Dual labeling of biomolecules by using click chemistry: A sequential approach

Article abstract of DOI:10.1002/anie.200804514

(Figure Presented) Click by click: Dual labeling of model compounds was carried out by using copper-free and copper-mediated click chemistry in a sequential manner. This method was used to introduce two labels onto biological targets or nanoparticles, thu

Full text of DOI:10.1002/anie.200804514

Communications
DOI: 10.1002/anie.200804514
Synthetic Methods
Dual Labeling of Biomolecules by Using Click Chemistry: A
Sequential Approach**
Pꢀter Kele,* Gꢁbor Mezꢂ, Daniela Achatz, and Otto S. Wolfbeis*
Dedicated to Professor Ferenc Sebestyꢀn on the occasion of his 70th birthday
Imaging biomolecules by means of fluorescent tags is an
important tool for the study of complex biological processes
both in vitro and in vivo. The introduction of these reporter
tags rely on their selective and efficient reaction, under
physiological conditions, with the available functional groups
on the biomolecule of interest. Bioorthogonal chemical
reporters which are “non-native, non-perturbing chemical
handles that can be modified in living systems through highly
selective reactions with exogenously delivered probes” have
drawn much attention lately.[1a] Among bioorthogonal tagging
reactions the Staudinger ligation[1] and the click reaction,
involving the copper(I)-catalyzed azide alkyne cycloaddition
(CuAAC),[2] are the most valuable. These two methods are
superior to other labeling techniques because of the inertness
of the chemical reporters and the exogenously delivered
probes, and the selective and efficient reaction between the
reporter and the probe. The extreme rareness of azide and
alkyne functions in biological systems additionally increases
the importance of tagging by the means of a copper catalyzed
azide/alkyne cycloaddition (CuAAC; a click reaction). This
reaction has been shown to be quite versatile in terms of
biological applications.[3] CuAAC also finds widespread
applications in the high throughput screening of libraries.[4]
Recently, Bertozzi et al. have proposed a copper-free version
of tagging by using click reactions of strained cycloalkynes.[5]
Boons et al. recently reported the synthesis of novel and
potent dibenzocyclooctynols which react with azides at very
high rate.[6] The efficiency of these strained cyclooctynes in
click reactions was found to be comparable to that of CuAAC,
which paves the way to bioorthogonal chemical reporters that
can be easily labeled with more than one fluorescent tag. This
option is particularly useful when these reporters are to be
transformed into resonance energy transfer (RET) or fluo-
rescence resonance energy transfer (FRET) systems, by using
the appropriate fluorescent tags.[7] The introduction of multi-
ple labels onto biomolecules still remains a challenging task.
During the preparation of this manuscript, Carell et al.
published modular labeling of DNA sequences with multiple
click labels using a combination of alkyne protecting groups.[8]
To tag peptide sequences post-synthetically with multiple
labels by using click chemistry, we first tried to differentiate
the terminal alkyne groups by using protecting groups. Our
efforts to introduce protected alkyne groups to appropriately
modified model peptide sequences failed, probably as a result
of the lability of the alkyne protecting groups. We also tried to
introduce protected alkyne groups to aryl-halide modified
peptides by using the Sonogashira reaction.[9] However, this
route was found to not be efficient as very low overall yields
were observed, so we then focused on the cyclooctyne moiety
known[5] to react with azides in an uncatalyzed click reaction,
which is in contrast to the behavior of terminal alkynes.
Herein we demonstrate the feasibility of sequential
tagging using click labels on bioorthogonal model systems in
solution by sequentially exploiting copper-free and copper-
mediated click chemistry. We believe that this is a very
versatile method that has numerous applications, especially
because the reactions proceed readily under conditions
similar to physiological conditions (i.e., room temperature,
aqueous solution, pH 7).
[*] Dr. P. Kele
Institute of Chemistry, Eꢀtvꢀs Lorꢁnd University
1117 Pꢁzmꢁny Pꢂter sꢂtꢁny 1a, Budapest (Hungary)
Fax: (+36)1-372-2909
The introduction of multiple labels onto a biomolecule
was first tested for simple model systems. Several cyclooctyne
derivative candidates are known,[5c] but for the sake of facile
and concise synthesis we chose compound A[5b] (Figure 1) to
elucidate the conditions for copper-free labeling using
fluorescent azides. Amongst the azido labels considered,
compound 2 (Figure 2) had the salient feature that only its
click products are fluorescent, thus eliminating background
fluorescence of unreacted starting material.[10] To introduce a
novel long wavelength azido label that emits in the red region
of the visible spectrum, we have prepared compound 3 (see
the Supporting Information for experimental details).[11] Its
design was derived from the Stokes dye family developed by
Czerney et al.[12]. This family possesses remarkable photo-
stability, high quantum yields, and large Stokes shifts.
E-mail: kelep@elte.hu
D. Achatz, Prof. Dr. O. S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors,
University of Regensburg
93040 Regensburg (Germany)
Fax: (+49)941-943-4065
E-mail: otto.wolfbeis@chemie.uni-regensburg.de
Prof. Dr. G. Mezꢀ
Research Group of Peptide Chemistry, Hungarian Academy of
Sciences, Eꢀtvꢀs L. University (Hungary)
[**] P.K. thanks the Humboldt Foundation for a Humboldt fellowship
(3.3-UNG/1126507). Financial support from GVOP-3.2.1.-2004-04-
0005/3.0, T 049814 (OTKA) and H07-B-74291 (NKTH-OTKA) are
greatly acknowledged. The authors wish to thank Dr. K. Hegyi and
Dr. Z. Novꢁk for helpful discussions.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200804514.
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Angew. Chem. Int. Ed. 2009, 48, 344 –347

