Rhodamine F: A novel class of fluorous ponytailed dyes for bioconjugation

Article abstract of DOI:10.1039/c3ob40267c

Incorporation of fluorous ponytails such as polyfluorinated alkyl residues (CH₂)_m(CF₂)_nCF₃ leads to a novel class of bright rhodamine-based fluorescence dyes. These dyes combine the excellent photophysical properties of the frequently used rhodamine dyes with the unique features of "light" fluorous molecules. One of those features is the possibility to separate substances utilizing fluorous solid-phase extraction (F-SPE), which is based on the specific intermolecular interaction between fluorous compounds. Thus, molecules, which are labeled with these new dyes, are not only accessible to fluorescence experiments, but can also be easily purified (via so-called FluoroFlash columns) prior to use. The dyes were bound to a cell penetrating peptoid (polycationic oligo(N-substituted) glycine) on solid supports. These conjugates were purified with F-SPE before their photophysical and biological properties were investigated. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40267c

Organic&
BiomolecularChemistry
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Volume 11 | Number 24 | 28 June 2013 | Pages 3927–4124
ISSN 1477-0520
PAPER
Stefan Bräse et al.
1477-0520(2013)11:24;1-9
Rhodamine F: a novel class of fluorous ponytailed dyes for bioconjugation

Organic &
Biomolecular Chemistry
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Rhodamine F: a novel class of fluorous ponytailed dyes
for bioconjugation†
Cite this: Org. Biomol. Chem., 2013, 11,
3954
Dominik K. Kölmel,a Birgit Rudat,a,b Delia M. Braun,a Christin Bednarek,a,c
Ute Schepersc and Stefan Bräse*a,c
Incorporation of fluorous ponytails such as polyfluorinated alkyl residues (CH2)m(CF2)nCF3 leads to a
novel class of bright rhodamine-based fluorescence dyes. These dyes combine the excellent photo-
physical properties of the frequently used rhodamine dyes with the unique features of “light” fluorous
molecules. One of those features is the possibility to separate substances utilizing fluorous solid-phase
extraction (F-SPE), which is based on the specific intermolecular interaction between fluorous com-
pounds. Thus, molecules, which are labeled with these new dyes, are not only accessible to fluorescence
experiments, but can also be easily purified (via so-called FluoroFlash columns) prior to use. The dyes
were bound to a cell penetrating peptoid (polycationic oligo(N-substituted) glycine) on solid supports.
These conjugates were purified with F-SPE before their photophysical and biological properties were
investigated.
Received 5th February 2013,
Accepted 2nd April 2013
DOI: 10.1039/c3ob40267c
www.rsc.org/obc
“fluorophobic pass”, all non-fluorous compounds are
removed. In the second step, the “fluorophilic pass”, all
Introduction
The purification of (bio)molecules obtained by synthesis is a fluorous compounds bound to the gel are eluted with an
very time-consuming process. In the last decade, fluorous organic solvent like water-free MeOH, CH3CN or THF.1 With
solid-phase extraction (F-SPE) as an alternative to common this separation technique being very specific for fluorous com-
chromatographic methods was proven as a reliable and simple pounds, further purification methods can often be completely
separation technique.1,2 The original or so-called standard replaced. Nevertheless, a few reports describe the use of an
F-SPE was introduced in 1997 by Curran et al.3 It is based on additional purification step like e.g. high-performance liquid
the exploitation of strong, specific fluorine–fluorine inter- chromatography (HPLC).5,6
actions and does not depend on polarity or molecular features
The method of F-SPE facilitates a wide range of applications
other than the fluorous part. The method uses a fluorous solid like separation, derivatization as well as identification of mole-
phase to bind fluorous molecules and to separate them from cules.7 For instance, it is advantageous for product isolation
non-fluorous compounds by a fluorophobic liquid phase. Typi- in Mitsunobu reactions8,9 or the synthesis of large libraries
