R.
Mizuta
ab,
J. M.
Devos
a,
J.
Webster
ab,
W. L.
Ling
c,
T.
Narayanan
b,
A.
Round
df,
D.
Munnur
abe,
E.
Mossou
af,
A. A.
Farahat
gh,
D. W.
Boykin
g,
W. D.
Wilson
g,
S.
Neidle
e,
R.
Schweins
a,
P.
Rannou
i,
M.
Haertlein
a,
V. T.
Forsyth
af and
E. P.
Mitchell
*bf
aInstitut Laue-Langevin, 71 Avenue des Martyrs, 38000 Grenoble, France
bEuropean Synchrotron Radiation Facility, 71 Avenue des Martyrs, 38000 Grenoble, France. E-mail: mitchell@esrf.eu
cUniv. Grenoble Alpes, CEA, CNRS, IBS, F-38000 Grenoble, France
dEuropean Molecular Biology Laboratory, 71 Avenue des Martyrs, 38000 Grenoble, France
eSchool of Pharmacy, University College London, Brunswick Square, London, WC1N 1AX, UK
fFaculty of Natural Sciences, Keele University, Staffordshire, ST5 5BG, UK
gDepartment of Chemistry, Georgia State University, Atlanta, GA 30303, USA
hDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
iUniv. Grenoble Alpes, CNRS, CEA, INAC-SyMMES, 38000 Grenoble, France
First published on 8th March 2018
We describe a novel self-assembling supramolecular nanotube system formed by a heterocyclic cationic molecule which was originally designed for its potential as an antiparasitic and DNA sequence recognition agent. Our structural characterisation work indicates that the nanotubes form via a hierarchical assembly mechanism that can be triggered and tuned by well-defined concentrations of simple alkali halide salts in water. The nanotubes assembled in NaCl have inner and outer diameters of ca. 22 nm and 26 nm respectively, with lengths that reach into several microns. Our results suggest the tubes consist of DB921 molecules stacked along the direction of the nanotube long axis. The tubes are stabilised by face-to-face π–π stacking and ionic interactions between the charged amidinium groups of the ligand and the negative halide ions. The assembly process of the nanotubes was followed using small-angle X-ray and neutron scattering, transmission electron microscopy and ultraviolet/visible spectroscopy. Our data demonstrate that assembly occurs through the formation of intermediate ribbon-like structures that in turn form helices that tighten and compact to form the final stable filament. This assembly process was tested using different alkali–metal salts, showing a strong preference for chloride or bromide anions and with little dependency on the type of cation. Our data further demonstrates the existence of a critical anion concentration above which the rate of self-assembly is greatly enhanced.
Previously reported systems have shown that such self-assembly typically arises from a subtle interplay between various non-covalent interactions including ionic interactions, hydrogen bonding, π–π stacking of aromatic groups, solvent interactions and steric hindrances.4–6 Therefore, a thorough understanding of these governing interactions is paramount in achieving the necessary control over the dynamics of the self-assembly and resulting morphology for potential applications. Owing to the extensive number of parameters that influence the assembly, it remains a difficult task to achieve such control. However, recent examples demonstrate promising developments in the field towards materials with tailored functionalities.7–10
Small organic molecules that recognise and bind specifically to the minor groove of DNA, often based on planar heterocyclic ring structures, have been studied extensively.11–15 Interest in these molecules arises from their strong biotechnological potential and as candidate anti-parasitic and DNA sequence recognition agents via specific curvature of the molecules fitting the grooves in DNA helices. However, to date none of these small molecules have been shown to possess self-assembling properties. The biscationic form of 2-(4′-amidinobiphenyl-4-yl)-1H-benzimidazole-5-amidine, one member of this family of compounds and named hereinafter as DB921, consists of three connected rings, terminated at each end of the planar molecule by charged amidinium groups, shown in Fig. 1.16,17
Here we report a novel hierarchical self-assembling system of nanotubes, formed by DB921 in the presence of suitable alkali-halide salts. We present a multi-disciplinary study conducted using complementary techniques of solution-state small-angle X-ray and neutron scattering (SAXS and SANS respectively), negative stain and cryo-transmission electron microscopy (cryo-TEM), UV-vis spectroscopy and X-ray fibre diffraction, and propose an assembly mechanism and a possible atomic-level model for the nanotubes.
The same protocol was used for the various salts tested in this study. The final samples were saturated solutions of DB921 with 4% (v/v) DMSO and the desired final concentration of the various salts tested in the experiments described in this paper. Regarding the sample prepared in D2O for the SANS experiment, the same protocol was followed apart from using deuterated DMSO and D2O.