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revealed that the cyclooctyne moiety is sensitive to the acidic
conditions of peptide cleavage from the Rink-amide MBHA
resin. Other resins requiring milder cleavage conditions are
potentially more adequate for the incorporation of the
À
CyOCO moiety onto the solid-phase. Alternatively, direct
labeling of support-bound sequences may also be possible
prior to cleavage from the resin.
Dual labeling of the peptides was carried out in acetoni-
trile/water (1:1). The solution containing the peptide was
treated with an equimolar quantity of the first label. The
solutions were stirred at room temperature for 12hours, and
then the crude products were analyzed by using HPLC
methods (Table 1). The monolabeled peptide was then
treated with an equimolar quantity of the second label in
the presence of CuSO4, ascorbic acid, and triethylamine, and
then stirred at room temperature for 12 hours. HPLC analyses
indicated that both coupling steps proceeded with good to
excellent yields (Table 1). MS analyses of the crude or
Figure 1. Model systems.
Table 1: Dual labeling of model compounds.[a]
Entry Model
compound
Label 1 Yield 1 [%][b,c] Label 2 Yield 2 [%][b,c]
Figure 2. Fluorophore-containing azides for click chemistry. 1 is a
nonfluorescent azide designed for use as a dark quencher, whereas 2
is a nonfluorescent azide that becomes fluorescent after undergoing a
click reaction. 3 is strongly fluorescent both as the azide and its click
product.
1
2
3
4
5
6
7
Pep1
2
2
3
2
2
3
2
96
98
1
3
–
1
3
–
2
71
73
–
>99 (65)
95 (60)
–
Pep1
Pep1
Pep2
Pep2
Pep2
Pep2
76 (66)
>99 (82)
98 (89)
78 (68)
98
96
Compound A readily reacted with azides, in the absence
of CuI and in aqueous media, to give both regioisomers of the
triazole products, which is in accordance with earlier obser-
vations.[5] Next we incorporated the cyclooctyne and the
terminal alkyne moieties into model compounds. Pep1 and
Pep2 were prepared on Rink-amide MBHA (MBHA = 4-
methylbenzhydrylamine) resin by using standard 9-fluorenyl-
methoxycarbonyl (Fmoc) chemistry. In a first attempt, the
cyclooctyne moiety was attached to resin-bound peptide
chains. However, the crude and purified peptides had mass
values that were different from the calculated mass values.
The CyOCO-Ala-dl-PrGly-NH2 showed 426.2 Da instead of
408.0, probably the result of the presence of one firmly bound
water molecule. The CyOCO-Gly-Pro-Leu-Gly-Val-Arg-dl-
PrGly-NH2 had a mass of 1056.3 Da which is higher by 140 Da
than expected. By using the ESI MS/MS method we found
that in both cases the cyclooctyne derivatives were modified
by the addition of the scavengers, applied during the cleavage
of the peptides from the resin (details not shown here), to the
triple bond.
[a] Click reactions in solution. [b] Determined by integration of the HPLC
traces of the crude products at 220, 410 and 500 nm after 12 h.
[c] Numbers in parentheses indicate yields of isolated products.
purified products were in complete agreement with the
expected data (see the Supporting Information). For Pep1
the second labeling proceeded with lower yields compared to
Pep2, potentially because of the increased steric hindrance
present on the smaller peptide.
The sequence of Pep2 represents a synthetic substrate for
the enzyme matrix metalloproteinase-2 (MMP-2) whose
elevated activity is a diagnostic indicator for tissue tumors.
The renown substrate sequence[13] of Gly-Pro-Leu-Gly-Val-
Arg-Gly-Lys-Cys-NH2 was slightly altered for our conven-
ience to give Pep2. Enzymatic scission occurs between Gly4-
Val5. To demonstrate the importance of double labeling we
used Pep2 to construct a FRET system by using an
appropriate combination of labels and our sequential labeling
technique.
The resulting substrate has not yet been optimized with
respect to cleavage by MMP-2, but despite this and the fact
that it is a mixture of diastereomers and regioisomers, the
dually labeled substrate 2–Pep2–3 is readily cleaved by the
enzyme as can be seen from the decrease in the efficiency of
FRET (Figure 3). The large Stokes shift of label 3 certainly is
