Click chemistry based method for the preparation of maleinimide-type thiol-reactive labels

Article abstract of DOI:10.1002/ejoc.201001085

Maleinimides are widely used to label proteins and to derivatize thiols. The so-called click reaction was shown to allow the efficient introduction of the maleinimido group into azido-modified fluorophores (benzoxazines) by reacting them with an alkyne-mo

Full text of DOI:10.1002/ejoc.201001085

FULL PAPER
DOI: 10.1002/ejoc.201001085
Click Chemistry Based Method for the Preparation of Maleinimide-Type
Thiol-Reactive Labels
Martin Link,[a] Xiaohua Li,[a,b] Jana Kleim,[a] and Otto S. Wolfbeis*[a]
Keywords: Click chemistry / Fluorescent probes / Imaging agents / Proteins / Thiols
Maleinimides are widely used to label proteins and to deriv- nimides starting from open-ring precursors require rather
atize thiols. The so-called click reaction was shown to allow
the efficient introduction of the maleinimido group into
azido-modified fluorophores (benzoxazines) by reacting
them with an alkyne-modified maleinimide to yield new
thiol-reactive fluorescent labels 5–7. The reaction proceeds
under mild experimental conditions and provides a new
strategy with which to introduce the maleimide group, as ex-
emplified here for fluorophores. Conceivably, it may be ex-
tended to radioactive, electro-active, isotopic, or spin labels.
Previously reported methods for the formation of such malei-
harsh conditions. The new benzoxazines 5–7 are charac-
terized by a fairly long and flexible linker between the chro-
mophore and the maleinimide functional group, and their
fluorescence can be photoexcited with diode lasers (which
are preferred light sources in fluorometry). They were conju-
gated to: (a) the aminothiol cysteamine, (b) the peptide gluta-
thione, and (c) to human serum albumin. The fluorescence of
the phenoxazinone 5 is strongly solvatochromic, which sug-
gests its use as a polarity-sensitive probe.
Introduction
tial interest in the development of labels for thiolated spe-
cies.[11] Although iodoacetamides and sulfochlorides may
play a certain role,[12] the maleinimides form by far the
largest group of thiol-reactive labels that are used for selec-
tive fluorescent,[9a,11] radioactive,[13] or isotopic labeling of
thiols.[14] However, such labels are typically synthesized in
only moderate yields,[11b] mainly because the cyclization of
the maleamic acids requires rather harsh conditions, which
is highly undesirable for chemically less stable markers (flu-
orophores) such as cyanines and (benz)oxazines. Moreover,
most thiol-reactive labels have a maleinimido group that is
attached directly to the label, often to the heteroaromatic
ring of a fluorophore,[9b,11b,11d,15] which is also undesirable
because the buried thiol groups are difficult to access in
such cases. We envisioned that the click reaction may pro-
vide a facile, novel method with which to introduce malein-
imides into a (fluorescent) label.
Among the long-wavelength emitting dyes, the oxazines
and the benzoxazines (such as Nile Red and Nile Blue) are
attractive because of their stability and brightness.[16] They
have been used mainly, for example, as biomarkers for nu-
cleic acid detection in histochemistry,[17] and for labeling
proteins.[18] Deeply colored oxazines have a mesomeric do-
nor–acceptor chromophoric system. Oxazinones of type A
(Scheme 1) are strongly solvatochromic (e.g., orange and
strongly fluorescent in non-protic solvents, red in methanol,
but purple and less fluorescent in water solution). Diamino-
substituted oxazines of type B are blue and exhibit almost
no solvatochromic effects. Being cationic, they have good
water solubility, often display an increase in fluorescence
So-called click chemistry has emerged as an extraordi-
narily versatile synthetic tool.[1] It involves the 1,3-dipolar
cycloaddition of azides to alkynes using CuI ion as the cata-
lyst. Click chemistry proceeds in water of pH 7 at room
temperature (thus making it a very useful tool in terms of
bioconjugation), is regioselective, efficient, simple, and bio-
orthogonal in that azido groups and alkyne groups are ra-
rely found in biomolecules. As a result, in vitro click chem-
istry has been used for labeling biomolecules,[2] hormones,[3]
and for in-vivo tumor cell targeting[4] with either fluoro-
phores, radiomarkers, spin labels, or nanoparticles made
from gold[5] or silica.[6] The success of click chemistry is
especially striking in the case of labeling glycans and glyco-
conjugates.[7]
Site-specific labeling of proteins is of particular interest
because it has several advantages over random labeling.[8]
One common approach for site-specific labeling is to label
solvent-accessible cysteine (Cys) using thiol-reactive rea-
gents.[4–8,9] Cysteine is most attractive in this respect be-
cause of its relative rarity throughout the proteome, and
because it may even be introduced into proteins that are
normally free of the amino acid.[10] Hence, there is substan-
[a] Institute of Analytical Chemistry, Chemo- and Biosensors,
University of Regensburg,
93040 Regensburg, Germany
[b] Beijing National Laboratory for Molecular Sciences,
Key Laboratory of Analytical Chemistry for Living Biosystems,
Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100190, P. R. of China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001085.
