Novel pyridinium dyes that enable investigations of peptoids at the single-molecule level

Article abstract of DOI:10.1021/jp103308s

Single-molecule microscopy is a powerful tool for investigating various uptake mechanisms of cell-penetrating biomolecules. A particularly interesting class of potential transporter molecules are peptoids. Fluorescence labels for such experiments need to

Full text of DOI:10.1021/jp103308s

J. Phys. Chem. B 2010, 114, 13473–13480
13473
Novel Pyridinium Dyes That Enable Investigations of Peptoids at the Single-Molecule Level
Birgit Rudat,†,‡ Esther Birtalan,† Isabelle Thome´,§ Dominik K. Ko¨lmel,† Viviana L. Horhoiu,§,|
Matthias D. Wissert,‡ Uli Lemmer,‡,| Hans-Ju¨rgen Eisler,‡ Teodor Silviu Balaban,§,|,⊥ and
Stefan Bra¨se*,†,|
Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6,
D-76131, Karlsruhe, Germany, and Institute for Nanotechnology (INT), Light Technology Institute (LTI), and
Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology, Karlsruhe, Germany
ReceiVed: April 13, 2010; ReVised Manuscript ReceiVed: August 17, 2010
Single-molecule microscopy is a powerful tool for investigating various uptake mechanisms of cell-penetrating
biomolecules. A particularly interesting class of potential transporter molecules are peptoids. Fluorescence
labels for such experiments need to comply with several physical, chemical, and biological requirements.
Herein, we report the synthesis and photophysical investigation of new fluorescent pyridinium derived dyes.
These fluorescent labels have advantageous structural variations and spacer units in order to avoid undesirable
interactions with the labeled molecule and are able to easily functionalize biomolecules. In our case, cell-
penetrating peptoids are successfully labeled on solid supports, and in ensemble measurements the photophysical
properties of the dyes and the fluorescently labeled peptoids are investigated. Both fluorophores and peptoids
are imaged at the single-molecule level in thin polymer gels. With respect to bleaching times and fluorescence
lifetimes the dye molecules and the peptoids show only slightly perturbed optical behaviors. These investigations
indicate that the new fluorophores fulfill well single-molecule microscopy and solid-phase synthesis
requirements.
1. Introduction
conjugate for a transporter needs to be highly fluorescent in
physiological media; (iv) the molecular mass (or size) should
be minimal in order to not burden the transporter; and (v) the
fluorophore needs to have a reactive functional group to allow
an easy and stable conjugation to biomolecules of interest. To
conduct experiments in living cells, it is also desirable that they
should absorb and especially fluoresce in the green or red
spectral region for diminishing the background due to the
cellular autofluorescence. Because of these stringent require-
ments, the development of new and possibly improved emitters
for single-biomolecule detection is presently an actively pursued
endeavor.8–10
Peptoids (oligo-N-alkylglycines) with positively charged side
chains are known for their ability to enable or enhance cellular
uptake therewith functioning as molecular transporters.1 Their
cargo can be either covalently linked or bound by supramo-
lecular interactions, whereas the latter ones are engineered in
order to deliver DNA or siRNA to cell nuclei in view on
nonviral gene therapies.2 Various studies show the influence of
peptoid structures on cellular uptake efficiency or intracellular
localization.3,4 For tailoring the transporters and thus for
optimization of the delivery, the exact mechanism of peptoid
internalization into cells needs to be understood. Single-molecule
imaging is a well-suited method for studying this.
Since the initial experiments on single molecules in a
biologically relevant environment,5 many groups have used
single-molecule methods for both in vitro and in vivo investiga-
tions.6 For example, Lee et al. have tracked the motion of peptide
transporters on plasma membranes and therefore obtained
detailed information about cellular uptake mechanisms.7
Fluorescent dyes have to fulfill several physical, chemical,
and biological requirements to be attractive for single-biomol-
ecule investigations: (i) They should be brilliant (i.e., display a
high molar extinction coefficient related to intense light absorp-
tion and a high fluorescence quantum yield); (ii) they need to
be particularly photostable; (iii) organic fluorophores usually
show fluorescence quenching in aqueous media, so an ideal
The majority of published single-molecule reports being
relevant to biological questions mainly addresses proteins or
DNA. Most labeling procedures of these water-soluble biomol-
ecules are carried out in aqueous solution.7,11,12 On the contrary,
smaller molecules such as peptides or peptoids are routinely
synthesized and labeled on solid supports using organic
solvents.1,3,4,13 Interestingly, within their synthetic protocols,
groups rarely use single-molecule suitable dyes.14,15 One of the
reasons is that the photophysical properties of fluorescent dyes
can be negatively affected by solid-phase synthesis (SPS).
Especially the cleavage of fluorescently labeled biomolecules
from the resin by the commonly used trifluoroacetic acid (TFA)
could be a critical step affecting the dye. Since the chemical
stability during the peptoid synthesis is a main issue, we here
report on fluorophores which satisfy the miscellaneous require-
ments of single-molecule methods and SPS.
* To whom correspondence should be addressed. Tel.: (+) 49 721 608
2902. Fax: (+) 49 721 608 8581. E-mail: braese@kit.edu.
We investigated new pyridinium derived dyes (in the fol-
lowing named as Pyr-dyes) as promising candidates. The
molecules are synthesized from pyrylium salts which Kostenko
et al. used as fluorescent dyes.16 Pyrylium salts fluoresce slightly
and are quite small labels that react under mild conditions with
various amino groups of proteins. Thereby they form in only
† IOC, KIT.
‡ LTI, KIT.
§ INT, KIT.
| CFN, KIT.
⊥ Present address: Institut de Sciences Mole´culaires de Marseille,
Chirosciences, Campus Saint Je´roˆme, Universite´ “Paul Ce´zanne” Aix-
Marseille III, Marseille, France.
