Stereochemistry-Controlled Supramolecular Architectures of New Tetrahydroxy-Functionalised Amphiphilic Carbocyanine Dyes

Article abstract of DOI:10.1002/chem.201905745

The syntheses of novel amphiphilic 5,5′,6,6′-tetrachlorobenzimidacarbocyanine (TBC) dye derivatives with aminopropanediol head groups, which only differ in stereochemistry (chiral enantiomers, meso form and conformer), are reported. For the achiral meso form, a new synthetic route towards asymmetric cyanine dyes was established. All compounds form J aggregates in water, the optical properties of which were characterised by means of spectroscopic methods. The supramolecular structure of the aggregates is investigated by means of cryo-transmission electron microscopy, cryo-electron tomography and AFM, revealing extended sheet-like aggregates for chiral enantiomers and nanotubes for the mesomer, respectively, whereas the conformer forms predominately needle-like crystals. The experiments demonstrate that the aggregation behaviour of compounds can be controlled solely by head group stereochemistry, which in the case of enantiomers enables the formation of extended hydrogen-bond chains by the hydroxyl functionalities. In case of the achiral meso form, however, such chains turned out to be sterically excluded.

Full text of DOI:10.1002/chem.201905745

A Journal of
Accepted Article
Title: Stereochemistry controlled supramolecular architectures of novel
tetrahydroxy functionalized amphiphilic carbocyanine dye
Authors: Boris Schade, Abhishek Singh, Virginia Wycisk, Jose Luis
Cuellar-Camacho, Hans v. Berlepsch, Rainer Haag, and
Christoph Böttcher
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Stereochemistry controlled supramolecular
architectures of novel tetrahydroxy function-
alized amphiphilic carbocyanine dyes
Boris Schade†,1 Abhishek Kumar Singh†*,2 Virginia Wycisk,2 Jose Luis Cuellar-Camacho,2 Hans
v. Berlepsch,1 Rainer Haag,2 and Christoph Böttcher*1
I.
Abstract
The synthesis of novel amphiphilic 5,5',6,6'-tetrachlorobenzimidacarbocyanine (TBC) dye
derivatives with amino-propanediol head groups is reported, which only differ in
stereochemistry (chiral enantiomers, meso-form, and conformer). For the achiral meso-form,
a new synthetic route towards asymmetric cyanine dyes was established. All compounds form
J-aggregates in water whose optical properties were characterized by spectroscopic methods.
The supramolecular structure of the aggregates was investigated by cryo-transmission
electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET), and atomic force
microscopy (AFM) revealing extended sheet-like aggregates for the chiral enantiomers and
nanotubes for the mesomer, respectively, while the conformer forms predominately needle-
like crystals. The experiments demonstrate that the aggregation behavior of compounds can
be controlled solely by head group stereochemistry which in case of the enantiomers enables
formation of extended hydrogen bond chains by the hydroxyl functionalities. In case of the
achiral meso-form, however, such chains turned out to be sterically excluded.
II.
Introduction
Cyanine dyes represent a class of organic dyes that are able to self-assemble in polar solvents
and on solid surfaces.[1] Strong dipole-dipole coupling between the dye monomers leads to an
electronically excited state shared by several monomers. Such exciton states cause a dramatic
change in optical properties of the dye assemblies as compared to the isolated molecules.[2]
Depending on the relative orientation between neighbouring monomers, different types of
assemblies can be observed. A so-called “head-to-tail” configuration of transition dipoles
leads to a red-shifted absorption band with large absorption cross section and
superradiance.[2] Such assemblies are called J- or Scheibe-aggregates, named after their
discoverers E. E. Jelly[1a] and G. Scheibe.[1b] The exceptional optical characteristics make J-
1
Forschungszentrum für Elektronenmikroskopie und Gerätezentrum BioSupraMol, Institut für Chemie und
Biochemie, Freie Universität Berlin, Fabeckstraße 36a, 14195 Berlin, Germany
2
Institut für Chemie und Biochemie, Organische Chemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin,
Germany
† These authors contributed equally to this work
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aggregates interesting tools for a lot of applications ranging from dye-sensitized silver halide
photography, light harvesting, photovoltaics, sensing, to biomedical imaging.[3]
Aggregation in polar solvents is a common feature of dye molecules containing extended pla-
nar π-electron systems. Further competing non-covalent interactions such as hydrogen
bonding, halogen bonding or solvophobic forces support the aggregation and control the
formation of complex supramolecular structures. Since dipole-dipole coupling strength and
thus photophysical properties of the aggregates are highly sensitive to the particular
arrangement of the chromophores, controlled structuring of dye assemblies by tuning
supramolecular interactions between individual building blocks facilitates the design of novel
materials with desired properties. Powerful approaches in this regard are specific alterations
of the chromophore side groups, incorporation of additives, or modifications of the solvent
polarity. In this context a class of derivatives of the well-known 5,5',6,6'-tetrachlorobenzimida-
carbocyanine chromophore (TBC)[4] has been investigated in detail by several groups.[5] The
chromophore was functionalized by attaching different polar or nonpolar substituents at the
nitrogen atoms in 1,1’ and 3,3’ position. This allows for the tailored design of a large variety
of supramolecular structures with interesting optical characteristics. By introducing 1,1’-
dioctyl substituents, Dähne and co-workers advanced a class of amphiphilic dyes.[5a]
A fundamental prerequisite for studying dye assemblies is the availability of appropriate
characterization methods. Cryogenic transmission electron microscopy (cryo-TEM) turned out
to be an excellent technique to elucidate their morphology in the native environment of the
solvent on the nanometre to micrometre scale.[6] Depending on the substituents of the TBC-
chromophore one-dimensional fibres, two-dimensionally extended sheet-like aggregates, and
single- or multi-layered tubular architectures with a helical molecular organisation were
detected. The latter became a matter of particular interest as shape and size of such tubular
aggregates resemble the natural light-harvesting system in green sulphur bacteria.[7]
Thorough optical characterizations by linear optical spectroscopy, usefully supplemented by
nonlinear techniques[8] or theoretical structure modelling[5e, 9] are mandatory for a prospective
use of photophysical characteristics. Although understanding of dye assemblies has made
excellent progress over the last years, a substantial challenge remains: how does a particular
molecular modification control the morphological and thus optical characteristics of the final
supramolecular structure? So far, empirical approaches are still necessary due to a missing
conclusive concept.
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Table 1:
Abbreviations for TBC based dyes
Z
n
m
TDBC (C2O4)
C8S3
-SO3
-SO3
1
4
7
7
3
3
C8O3
-COOH
The most extensively investigated representatives of tube forming TBC based cyanine dyes
are amphiphilic C8O3 bearing two carboxylic acid groups and C8S3 bearing two sulfonic acid
groups.[5a, 10] (For abbreviations of TBC based dyes see Table 1) In aqueous media they are
converted into their conjugate bases yielding negatively charged aggregate surfaces.
Moreover, previous studies had revealed that the helicity of C8O3 tubes could be tuned upon
addition of chiral alcohols.[11]
The goal of the present study was to prevent surface charge effects and to investigate the
impact of chiral head groups on the supramolecular architecture of assemblies.
Scheme 1: Chemical structure of parent dye C8O3 and the family of newly synthesized amino-propanediol
isomers.
To achieve this goal, we selected C8O3 as parent TBC-derivative and functionalized both its
carboxyl groups with amino-propanediol which provided a well-balanced amphiphilic charac-
ter to allow for the formation of aggregates. Since there exist two conformations of amino-
propanediol, chiral 1-amino-2,3-propanediol and non-chiral 2-amino-1,3-propanediol,
3 (34)
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amidation of the two symmetrically situated carboxyl groups in C8O3 renders the formation
of four different isomers possible, i.e., two enantiomers with either R,R or S,S configuration, a
meso-form with R,S configuration and the non-chiral conformer derived from 1-amino-2,3-
propanediol (Scheme 1). Hence, our approach allowed for versatile molecular alterations
while the hydrophilic-lipophilic-balance (HLB) of the dyes and the spatial demand of the head
groups remain unchanged. Moreover, it enables studies on the impact of the molecular
chirality. With this structural diversity, we were able to elucidate the specific influence of head
group stereochemistry and/or conformation on the aggregation behaviour of the novel
amphiphilic cyanine dyes.
