Self-assembly and stability of double rosette nanostructures with biological functionalities

Article abstract of DOI:10.1039/b508449k

The syntheses of calix[4]arene dimelamines that are functionalized with alkyl, aminoalkyl, ureido, pyridyl, carbohydrate, amino acid and peptide functionalities, and their self-assembly with barbituric acid or cyanuric acid derivatives into well-defined h

Full text of DOI:10.1039/b508449k

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A R T I C L E
Self-assembly and stability of double rosette nanostructures with
biological functionalities†
Mattijs G. J. ten Cate, Merdan Omerovic´, Gennady V. Oshovsky, Mercedes Crego-Calama*
and David N. Reinhoudt*
Laboratory of Supramolecular Chemistry and Technology, MESA+ Institute for
Nanotechnology, University of Twente, P.O. Box 217, 7500, AE Enschede, The Netherlands.
E-mail: m.cregocalama@utwente.nl; Fax: +31 53 489 4645; Tel: +31 53 489 2978
Received 15th June 2005, Accepted 2nd August 2005
First published as an Advance Article on the web 24th August 2005
The syntheses of calix[4]arene dimelamines that are functionalized with alkyl, aminoalkyl, ureido, pyridyl,
carbohydrate, amino acid and peptide functionalities, and their self-assembly with barbituric acid or cyanuric acid
derivatives into well-defined hydrogen-bonded nanostructures are described. The thermodynamic stability of these
hydrogen-bonded assemblies was studied by CD spectroscopy in mixtures of CHCl3 and MeOH. The stability of the
assemblies depends on several steric factors and the polarity of the functional groups connected to the assembly
components.
Introduction
Natural receptors exhibit extraordinary high binding affinity
and selectivity for a particular substrate. The binding affinity
and selectivity arises from the cooperative action of many chain
functionalities oriented in a three-dimensional fashion around
the target molecule.
In general, a rational-based design of synthetic molecules
is used to mimic the binding properties of natural receptors.
However, large covalent systems are difficult to synthesize and
structural diversity can only be introduced into the scaffolds
at the expense of challenging and time consuming synthesis.
Therefore, self-assembly of nanostructures using multiple weak
noncovalent interactions is considered a powerful tool for the un-
derstanding, modelling, and mimicking of biological systems.1 A
major advantage is that self-assembled structures are generally
formed under thermodynamically controlled conditions, often
giving rise to quantitative yields of product.2
The concave 3D positioning of different functionalities
around a central cavity by self-assembly provides the basis for
natural receptors to bind substrates and to catalyze biochem-
ical transformations with exquisite regio- and stereoselectivity.
Natural antibodies provide a perfect example of self-assembled
receptors. They consist of four different peptide chains, two
heavy (H) and two light (L) chains which are held together
mainly by noncovalent interactions, reinforced by covalent
disulfide bonds.3 Natural antibodies have a hypervariable region
(Fab) and a constant region (Fc). Because differences between
antibodies arise from differences in the Fab fragments, it is
not surprising that specific antigen recognition sites are located
in this part of the antibody. Such self-assembled systems
provide nature with a powerful tool to generate many different
antibodies.
Fig.
1 Self-assembly of small functional groups via synthetic
self-assembled platforms (rosettes).
guest-binding region (variable region). Furthermore, to mimic
natural antibodies structural diversity has to be introduced to
the double rosette assemblies. Structural diversity in the guest-
binding region of these synthetic receptors can be generated
both at a covalent level (variation in functional groups) and at
a supramolecular level (statistical combination of fixed number
of molecular components, Fig. 1).
In general, a major drawback in the use of hydrogen-bonded
supramolecular systems is their low stability in polar solvents,
because these solvents compete with the hydrogen-bonding
motif that holds the system together. On the other hand,
natural host–guest recognition events take place in aqueous
environments or at the cell membranes. Therefore, the study
of the formation and the stability of self-assembled hydrogen
bonded systems in polar solvents is very important for the
understanding and the mimicking of natural systems.
Here, we focus on the formation and stability of differently
functionalized hydrogen-bonded assemblies in competitive sol-
vent mixtures of different polarity. Both steric hindrance induced
by linear or branched alkyl chains and the effect of simple polar
functionalities, such as amino alkyl, ureido, and pyridyl moieties
on the stability of double rosettes are investigated. Subsequently,
the stability of more complex assemblies bearing sugars, amino
acids or small peptide fragments is examined.
For the design of synthetic receptors, a strategy was developed
in our group that is similar to the assembly of antibodies.4
This approach involves the formation of a synthetic self-
assembling platform in which a double rosette motif (constant
region) is formed based on multiple hydrogen bonds (Fig. 1).5
Functionalization of the individual components with small
functional groups (X or Y in Fig. 1) gives noncovalent assemblies
with diverse molecular fragments generating a potential 3D-
Results and discussion
The thermodynamic stability of the designed double rosette as-
semblies (Fig. 2) might be affected when different functionalities
are introduced in the building blocks. For example, hydrogen
bond donating or accepting groups can possibly interfere with
the rosette platform formation, while bulky groups can cause
steric strain in the assembly and decrease their thermodynamic
stability.6
† Electronic supplementary information (ESI) available: experimental
procedures and compound characterization. See http://dx.doi.org/
10.1039/b508449k
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 2 7 – 3 7 3 3
3 7 2 7
©

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Fig. 2 Formation of the double rosette assemblies 1a3·(DEB/BuCYA)6 from three calix[4]arene dimelamines 1a and six 5,5-diethylbarbiturate
(DEB) or n-butyl cyanurate (BuCYA) molecules.
