Benzene tricarboxamide derivatives with lipid and ethylene glycol chains self-assemble into distinct nanostructures driven by molecular packing

Article abstract of DOI:10.1039/d1cc03437e

The self-assembly in aqueous solution of benzene-1,3,5-tricarboxamide (BTA) bearing one alkyl chain and two PEG (polyethylene glycol) chains or two alkyl chains and one PEG chain yields completely distinct nanostructures. Two series of derivatives were sy

Full text of DOI:10.1039/d1cc03437e

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Benzene tricarboxamide derivatives with lipid and
ethylene glycol chains self-assemble into distinct
nanostructures driven by molecular packing†
Cite this: DOI: 10.1039/d1cc03437e
Received 28th June 2021,
Accepted 27th July 2021
Nada Aljuaid,a Mark Tully,b Jani Seitsonen,c Janne Ruokolainenc and
a
Ian W. Hamley
*
DOI: 10.1039/d1cc03437e
rsc.li/chemcomm
The self-assembly in aqueous solution of benzene-1,3,5-tricarboxamide organocatalysts,7 stimuli-responsive materials,8 among others,
(BTA) bearing one alkyl chain and two PEG (polyethylene glycol) chains and as model systems to study supramolecular polymerization.8,9
or two alkyl chains and one PEG chain yields completely distinct
In a recent paper, the self-assembly behavior was investi-
nanostructures. Two series of derivatives were synthesized and exten- gated for BTA derivatives in which two of the three arms
sively characterized and electron microscopy and small-angle X-ray are alkyl ethoxylates [12 methylene units connected to
scattering (SAXS) reveal micelle structures for derivatives with one alkyl tetra(ethylene glycol)] and the third arm was a carboxylic acid
and two PEG chains, but nanotapes and nanoribbons for the series with with 1, 6 or 10 methylene units (or as a control a sample
two alkyl and one PEG chain.
containing a tetra(ethylene glycol) carboxylate).9 These mole-
cules have hydrophobic chains around the benzene core and
Supramolecular polymerization is attracting great interest as a hydrophilic outer arm units. Nanostructures including fibers,
means to create innovative nanomaterials with unique proper- membranes and nanotubes were observed via cryo-TEM and
ties stemming from the non-covalent self-assembly processes.1 SAXS for the different BTA derivatives, depending also on
These can be either isodesmic self-assembly (equal association concentration and temperature.9
constants for the first and subsequent assembly steps) or
In the present paper, we investigate for the first time the self-
nucleation/elongation or co-operative self-assembly.2,3 Supra- assembly in aqueous solution of BTA derivatives bearing simple
molecular polymerization is driven by a variety of intermole- combinations of one or two alkyl chains or ethylene glycols
cular interactions including hydrogen bonding, p-stacking, (oligo-ethylene glycols OEGs, or short polyethylene glycols,
metal–ligand interactions, hydrophobic or solvophobic interac- PEGs) as shown in Scheme 1. Table 1 summarizes the molecules
tions etc. It can occur in aqueous or non-aqueous solvents and synthesized. The alkyl chains were selected in the range C10 (decyl)
the self-assembly process depends on these intermolecular to C18 (octadecyl), corresponding to the typical lengths of biolo-
interactions as well as solute–solvent interactions.2
gical lipids which should provide self-assembly properties in
Benzene-1,3,5-tricarboxamide (BTA) derivatives have recently molecules also bearing suitable short PEG chains, which were
been extensively studied by the groups of Meijer and Palmans4,5 selected in the range (EG)4 [tetra(ethylene glycol)] to (EG)15.
(as well as others) as model supramolecular polymers with a rigid Sample names are of the form DAmMPEGn (m = number of
p-stacking benzene motif surrounded by three hydrogen-bonding carbon atoms in alkyl chain, n = number of ethylene glycol
amide groups forming three-arm self-assembling molecules. repeats) for the derivatives with two alkyl chains and a single
BTA derivatives show promising properties as biomaterials,6
a Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD,
UK. E-mail: I.W.Hamley@reading.ac.uk
b ESRF-The European Synchrotron, 71 Avenue des Martyrs, Grenoble 38000, France
c Nanomicroscopy Center, Aalto University, Puumiehenkuja 2, Espoo FIN-02150,
Finland
† Electronic supplementary information (ESI) available: Experimental procedures
including materials and methods, synthesis details and Schemes and character-
Scheme 1 Schematic of the scaffold of the BTA derivatives studied. Here
R1, R2 and R3 correspond to two PEG chains + one alkyl chain or two alkyl
chains + one PEG chain as shown in Table 1.