The studies of the C8O3 and C8S3 derivatives were used as guidelines for the present
investigations and provided the basis for comparisons. The new derivatives were first
characterized as monomers in organic solvents by absorption and fluorescence spectroscopy.
Subsequently, aggregation in pure water was followed by absorption-, circular dichroism (CD),
linear dichroism (LD), and fluorescence spectroscopy and structurally monitored by cryo-TEM,
cryogenic electron tomography (cryo-ET) and atomic force microscopy (AFM). Structure
models are presented and discussed.
III.
Results and Discussion
Synthesis
Scheme 2: Synthesis of cyanine dye tetrahydroxy derivatives. i) PPh3, phthalimide, DEAD, THF, r.t., 20 h; ii)
NH2-NH2, H2O, MeOH, reflux, 4-5 h; iii) HATU, DIPEA, DMF, r.t., 2 h; iv) HCl, MeOH, r.t., 2-5 h.
4 (34)
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Synthesis of amino-propanediol cyanine dyes (compounds 1a, 1b, 1c and 2) has been achieved
by two different synthetic approaches. A straight forward synthetic route was followed to
obtain the isomers with uniform head groups, i.e. the R,R or S,S enantiomer from 3-amino-
1,2-propanediol (Scheme 2), and the non-chiral symmetric serinol derivative (Scheme SI 4). To
achieve and preserve the desired stereo chemistry and conformation of the chiral head
groups, commercially available enantiopure solketal, i.e. the acetal of 1,2,3-propane diol, was
chosen as starting material. This solketal (R or S) was converted into the respective solketal
amine 4 in two steps (Scheme 2) and was then coupled to the acid groups of C8O3, followed
by deprotection of the acetal groups under mild acidic conditions. The achiral serinol
derivative 2 was synthesized accordingly using commercially available 2-amino-1,3-
propanediol (serinol) without protection of the OH-groups following the same coupling
procedure (Schemes SI 1-3).
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Scheme 3: Synthesis of asymmetrical benzimidazoles; i) Ethyl-bromo acetatae, NaOH, DMSO, r.t., 48 h; ii) 1-
bromodecane, 150°C, 6 h; iii) HBr (48%), water, 120°C; iv) KOH, ethanol, reflux, 12 h; v) EDC.HCl,
DMAP, DMF, 24 h, rt; vi) 1-bromodecane, 150°C; vii) DBU, CH3I, methanol, r.t., 48 h; viii) R-solketal,
EDC.HCl, DMAP, DMF, 24 h, r.t.; ix) HCl, methanol.. (see the complete synthesis Scheme in SI)
Despite this convenient synthetic route above, a different strategy was necessary to synthe-
size the enantiopure meso-form (1c, Scheme 3). This mirror-symmetrical dye derivative was
synthesized from two different monomers to attain its specific R,S conformation selectively.
For that, 5,6-dichloro-2-methylbenzimidazole was subsequently treated with (i) ethyl 4-
bromobutanoate and (ii) 1-bromo octane followed by hydrolysis in the presence of aq. HBr
(iii) to yield benzimidazole 8. The benzimidazole 11 was obtained by ester hydrolysis of 6 (iv),
amidation with S-solketal amine (v), and finally N-alkylation with 1-bromo octane (Scheme 3).
Both benzimidazoles 8 and 11 were coupled in methanolic solution in the presence of DBU
and CH3I. As expected, three different combinations of these two monomers led to the
formation of three different dyes (Scheme SI 3). From their polarity order, however, it was
possible to extract the desired cyanine dye 12 by performing column chromatography.
Amidation with R-solketal amine and following acetal deprotection finally yielded the
mesomer (1c).
Spectroscopic characterization
Figure 1:
Normalized absorption (blue) and fluorescence spectra, excitation (green) and emission (red ) of
the new amino-propanediol R-enantiomer (1a) in methanol. The spectra are representative for all
four compounds 1a-c and 2. Concentrations are 0.1 mM for UV and 0.01 mM for fluorescence
measurements. The excitation spectrum was collected at 550 nm; the emission spectrum was
collected after excitation at 480 nm.
The amino-propanediol dyes (1a-c and 2) are readily soluble in MeOH and DMSO. In MeOH
they all show an identical absorption band peaked at 520 nm (fwhm = 932 cm-1) with a
vibronic shoulder at around 485 nm (see Fig. 1 for the R-enantiomer (1a)). The fluorescence
emission spectrum reveals a mirror image of the absorption band with a maximum at 544 nm,
yielding a Stokes-shift of 24 nm. The corresponding excitation spectrum resembles the
absorption spectrum. The spectra are in good agreement with those of the parent derivative
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C8O3, indicating that the introduction of the non-ionic amino-propanediol head groups with
their particular chirality does not affect the spectroscopic properties of the chromophore. The
spectroscopic features are typical for cyanine dye monomers.[12]
Figure 2:
Absorption (blue) and fluorescence spectra, excitation (green) and emission (red) of (A) the R-en-
antiomer (1a), (B) the mesomer (1c), and (C) the conformer (2) in water, respectively. Matured
stock solutions were diluted to concentrations of 0.1 mM for UV/vis and 0.01 mM for fluorescence
on the verge of measurements. The fluorescence excitation spectra were collected at (A) 527 nm,
(B) 604 nm, (C) 604 nm and the emission spectra were collected after excitation at (A) 488 nm, (B)
530 nm, (C) 550 nm, respectively.
The spectra of the dyes in water differ significantly from the monomer spectra in organic
solvents (Fig. 2). Remarkable bathochromic shifts of the absorption band indicate the
formation of J-aggregates[1c] in all cases (1a-c, 2) and the influence of the compounds chirality
becomes clearly visible by the differing absorption spectra of the enantiomers (1a and 1b) and
of the meso-form (1c).
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Spectra of both enantiomers (1a and 1b) are identical. The absorbance is characterized by a
single narrow band at 583 nm (fwhm = 413 cm-1) with a shoulder at 535 nm (shown
exemplarily for the R-enantiomer (1a) in Fig. 2A). The fluorescence emission has its maximum
at 585 nm. This nearly resonant emission is another typical feature of J-aggregates.[1c] The
related excitation spectrum is in good agreement with the absorption spectrum. With respect
to their shape all these spectra are similar to those found for the J-aggregates of the closely
related cyanine dyes TDBC (C2S4)[5b] and C8O4.[13]
In contrast, the mesomer (1c) shows a split absorption spectrum (Fig. 2B) with a sharp and
intense sub-band at 603 nm (fwhm = 165 cm-1) and a second narrow and intense sub-band at
572 nm; a third broader band of lower intensity is located at around 543 nm. The emission
spectrum shows only one sharp peak in resonance with the absorption band at 603 nm (fwhm
= 247 cm-1) regardless of the excitation wavelength.
Figure 3:
Three individual preparations from three different batches of the mesomer (1c) in Milli-Q water
(yellow, green and blue) show a differently strong third sub-band at ~ 543 nm, while the two
longer-wavelength bands remain almost stable (A). The individual spectra are normalized to the
same absorbance at ~ 603 nm. The dotted spectrum was derived from a solution of 1c in HCl at a
pH < 4. After appropriate scaling and subtraction of this low pH spectrum from the three spectra
in Milli-Q water, two-banded spectra are obtained for all preparations as shown in the lower panel
(B).
Fig. 3A shows a family of spectra from three individual mesomer (1c) sample preparations.
The comparison reveals differences in the intensity of the third absorption band at 543 nm,
which clearly exceed the experimental error limits. Also, the maximum position of the longest
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wavelength band varies slightly from sample to sample but to a much lesser extent (between
603 and 606 nm). It is noticeable, however, that the intensity ratio of the first and second sub-
band remains almost constant. When the mesomer was dissolved in aqueous HCl at low pH
(lower than 4) the solution displays a spectrum with a maximum at 543 nm (purple dotted
line), which resembles that of monomers (Fig. 1), but is clearly broader (fwhm = 1295 cm-1)
and red shifted by about 23 nm. After appropriate scaling and subtraction of this band from
the spectra in Milli-Q water (Fig. 3A), roughly identical, two-banded spectra are obtained for
all three preparations (Fig. 3B). This simple fit procedure suggests that the remaining longer-
wavelength bands belong to the aggregate while the variable third band around 543 nm might
indicate the presence of a further independent species (probably dimers or smaller
oligomers).
