Brightly fluorescent purple and blue labels for amines and proteins

Article abstract of DOI:10.1016/j.bmcl.2011.06.133

Novel amino-reactive phenoxazines were obtained by reasonably simple synthetic protocols and characterized in terms of their use as fluorescent labels for amines, amino acids and proteins in general. Purple labels (alternatives to Texas Red) and blue labe

Full text of DOI:10.1016/j.bmcl.2011.06.133

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5538–5542
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Brightly fluorescent purple and blue labels for amines and proteins
⇑
Martin Link, Péter Kele , Daniela E. Achatz, Otto S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93053 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel amino-reactive phenoxazines were obtained by reasonably simple synthetic protocols and charac-
terized in terms of their use as fluorescent labels for amines, amino acids and proteins in general. Purple
labels (alternatives to Texas Red) and blue labels (alternatives to Cy-1) were obtained by this strategy.
The absorption/emission maxima, in aqueous solution, are at around 589/630 nm and 648/670 nm,
respectively, thus indicating larger Stokes’ shifts than those of common cyanine-type of labels. The
new labels are compatible with commercial diode laser light sources.
Received 23 May 2011
Revised 20 June 2011
Accepted 20 June 2011
Available online 13 July 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Amino reactive label
Fluorescent label
Oxazine
Protein label
Bioconjugation
Labeling of biological matter such as amino acids, proteins, cells
or viruses with synthetic fluorescent labels has become an indis-
pensible tool in bioanalytical sciences. Applications are ranging
from simple immunoassays and histochemical techniques to
high-throughput screening and cell biology.1,2 Fluorescence label-
ing is also a preferred method in separation techniques such as
electrophoresis and chromatography.3 The widespread use of fluo-
rescent labels can be owed to the remarkable sensitivity of fluores-
cence detection that can even go down to the single molecule level
in case of certain techniques such as laser-assisted fluorometry.4–6
In recent years, the growing interest in tumor imaging has inspired
important and developing fields in fluorescence spectroscopy, for
example, laser-induced fluorescence microscopy of (tumor) cells.7
Fluorescence imaging in the spectral range between 600 nm and
1000 nm is particularly appealing (compared to the UV and short-
wave visible part)8–10 as biomolecules display less background
fluorescence and straylight,11 and for the better penetration prop-
erties of longwave radiation into tissue in this regime. Green, yel-
low and (deep) red laser diodes are preferred light sources for their
affordable and compact nature. Such laser diodes can be battery-
powered and easily driven with high efficiency in terms of conver-
sion of electrical energy into light.12 Hence, there is a substantial
interest in fluorescent labels for use in bioanalytical sciences and
in combination with diode laser light sources.
There is a vast number of fluorescent labels in the literature
with excitation maxima above 580 nm, some of them being avail-
able from commercial sources (e.g., from Invitrogen; www.probes.-
com), Dyomics (www.dyomics.de), Attotec (www.atto-tec.com),
ActiveMotif Chromeon (www.chromeon.de) or Amersham
(www.amershambiosciences.com). The state of the art of the syn-
thesis and application of oxazine-type labels has been reviewed,
for example, by Drexhage,13 Hartmann,14 Tung,15 and Simmonds
and Briggs.16 Unfortunately, some of these methods are either
cumbersome or lead to products that are covered by patents.
Among the long-wavelength emitting dyes, oxazines and benzoxa-
zines (such as Nile Red and Nile Blue) are particularly attractive for
their extraordinary stability and brightness.17 Oxazine based labels
have been used in a wide range of applications, for example, in nu-
cleic acid detection, histochemistry,18,19 protein labeling20 or envi-
ronmental analysis.21 The deeply colored oxazines possess a
mesomeric donor–acceptor chromophoric system.
Oxazinones, like the purple label in Scheme 1, are strongly sol-
vatochromic (e.g., orange and strongly fluorescent in nonprotic sol-
vents, red in methanol, purple and weaker fluorescent in aqueous
solution). Diamino-substituted oxazines (the blue oxazine in
Scheme 2) are blue and less prone to solvatochromism. Due to
their charged nature, they display improved water solubility and
often undergo an increase in fluorescence quantum yield upon
conjugation. Another feature that attracts attention is the relative
ease of structural modification for introducing various functional
groups for bioconjugation. Recently, we have reported on the syn-
thesis of clickable (azide or alkyne functionalized) and thiol reac-
tive (maleimide functionalized) oxazine derivatives.22,23 Herein
we present the synthesis of purple and blue oxazine labels that
are accessible in a few synthetic steps and to the best of our knowl-
⇑
Corresponding author. Tel.: +49 941 943 4065, fax: +49 941 943 4064.
E-mail addresses: otto.wolfbeis@chemie.uni-r.de, otto.wolfbeis@chemie.uni-re-
gensburg.de (O.S. Wolfbeis).
Present address: Institute of Chemistry, Eötvös Loránd University, H-1117
Budapest, Hungary
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.133