cally, the solid phase is composed of silica gel with a fluoro- of small molecules.6,10 Moreover, it was successfully utilized
carbon modified phase (–SiMe2(CH2)2(CF2)7CF3) and can be for synthesis and/or purification of several biomolecules
purchased for example as prepacked FluoroFlash columns.4 like oligonucleotides,11,12 peptides,5,13–16 glycolipids,17 and
For purification the crude mixture is loaded onto the gel and oligosaccharides,18–20 as well as for the enrichment of pep-
the solid phase is washed with a fluorophobic solvent (e.g. tides21 or parts of the proteome.22 Other life science appli-
70–80% aqueous MeOH, 80–90% aqueous DMF, 50–60% cations of fluorine–fluorine interactions involve enzyme assays
aqueous CH3CN or 100% DMSO). Thus, during this so-called and identification of small molecules.23–25
The target products purified by F-SPE have to be “light”
fluorous molecules, meaning that they contain 9–17 fluorine
aKarlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-
atoms.1,26 Usually, such compounds are readily available,
Weg 6, D-76131 Karlsruhe, Germany. E-mail: braese@kit.edu;
cheap, soluble in common organic solvents and their reactivity
Fax: (+49) 721 608-48581; Tel: (+49) 721 608-42902
bKarlsruhe Institute of Technology (KIT), Light Technology Institute, Engesserstraße
is comparable to their non-fluorous analogues. In recent years,
several groups developed an array of fluorinated handles with
respect to biomolecule synthesis (e.g. t-Bu,27 Boc,28 Bn,29
THP,30 Z13,31 and other protecting groups14,32–36), which are
typically cleaved after tagging, reaction and purifi-
cation.5,6,12,14,16,19,20,26 However, if a biomolecule’s activity is
13, D-76131 Karlsruhe, Germany
cKarlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics,
Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany
†Electronic supplementary information (ESI) available: Experimental and spec-
tral data for all new compounds. HPLC traces and mass spectra of the dye-
labeled peptoids. See DOI: 10.1039/c3ob40267c
3954 | Org. Biomol. Chem., 2013, 11, 3954–3962
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not changed by the fluorous handle or if the fluorous label
is useful for further analysis, it is also possible to omit the
cleavage step. In this case the fluorous handle is referred to as
a fluorous “ponytail” or just ponytail to distinguish it from a
cleavable tag.37
For many applications such as fluorescence microscopy,
fluorescence assisted flow cytometry (FACS) or other fluore-
scence based analysis methods, it is important to incorporate
or add fluorophores to newly synthesized compounds, proteins
or other biomacromolecules. So far, only a few groups have
reported the combination of F-SPE and fluorescent molecules.
Miura et al. synthesized fluorinated oligosaccharides, which
were eventually purified by F-SPE. However, they introduced
the fluorophore after the purification and removal of the
fluorous marker.19 Todoroki et al. developed a fluorescent deri-
vatization reagent for amines containing the fluorophore con-
nected to a fluorous tagged amine-reactive group. Upon
reaction with the amino group the fluorous tag was released
and the reaction byproduct could be simultaneously removed
by F-SPE, while the fluorescent target molecules were not
fluorinated.38 One year later, they described a similar fluorous
scavenging-derivatization method for carboxylic acids.39
Lo et al. recently developed a fluorous label for bioconjugation
that can be used for imaging and purification.40 However, they
used luminescent rhenium(I)-complexes instead of organic
dyes.
Fig. 1 Structures of rhodamine F dyes 1a–f, 2 and 3. Rfn = (CF2)n−1CF3.
amino groups were used.47,60,61 However, only few reports
describe the use of xanthene fluorophores with “light”
fluorous labels: Li et al. applied a “light” fluoroponytailed
fluorescein derivative for anti-stick coating62 and Vegas et al.
synthesized a rhodamine derivative for microarray analysis.23
Herein we extend the latter approach. We synthesized eight
new rhodamine-based fluorophores with incorporated, perma-
nently affixed fluorous ponytails, called rhodamine F (Fig. 1).
