Communication
soluble in water and related aqueous buffers ranging in con-
centration from 1 mm to 20 mm, thus emphasizing the rele-
vance of our “SR101-hybrid” approach to rapidly obtain water-
soluble xanthene-based fluorescent dyes, which could poten-
tially be used in biological media. Conversion of these dyes
(except those bearing a primary aniline or phenol group) into
amine-reactive reagents for labeling biomolecules, may be
readily achieved by derivatization of their “remote” sulfonic
acid residue with an unusual amino acid, namely, isonipecotic
acid, and through the corresponding sulfonyl chloride inter-
mediate.[22] Indeed, this is the most common way to avoid un-
desired ring-chain tautomerism of sulforhodamine-amine con-
jugates, a transformation that leads to the formation of the
colorless and nonfluorescent sultam.[23] Further conversion of
the carboxylic acid into the corresponding N-hydroxysuccini-
midyl (NHS) ester should enable stable bioconjugatable fluoro-
phores to be obtained.
moments of this dye.[24] As revealed by fluorescence emission
profiles shown in Figure 1 (and those available in the Support-
ing Information), all these unsymmetrical sulforhodamines ex-
hibit an intense emission peak with a maximum in the range
567–638 nm. In all cases, the quantum yields are high, and to
our great satisfaction, are especially high for compounds in si-
mulated physiological environments (Table 1, entries 4, 16, 24
and 28). These results support the beneficial effects of intro-
ducing negatively charged ionizable groups (that is, sulfonic
acids) onto the meso-phenyl ring of these xanthene dyes, both
to impart water solubility and to promote dye–dye repulsion
and resistance to aggregation-induced fluorescence quenching
in aqueous media. Indeed, a perfect match between the ab-
sorption and excitation spectra recorded in PBS (see the Sup-
porting Information) was observed, except for the more hydro-
phobic p-extended derivative SR101-NaphtNH2, the two sulfo-
nate groups of which are not sufficient to completely prevent
dye–dye aggregation, which causes a subsequent decrease in
the quantum yield (Table 1). However, the strong red fluores-
cence of SR101-NaphtNH2 is recovered by adding 5% (w/v) of
bovine serum albumin (BSA), an additive often used in buffers
in order to mimic body fluids (FF =25%). Indeed, this protein
is known to enhance the emission of many fluorophores
owing to a combination of rigidification, reduction in the po-
larity of the dye’s microenvironment (binding in the hydropho-
bic BSA pocket), and deaggregation.[25] As a final matter, small
Stokes’ shifts, all within the range 20–30 nm, are observed and
are comparable to those of traditional symmetrical rhodamine
dyes. For the two sulforhodol derivatives, SR101-OH and
SR101-NaphtOH, some interesting differences in their spectral
behavior at physiological pH are noted (Figure 1 and Table 1).
The fluorescein–SR101 hybrid was found to display an intense
orange fluorescence assigned to its anionic form (that is, a phe-
nolate species) that may be the predominant form of SR101-
OH in PBS. The fluorescence quantum yield (35%) is of the
same order of magnitude as that of a similar rhodol dye bear-
ing a single carboxylic acid instead of the two sulfonic acid
moieties on the meso-phenyl ring, known as Peroxy Orange 1
(FF =46% in 20 mm HEPES, pH 7.0).[26] In contrast, absorption
and emission spectra of the naphthol derivative appear as
broad red-shifted bands that
The spectroscopic properties of these sulforhodamines/sulfo-
rhodols were characterized both in polar aprotic solvents
(MeCN and DMSO) and in protic solvents (MeOH and phos-
phate-buffered saline (PBS), pH 7.5). The results are summar-
ized in Table 1 and representative examples of absorption/fluo-
rescence spectra recorded for samples in PBS are shown in
Figure 1. All sulforhodamines display a broad and intense ab-
sorption band with a maximum in the range 542–599 nm, de-
pending on the bis(amino)xanthene substitution pattern and
the solvent used, and assigned to the 0–0 band of the S0!S1
transition. A less pronounced shoulder peak at the higher
energy side is also observed and is attributed to the vibronic
relaxation (the 0–1 vibrational band). Furthermore, the batho-
chromic shift observed in both absorption and fluorescence
spectra (see Table 1 and the Supporting Information) with in-
creasing solvent polarity (compare spectra for solvents that do
not donate hydrogen bonds, such as MeCN and DMSO, or
compare spectra for different protic solvents, such as MeOH
and aqueous buffer) suggests that the fluorescence involves
the p!p* transition. Such a solvent effect on the spectral
properties of sulforhodamines has been recently reported for
the commercial sulforhodamine B and meaningfully interpret-
ed through the evaluation of ground- and excited-state dipole
suggest the presence of several
distinct absorbing/emissive spe-
cies at pH 7.5, including mainly
the protonated and deprotonat-
ed forms of SR101-NapthOH.
The presence of two local
maxima at 537 and 580 nm in
the UV/Vis absorption spectrum
also supports the presence of
two predominant species in PBS
even if the formation of H-type
dimers (known to lead to a re-
markable blue-shift of the ab-
sorption peak), which may
partly explain the moderate
fluorescence quantum yield of
Figure 1. Normalized absorption (left) and emission (right) spectra of unsymmetrical sulforhodamine/sulforhodol
dyes recorded in PBS (pH 7.5) at 258C.
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Chem. Eur. J. 2014, 20, 1 – 9
4
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Chevalier, Arnaud
