Aqueous Assembly of Zwitterionic Daisy Chains

Article abstract of DOI:10.1002/chem.201803944

The synthesis and characterization of zwitterionic molecular [c2]- and [a2]-daisy chains are described, relying on recognition of a positively charged cyclophane and a negatively charged oligo(phenylene-ethynylene) (OPE) rod in aqueous medium. For this pu

Full text of DOI:10.1002/chem.201803944

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Accepted Article
Title: Aqueous Assembly of Zwitterionic Daisy Chains
Authors: Yves Aeschi, Sylvie Drayss-Orth, Michal Valášek, Daniel
Häussinge, and Marcel Mayor
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10.1002/chem.201803944
Chemistry - A European Journal
Aqueous Assembly of Zwitterionic Daisy Chains
Yves Aeschi, Sylvie Drayss-Orth, Michal Valášek, Daniel Häussinger, and Marcel Mayor*
[*]
Y. Aeschi, Dr. S. Drayss-Orth, PD Dr. D. Häussinger, Prof. Dr. M. Mayor
Department of Chemistry
University of Basel
St. Johanns-Ring 19
4056 Basel, Switzerland
E-mail: marcel.mayor@unibas.ch
Y. Aeschi, Prof. Dr. M. Mayor
Swiss Nanoscience Institute
University of Basel
Klingelbergstrasse 82
4056 Basel, Switzerland
Dr. M. Valášek, Prof. Dr. M. Mayor
Institute for Nanotechnology (INT)
Karlsruhe Institute of Technology (KIT)
P. O. Box 3640
76021 Karlsruhe, Germany
Prof. Dr. M. Mayor
Lehn Institute of Functional Materials (LIFM)
School of Chemistry
Sun Yat-Sen University (SYSU)
Guangzhou 510275, China
1
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TOC Graphic:
Abstract
The synthesis and characterization of zwitterionic molecular [c2]- and [a2]- daisy chains is
described, relying on recognition of a positively charged cyclophane and a negatively charged
oligophenyleneethynylene (OPE) rod in aqueous medium. For this purpose, syntheses of an
acetylene-functionalized macrocyclic receptor and a water-soluble OPE-rod as guest component
are presented, from which a heteroditopic daisy chain monomer is prepared. This monomer
aggregates strongly in water/methanol 4:1 and forms molecular daisy chains, which are isolated
as interlocked species from a stoppering reaction at 1 mM concentration. The cyclic dimer ([c2])
is the main product with an isolated yield of 30 % and consists of a mixture of diastereomers,
which is evidenced by 1H-NMR spectroscopy.
Introduction
Synthesis of rotaxanes, catenanes and derived structures thereof from dynamic host-guest
assemblies has been accomplished by employment of various strategies.[1] A plethora of such
mechanically interlocked super-structures are known up to date, which upon introduction of
stimuli-responsive features enabled chemists to obtain elaborate molecular machines.[2–4] The
2
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stupendous development in this field was recently appreciated by awarding a Nobel Prize for the
achievements in this research area. Introduction of rotaxane-type supramolecular subunits or
even supramolecular machinery into materials chemistry[4,5] and molecular electronics[6–8] may
give rise to the development of functional materials with unprecedented physical and/or
chemical properties.
Covalently bound host-guest conjugates result in the formation of supramolecular daisy
chains[9–16] if the molecular structures are designed appropriately to rule out unimolecular self-
complexation and formation of non-interlinked, intermolecular aggregates.[16] Due to the
hermaphroditic nature of daisy chains, only one component is required for supramolecular
complex formation, which is appealing for the construction of molecular devices as it is possible
to obtain aggregates of different sizes by rational design, which was demonstrated by the
synthesis of linear and cyclic molecular muscles,[12–14,17–19] polymers[11,20–22] and gels.[13,23,24] Most
daisy chains are based on recognition features which have only limited applicability in water,
either due to limited solubility or functional disruption of the recognition site. This is striking as
hydrophobic self-assembly is widespread in the biological realm, often in combination with other
driving forces, such as hydrogen-bonding, donor-acceptor- and electrostatic interactions,[25–27]
which generates a sheer limitless, modular pool of supramolecular assemblies. Furthermore,
exploitation of the hydrophobic effect as a driving force for supramolecular assembly is
particularly appealing as no specialized recognition site is required, thus any hydrophobic surfaces
will aggregate in aqueous environment. Limitations for hydrophobic assembly are mainly the
solubility of host-guest complexes in aqueous environment in combination with sterically
matching hydrophobic sites - two parameters which are often challenging to be both
implemented. Furthermore, the lack of inherent specificity of hydrophobic interactions requires
that lipophilic und hydrophilic properties are carefully balanced to avoid undesired aggregation
modes.