The double rosette assemblies with dimensions of ca.
3.0 × 3.3 × 1.2 nm are formed spontaneously by mixing of
calix[4]arenes diametrically substituted at the upper rim with
two melamine units (for example 1a, Fig. 2) with 2 equivalents of
either barbituric acid (BAR) or cyanuric acid (CYA) derivatives.
The assemblies 13·(DEB/CYA)6 consist of two rosette motifs,7
connected via three calix[4]arene molecules. The assemblies
are held together by 36 hydrogen bonds and are stable in
apolar solvents like chloroform, benzene and toluene even at
10−4 M.5a,8
With the aim to generate stable and structural diverse
double rosettes, calix[4]arene dimelamines bearing alkyl (1a–
c), aminoalkyl (2a–g), ureido (3), pyridyl (4a–c), sugar (5a–
e), amino acid (6a–f), dipeptide (7a–e), and tripeptide (8a–b)
functionalities were synthesized (Chart 1). Subsequently, the
thermodynamic stability of the corresponding rosette with these
dimelamines was determined after their self-assembly with DEB,
BuCYA, BzCYA, (R)-PhEtCYA or (S)-PhEtCYA in a mixture
of chloroform and an increasing percentage of a competing
solvent for hydrogen bonding.
Synthesis and characterization of assemblies with different
functionalities
Compounds 1–8 were synthesized starting from calix[4]arene
bis(chlorotriazine) 9.8 Dimelamines 2 were synthesized by
reaction of 9 with an excess of the corresponding diaminoalkane
◦
at 90 C (2a–g, 53–92%). Reaction of 2g with excess of propyl
isocyanate in THF at room temperature afforded dimelamine 3
(78%).9 Syntheses of dimelamines 4 are described elsewhere.10
Reaction of 2g with tetra-O-acetyl-b-D-glucopyranosyl-
isothiocyanate and hepta-O-acetyl-b-D-cellobiosyl-isothio-
cyanate11 in THF gave dimelamines 5a (36%) and 5c (88%), res-
pectively. Successive deacetylation of 5a and 5c with a catalytic
amount of a sodium methoxide solution afforded dimelamines
5b and 5d, respectively. Compound 5e was obtained in 41%
yield by coupling of 2g and gulonic acid with HBTU in the
presence of HOBT and DIPEA in CH2Cl2 at room temperature.
Dimelamines 6a–d were obtained by reaction of 9 with excess
of glycine ethyl ester (6a, 74%), L-alanine methyl ester (6b, 74%),
L-methionone methyl ester (6c, 69%), or L-lysine(Boc) methyl
Chart 1 Molecular structures of dimelamines 1–9 and BAR/CYA derivatives.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 2 7 – 3 7 3 3
3 7 2 8

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ester (6d, 84%) in presence of DIPEA in THF. Deprotection of
the e-amino group of dimelamine 6d with TFA–CH2Cl2 (1 : 1)
afforded compound 6e in 24% yield. Hydrolysis of compound
6d with sodium hydroxide in THF–MeOH (1 : 1) gave 6f (83%).
Dimelamine 7a (85%) was synthesized by reaction of 9 with
excess of gly–gly ethyl ester in presence of DIPEA in THF. Hy-
drolysis of 6a with sodium hydroxide in THF–MeOH (1 :
1) and coupling of L-alanine methyl ester, L-serine methyl ester
or gly–gly ethyl ester using standard peptide coupling conditions
(HATU, DIPEA in CH2Cl2) afforded 7b (50%), 7c (23%) and 8a
(68%), respectively.
Coupling of Boc-L-glu(OcHx)–OH with isopropyl amine
using peptide coupling conditions (EDC, HOBT, DIPEA in
CHCl3) gave Boc-L-glu(OcHx)–NHiPr (88%), which after de-
protection with TFA–CH2Cl2 (1 : 1) was coupled with 6f using
standard peptide coupling conditions (EDC, HOBT, DIPEA in
CH2Cl2) giving 7d (48%). Dimelamine 7e (66%) was obtained
similarly to 7d but using L-tyrosine methyl ester.
Fmoc-L-gln(Trt)–L-ser(OtBu)–OMe was prepared by cou-
pling of Fmoc-L-gln(Trt)–OH and L-ser(OtBu)–OMe with
HBTU and DIPEA in acetonitrile. Successive Fmoc cleavage
with diethylamine gave L-gln(Trt)–L-ser(OtBu)–OMe (90%).
Coupling of 6f with L-gln(Trt)–L-ser(OtBu)–OMe using stan-
dard peptide coupling conditions (EDC, HOBT, DIPEA in
CH2Cl2) afforded 8b (45%).