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Maleinimide-Type Thiol-Reactive Labels
quantum yield upon conjugation, and are amenable to diode laser, which is in widespread use in analytical fluores-
structural modification so that various functional groups cence. The three basic chromo(fluoro)phores 1–3 were pre-
can be introduced.
pared as outlined below and detailed in the Supporting In-
formation. The compounds carry azido groups and – in
terms of synthesis – are fairly easily accessible. More specifi-
cally, the synthesis of the purple oxazine 1 was ac-
complished in one step by alkylating 7-(dimethylamino)-1-
hydroxyphenoxazin-3-one[19] with 1-azido-3-bromopro-
pane. Oxazine 2 was obtained in four steps by reacting
piperidine with phloroglucinol to give 5-(piperidin-1-yl)-
benzene-1,3-diol. This was azido-functionalized by (a) alk-
ylation with 1-bromo-3-chloropropane, (b) exchanging the
chloro atom with iodine, and (c) converting the iodide into
an azide with NaN3. The resulting 3-(3-azidopropoxy)-5-
(piperidin-1-yl)phenol was condensed with N,N-dimethyl-4-
nitrosoaniline to give the blue oxazine 2.
The blue oxazine 3 was obtained by a different route;
in this case the azido functionality was introduced by first
alkylating 1-naphthylamine with 3-bromo-1-propanol to
give 3-(naphthalene-1-ylamino)propan-1-ol, the hydroxy
group of which was then converted into an azido group
with NaN3. The resulting 3-azido-propyl-1-naphthyl-amine
Scheme 1. Chromophores of the purple oxazines A and the blue
oxazines B used in this work.
The presence of a flexible linker between the maleinimide
group and the fluorophore is known to facilitate label-
ing.[11b] We hypothesized that an application of click chem-
istry to the preparation of thiol-reactive labels according to
Scheme 2 may be an elegant way to introduce the malein-
imide group in the final step. The results are presented here.
Scheme 2. Strategy for preparation of fluorescent thiol-reactive lab-
els with spacer groups by click chemistry. Fl stands for the respec-
tive fluorophore.
Results
Synthesis of Azido-Modified Oxazines
Oxazine chromophores were chosen as the fluorophores
because they can emit at both red and blue wavelengths,
have reasonably good quantum yield (QY; at least in or-
ganic solvent) and brightness (defined as molar absorbance
multiplied by QY), and are adequately photostable.[15] The
blue oxazines are cationic (and thus display good solubility
Figure 1. Chemical structures of the oxazine azides 1–3, and of the
in aqueous solutions), and are compatible with the 635-nm alkyne maleinimide 4 to which the azides were ‘clicked’.
Figure 2. Chemical structures of the thiol-reactive oxazines 5–7.
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O. S. Wolfbeis et al.
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was condensed with 5-(dimethylamino)-2-nitrosophenol in
acidic medium to give the azidophenoxazine 3. Compounds
1–3 were then ‘clicked’ to the maleinimide 4 as outlined
below (Figure 1).