In a subsequent reaction we coupled them in a solid phase
approach to cell penetrating peptoids (CPPos, polycationic
oligo(N-substituted) glycines).63–69 Spectral properties of all
derivatives were studied. Moreover, we demonstrated their sim-
plified purification via F-SPE as well as their use in live cell
fluorescence microscopy.
Yet, it should be possible to synthesize fluorescent mole-
cules, which simultaneously act as markers and contain
“light” fluorous labels. They would enable the labeling of bio-
molecules and an easy separation from unfunctionalized sub-
stances after just one modification. Moreover, the detagging
step after F-SPE could be omitted, if the fluorous handles
do not influence the label’s photophysical properties, as is
Results and discussion
Design and synthesis
already reported for some fluorescent compounds containing The eight rhodamine F dyes 1a–f, 2 and 3 (Fig. 1) were syn-
fluorous tags.41
thesized from different m-hydroxyanilines according to
In order to develop such novel fluorous fluorophores, common reaction conditions, published by e.g. Hell et al.47
rhodamine derivatives were chosen. They are widely used as The main difference between the rhodamine F dyes are the
fluorescent labels for lipids, proteins, peptides, nucleic acids, substituents at the amine functions. Every dye contains two
and other biomolecules.42–46 The synthesis of rhodamines “light” fluorous ponytails ((CH2)m(CF2)n−1CF3 = (CH2)mRfn).
with different substitution patterns is well described.47–50 And The ponytails differ in the length of the perfluorinated part Rfn
in particular, they display high absorption coefficients (εmax), and the length of the alkylene spacer. Optionally some dyes
absorption (λmax,abs) and emission (λmax,em) in the visible contain N-ethyl substituents to bathochromically shift the
region, high fluorescence quantum yields (ΦF), and high spectroscopic properties. The double ponytailed dyes can be
chemical stability and photostability.51,52 In addition, their considered as relatively “light” fluorous (fluorine content of
photophysical properties according to the chemical structure ≤50% by molecular weight), which preserves reasonable solu-
and the environment are well described.53,54
bility in many organic solvents.
The development of several novel high resolution and high
The rhodamine F dyes 1a–c, which contain perfluoroalkyl-
speed live imaging techniques and other fluorescent techno- methyl substituents CH2Rfn, were synthesized from m-anisidine
logies during the last few years increased the demand for (4-H) or N-ethyl-m-anisidine (4-Et) and a perfluoroalkanoyl
novel fluorogenic dyes with high quantum yields and decent chloride (Scheme 1). After acylation, the amides 5-Rf6-H, 5-Rf6-
photostabilities, which has been proven for several rhodamine Et and 5-Rf7-H were reduced to the corresponding amines
dyes.47,54–59 Furthermore, fluorinated fluorophores have been 6-Rf6-H, 6-Rf6-Et and 6-Rf7-H by using BH3·THF.
synthesized and utilized. For example, perfluorination of the
This step is crucial for the preparation, because N-acylated
carboxyphenyl ring in order to generate bathochromic shifts, rhodamine derivatives would be locked in the spirolactone
fluorination of the xanthene fragment for higher photostabil- form, which is non-fluorescent.70 Next, the aryl methyl ethers
ities or utilization of CF3CH2 as a protecting group for the 6-Rf6-H, 6-Rf6-Et and 6-Rf7-H were cleaved. One method to
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Scheme 3 Synthesis of the rhodamine
F 2. Reagents and conditions:
(i) BH3·THF, THF, reflux, overnight; (ii) Rf6(CH2)2I, DMF, 140 °C, 2 h; (iii) phthalic
anhydride, TsOH, propionic acid, 160 °C, 24 h.
Scheme 1 Synthesis of the perfluoroalkylmethylated rhodamine F dyes 1a–c.