ization data (MS and NMR), UV and FTIR spectra and tables listing the
parameters extracted from the fitting to the SAXS data. See DOI: 10.1039/
d1cc03437e
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Table
1
Molecules synthesized for which conformation and self-
assembly were studied
(a) Derivatives with variable PEG chain length with C16 alkyl chain
(C16)2PEGn
C16(PEGn)2
PEG4
PEG8
PEG12
PEG15
DA16MPEG4
DA16MPEG8
DA16MPEG12
DA16MPEG15
MA16DPEG4
MA16DPEG8
MA16DPEG12
MA16DPEG15
(b) Derivatives with variable alkyl chain length with PEG12 chain(s)
(Cn)2PEG12
Cn(PEG12)2
C10
C12
C16
C18
DA10MPEG12
DA12MPEG12
DA16MPEG12
DA18MPEG12
MA10DPEG12
MA12DPEG12
MA16DPEG12
MA18DPEG12
Fig. 1 Cryo-TEM images for the MAmDPEGn series (all 1 wt% aqueous
solutions). For each sample, lower and higher magnification images are
presented, to more clearly see the micelle structures.
regular micelle structures with a defined diameter (ca. 6–8 nm) are
observed extensively over the cryo-TEM grid (Fig. 1).
PEG chain and MAmDPEGn for derivatives with one alkyl chain
and two PEG chains (Table 1). All samples were found to be
soluble at pH 3, 7 and 12. In the following, data is presented at
native pH (approximately pH 7). We used a combination of cryo-
TEM and SAXS to elucidate the self-assembled nanostructures in
aqueous solution. Our results show distinct modes of self-
assembly for BTA derivatives bearing one lipid chain and two
PEG chains (if sufficiently short) compared to those with two PEG
chains and one lipid chain.
Detailed schemes of the synthesis methods along with
extensive characterization data are provided in the (ESI†
Schemes S1–S4 and ESI† Fig. S1–S32). In brief, the DAmMPEGn
BTA compounds were synthesized following the route reported
by Meijer’s group,10 i.e. synthesis of 3,5-(methoxycarbonyl)
isophthalic acid 3 (ESI† Scheme S1) through dihydrolysis of
trimethyl benzene-1,3,5-tricarboxylate 2 followed by conversion
to acyl chloride. The alkyl amine chain was reacted with acyl
chloride forming a secondary amide in two positions. Methyl
ester groups were used to protect the third position in com-
pound 6 before reaction with PEGn amine followed the same
procedure. This synthetic strategy used the desymmetrised BTA
synthesis.4,10 The MAmDPEGn compounds were prepared
from benzene-1,3,5-tricarboxylic acid 1,3-dimethylester 8 (ESI†
Scheme S2) through monohydrolysis of trimethyl benzene-
1,3,5-tricarboxylate 2 before conversion to the acyl chloride by
an acylation reaction. The alkyl amine chain was reacted with
acyl chloride forming a secondary amide in one position. The
methyl ester group in this case was used to protect the other
two positions before the hydrolysis and acylation reaction with
two equivalents of PEGn to produce compounds 12.
In complete contrast to the micelle structures formed by the
MAmDPEGn series of samples, the micrographs for the DAmM-
PEGn samples shown in Fig. 2 reveal the presence of extended
tape-like structures when the alkyl chain is sufficiently long,
with some differences in morphology from sample to sample.
The two BTA derivatives with shorter alkyl chains, DA10M-
PEG12 and DA12MPEG12 do not show a large population of
self-assembled structures (only occasional structures were
observed over the cryo-TEM sample grids, however nanostruc-
tures were detected by SAXS, vide infra). Solutions of DA18M-
PEG12, DA16MPEG4, DA16MPEG8, and DA16MPEG12 all show
nanotape morphologies in aqueous solution, with variation in
nanotape width and curvature. Distinctly, DA16MPEG15 clearly
self-assembles into remarkable twisted nanotape (nanoribbon)
structures. However, all DAmMPEGn samples show the com-
mon feature that the self-assembled structures are based on
planar 2-dimensional self-assemblies, a conclusion supported
by detailed analysis of SAXS data discussed in the following.
The presence of intramolecular H–bonding was assessed by
FTIR. IR spectroscopy shows vibrations for the N–H stretch at
3238 cmÀ1, the CQO stretch at 1632 cmÀ1 and the amide II at
1564 cmÀ1 for DAmMPEGn (ESI† Scheme S31) which indicate
the presence of intermolecular H–bonding. In contrast, the N–
H stretch at 3344 cmÀ1, the CQO stretch at 1651 cmÀ1 and the
amide II peak at 1534 cmÀ1 for MAmDPEGn (ESI† Scheme S32)
suggests the absence of threefold intermolecular H-bonds.
Cryo-TEM was used to image nanostructures in aqueous
solutions as it provides information on self-assembled struc-
tures, avoiding sample drying which occurs during sample
preparation for conventional TEM and AFM. The images reveal
that solutions of derivatives with mono-alkyl and di-PEG
chains, i.e. MAmDPEGn form globular micelle-like structures
in aqueous solution as shown in the cryo-TEM images in Fig. 1.
The mono-alkyl derivatives with short alkyl chains (C10 or C12
)
with DPEG12 do not show regular structures over the grid, these
are only observed for longer alkyl chains (C16 in particular) where
Fig. 2 Cryo-TEM images for the DAmMPEGn series (all 1 wt% aqueous
solutions).
