Water-soluble monofunctional perylene and terrylene dyes: Powerful labels for single-enzyme tracking

Article abstract of DOI:10.1002/anie.200705409

All in one: Exceptionally photostable, highly fluorescent, water-soluble, and monofunctional perylene and terrylene dyes bearing reactive groups for covalent attachment to biomolecules have been synthesized (see picture). Single-molecule enzyme tracking r

Full text of DOI:10.1002/anie.200705409

Communications
DOI: 10.1002/anie.200705409
Ultrastable Dyes
Water-Soluble Monofunctional Perylene and Terrylene Dyes: Powerful
Labels for Single-Enzyme Tracking**
Kalina Peneva, Gueorgui Mihov, Fabian Nolde, Susana Rocha, Jun-ichi Hotta,
Kevin Braeckmans, Johan Hofkens, Hiroshi Uji-i, Andreas Herrmann, and Klaus Müllen*
Fluorescence microscopy is the most widely used tool for
visualizing subcellular structures and for localizing proteins
within cells.[2] Single-molecule spectroscopy (SMS)has gone
beyond that, and has revealed information about complex
biological molecules and processes which are difficult to
obtain from ensemble measurements.[3] Single proteins,
virions, drugs, and other single bioparticles have been labeled
and their pathway and interactions followed inside living
cells.[4,5] One critical issue in observing biological entities at
the single-molecule level is the label: It should be water-
soluble, highly fluorescent in an aqueous environment, and
have a reactive group for attachment to the biomolecule, for
example, a protein or enzyme. Moreover, the attachment
should not affect the structure or function of the biomole-
cules, or in the case of an enzyme, its activity. Finally, an
exceptional photostability of the label is needed for visual-
ization or tracking over a sufficient period of time. Is there a
chromophore that has it all? It appears that rylene dyes fulfil
all of these requirements. Rylene chromophores have proven
to be a remarkable class of dyes that are characterized by an
exceptional thermal and photochemical stability as well as
having fluorescence quantum yields close to unity in organic
solvents.[6,7] The extreme photostability of several members of
the rylene family has been shown in numerous single-
molecule experiments.[8–11] To covalently attach these out-
standing chromophores to proteins, we focused on the
synthesis of water-soluble, monofunctional perylene and
terrylene chromophores possessing N-hydroxysuccinimide
ester and maleimide groups. The corresponding rylene–
protein conjugates were employed for tracking single phos-
pholipases on their native substrates by real-time wide-field
fluorescence microscopy. Enzyme kinetics at the single-
molecule level have been previously analyzed by monitoring
the conversion of a nonfluorescent substrate into a fluores-
cent product.[12–17] The observed fluctuations in the kcat value
over time could be correlated with different conformations of
the enzyme molecule, with each conformation having its own
kcat value. Measurement of enzyme activity on native sub-
strates has also been performed at the single-molecule level,
namely on DNA-processing enzymes and motor proteins, by
using confocal SMS.[18–20] However, tracking of single inter-
facial enzymes such as phospholipases that have complex
catalytic pathways and modes of operation has not yet been
performed.
The synthesis of the herein described perylene derivatives
starts from the symmetrical perylene diimide (PDI) 1. One of
the imide groups was saponified under strong basic conditions
and then further treated with 1,2-ethylenediamine or
3-aminopropanoic acid to give the corresponding monofunc-
tionalized perylenes. Water solubility was achieved by intro-
ducing four sulfonyl substituents at the phenoxy groups in the
bay regions.[21] Compound 2 was treated with 4-maleimido-
butyric acid N-succinimidyl ester (GMBS)in dry DMF in the
presence of triethylamine to give 3 in high yield (Scheme 1a).
N-hydroxysuccinimide ester 5 was obtained from 4 by using
N,N,N’,N’-tetramethyl(succinimido)uronium tetrafluorobo-
rate (TSTU)and diisopropylethylamine (DIPEA)at RT
(Scheme 1a).[22–24] Compounds 3 and 5 are highly soluble in
water and have high fluorescence quantum yields (Ff)in
aqueous media (Table 1).[25,26] The higher homologue terryl-
enediimide (TDI)was synthesized by formally introducing
one additional naphthalene unit into the perylene scaffold.