10.1021/jp103308s  2010 American Chemical Society
Published on Web 10/05/2010

13474 J. Phys. Chem. B, Vol. 114, No. 42, 2010
Rudat et al.
one step positively charged N-substituted pyridinium derivates
which fluoresce strongly. This property explains the use of
pyrylium salts for several biochemical applications.17–19 Fur-
thermore, they are readily obtained with practically any substitu-
tion pattern in preparatively useful yields from cheap starting
materials.20 Thereby it is also straightforward to change the Pyr-
dyes photophysical properties by structural variations.21
Herein, we report the synthesis of new fluorescent Pyr-dyes
and their suitability for investigating labeled peptoids by single-
molecule fluorescence microscopy. Compared to previous work
using pyridinium salts,17 the present fluorophores have advanta-
geous structural variations and spacer units in order to avoid
undesirable interactions with biomolecules which usually lead
to fluorescence quenching. We then successfully labeled cell-
penetrating peptoids with these dyes on solid support, and the
photophysical properties of the Pyr-dyes and the fluorescently
labeled peptoids were determined in ensemble and single-
molecule measurements. To the best of our knowledge, this
represents the first report describing investigations of fluores-
cently labeled peptoids at a single-molecule level. In thin
poly(methyl methacrylate) (PMMA) gels, both Pyr-dyes and
peptoids could be successfully imaged at nanomolar concentra-
tions. We analyzed both bleaching times as well as fluorescence
lifetimes. The fluorophores and the peptoids showed only
slightly perturbed optical behavior which indicates that Pyr-
dyes fulfill well the requirements of single-molecule microscopy
and SPS.
considerable amounts of 2- and/or 6-styryl-substituted pyrylium
salts. Second, the pyrylium to pyridinium conversion is not
quantitative (80% at best) with anilines being the most prominent
secondary products.22 Inhomogeneous materials could thus be
obtained by coupling to peptoids. And importantly, anilines can
act as potent fluorescence quenchers as they can undergo facile
photoinduced electron transfer. Additionally, the tetrafluorobo-
rate anion is incompatible with most cell lines as its cytotoxic
activity has been proven especially when associated with ionic
liquids.23
To exploit the advantages of solid-phase synthesis,24 an
alternative synthetic strategy was developed. First the two
methyl groups in position 2 and 6 within the pyrylium salt were
replaced by two 2-propyl groups. This simple modification,
without increasing significantly the mass of the molecule, has
three evident advantages: (i) The two isopropyl groups do not
react with benzaldehydes. Thus the latter can selectively attack
the 4-methyl group forming the corresponding 4-styrylpyrylium
salt in high yields; (ii) albeit being bulkier than methyl groups
the two isopropyl groups react in excellent yields with primary
amines to afford the corresponding pyridinium salts due to their
Janus-type behavior (the Janus facet is based on what an
incoming reagent experiences: either a small methyl group or a
bulky tert-butyl group depending on which conformation of the
isopropyl group is preferred);25 (iii) the two isopropyl groups
impart an increased solubility in organic solvents used for the
SPS.
Two synthetic methods using low-cost starting materials such
as either tert-butanol, isobutyric anhydride, and hexafluoro-
phosphoric acid, or tert-butyl chloride, aluminum chloride, and
isobutyryl chloride have been described recently for the mul-
tigram synthesis of the starting pyrylium salt 1.26 This was
reacted further with either 4-N,N-dimethylaminobenzaldehyde
2 or the benzaldehyde derived from julolidine 5 to give the
pyrylium salts 3 and 6, respectively.
2. Results and Discussion
2.1. Synthesis. “Chameleon dyes” derived from styryl pyry-
lium salts have been reported.17 In this previous approach,
commercial 2,4,6-trimethylpyrylium tetrafluoroborate was re-
acted with 4-(N,N-dimethylamino)benzaldehyde to afford the
4-styryl-substituted salts. Subsequently, these compounds were
reacted with proteins where ε-amino groups of lysines for
instance directly form unspecifically the corresponding pyri-
dinium salts. For our purpose this synthetic strategy is not
suitable. First, the condensation with benzaldehyde affords
In a second synthetic step, these pyrylium salts were
converted to the corresponding N-(2-aminoethyl)pyridinium salts
4 and 7 in good yields and multigram quantities. The 2-ami-
SCHEME 1: Synthesis of Pyr-Dyes 4 and 7a
a (I) Reagents, conditions and yields: (a) ethanol, reflux, 15 min, 74%; (b) 2.7 equiv of ethylenediamine, ethanol, reflux, 2 h, 58%. (II) Reagents,
conditions and yields: (c) ethanol, reflux, 30 min, 82%; (d) 1.5 equiv of ethylendiamine, ethanol, reflux, 2 h, 94%.

Single-Molecule Imaging of Pyr-Conjugated Peptoids
J. Phys. Chem. B, Vol. 114, No. 42, 2010 13475
Figure 2. Hammett plot showing the absorption maxima in dry
dichloromethane for N-(2-hydroxyethyl)-4-styryl pyridinium salts hav-
ing various 4′-R-substituents. σp represents the Hammett substituent
constant for the para-position.
Figure 1. Fluorescently labeled peptoids after deprotection and
cleavage from the solid support.
in cells. Excitation above 500 nm is effectively possible, where
autofluorescence problems in a cell environment are suppressed.
Furthermore, the spectral adjustability of the Pyr-dyes allows a
choice of the right fluorescent molecule for a specific application
and microscope setup.
Second, local environments influenced the absorption and
fluorescence maxima of the Pyr-dyes. Changing the dielectric
environments from water toward the less polar solvent dichlo-
romethane, both dyes, 4 and 7, showed significant bathochromic
shifts (54 and 48 nm, respectively) in absorption and hypso-
chromic shifts (14 and 8 nm, respectively) in fluorescence.
Hence, the Stokes shifts in water are higher than in dichlo-
romethane and above all remarkable (164 nm for 4, 143 nm
for 7). Thereby an effective separation of fluorescence signals
from the excitation light in imaging experiments is guaranteed,
promoting thus Pyr-dyes as easily available and interesting
candidates for single-molecule experiments.