It should be stated at this point that these azides are also
useful click labels, for example, for labeling proteins[20] or
saccharides[21] containing azido groups, or when labeling
alkyne modified nanoparticles.[6,20]
The known synthon 4 was then used to link the malein-
imido group to the azido-modified fluorophores. The reac-
tion of 4 with the azidophenoxazines 1–3 is characterized
by high yields, easy work-up, and proceeds in aqueous sol-
vent at room temperature to give the thiol-reactive labels 5–
7 shown in Figure 2. The high yield and ease of work-up in
the final step makes this approach very powerful in terms
of ligating maleimide groups to a fluorophore. We presume
that this is a generally applicable approach in that almost
any fluorophore containing an azido group can be linked
to a maleinimide (and thus become a thiol-reactive label)
using the alkyne reagent 4. Previously reported methods are
often tedious.[22] Furthermore, the long linker between the
fluorophore and the maleinimide group provides the latter
with more flexibility so that thiol groups within biomole-
cules are more easily accessed.[11b]
Figure 3. Absorption (left) and emission (right) of label 5 in water.
The line of the 594-nm helium/neon laser is also shown.
Figure 4. Absorption (left) and emission (right) of label 6 in water.
The lines of the 635- and 650-nm laser diodes are also shown.
Spectral Properties of Labels
The absorption and emission maxima of labels 5–7 in
buffer solution of pH 7 are summarized in Table 1, and the
spectra of 5 and 6 are shown in Figures 3 and 4, respec-
tively. The compounds usually display broad absorption
bands and a longwave shoulder. The blue oxazines 6 and 7
almost perfectly match the 635-nm line of the red diode
lasers often found in commercial fluorescence instrumen-
tation. The small Stokes’ shifts (typically 20–25 nm) are
characteristic of such rigid fluorophores.[16c]
both the absorption and emission maxima in ethanol, meth-
anol, and water are very different, as are the quantum
yields. Some data are given in Table 1 for compounds 1–3
and 5–7. The bathochromic shift and the lower quantum
yield on going from methanolic to aqueous solutions are
particularly significant. By analogy to previous studies,[26]
lifetimes can be estimated to be in the order of 1–2 ns and
are likely to be multi-exponential in the presence of other
biomolecules or when other processes (such as electron
transfer) can occur.
Table 1. Absorption and emission maxima (nm), molar ab-
sorbances [L/(mol·cm)], and quantum yields (QY; Ϯ20%) of oxaz-
ine azides and of oxazine labels (in aqueous buffer of pH 7.0) and
in methanol (labels 3 and 7 only). Quantum yields were determined
by using the dyes oxazine 1 (QY = 0.11 in ethanol[23]), Nile Red
Stability and Effects of pH on Fluorescence
(QY = 0.08 in methanol[24]), and Nile Blue (QY = 0.004 in water[25]
as standards.
)
The fluorescence intensity of the maleinimides 5–7 in
aqueous buffer slowly increased over time. This may be at-
tributed to the known[27] tendency of maleinimides to un-
dergo hydrolysis to give the corresponding (open-ring) ma-
leamic acid. The increase in fluorescence is due to the fact
that the (cyclic) maleinimides are efficient triplet quenchers
of many fluorophores, whereas (non-cyclic) maleic acid
monoamides and the thiol conjugates of maleinimides, be-
ing succinimides and cyclic, are not.
Dye (solvent)
Absorption (ε)
Emission (QY)
1 (water)
595 nm (17,300)
649 nm (53,000)
636 nm (43,500)
630 nm (49,600)
597 nm (22,000)
557 nm (21,400)
650 nm (58,000)
636 nm (45,000)
628 nm (46,200)
629 nm (0.012)
669 nm (0.014)
672 nm (0.044)
664 nm (0.21)
628 nm (0.023)
620 nm (0.16)
668 nm (0.014)
669 nm (0.015)
662 nm (0.033)
2 (water)
3 (water)
3 (methanol)
5 (water)
5 (methanol)
6 (water)
7 (water)
7 (methanol)
The effect of pH on the fluorescence of the labels was
studied in solutions (ca. 3 μm) with pH values of between 5
It is known[16] that the absorption and emission profiles and 10. The red emission of the blue oxazines 6 and 7 did
of such oxazine fluorophores are solvatochromic, and that not depend on the pH value in the range tested. The yellow
even slight variations in the substitution pattern can have fluorescence of the purple oxazinone label 5, in contrast,
marked effects on their photophysical properties. Indeed, started to decrease at pH values of below pH 6. This is
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Maleinimide-Type Thiol-Reactive Labels
clearly due to the fact that the oxazinone is uncharged, of cysteamine to 7. Addition of GSH also caused an in-
whereas compounds 6 and 7 are positively charged and are crease (by 73%) in fluorescence while control reactions per-
thus unlikely to be protonated.
formed in parallel (label 7 in the same concentration but
without cysteamine or GSH added) showed virtually no
change in fluorescence. The product of the reaction with
cysteamine was subjected to LC-MS analysis (see MS spec-
tra in the Supporting Information), and the results con-
firmed that the addition reaction occurred as outlined in
the Scheme shown in the Supporting Information.