The conformer (2) displays a split absorption spectrum in water (Fig. 2C, blue) just as described
for the mesomer (1c). In case of the conformer (2), the bands became broader and their
intensity weaker within a period of minutes indicating precipitation. The fluorescence
behaviour, however, is similar to the mesomer. Due to the rapid precipitation the conformer
was not investigated in more detail by spectroscopy.
Figure 4:
Circular (red line) and linear dichroism (green line) spectra of an aqueous solution of achiral
mesomer (1c). To compare the peak positions, a normalized isotropic absorption spectrum (blue
broken line) is added to the graph without a dedicated ordinate.
In former studies we demonstrated that the achiral parent dye C8O3 forms optically active
helical J-aggregates by applying circular dichroism (CD) spectroscopy and cryo-TEM.
Moreover, we were able to tune the handedness of aggregates by adding chiral alcohols.[10a,
11]
Therefore, we were interested to see whether the use of chiral head groups affects the
supramolecular assembly in a similar way. The absence of CD signals from methanolic
solutions of the dyes indicates that the chirality of head groups has at the best negligible effect
on the conformation of the monomeric (non-aggregated) chromophores.[14] In contrast, the
aggregated mesomer (1c) shows negative and positive Cotton effects at 577 and 605 nm,
respectively (Fig. 4, red line). Phenomenological, this behaviour is in line with corresponding
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CD results of the parent C8O3 dye and points to a similar molecular architecture of aggregates.
For this dye a tubular architecture was detected, with the tube wall being composed of two
helically twisted chromophore layers.[5b] The observed Cotton effects were ascribed to
molecular excitons. In the case of the related C8S3 dye the helical architecture of tubes was
even proven directly by highly resolved cryo-TEM images.[6, 15] For the enantiomers (1a and
1b), however, we did not obtain spectra with mirror symmetry, indicating the absence of a
chiral supramolecular organisation. Morphological investigations proved this claim (see
below).
Linear dichroism (LD) spectroscopy is a highly sensitive method that can give valuable
information about the molecule packing orientation within aggregates. Isotropically oriented
molecules give no LD signal, while anisometric aggregates with high aspect ratio give
characteristic LD signals. To measure the linear dichroism the aggregates dispersed in the
sample have to be aligned.[16] For fibrous aggregates (cf. Fig. 8 and Fig. 9), this can simply be
accomplished in the streaming field of a Couette flow cell. Solutions of the mesomer (1c) show
a LD spectrum (Fig. 4, green line) consisting of two strong sub-bands, which locations coincide
with the two strongest absorption bands. The positive LD signal of the longest-wavelength
band (604 nm) indicates a parallel polarization of the associated transition with respect to the
aggregates’ long axis. The other band (571 nm) is polarized perpendicularly. This polarization
pattern is characteristic for single-layered tubular J-aggregates.[5e, 17]
Analog LD measurements of the enantiomer solutions gave only one small positive band. This
finding is difficult to interpret because the sheet-like structure of these aggregates (cf. Fig. 6
as evidence) casts serious doubts on their ability to be directionally oriented in the Couette
cell.
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Figure 5:
Disaggregation of J-aggregates of (A) the R-enantiomer (1a) and (B) the mesomer (1c) upon MeOH
titration. The equilibration time between each titration step was 5 min. Start solution: 0.1 mM dye
in pure water. Arrows indicate the effect of increasing MeOH concentration: (A): [MeOH]: 0, 10,
20, 30, 40, 50, and 60%; (B): [MeOH]: 0, 10, 20, 30, 40, 45, 50, and 60%.
Due to the solubility of dyes in methanol, disaggregation can simply be monitored by absorp-
tion spectroscopy upon methanol titration. Such measurements provide additional
information about the stability of aggregates and the kinetics of disaggregation. In the
experiments reported here, we added a methanolic dye solution to the respective aqueous
solution of the aggregated dyes in order to keep the dye concentration constant.
Fig. 5 displays sets of absorption spectra for the R-enantiomer (1a, Fig. 5A) and the mesomer
(1c, Fig. 5B). The absorbance of 1a remains almost unchanged up to 30 vol% methanol
admixture. Further admixture of up to 60 vol% promotes the gradual formation of monomers
at the expense of J-aggregates. A defined isosbestic point at 540 nm indicates disassembly of
J-aggregates directly into monomers without the appearance of intermediates.
A slightly different result was obtained for the mesomer (Fig. 5B). Here, the isosbestic point is
located at 535 nm. In contrast to the R-enantiomer, the J-bands remain almost unchanged up
to 20 vol% of methanol. Complete disassembly of the aggregates is accomplished at 50 vol%.
Noticeable is the appearance of a hump at around 540 nm prior to complete disassembly. By
deconvolution of the spectra into monomer and aggregate components (not shown) a
corresponding band could indeed be extracted, but an associated aggregate species could not
be detected by cryo-TEM.
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Structural Characterization
Spectroscopic investigations of all aqueous amino-propanediol dye solutions (1a-c, 2) were
complemented by direct structural characterisation using cryo-TEM, cryo-ET, as well as AFM
imaging techniques.
Sheet-like assemblies for the enantiomers
Figure 6
The cryo-TEM micrograph of a 0.1 mM solution of the R,R enantiomer 1a in pure water discloses
the formation of sheet-like aggregates. Their approximate thickness can be roughly estimated from
wrinkles (arrow heads) measuring 6 ± 1 nm. Bar represents 200 nm.
Both enantiomers (1a and 1b) assemble into sheets with dimensions in the micrometre range
(Fig. 6). The sheets are separated without any tendency to form stacks. Occasionally emerging
wrinkles (dark lines marked by white arrow heads) were used to estimate a sheet thickness of
approximately 6 nm. This value, however, is only a rough estimate due to inhomogeneous
folding events and blurring at the edges of the folds.
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Figure 7:
A) AFM reveals the deposition of the sheet-like aggregates from the R-enantiomer (1a) on mica. B)
The height profiles measured along the trajectories depicted by dashed lines in the top view image
show recurring 4.5 nm steps, even when smaller aggregates are deposited on top of the large
sheets (red and green line). C) Space filling model of the amino-propanediol cyanine dye and a
schematic representation of a J-type bilayer arrangement thereof. The model reproduces the dis-
tance between opposing chromophores and the overall thickness of the sheet-like aggregates from
the enantiomers as elucidated by the AFM measurements. TBC chromophores in a brickwork
arrangement are represented by green blocks, diol head groups by red ovals, and hydrocarbon
chains by grey rods.
For a more accurate thickness determination, AFM measurements were exemplarily
performed on R-enantiomer sheets deposited on mica support in a water-filled liquid
chamber. Sheets with a thickness of 4 - 4.5 nm were imaged in PeakForce mode (Fig. 7A and
B). Even smaller sheets deposited on top provided additional ~ 4.5 nm high plateaus. The
value corresponds approximately to twice the length of the molecule and thus indicates a
bilayer arrangement of dye molecules (Fig. 7C). Despite a high level of order as suggested by
the narrow absorption band (cf. Fig. 2), ultra-structuring of the sheets’ surface could not yet
be resolved.
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Tubular assemblies of the mesomer
Figure 8
Left: The cryo-TEM micrograph of a two days old 1 mM solution of the mesomer (1c) in pure water
displays uniform individual tubes. Their length often exceeds several micrometres. The line plot
(right) across the highlighted tube section provides the cross-sectional density and thus allows for
the precise characterization of the tubes’ double layer structure and its dimensions. Bar represents
50 nm.
Other than the enantiomers, the mesomer (1c) forms tubular supramolecular aggregates. In
one particular preparation we found almost exclusively individual tubes with maximum
lengths reaching several micrometres, while other preparations predominantly showed tube
bundles with varying degrees of twist. Once formed, the particular proportions of tubes and
bundles in either sample persisted over the time (Fig. SI 2). The heterogeneity in preparation-
dependant structural varieties, however, remains unexplained.