Reagents and conditions: (i) pyridine, CH2Cl2, RT, overnight; (ii) BH3·THF, THF,
reflux, overnight; (iii) BBr3, CH2Cl2, RT, overnight; (iv) phthalic anhydride, TsOH,
propionic acid, 160 °C, 24 h.
the phenols 7-CH2Rf6-H, 7-CH2Rf6-Et and 7-(CH2)2Rf8-H with
phthalic anhydride, rhodamine F dyes 1d–f were obtained.
The tetrahydroquinoline-containing rhodamine F 2 was
synthesized from amide 8, which again was reduced by
BH3·THF to yield amine 9 (Scheme 3).61 Next, the fluoropony-
tail was introduced by N-alkylation with Rf6(CH2)2I. The result-
ing tetrahydroquinoline 10 and phthalic anhydride were
converted to rhodamine F 2.
The dihydroquinoline scaffold of rhodamine F 3 was built
up from m-anisidine (4-H) via a double condensation with
acetone (Scheme 4).47 The resulting dihydroquinoline 11 was
demethylated with glacial acetic acid and 48% aqueous HBr,
as BBr3 was not suitable for this reaction. Dihydroquinoline 12
was N-alkylated with Rf6(CH2)2I to give fluorous compound 13.
The corresponding rhodamine F 3 was obtained after the reac-
tion of 13 with phthalic anhydride.
All rhodamine F dyes were obtained as hydrochlorides in
the fluorescent, open form (Scheme 5), which arises, besides
their intense colour, from the obtained 13C NMR spectra. If
the rhodamine F dyes would be present in the closed, non-
fluorescent spirolactone form, the quaternary aliphatic spiro
carbon atom would give a signal at about δ = 84 ppm. In the
open form the signal shifts downfield to the densely populated
region between δ = 110 and 140 ppm.47
achieve this is to reflux the m-methoxyaniline derivatives in
glacial acetic acid and 48% aqueous HBr.47 But due to the
highly hydrophobic polyfluorinated residues, demethylation
with BBr3 in CH2Cl2 is more appropriate. Finally, the resulting
phenols 7-Rf6-H, 7-Rf6-Et and 7-Rf7-H were condensed with
phthalic anhydride to yield the fluorescent rhodamine F dyes
1a–c.
Furthermore, it was possible to introduce fluoroponytails
via polyfluorinated iodides (Scheme 2).71,72 The perfluoro-
hexyl-substituted dyes 1d, 1e and the perfluorooctyl-substi-
tuted dye 1f were synthesized by using this method. Again, m-
anisidine (4-H) was used as starting material, which was alkyl-
ated with the appropriate fluorous alkyl iodide to provide
amines 6-CH2Rf6-H and 6-(CH2)2Rf8-H. Afterwards 6-CH2Rf6-H
may be alkylated with ethyl iodide to tertiary amine 6-CH2Rf6-Et.
Demethylation of compounds 6-CH2Rf6-H, 6-CH2Rf6-Et and
6-(CH2)2Rf8-H was carried out with BBr3. After condensation of
Scheme 2 Synthesis of the rhodamine F dyes 1d–f. Reagents and conditions:
(i) 140 °C, 3 h; (ii) ethyl iodide, Na2CO3, acetone, reflux, 30 h; (iii) BBr3, CH2Cl2,
RT, overnight; (iv) phthalic anhydride, TsOH, propionic acid, 160 °C, 24 h.
Scheme 4 Synthesis of the rhodamine F 3. Reagents and conditions: (i) Yb(OTf )3,
acetone, RT, 24 h; (ii) 48% aqueous HBr, AcOH, reflux, 6 h; (iii) Rf6(CH2)2I, DMF,
140 °C, 3 h; (iv) phthalic anhydride, TsOH, propionic acid, 160 °C, 24 h.
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Scheme 5 Thermodynamic equilibrium between the fluorescent, open form
and the non-fluorescent, closed spirolactone form.