M. Link et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5538–5542
5539
NO
CO2Et
CO2Et
OH
O
4
O
Br
4
CO2Et
Me2N
N
O
4
HO
OH
HO
OH
Me2N
O
1
2
CO2H
CO2NHS
O
O
4
4
H+ /H2O
NHS, DCC
N
N
Me2N
O
O
Me2N
O
O
3
4
Scheme 1. Synthesis of the purple phenoxazine label 4.
OH
N
H
OH
HO
N
HO
OH
5
NO
CO2Et
CO2Et
OH
O
O
Br
4
4
CO2Et
Me2N
N
O
4
HO
N
HO
N
Me2N
N
6
7
CO2H
CO2NHS
O
O
4
4
H
+ /H2O
NHS, DCC
N
N
O
Me2N
O
N
Me2N
N
8
9
Scheme 2. Synthesis of the blue phenoxazine label 9.
edge are not protected by any existing patents. Both can be used in
combination with commercial diode lasers and are comparable, in
terms of brightness and photostability, with other probes in this
spectral range, therefore offering good alternatives to them.
The oxazine framework was chosen on the basis of its synthetic
conciseness and spectral properties. We anticipated that the dyes
can be synthesized in relatively few steps and that they exert all
the aforementioned advantages of labels operating in this highly
desirable spectral region. The hydroxyphenoxazine can be easily
derivatized with a C-6 linker carrying a carboxylic function. Subse-
quent in situ activation by converting to its N-hydroxysuccinimide
ester offers a ready-to-use amine reactive label.24
troso-N,N-dimethylaniline provided phenoxazine 7. Similarly to
the previous protocol, acidic hydrolysis of the ester afforded the
carboxylic acid functionalized dye 8. Both carboxy-functionalized
dyes can be converted into their ready-to-use active ester form,
for example, N-hydroxysuccinimide (NHS) esters, using standard
in situ protocols prior to their use in amino-group modification
reactions.
The absorption spectrum of the purple phenoxazine (free acid)
3 exhibits a broad band cantered at 598 nm with a shoulder at
around 565 nm. The emission spectrum shows a maximum at
630 nm (Fig. 1). It was found that dye 3––similar to other Nile
Red type of dyes––displays strong solvatochromism. The absorp-
tion/emission maxima in water (589/630 nm) are blue-shifted to
557/619 nm in methanol and 510/580 nm in toluene. We therefore
believe that the respective label can also be utilized as an intra-
protein (local) polarity probe,25 comparable in its response to that
of certain ketocyanines.26 The molar absorbance of 3 is 38,000 L/
(mol cm) at the peak wavelength. The quantum yield in aqueous
solution is 0.05 using Nile Red (QY 0.018)27 as a reference standard.
Compared to phenoxazine 3, the absorption and emission spec-
tra of dye 8 are distinctly red-shifted due to the presence of the
iminium group (Fig. 2).28 The absorption maximum is located at
648 nm in aqueous solution, with a shoulder at around 598 nm,
while the emission peaks at 670 nm. Compared to 3, the spectral
properties of 8 are much less affected by the polarity of the solvent
When designing the synthetic routine to reach the purple and
the blue phenoxazine labels, we intended to minimize the neces-
sary synthetic steps. Synthesis in both cases started from commer-
cially available phloroglucinol. In case of dye 3, phloroglucinol was
first functionalized with ethyl
x-bromohexanoate to afford mono-
substituted intermediate 1. This intermediate was then condensed
with p-nitroso-N,N-dimethylaniline to give phenoxazine 2, which
was subsequently hydrolyzed under acidic conditions to result in
the purple carboxylic acid 3.
As outlined in Scheme 2, the synthesis of the blue phenoxazine
8 also started from phloroglucinol, which was first converted to 5
by reaction with piperidine. Compound 5 was then reacted with
x-
bromohexanoate to furnish 6. Subsequent condensation with p-ni-