The water-soluble terrylene dye 6 absorbs above 600 nm, and
thus is ideal for single-molecule and live-cell-imaging experi-
ments because autofluorescence of cellular components at
that wavelength region is minimal. The synthesis of the
monocarboxylic acid functionalized TDI was recently
reported,[9] and its transformation into the activated ester
7 was accomplished by its treatment with TSTU and DIPEA,
as described for 5 (Scheme 1b). Compound 7 was employed
as the starting material for the maleimide-functionalized TDI
[*] K. Peneva, Dr. G. Mihov, F. Nolde, Prof. Dr. K. Müllen
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
Fax: (+49)6131-379-350
E-mail: muellen@mpip-mainz.mpg.de
Homepage: http://www.mpip-mainz.mpg.de/groups/muellen
Prof. Dr. A. Herrmann
University of Groningen
Zernike Institute for Advanced Materials
Department of Polymer Chemistry
Nijenborgh 4, 9747 AG Groningen (Netherlands)
S. Rocha, Dr. J. Hotta, Prof. J. Hofkens, Dr. H. Uji-i
Chemistry Department
Katholieke Universiteit Leuven
Celestijnenlaan 200F, 3001 Heverlee-Leuven (Belgium)
K. Braeckmans
Laboratory of GeneralBiochemistry & Pharmacy
Ghent University
Harelbekestraat 72, 9000 Ghent (Belgium)
[**] Support from the FWO (grant G.0366.06), the KULeuven Research
Fund (GOA) 2006/2, Centre of Excellence INPAC, Centre of
Excellence in Catalysis (CECAT), the Flemish Ministry of Education
(ZWAP 04/007), the Portuguese Foundation for Science and
Tecnhology (FCT), the Federal Science Policy of Belgium (IAP VI),
and the EU Sixth Framework Programme (STREP Bioscope) is
acknowledged.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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such resins are usually
employed for solid-phase
organic synthesis. Here the
unreacted dye was cova-
lently captured after addi-
tion of the support to the
labeling solution, and the
labeled enzyme was isolated
by filtration. Gel electropho-
retic analysis confirmed that
the unreacted label was
removed very efficiently
(see Figure S2 in the
Supporting Information).
Further evidence for the
effective
removal
of
unreacted dye was obtained
by performing fluorescence
correlation
measurements
spectroscopy
(see the
Supporting Information). A
diffusion coefficient of 2.3 ”
10À6 cm2 sÀ1 was measured
for the free dye, whereas a
value of 1 ” 10À6 cm2 sÀ1 was
found for the labeled pro-
tein. The values are in agree-
ment with calculations based
on the molecular weight of
the
protein
(see
the
Supporting
Information).
Labeling of the enzyme
with TDI derivative 7 was
achieved in a similar way.
This novel strategy allows
the convenient and fast sep-
aration of labeled enzymes
without the need to perform
time-consuming chromato-
graphic or electrophoretic
purification steps.
The bulk activity of the
Scheme 1. Synthesis of perylene and terrylene labels: a) Reagents, conditions and yields: 1) KOH, isopropa-
nol, 1408C, 12 h, 24%; 2) ethylenediamine, toluene, 608C, 3 h, 60%; 3) H2SO4, RT, 16 h, 97%; 4) 3-amino-
propanoic acid, propionic acid, 1408C, 2 h, 65%; 5) GMBS, triethylamine (TEA), DMF, 5 h, 95%; 6) TSTU,
DIPEA, DMF, water, 1,4-dioxane, 1 h, 85%. b) Reagents, conditions, and yields: 1) TSTU, DIPEA, DMF, water,
1,4-dioxane, 1 h, 80%; 2) 1-(2-aminoethyl)pyrrol-2,5-dione, DMF, TEA, 5 h, 90%.
labeled enzyme was tested.