The fluorescence spectra recorded in the absence and
presence of cysteamine were also acquired in presence of
histamine, tyramine, dopamine, and tryptamine, none of
which carried thiol groups. No further increase in fluores-
cence intensity was detectable. This is additional proof that
label 7 reacts with the thiolated amine selectively, and not
with other amines.
Labeling of Thiols
The maleinimides 5–7 were first used to label human se-
rum albumin (HSA; a 65 kD protein), which is known to
have one single thiol group [along with more than 30 dithio
(–S–S–) groups, which do not react with maleinimido
groups]. Labeling was accomplished in buffer solutions of
pH 7 by adding the labels as concentrated solutions in
either N,N-dimethylformamide (DMF) or dimethyl sulfox-
ide (DMSO). After reacting HSA with either of the labels
for 3–6 h at room temperature, the labeled protein was puri-
fied by size exclusion chromatography (SEC). In some
cases, they were submitted to capillary electrophoresis.
The dye-to-protein ratio (DPR), which is defined as the
number of labels statistically bound to one HSA, was deter-
mined by spectrophotometric analysis to be 1.0. This is in
agreement with the fact that native HSA has one single
thiol group. The fluorescence spectra of the conjugate re-
vealed that it is stable for at least one week in buffered solu-
tion at room temperature, which indicates both the stability
of the fluorophore and the spacer (in essence the triazole).
The MALDI-TOF mass spectrum of the HSA conjugate
also confirmed that single-labeling had occurred (see the
Supporting Information).
Label 7 also was conjugated to two smaller thiols: cyste-
amine (an amine) and l-glutathione (GSH; a peptide). Both
were found to undergo quantitative reaction in pH 7 buffer
after a one hour reaction, whereas labeling of HSA required
at least three hours. The reaction was monitored by spectro-
photometric analysis. Addition of cysteamine or GSH to
label 7 in pH 7 buffer resulted in a small increase in absorp-
tion at around 600 nm and a moderately strong increase in
emission (Figure 5).
Discussion
The method presented here for the introduction of a ma-
leinimido group involves a small number of synthetic steps
and the critical step, whereby the preformed fluorophore is
linked to a maleimide functional group, occurs in the final
step of synthesis using the efficient click reaction. Although
not shown here, we presume that the method can be ex-
tended to other fluorescent labels, such as rhodamines, fluo-
resceins, and dipyrromethenones (e.g., the Bodipy™ dyes)
or to other popular labels (such as the Alexa™ dyes), and
to ketocyanines.[28] Moreover, this approach may be ex-
tended to labels other than fluorescent compounds, for ex-
ample to spin labels, radiomarkers, or markers for use in
NMR spectroscopy.
Maleinimides may also be obtained by reacting fluoro-
phores with amino-reactive functions, such as N-hydroxy-
succinimide esters, with 2-aminoethylmaleinimide. This re-
action, although proceeding smoothly, requires more te-
dious work up and, in particular, requires a carboxy deriva-
tive of the dye to be prepared and then made amino-reac-
tive, typically by activation with a carbodiimide and N-hy-
droxysuccinimide (NHS) (or its sulfo derivative) to yield the
NHS ester, which is sensitive to moisture and has limited
storage stability. Synthesis of maleinimides by using NHS
esters is therefore deemed to be more tedious (at least in
most cases) and less straightforward.
Labeling of typical small thiols was found to proceed un-
der the conditions used to introduce the maleimide labels
and yielded conjugates with longwave absorption and fluo-
rescence maxima. This characteristic is useful if background
fluorescence from biological samples is to be suppressed. In
the case of cysteamine, labeling with 7 may also be consid-
ered to be an example of the derivatization of thiols, which
then may be quantified following separation techniques
such as HPLC or CE (capillary electrophoresis).