In addition to the spectral shifts a drastic increase in quantum
yield occurred in dichloromethane compared to water. This
effect can be due to inhibition of unspecific aggregation. Such
aggregation phenomena drastically reduce the fluorescence,
because of concentration quenching. It is the main reason why
“sticky” organic, hydrophobic fluorophores, which are extremely
brilliant in their monomeric form, only dully fluoresce in water.
Additionally, for related structures (e.g., 9-(2-carboxy-2-cy-
anovinyl)julolidine) a twisted intramolecular charge-transfer
(TICT) by rotation around the CdC bond is known as a
nonradiative relaxation pathway.29,30 Moreover, TICT as well
as planar ICT (PICT) processes involving the N-stilbenyl bond
are discussed.31,32 We assume that similar excited-state events
are occurring for the Pyr-dyes: After excitation, the molecule
can relax to an ICT state. This state is characterized by an
increased charge separation and is especially well-stabilized in
noethyl handle assures the reactive linking group of the
fluorophore onto peptoids being compatible with SPS protocols
and acts as short spacer. Scheme 1 details the syntheses, reaction
conditions and yields. Furthermore, the introduction of other
anchoring groups such as maleimides or ethanolamine was
possible as well.27 Hence, Pyr-dyes could label various biomol-
ecules also by thio or ester group linkages.
The synthesis and the labeling procedure as well as purifica-
tion of fluorescently labeled peptoids are based on our previously
described protocols.3,4 All steps were performed on solid
supports. Peptoids (Figure 1) were obtained in satisfying yields
and excellent purities.
2.2. Photophysical Properties in Ensemble Measurements.
We further investigated if the Pyr-dyes are suitable for studies
at the single-molecule level of biological probes, namely, cell-
penetrating peptoids. In our standard protocols we incubate cells
with these biomolecules in aqueous solution.3,4 Therefore we
initially determined the bulk photophysical properties of free
Pyr-dyes as well as of fluorescently labeled peptoids in water.
Additionally, the stationary absorption and fluorescence spectra
of the fluorophores were measured in dichloromethane. Table
1 summarizes the photophysical data.
First of all, it was evident that the auxochromic groups on
the styryl moiety of the dyes strongly influenced their spectral
properties (Figure 2). Independent of the solvent, the absorption
as well as the emission bands of Pyr-dye 4 shifted toward the
blue region (35-50 nm) in comparison to Pyr-dye 7. The same
result was observed after coupling to the peptoid structures due
to the effect of the rigid julolidine rings in Pyr-dye 7 compared
to the N,N-dimethylaniline part in Pyr-dye 4.28 This makes
especially Pyr-dye 7 a favorable candidate for measurements
TABLE 1: Summary of the Photophysical Properties of Chromophores in Free Form and of Fluorescently Labeled Peptoids
εmax (M-1cm-1
)
λmax,em (nm)
ΦF
τ (ns)
a
b
c
molecule
solvent
λmax,abs (nm)
4
CH2Cl2
H2O
H2O
H2O
CH2Cl2
H2O
498
444
460
453
545
497
501
47000 ( 1000
12600 ( 300
12000 ( 400
8600 ( 100
50000 ( 3000
23000 ( 3000
2290 ( 50
594
608
606
604
632
640
641
0.5 ( 0.01
0.0094 ( 0.0004
0.054 ( 0.005
0.045 ( 0.006
0.08 ( 0.007
0.0045 ( 0.0004
0.022 ( 0.002
0.235 ( 0.007
0.611 ( 0.005
1.03 ( 0.06
8
9
7
0.985 ( 0.008
0.29 ( 0.02
0.66 ( 0.02
0.776 ( 0.007
10
H2O
a Absorption maxima have a (1 nm imprecision. b For the peptoids the calculation is based on the molar mass of the free amines without a
counter ion. c Fluorescence maxima are reproducible within a (2 nm range.

13476 J. Phys. Chem. B, Vol. 114, No. 42, 2010
Rudat et al.
for single-molecule imaging in cells, have comparable values
in water. For example Lord et al. present dicyanomethylene-
dihydrofuran fluorophores which show quantum yields < 0.003
in water that were sufficient for single-molecule imaging within
cell membranes.9 Thus, the low quantum yields of Pyr-dyes and
their derived labeled peptoids are not necessarily an obstacle
for cellular imaging. Fluorescence lifetime curves for ensemble
measurements showed monoexponential decays (data not shown).
The fitted values were around 0.6 ns for the fluorophores and
between 0.7 and 1 ns for the peptoids and thus similar to related
structures.18
2.3. Single-Molecule Properties. For future single-molecule
tracking of fluorescently labeled peptoids in cells we investigated
Pyr-dye 7 and peptoid 10 more extensively. These molecules
fluoresce at higher wavelengths and are more advantageous for
biological experiments.
Figure 3. Normalized absorption and fluorescence spectra of Pyr-dye
7 and peptoid 10 in water.
First of all, a fluorescent dye is applicable in single-molecule
microscopy experiments if imaging is possible. Initially, judging
from their extinction coefficients and quantum yields at the
ensemble level, it was not obvious that Pyr-dyes are suitable
for such experiments. Nevertheless, our setup allowed us to
obtain single-molecule images of immobilized Pyr-dye 7
molecules as well as fluorescently labeled peptoid 10 (Figure
4A) at nanomolar concentrations (5-10 nM) in thin films of
PMMA. Fluorescence intensity traces give an indication of
single-molecule observation33 (Figure 4B). Additionally, the
resistance to enter long-lived dark states is visible in the
exemplary trace. Both the free Pyr-dye 7 and the peptoid 10
exhibited little or no blinking on the time scale of our
experiments (2-100 ms).
We recorded 166 traces of Pyr-dye 7 molecules and also of
127 molecules of peptoid 10. By fitting the histogram for the
duration of transients (Figure 5), it was possible to obtain the
mean survival times. For the free dye it was 1.87 ( 0.15 s and
for the peptoid 1.9 ( 0.3 s, respectively, the values being thus
identical within the experimental error. The observed survival
times were not as high as those reported for other fluorescent
dyes.8 However, a comparison is not unambiguously possible
as experimental setups and measurement conditions in diverse
polar solvents such as water. On the contrary, in less polar
solvents the ITC stage is less favorable. Thus, the nonradiative
relaxation pathway is reduced and the quantum yield is
increased. Such solvent/aggregation dependency can be a very
useful feature for spatially and spectrally resolved imaging of
biological probes with different polarities.