PLA1 catalyzes the saponifi-
cation of the acetate groups
of the fluorescein derivative,
which is nonfluorescent (Fig-
ure 1a). The hydrolysis of
the substrate can be followed
derivative 8. The synthesis of 8 was realized by treating 7 with
1-(2-aminoethyl)pyrrole-2,5-dione in DMF.
by the increase in the fluorescence intensity arising from the
formation of the fluorescent product by the enzymatic
reaction. Figure 1b shows the changes in the fluorescence
intensity of a similar solution of pro-fluorescent substrate as a
function of time without the enzyme, after adding non-labeled
enzyme, and after adding the labeled enzyme. The rate
constants of the hydrolysis were estimated to be (3001.3 Æ
29.1)s À1 and (3083.1 Æ 38.1)s À1 for the labeled and the
nonlabeled enzyme, respectively. The autohydrolysis of
5-CFDA was measured as (110 Æ 17.9)s À1. The observed
The perylene derivative 5 was used for the labeling of
phospholipase (PLA1). As the seven solvent-exposed lysine
residues of the enzyme were targeted, 5 was applied in 20-fold
excess to the enzyme. The purification of the conjugates was
accomplished by coupling the unreacted dye to a solid support
(see the Supporting Information). The resin consisted of a
low-cross-linked polystyrene matrix onto which polyethylene
glycol containing a free terminal amino group was grafted;
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Table 1: Absorption (lmax, abs) and fluorescence maxima (lmax, flu), as well
as fluorescence quantum yields (Ff)[a] in water for 2–5, 7, and 8.
Compound lmax,abs [nm] (e[mÀ1 cmÀ1])
lmax,flu [nm] Ff
2
3
4
5
450 (10850), 534 (21825), 562 (22243) 620
0.39
0.40
0.66
0.58
–
450 (8367), 534 (16199), 566 (16569) 620
450 (11454), 534 (22510), 564 (25059) 620
450 (9951), 534 (18867), 566 (21071) 622
7[b]
8[b]
426 (4569), 638 (24468)
426 (2782), 638 (16633)
–
–
–
Figure 2. Fluorescence image of individual labeled enzymes on POPC
layers. The phospholipid bilayers were prepared by the rehydration
method using mica as a support[1] a) PLA1 labeled with PDI 5 on a
nonlabeled POPC layer; b) magnification of the area in image (a)
indicated by the white square. The arrows indicate clearly distinguish-
able individual enzymes located on the edge of the layer. Note that the
signal-to-noise is much higher than for the non-adsorbed molecules
diffusing in solution (outlined). Exposure time for each frame 50 ms;
the excitation wavelength for the dye was 532 nm.
[a] Ff was measured at room temperature using cresylviolet in methanol
(Ff =0.54) as a reference.[25] [b] The water-soluble terrylene derivatives
form nonfluorescing H aggregates in water under the conditions
employed here. However, when highly diluted they are well suited for
single-molecule studies.[9]
between PLA1 interacting with the edge of the POPC layers
and ones diffusing on the top of the layer (Figure 2b).
Individual enzymes interacting with the POPC layers edge
show up as diffraction-limited spots in the image (Figure 2b),
whereas freely diffusing enzymes appear as blurry spots
(Figure 2b). Similar results were obtained using phospholi-
pase labeled with terrylene derivative 7 (data not shown).
To correlate the diffusion of the labeled enzyme with
hydrolysis of the POPC layers, 3,3’-dioctadecyloxacarbocya-
nine perchlorate (DiO)was incorporated into the phospho-
lipid layers to render them fluorescent, albeit at a different
emission wavelength than the label. The use of labeled
phospholipid layers allows a clear visualization of the edge
between two consecutive layers or between a bilayer and the
support (Figure 3a). Furthermore, hydrolysis of the layers can
also be followed by the drop in the quantum yield of DiO as a
result of the increase in cis–trans isomerization when it is
freely diffusing in aqueous solution. Even in these difficult
conditions for SMS (introduction of a fluorescent back-
ground), the new labels allow us to obtain a sufficient signal-
to-noise ratio to discriminate and track individual enzymes on
the labeled POPC layers (Figure 3a,b).
Figure 3c shows the trajectory of an enzyme diffusing
along the edge of the layer (see the movie in the Supporting
Information). The diffusion coefficient can be quantified by
calculating the mean-square displacement from these trajec-
tories. The diffusion constant of the phospholipase molecule
shown by the trajectory in Figure 3 is (1.276 Æ 0.014) mm2 sÀ1.