Fig. 8 displays a sample of 1c in which almost exclusively individual tubes were formed. The
high spatial image resolution discloses the double layer architecture of the tube walls. The line
plot (right) averages the grey values of a 41 nm long straight tube section (red area) along its
central axis and thus provides the tubes’ cross-sectional electron density with enhanced
signal-to-noise ratio. The labelled tube has an outer diameter of ~ 12.9 nm and an inner
diameter of ~ 4.9 nm. The total thickness of the wall is about 4.0 nm (dark dashed lines) and
the peak to peak distance (highest densities) between the layers measures 2.1 nm (light
dashed lines) and corresponds to previous data of C8O3 tubes.[5b, 18] The peaks mark the
positions of the electron rich dye skeleton (chromophore). Thus, the profile of the bent double
layer of the tubes is in good agreement with that of the flat double layer (cf. Fig. 7C) and
matches the molecular dimensions.
To look for long-time changes of the morphology we re-examined the 1 mM solution after 2,
21, and 125 days. The statistical analysis of in total 344 line plots as in Fig. 8, i.e. approximately
100 from each sample, yielded mean diameters of 12.7, 12.5, and 12.7 nm, respectively, with
standard deviations of 0.76, 0.97, and 0.64 nm for each subset. This proves a robust geometry
of the individual tubes even upon long-time storage.
The (outer) tube diameter is a little larger than for the parent dye C8O3 (for pure dye: ~ 10.0
nm[5b], for PVA stabilized tubes: 11.5 nm[18]) and comparable with that of the sulfonic acid
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analogue C8S3 prepared by the alcoholic route (13 nm[9a, 19]). The wall thickness of about 4 nm,
however, is almost identical for all tubes.
Figure 9
(A) Cryo-TEM micrograph of mesomer (1c) in 1mM aqueous solution which was subjected to cryo-
ET showing individual tubes and twisted tube bundles. Bar represents 200 nm. (B) A sum-image of
aligned slices extracted perpendicular to the long axis of the labelled bundle from the recon-
structed volume provides the mean cross-sectional density with enhanced signal-to-noise ratio
(note the inverted contrast necessary for image processing). Due to the rotation of the motif upon
following the bundles’ long axis the missing wedge artefact of the tomography reconstruction can
be overcome. Bar represents 50 nm (C) The averaged cross-section was used to reconstruct a 3D-
model of the bundle which is presented in voltex[20] representation to illustrate the twisted course
of the constituting tubes.
Fig. 9A displays a cryo-TEM micrograph of a 1 mM solution of the mesomer (1c). Here, next to
short individual tubes also tube bundles such as often found for the parent dyes C8O3[5b, 21]
and C8S3[5e, 19] are obtained.
To elucidate the three-dimensional volume structure of the twisted bundles, we subjected the
sample to cryo-ET. The tomogram was reconstructed from 65 single exposures taken at angles
from -65° to 63° in two-degree tilt increments. Slices of the tomogram along the boxed bundle
(Fig. 9A) provide 5 tubes which are hexagonally arranged around a sixth central tube (B). This
composition leaves a void at the outer face of the aggregate which permits to follow the
continuous displacement of this pattern along the bundle long axis by scrolling through the
slices of the tomogram (see Fig. SI 4). A complete 360° cycle corresponding to the bundles’
pitch is 400 nm. 232 extracted slices were used to calculate the mean cross-section of the
bundle with enhanced signal-to-noise ratio (B). Due to the complete 360° rotation of the motif
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along the bundle long-axis a complete and accurate density profile is averaged without
restriction of the missing wedge artefact. This motif was used for the reconstruction of the
complete volume of the bundle (Fig. 9C). It becomes apparent that the wall thickness between
the tubes and towards the exterior of the bundle is the same, disclosing that the tube walls
double-layer construction is absent in the interior of the bundles, where tubes should form
two double-layers at their contact faces.
Recently, Eisele et al.[5e] elucidated the same tube layer deviation for bundles of C8S3 by using
cryo-ET volume reconstructions. They assumed that upon bundling the originally bilayered
tubes lose their outer layer. In the following we describe an image-based approach to reveal
an even more detailed insight into this unusual molecular tube layer organisation.
Modelling of tube bundles
Due to the limited resolution in cryo-ET data, more detailed information about the
supramolecular construction can be obtained by analysing individual cryo-TEM projection
images recorded at higher magnification by a geometry-based simulation approach. We were
in particular interested in the structural organization at the interfaces of adjacent tubes.
Hereby, the hexagonal arrangement of tubes as observed by cryo-ET (cf. Fig. 9B) provided the
basic motif.
Figure 10:
Modelling of a twisted tube bundle. (A) Cross-sectional view of a model tube with a mono layered
wall. Constituting dye molecules are oriented with their head groups (red) towards a central
channel whereas octyl chains (grey) point outwards. (B) Packing motifs for the tubes in a bundle.
16 (34)
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Side-by-side arrangement of the tubes (left) leaving voids at the interfaces. Compressed packing of
the tubes (right) diminishes interspaces. (C) Cross-section of a bundle of mono layered tubes (cf.
A) reproducing the number and orientation of the tubes in the original bundle in (D). This cross-
section was used to generate a 3D model of the respective bundle (cf. Fig. SI 6). (D) Cryo-TEM
image of twisted bundle with the marked positions of characteristic line patterns. (E) Back
projection patterns in 10° steps of 3D models calculated for the side-by-side arrangement (row 1),
the compressed packing (row 2) and the experimental cryo-TEM data (row 3). Projection images
of the compressed arrangement reveal a much better match with the experimental data.
The projection pattern along the twisted bundle in Fig. 10D was used as experimental
reference. As the changes in the pattern along the bundle axis are a structural equivalent for
a rotation around the bundle axis (as proven by the cryo-ET data), we compared the systematic
variation in the experimental projection pattern with that of a rotated hexagonally packed
bundle of tubes, whose multiplicity and tube diameter can be determined directly from the
cryo-TEM image (cf. Fig. SI 5). For the case at hand (see Fig. 10D), we determined multiplicities
of 4, 4, and 5, layers in the directions of 0°, 60°, and 120°, respectively, and a tube diameter
of 8.4 nm from the line distances at 30°, 90°, or 150°. These values indicate a tube
arrangement as shown in Fig. 10C.
We calculated the back-projections of the respective 3D-volume and compared them with the
experimental data. The back-projection patterns of the side-by-side arranged tube bundle
(Fig. 10E, row 1) coincide fairly well with the data. A compressed (interdigitated) arrangement
of the monolayered tubes (Fig. 10B, right), however revealed a much better fit (Fig. 10E, row
2). Moreover, in this arrangement the unfavourable voids at the trigonal contact interfaces of
the tubes (arrow in Fig. 10B, left) are largely diminished (right).
Figure 11
Twisting and bundling starts at the early state of the aggregation process. This micrograph shows
a vitrified sample prepared six hours after the dissolution of the solid matter in water. Next to short
individual tubes a short (~ 100 nm length) twisted bundle can be found. Bar represents 100 nm.
To obtain information about the assembly process of the tube bundles we prepared early
states of aggregation. A cryo-preparation 6 hours after dissolution of the dye shows short only
50 - 100 nm long bundles (Fig. 11) which already exhibit the typical patterns of a twisted
bundle (cf. Fig. 9). Its existence at this early stage of the aggregation process as well as the
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persistence of individual tubes indicates a new aspect of the assembly growth, i.e. the hierar-
chical organization of tubes into bundles obviously results from intrinsic supramolecular
interactions at the start of aggregation and contravenes the assumption that bundling and
twisting is caused in a multi-stage process over time.
Needle-shaped crystals of the conformer
Conformer (2) showed macroscopically visible needle like aggregates, too large to be
preparable for cryo-TEM. Upon addition of methanol, however, needles became significantly
smaller and thus enabled cryo-TEM investigations.