F-SPE purification
As a proof of principle, a mixture of commercial rhodamine B
(red) and rhodamine F 1c (yellow) was loaded onto a Fluoro-
Flash cartridge and separated by F-SPE (Fig. 2). At first, non-
fluorous rhodamine B was eluted by using 2 M aqueous HCl–
MeOH (20 : 80) as the solvent. Afterwards, acidic MeOH
released the rhodamine F again from the fluorous phase. Both
washing steps can be observed optically by the different
colours of the dyes.
To test whether purification of rhodamine F labeled mole-
cules via F-SPE is suitable, the dyes were coupled to a peptoid
(oligo(N-substituted) glycine) on solid supports. The corre-
sponding peptoids 14a–d, 15 and 16 (Fig. 3) are cell penetrable
because of their polycationic character so that these conjugates
can be used for live cell imaging afterwards. The cell penetrat-
ing peptoid (CPPo) was synthesized on solid supports by using
standard protocols.65–69 After the synthesis of the hexameric,
immobilized peptoid scaffold, the rhodamine F dyes 1a–f,
2 and 3 were coupled directly with the secondary amine of the
peptoidic backbone, using diisopropylcarbodiimide (DIC) and
1-hydroxybenzotriazole (HOBt) as coupling reagents. Labeling
of structures during solid phase synthesis is perfect, because
Fig. 3 Structures of the peptoid conjugates 14a–d, 15 and 16.
the unreacted dye can easily be removed by a simple washing
step. Otherwise the unreacted dye and the labeled target
would both be retained during the F-SPE. Most rhodamine F
dyes performed well, but the methylene-spaced rhodamine F
dyes (1a–c) proved to be quite unreactive. Only rhodamine F 1b
could be coupled, but just in low yield. We assume that in
these cases the alkylene spacer is too short to enable good
coupling yields. Due to the electron withdrawing effect of the
perfluoroalkyl residues, the ponytail of a suitable rhodamine F
dye should have a spacer of at least two methylene groups to
allow conjugation.
Previous studies showed that introduction of a spacer (e.g.
6-aminohexanoic acid (Ahx)) between the peptoid and the dye
is not necessary to preserve the fluorophore’s photophysical
properties.73 As some rhodamine conjugates can reversibly
form non-fluorescent spirolactams74,75 (a feature that can
be used for specific microscopic techniques),47,54,55,75–79 this
should be suppressed to maintain fully functional fluorous
fluorophores. The peptoid backbone provides a secondary
amine, so that the corresponding conjugates are not able to
form the non-fluorescent spirolactam.75
Fig. 2 Separation of a mixture of rhodamine B (red) and rhodamine F 1c
(yellow) by F-SPE. The upper row displays the FluoroFlash cartridge at different
points of the separation. The lower row displays test tubes with the dye-contain-
ing washing solutions. Rhodamine B was eluted first by washing with 2 M
HCl(aq)–MeOH (20 : 80). Afterwards, HCl in MeOH was used to release the
fluorous dye.
To prove that the final F-SPE is working, only 0.5 equivalent
of the corresponding rhodamine F dyes were used, which
resulted in a mixture of labeled and unlabeled peptoids. Due
to the final F-SPE even a great excess of unlabeled starting
material can be easily separated during the fluorophobic pass.
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After the acidic cleavage step, the crude product was lyophi-
lized and loaded on a fresh FluoroFlash cartridge. The F-SPE
was carried out using a slightly modified standard protocol.4
Because of the high hydrophilicity of the peptoid conjugates,
the crude products could be loaded as aqueous solutions for
maximum absorption on the stationary phase. The fluoropho-
bic wash was done with MeOH–H2O (60 : 40). The relatively
high water content was no problem, because the fluorous
labeled CPPos as well as the non-fluorous byproducts pos-
sessed good solubility in water. Unlike standard protocols, the
fluorophilic wash was done with acidic MeOH (0.1 M HCl in
MeOH). This proved to be a much better fluorophilic washing
solution for the cationic rhodamine F dyes and their conju-
gates than pure MeOH.