In the context of daisy chains, aqueous assembly is so far mostly limited to systems based on
cyclodextrin receptors.[16] In spite of the wide availability and well-established chemistry,[28,29]
their synthetic modifiability is limited. An alternative, synthetically modifiable receptor for
aqueous media is therefore desirable. The series of cyclophanes synthesized and extensively
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characterized by Diederich et.al.[30,31] bind strongly to hydrophobic guests and show a high degree
of synthetic flexibility.[32–35] Rotaxane syntheses with these cyclophanes have been
accomplished[36–39] and high association constants with several guest molecules[37,39,40] were
reported. Due to their cationic nature, an additional electrostatic driving force can be expected
by choosing guests with a complementary charge.[37] Appropriate coupling chemistry is required
to obtain interlocked species from the dynamic equilibrium, for which copper-catalyzed azide-
alkyne cycloaddition (CuAAC) click-chemistry is an ideal choice, allowing modular and efficient
coupling in aqueous solutions as well as in organic solvents.[41,42]
We became interested in aqueous soluble daisy chain dimers as potential subunit of
mechanically interlocked superstructures. The molecular design is based on the combination of a
“Diederich”-type cyclophane and an oligophenyleneethynylene (OPE)-type rod comprising
terminal salicyl-type synthons (see Figure 1 a). Both structures receive their water solubility from
their peripheral ionic subunits and the attraction of the opposed charges further stabilize the
pseudorotaxane complex. To obtain a monomer suitable for the formation of a daisy chain dimer
however, the macrocyclic cyclophane and the OPE-type rod have to be tethered tightly to avoid
the formation of an intramolecular “lasso-type” complex.[43,44] The here presented approach is
sketched in Figure 1 b) and starts with the introduction of suitable coupling groups at both
structures, the cyclophane and the OPE-rod. Their covalent linkage results in the desired
monomer, which is expected to dimerize in aqueous solution to the daisy chain dimer in order to
optimize the solvation conditions of all subunits. In a following step, similar coupling chemistry
that allowed for the linkage of the subunits enables the introduction of bulky stoppers in order to
freeze the supramolecular complex for its subsequent isolation and characterization.
Here we report the successful implementation of this concept by decorating the involved
building blocks with functional groups enabling their covalent linking by CuAAC. The formation of
supramolecular complexes is studied and the stoppered [c2]-daisy chain was isolated and
characterized.
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Figure 1. a) Pseudorotaxane formed by the combination of CyOMe8, a “Diederich”-type cyclophane, and an OPE-
rod with salicyl-type termini. b) Conceptual approach to water soluble and stable [c2]-daisy chain structures: The
covalent linkage of axle and macrocycle results in a monomer, which forms [c2]-daisy chain dimers to satisfy the
solvation requirements programed in the structure. The covalent attachment of bulky stoppers interlocks the
mechanically interlinked structures.
Results and Discussion
Rigid, hermaphroditic receptor-guest-conjugate 1 can be obtained by tethering receptor 2 to rigid
OPE rod 3 by CuAAC (Scheme 1). The rigidity of the OPE-subunit is expected to reduce the entropic
penalty upon daisy chain formation,[16] and disfavor intramolecular self-complexation. Such a
unimolecular complexation mode would induce significant strain, while still exposing the
hydrophobic parts of the OPE rod to the bulk water. 2 was designed on the basis of CyOMe8,
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exposing a peripheral alkyne group, enabling its further decoration employing CuAAC strategies.
Similar to the original design, the periphery is still decorated with methoxy groups which reduce
the tendency of self-aggregation,[31] while two quaternary ammonium centers provide solubility
in water. Hydrophobic and hydrophilic properties of OPE-rod 3 are balanced by the presence of
the two anionic carboxylate groups while its central phenylene unit remains as a highly
hydrophobic station remote from the hydration shell of the carboxylate functions. Furthermore,
the salicylate precursor can be functionalized with peripheral azide groups. Masking of the
carboxylic acid as alkyl esters can be accomplished by standard esterification protocols, which
ensures good processability during synthesis. Water-soluble stopper 4 allows to obtain either
cyclic or acyclic interlocked daisy chains (5-[a2], 5-[c2]) from the dynamic equilibrium in aqueous
solution. These can subsequently be separated and characterized as discrete entities. This stopper
is a derivative of our precedent work, allowing to interlock supramolecular assemblies which
incorporate a cyclophane with a comparable cavity size to CyOMe8.[39]
Scheme 1. Molecular structures of all building blocks for the assembly of daisy chains 5-[a2], 5-[c2] and stoppered
monomer 5-[a1]. Molecular structures of daisy chains are given in Scheme 4.