5540
M. Link et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5538–5542
3 drops drastically at pH values below pH 8, whilst the fluores-
cence of dye 8 remains virtually unchanged. These findings can
be explained by the chemical structure of the two dyes: dye 3 is
a slightly basic molecule whose nitrogen atoms (dimethylamino
group and heterocycle) are prone to protonation. Upon proton-
ation, the molecule (that is electrically neutral in its nonprotonated
form) becomes charged. The resulting positive charge is most likely
mesomerically distributed over the whole
p-electron system and
thus affects emission intensity. Contrary to this, the positive charge
present in compound 8 makes it less prone to protonation. These
structural differences are reflected in the response of emission
intensities to the pH of the environment for the two dyes.
In order to elaborate the labeling potential of the oxazines, dyes
3 and 8 were converted to their active esters using DCC and NHS in
dry DMSO (see Schemes 1 and 2 and the supplementary data). The
resulting solutions of label 4 and 9, respectively, were then directly
used to label amino acids, peptides, and proteins. The pre-formed
active esters were found to be stable if stored dry and can be stored
at À18 °C at least for 1 week without any change in activity.
Figure 1. Absorption (A) and emission (B) spectra of the purple dye 3 in water. The
line of the 594-nm helium–neon laser is also shown.
The amino acids L-lysine, L-serine, L-glycine, L-glutamic acid, L-
aspartic acid were labeled by treating them with solutions of either
4 or 9 overnight at room temperature.30 In each case, the labeling
process was monitored via TLC. As a model system for peptide
labeling we have chosen bradykinin. Treatment of the amine con-
taining media with solutions of labels 4 or 9 overnight at room
temperature resulted in efficient staining of the samples as indi-
cated by complete conversion of the starting materials into their
red or blue conjugates.
Labeling of proteins was studied on BSA, a 65 kDa protein that
often serves as a model protein in labeling experiments. Like in
the case of amino acids, labeling was carried out by treatment with
the active ester solutions overnight at room temperature. The pro-
tein was purified by size exclusion chromatography and submitted
to TOF-MS analysis (see data in the Supplementarydata). The effect
of BSA on the fluorescence intensity of the dyes was checked via
Figure 2. Absorption (A) and emission (B) spectra of the blue dye 8 in water. The
lines of the 635-nm and 650-nm diode lasers are also shown.
BSA titration e.a. 5 lL (c = 0.054 M) of nonreactive dye (3 or 8) in
buffered medium using BSA in concentrations between 0 mg/L
and 1000 mg/L. Results have shown that no significant change in
fluorescence intensity or emission maxima could be observed,
indicating that there is no unspecific binding of the dyes on the
protein surface. Contrary to this, obvious changes in the positions
of absorption and emission maxima as well as quantum yields
were seen in case of covalent modification of BSA with the reactive
dyes. Characteristic photophysical properties of labels and their
conjugates to BSA are summarized in Table 1.
Conjugation also resulted in remarkable changes in the shapes
of the bands, especially for compound 9 (Fig. 4). This effect is vis-
ible only on the covalent attachment of the fluorophore to the pro-
tein. The absorption spectra remain unchanged if nonreactive dyes
are added to BSA. The changes shown in Figure 4 in the absorption
spectra clearly indicate covalent attachment of the labels to BSA.
In order to demonstrate the wider scope of the labels, we have
also labeled amino-modified silica nanoparticles (SiNPs) with the
purple dye 4. SiNPs are a new and interesting class of materials.
They are readily available in defined sizes and––unlike nonencap-
(e.g., 639/663 nm in methanol). The molar absorbance of 8 is
71,000 L/(mol cm) at the peak wavelength, whilst the quantum
yield in water is 0.004 using Nile Blue (QY 0.00429 in water) as
the reference standard.
Next, we have investigated the pH dependency of the two phen-
oxazine dyes. Fluorophores 3 and 8 were excited at their respective
absorption maxima, and the change of their fluorescence intensity
with pH was measured at their emission maxima. Figure 3 depicts
the changes of emission intensities at the emission maxima of the
new oxazine dyes as a function of pH. The emission intensity of dye
Table 1
Photophysical properties of the dyes and their BSA conjugates at pH 7.2
a
kabs
(nm)
kem
(u
(max)
)
(nm)
(max)
4
598 (565)
550 (589)
648 (598)
592 (658)
630 (0.05)
627 (0.04)
670 (0.004)
677 (0.003)
4-BSA
9
9-BSA
a
Quantum yields were determined in phosphate buffered saline (pH 7.2; 50 mM)
using Nile Red and Nile Blue, respectively, as standards.
Figure 3. Normalized pH dependencies of the emission of dye 3 (A) and 8 (B).