Table 1 Spectral properties of rhodamine F dyes 1a–f, 2 and 3. All spectra
were measured in MeOH
Dye λmax,abs a [nm] εmax [M−1 cm−1
]
λmax,em b [nm] ΦF
1a
1b
1c
1d
1e
1f
2
497
526
503
511
537
516
543
571
23 700 1400
40 000 2000
39 000 3000
85 000 5000
80 000 5000
85 000 5000
96 000 6000
88 000 5000
522
545
522
535
559
539
565
596
1.00 0.02
0.94 0.13
0.87 0.07
0.88 0.03
0.97 0.04
0.85 0.05
1.00 0.02
1.00 0.02
3
a Absorption maxima have
a
1
nm imprecision. b Fluorescence
maxima are reproducible within a 2 nm range.
In some cases, the fluorous product partially broke through
at the end of the fluorophobic pass, which could be detected
optically. However, the major part was well retained. The loss
was absolutely negligible and thus the separation successful.
The final elution with acidic MeOH could be easily monitored,
due to the intensive colours of the labeled structures. However,
it should be mentioned that the columns were still slightly
coloured once the elution was completed.
After the F-SPE, all peptoids except 14a could be obtained
in medium to high purity (up to 96%), which was determined
by analytical HPLC and MALDI-TOF-MS (see the ESI†). The
unlabeled peptoid could no longer be detected in any case.
Peptoid 14a, deriving from rhodamine F 1b, was not only
obtained in rather low yield but also the F-SPE did not work as
well as for the other peptoids.
The peptoids 14c and 15 were essentially pure after the
F-SPE (96% HPLC purity) and thus HPLC purification was
completely skipped. The other peptoids were again purified by
semi-preparative HPLC to further improve the purities. The
mass spectra of the peptoids were essentially pure after the
F-SPE and in all cases the remaining impurities could be easily
separated from the prepurified peptoids by HPLC.
In summary, the rhodamine F dyes allowed for easy and
quick F-SPE purification of the labeled compounds so that in
some cases HPLC purification could be completely omitted.
The F-SPE purification could be done within a few minutes
and with very little solvent (about 30 mL total volume).
Although an additional HPLC purification step was performed
for some examples, the F-SPE was still beneficial, because the
amount of impurities could be reduced drastically.
Fig. 4 Normalized absorption and fluorescence spectra of rhodamine F dyes in
MeOH. Cyan: 1a; orange: 1b; green: 1c; light green: 1d; red: 1e; yellow: 1f; dark
red: 2; violet: 3.
coefficients ranged between 2.4–9.6 × 104 M−1 cm−1, which
was of the magnitude of commercially available rhodamines
(e.g. rhodamine 110: 11.6 × 104 M−1 cm−1, and rhodamine B:
11.4 × 104 M−1 cm−1). The quantum yields are in the range of
0.85–1.00, which is characteristic for many rhodamines (rhod-
amine 110: 0.99; rhodamine B: 0.74). The dyes 1a and 1c
proved to be fluorous analogues of rhodamine 110. This is due
to the fact that perfluoroalkylmethyl residues (CH2Rfn) have a
comparable inductive effect like hydrogen atoms, which was
proposed by Hell et al. and proven by their bis-N,N′-trifluoro-
ethylated rhodamine.47
Spectral properties
The spectral properties of the new rhodamine F dyes were
studied and compared with selected commercially available
and frequently used rhodamines (rhodamine 110 chloride and
rhodamine B). All spectra were recorded in MeOH (Table 1),
because it is known to be a good fluorophilic solvent. The
obtained absorption and fluorescence spectra are shown in
Fig. 4.