The detailed experimental procedures and compound char-
acterization can be found in the electronic supplementary
information.
formation of 36 hydrogen bonds. The change in entropy is
unfavorable, because upon assembly formation the translational
and rotational degrees of freedom of 9 molecular components
are severely reduced.15 The formation of hydrogen bonds favors
the assembly of the double rosettes enthalpically. Hydrogen-
bonding solvents lower this enthalpy, causing double rosettes
eventually to dissociate. The amount of polar solvent required
for dissociation can be taken as a measure for the stability of the
double rosettes.16
The effect of linear (1a) and branched (1b–c) alkyl functionali-
ties on the thermodynamic stability of the corresponding double
rosettes was investigated by CD spectroscopy. Double rosettes
with chiral building blocks display very intense and character-
istic CD spectra5a,8,17 that are not observed for the individual
components. The CD signal is therefore a direct measure of
assembly formation. Since calix[4]arene dimelamines 1a–c are
not chiral, (R)-PhEtCYA was used as chiral building block.
For the CD titration experiments the percentage of methanol
in chloroform was increased while the total concentration of
dimelamine and of (R)-PhEtCYA was kept constant at 3 mM
and 6 mM, respectively. Under these conditions the maximum
concentration of double rosette assemblies is 1 mM. Unless men-
tioned otherwise, CD titrations were started with rosette solu-
tions in neat CHCl3, and subsequently the amount of MeOH was
increased up to MeOH–CHCl3 (1:1). The parameter vpolar solvent
was used to quantify the thermodynamic stability of double
rosette assemblies. vpolar solvent is defined as the percentage of polar
solvent in chloroform at which 50% of the assembly is still intact.
Fig. 4 shows the results of CD titration experiments with
assemblies 1a–c3·((R)-PhEtCYA)6. At MeOH–CHCl3 (1 : 1)
the double rosette 1a3·((R)-PhEtCYA)6 is still present for 58%.
Though vMeOH lies beyond the range of the titration experiment
a vMeOH of 60% was estimated (see Fig. 4). The branched
isopropyl functionalities in 1b3·((R)-PhEtCYA)6 resulted in less
stable double rosettes (vMeOH = 17%). Further branching by tert-
butyl moieties in 1c3·((R)-PhEtCYA)6 gave a further decrease of
stability (vMeOH = 7%). The decrease in thermodynamic stability
is obviously due to a larger steric strain of the rosette platforms
induced by the more voluminous branched alkyl functionalities.
For the series of amino alkyl functionalized double rosette
assemblies 2a–f3·((R)-PhEtCYA)6, no significant change in ther-
modynamic stability was observed when the length of the amino
alkyl chain was increased from aminoethyl to aminoheptyl
(Fig. 5). The vMeOH values are in the range of 39 to 48%.
The amino group present in assemblies 2a–f3·((R)-PhEtCYA)6
The double rosette formation with dimelamines 1–8 and vari-
ous BAR/CYA was confirmed by 1H NMR spectroscopy.12 The
1H NMR spectra of assemblies 1–83·(BAR)6 exhibit diagnostic
signals between d = 14.5–14.0 ppm (Ha), 13.5–13.0 ppm (Hb),
8.5–8.0 ppm (Hc), 7.4–7.2 ppm (Hd), 7.0–6.5 ppm (He,f ), and
1
ca. 6.0 ppm (Hh) (Fig. 3a). H NMR spectra of assemblies 1–
83·(CYA)6 exhibit diagnostic signals between d= 15.0–14.0 ppm
(Ha), 14.0–13.0 ppm (Hb), 8.8–8.3 ppm (Hc), 7.7–7.3 ppm (Hd),
7.2–6.6 ppm (He,f ), and ca. 6.0 ppm (Hh) (Fig. 3b). Formation of
assemblies 1–83·(BAR/CYA)6 was quantified by integration of
the appropriate 1H NMR signals13 and confirmed by MALDI-
TOF mass spectrometry using the Ag+-labelling technique.5b,14
Thermodynamic stability of assemblies with alkyl (1a–c),
aminoalkyl (2a–g), ureido (3) and pyridyl (4a–c) functionalities.
The spontaneous assembly of double rosettes in apolar solvents
is an enthalpy driven process that involves the cooperative
Fig. 3 1H NMR spectra of assembly (a) 1a3·(DEB)6 and (b) 6d3·(BuCYA)6. Spectra were recorded at 300 MHz in CDCl3 at 298 K. (For clarity only
the molecular structure of part of one rosette floor of the assemblies is shown).
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 2 7 – 3 7 3 3
3 7 2 9

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Fig.