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maxima, indicating distinct nanostructures to those for the
MAmDPEGn samples. Motivated by these features and the
presence of nanotape structures in the cryo-TEM images
(Fig. 2), the SAXS data were fitted to form factors of bilayer
structures (some samples gave weak SAXS profiles indicating
low populations of the nanostructures imaged by cryo-TEM).
This form factor fits the data very well. The model is based on
that used for lipid bilayers12 and represents the electron density
profile across the bilayer as a sum of three Gaussian functions,
one representing the hydrocarbon centre of the bilayer (with a
negative electron density relative to the solvent) and the other
two (positive electron density contrast) Gaussians symmetri-
cally representing the outer surfaces. SAXS is able to accurately
determine the layer thickness, which cannot reliably be
obtained from the cryo-TEM images. The fit parameters are
detailed in ESI† Table S2. The results indicate that the chains in
the bilayer are highly interdigitated, since the layer thicknesses
are less than twice the estimated extended length of the alkyl
chains, for example for C18 chains, the chain length is approxi-
mately 2.0 nm whereas the layer thickness t = 2.47 nm accord-
Fig. 3 Measured SAXS data (open symbols) and fitted form factors
(models and fitted parameters listed in ESI† Tables S1 and S2). (a) MAmD-
PEGn series. (b) DAmMPEGn series. Data has been scaled to enable
visualization.
This observation suggests that the lack of planar order in ing to the SAXS form factor fitting for DA18MPEG12 (ESI† Table
MAmDPEGn may be influenced by the absence of H–bonding S2) and in addition the PEG chain outer layers will have finite
molecular interactions.
thickness (ca. 0.97 nm radius of gyration assuming Gaussian
SAXS is a powerful method to probe self-assembled nanos- coil conformation).
tructures in situ11 and was applied to quantitatively determine
Based on the results from cryo-TEM and SAXS, we propose
the form factors and hence dimensions and scattering (electron the model shown in Fig. 4, which is consistent with simple
density) contrasts of the assemblies formed by the BTA deriva- molecular packing argument, i.e. applying the surfactant pack-
tives. The measured data along with form factor fits are ing parameter model13–15 to the case of the BTA derivatives
presented in Fig. 3. The SAXS profiles in Fig. 3a show that all which have a highly constrained tri-functional linking unit
MAmDPEGn samples form micelles to a certain extent, except compared to conventional amphiphiles. Samples MAmDPEGn
MA10DPEG12 which shows features of a monomeric form factor with mono-alkyl chains of sufficient length and two PEG chains
(data fitted using a generalized Gaussian coil form factor model, form micelle structures due to packing constraints of the
allowing for swelling or collapse via a variable Flory exponent in hydrophilic di-PEG headgroup and hydrophobic mono-alkyl
the radius of gyration). SAXS is able to detect micelle formation for core group (Fig. 4). In the surfactant packing parameter
some samples for which only a low population of micelles was description, the molecules would be considered to have a
observed by cryo-TEM. The parameters from the fitted spherical cone-like shape. In contrast, the samples with di-alkyl chains
core–shell particle form factor are listed in ESI† Table S1. These of sufficient length (and appropriate single PEG chains) pack
show that the outer radius lies in the range 3.1–3.8 nm, which is into nanotape structures based on layered stacking of the BTA
both consistent with the cryo-TEM images in Fig. 1 and physically
realistic, considering estimations of the length of the molecules,
for example for MA16DPEG15, the extended alkyl chain length is
approximately 1.8 nm and the PEG radius of gyration is about
1.5 nm. The obtained inner core radii values listed in ESI† Table
S1 are actually slightly lower than the extended alkyl chain
lengths, which may indicate folding, however in addition the
boundaries between core and shell will be diffuse and it also has
to be considered that the BTA residues must presumably reside at
the outer part of the core. No general significant trends in the core
or shell radius were observed across the samples, the values
reflecting the length of both hydrophobic and hydrophilic chains
and the balance of these that affects the core–shell interfacial area
and hence molecular packing. This is also influenced of course by
the positioning of the BTA residues at the interface between the
inner lipid-rich core and the outer PEG corona.
Fig. 4 Proposed models for self-assembly. Top: MAmDPEGn series with
The SAXS data in Fig. 3b for DAmMPEGn solutions have a
different scaling at low wavenumber q and shape of form factor
mono-alkyl chain and di-PEG, Bottom: DAmMPEGn series with di-alkyl
chains and mono-PEG.
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derivatives into bilayers with a core of hydrophobic di-alkyl Notes and references
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N. A. acknowledges support from the Saudi Arabian Cultural
Bureau. IWH was supported by EPSRC Platform grant EP/
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(ref. MX-2345). We are grateful to Dr Lewis Hart for advice
concerning synthesis.
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Conflicts of interest
There are no conflicts to declare.
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