Monovalent, clickable, uncharged, water-soluble perylenediimide-cored dendrimers for target-specific fluorescent biolabeling

Article abstract of DOI:10.1021/ja2009136

Herein we report the synthesis of water-soluble polyglycerol-dendronized perylenediimides with a single reactive group that undergoes high-yielding click reactions. Single-molecule studies and target-specific biolabeling are reported, including the highly

Full text of DOI:10.1021/ja2009136

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pubs.acs.org/JACS
Monovalent, Clickable, Uncharged, Water-Soluble
Perylenediimide-Cored Dendrimers for Target-Specific Fluorescent
Biolabeling
||
Si Kyung Yang,† Xinghua Shi,‡,§,|| Seongjin Park,‡ Sultan Doganay,# Taekjip Ha,‡,§, ,# and
Steven C. Zimmerman*,†
†Department of Chemistry,‡Department of Physics, §Institute for Genomic Biology, Howard Hughes Medical Institute, and
#Center for Biophysics and Computational Biology, University of Illinois at UrbanaÀChampaign, Urbana, Illinois 61801, United States
S Supporting Information
b
ABSTRACT: Herein we report the synthesis of water-
soluble polyglycerol-dendronized perylenediimides with a
single reactive group that undergoes high-yielding click
reactions. Single-molecule studies and target-specific biolabel-
ing are reported, including the highly specific labeling of pro-
teins on the surface of living bacterial and mammalian cells.
luorescent compounds are useful reporters in biology be-
cause they signal their environment and probe a wide range of
F
biological processes, including enzyme catalysis and protein fold-
ing and trafficking.1 The performance of these probes is enhanced
when the following criteria are met: (i) high brightness and
photostability, (ii) water-solubility, (iii) biocompatibility, and
(iv) absorption and emission maxima above 500 nm to minimize
the autofluorescence background of cells.2 In this regard, perylene
tetracarboxylic acid diimides (perylenediimides, PDIs) are an
attractive class of fluorophores because of their high thermal and
photochemical stability with high fluorescence quantum yields in
organic solvents.3 The many successful efforts to prepare soluble
and functional PDIs have been reviewed by Langhals, M€ullen,
W€urthner, Nagao, and others.3,4
Perhaps the largest challenge with PDI chromophores is
developing water-soluble, nonaggregating analogues that retain
their exceptional properties. One of the most powerful methods,
introduced by M€ullen and co-workers, is the incorporation of
ionic moieties such as sulfonate, pyridinium, and quaternary
ammonium groups into the bay regions of PDI chromophores.5
The resulting highly charged PDIs are very effective at staining
membranes, have the added advantage that the donor groups in
the bay region cause a significant red shift in the absorption
maximum and an increased Stokes shift, and recently have been
prepared with a single reactive group for protein conjugation.6
Our interest in developing stable fluorophores for bioimaging
led us to seek a neutral and biocompatible PDI for targeted
imaging.7 While this work was in progress, Haag and co-workers
reported neutral, water-soluble PDI chromophores prepared by
the reaction of amine-cored polyglycerol dendrons (PGDs) and
perylene tetracarboxylic acid bisanhydride. The dendritic PDIs exhibited
high fluorescence quantum yields in water as a result of reduced
aggregation.8 By combining the advantages of the approaches
pioneered by M€ullen and Haag, we have prepared neutral PDIs
Figure 1. Structures of PGDÀPDIs 1À7 and PDI 8.
that are bay-substituted with PGDs and contain a single azide
group having high reactivity in the click reaction, despite the dense,
globular PGD shell.
The key structural feature of the PGDÀPDIs reported in this
study is a fluorescent PDI core that is linked through amides to
four (1 and 3) or eight (2) PGDs, giving 64 and 128 hydroxyl end
groups, respectively (Figure 1). PGDÀPDIs 1À3 were prepared
by HATU-mediated amide coupling of the PDI cores with
amine-functionalized PGDs containing eight allyl groups fol-
lowed by dihydroxylation. The synthetic pathway is described in
the Supporting Information (SI). Purification was achieved by a
combination of column chromatography, size-exclusion chroma-
tography (SEC), and dialysis, and the purity was determined by
analytical SEC, 1H NMR spectroscopy, and matrix-assisted laser
Received: January 29, 2011
Published: June 14, 2011
r
2011 American Chemical Society
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Figure 3. Normalized absorption and emission spectra of PGDÀPDIs
1À3 and PDI 8 in water. λabs,max (nm), λem,max (nm), Φ (in water): 567,
612, 0.62 (1, black); 569, 612, 0.83 (2, red); 559, 612, 0.57 (3, pink);
561, 612, 0.39 (8, cyan).
absence of preincubated neutravidin. The optics used were
modified from those that are normally implemented in Cy3/
Cy5 fluorescence resonance energy transfer (FRET) experi-
ments10 by removing the dichroic beamsplitter (see the SI).