Figure 3 shows normalized absorption and fluorescence
spectra of Pyr-dye 7 and peptoid 10 in water. Slight shifts in
absorption maxima to higher wavelengths occurred after cou-
pling Pyr-dyes to peptoids. Nevertheless, in comparison with
the above-discussed shifts, these differences are negligible.
Noticeable is that the emission curves were almost identical.
Thus, the spectral properties of the Pyr-dyes were only slightly
perturbed, even after the coupling steps on solid supports.
Molar extinction coefficients in water decreased for Pyr-dyes
after coupling to peptoids. In the case of Pyr-dye 4 the effect
was moderate and values were still in the range of other single-
molecule dyes such as perylenes.12 Worth mentioning is that
the quantum yields increased by almost 1 order of magnitude
but were still lower than the quantum yields of perylenes (ΦF
in water ) 0.39-0.66).12 Nevertheless, other emitters, suitable
Figure 4. (A) Typical fluorescence image of single copies of peptoid 10 in PMMA (c ) 10 nM). (B) Fluorescence intensity as a function of time
for a single molecule. (C) Fluorescence lifetime image of the same area as in A. (D) Distribution of lifetimes from the fluorescence lifetime image
with a maximum around 2.1 ns.

Single-Molecule Imaging of Pyr-Conjugated Peptoids
J. Phys. Chem. B, Vol. 114, No. 42, 2010 13477
structures related to Pyr-dyes9 as well as other emitters such as
dicyanomethylenedihydrofuran fluorophores29,30 and can be
once again explained with reduced nonradiative relaxation
pathways. Because a CdC bond twist to the TICT state is less
favorable in rigid environments, the fluorescence quantum yield
and thus the fluorescence lifetime (which is in general propor-
tional to the quantum yield) could increase. Besides, fluoro-
phores chemically related to Pyr-dyes show such strong changes
in fluorescence lifetime (from 1.0 up to 2.4 ns) upon conjugation
to bulky proteins, a fact which was successfully used in an
affinity assay.18 This sensitivity to local environment is a useful
tool for, e.g., fluorescence lifetime imaging as well as to follow
dynamic changes even on the single-molecule level.
3. Conclusions
We presented the synthesis and spectral investigation of new
functionalized fluorescent dyes and fluorescently labeled pep-
toids. Fluorophores based on new styrylpyridinium salt structures
have a major advantage: the possibility to modify their spectral
properties by varying the p-auxochromic groups on the ben-
zaldehyde synthon. Furthermore, their remarkably high Stokes
shifts in comparison to other commonly used fluorophores allow
an effective separation from excitation sources and represent
an advantage especially for single-molecule experiments. Be-
cause of optimized synthetic protocols, the fluorescent dyes were
available in large quantities and high purity. In reaction with
anchor molecules, they formed highly fluorescent pyridinium
salts. These were chemically compatible with functional groups
in biomolecules, such as peptoids. Rapid and efficient biocon-
jugation was possible by optimized solid-phase techniques. As
notably small fluorophores, they minimally burden the conju-
gate, thus enabling efficient cargo trafficking. Due to these facts,
Pyr-dyes have a great potential as labels for peptoids and for
many other molecules, e.g., peptides, proteins, and sugars.
We presented the first single-molecule investigation of
fluorescently labeled peptoids. In PMMA gels, both pyridinium
derived dyes and peptoids were imaged at the single-molecule
level and showed similar photophysical properties with respect
to bleaching times and fluorescence lifetimes. Consequently,
Pyr-dyes were stable enough for labeling on solid supports. Both
free dyes and peptoid molecules undergo minimal blinking on
the single-molecule levelsan advantage for tracking experi-
ments. Moreover, the sensitivity to the viscosity of the environ-
ment makes them promising candidates for further analytical
investigations.
Figure 5. Distribution of the duration of transients for Pyr-dye 7 (A)
and peptoid 10 (B) molecules. Data were fitted as a monoexponential
function.
reports differ. Fluorophores, such as, e.g., the fluorescent protein
DsRed, show different survival times in different polymer
gels.34,35 Furthermore, the measured survival times of the Pyr-
dye and the labeled peptoid were on similar or even longer time
scales as residence times and dynamic events of cell-penetrating
molecules on membranes. For example Lee et al. analyzed
oligoarginine transporter conjugates on the plasma membrane
of cells which all showed residence times lower than 1.6 s.7
Ciobanasu et al. successfully tracked the cell-penetrating peptide
HIV TAT1 within biological model membranes. In this case,
analyzing the binding and mobility was possible out of movie
frames no longer than 500 ms in total.36 For this reason we
assume that the photobleaching time of peptoid 10 should be
sufficient and adequate for cellular imaging. Peptoids labeled
with Pyr-dyes could in fact already successfully be imaged in
cells in higher concentrations (c ) 10-50 µM, data not shown).
We also expect that upcoming single-molecule experiments on
living cells will give us further insight into internalization
mechanisms of peptoids.
As pulsed laser light excited the samples, fluorescence lifetime
images could also be obtained. Figure 4C shows the same area
as Figure 4A, but with lifetime information. Values were plotted
against their frequency and fitted by a Gaussian (Figure 4D).
The measured fluorescence lifetime was 2.1 ( 0.1 ns for Pyr-
dye 7 and 2.0 ( 0.1 ns for peptoid 10, again identical values
within the error margins. This and the identical values for
photobleaching statistics show an important fact: in single-
molecule experiments, the fluorescence behavior of the dyes is
almost independent of the conjugated peptoid. Additionally, the
Pyr-dyes were stable enough to overcome SPS procedures.
Fluorescence lifetimes for both, Pyr-dye 7 and peptoid 10
were longer than those in solution, what might be due to the
change in viscosity. Such sensitivity is known for chemical
We thus assume that the new fluorophores are promising
candidates for upcoming single-molecule tracking experiments.