4
CD titrations of ᭜: 1a3·((R)-PhEtCYA)6, ꢀ: 1b3·((R)-
PhEtCYA)6, and ꢁ: 1c3·((R)-PhEtCYA)6. Spectra were recorded
in MeOH–CHCl3 mixtures (f MeOH) at 298 K. [1a–c] = 3 mM,
[(R)-PhEtCYA6] = 6 mM.
enhances the solubility in more polar solvents (mixtures)
compared to double rosettes 1a–c3·((R)-PhEtCYA)6, but it does
not enhance the stability. When a CD titration with assembly
2e3·((R)-PhEtCYA)6 was performed in MeOH–CHCl3 mixtures
ranging from neat chloroform to neat methanol (Fig. 5) a
linear decrease of intact assemblies with increasing amount of
methanol was found between ratios 1 : 9 and 8 : 2 (MeOH–
CHCl3). Above a ratio of 8 : 2, complete dissociation of double
rosette 2e3·((R)-PhEtCYA)6 was observed.
Fig. 6 Part of the molecular structure of assembly 33·(DEB)6 where the
back folding of the ureido moieties is shown.17
a large variety of biological molecular recognition processes.18
Therefore, the introduction of carbohydrate oligomers in double
rosette assemblies is very interesting for the mimicking of natural
recognition events. However, before studying the properties of
these assemblies as receptors it is important to assess their
thermodynamic stability.
Double rosette assemblies 5a3·(BuCYA)6 and 5c3·(BuCYA)6,
bearing protected glucosyl and cellobiosyl moieties, respec-
tively, are formed for 80% in CDCl3 as estimated by 1H
NMR spectroscopy.13 Furthermore, only one set of singlets
was observed within the region from 13 to 15 ppm for both
assemblies, indicating the presence of only one diastereomer in
solution.17b,c CD spectroscopy revealed that both 5a3·(BuCYA)6
and 5c3·(BuCYA)6 exhibit P-helicity.
CD titration experiments with 5a3·(BuCYA)6 in MeOH–
CHCl3 gave a vMeOH value of 18% (Table 1). Assembly
5c3·(BuCYA)6 is formed in pure chloroform, but addition of
methanol results in rapid dissociation. Free dimelamine 5c
exhibits a significant CD intensity induced by its 16 chiral
centers that coincide with double rosette 5c3·(BuCYA)6 but the
CD spectrum differs in shape. The observed CD signals are
the sum of the CD intensities attributed to free dimelamine
5c and to assembly 5c3·(BuCYA)6. CD titrations showed that
above MeOH–CHCl3 (2 : 8) the observed CD spectrum matches
the spectrum of 5c, indicating that assembly 5c3·(BuCYA)6 is
completely dissociated. CD titrations indicated that vMeOH is
<5% for 5c3·(BuCYA)6.
Fig.
5
CD titrations of ᭜: 2a3·((R)-PhEtCYA)6, ꢀ: 2b3·((R)-
PhEtCYA)6, ꢁ: 2c3·((R)-PhEtCYA)6, ♦: 2d3·((R)-PhEtCYA)6, ꢂ:
2e3·((R)-PhEtCYA)6, and D: 2f3·((R)-PhEtCYA)6. Spectra were recorded
in MeOH–CHCl3 mixtures (f MeOH
[(R)-PhEtCYA6] = 6 mM.
)
at 298 K. [2a–f]
=
3 mM,
CD titrations with ureido functionalized double rosette
33·((R)-PhEtCYA)6 performed in MeOH–CHCl3 mixtures rang-
ing from neat methanol to chloroform gave a vMeOH of 82%. Thus,
the ureido functionalities in 33·((R)-PhEtCYA)6 increase consid-
erably the stability compared to assembly 1a3·((R)-PhEtCYA)6
bearing butyl moieties. This increase is the result of a very rigid
conformation adopted by the 2,2-dimethylpropyl side chain
allowing the ureido moiety to fold back over the calix[4]arene
aromatic rings and to form an extra hydrogen bond with one of
the nitrogen atoms of the triazine ring (Fig. 6).17
1H NMR spectroscopy in chloroform indicated that assem-
blies 5b3·(BuCYA)6 and 5d3·(BuCYA)6, bearing unprotected
glucosyl and cellobiosyl moieties, are formed for >95 and 76%,
respectively. CD titrations with 5d3·(BuCYA)6 were performed in
a solvent mixture ranging from MeOH–CHCl3 (2 : 8) (where 34%
assembly is formed) to neat MeOH (vMeOH is <20%). As observed
for CD titrations with 5c3·(BuCYA)6, free 5d also contributes
significantly to the observed CD signal. Because the shape
For pyridyl functionalized assemblies 4a–c3·((R)-PhEtCYA)6
a vMeOH of 53% was found, comparable to the stability of
aminoalkyl funtionalized assemblies 2a–f3·((R)-PhEtCYA)6.
In summary, branching of alkyl functionalities cause larger
steric strain of the rosette platforms and decreases the thermody-
namic stability. More polar aminoalkyl or pyridyl functionalities
decrease the thermodynamic stability compared to n-alkyl func-
tionalities but enhance the solubility in polar solvents (mixtures).
The double rosette assembly with ureido functionalities is the
most stable due to the formation of extra hydrogen bonds of the
ureido groups with the triazine rings.