Figure 5. Absorption spectra (a–c) and emission spectra (aЈ–cЈ) of
label 7 after addition of cysteamine and glutathione in pH 7 buffer;
(a, aЈ): label only; (b, bЈ): label plus cysteamine; (c, cЈ): label plus
glutathione.
The merits of labeling a thiol group are particularly clear
It can be seen in Table 1 that the maleimide 7 has a rather in case of HSA. This protein contains 35 cysteines, 34 of
small QY (0.015), whereas Figure 5 shows that the fluores- which are present in the disulfide form, whereas only one is
cence increases by around 69% within 5 min after addition freely available for labeling.[29] This is in contrast to the
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O. S. Wolfbeis et al.
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number of amino groups: HSA contains 60 lysines, 35 of which is shown here for fluorescent labels of the benzox-
which are freely accessible.[30] Labeling of amino groups not azine group, can be extended to various other fluorescent
only occurs randomly,[31] but may also lead to a range of labels, to electrochemical labels, and to radiomarkers.
DPRs, whilst thiol labeling of HSA results in highly site-
specific and strictly single labeling as demonstrated by a
DPR of 1. Thiol labeling also eliminates the risk of self-
quenching of fluorophores due to multiple labeling. More-
over, the functionality of the protein is likely to be better
retained in the case of thiol labeling. In cases where no free
cysteine is available, disulfide bonds (if present) may first
be reduced to thiols. Alternatively, site-directed mutagenesis
Experimental Section
Chemicals and Reagents: All reagents were purchased from Sigma–
Aldrich unless noted otherwise. Buffer salts and solvents were pur-
chased from Merck. Deuterated solvents, such as [D6]dimethyl sulf-
oxide were obtained from Deutero GmbH (www.deutero.de). Tet-
ramethylsilane was used as an external standard for NMR mea-
or heterobifunctional crosslinkers may be applied to intro-
duce a cysteine group. Hence, thiol labeling has a wide
scope and is generally to be preferred over amine labeling.
The labels presented here (compounds 5–7) have the typi-
cal properties of oxazine dyes in terms of molar ab-
sorbances and fluorescence emission profiles. The effect of
substituents on the molar absorbances of the oxazines un-
der consideration is worth noting (Table 1). The molar ab-
sorbances of the blue diamino-substituted oxazine labels of
type B are significantly smaller if the nitrogen of the meso-
meric system is directly substituted than if the label is sub-
stituted at position 3. For example, the molar absorbance
of label 6 (58,000 L/mol·cm) is much higher than that of
label 7 (46,000 L/mol·cm) although both possess similar
mesomeric π-electron systems. This effect is corroborated
by Frade et al.,[16d,32] who synthesized various derivatives
of the Nile Blue compound functionalized at the nitrogen
atom of the mesomeric system. The molar absorptions of
these dyes are in the range 13,000–22,000 L/mol·cm, which
is much smaller than the molar absorption of Nile Blue
(76,800 L/mol·cm in ethanol[33]). The beneficial effect of an
increase in the quantum yield of maleimide fluorophores
surements.
Instruments: Fluorescence spectra were recorded with a Hitachi F-
2500 fluorometer in 1ϫ1ϫ3 cm quartz cuvettes, with excitation
and emission slit widths set to 10 nm. Absorption spectra were re-
corded in glass cuvettes of the same size with a Techcomp UV-8500
spectrophotometer (Shanghai, China; www.techcomp.com.cn). ESI
mass spectra were acquired with a ThermoQuest TSQ 7000 mass
spectrometer (www.thermo.com), and the MALDI mass spectra
with a model 4700 Proteomics Analyzer (Applied Biosystems;
www.appliedbiosystems.com). 1H NMR spectra were recorded with
an Avance 300 MHz NMR spectrometer (Bruker-BioSpin;
www.bruker-biospin.com). Emission spectra were acquired with a
635-nm diode laser as the excitation light source. IR spectroscopic
analysis of the intermediates and new labels were carried out in
the attenuated total reflection mode on an Excalibur FTS 3000
spectrometer (Biorad; www.bio-rad.com), equipped with a Golden
Gate Diamond Single Reflection ATR System (Specac; www.
specac.com).