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Scheme 2: Synthetic route towards cyclophane 2. (a) 1,4-dichlorobutane, K2CO3, DMF, 110 °C, 3 h, 94 %; (b) i-PrMgCl,
n-BuLi, 1-acetyl-4-piperidone, THF, -5 °C, 20 min, 52 %; (c) o-cresol, BF3 × Et2O, r.t., DCM, 18 h, 90 %; (d) ICl
(NaI/NaOCl), NaOH, MeOH/H2O, 0 °C, 76 % (quant.); (e) 1,4-dichlorobutane, NaH, DMF, 50 °C, 6 h, 92 %; (f) Cs2CO3,
DMF, 100 °C, 18 h, 41 %; (g) TMS-acetylene, THF, Et3N, Pd(PPh3)2Cl2, PPh3 CuI, r.t., 20 h, 89 %; (h) LiAlH4, DIBAL-H,
THF, -10 °C, 30 min, r.t., 30 min, then K2CO3, MeOH/DCM, r.t., 30 min, 91 %; (i) EtI, THF, 4 d, r.t., then KPF6, H2O,
97 % .
Receptor synthesis (Scheme 2): Synthesis of cyclophane 2 starts by alkylation of 4-bromosyringol
(6) in DMF with 1,4-dichlorobutane in presence of potassium carbonate, furnishing 7 in 94 % yield.
Initial attempts to prepare 8 by lithiation of 7 followed by nucleophilic addition to 1-acetyl-4-
piperidone resulted in rather low and poorly reproducible yields of the addition reaction
regardless of the reaction temperature. Therefore, a Grignard reaction was attempted, starting
with a bromine/magnesium exchange using the (n-Bu)2(i-Pr)MgCl magnesate complex [45] in THF
solution. Attempts to perform this reaction with i-PrMgCl × LiCl [46] did not result in any metalation
of the aryl bromide. The nucleophilic addition then resulted in formation of 8 in an acceptable
and reproducible yield of 52 %. The following Friedel-Crafts alkylation of o-cresol with 8 in DCM
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with BF3 × Et2O as a Lewis acid furnished 9 in 90 % yield. The choice for o-cresol over 2-
methoxyphenol in this step was simply due to the high regioselectivity of the Friedel-Crafts-
reaction compared to 2-methoxyphenol. For the latter, substitution in the 5-position (para to the
methoxy group) always occurred to a small extent. This resulted in a mixture of regioisomers
which could not be purified on a preparative scale. Ortho-iodination of the cresyl moiety in
methanol/water under basic conditions with ICl afforded 10 in 76 % yield after column
chromatography (CC). On a small scale, 10 could be obtained in quantitative yield by in situ-
generation of the electrophilic iodonium species, using sodium iodide and sodium
hypochlorite.[47] This reaction unfortunately tended to form poorly separable chlorinated species
and oxidation products upon upscaling of the batch size. The following alkylation with an excess
of 1,4-dichlorobutane was conducted in DMF solution using NaH as a base, which resulted in 92 %
yield for cleft 11. Intermolecular cyclization of 11 and bisphenol 12 in DMF in the presence of
Cs2CO3 yielded 13 in 41 % on a multigram scale, which is a typical yield for this type of
cyclization.[35,36] It was found that preheating of the oil bath and immediate, vigorous stirring is
essential to obtain reproducible yields. Introduction of a TMS-protected acetylene by
Sonogashira-cross-coupling in DCM/Et3N proceeded in a yield of 89 % for 14. This was followed
by a reduction with a combination of LiAlH4/DIBAL-H in THF at -10 °C. The combination of both
reagents and careful temperature control resulted in a clean conversion, avoiding incomplete
reduction or N-deacetylation which sometimes occurred when using DIBAL-H or LiAlH4 alone. As
the silyl group was only partially cleaved during basic workup, complete desilylation was achieved
in DCM/MeOH solution in the presence of K2CO3, yielding 91 % for 15 over both steps.
Quaternization with ethyl iodide and subsequent precipitation from aqueous solution by addition
of KPF6 afforded 2 * 2 PF6 in an excellent yield of 97 %.
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Scheme 3. Synthetic routes towards OPE 3, stopper 4 and daisy chain monomer 1. (a) n-BuOH, H2SO4, cyclohexane, reflux, 2 h,
78 %; (b) Br(CH2)2Cl, K2CO3, DMSO, r.t., 3 d, 83 %; (c) NaN3, DMSO, 80 °C, 2 d, 88 %; (d) 1,4-diethynylbenzene, Pd(PPh3)2Cl2, CuI,
THF/Et3N, r.t. 30 min, 55 %; (e) NaN3, DMSO/PhMe, 120 °C MW irradiation, 2 h, 58 %; (f) 1,4-diethynylbenzene, Pd(PPh3)2Cl2, CuI,
THF/Et3N, 50 °C, 1 d, 63 %; (g) NaOH, H2O/MeOH/THF, r.t., 20 h, then HCl, 99 %; (h) NaOH, i-PrOH/THF, H2O, r.t. 90 min, 74 %; (i)
H(OC2H4)4N3, TBTA, [Cu(CH3CN)4]PF6, acetone, r.t., 30 min, 93 %; (j) TBAF, THF, r.t., 6 h, 79 %; (k) TBTA, [Cu(CH3CN)4]PF6, DMF,
Et3N, r.t., 24 h, 30-64 %.