Table 1 Thermodynamic stability for the self-assembly of double
rosettes 5a–e3·(BuCYA)6 in MeOH–CHCl3 mixtures determined by CD
titration experiments (298 K). The maximum possible concentration of
intact assembly over the whole titration is 1.0 mM
R
Assembly
vMeOH (solvent range)
Glucopyranosyl(OAc)4
Glucopyranosyl(OH)4
Cellobiosyl(OAc)7
Cellobiosylsyl(OH)7
Gulonyl
5a3·(BuCYA)6
5b3·(BuCYA)6
5c3·(BuCYA)6
5d3·(BuCYA)6
5e3·(BuCYA)6
18% (0–50% MeOH)
N.d.a
<5% (0–50% MeOH)
<20% (20–100% MeOH)
10% (0–50% MeOH)
Formation and thermodynamic stability of assemblies with
carbohydrate moieties (5a–e). Carbohydrates are involved in
a Not determined.
3 7 3 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 2 7 – 3 7 3 3

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of the CD spectra varies between free 5d and 5d3·(BuCYA)6
it is possible to quantify the double rosette formation. CD
intensities at 260 nm were used because the assembly exhibit
strong CD intensity at this wavelength, while the CD intensity
for free 5d is minimal. From these data De260 values of 147.1
and 0.4 l mol−1 cm−1 were calculated for 5d3·(BuCYA)6 and
free 5d, respectively. In addition, 5d3·(BuCYA)6 was formed
for 25 and 7% in mixtures of MeOH–CHCl3 in 3 : 7 and 4 :
6 volume ratio, respectively. At higher MeOH–CHCl3 ratios,
assembly 5d3·(BuCYA)6 is not formed.
1H NMR spectroscopy indicated that assembly 5e3·
(BuCYA)6, bearing L-gulose moieties, was formed for 65% in
CDCl3. This assembly forms the M-diastereomer. CD titration
measurements in MeOH–CHCl3 gave a vMeOH of 10% (Table 1).
Fig.
7
CD titrations of assemblies 6a3·((R)-PhEtCYA)6 (♦),
Formation and thermodynamic stability of assemblies with
amino acid functionalities (6a–f). For assembly (P)–6a3·((R)-
PhEtCYA)6 a vMeOH value of 43% was obtained (Table 2).
Dimelamine 6a has the achiral amino acid Gly, thus (R)-
PhEtCYA was used for the CD titration experiments to induce
the formation of only one diastereomer.
6b3·((R)-PhEtCYA)6 (D), 6b3·(BuCYA)6 (ꢂ), 6c3·(BzCYA)6 (ꢀ),
6d3·(DEB)6 (᭹), 6d3·(BuCYA)6 (᭜), and 6d3·(BzCYA)6 (ꢁ). Spectra
were recorded in MeOH–CHCl3 (f MeOH) mixtures at 298 K where the
maximum assembly concentration was kept constant (1.0 mM).
Previously, our group has reported19 that the assembly of
one melamine to one cyanurate (CYA) gives a complex that
is much stronger than the complex between one melamine
and one barbiturate (BAR) due to the higher acidity of the
CYA. Association constants of 102 and 104 M−1 were reported for
melamine–BAR and melamine–CYA, respectively. In agreement
with this, we found a very low stability (vMeOH = 3.2%; Table 2) for
assembly 6d3·(DEB)6, formed with diethyl barbiturate instead of
cyanurate derivatives.
For 6e3·(BuCYA)6, bearing unprotected (side chain) L-lysine
methyl ester moieties the vMeOH is 49% (Table 2). This indicates
that the free e-amino group of the lysine gives a less stable double
rosette assembly 6e3·(BuCYA)6 compared to 6d3·(BuCYA)6
(formed quantitatively in 0–50% MeOH in CHCl3 at 1 mM),
in which the e-amino group of the lysine is Boc-protected. For
assembly 6f3·(BuCYA)6, with free carboxylic acid groups the
vMeOH is 40%. Assembly 6f3·(BuCYA)6 is less stable than its
methyl ester analogue 6d3·(BuCYA)6. Assembly 6f3·(DEB)6 was
not formed at any ratio MeOH–CHCl3 at 1.0 mM concentration,
emphasizing the weaker association between melamine·BAR
compared to melamine·CYA. Previous results indicate that the
stability of double rosettes was mainly affected by the molecular
structure of the cyanurates and not by the side chain function-
ality of the amino acid esters. Because dimelamine 6d is bulkier
than dimelamines 6e and 6f, charge or electronic influences of
the e-amino or carboxylic acid groups in 6e3·(BuCYA)6 and
6f3·(BuCYA)6, respectively, rather than steric hindrance seem to
decrease their stability.
Assembly 6b3·(BuCYA)6, bearing alanine methyl ester moi-
eties, is stable upon addition of 50% of MeOH (Fig. 7, Table 2).
Further increase of the MeOH concentration was not possible
due to solubility limitations. CD titrations with assembly
6b3·((R)-PhEtCY)6 gave a vMeOH of 39%, a value comparable
with that for 6a3·((R)-PhEtCYA)6. These results indicate that
the methyl side chain moiety of alanine does not interfere
with the stability of assembly 6b3·(BuCYA)6. Futhermore,
assembly 6b3·(BuCYA)6 is more stable than assembly 6a3·((R)-
PhEtCYA)6. It is possible that branching of the cyanurate
side chain in (R)-PhEtCYA close to the rosette core causes
more steric hindrance than BuCYA. CD titrations showed
that assemblies 6c3·(BzCYA)6, and 6d3·(BzCYA)6, bearing L-
methionine methyl ester and L-lysine(Boc) methyl ester moieties,
are stable up to 50% of methanol (Table 2). Also, further
increase of the methanol concentration was not possible due to
solubility limitations. These results indicate that branching of the
cyanurate side chain, as in (R)-PhEtCYA is indeed unfavorable.
