. V.; Briand, J. P.; Plaue, S. Laboratory
Techniques in Biochemistry and Molecular Biology Vol. 19: Synthetic
Polypeptides as Antigens; Elsevier: 1988.
4776
Org. Lett., Vol. 14, No. 18, 2012

Figure 3. Emission spectra of Bovine Serum Albumin labeled
with Boranil 10, and mixture of BSA with Boranil 9, in PBS at
pH = 7.4. λexc = 370 nm for the Boranil 9 and λexc = 380 nm for
the labeled BSA .
Figure 2. Absorption spectra of Bovine Serum Albumin labeled
with Boranil 10, and mixture of BSA with Boranil 9, in PBS
at pH = 7.4.
made of the emission spectrum of 10 with that of a simple
mixture of dye 9 and BSA. For mixtures of 9 in various
proportions with BSA, a weak emission is observed at
508 nm for the 1:1 mixture and at 535ꢀ539 nm for a larger
amount of dye. The bathochromic shift of the emission
compared to that of 9 in CH2Cl2 could be explained by a
higher dipole moment of the solvent in the first case,
concomitant with a probable surfactant action of BSA,
avoiding aggregate formation. With a larger amount of
dye, some emissive aggregates of 9 are probably formed,
which explains the greater red shift of the emission (Figure 3)
and also the presence of two decay times.
When several boranil dyes were covalently grafted onto
BSA, via thiourea formation, the solution exhibited an
intense fluorescence at 513 nm and a quantum yield of
47%. Notice that similar emission wavelength and excited
state lifetimes are measured with respect to 9 in the
presence of genuine BSA (Table 2). The bathochromic
shift of 39 nm for 10 compared to 9 in organic solvents is
likely due to the solvent polarity and protein environment
which favors dye interaction. This clearly demonstrates
that Boranil-ITC 8 is a good candidate for biological
labeling. Moreover, despite its water insolubility, once
grafted onto a protein, the luminescence is strongly en-
hanced. This is a clear indication that the Boranil dye is
likely lying in hydrophobic pockets resulting from the
folding of the protein after grafting.
In conclusion, we have been successful in the high yield
reduction of nitrophenyl-Boranil to anilino-Boranil, thus
opening an avenue to new useful derivatives. We demon-
strate that imino and phenol functions coordinated to a
boron center are protected against additional reactions.
This strategy allowed preparation of various amide, imine,
urea, and thiourea derivatives without decomplexation of
a boron center. Isolation of the pivotal isothiocyanate
derivative allowed us to label a model protein and to observe
strong fluorescence in an aqueous solution. Further studies
are currently underway to solubilize these dyes in water and
extend their emission to the red part of the electromagnetic
spectrum.
Acknowledgment. We acknowledge the CNRS for pro-
viding research facilities and financial support and the
ꢁ
ꢀ
Ministere de l’Enseignement Superieur et de la Recherche
for an MENRT fellowship for D.F. We also thank Dr.
J. Massue andProf. J. Harrowfield(ISIS inStrasbourg) for
reading the manuscript before publication.
Supporting Information Available. Synthetic proce-
dures and analytical data. This material is available free
of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 18, 2012
4777