Future work will address the single-molecule analysis of
peptoids in aqueous polymers and the investigation of hydro-
dynamic radii for Pyr-dyes and fluorescently labeled peptoids.
Single-molecule experiments will give further insight into
internalization mechanisms of peptoids into cells and will allow
one to fine-tune their cargo delivering ability.
4. Experimental Methods
4.1. Synthetic Procedures. All reagents were purchased from
commercial sources and used without further purification.
Solvents of technical quality were distilled before using; solvents
with the quality per analysis (p.a.) were purchased from
commercial sources. Dichloromethane was dried over CaCl2,
refluxed over CaH2, and distilled. For microwave assisted
syntheses the single-mode CEM Discover microwave was used.
Melting points were recorded with Stuart melting point
apparatus (Bibby Sterilin). Routine NMR spectra were measured

13478 J. Phys. Chem. B, Vol. 114, No. 42, 2010
Rudat et al.
1
with a Bruker DPX 300 Avance spectrometer at 300 MHz and
a Bruker AM 400 spectrometer at 400 MHz. All spectra are
referenced to the respective solvent signals (δ ) 0 ppm):
yield). Mp 215 - 217 °C. H NMR (300 MHz, acetone-d6): δ
7.94 (s, 2 H, 3-, 5-CH), 7.60 (d, J ) 8.8 Hz, 2 H, 19-, 23-CH),
7.16 (d, J ) 16.2 Hz, 2 H, 16-, 17-CH), 6.78 (d, J ) 8.8 Hz,
1 H, 20-, 22-CH), 4.97 (t, J ) 5.9 Hz, 2 H, 7-CH2), 4.04 (sept,
J ) 6.7 Hz, 2 H, 10-, 13-CH), 3.84 (t, J ) 5.8 Hz, 2 H, 8-CH2),
3.05 (s, 6 H, 25-, 26-CH3), 2.80 (s, 2H, 9-NH2), 1.46 (d, J )
6.8 Hz, 12 H, 11-, 12-, 14-, 15-CH3). 13C NMR (300 MHz,
acetone-d6): δ 165.7 (2-, 6-C), 155.6 (21-C), 154.0 (4-C), 143.4
(17-C), 131.3 (19-, 23-C), 124.7 (16-C), 120.3 (3-, 5-C), 119.0
(18-C), 113.7 (20-, 22-C), 52.7 (7-C), 51.9 (8-C), 41.0 (25-,
26-C), 32.8 (10-, 13-C), 23.7 (11-, 12-, 14-, 15-C); IR (KBr):
ν ) 3404 (vw), 2977 (w), 2944 (w), 2807 (w), 1588 (m), 1530
(m), 1369 (m), 1192 (m), 1162 (m), 843 (s), 558 cm-1 (m).
UV/vis (CH2Cl2): λmax (log εmax) ) 498 (4.67), 286 (2.43), 254
nm (2.36). FAB-MS (m/z): 369.2 (100) [Matrix], 352.4 [M+],
309.4 [M+ + H - C2H6N•]. HRMS [ESI]: [M + H]+ calcd for
C23H34N3, 352.2753; found, 352.2748.
1
chloroform-d1 with 7.26 ppm for H NMR and 77.0 ppm for
1
13C NMR, acetone-d6 with 2.05 ppm for H NMR ppm and
206.0 ppm for 13C NMR. Multiplicities of signals are described
as follows: s ) singlet, d ) doublet, t ) triplet, sept ) septet,
and m ) multiplet. Coupling constants (J) are given in hertz.
FT-IR spectra were measured on IFS 88 spectrometer (Bruker).
Intensities of transmission (T) are described as follows: vs, very
strong (0-10% T); s, strong (10-40% T); m, mean (40-70%
T); w, weak (70-90% T); vw, very weak (90-100% T). Matrix
assisted laser desorption/ionization time of flight mass spectra
(MALDI-TOF-MS) of the Pyr-dyes were recorded with Per-
Septive Biosystems Voyager-DE Pro with 100 laser pulses and
an acceleration voltage of 20 kV in isovanillin (3-hydroxy-4-
methoxybenzaldehyde) as matrix. For the peptoids we used
Bruker Biflex IV with a nitrogen laser (λ ) 337 nm) and
FlexControl version 1.1 and XMASS-XTOF version 5.1.1 as
software. Each sample was shot 100-300 times with a repetition
rate of 1-3 Hz. We used Bruker standard targets of aluminum
386 “spots” and 2,5-dihydroxybenzoic acid (DHB) 50% aceto-
nitrile and 0.1% TFA in water as matrix. Fast atom bombard-
ment mass spectra (FAB-MS) were measured with MAT 95
(Finnigan). Elemental analyses were recorded on the CHN rapid
instrument from Elementar.
4.3. Synthesis of the Julolidine Derived Dye. Samples of
2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline (julolidine)
and 2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-car-
baldehyde (5) were synthesized according to literature.37
2,6-Diisopropyl(pyrylium-4-yl)Winyl[2,3,6,7-tetrahydro-1H,5H-
pyrido[3,2,1-ij]quinoline] Hexafluorophosphate (6). 2,6-Di-
isopropyl-4-methylpyrylium hexafluorophosphate (1, 2.00 g,
6.17 mmol, 1.00 equiv) was dissolved in ethanol (100 mL) and
treated in an ultrasonic bath. A saturated solution of julolidine-
carbaldehyde 5 (1.24 g, 6.17 mmol, 1.00 equiv) in ethanol
(approximately 60 mL) was added, and the color changed from
green to blue. The mixture was stirred for 30 min under reflux.