The eight rhodamine F dyes 1a–f, 2 and 3 had absorption
maxima in the range between 500 and 570 nm and fluo-
rescence maxima between 520 and 600 nm. The extinction
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If the alkylene spacer is enlarged, the electron withdrawing
effect of the fluorous ponytail is reduced and thus the spectro-
scopic properties shift bathochromically. However, the length
of the fluorous part of the ponytail (Rfn) had only a minor
influence on the spectroscopic properties (absorption spectra
of 1a and 1c were very similar and the fluorescence spectra
were almost identical). If the alkylene spacer was elongated
from a methylene (1a or 1c) to an ethylene unit (1d), the
fluorescence maximum shifted 13 nm. The next elongation to
a propylene-spaced rhodamine F (1f) provided only a minor
4 nm shift, which implied that the spectral properties were
less influenced by additional elongation of the spacer. As is
well known from other rhodamines, a further bathochromic
shift can be provided by N-ethylation (compare 1a/1b and 1d/
1e). The dyes 1b and 1e are structurally quite similar to rhod-
amine B and therefore a complete insulation from the electron
withdrawing perfluoroalkyl groups should provide the same
spectral maxima. The difference between the fluorescence
maxima of 1b/1e and 1e/rhodamine B was 14 and 9 nm,
respectively. The fluorescence maximum of rhodamine B in
MeOH was found to be at 568 nm, which is in good accord-
ance with the literature.80 Although the two methylene groups
of 1e insulate the dye incompletely (for complete insulation,
about 7–8 methylene groups are needed),81 it is evident that
the spectral maxima approached exponential rather than
linear. Despite the fact that the rhodamine F dyes were all
influenced by their ponytails, this influence was rather neg-
ligible if the spacer consists of at least two methylene groups.
The rhodamine F dyes 2 and 3 had an additional ring
and therefore rigid amino groups. Within the accuracy of the
measurements, the quantum yields of these dyes are near
unity, which is due to the inhibition of eventual C–N-rotations.
Such rotations are known for dissipating energy of excited
states, which lowers quantum yields.53,54 The extra double
bonds of rhodamine F 3 provided a further bathochromic shift
of its spectral properties so that the fluorescence maximum
was almost at 600 nm.
Fig. 5 Normalized absorption and fluorescence spectra of rhodamine F-
labeled peptoids in water. Orange: 14a; green: 14b; red: 14c; yellow: 14d; dark
red: 15; violet: 16.
maxima 12–20 nm bathochromically. The extinction coeffi-
cients were still quite high (2.0–8.3 × 104 M−1 cm−1) with the
exception of peptoid 14a (0.7 × 104 M−1 cm−1). The quantum
yields of rhodamine F 1d and 1f were barely influenced by the
peptoid and thus remain extremely high (>0.9). The quantum
yields of the other conjugates decreased slightly, but were still
around 0.5–0.7. Therefore, all conjugates were still remarkably
bright, which is not at all self-evident for fluorescently labeled
peptoids.73
The purified peptoid conjugates 14a–d, 15 and 16 were
examined in aqueous solution (Table 2, Fig. 5) to provide
optimal comparison with the results of biological tests. The
conjugation with the peptoid structure in combination with
Confocal cell imaging
the changed solvent shifted the absorption and emission The rhodamine F dyes should be tested for their applicability
in live cell imaging. As is already known, peptoids with posi-
tively charged amino side chains act as molecular transporters,
which means that they are able to overcome the cell membrane
Table 2 Spectral properties of peptoid conjugates 14a–d, 15 and 16. All
and therefore mediate cellular uptake.65–69 Furthermore, this
spectra were measured in water
transport through the cell membrane works for conjugates of
a
b
λmax,abs
[nm]
εmax
λmax,em
[nm]
such peptoids and a variety of cargoes can be applied. Because
fluorous compounds are highly hydrophobic, the rhodamine F
dyes themselves were poorly water-soluble. Due to the fact that
the peptoid scaffold of the dye conjugates 14a–d, 15 and 16 is
polycationic, it serves not only as a molecular transporter, but
also provided the required water solubility for biological
assays.