Figure 12
Cryo-TEM micrograph of an aqueous solution of the conformer (2) containing 30 vol% of methanol
displaying a few tube bundles (black arrow heads) and predominantly wide elongated and non-
twisted crystalline assemblies. Bar represents 100 nm.
In contrast to the mesomer (1c) cryo-TEM micrographs of aqueous solutions of the conformer
(2) revealed only very few individual tubes or tube bundles (Fig. 12, black arrowheads), while
predominantly formation of smooth elongated structures occurs. The latter can reach lengths
of several micrometres and widths of hundreds of nanometres. Despite their dimension, the
large assemblies seem to preserve some flexibility as many of them are slightly bent.
Nevertheless, frequently occurring very narrow line patterns suggest a repetitive crystalline
order. These morphological findings are in line with the observed spontaneous precipitation
of dye 2 and the remarkably broad absorption bands. Although tube-like aggregates were
formed in negligible amounts, crystallization seems to dominate over supramolecular
aggregation in case of the conformer (2).
IV.
Discussion
Monomer spectra of all novel dyes coincide with the respective data of the parent dye C8O3
proving that the modifications in the head group chemistry have no effect on the electronic
properties of the chromophore. In water red shifted absorption spectra indicate the formation
of J-aggregates for all four dyes. Spectral differences among the new dyes as well as in relation
to C8O3 or C8S3, which only differ in the hydrophilic head groups, indicate different molecular
organizations.
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The structural investigations showed that the enantiomers (1a and 1b) form sheet-like aggre-
gates as observed earlier for the related dyes TDBC (C2S4) and C8O4.[8] Absorbance and
fluorescence spectra are quite similar and in line with the supramolecular structure of the
aggregates. Brickwork-like arrangements of molecules in flat two-dimensionally extended
sheets can explain this behaviour.[22] In contrast, spectra of the mesomer (1c) resemble those
of special preparations of the reference dyes C8O3 and C8S3 where bundles of tubes
dominate.
Isolated tubes show two longitudinally polarized transitions on the low-energy side of the
absorption spectrum. Thereby, both transitions are radiative. In addition, perpendicularly
polarized transitions are observed at higher energy. This results from the double layer
architecture of the tube walls.[5f, 18, 23] In contrast, single-layered tubes show only one
longitudinally polarized low-energy transition accompanied by a perpendicularly polarized
transition at higher energy and only one emission band in resonance with the low-energy
absorption band.[17] Eisele et al.[5e] explained this quite interesting effect by the loss of the
outer dye monolayer during the bundling process. A similar spectral single-layer signature was
observed for preparations of the mesomer (cf. Fig. 2B and Fig. 4). Since tube bundles were
also detected for the mesomer (1c), we suggest the same mechanism being valid for this
derivative. Volume reconstruction from cryo-ET data and modelling of highly resolved tube
bundle projection images support this concept and add additional details. The observation of
a spectral single-layer signature for the preparation with exclusively isolated double-layered
tubes, however, cannot be explained at present.
The amphiphilic dye aggregation in water is driven by a multitude of different forces:
amphiphilic and dispersive interactions of alkyl chains, chromophores, and the extended
planar π-systems as well as interactions and steric demand of the head groups. Since both,
the hydrophobic part of the molecules and the chemical composition of the head groups are
identical for all the new dyes, the differing aggregation behaviour must result from differing
interactions of the head groups due to their stereochemistry. Our results reveal, that inversion
of one stereo centre (enantiomer vs. mesomer) induces the formation of completely different
architectures, i.e. sheets (Fig. 6) and tubes (Fig. 8 and Fig. 9), respectively, associated with
particular spectroscopic characteristics.
The chiral enantiomers (1a and 1b) form planar, hence achiral aggregates, which is a rather
unexpected result given the multitude of chiral ultra-structures reported for chiral compounds
in literature. Thickness measurements indicate that the sheets of the enantiomers are formed
by molecular double layers, as expected from the amphiphilic character of the dyes.
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Figure 13:
The brickwork arrangement of cyanine dyes constitutes the basic arrangement of the molecules in
sheet-like J-aggregates (top). If rolled up into tubes (bottom) a helical arrangement of the constitu-
ent dyes results which causes a strong CD signal of the superficially achiral structure.
According to theory, the spectroscopic properties of J-aggregates can be explained by a so-
called “head-to-tail” arrangement of the transition dipoles of the chromophores. Such an
arrangement is realized in a two-dimensional layer of dye molecules, where neighbouring
molecules are shifted against each other in a brickwork-like pattern (Fig.13A).[2, 22] The model
explains the occurrence of a single sharp absorption band and the quasi-resonant emission.
This simple spectroscopic behaviour is observed for many sheet-like J-aggregates including the
related cyanine dyes TDBC (C2S4) and C8O4, which form extended monolayers and double-
layered sheets, respectively.[5b] Considering their similar spectroscopic and structural features
we assume a comparable brickwork arrangement for the enantiomers (Fig. 13).
The fact that the chiral enantiomers (1a and 1b) form planar structures upon aggregation
seems all the more surprising because the related achiral dyes C8O3 and C8S3 form double-
layered tubular structures instead. Some of these tubular structures reveal even helical
packing patterns[6, 15] and the tubes show generally a strong tendency to assemble into twisted
rope-like bundles.[5b] Hence, such tubes possess a chiral structure and their absorption
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behaviour can be modelled on this basis.[5f, 8c, 9a, 23] The ability of the presented non-chiral
derivatives, i.e. the achiral mesomer (1c) and the non-chiral conformer (2), to form tubes
suggests a comparable architecture. Considering the Cotton effect of their CD spectra (Fig. 4),
at least the tubular assemblies of the achiral mesomer (1c) are organized in a chiral molecular
arrangement (Fig. 13B).
The obvious inverse relationship between molecular chirality and helicity of supramolecular
architecture asks for a model that can explain the various structures and is consistent with the
detected spectra.
The formation of chiral supramolecular structures from achiral or non-chiral molecules in
solution has been reported but remains rare.[24] This may derive from the even probabilities
to form right- or left-handed assemblies resulting in a net zero CD-signal. Only homochirality
can be detected unambiguously by CD spectroscopy. Examples include the non-chiral cyanine
dye C8O3[10a, 11] or the generation [1.0] polyglycerol amphiphile reported by Kumar et al.[25] In
the latter case, chiral ultra-structuring of the self-assembled tubes was detected by CD only in
the case of the meso-form. The origin of this supramolecular chirality was explained to result
from the cooperation of a fixed lateral arrangement of molecules and specific intermolecular
hydrogen bonding patterns of the hydroxyl groups, which produces a strong helical twist of
the molecular stacks only in case of the meso-form.
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Figure 14
(Top) The brickwork arrangement of the S enantiomers gives rise to continuous strings of hydroxyl
groups (red) running transversely to the chromophore skeletons (coloured in background). The
close up (below) shows the OH groups properly situated to form endless hydrogen bridge chains.
Hence, all dye molecules are connected to four neighbouring molecules (coloured). This stable two-
dimensional arrangement is the unique feature for the chiral enantiomers and cannot be realised
by the meso-form (bottom) due to the different orientation of the respective OH group at the chiral
centre (red circles).
We consider a quite similar reason here. In a brickwork arrangement of the enantiomers form-
ing the planar double-layer, the rows of molecules are positioned in a way that neighbouring
rows are staggered approximately by half of the chromophore length (Fig. 14). The distance
between the rows results from the space demand of the chromophore skeletons which is 0.35
– 0.4 nm. By placing the enantiomers accordingly, continuous strings of hydroxyl groups can
be observed running transversely in respect of the rows of the chromophores (top left). A
more detailed presentation (bottom left) shows that all OH groups within a row are connected
by endless hydrogen bond chains. Thus, each dye molecule interacts strongly with four
neighbouring molecules stabilizing the planar arrangement. In case of the mesomer, however,
the inversion of one of the chiral centres prevents linear hydrogen bonds of hydroxyl groups
due to their alternating orientation (marked by a red circle). The curved molecular
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arrangement upon rolling of the sheets into tubes is stabilized by enhanced chromophore
interactions.