After cooling to room temperature the precipiate was filtered
off, washed with a little amount of diethyl ether, and dried in
vacuo. The product was recrystallized from ethanol to give a
4.2. Synthesis of the 2,6-Diisopropylpyridinium Derived
Dye. 2,6-Diisopropyl(pyrylium-4-yl)Winyl-N,N-dimethylbenz-
amine Hexafluorophosphate (3).25 N,N-Dimethylaminobenzal-
dehyde (2; 0.69 g, 4.63 mmol, 1.50 equiv) and 2,6-diisopropyl-
4-methylpyrylium hexafluorophosphate (1; 1.00 g, 3.09 mmol,
1.00 equiv) were dissolved in ethanol (70 mL) and stirred for
15 min under reflux. The blue mixture was then allowed to cool
to room temperature. The precipitate was filtered and dried in
vacuo. The product was recrystallized from 2-propanol (1.04
g, 74% yield). Mp 190-193 °C. 1H NMR (300 MHz, chloroform-
d1): δ 8.12 (d, J ) 15.2 Hz, 1 H, 13-CH), 7.73 (d, J ) 8.8 Hz,
2 H, 16-, 20-CH), 7.31 (s, 2 H, 3-, 5-CH), 6.91 (d, J ) 15.2
Hz, 1 H, 14-CH), 6.60 (d, J ) 9.1 Hz, 2 H, 17-, 19-CH), 3.11
(s, 6 H, 22-, 23-CH3, 1.35 (d, J ) 6.9 Hz, 12 H, 8-, 9-, 11-,
12-CH3). 13C NMR (300 MHz, chloroform-d1): δ 177.0 (2-,
6-C), 160.8 (4-C), 154.6 (18-C), 152.8 (13-, 14-C), 134.5 (16-,
20-C), 122.9 (15-C), 112.3 (17-, 19-C), 111.3 (3-, 5-C), 39.9
(22-, 23-C), 33.3 (7-, 10-C), 19.8 (8-, 9-, 11-, 12-C). IR (KBr):
ν ) 2977 (w), 2939 (w), 1655 (m), 1617 (m), 1533 (m), 1374
(m), 1334 (m), 1274 (m), 1172 (m), 937 (m), 839 cm-1 (m).
UV/vis (CH2Cl2): λmax (log εmax) ) 603 (3.12), 297 nm (2.63).
FAB-MS (m/z): 455.3 [M+ + PF6-], 310.4 [M+] (100), 191.5
[M+ + H - C8H10N·]. HRMS [ESI]: [M + H]+ calcd for
C21H28NO, 310.2213; found, 310.2220.
1
blue powder (1.16 g, 82% yield). Mp 208-210 °C. H NMR
(400 MHz, acetone-d6): δ 8.26 (d, J ) 14.9 Hz, 1 H, 15-CH),
7.25-7.58 (m, 11-, 13-, 17-, 21-CH), 7.04 (d, J ) 14.9 Hz, 1
H, 14-CH), 3.55 (t, 4 H, 2-, 10-CH2), 3.15 (sept, J ) 6.9 Hz, 2
H, 22-, 25-CH), 2.79 (t, J ) 6.2 Hz, 4 H, 4-, 8-CH2, 2.02-1.96
(m, 4 H, 3-, 9-CH2), 1.38 (d, J ) 6.9 Hz, 12 H, 23-, 24-, 26-,
27-CH3). 13C NMR (400 MHz, acetone-d6): δ 177.5 (18-, 20-
C), 160.6 (16-C), 153.6 (15-C), 152.6 (6-C), 134.4 (11-, 13-,
17-, 21-C), 133.8 (12-C), 124.9 (5-, 7-C), 115.8 (14-C), 52.5
(2-, 10-C), 35.2 (22-, 25-C), 29.0 (4-, 8-C), 22.5 (3-, 9-C), 21.3
(23-, 24-, 26-, 27-C). IR (KBr): ν ) 3428 (vw), 2975 (m), 2941
(m), 2874 (w), 1650 (m), 1557 (s), 1522 (s), 1435 (m), 1276
(s), 1252 (s), 1166 (s), 1079 (m), 936 (m), 840 (s), 558 cm-1
(m). UV/vis (CH2Cl2): λmax (log εmax) ) 646 (3.18), 299 nm
(2.21). FAB-MS (m/z): 508.3 [M+ + H+ + PF6-], 507.3 [M+
+ PF6-], 362.3 [M+] (100). HRMS [ESI]: [M + H]+ calcd for
C25H32NO, 362.2526; found, 362.2524.
[1-Ethylamine(2,6-disopropyl(pyridinium-4-yl)]Winyl[2,3,6,7-
tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline] Hexafluorophos-
phate (7). Pyrylium salt 6 (1.00 g, 1.97 mmol, 1.00 equiv) was
dissolved in ethanol (80 mL) under nitrogen atmosphere and
was heated to reflux. Then ethylendiamine (0.18 g, 0.20 mL,
2.99 mmol, 1.50 equiv) was added quickly, and the blue solution
changed its color to red. The mixture was stirred for 2 h under
reflux, and after cooling to room temperature the solvent was
removed under reduced pressure. The precipitate was taken up
in a small amount of dichloromethane and added to a mixture
of n-hexane and diethyl ether (2:1). The product precipitated
as a red solid and was recrystallized from ethanol and dried in
[1-Ethylamino(2,6-diisopropylpyridinium-4-yl)]Winyl-N,N-
dimethylbenzamine Hexafluorophosphate (4). Pyrylium salt (3;
0.50 g, 1.10 mmol, 1.00 equiv) was dissolved in ethanol (15
mL) under nitrogen atmosphere and heated to reflux. Then
ethylendiamine (180 mg, 0.20 mL, 3.00 mmol, 2.7 equiv) was
added fastly, whereas the blue solution changed its color to red.