Peptoid
[M−1 cm−1
]
ΦF
14a
14b
14c
14d
15
539
528
556
533
562
601
7000 1800
45 000 7000
83 000 3000
56 000 9000
70 000 2000
20 000 4000
561
549
575
549
582
616
0.47 0.02
0.950 0.013
0.574 0.010
0.912 0.003
0.685 0.009
0.65 0.03
16
a Absorption maxima have
maxima are reproducible within a 2 nm range.
a
1
nm imprecision. b Fluorescence
The tests were performed on human cervix carcinoma
(HeLa) cells, which were incubated with different
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synthesized in relatively few steps. Different substituents at the
amino functions allowed altering of the dyes’ photophysical
properties. The photophysic was not negatively affected by the
polyfluorinated alkyl residues. The length of the perfluorinated
part of the ponytails had only minor effects on the dyes,
whereas elongation of the alkylene spacer provided better insu-
lation from the electron withdrawing perfluoroalkyl residue
and thus minimized the influence on the dyes. All dyes pro-
vided medium to high extinction coefficients and excellent
quantum yields. Conjugating the dyes to a cell penetrating
peptoid on solid supports using standard coupling reagents
(DIC and HOBt) was successful. Only the methylene-spaced
rhodamine F dyes 1a–c lacked reactivity. Rhodamine F 1b
could be coupled only with low yield and was just to a limited
extent suitable to F-SPE. Therefore we subsume that fluorous
rhodamines should have at least ethylene spacers to preserve
reactivity and good F-SPE compatibility.
The F-SPE purification of the rhodamine F dyes 1e and 2
worked perfectly and revealed very high purities of the labeled
structures. In these cases HPLC purification could be comple-
tely omitted. The other peptoid conjugates, which were
obtained with medium purity after F-SPE, were purified again
by HPLC. However, the F-SPE was still beneficial, because the
amount of impurities was drastically reduced and moreover
the separations were quite simple. In summary, F-SPE proved
to be an easy, quick and reliable separation technique for the
presented rhodamine F dyes. Even if the HPLC purification
could not be omitted generally, the labeled structures can be
greatly enriched and thus the F-SPE is well worth the effort. In
addition, this strategy of purifying rhodamine F-labeled struc-
tures should be adaptable for numerous other (bio)molecules,
because of its independence of the marked structure.
Due to the cell penetrating character of the peptoid conju-
gates, the rhodamine F dyes could easily be applied to live cell
imaging. The peptoid–dye conjugates proved to be very bright
and biocompatible markers. Thus, we believe that rhodamine
F dyes may also be of particular interest to a large number of
research groups who are working on the labeling and purifying
of (bio)molecules.
Fig. 6 HeLa cells were incubated with a 1 μM peptoid solution. A, 15 (yellow);
B, 14a (red); C, 16 (violet) for 24 h at 37 °C and subjected to laser scan confocal
microscopy (LSM) for the determination of the intracellular localization. For
colocalization studies mitochondria were stained with MitoTracker® Green FM
(green) and nuclei were stained with Hoechst 33342 stain (blue). For details of
microscopy see the experimental section. The peptoids are found in vesicular,
most likely endosomal structures in the perinuclear region. Scale bar: 25 μm.
concentrations (0.1–100 μM) of the peptoids. After an incu-
bation of 24–48 h, live cell images were taken by fluorescence
confocal laser scanning microscopy. Independent of the con-
centration, the CPPos were always found to be perinuclear in
endosomal structures (Fig. 6). Even low concentrations of 1 μM
afforded clear and bright images for all the used rhodamine F
conjugates. In summary, the rhodamine F dyes proved to have
good biocompatibility and can be used as versatile labels for
synthetic molecules or biomolecules.
Acknowledgements
This work was supported by the Center of Functional Nano-
structures of the German Research Council (project E1.1), the
Fonds der Chemischen Industrie (fellowship to D.K.K.), the
Karlsruhe School of Optics & Photonics (KSOP) (fellowship to
D.K.K. and B.R.), the Carl Zeiss Foundation (fellowship to
C.B.) and the Helmholtz Program “Biointerface”.
Conclusions
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