This comparison gives a good explanation not only on how the introduction of hydrogen
bonding OH groups can direct the formation of planar sheets in case of a chiral compound but
also on why the formation of tubular aggregates is preferred by the achiral mesomer. In this
respect as well as regarding the aggregation behaviour of the parent C8O3/S3 dyes, which
both form only tubular aggregates, one can suspect that the formation of tubes may be an
intrinsic property of the hydrophobic dye skeletons with their hydrocarbon chains rather than
being provoked by the hydrophilic head groups. The observation that the diameter of
monolayered tubes (of the bundles) and of the inner cylinders of the bilayered tubes are
identical points at comparable chromophore interactions in both architectures. The initial
twist in the monolayered inverted tube bundles, however, gives direct structural evidence for
a chiral packing principle in both tubular assembly structures. It is, however, still not clear
what drives the assembly process of the mesomer into two structurally different species
(individual tubes and bundles).
V.
Conclusion
We have synthesized a new group of TBC based amphiphilic cyanine dye derivatives by
converting the anionic carboxylic acid groups into non-ionic hydroxyl functionalities. The
compounds contain amino-propanediol groups which provide sufficient water solubility and
defined chirality. To produce the achiral meso-form of the dye a new synthetic route towards
asymmetrically functionalized TBC dyes was established.
All compounds form J-aggregates in water. Cryo-TEM revealed the formation of extended
sheet-like aggregates for the chiral enantiomers (1a and 1b) and individual tubes with 12 -
13 nm diameter as well as tube bundles for the mesomer (1c). In case of the conformer (2)
needle-like crystals predominate by far.
Our findings demonstrate that the supramolecular aggregation behaviour of cyanine dyes can
be controlled solely by stereochemistry. The surprising aspect is the inverse relationship
between molecular chirality and helicity of the supramolecular architecture. The reason is
obviously the special interaction of the non-ionic hydroxyl functionalities. In case of the chiral
enantiomers (1a and 1b) extended hydrogen bond chains can interconnect molecules in a
brickwork arrangement. This yields a two-dimensional planar network which prevents the
spontaneous formation of curved assemblies like the tubes and tubes bundles formed by the
meso-form (1c) which is not capable of forming comparable hydrogen bond chains.
Absorption, LD and fluorescence spectra of the tubes point to a monolayered architecture,
although single tubes clearly show a double-layer wall geometry. In recent literature[5e] this
apparent contradiction has been resolved by the evidence of a dominating population of tube
bundles. Supported by cryo-ET it was observed that during the hierarchical assembly of the
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bilayered C8S3 tubes into bundles the outer monolayer is lost. The present cryo-ET investiga-
tion and the structural analysis of similar bundled aggregates not only affirms this
interpretation but adds evidence for a tight packing of the hydrophobic cylinders.
The new insights into the interdependence of stereochemistry and supramolecular
aggregation behaviour of cyanine dyes might contribute to the development of specialised,
dye-based materials with predictable properties.
VI.
Experimental Section
Materials and methods
Materials. Dry solvents and chemicals were purchased from Sigma-Aldrich, TCI and abcr
Chemicals. Ethyl acetate, hexane, and dichloro methane were distilled before utilisation in
reactions as well as in compound purifications. Enantiopure R- and S-solketal were purchased
from TCI with 98 % ee optical purity. C8O3 was obtained from FEW chemicals (Wolfen,
Germany). Reactions were monitored using thin layer chromatography on silica coated
aluminium sheets using 60 F254 silica gel or 60 RP-18 F254S silica gel for reverse phase
analysis. All intermediates were purified using normal phase column chromatography and
automated flash chromatography on a combi Flash Rf column (Teledyne ISCO) packed with
normal silica gel (30 µm). The final products were purified using reverse phase preparative
HPLC.
1
NMR-spectroscopy. H NMR and 13C NMR were recorded on a JEOL ECP 500 spectrometer
(500 MHz); mass spectra were recorded using an Agilent 6210 ESI-TOF spectrometer.
Sample preparation. Dye stock solutions were prepared from dried solid matter and the
respective solvent by vigorously shaking the samples. For spectroscopic measurements, the
solutions were diluted, if necessary.
Spectroscopic measurements. Isotropic absorption spectra (UV/vis) were measured on a
Varian Cary® 50 spectrophotometer (Agilent Technologies Inc., Santa Clara, USA),
fluorescence spectra on a luminescence spectrometer LS 50B (Perkin Elmer, Rodgau,
Germany). Circular dichroism (CD) and linear dichroism (LD) were measured on a J-810
spectropolarimeter (Jasco Corp., Tokyo, Japan), which could be equipped with a microvolume
Couette flow LD cell (Dioptica Scientific Limited, Rugby, Warwickshire, UK) with 0.5 mm optical
path length for the LD measurement.[26] The LD spectra were independent of the angular
velocity of the rotating cell. Rotating velocities up to 3000 rpm, corresponding to shear forces
of ~ 1200 s-1, were used. CD measurements were carried out at 20°C, all other spectroscopic
measurements at room temperature (22 ± 1°C). Cuvettes for UV/vis, fluorescence, and CD
were siliconized before measurements according to the “Siliconization of Glasware“-protocol
using a silicone solution in isopropanol (SERVA Electrophoresis GmbH, Heidelberg, Germany).
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Cryo-Transmission Electron Microscopy (cryo-TEM). 200 mesh grids covered with perforated
carbon film (R1/4 batch of Quantifoil, MicroTools GmbH, Jena, Germany) were cleaned with
chloroform and hydrophilized by 60 s glow discharging at 8 W in a BAL-TEC MED 020 device
(Leica Microsystems, Wetzlar, Germany). After applying 5 µl aliquots of the dye solution to the
grids the samples were vitrified by automated blotting and plunge freezing in to liquid ethane
by use of a FEI Vitrobot Mark IV (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).
The vitrified specimens were transferred under liquid nitrogen to a FEI TALOS L120C electron
microscope (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) using a Gatan cryo-
holder and stage (model 626, Gatan, Inc., Pleasanton, California, USA). The microscope was
equipped with a LaB6 cathode and operated at 120 kV accelerating voltage. Micrographs were
acquired on a FEI Ceta CMOS camera (Thermo Fisher Scientific Inc., Waltham, Massachusetts,
USA) at a nominal magnification of 36,000 , corresponding to a calibrated pixel size of
4.09 Å/pixel.
Cryo-Electron Tomography (cryo-ET). Vitrified specimen (see above) intended for cryo-ET
were transferred to the autoloader of a FEI TALOS ARCTICA electron microscope (Thermo
Fisher Scientific Inc., Waltham, Massachusetts, USA). This microscope is equipped with a high-
brightness field-emission gun (XFEG) operated at an acceleration voltage of 200 kV. Micro-
graphs were acquired on a FEI Falcon 3 direct electron detector (Thermo Fisher Scientific Inc.,
Waltham, Massachusetts, USA) at a nominal magnification of 28,000 , corresponding to a
calibrated pixel size of 3.64 Å/pixel.
Tomography series were recorded in the context of FEI Tomography Software V 4.3.1. 4096
 4096 pixel images were recorded in the tilt angle range of ± 65° in 2° increments with a total
electron dose of 180 e/Å2. 3D volume reconstructions were calculated with the help of
INSPECT3D Software V4.4 (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) and
visualized with Imod V4.9.10[27]
.
Image processing. Each three consecutive slices of the tomogram along the tube bundle were
summed and cropped using the slicer of Imod V4.9.10.[27] The cross-sectional motif of the
bundle was boxed off these images using the boxer module of the EMAN software package.[28]
Alignments, multivariate statistical analysis (MSA) of a total of 232 such motifs, summing, and
finally construction of a twisted 3D volume of the bundle with a pitch of 400 nm was
performed with the Imagic 5 Software package (Image Science Software GmbH, Berlin,
Germany). The resulting 3D volume was visualized using AMIRA Software V2019.1 (Thermo
Fisher Scientific Inc., Waltham, Massachusetts, USA).