Protocol for Labeling HSA: HSA (3.0 mg, 4.6 nmol) was dissolved
in phosphate buffer (300 μL, 22 mm, pH 7). Labels 5, 6, or 7
(0.2 mg) were dissolved in DMSO or DMF (20 μL). The two solu-
tions were mixed and allowed to react for 6 h at r.t. in the dark
with gentle stirring. The labeled HSA was purified by size exclusion
upon conjugation to a thiol is also observed in this case. chromatography on a Sephadex G-25 column with an i.d. of 2 cm
using phosphate buffer (22 mm, pH 7) as the eluent. The eluted
fractions containing the label were dialyzed for five days at 4 °C
(two days with 5 L of 10 mm phosphate buffer of pH 7; three days
with 5 L of a 1 mm phosphate buffer of pH 7) to remove free and
non-covalently bound label. MALDI-TOF spectra of the labeled
proteins are given in the Supporting Information.
Maleinimides have a low-lying triplet state that is capable
of deactivating the excited singlet states of fluorophores by
enhancing radiationless dissipation of excited-state energy.
This results in rather poor quantum yields (3.3% for label
7 in air-equilibrated methanol). Upon conjugation, the
π-electron system of the maleimide is converted into a suc-
cinimide (as shown in the Supporting Information). As a
result, the level of the triplet state increases and leads to
less efficient radiationless deactivation, i.e., an increase in
fluorescence. This is reflected by a QY of 3.8% for
the conjugate to cysteamine, 4.1% for labeled GSH (both
in air-equilibrated methanol), and 3.4% for HSA (in
water).
Determination of the Dye-to-Protein Ratio (DPR): The HSA con-
centrations and DPRs of the HSA conjugates were determined by
assuming the additivity of the absorbances at 280 nm of both the
protein and the labels. Dye concentrations in solutions of labeled
HSA were determined according to the Lambert–Beer law from
the absorbance of the label at (or near) the peak wavelength (590–
630 nm). It was assumed that the bound and the unbound labels
have identical molar absorbances.
The selectivity of maleinimide for thiol groups over
amino groups is more than 1000:1 at pH 7, and the best
specificity is found[34] at pH values of between 6.5 and 7.5.
This enables selective labeling of thiol groups in most cases.
However, it needs to be kept in mind that maleinimides
often suffer from hydrolysis, which yields a luminescent
product that is not conjugated to the thiol.
Protocol for Labeling Cysteamine and
L-Glutathione: Label 7
(0.2 mmol) was dissolved in methanol (100 μL) and mixed with cys-
teamine or glutathione (30 μmol) in pH 7 buffer (0.5 mL). The
solution was stirred for 20 min and then loaded onto a short C18
reverse-phase column. After chromatography, the volume of the
solution was adjusted to 3 mL, transferred to a quartz cuvette and
submitted to absorptiometric and fluorometric analyses.
In conclusion, we have developed a versatile method for
introducing the widely used maleimido group, which, pre-
viously, has only been introduced by using thermal methods
Supporting Information (see also the footnote on the first page of
this article): The synthetic route to N-propargylmaleimide (4), and
to labels 5, 6, and 7. MALDI-TOF characterization of unlabeled
and then only in poor yield. We believe that this reaction, and labeled HSA. Schematic of the reaction of label 7 with cyste-
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Maleinimide-Type Thiol-Reactive Labels
[13]
[14]
[15]
[16]
M. Berndt, J. Pietzsch, F. Wuest, Nucl. Med. Biol. 2007, 34, 5–
15.
R. Tressl, G. R. Tressl, G. Wondrak, E. Kersten, J. Agric. Food
Chem. 1994, 42 , 2692–2697
a) R. P. Haugland, Molecular Probes, Eugene, 2003, Oregon;
b) See: www.probes.com; www.attotech.de.
a) T. Schoetzau, U. Koert, J. W. Engels, Synthesis 2000, 707–
713; b) J.-P. Knemeyer, D.-P. Herten, M. Sauer, Anal. Chem.
2003, 75, 2147–2153; c) J. Jose, K. Burgess, Tetrahedron 2006,
62, 11021–11037; d) V. H. J. Frade, P. J. G. Coutinho, J. C. V. P.
Moura, M. S. T. Goncalves, Tetrahedron 2007, 63, 1654–1663;
e) X. Song, D. S. Kassaye, J. W. Foley, J. Fluoresc. 2008, 18,
513–518; f) J. Fries, E. Lopez-Calle, K. H. Drexhage, US Patent
App. 2006, 11/379,433.
a) J. S. Kang, J. R. Lakowicz, G. Piszczek, Arch. Pharmacal.