Synthesis of hermaphroditic monomer 1, OPE-rod 3 and stopper 4 (Scheme 3): Esterification of
commercially available 5-iodosalicylic acid (16) with n-butanol gave the butyl ester 17 in 78 %
isolated yield. Alkylation of 17 with 1-bromo-2-chloroethane provided 18 in a yield of 83 %.
Chloroethyl substituted OPE 20 was obtained by Sonogashira cross-coupling of 18 with 1,4-
diethynylbenzene in 55 % yield. A nucleophilic substitution with sodium azide in DMSO/Toluene
solution under microwave irradiation at 120 °C afforded azidoethyl-substituted OPE 21 in 58 %
yield. Purification of this reaction mixture required extensive CC due to incomplete substitution
and formation of byproducts with very similar retention times on silica gel. Fortunately, the
situation improved considerably by treating 18 with sodium azide in DMSO, resulting in the azide-
functionalized OPE precursor 19 in an isolated yield of 88 %. Sonogashira cross-coupling of 19
with 1,4-diethynylbenzene afforded OPE-rod 21 in a yield of 63 % after a short CC to remove
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catalyst residues. Subsequent basic ester hydrolysis in a mixture of water, methanol and THF
followed by precipitation with hydrochloric acid afforded 3 in 99 % yield. The sodium salt was
obtained by reacting 3 in i-PrOH/THF suspension with aqueous NaOH and subsequent
recrystallization from H2O/EtOH.
Acetylene-functionalized, water-soluble stopper 4 was obtained starting by a click
reaction of 22 with tetra(ethylene glycol) monoazide in acetone, using tetrakis(acetonitrile)-
copper(I)-hexafluorophosphate with tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) as
Cu(I)-stabilizing ligand.[48] This procedure gave 23 in an excellent yield of 93 % after CC.
Subsequent desilylation using TBAF in THF solution afforded 4 in 79 % yield. The assembly of daisy
chain monomer 1 was achieved in DMF by CuAAC of 2 with an excess of 3, using again
tetrakis(acetonitrile)copper(I)-hexafluorophosphate and TBTA. Addition of triethylamine was
crucial for an efficient transformation, presumably due to protonation of the Cu(I)-stabilizing
TBTA ligand by 3 in absence of a base. Surprisingly, attempts to isolate the reaction products by
size exclusion chromatography on Sephadex LH-20 or C-25 failed. It was however possible to
remove a major fraction of excess 3 prior to rigorous purification by preparative C-18 reversed-
phase chromatography. 1 * 2 HCOOH was obtained in varying yields between 30 % and 64 %
depending on the extent of purification required. 0.3 % Formic acid was employed as buffer acid
for the HPLC gradient (5 % to 90 % CH3CN in H2O in 13 minutes), fractions could be conveniently
evaporated to dryness by rotary evaporation at heating bath temperatures of 25 - 30 °C. When
using trifluoroacetic acid the formation of decomposition products was observed during
evaporation or upon standing. Usage of a basic additive such as triethylammonium bicarbonate
hampered chromatographic resolution and was therefore no acceptable option. The quality of all
fractions were analyzed prior and after evaporation. If any sign of impurities was detected by
HPLC/ESI-MS, the purification procedure was either repeated or the fractions were discarded.
The excessive purification of 1 was mandatory to avoid the formation of complex libraries of
products in the synthesis of interlocked daisy chains. In order to obtain reproducible analytical
HPLC chromatograms it was essential to employ a RP-C18 column which has either been in use
for several weeks or to artificially age a new column. For reasons that we were not able to
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rationalize completely so far, 1 could either not be eluted or excessively high retention times were
observed from new, previously unused columns. Interestingly, this peculiar behavior was
exclusively observed for 1, while neither 2 nor 3 displayed a particular sensitivity to the history of
the column.
Aggregation behavior: To gain insight into the association behavior of the subunits of 1, the
complexation behavior of OPE 3 * 2Na with CyOMe8 * 2 Cl was investigated as model system. An
association constant of 4 × 106 M-1 was determined by isothermal titration calorimetry, which was
the expected high value considering the combination of hydrophobic complexation and the
additional electrostatic contribution to the total binding strength (Figure SI 1).[37] 1H-NMR spectra
of 3 * 2 Na at 0.5 mM concentration in D2O (Figure SI 2) display a typical complexation-induced
upfield shift of the phenylene protons from δ = 7.6 to 5.5 ppm upon addition of 1 eq of CyOMe8
* 2 Cl as host. The more than 2 ppm shift is typical for aromatic protons encircled by CyOMe8 * 2
Cl and structurally related hosts at saturation binding in aqueous solution.[22] As consequence of
the high association constant, complexation reached saturation in this NMR experiment and no
significant changes of the guest protons were observed upon further host addition, pointing at
the expected predominant 1:1 host/guest stoichiometry. In the NMR sample with an excess of
host, two separated sets of aryl signals for the complexed and uncomplexed host are observed,
1
pointing at a slow exchange of the complexing host on the H-NMR timescale (500 MHz). In
conclusion, the complexation behavior of the subunits was promising and in analogy, a strong
self-aggregation of 1 was expected in water displaying a slow exchange kinetics. Also promising
was the conclusion, that an aggregation mode with more than one receptor moiety per OPE rod
seemed unlikely for 1.