Furthermore, these results show that the methylsulfide and
tert-butoxycarbonyl aminobutyl side chain functionalities of
6c and 6d, respectively, do not affect the stability of as-
semblies 6c3·(BzCYA)6, and 6d3·(BzCYA)6. CD titrations with
6d3·(BuCYA)6 showed that this assembly is also stable in 50% of
MeOH (Table 2). Fig. 7 clearly indicates that small differences in
cyanurate side chain functionalities can decrease the stability of
double rosette assemblies, while the effect of amino acid side
chain functionalities at the calix[4]arene dimelamine units is
almost negligible.
Chiral induction by the lysine group
Table 2 Thermodynamic stability for the self-assembly of double
rosettes 6a–f3·(BAR/CYA)6 in MeOH–CHCl3 mixtures determined by
CD titration experiments (298 K). The maximum possible concentration
of intact assembly over the whole titration is 1.0 mM
Dimelamines 6b, 6c, 6d and 6e, having L-alanine methyl ester,
L-methionine methyl ester, e-N-Boc-L-lysine methyl ester or L-
lysine methyl ester moieties, respectively, all induce (P)-helicity
in the assembly formation. However, the CD spectrum observed
for 6f3·(BuCYA)6, bearing the L-lysine amino acid with a free
carboxylic acid group, is inverted compared to the double
rosettes 6b–e3·(DEB/CYA)6 with the amino ester functionalities.
This indicates that dimelamine 6f induces (M)-helicity for
the double rosette 6f3·(BuCYA)6, even though the chirality
of dimelamine 6f (L-lys) is the same as of the other lysine
functionalized dimelamine derivatives 6d and 6e.
R
Assembly
vMeOH (solvent range)
Gly–OEt
Ala–OMe
6a3·((R)-PhEtCYA)6 43% (0–50% MeOH)
6b3·(BuCYA)6
Quantitative (0–50% MeOH)a
6b3·((R)-PhEtCYA)6 39% (0–50% MeOH)
Met–OMe
Lys(Boc)–OMe 6d3·(DEB)6
6c3·(BzCYA)6
Quantitative (0–50% MeOH)a
3.2% (0–50% MeOH)
Quantitative (0–50% MeOH)a
Quantitative (0–50% MeOH)a
49% (0–100% MeOH)
No rosette
6d3·(BuCYA)6
The formation of assemblies 6f3·((R)-PhEtCYA)6 and 6f3·((S)-
PhEtCYA)6 was studied to confirm that in all cases L-6f induces
the M-helicity. Generally, when chiral dimelamines and chiral
cyanurate derivatives have an opposite preference for induction
of (M)- or (P)-helicity, the formation of double rosettes is not
observed.20,21 Assembly 6b3·((R)-PhEtCYA)6 is only formed in
the (P)-helical form, thus (R)-PhEtCYA induces only the (P)-
6d3·(BzCYA)6
Lys–OMe
Lys(Boc)–OH
(M-Isomer)
6e3·(BuCYA)6
6f3·(DEB)6
6f3·(BuCYA)6
40% (0–100% MeOH)
6f3·((R)-PhEtCYA)6 No rosette
6f3·((S)-PhEtCYA)6 Rosette <15% in CDCl3
a Assembly formation is quantitative over the whole solvent range.
1
helicity. H NMR and CD spectroscopy studies indicate the
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 2 7 – 3 7 3 3
3 7 3 1

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formation of the assembly 6f3·((S)-PhEtCYA)6 with the (M)-
helicity but assembly 6f3·((R)-PhEtCYA)6 is not formed. Thus,
L-6f induces only the (M)-helicity.
It is known that the induction of helicity by amino acid
residues could depend on the solvent polarity and on the pH.22
Though it is difficult to explain this change in helicity it is
likely that differences in polarity between the different lysine
moieties of 6d, 6e and 6f are playing a role. The complete
inversion of helicity as observed for 6f3·(BuCYA)6, compared to
other amino acid functionalized assemblies, indicates that small
structural changes within the building blocks could largely affect
the structure of the assembly.
Formation and thermodynamic stability of assemblies with
di-(7a–e) and tripeptide (8a–b) functionalities. 1H NMR
spectroscopy showed that assemblies 7a3·((R)-PhEtCYA)6,
7b3·(BuCYA)6 and 7c3·(BuCYA)6, bearing gly–gly–OEt, gly–
L-ala–OMe and gly–L-ser–OMe functionalities, respectively,
were formed quantitatively in CDCl3, while 7d3·(BuCYA)6 and
7e3·(BuCYA)6, bearing L-lys(Boc)-L-glu(OcHx)-NHiPr and L-
lys(Boc)–L-tyr–OMe functionalities were formed only for 84 and
65%, respectively.