Moreover, this image clearly shows that the fluorescence
intensity of single PGDÀPDI molecules can easily be distin-
guished from the background signal with an integration time
of 100 ms (Figure 4B). Thus, individual PGDÀPDI molecules
are bright enough for single-molecule detection under typical
imaging conditions.
Figure 2. MALDIÀTOF mass spectra of PGDÀPDIs 1À6.
desorption ionizationÀtime of flight (MALDIÀTOF) mass
spectrometry. PGDÀPDIs 1 and 3 appeared to be homogeneous
with a complete attachment of four dendrons in each, while the
MALDIÀTOF mass spectrum of PGDÀPDI 2 revealed a
mixture of PDIs having seven and eight attachments, with the
latter predominating (Figure 2).
All of the synthesized PGDÀPDIs were fully water-soluble
and highly fluorescent in water with quantum yields ranging from
0.57 to 0.83. The absorption and emission spectra (Figure 3 and
Figure S1 in the SI) showed maxima at 559À569 and 612 nm,
respectively, consistent with those of the nondendronized PDI
analogues reported in the literature.5 These results indicate that
the attachment of bulky PGDs not only retains the excellent
photophysical properties of the original PDI chromophores but
also improves their water solubility with minimal aggregation in
water, making them useful in bioapplications.
Furthermore, the reactivities of monofunctional PGDÀPDI 3
with alkyne-functionalized biotin and maleimide were investi-
gated using click chemistry.9 Quantitative conversions to PGDÀ
PDIs 4 and 5 containing single biotin and maleimide functions,
respectively, were confirmed by comparison of the MALDI mass
spectra of 3À5 (Figure 2), demonstrating the potential of our
clickable monofunctional PGDÀPDIs for conjugation to bio-
molecules.
The use of copper in the click reaction and the known
quenching of fluorescence by metal ions raised the question of
whether the fluorescence of the PGDÀPDIs might be affected by
copper ion. At concentrations higher than those likely to be
present from the click reaction, the fluorescence of PGDÀPDIs
was almost unchanged, whereas that from nondendronized PDI
analogues was significantly quenched (see the SI).
The intensity time traces of hundreds of isolated molecules
were obtained in T50 buffer10b containing 10 mM Tris and
50 mM NaCl at pH 8.0 (Figure 4C). As expected, the average
intensity of these molecules decayed with time (Figure 4D), and
this decay was fit to a single exponential in which the fitted
lifetime was defined as the single-molecule photobleaching time,
τphotobleaching. From six repeated measurements in different
imaging areas, τphotobleaching was determined to be 9.6 ( 2.0 s for
4, while the nondendronized analogue 8 exhibited τphotobleaching
=
7.8 (1.2 s under the same conditions (see the SI). The fluorescence
intensities of single molecules of 4 and 8 were also determined by
the average numbers of detected photons as 21000 ( 2000 and
17000 ( 2000 photons, respectively. This result indicates that
the original photostability of the PDI can be retained despite
substitution with the bulky PGDs.
To illustrate the usefulness of PGDÀPDI 4 in targeted bio-
logical imaging, the fluorescent labeling of λ receptors on the
surface of living bacterial cells using 4 was explored. These
receptor proteins, also known as LamB or maltoporin, function
as receptors for bacteriophage λ and also facilitate the transport
of maltodextrins across the outer membrane of Escherichia coli.11
In vivo, the E. coli strain used in this study, called pLO16,
produces biotinylated λ receptors with the biotin on the extra-
cellular side.12 The low efficiency of biotinylation in this strain
allowed for the detection of the λ-receptor localization pattern in
these cells. As shown in Figure 5A, PGDÀPDI 4 produced bright
fluorescent spots only at the surfaces of the cells that had been
preincubated with streptavidin. Furthermore, in some bacteria, a
helical pattern of the spots along the long axis of the bacterium
could be discerned, consistent with the surface arrangement of
To characterize the photostability of PGDÀPDIs for single-
molecule applications, biotinylated PGDÀPDI 4 was immobi-
lized on a PEG-passivated surface through biotinÀneutravidin
linkages (Figure 4A). This immobilization was indeed highly
specific because only negligible binding of 4 was observed in the
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the proteins.13 This labeling was highly target-specific, as evi-
denced by control experiments wherein labeling with 4 in the
absence of streptavidin showed no detectable staining of
the bacteria cells, either on the surface or inside the bacteria
(Figure 5B).
The importance of the dendronized, neutral character of PDI
4 is demonstrated by comparison with nondendronized analogue
8. Although 8 carries a biotin unit, it exhibited only nonspecific
staining of bacteria at the same concentration as used for 4 and in
the presence of streptavidin. As shown in Figure 5C, cells stained
with 8 exhibited a largely homogeneous distribution in fluores-
cence signal throughout the entire bacterium that could not be
removed even after extensive washes. That ionic interactions
dominated the cell staining was further shown in experiments
where 8 was used without preincubation with streptavidin. The
cells in this case exhibited a comparable level of staining
throughout the bacterium (Figure 5D). These studies demon-
strate the importance of the neutral, dendronized PDI in achiev-
ing target-specific biolabeling.