In spite of its tetraionic nature, 1 required addition of a significant proportion of an organic
co-solvent as it precipitates from pure water. A water to methanol ratio of 4:1 turned out to be a
good compromise for complexation studies. Qualitative interpretation of concentration
1
dependent H-NMR spectra in 4:1 D2O/MeOD (Figure 2) revealed that below 1 mM, distinct
resonances at about δ = 5.4 ppm occur. In analogy to the shifts observed in the model system
3CyOMe8, these signals can be attributed to the phenylene moiety of the OPE rod being
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encircled by the cyclophane moiety. To get the chemical shifts of these hydrogens in an
uncomplexed state, a sample of 1 with a concentration of 26 mM in a low polarity solvent mixture
(THF-d8/D2O = 4:1) was recorded. In that case no signals at δ = 5.4 ppm were detectable, but a
singlet at δ = 7.3 ppm pointed at the complete dissociation of intermolecular complexes. Again,
the complexation induced shift of approx. 2 ppm matches well with the observations in the model
system (3CyOMe8). In contrast to the model system however, the appearance of coupling
patterns upon complexation reflects the unsymmetrical environment of the encircling receptor.
Furthermore, the occurrence of more than one set of multiplets indicates the presence of more
than one aggregate in the equilibrium. On the basis of the signals at 5.4 ppm, it seems that even
at low concentrations like 50 μM, a significant degree of complexation occurs. Above a
concentration of about 1 mM, all peaks become increasingly broad, pointing at larger aggregates,
either of longer interlinked daisy chain oligomers or of unspecific hydrophobic aggregates.
In conclusion, the aggregation studies corroborate the expected efficient assembly and
aggregation of the molecular species involved. However, the structural and stoichiometric variety
1
of possible aggregates does not allow to determine an association constant from H-NMR data
for the desired supramolecular daisy chain dimer 1-[c2]. In order to gain insight into the structural
variety of the species formed, the “stoppering” strategy displayed in figure 1 was applied to freeze
the transiently formed superstructures mechanically.
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Figure 2. Concentration-dependent 1H-NMR spectra (500 MHz. expanded aromatic region) displaying the self-inclusion or
aggregation behavior of 1 in D2O/MeOD (4:1) at 298 K. For comparison, a 1H-NMR spectrum in an uncomplexed state (600 MHz,
4:1 THF-d8/D2O) is shown at the bottom.
Synthesis and characterization of interlocked daisy chains: Daisy chains were synthesized in a
stoppering click reaction of 4 with 1 in 1 mM H2O/MeOH (4:1) solution, using 1.5 equivalents of
the stopper 4 (Scheme 4). As catalytic system, 50 mol% copper(II) sulfate/tris{[1-(3-
hydroxypropyl)-1H-1,2,3-triazol-4-yl]methyl}amine (THPTA)[48] in the presence of 15 mM sodium
ascorbate were employed. The presence of 15 mM sodium carbonate served for pH stabilization.
After 1 day of stirring, analysis of the crude mixture by HPLC/ESI-MS revealed first evidence for
the formation of interlocked aggregates.
The HPLC/ESI-MS set-up allows not only the separation of the crude mixture into different
fractions, it enables also to analyze the contribution of each band to a particular ion current. The
UV signal and the total ion current displayed as the two top chromatograms of figure 3 display 4
peaks, from which the three later ones are labeled with colors and seem to have some broader
signals as background. The first signal at about 6.5 minutes belongs to the residual unreacted
stopper 4. The identity of the remaining 3 peaks can be assigned by analyzing their contribution
to a particular ion current. The panel representing the extracted ion current (EIC) 1052+
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represents all structures able to make a signal with m/z = 1052. These are all (super)structures
consisting exclusively of the stoppered and doubly charged monomer, a criterion that can be
satisfied by the doubly protonated monomer (5-[a1]+2H)2+ itself, but also by the correspondingly
fourfold protonated mechanically interlocked dimers (5-[a2]+4H)4+ and (5-[c2]+4H)4+. As
consequence, the EIC 1052+ chromatogram is fed by contributions from all 3 peaks of interest. Of
course these are only the most prominent examples, any higher interlocked oligomer consisting
of doubly protonated monomers also contribute to the EIC 1052+ chromatogram, what explains
the additional weak and broad background in the panel. More selective is the next panel
representing the contributors to EIC 701+. The signal represents the triply protonated monomer
(5-[a1]+3H)3+ and mechanically interlinked superstructures consisting thereof. However, a sixfold
protonation of the dimeric superstructures is considerably less likely, and thus the contribution
of (5-[a2]+6H)6+ and (5-[c2]+6H)6+ to the 701+ signal are existing, but very minor. It thus becomes
apparent that the last peak in the chromatogram (green in Fig. 3) belongs to the stoppered
monomer 5-[a1]. For the peak assignment instrumental is the EIC 842+ signal, which belongs to
fivefold protonated and interlocked dimers, as there is no charging state allowing the monomer
to contribute. As displayed in figure 3, only the dark yellow and brown peak are contributing to
this signal and thus, these two peaks are the mechanically interlocked dimers 5-[a2] and 5-[c2].