Res. 2002, 25, 143–150; b) Q. Chen, D. Li, H. Yang, Q. Zhu,
J. Xu, Y. Zhao, Analyst 1999, 124, 901–906.
a) S. F. Abu-Absi, J. R. Friend, L. K. Hansen, W. Hu, Exp. Cell
Res. 2002, 274, 56–67; b) S. Chen, X. Li, H. Ma, ChemBio-
Chem 2009, 10, 1200–1207.
M. Kotoucek, M. Martinek, E. Ruzicka, Monatsh. Chem.
1965, 96, 1433–1434.
P. Kele, G. Mezö, D. E. Achatz, O. S. Wolfbeis, Angew. Chem.
Int. Ed. 2009, 48, 344–347.
a) P. Kele, X. Li, M. Link, K. Nagy, A. Herner, K. Lörincz, S.
Beni, O. S. Wolfbeis, Org. Biomol. Chem. 2009, 7, 3486–3490;
b) N. J. Agard, J. M. Baskin, J. A. Prescher, A. Lo, C. R.
Bertozzi, ACS Chem. Biol. 2006, 1, 644; c) S. T. Laughlin, J. M.
Baskin, S. L. Amacher, C. R. Bertozzi, Science 2008, 320, 664–
667.
H. J. Gruber, G. Kada, B. Pragl, C. Riener, C. D. Hahn, G.
Harms, W. Ahrer, T. G. Dax, K. Hohenthanner, H. Knaus, Bio-
conjugate Chem. 2000, 11, 161–166.
amine, and mass spectrum of the product. Four additional refer-
ences are also included.
[1] a) V. R. Sirivolu, P. Chittepu, F. Seela, ChemBioChem 2008, 9,
2305–2316; b) Q. Shi, X. Chen, T. Lu, X. Jing, Biomaterials
2008, 29, 1118–1126; c) S. Ciampi, T. Bocking, K. A. Kilian,
M. James, J. B. Harper, J. J. Gooding, Langmuir 2007, 23,
9320–9329.
[2] a) O. S. Wolfbeis, Angew. Chem. Int. Ed. 2007, 46, 2980–2982;
b) P. Kele, G. Mezö, D. Achatz, O. S. Wolfbeis, Angew. Chem.
2009, 48, 348–347; c) S. Berndl, N. Herzig, P. Kele, D. Lach-
mann, X. Li, O. S. Wolfbeis, H.-A. Wagenknecht, Bioconjugate
Chem. 2009, 20, 558–564; d) J. Kalia, R. T. Raines, ChemBio-
Chem 2006, 7, 1375–1383.
[17]
[18]
[3] H. Langhals, A. Obermeier, Eur. J. Org. Chem. 2008, 6144–
6151.
[4] G. Maltzahn, Y. Ren, J. H. Park, D. H. Min, V. R. Kotamraju,
J. Jayakumar, V. Fogal, M. J. Sailor, E. Ruoslahti, S. N. Bhatia,
Bioconjugate Chem. 2008, 19, 1570–1578.
[5] J. L. Brennan, N. S. Hatzakis, T. R. Tshikhudo, N. Dirvian-
skyte, V. Razumas, S. Patkar, J. Vind, A. Svendsen, R. J. M.
Nolte, A. E. Rowan, M. Brust, Bioconjugate Chem. 2006, 17,
1373–1375.
[6] a) H. Mader, X. Li, S. Saleh, M. Link, P. Kele, O. S. Wolfbeis,
Ann. N. Y. Acad. Sci. 2008, 1130, 218–223; b) H. S. Mader, M.
Link, D. E. Achatz, K. Uhlmann, X. Li, O. S. Wolfbeis, Chem.
Eur. J. 2010, 16, 5416–5424.
[7] a) J. A. Codelli, J. M. Baskin, N. J. Agard, C. R. Bertozzi, J.
Am. Chem. Soc. 2008,130, 11486–11493; b) T. L. Hsu, S. R.