The mixture was stirred for 2 h under reflux. After cooling to
room temperature the solvent was removed under reduced
pressure. The precipitate was taken up in a small amount of
dichloromethane and added to a mixture of n-hexane and
diethylether (2:1). The product precipitated as a red solid, it
was filtered, washed with a small amount of n-hexane, and dried
under reduced pressure. It was recrystallized from ethanol and
the product was obtained as an orange powder (320 mg, 58%
1
vacuo (1.02 g, 94% yield). Mp 158 - 160 °C. H NMR (400
MHz, acetone-d6): δ 7.88 (s, 2 H, 17-, 21-CH), 7.83 (d, J )
16.0 Hz, 1 H, 15-CH), 7.13 (s, 2 H, 11-, 13-CH), 7.04 (d, J )

Single-Molecule Imaging of Pyr-Conjugated Peptoids
J. Phys. Chem. B, Vol. 114, No. 42, 2010 13479
16.0 Hz, 1 H, 14-CH), 4.95 (t, J ) 5.9 Hz, 2 H, 22-CH2), 3.90
(sept, J ) 6.6 Hz, 2 H, 25-, 28-CH), 3.74 (t, J ) 5.9 Hz, 2 H,
23-CH2), 3.28 (t, J ) 6.2 Hz, 4 H, 2-, 10-CH2), 2.72 (t, J ) 6.3
Hz, 4 H, 4-, 8-CH), 1.90-1.97 (m, 4 H, 3-, 9-CH2), 1.45 (d, J
the cleavage solution was colorless. Water was added, and the
mixture was lyophilized to give the crude peptoid 9. After HPLC
purification (method 1, Rt ) 22.6 min) 1.44 mg of the product
was obtained with a purity of 93%. MALDI-TOF-MS [m/z]:
[M]+ calcd for C79H135N16O8+, 1437.02 (average mass); found,
1437.1.
Peptoid 10. A solution of 7 (52.8 mg, 96.0 µmol, 3.00 equiv)
in DMF (1.00 mL), HOBt ·H2O (14.7 mg, 96.0 µmol, 3.00
equiv), and DIC (24.5 µL, 20.0 mg, 96.0 µmol, 3.00 equiv)
was added to the resin synthesized as described above (32.0
µmol, 1.00 equiv). From here on the same procedure was used
as reported for peptoid 9 to give the crude peptoid 10. After
HPLC purification (method 2, Rt ) 33.2 min) 2.39 mg of the
product was obtained with a purity of 91%. MALDI-TOF-MS
[m/z]: [M + H]+ calcd for C83H139N16O8+, 1489.09 (average
mass); found, 1490.3.
) 6.6 Hz, 12 H, 26-, 27-, 29-, 30-CH3), 0.94 (t, 2 H, NH2). 13
C
NMR (300 MHz, acetone-d6): δ 165.3 (18-, 20-C), 155.7 (16-
C), 147.1 (6-C), 143.9 (14-C), 129.6 (11-, 13-C), 123.7 (12-C),
122.8 (14-C), 119.8 (17-, 21-C), 117.6 (5-, 7-C), 52.8 (22-C),
51.7 (2-, 10-C), 37.5 (23-C), 32.7 (4-, 8-C), 29.1 (25-, 28-C),
23.7 (26-, 27-, 29-, 30-C), 23.0 (3-, 9-C). IR (KBr): ν ) 3591
(w), 2945 (w), 2843 (w), 1582 (m), 1524 (w), 1315 (m), 1259
(w), 1158 (w), 842 (m), 559 (w), 1257 (m), 1155 (m), 1126
(m), 1096 (m), 977 (m), 840 (s), 558 cm-1 (m). UV/vis
(CH2Cl2): λmax (log εmax) ) 544 nm (4.70). FAB-MS (m/z): 549.3
[M+ + PF6], 518.4 (100) [M+ + H+ + PF6 - CH4N], 458.4
(100), 404.3 [M+], 360.3 [M+ - C2H6N]. HRMS [ESI]: [M +
H]+ calcd for C27H38N3, 404.3107; found, 404.3112.
Peptoid 8. A solution of 4 (47.8 mg, 96.0 µmol, 3.00 equiv)
in DMF (1.00 mL), HOBt ·H2O (14.7 mg, 96.0 µmol, 3.00
equiv), and DIC (24.5 µL, 20.0 mg, 96.0 µmol, 3.00 equiv)
was added to the resin synthesized as described above (32.0
µmol, 1.00 equiv). From here on the same procedure was used
as reported above to give again the crude peptoid 9. A solution
of 1H-pyrazole-1-carboxamidine hydrochloride (128 mg, 870
µmol, 20.0 equiv) in N,N-dimethylacetamide (DMA; 3.00 mL)
and N,N-diisopropyl-N-ethylamine (DIPEA; 303 µL, 225 mg,
1.74 mmol, 40.0 equiv) was added to the peptoid 9. The reaction
vessel was subjected to microwave irradiation to keep the
constant temperature at 60 °C for 120 min while being stirred.
Water was added and the mixture was lyophilized to give
peptoid 8. After HPLC purification (method 3, Rt ) 19.1 min)
3.19 mg of the product was obtained with a purity of 99%.
4.4. Synthesis of the Peptoid Conjugates. Construction of
the Peptoids:38 The amino-functionalized Rink amide resin (200
mg, 128 µmol, 1.00 equiv) was covered with 5 mL of N,N-
dimethylformamide (DMF) and swollen for 60 min at room
temperature. The resin was washed with DMF and treated with
20% piperidine in DMF (3 × 5 min with 3 mL each), and
thoroughly washed with DMF. A solution of N-(8-(tert-
butoxycarbonylamino)hexyl)-N-(9H-fluorene-9-ylmethoxycar-
bonyl)amino acetic acid (191 mg, 384 µmol, 3.00 equiv,
synthesized according to published methods with slight modi-
fications3) in DMF (3.80 mL), 1-hydroxybenzotriazole hydrate
(HOBt·H2O; 58.9 mg, 384 µmol, 3.00 equiv), and N,N′-
diisopropylcarbodiimide (DIC; 97.8 µL, 79.7 mg, 384 µmol,
3.00 equiv) was added to the preswollen resin (128 µmol, 1.00
equiv). The reaction vessel was subjected to microwave irradia-
tion to keep the temperature at 60 °C for 30 min while being
stirred. The reaction solution was filtered and the resin treated
a second time with freshly prepared reaction solution using the
above-mentioned conditions (double coupling). The resin was
washed with DMF (3 × 5 mL each). This procedure was
repeated five times to obtain the yellowish resin bound hexamer.