The assignment of the more intense dark yellow peak to the desired 5-[c2] is based on the isolated
yields (see next section) and the rational that the dimer 5-[a2] resembles in its interaction with
the column more the monomer 5-[a1] and is thus expected belonging to the peak with a retention
time closer to the monomer. A similar argument can be made for 7 fold protonated trimeric
superstructures resulting in an EIC of 902+. And indeed, the peaks contributing to the EIC 902+
suggest that the broad background signals of the chromatogram are mainly due to traces of
mechanically interlocked higher oligomers.
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Figure 3. Analytical HPLC/ESI-MS traces of the
crude stoppering reaction. From top to
bottom: HPLC chromatogram (UV-Vis, 200-600
nm; positive total ion current (TIC+); extracted
positive ion currents (EIC, m/z +). The colored
peaks were separated by preparative HPLC and
the corresponding products were character-
ized. At ~ 6.5 min retention time residual
unreacted stopper is eluted.
The promising HPLC/ESI-MS analysis of the reaction mixture moved the isolation of the major
superstructures into the focus of interest. Tedious purification by preparative C-18 reversed-
phase HPLC provided 5-[c2] * 4 HCOONH4 in 30 % yield, along with 5-[a2] * 4 HCOONH4 (3.0 %)
and 7.3 % of 5-[a1] * 2 HCOONH4 (From this point, the ammonium formate content will be
neglected in the product designations). Two impure fractions with trace amounts probably of a
trimeric daisy chains were obtained as judged from LC/MS analysis. A total of 6 consecutive
preparative HPLC runs were required to provide sufficient quantities for characterization. During
each run (H2O/CH3CN gradient 20 – 70 % in 25 min, 0.3 % formic acid) 30 to 40 fractions were
collected. All of them were analyzed by HPLC/ESI-MS and subjected to further purification if
required. Rotary evaporation at 25 °C and neutralization of the dry product with ammonia
solution allowed to maintain a fast workflow to avoid the appearance of decomposition products.
After evaporation, all products required a mixture of THF or CH3CN and water to be readily
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soluble. They tended to form poorly soluble films on glass walls, which sometimes required
several minutes to be dissolved. Similar to 1, the daisy chains were susceptible to decomposition
in the presence of trifluoroacetic acid and required aged HPLC columns to show reproducible
retention times. No dissociation of the daisy chains to the monomeric species was observed
during evaporation. 6-[a1] was equally susceptible to decomposition as 6-[a2]/[c2].
Characterization of the daisy chains: HRMS analysis confirmed the dimeric nature of 5-[a2],
5-[c2] and the monomeric nature of 5-[a1]. The identity of the compounds which were suspected
to be trimers could unfortunately not be confirmed by HRMS. The two interlocked daisy chain
1
species 5-[a2], 5-[c2] and the stoppered monomer 5-[a1] show distinctly different H-NMR
spectra in the aromatic region (Scheme 4). As a global feature, all spectra comprise the expected
signals from the stopper moiety between δ = 8 - 9 ppm. 5-[c2] could be identified by the absence
of phenylene proton resonances at δ = 7.3 – 7.6 ppm and the appearance of signals between δ =
5.5 – 5.8 ppm, which occur as two separate sets of AB coupling patterns. Their chemical shift and
the appearance of coupling patterns strongly resemble the phenylene resonances in the dilution
series of 1 in D2O/MeOD (Figure 2). A 4:1 integration ratio of the combined phenylene resonances
to the lowest-field triazole resonance of the stopper moiety confirms the cyclic dimeric structure.