To further illustrate the usefulness of PGDÀPDIs in targeted
biological imaging, fluorescent labeling of ACP-tagged fusion
proteins on the surface of living mammalian cells was explored
using PGDÀPDI 7, which was synthesized by maleimideÀthiol
coupling between 5 and coenzyme A (CoA). This fusion protein
contains a surface-localized glycosylphosphatidylinositol (GPI)
anchor upstream of the ACP tag,14 exposing this tag on the outer
surface of the plasma membrane. The ACP tag used here was
based on the E. coli acyl carrier protein, which can be conjugated
to fluorophore derivatives of CoA in the presence of an enzyme
named SFP synthase (Figure 6A).15 As shown in Figure 6B,
PGDÀPDI 7 clearly produced a bright, surface-localized
Figure 4. Single-molecule analysis of PGDÀPDI 4. (A) Experimental
scheme, in which 4 was immobilized on a PEG-coated surface through
biotinÀneutravidin linkages. (B) Fluorescence image of immobilized
molecules with pseudo colors. (C) Typical fluorescence intensity time
traces of individual molecules in T50 buffer. (D) Average fluorescence
intensity as a function of time in T50 buffer.
Figure 6. Fluorescent labeling of GPIÀACP fusion proteins on the
surface of living mammalian cells. (A) Scheme of enzymatic labeling. (B)
Fluorescence image of HeLa cells labeled with PGDÀPDI 7 after
incubation in the presence of SFP synthase. (C) As a control, cells were
labeled without SFP synthase.
Figure 5. Fluorescent labeling of λ receptors on the surface of living bacteria cells. (A) Bright-field and fluorescence images of E. coli cells labeled with
PGDÀPDI 4 after preincubation with streptavidin. The helical pattern of λ receptors is highlighted for one cell in the right panel, which shows an
enlargement of the white box in the middle panel. (B) As a control, cells were labeled with 4 in the absence of streptavidin. In (C) and (D), cells were
labeled with nondendronized PDI 8 with and without streptavidin preincubation, respectively. All of the images were processed using ImageJ software
(NIH), and an intensity range of (10133, 23589) and (28856, 65378) was used for the fluorescence images of 4 and 8, respectively. It should be noted
that a scale greater in intensity value was used for 8 because of the higher background and absolute intensity therein.
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fluorescence signal on live HeLa cells that had been transiently
transfected and incubated with SFP synthase. This labeling was
also target-specific, as shown by control experiments in which
labeling with 7 in the absence of SFP synthase or transfection
with the plasmid encoding the above-mentioned fusion protein
showed no apparent fluorescent labeling on the cell surface
(Figure 6C and Figure S10).
The methodology for specific protein labeling can be general-
ized by employing PGDÀPDI 5 functionalized with a single
maleimide that enables efficient labeling of cysteine residues in
proteins in a specific fashion. In this study, 5 was reacted with
bovine serum albumin (BSA), which contains a free surface
cysteine at amino acid 34, and the formation of PGDÀPDIÀBSA
conjugates was confirmed by the MALDI mass spectrum of 6,
which showed two intense peaks corresponding to native BSA
and PGDÀPDI 6 at m/z 66 431 and 72 400, respectively
(Figure 2). Unreacted BSA was observed because partial oxida-
tion at Cys-34 resulted in only ∼50% of the cysteine residues
being available for reaction.16 Quantification using Ellman’s
assay17 revealed 46 and ∼0% availability of cysteine sites on
BSA before and after reaction with PGDÀPDI 5, respectively,
indicating quantitative labeling of BSA with 5. These results
demonstrate the versatility of the monofunctional PGDÀPDIs
for efficient, specific protein labeling.
In conclusion, we have reported the synthesis of a series of
highly water-soluble and fluorescent PGDÀPDIs and the ability
of a clickable monofunctional PGDÀPDI to be singly linked to a
biomolecule in a specific fashion. Single-molecule and live-
bacteria imaging were performed using a single biotinylated
PGDÀPDI. The key finding was the ability of uncharged, site-
isolated PGDÀPDIs to serve as highly specific protein labels on
the surfaces of living bacteria cells, which is not possible with
previous ionic nondendronized PDI analogues. This approach
using dendrimers to encapsulate dyes clearly enhances the
performance of the dyes and thereby opens up a new generation
of biolabels wherein the design would couple a monofunctional
fluorescent core with a multivalent periphery containing cellular
receptors, therapeutic agents, targeting groups, or ligands.
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S
Supporting Information. Synthetic details, characteriza-
b
tion data, experimental procedures, additional data for single-
molecule and live-bacteria imaging, and movies (AVI) showing
the images acquired at z positions 1À6 in Figures S4, S6, and S8.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
sczimmer@illinois.edu
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’ ACKNOWLEDGMENT
We thank the National Institutes of Health and the National
Science Foundation for financial support of this research and Kyung
Suk Lee for helpful discussions on single-molecule data processing.
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