To a solution of terephthalic acid (63.8 mg, 384 µmol, 3.00
equiv) in DMF (3.80 mL) were added HOBt ·H2O (118 mg,
768 µmol, 6.00 equiv) and DIC (100 µL, 81.5 mg, 588 µmol,
4.59 equiv) before being added to the resin (128 µmol, 1.00
equiv). The reaction vessel was subjected to microwave irradia-
tion to keep the constant temperature at 60 °C for 30 min while
being stirred. The reaction solution was filtered off and the resin
treated a second time with freshly prepared reaction solution
using the above-mentioned conditions (double coupling). The
resin was washed with DMF (3 × 5 mL each). The resin was
split into four (128 µmol/4 ) 32 µmol) portions.
Labeling of the Peptoids. Peptoid 9. A solution of 4 (47.8
mg, 96.0 µmol, 3.00 equiv) in DMF (1.00 mL), HOBt ·H2O
(14.7 mg, 96.0 µmol, 3.00 equiv), and DIC (24.5 µL, 20.0 mg,
96.0 µmol, 3.00 equiv) was added to the resin synthesized as
described above (32.0 µmol, 1.00 equiv). The reaction vessel
was subjected to microwave irradiation to keep the temperature
at 60 °C for 30 min while being stirred. The reaction solution
was filtered off and the resin treated a second time with freshly
prepared reaction solution using the above-mentioned conditions
(double coupling). The resin was washed with DMF (3 × 5
mL each). To cleave the peptoid from solid support, a solution
of 1 mL of TFA in dichloromethane (50:50) was added to the
resin and left for 2 h at room temperature. The resin was rinsed
two times with 0.5 mL of methanol. This procedure of adding
cleavage solution and rinsing with methanol was repeated until
+
MALDI-TOF-MS [m/z]: [M]+ calcd for C85H147N28O8 , 1689.26
(average mass); found, 1688.6.
HPLC Purification. Reverse-phase analytical HPLC was
performed using Agilent Series 1100, employing a C18 column
PerfectSil Target (MZ-Analytik, 3-5 µm, 4.0 × 250 mm).
Reverse-phase semipreparative HPLC was performed using Agilent
Series 1200, using a C8 Zorbax 300SB-C8 column (Agilent, 5 µm,
9.4 × 250 mm). Flow rate: 1.5 mL; A, 0.1% TFA (trifluoroacetic
acid) in H2O; B, 0.1% TFA in acetonitrile.
Method 1 (peptoid 9): 25% B to 55% B over 30 min at 15
°C, Rt ) 22.6 min.
Method 2 (peptoid 10): 5% B to 77% B over 38 min at 50
°C, Rt ) 33.2 min.
Method 3 (peptoid 8): 25% B to 65% B over 20 min at 60
°C, Rt ) 19.1 min.
Separation of the peptoids was monitored with UV-detection
in the range of 200-650 nm, and UV spectra along with
MALDI-TOF-mass spectrometry were used to identify the
product peaks. Manually collected fractions of the semiprepara-
tive runs were freeze-dried and immediately used in the assays.
Prior to lyophilization, fraction aliquots were directly reinjected
onto the analytical column to quantify purity, which was
determined by integration of the respective single peak area from
the chromatograms at 218 nm.
4.5. Bulk Experiments. All bulk solution UV/vis spectra
were recorded with a Cary 500 scan (Varian) or Lambda 5 UV/
vis spectrometer in 1 cm cuvettes. All bulk solution fluorescence
spectra were obtained using quartz cuvettes in a Cary Eclipse
fluorescence spectrometer (Varian). Quantum yields were measured
at room temperature against a perylene (N-(2,6-diisopropylphenyl)-
N′-[3-(N-succinimidyl)carboxyethyl]-1,6,7,12-tetra(4-sulfophenox-
y)perylene-3,4:9,10-tetracarboxydiimide) with known quantum
yield.12 Fluorescence lifetimes were measured on samples prepared

13480 J. Phys. Chem. B, Vol. 114, No. 42, 2010
Rudat et al.
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Versa, 80 MHz, Koheras) was used. The laser light passed bandpass
filters to selectively illuminate the samples with light in the range
of 510-560 nm (Filter set 14, Zeiss). The emission light passed
an objective (40×, 0.6 NA, Zeiss), a dichroic mirror, and a filter
to remove scattered excitation light. It was detected by single-
photon avalanche photodiodes (APD; SPCM-AQRH, PerkinElm-
er). Acquisition was done using a time correlated single-photon
counting (TCSPC) PC card (SPC 830, Becker & Hickl GmbH).
Signals were recorded in first in, first out (FIFO) mode, where time
intervals of each photon with respect to the excitation pulse as well
as the time lag with respect to the previously detected photon were
recorded. The decay curves were fitted exponentially.
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Glass coverslips (170 µm thickness) were cleaned to remove
sources of fluorescence. They were baked for 8 h at 510 °C
followed by a cleaning procedure: five parts deionized water and
one part 27% ammonia solution (Carl Roth) were heated to 70 (
5 °C. The solution was removed from the hot plate, and one part
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spectroscopy measurements were prepared in pure solvents.
Samples in poly(methyl methacrylate) (PMMA; AR-P649.04, MW
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Measurement Procedure. Samples were studied using the
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Acknowledgment. This work has been partially supported by
the Center for Functional Nanostructures (CFN) of the German
Research Council (Deutsche Forschungsgemeinschaft, DFG) through
Projects C3.2, C3.5, and E1.1, the Karlsruhe School of Optics and
Photonics (KSOP) at the Karlsruhe Institute of Technology (KIT,
fellowship to B.R.) and the German Business Foundation (Stiftung
der Deutschen Wirtschaft, fellowship to E.B.). The skillful experi-
mental assistance of Danny Wagner, Natalie Ka¨ser, and Katharina
Go¨tz is acknowledged just as much as the assistance of Karolin
Niessner and Daniel Biedermann during the HPLC separations and
purifications. Finally, we gratefully acknowledge Prof. Klaus
Mu¨llen (Mainz) who kindly provided the perylene dye, while Prof.
Alexandru T. Balaban (Galveston, TX) provided valuable advice.
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