Atomic Force Microscopy (AFM). Atomic force microscopy was carried out with a Multimode
8 Nanoscope with Nanocontroller V (Bruker, Billerica, Massachusetts, USA) and equipped with
ultra-sharp PEAKFORCE-HIRS-F-B tips (Bruker) providing a nominal radius of 1 nm and
maximum radius of 2 nm. The sample was prepared by deposition of 10 µl of the 0.1 mM
aqueous dye solution on cleaved mica which was glued on a circular metal disk with double
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sided tape. The dye assemblies were allowed to settle for about 5 minutes before the solution
was blotted with filter paper until only a thin solution film was left. The sample was then
immediately mounted on the AFM scanner and a liquid chamber was assembled and carefully
filled with Milli-Q water to prevent drying of the sample. Imaging was performed with a
calibrated cantilever[29] in PeakForce Quantitative NanoMechanics (QNM) mode,[30] in order
to control the loading force on the sample at all times. The maximum loading force used was
500 pN, the resolution was 512 points per line, and the scan rate was 0.7 Hz.
Synthesis
Compounds 3a and 3b. Enantiopure Solketal (R or S) was converted into the corresponding
phthalimide according to the published procedure from Goubert et al.[31]
Compound 3a. Using (R)-(−)-2,2-dimethyl-1,3-dioxolane-4-methanol (4.2 g, 31.8 mmol) the
1
corresponding phthalimide 3a was obtained as a colorless solid (7.6 g, 92 %). H NMR (500
MHz, Chloroform-d) δ 7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.71 (dd, J = 5.5, 3.0 Hz, 2H), 4.49-4.38 (m,
1H), 4.06 (dd, J = 8.7, 6.2 Hz, 1H), 3.92 (dd, J = 13.8, 6.9 Hz, 1H), 3.84 (dd, J = 8.7, 5.1 Hz, 1H),
3.71 (dd, J = 13.8, 5.3 Hz, 1H), 1.43 (s, 3H), 1.30 (s, 3H); 13C NMR (126 MHz, Chloroform-d) δ
168.33, 134.16, 132.12, 123.49, 109.95, 73.43, 67.49, 41.09, 26.95, 25.49. MS m/z 284.0898
+
(calcd. C14H15NNaO4 284.0893).
Compound 3b. Using (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (2.0 g, 15.1 mmol) the
corresponding phthalimide 3b was obtained as a colorless solid (3.3 g, 83 %). 1H NMR (500
MHz, Chloroform-d) δ 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.71 (dd, J = 5.5, 3.0 Hz, 2H), 4.49-4.38 (m,
1H), 4.06 (dd, J = 8.7, 6.2 Hz, 1H), 3.92 (dd, J = 13.8, 6.9 Hz, 1H), 3.84 (dd, J = 8.7, 5.1 Hz, 1H),
3.71 (dd, J = 13.8, 5.3 Hz, 1H), 1.43 (s, 3H), 1.30 (s, 3H); 13C NMR (126 MHz, Chloroform-d) δ
168.33, 134.16, 132.12, 123.49, 109.95, 73.43, 67.49, 41.09, 26.95, 25.49. MS m/z 284.0891
+
(calcd. C14H15NNaO4 284.0893).
Compounds 4a and 4b. 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)isoindoline-1,3-dione 3 (a
or b) were reacted according to the procedure published in literature.[31]
Compound 4a. Using S-phthalimide 3a (1.4 g, 5.4 mmol) the corresponding amine 4a was
obtained as a pale-yellow liquid (0.3 g, 43 %). 1H NMR (500 MHz, Methanol-d4) δ 4.14 (qd, J =
6.4, 4.8 Hz, 1H), 4.05 (dd, J = 8.3, 6.4 Hz, 1H), 3.65 (dd, J = 8.3, 6.3 Hz, 1H), 2.81 – 2.64 (m, 2H),
1.39 (s, 3H), 1.33 (s, 3H); 13C NMR (126 MHz, Methanol-d4) δ 110.33, 78.06, 68.04, 45.21,
+
27.18, 25.57. MS m/z 132.1008 (calcd. C6H14NO2 132.1019).
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Compound 4b. Using R-phthalimide 3b (1.0 g, 3.9 mmol) the corresponding amine 4b was
obtained as a pale-yellow liquid (0.28 g, 55 %). 1H NMR (500 MHz, Methanol-d4) δ 4.13 (qd, J
= 6.4, 4.8 Hz, 1H), 4.04 (dd, J = 8.2, 6.4 Hz, 1H), 3.65 (dd, J = 8.2, 6.4 Hz, 1H), 2.80 – 2.64 (m,
2H), 1.39 (s, 3H), 1.33 (s, 3H); 13C NMR (126 MHz, Methanol-d4) δ 110.30, 78.16, 68.05, 45.25,
+
27.17, 25.57. MS m/z 132.1037 (calcd. C6H14NO2 132.1019).
Compounds 5a and 5b. To a solution of 30 mg (0.037 mmol) C8O3, 48 mg (0.126 mmol) HATU
and 48.5 mg (0.370 mmol) solketal-amine 4 (a or b) in 3 mL DMF was added (0.518 mmol)
DIPEA and the mixture was stirred at r.t. for 2 h. After removal of the solvent in vacuum, the
residue was dissolved in CH2Cl2 and washed with water three times. The combined organic
phases were concentrated, and the residue was purified by automated column chromato-
graphy (CH2Cl2/methanol, 0-5 %).
Compound 5a. Solketal amine 4a was reacted according to the above-described procedure
and dye 5a was obtained as a red solid (36 mg, 91 %). 1H NMR (700 MHz, Methanol-d4) δ 8.00
(s, 1H), 7.76 (s, 2H), 7.73 (s, 2H), 4.31 (dt, J = 14.9, 7.5 Hz, 8H), 4.10 (p, J = 5.8 Hz, 2H), 3.97 (dd,
J = 8.4, 6.3 Hz, 2H), 3.60 (dd, J = 8.4, 5.9 Hz, 2H), 3.28-3.20 (m, 4H), 2.39 (t, J = 6.7 Hz, 4H), 2.14
(p, J = 6.9 Hz, 4H), 1.87 (p, J = 7.4 Hz, 4H), 1.45 – 1.21 (m, 32H), 0.86 (t, J = 7.0 Hz, 6H); 13C NMR
(176 MHz, Methanol-d4) δ 174.41, 151.36, 144.06, 133.78, 133.60, 128.69, 116.58, 112.56,
110.48, 75.88, 68.15, 46.31, 45.74, 43.00, 32.93, 32.73, 30.76, 30.35, 29.09, 27.66, 27.20,
+
25.56, 24.62, 23.68, 14.42. MS m/z 1035.4713 (calcd. C53H77Cl4N6O6 1035.4624).
Compound 5b. Solketal amine 4b (48.5 mg, 0.370 mmol) was reacted according to the above-
described procedure and dye 5b was obtained as a red solid (32 mg, 81 %). 1H NMR (700 MHz,
Methanol-d4) δ 8.01 (s, 1H), 7.76 (s, 2H), 7.73 (s, 2H), 5.82 (d, J = 13.3 Hz, 2H), 4.31 (dt, J = 14.0,
8.0 Hz, 8H), 4.10 (p, J = 5.8 Hz, 2H), 3.97 (dd, J = 8.4, 6.3 Hz, 2H), 3.60 (dd, J = 8.4, 5.9 Hz, 2H),
3.26 – 3.21 (m, 4H), 2.38 (t, J = 6.8 Hz, 4H), 2.14 (p, J = 7.0 Hz, 4H), 1.87 (p, J = 7.3 Hz, 4H), 1.45
– 1.21 (m, 32H), 0.85 (t, J = 6.9 Hz, 6H); 13C NMR (176 MHz, Methanol-d4) δ 174.46, 151.29,
133.79, 133.56, 128.90, 128.64, 112.51, 110.47, 75.85, 68.16, 46.31, 45.66, 42.99, 32.91,
32.71, 30.39, 30.27, 29.07, 27.62, 27.19, 25.57, 24.62, 23.67, 14.42. MS m/z 1035.4797 (calcd.
+
C53H77Cl4N6O6 1035.4624).