an additional advantage in terms of signal separation; in fact,
the fluorescence at 620 nm could be easily measured even
without the use of optical filters.
The synthetic strategy was therefore partially modified
such that the peptides were built up on the resin and then the
cyclooctyne derivative was attached in solution. This synthetic
route was successful and the expected compounds were
identified by mass spectrometry (m/z 408.0 and 916.4, respec-
tively). Although the application of the racemic forms of
À
PrGly and CyOCO may lead to four diastereomers, HPLC
analysis revealed only two peaks for each of the peptides (see
the Supporting Information). At this point there were no
attempts to separate the isomers. This synthetic approach also
Angew. Chem. Int. Ed. 2009, 48, 344 –347
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Communications
Figure 4. Schematic representation of dual labeling of alkyne modified
BSA.
Figure 3. Spectral changes of 2–Pep2–3 (1 mm) in the presence of
activated MMP-2 (4 nm) in TCNB buffer at 378C; inset: changes in the
ratio of the monitored wavelengths. TCNB=tris/calcium/sodium
buffer.
To provide additional examples of sequential labeling by
using copper-free and copper-mediated 1,3-dipolar cyclo-
additions, we attempted to label bovine serum albumin (BSA)
with our FRET pair. BSA was treated first with a CyOCO-
modified maleimide linker (see the Supporting Information)
to create a copper-free anchor through the free sulfhydryl
group of BSA. This step was then followed by treatment with
pentynoic acid succinimide ester[14] to provide numerous sites
on the protein for the copper-mediated click reactions. The
modified BSAwas then separated from excess reagents by gel
filtration and then label 3 was introduced. After removal of
the unreacted dyes the monolabeled BSA was reacted with
label 2 in the presence of CuI (Figure 4). As expected, the
intensity of both emission bands increased as the second click-
reaction proceeded. Changes of the second band at 620 nm
also indicated the presence of FRET (see the Supporting
Information).
A third piece of evidence for the versatility of the
sequential labeling is presented by using silica nanoparticles
doped with 2.[11] Pep1 was linked to the doped particles
bearing azido functions by using the copper-free click
chemistry. The Pep1-functionalized fluorescent particles
were then separated from unreacted peptides. Subsequent
labeling by using a copper-mediated click-reaction with 3
yielded dually labeled nanoparticles (Figure 5, see details in
the Supporting Information). This method can be used to
efficiently functionalize doped nanoparticles by using click
chemistry with biologically active, labeled motifs.
Figure 5. Loading biomolecules onto azido-functionalized silica nano-
particles and subsequent labeling.
tions (both are highly selective and efficient) represents a
viable tool to sequentially labeling biological targets with two
labels without protecting the functional groups. Given the
mild experimental conditions, the method is likely to work
in vivo, provided that the alkyne- or azide-modified building
blocks are taken up by cells through metabolic pathways.
Subsequent sequential labeling with fluorescent tags bearing
the counterpart functionality enables one to introduce multi-
ple labels into these modified biological targets. An extension
of the method towards labeling even more complex systems,
as well as labeling doped silica nanoparticles with an MMP-2
substrate is in progress and will be reported in due course.
Received: September 13, 2008
Published online: December 9, 2008
We presume that the method for sequential labeling
presented herein can be extended to various systems includ-
ing small synthetic protein oligomers, bioorthogonalized
proteins, organic messengers, polysaccharides, or nucleic
acids and nanoparticles. Its efficiency allows the whole
labeling process to take place in the same solution by
administering the labels sequentially to the appropriate
acceptors.
Keywords: click chemistry · copper · FRET · peptides
.
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