NOESY cross-peaks of the phenylene protons to the cavity aryl- and methoxy protons further
corroborate the nature as an interlocked species. The occurrence of an AB coupling pattern of the
phenylene moiety can be explained by a fast axial rotation within the unsymmetrical environment
of the cyclophane. The two separate sets of phenylene AB resonances suggest that 5-[c2] was
isolated as a mixture of its meso form and chiral diastereomers.[9,49] This was found to occur in
other examples of daisy chains composed of unsymmetrical subunits.[50–52] A 1:1 integration ratio
between the two AB-systems reveals that no diastereoselective assembly occurs despite the
relatively rigid nature of 1. The different chemical shift of the complexed phenylene protons
indicates a different spatial arrangement of the OPE rods within the cavity. Thus it can be
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concluded that the alkyl linkage between OPE-rod and cyclophane provides enough flexibility to
allow the formation of two equally stable [c2]-daisy chains. Other doubled signals with equal
integration ratios but less pronounced differences in chemical shift were observed for the
stopper- and salicyl moieties of the OPE rod (See Figure SI-45 - 48). Most remarkable is a set of
split resonances belonging to the diastereotopic methylene protons of the two methylene units
between cyclophane and OPE rod. This can be explained by a hindered rotation within the
sterically crowded, unsymmetrical environment between the cyclophane cavities. In contrast, the
rotation of the methylene units between OPE rod and stopper is unrestricted, therefore the
diastereotopic protons are indistinguishable. 5-[a2] could be identified by the presence of one
set of doublets of the OPE resonances between δ = 5.5 and 5.7 ppm as well as of a singlet
remaining at 7.41 ppm. These signals affirm the presence of one complexed and one
uncomplexed OPE rod. In addition to this, the resonances of the cavity aryl protons are split into
one set of occupied, low-field resonances and unoccupied, high field resonances. Due to the small
amount of this substance available, no satisfactory NOESY/ROESY spectra could be recorded. 5-
[a1] can clearly be distinguished from the interlocked species by the absence of any phenylene
signals around δ = 5.5 ppm. The phenylene proton resonance occurs as a singlet at a comparable
shift (7.44 ppm) to the spectrum of 1. Diffusion constants obtained from DOSY NMR reveal similar
diffusion constants for 5-[a2] and 5-[c2] which were much smaller than for 5-[a1]. The active
volumes obtained from the diffusion constants provided a 3.2 times larger value for the
mechanically interlocked dimer 5-[c2] and 3.7 times larger volume for 5-[a2] compared to the
stoppered monomer 5-[a1]. While the slightly more compact nature of 5-[c2] compared to 5-[a2]
is reflected in the slightly smaller hydrodynamic radius, the active volumes for both dimers are,
based on the monomer, considerably larger than expected. For the compact arrangement in the
interlocked superstructures a smaller value than twice the monomer volume was anticipated.
While the measurement confirms a larger hydrodynamic radius of the dimeric daisy chains, the
surprising extent of the increase might be caused by concentration effects and/or differences in
solvation. The interlocked daisy chains did not exhibit any deslipping behavior upon storage in
the NMR solvent (CD3CN/D2O 10:1) at room temperature for several days, confirming the efficacy
of stopper 4 to disrupt supramolecular exchange.
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Scheme 4.Top: molecular structures of 5-[a1] (meso form shown representative for all 3 isomers), 5-[a2] and 5-[c2]. Bottom: 1H-
NMR spectra (600 MHz, CD3CN/D2O = 10:1) of the three species above. Assignments of the peaks for 5-[a1] and 5-[c2] are based
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on NOESY spectra and integration ratios. For 5-[a2] it was required to rely on integration ratios and comparison to the first two
compounds.
The UV-Vis and fluoresence spectra are very comparable for all three isolated stoppered
compounds 5-[a1], 5-[a2], and 5-[c2]. As displayed in figure 4, the UV-Vis spectra of all three
compounds have the absorption maxima at 332 nm and a shoulder at 359 nm is clearly visible for
both mechanically interlocked dimers (5-[a2] and 5-[c2]) but is also adumbrated for the
stoppered monomer 5-[a1]. In analogy, also the fluorescence spectra of the three compounds
are comparable and display emission maxima at 367 nm with a shoulders at 385 nm (Figure 4,
solid lines). While no shift of the emission peaks was observed upon complexation, the intensity
of the emission signal increases with the number of complexed OPE subunits. The symmetric 5-
[c2] with both rods inside a cyclophane displayed the most intense signal and the monomeric 5-
[a1] with its OPE rod exposed to the solvent displayed the weakest emission signal. While the
variation of the emission intensity was expected, the complete absence of a shift of the signal
came as a surprise. During the preliminary investigations, the subunit OPE 3 * 2 Na was titrated
with CyOMe8 * 2 Cl (Figure SI 4) and displayed both, a decrease of approximatealy 50 % of the
emission intensity of the broad signal at 387 nm and the emergence of a hypsochromically shifted
peak at 381 nm. Concentration-dependent emisson specta were also recorded from the
unstoppered precursor 1 in H2O/MeOH = 4:1 (Figure SI 18). These spectra resemble those of the
stoppered daisy chains, displaying an emission maximum at 367 nm with a shoulder at 383 nm.
After correcting the recorded spectra for the concentration dependent optical density, no
additional variation of the emission intensities due to aggregation was observed for 1. In
summary, both, the mechanically fixed isolated model compounds 5-[a2] and 5-[c2] as well as
the free and stoppered monomer 1 and 5-[a1] showed only very minor aggregation induced
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variations of their optical spectra in the solvent mixture of interest (H2O/MeOH = 4:1) and are
thus not suited for titration experiments monitored by optical spectroscopy.