M. Link et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5538–5542
5541
Figure 4. Absorption spectra of dye–BSA conjugates (A) and free dyes (B) for phenoxazine 4 (I) and 9 (II) in PBS (50 mM, pH 7.2).
sulated quantum dots––are biocompatible.31,32 Fluorescent SiNPs
are either prepared by post-synthetic direct tagging of the ami-
no-modified silica surface (method A), or by doping fluorophores
into the outer coating of silica nanoparticles during nanoparticle
fabrication (method B).33 In method A (Fig. 5) silica nanopowder
was treated with aminopropyltriethoxysilane (APTES) in order to
create an amino-modified surface. Next, label 4 was added to a sus-
pension of the amino-modified silica nanopowder in ethanol and
the reaction mixture was stirred overnight at rt. The labeled red
particles were collected by centrifugation and washed with etha-
nol to remove unreacted label. Parallel to this, a blank sample
was prepared by combining plain silica nanoparticles (without
amino groups) with activated fluorophore 4. The blank samples
showed virtually no color after centrifugation and washing,
whereas the amino-labeled particles displayed the characteristic
red color.
4 were stirred overnight in order to obtain silane 10. This dye-si-
lane conjugate was then directly used to coat the SiNPs with a
dye doped shell around the silica core. The labeled SiNPs were
purified by size exclusion chromatography (SEC) on SephadexÒ
LH-20. The labeled particles were obtained by collecting the first
colored fraction. These particles are more homogeneous in terms
of size distribution. We find method B to be easier and to give more
strongly colored SiNPs, probably because the label is contained in
the complete outer shell, and not only on the surface as in SiNPs
prepared by method A.
The emission spectra of the labeled SiNPs and of the unreacted
reagent were acquired in ethanolic colloidal solution. The peak of
the emission of the labeled SiNPs is red-shifted (kem = 626 nm) in
comparison to the maximum of the unreacted label in ethanol
(kem = 619 nm), indicating its presence in a more polar microenvi-
ronment. A blank sample was also prepared to elucidate unspecific
binding of the fluorophore to the particle surface. The alcosol con-
taining the particles was diluted with ethanol and treated with an
ethanolic solution of nonactivated dye 3 (free acid). The mixture
In method B (Fig. 5), plain silica nanoparticles (SiNPs) were first
prepared by the Stöber method using tetraethylorthosilicate
(TEOS).34 Parallel to this, a mixture of APTES in ethanol and label
Method A
EtO
H2N
H2N
NH2
EtO Si
NH2
EtO
APTES
4
SiO2
SiO2
NH2
NH2
SiO2
SiO2
TEOS
TEOS
H2N
H2N
H2N
Method B
(EtO)3Si
NH2
O
O
+
O
N
Si(OEt)3
4
H
N
O
CO2NHS
4
N
Me2N
O
Me2N
O
O
10
4
10
SiO2
SiO2
Figure 5. Modification of SiNPs with reactive label 4.

5542
M. Link et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5538–5542
was stirred overnight and purified by SEC as described above. The
SiNPs obtained exhibited no significant fluorescence. See the Sup-
plementary data for spectra and further details of the labeling
procedures.
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excited by the 635-nm laser diode which is a small, affordable and
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38,000 L/(mol cm) for the purple phenoxazine
71,000 L molÀ1cmÀ1 for the blue phenoxazine 8 are comparable
to those of Nile Red ( and Nile Blue
= 38,000 L molÀ1cmÀ1 35
= 76,000 L molÀ1cmÀ1 36
which are well-established stains for
3
and of
e
)
(e
)
biological samples.37,38 BSA was chosen as an easily accessible
and well-known protein for demonstrating the utility of the two
amino reactive dyes. At this juncture, activated label 4 and espe-
cially label 9, which is positively charged, have been shown suit-
able for use in aqueous media. Similar labels were described by
the group of Briggs39 and in a patent.40 However, the dyes prepared
by Briggs et al. are related to the purple label 4 and distinctly larger
in size. We also applied our strategy to synthesize label 9 which ex-
tends the set of oxazine labels into the longwave part of the visible
spectrum. Compared to the blue phenoxazines described in a pat-
ent,40 our labels are not functionalized at the push and pull system
of the chromophoric core, which can be disadvantageous in terms
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}
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e and
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A. Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.133.