Fig.
8
CD titrations of assemblies 7a3·((R)-PhEtCYA)6 (ꢀ),
7b3·(BuCYA)6 (♦), 7d3·(BuCYA)6 (D) and 7e3·(BuCYA)6 (᭹). Spectra
were recorded in MeOH–CHCl3 (f MeOH) mixtures at 298 K where the
maximum assembly concentration was kept constant (1.0 mM).
of the third amino acid does not further decrease the stability.
The 1H NMR spectrum of the assembly 8b3·(BuCYA)6, bearing
L-lys(Boc)–L-gln(Trt)–L-ser(OtBu)–OMe moieties showed the
diagnostic signals for double rosette formation in chloroform.
However, it was not possible to quantify the rosette formation
due to peak broadening in the 1H NMR spectrum. To estimate
the double rosette formation, the CD signal (at 300 nm)
obtained for 8b3·(BuCYA)6 in chloroform was compared with
the intensity of the CD signal of 6d3·(BuCYA)6 in the same sol-
vent. This resulted in an estimated formation of 8b3·(BuCYA)6
of 40% in chloroform. CD titrations showed that increased
polarity led also here to dissociation. Assembly 8b3·(BuCYA)6
was formed about 15–20% in neat MeOH (Table 3) which is
comparable with assemblies 7d3·(BuCYA)6 and 7e3·(BuCYA)6
(20% of rosette formation also in neat MeOH). It seems that
bulky and polar amino acids at the second and possibly at
the third position increase the stability of double rosettes in
neat MeOH, contrary to what is observed for MeOH–CHCl3
mixtures with high content of CDCl3. These could suggest that
with larger and bulkier peptide functionalities at the assemblies
solvophobic interactions similar to the hydrophobic effects for
the self-assembly of amphiphilic molecules in water, might start
to play a role in the self-assembly process.
In summary, amino acids at the first position of the peptide
functionalities do not affect the formation of double rosettes
at 1 mM concentration in MeOH–CHCl3 solvent mixtures,
while amino acids at the second position in di- and tripeptide
functionalities decrease the stability of double rosette assemblies
in the same solvent mixtures. This decrease in stability becomes
larger when more bulky amino acids are used in the second
position. Interestingly, it seems that the introduction of the
third amino acid hardly affects the stability of double rosettes
when compared with the dipeptides. Surprisingly, in neat MeOH
assembly formation (ca. 20%) is only observed when bulky and
polar amino acids are present at the second (and third) position.
CD titration experiments were performed with 7a3·((R)-
PhEtCYA)6, 7b3·(BuCYA)6, 7d3·(BuCYA)6 and 7e3·(BuCYA)6
in MeOH–CHCl3 (Table 3). Because dipeptide functionalized
dimelamines 7a–e are soluble in methanol, titrations could
be performed from neat chloroform to neat methanol. For
these dipeptide functionalized double rosettes, it was found
that an increase of the ratio MeOH–CHCl3 leads to the dis-
sociation of the double rosettes. Assembly 7a3·((R)-PhEtCYA)6
(vMeOH = 25%) is less stable than the corresponding (mono)
glycine derivative 6a3·((R)-PhEtCYA)6 (vMeOH = 43%; Table 2).
These results indicate that the presence of a dipeptide instead of a
single amino acid decreases severely the stability of the assembly.
Assembly 7b3·(BuCYA)6 with gly–L-ala functionalities
(vMeOH = 50%) was formed for 50% in MeOH–CHCl3 (1 : 1), while
6b3·(BuCYA)6, bearing only L-ala, was formed quantitatively
(Table 2). Furthermore, the double rosettes 7d3·(BuCYA)6 and
7e3·(BuCYA)6, with bulkier dipeptides are less stable (vMeOH
values of 7 and 8%, respectively) than 7b3·(BuCYA)6. Because
the lysine at the first position did not affect the formation
of 6d3·(BuCYA)6 (formed quantitatively in MeOH–CHCl3
(1 : 1); Table 2), these results indicate that bulkier amino acids
in the second position decrease the stability in MeOH–CHCl3
solvent mixtures. However, above MeOH–CHCl3 (2 : 8) no
further dissociation of 7d3·(BuCYA)6 and 7e3·(BuCYA)6 was
observed as it can be seen by CD titrations. The percentage
intact assemblies for 7d3·(BuCYA)6 and 7e3·(BuCYA)6 remained
around 20% (Fig. 8), even in pure methanol!