The interaction between enzymes and the substrate layers
results in the value of the diffusion constant being lower than
the usual values obtained for the free diffusion of proteins in
solution (ca. 102 mm2 sÀ1).
Figure 1. Relative enzymatic activity in the bulk phase. By using a pro-
fluorescent substrate it is possible to follow the reaction kinetics
through the increase in the fluorescent intensity. a) Hydrolysis of
nonfluorescent 5-carboxyfluorescein diacetate (5-CDFA) to the fluores-
cent product 5-carboxyfluorescein (5-FAM). b) Fluorescence intensity
of 5-FAM as a function of time. For the activation of the surface
enzyme in solution (for example, to open the lid protecting the active
site), tritonX-100 was added to the solution; 0.7 mm 5-CDFA, 0.5 mm
~
tritonX-100. ”: nonlabeled PLA1 (3E-8M), (3001.3Æ29.1) sÀ1
; : PLA1
labeled with N-hydroxysuccinimideperylene derivative 5 (3E-8M),
(3083.1Æ38.1) sÀ1
;
: 5-CFDA autohydrolysis (110.54Æ17.9) sÀ1. The
*
labeling of the enzyme has no affect on its activity towards 5-CFDA.
rate constants clearly show that no activity was lost by the
labeling.
In the next step, single-enzyme tracking was performed by
using wide-field fluorescence microscopy. For this, the labeled
PLA1 was added on to 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC)supported bilayers and a snapshot
was taken every 50 ms. Figure 2a shows the fluorescence
image obtained with the enzyme labeled with PDI 5. The
individual labeled enzymes can be clearly observed as bright
spots (Figure 2). Furthermore, it is possible to distinguish
Most of the enzymes in the recorded movies stayed on the
layers for less than 0.3 s. It has been shown that the water-
soluble PDI label 5 has a survival time of 120 s when
immobilized in poly(vinyl alcohol).[27] Therefore, the disap-
pearance of the labeled enzymes after 0.3 s is probably
associated with enzyme activity as well as its mode of action
(nonprocessive hydrolysis of the layers).
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of the influence of the layer composition, fluidity, etc. on both
parameters (enzyme mobility and activity).
Received: November 26, 2007
Revised: February 11, 2008
Published online: March 26, 2008
Keywords: dyes/pigments · enzymes · fluorescent probes ·
.
kinetics · single-molecule studies
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Figure 3. Fluorescence image of individual enzymes labeled with PDI 5
on POPC layers labeled with DiO. The PDI label allows sufficient
photons to be obtained to discriminate individualenzymes even with a
fluorescence background from the layers. a) Labeling of the layers
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adsorption of the enzyme molecules at the steps. The image was
obtained after accumulating 8 frames (400 ms) b) Magnification of the
area indicated by the white square in image (a). Single enzyme
molecules are indicated by arrows. The exposure time for each frame
was 50 ms; the excitation wavelengths for PDI and DiO were 532 and
488 nm, respectively. The fluorescence from both dyes is detected
through the same filters, see the Supporting Information. c) Trajectory
described by the enzyme indicated by the orange arrow in (b) which is
on the edge of a POPC layer labeled with DiO. d) Plot of the mean-
square displacement (MSD) of the enzyme for the first points of the
track. From these data it is possible to calculate the diffusion constant
of a single active enzyme as (1.28Æ0.02) mm2 s.
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In summary, new water-soluble, fluorescent, monofunc-
tional perylene- and terrylenediimide derivatives have been
introduced. These dyes can be attached to a variety of
proteins through their reactive functional groups. In regard to
protein labeling, a convenient procedure for the removal of
unreacted dye from labeled enzymes has been developed
which involves capturing excess dye with a solid support.
The performance of the new fluorescent probes was
assessed by single-particle tracking. The measurements
revealed that single enzymes could even be visualized on a
fluorescently labeled substrate. The outstanding photostabil-
ity of the dyes and their extended survival times under strong
illumination conditions allow the actions of enzymes to be
characterized on their natural substrates, in this case, phos-
pholipase acting on phospholipid-supported layers. By using
this approach, enzyme mobilities could be correlated with the
catalytic activity. Furthermore, this assay allows the validation
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