Hanson, K. Kishikawa, S. K. Wang, S. Sawa, C. H. Wong,
Proc. Natl. Acad. Sci. USA 2007, 104, 2614–2619; c) J. M. Bas-
kin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang,
I. A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc. Natl.
Acad. Sci. USA 2007, 104, 16793–16797; d) V. Hong, S. I. Pre-
solski, C. Ma, M. G. Finn, Angew. Chem. 2009, 121, 10063–
10067.
[8] a) K. L. Holmes, L. M. Lantz, Methods Cell Biol. 2001, 63,
185–204; b) S. Wang, X. Wang, W. Shi, K. Wang, H. Ma, Bio-
chim. Biophys. Acta Proteins Proteomics 2008, 1784, 415–422;
c) Z. Bao, S. Wang, W. Shi, S. Dong, H. Ma, J. Proteome Res.
2007, 6, 3835–3841.
[9] a) X.-C. Su, T. Huber, N. E. Dixon, G. Otting, ChemBioChem
2006, 7, 1599–1604; b) J. Weh, A. Duerkop, O. S. Wolfbeis,
ChemBioChem 2007, 8, 122–128; c) I. M. Riederer, R. M. Her-
rero, G. Leuba, B. M. Riederer, J. Proteomics 2008, 71, 222–
230.
[10] a) T. Kurpiers, H. D. Mootz, ChemBioChem 2008, 9, 2317–
2325; b) V. Tolmachev, H. Xu, H. Wallberg, S. Ahlgren, M.
Hjertman, A. Sjöberg, M. Sandström, L. Abrahmsén, M. W.
Brechbiel, A. Orlova, Bioconjugate Chem. 2008, 19, 1579–1587.
[11] a) X. Wang, S. Wang, H. Ma, Analyst 2008, 133, 478–484; b)
J. E. T. Corrie, J. Chem. Soc. Perkin Trans. 1 1994, 2975–2982;
c) H. Stapelfeldt, C. E. Olsen, L. H. Skibsted, J. Agric. Food
[19]
[20]
[21]
[22]
[23]
E. Terpetschnig, J. D. Dattelbaum, H. Szmacinski, J. R.
Lakowicz, Anal. Biochem. 1997, 251, 241–245.
[24] N. Ghoneim, Spectrochim. Acta Part A 2000, 56, 1003–1010.
[25] K. Das, J. Jain, H. S. Patel, Spectrochim. Acta Part A 2004, 60,
2059–2064.
[26] T. Heinlein, J. P. Knemeyer, O. Piestert, M. J. Sauer, Phys.
Chem. B 2003, 107, 7957–7964.
[27] J. Gierlich, G. A. Burley, P. M. Gramlich, D. M. Hammond, T.
Carell, Org. Lett. 2006, 8, 3639–3642.
[28] M. A. Kessler, O. S. Wolfbeis, Spectrochim. Acta Part A 1991,
47, 187–192.
[29] B. Wetzl, M. Gruber, B. Oswald, A. Duerkop, B. Weidgans, M.
Probst, O. S. Wolfbeis, J. Chromatogr. B 2003, 793, 83–92.
[30] R. Sens, K. H. Drexhage, J. Lumin. 1981, 24, 709–712.
[31] C. Sun, J. Yang, L. Li, X. Wu, Y. Liu, S. Liu, J. Chromatogr.
B 2004, 803, 173–190.
[32] V. H. J. Frade, S. A. Barros, J. C. V. P. Moura, M. S. T. Gon-
çalves, Tetrahedron Lett. 2007, 48, 3403–3407.
[33] H. Du, R. A. Fuh, J. Li, A. Corkan, J. S. Lindsey, Photochem.
Photobiol. 1998, 68, 141–142.
Chem. 1999, 47, 3986–3990; d) T. Matsumoto, Y. Urano, T. [34] B. Karlen, B. Lindeke, S. Lindgren, K.-G. Svensson, R.
Shoda, H. Kojima, T. Nagano, Org. Lett. 2007, 9, 3375–3377.
[12] a) Ch. Stuhlfelder, F. Lottspeich, M. J. Mueller, Phytochemistry
2002, 60, 233–240; b) M. Brinkley, Bioconjugate Chem. 1992,
3, 2.
Dahlbohm, D. J. Jenden, J. E. Giering, J. Med. Chem. 1970, 13,
651–657.
Received: August 2, 2010
Published Online: November 12, 2010
Eur. J. Org. Chem. 2010, 6922–6927
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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