Compounds 1a and 1b. Hydrogen chloride (1.301 mmol) was added to a solution of dye 5 (a
or b) (0.033 mmol) in 100 mL methanol and the mixture was stirred at r.t. for 3 h. The solvents
27 (34)
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10.1002/chem.201905745
Chemistry - A European Journal
were removed in vacuum by distillation with toluene and the crude product was lyophilized.
The obtained residue was purified by reversed-phase column chromatography using
water/acetonitrile (75%) and 50mM ammonium formate.
Compound 1a. Dye 5a (35 mg, 0.033 mmol) was reacted according to the procedure described
above. Dye 1a was obtained as a red solid (31.4 mg, 96 %). 1H NMR (700 MHz, Methanol-d4) δ
8.46 (m, 1H), 8.40-8.38 (m, 2H), 8.36 (m, 1H), 7.25 (s, 1H), 4.71-4.53 (m, 8H), 3.65-3.58 (m,
3H), 3.49-3.43 (m, 3H), 3.20 (m, 2H), 3.01-2.99 (m, 1H) 2.88-2.86 (m, 1H), 2.52-2.41 (m, 4H),
2.29-2.15 (m, 4H), 2.04-1.91 (m, 4H), 1.42-1.30 (m, 20H), 0.92-0.88 (m, 6H). 13C NMR (176
MHz, MeOD) δ = 173.24, 150.54, 148.03, 141.40, 136.49, 131.82, 131.55, 131.35, 131.20,
131.08, 130.67, 115.35, 115.05, 70.51, 63.62, 55.76, 42.01, 31.53, 29.15, 28.78, 26.25, 22.43,
+
13.13. MS m/z 955.4044 (calcd. C47H69Cl4N6O6 955.3998).
Compound 1b. Dye 5b (30 mg, 0.028 mmol) was reacted according to the procedure described
1
above. Dye 1b was obtained as a red solid (26.1 mg, 94 %). H NMR (700 MHz, Methanol-d4)
δ 8.44 (s, 1H), 8.38 (s, 1H), 8.37 (s, 1H), 8.33 (s, 1H), 7.23 (s, 1H), 4.68-4.49 (m, 8H), 3.62-3.56
(m, 2H), 3.44-3.42 (m, 4H), 3.26-3.25 (m, 1H) 3.17-3.14 (m, 1H), 3.00-2.97 (m, 1H), 2.86-2.83
(m, 1H), 2.52-2.42 (m, 4H), 2.25-2.13 (m, 4H), 1.99-1.90 (m, 4H), 1.52-1.29 (m, 20, H), 0.90-
0.88 (m, 6H). 13C NMR (176 MHz, Methanol-d4) δ 174.61, 174.05, 151.87, 149.39, 142.97,
132.92, 132.89, 132.78, 132.72, 132.44, 132.38, 132.36, 132.33, 116.44, 116.29, 116.23, 71.93,
71.83, 65.02, 47.92, 47.34, 46.95, 43.37, 43.07, 32.95, 32.39, 32.14, 30.46, 30.41, 30.37, 30.34,
+
27.69, 25.90, 25.40, 23.70, 14.41. MS m/z 955.4044 (calcd. C47H69Cl4N6O6 955.3998).
Compound 2. To a solution of 30 mg (0.037 mmol) C8O3, 48 mg (0.126 mmol) HATU, and
34 mg (0.370 mmol) serinol in 3 mL DMF 88.2 µL (0.518 mmol) DIPEA was added and the
mixture was stirred at r.t. for 2 h. After removal of the solvent in vacuum, the residue was
purified by reversed-phase column chromatography using water/acetonitrile (75%) and
50mM ammonium formate to give compound 2 as red solid (16 mg, 44 %). 1H NMR (700 MHz,
Methanol-d4) δ 8.50 (s, 1H), 7.99 (s, 1H), 7.78 (s, 2H), 7.74 (s, 2H), 4.35 – 4.28 (m, 12H), 3.92
(p, J = 5.6 Hz, 2H), 3.60 (dd, J = 11.0, 5.4 Hz, 4H), 3.57 (dd, J = 11.1, 5.8 Hz, 4H), 2.42 (t, J =
6.9 Hz, 4H), 2.14 (p, J = 7.2 Hz, 4H), 1.87 (p, J = 6.9 Hz, 4H), 1.44 – 1.21 (m, 20H), 0.86 (t, J =
6.9 Hz, 6H); 13C NMR (176 MHz, Methanol-d4) δ 174.43, 170.27, 151.33, 151.27, 133.81, 133.55,
128.73, 112.57, 62.09, 54.50, 46.33, 45.67, 33.00, 32.92, 30.38, 30.29, 29.06, 27.65, 24.78, 23.67,
+
14.41. MS m/z 955.4007 (calcd. C47H69Cl4N6O6 955.3998).
28 (34)
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Compound 6. To a stirred solution of 5,6-dichlorobenzimidazole (5 g, 24.8 mmol, 1 eq) in
DMSO the required amount of NaOH (1.09 g, 27.3 mmol, 1.1 eq) was added. The reaction so-
lution was stirred for 2 h at room temperature. Thereafter, the calculated amount of ethyl 4-
bromobutanoate (5.82 g, 28.5 mmol, 1.2 eq) was added and the reaction mixture was left
stirring for 48 h at room temperature. Progress of the reaction was monitored by TLC using
methanol/dichloromethane. On completion of the reaction, the mixture was suspended in
water and ethyl acetate (3x30 mL). The combined organic layers were dried over anhydrous
sodium sulphate and the solvent was evaporated to yield the crude product, which was purified
through column chromatography using DCM and methanol to give compound 6 as a white solid
in 95% yield. 1H NMR (500 MHz, methanol-d3) δ 7.66 (s, 1H), 7.61 (s, 1H), 4.20 (t, J = 7.45, 3H),
4.07-4.02 (q, 2H), 2.59 (s, 3H), 2.41 (t, J = 6.70, 2H), 2.08-2.02 (m, 2H), 1.20 (t, J = 7.15, 3H); 13C
NMR (126 MHz, methanol-d3) δ 174.23, 156.04, 142.27, 135.63, 127.11, 126.82, 120.08,
+
112.66, 61.72, 44.16, 31.48, 25.41, 14.43, 13.52. MS m/z 315.0589 (calcd. C14H17Cl2N2O2
315.0361).
o
Compound 7. Compound 6 (2 g, 6.34 mmol, 1 eq) was liquefied at 150 C. 1-Bromodecane
(7.0 g, 31.7 mmol, 5 eq) was added to the reaction flask and the reaction mixture was left
stirring at 150 oC for 6 h. After completion of the reaction (indicated by TLC using
methanol/dichloromethane) the reaction mixture was extracted with water and
dichloromethane (3 × 50 mL). The combined organic layers were dried over anhydrous sodium
sulphate and the solvent evaporated to yield the crude product, which was further purified
through column chromatography using DCM and methanol as eluent to give compound 7 as a
light yellowish solid in 72% yield. 1H NMR (500 MHz, Chloroform-d3) δ 8.22 (s, 1H), 7.81 (s, 1H),
4.66 (t, J = 7.75, 3H), 4.44 (t, J = 7.45, 3H), 3.99-3.95 (q, 2H), 2.55 (t, J = 6.50, 2H), 2.13-2.07 (m,
2H), 1.82-1.76 (m, 2H), 1.36-1.30 (m, 2H), 1.25-1.11 (m, 13H), 0.77(t, J = 4.55, 3H); 13C NMR
(126 MHz, Chloroorm-d3) δ 172.68, 153.09, 131.36, 131.30, 130.30, 130.14, 114.91, 114.04,
77.41, 77.16, 76.90, 60.71, 47.08, 45.87, 31.57, 30.22, 28.99, 28.90, 26.61, 23.91, 22.44, 14.03,
+
13.93, 12.96. MS m/z 428.1876 (calcd. C22H34Cl2N2O2 428.1914)
Compound 8. Compound 7 (1 g 1.8 mmol, 1 eq) was stirred with a 1:1 mixture of HBr (48%)
and water at 120 oC for 15 h. Progress of the reaction was monitored by TLC using
methanol/dichloromethane. On completion of the reaction, the reaction mixture was cooled to
room temperature. The precipitate was filtered and washed with aq. hydrobromic acid (5%
29 (34)
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