Figure 4. UV-Vis absorption spectra (dotted lines) and emission spectra (solid lines) of 0.1 μM 5-[a2], 0.1 μM 5-[c2], and 0.2 μM
5-[a1] in H2O/MeOH = 4:1, measured with λex = 332 nm. Absorption spectra are normalized and offset in line with the emission
maxima. The relative intensities of the emission spectra were corrected for differences in the optical density at 332 nm. Absorption
coefficients at λ=332 nm ε (L mol-1 cm-1) = 5-[a1], 0.5 × 105; 5-[a2], 1.0 × 105; 5-[c2], 1.3 × 105.
Conclusion
Interlocked molecular daisy chains 5-[c2] and 5-[a2] were assembled from zwitterionic precursor
1 and stopper 4. As a consequence of the short, flexible linkage between cyclophane and OPE,
entropically favored [c2]-daisy chains were obtained as the main product. By NMR spectroscopy,
the interlocked nature of both daisy chains could be verified and further corroborated by HRMS.
The [c2]-daisy chains were obtained as a 1:1 mixture of meso/chiral diastereomers as evidenced
by NMR analysis. 1 represents one of the few examples from which interlocked daisy chains
comprising a rigid, extended π-conjugated rod were synthesized.[14] To the best of our knowledge
this is the only example where this could be accomplished in aqueous environment. A small
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10.1002/chem.201803944
Chemistry - A European Journal
amount of organic additive was still required to achieve significant solubility which is a
consequence of the large organic structure combined with the zwitterionic character of the
molecule. The yield of [c2]-daisy chains in this work was respectable, though isolation was
handicapped due the extensive purification protocol and challenging stability features of the
compounds. Strong associations are recorded for both, the daisy-chain precursor 1 and for the
separated subunits 3 and CyOMe8. The combination of high association constants and peripheral
azide- and alkyne functionalization makes the building blocks presented herein promising building
blocks for the assembly of mechanically interlocked molecules in aqueous environments.
Acknowledgments
Support for NMR measurements from Claudio Meyer and Thomas Müntener is gratefully
acknowledged. Felix Fleckenstein is acknowledged for synthetic support during his practical
course and Dr. Loïc LePleux for proofreading the manuscript. Generous financial support by the
Swiss Nanoscience Institute (SNI grant number P1303) and the Swiss National Science Foundation
(SNF grant number 200020-178808) are gratefully acknowledged.
Experimental
Experimental details and synthetic procedures of all daisy chain precursors are given in the
electronic supporting information.
Synthesis of daisy chains 5-[a1], 5-[a2] and 5-[c2]:
Sodium carbonate (26.2 mg, 0.248 mmol) and 1 * 2 HCOOH (26.5 mg, 16.5 μmol) were dissolved
in 15 mL H2O/MeOH 4:1, sonicated for 1 min and stirred for 10 min giving a clear solution. The
catalyst was prepared separately by dissolving copper(II)-sulfate heptahydrate (2.1 mg, 8.3 μmol,
0.5 eq), sodium-L-ascorbate (6.5 mg, 33 μmol, 2 eq) and tris{[1-(3-hydroxypropyl)-1H-1,2,3-
triazol-4-yl]methyl}amine (3.6 mg, 8.3 μmol 0.5 eq) in H2O/MeOH 4:1 (1.5 mL). Both solutions
were degassed with a stream of Ar for 2 min and then vigorously stirred. It was stirred for 3 days
at room temperature in the absence of light. The solvent was removed in vacuo at a heating bath
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temperature of 30 °C. The crude mixture was redissolved in H2O/CH3CN 5:1 + 0.3 % formic acid
(4.5 mL) and subjected to preparative RP-C18 HPLC (from 20 to 70 % CH3CN in H2O 25 minutes)
in 2 runs in equal portions. Final purification was then achieved by an additional 4 consecutive
preparative HPLC runs. Before each run, the pH of the solution was checked to match the starting
conditions on the HPLC column. The fractions were analyzed by HPLC/ESI-MS, combined and
evaporated to dryness as fast as possible by rotary evaporation with a bath temperature of 25-30
°C. After evaporation, the product was redissolved in a small amount of CH3CN/H2O (2:1),
neutralized by a few drops of 25 % ammonia solution and then evaporated to give the daisy chains
5-[a2] * 4 HCOONH4 (1.1 mg, 3.0 %) and 5-[c2] * 4 HCOONH4 (11 mg, 30 %) and the stoppered
monomer 5-[a1] * 2 HCOONH4 (2.7 mg, 7.3 %), total yield 40.3 %. HPLC chromatograms, detailed
NMR- and HR-mass spectra are given in the supporting information (Pages SI-24-45).
Keywords: daisy chain, rotaxane, cyclophane, hydrophobic complexation, click-chemistry
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