For tripeptide functionalized double rosettes, 1H NMR
spectroscopy indicated that the double rosette assembly 8a3·((R)-
PhEtCYA)6, bearing gly–gly–gly–OEt moieties, was formed
quantitatively in chloroform. CD titrations gave vMeOH = 23%
which is similar to 7a3·((R)-PhEtCYA)6 bearing the correspond-
ing dipeptide gly–gly–OEt. This indicates that the introduction
Table 3 Thermodynamic stability for the self-assembly of double rosettes 7a–e3·((R)-PhEtCYA/BuCYA)6 and 8a–b3·((R)-PhEtCYA/BuCYA)6 in
MeOH–CHCl3 mixtures determined by CD titration experiments (298 K). The maximum possible concentration of intact assembly over the whole
titration is 1.0 mM
R
Assembly
vMeOH (solvent range)
Gly–gly–OEt
Gly–L-ala–OMe
Gly–L-ser–OMe
L-Lys(Boc)–L-glu(OcHx)–NHiPr
L-Lys(Boc)–L-tyr–OMe
Gly–gly–gly–OEt
7a3·((R)-PhEtCYA)6
7b3·(BuCYA)6
25% (0–100% MeOH)a
50% (0–100% MeOH)a
N.d.b
7c3·(BuCYA)6
7d3·(BuCYA)6
7% (0–100% MeOH)a
8% (20–100% MeOH)a
23% (0–100% MeOH)a
Rosette formation ca. 40% in CDCl3
7e3·(BuCYA)6
8a3·((R)–PhEtCYA)6
L-Lys(Boc)–L-gln(Trt)–ser(OtBu)–OMe
8b3·(BuCYA)6
a CD titrations were performed from neat CHCl3 to neat MeOH due to the increased solubility of dimelamines 7 and 8 in methanol. b Not determined.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 2 7 – 3 7 3 3
3 7 3 2

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Royal Netherlands Academy of Arts and Science (KNAW) for
financial support.
Conclusions
The results shown in here demonstrate that noncovalent
hydrogen-bonded double rosettes with different functionalities
in the building blocks can be formed in MeOH–CHCl3 solvent
mixtures. In general, increase of the solvent polarity leads to
destabilization of the assemblies.
Furthermore, the functionalities strongly influence the stabil-
ity of the double rosettes. Steric hindrance close to the rosette
platform exerted by the functional groups of the building blocks
decreases the stability. Aminoalkyl groups decrease the stability
compared to n-alkyl functionalities. For linear aminoalkyl
groups, it was observed that the chain length does not affect
the stability. The stability of the double rosettes bearing pyridyl
groups is comparable to the stabilities of assemblies with
aminoalkyl groups. The ureido functionalized double rosette
is the most stable.
Double rosettes with protected glucosyl and cellobiosyl
moieties are also formed quantitative in neat chloroform, while
assemblies with unprotected glucosyl and cellobiosyl moieties
are formed for only ca. 80%. Carbohydrate moieties lead to a
dramatic decrease in stability of double rosettes in polar solvents
when compared to double rosettes having alkyl, aminoalkyl,
pyridyl or ureido moieties.
Double rosettes with amino acid or peptide functionalities
are more stable than double rosettes with carbohydrate moieties
in polar solvents. The stability of double rosettes with different
amino acid functionalities in polar solvents strongly depends on
the barbiturate or cyanurate derivative. In all cases, assemblies
with DEB are less stable than those with cyanurates. Assemblies
of amino acid ester functionalized dimelamines 6a–d with
BuCYA or BzCYA are stable up to at least 50% of methanol
in chloroform, while double rosettes with the more bulky
branched (R)-PhEtCYA have vMeOH values of 40%. Assemblies
with different lysine derivatives showed that free amino groups
or free carboxylic acid groups within the assembly decrease the
stability, probably due to ionic or electronic influences.
In apolar solvents, di- and tripeptide peptide functionalized
assemblies are less stable compared to single amino acid
functionalized assemblies. Amino acids at the first position of
the peptide functionalities do not seem to affect the stability
of double rosettes (at 1 mM), while amino acids at the second
position decrease the stability of double rosette assemblies. This
decrease in stability becomes larger when bulkier amino acids
are present in the second position. The introduction of a third
amino acid hardly influences the stability of the double rosettes.
Assembly formation in neat MeOH is observed only when
bulky amino acids are present at the second (and third) position.
This would indicate that the introduction of larger and/or
bulkier peptides might stabilize double rosettes in polar solvents.
This behavior is important for host–guest recognition in aqueous
solvents.
Overall, the large range of functionalities that can be intro-
duced on the rosette platform allows a large molecular diversity.
This is useful to generate hydrogen-bonded receptor assemblies
that resemble the natural antibodies. Introduction of large
diverse peptides to the double rosettes seems therefore especially
attractive because they might mimic the binding sites of these
natural receptors. Recently we showed that it is possible to form
double rosettes also in bilayer membranes.23 The stability of the
double rosettes is larger in the bilayer membranes compared
to polar solutions. Efforts to integrate the hydrogen-bonded
assemblies studied here as bilayer membrane receptors are
currently ongoing in our laboratory.
References
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13 The percentage of assembly was determined by comparison of the
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21 Occasionally, double rosette formation could be observed with 1H
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because either the opposite chirality inducing components strongly
affect the orientation of the chromophores responsible for the CD
activity or coincidentally cause opposite CD activity of identical
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Acknowledgements
Dr M. G. J. ten Cate and Dr M. Crego-Calama acknowledge
the Technology Foundation of the Netherlands (CW-STW) and
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 2 7 – 3 7 3 3
3 7 3 3