For the tests on assembly at different pH, both DB921 stock solution and NaCl solution were pH-corrected; different buffers were used for different pH ranges. For pH 4–6: acetate buffer. For pH 7.8: HEPES buffer. For pH 9: glycine buffer.
For cryo-TEM, ∼4 μl of the sample was applied to a glow-discharged Quantifoil grid and plunge frozen in liquid ethane using a Vitrobot. Frozen grids were observed on an FEI F20 microscope at 200 kV or on an FEI Polara microscope at 300 kV. Images on the Polara were recorded on an Ultrascan 4000 CCD camera. Nanotube dimensions were based on an average of 100 nanotube measurements from the cryo-TEM images.
Higher resolution SAXS measurements over an extended Q range were performed on selected samples at the ESRF ID02 beamline.20 Two sample-to-detector distances of 1.2 m and 10 m were used to cover the Q range from 0.007 nm−1 to 7 nm−1. The data reduction procedure was similar to BM29 measurements and intensities were normalised to an absolute scale. Fitting of the one-dimensional data was performed using the software SASView version 2.2.1. with the core cylinder model.21 Parameters derived from the fitting were inner and outer cylinder radii and the polydispersity of the outer radius defined as the standard deviation divided by the mean value. Here, the polydispersity was better described by a log-normal size distribution of outer radius and the wall thickness defined by the chemical structure was assumed to be uniform.
Characterisation technique | D I [nm] | D O [nm] | Wall Thickness [nm] |
---|---|---|---|
SAXS NaCl | 22.4 | 26.1 | 1.85 |
SANS NaCl (H2O solution) | 22.4 | 26.2 | 1.9 |
SANS NaCl (D2O solution) | 23.0 | 26.8 | 1.9 |
Cryo-TEM NaCl | 20 | 26 | 3 |
SAXS NaBr | 27.8 | 32.0 | 2.1 |
SANS data on mature nanotubes formed in the presence of NaCl were recorded on D11 at ILL in both H2O and D2O solutions as shown in Fig. 2(b). Both sets of SANS data could be fitted as a core–shell cylinder model and were consistent with the SAXS data analysis shown in Fig. 2(a). The incoherent background in the high Q region makes an independent determination of the wall thickness uncertain and therefore it was fixed at the SAXS value (1.9 nm). As can be seen in Table 1, the outer diameter deduced from the SANS model is consistent with the SAXS value.
The results highlight the remarkable monodispersity in nanotube cross-section, which is also evident in the cryo-TEM image of the nanotubes shown in Fig. 2(c). High degrees of monodispersity in cross-sectional dimensions have previously been reported in other similar self-assembled tubular systems.25,26 In such systems, the wall-thicknesses often correspond to a monolayer formed of the constituent molecules. This is particularly true for bolaamphiphilic molecules that enable hydrophilic groups to arrange on the inner and outer tube surfaces, with hydrophobic groups encased within the wall.26,27 In a similar manner, DB921 molecules may adopt an orientation with the hydrophilic, terminal amidinium groups pointing towards the inner and outer walls of the tube, thus sequestering the hydrophobic aromatic backbone. Furthermore, halide counterions are expected to condense around the positively charged amidinium groups at each end to achieve charge neutralisation.7 Therefore, we propose that the bulk of the nanotube wall is composed of a monolayer of DB921, with additional layers of halide ions decorating the inner and outer surfaces, screening the electrostatic repulsion while potentially facilitating π-stacking by bridging across adjacent amidinium ions.
For the present system, the calculated length of a single DB921 molecule of 1.92 nm, combined with the diameter of two chloride ions (2 × 0.36 nm) associated at either end, would suggest a wall-thickness value of 2.64 nm. The wall thickness deduced from SAXS in solution of 1.85 nm, where the counterion profile may be more diffuse, is comparable to the size of the DB921 molecule. The TEM measurements suggest a larger thickness of 3 nm. The larger wall thickness measured from cryo-TEM might be due to the subjectivity in determining the wall boundary during the measurements and the defocusing necessary to obtain sufficient contrast to clearly discerning the nanotubes. The SAXS/SANS dimensions were therefore used for modelling. SAXS measurements (ESRF beamline BM29) were made on samples aged by 1 month at 4 °C, which confirmed long-term stability of the nanotubes (data not shown).
Nanotube formation was also tested using various cation/anion combinations from the alkali metal/halide element groups. The results show that nanotube formation is independent of the alkali metal used, where the closely aligned scattering features (BM29 beamline) shown in Fig. 3(a) indicate very similar nanotube cross sections. Negative stain TEM also shows the consistency of the nanotube morphologies over the alkali–metal series (data not shown). Formation was also investigated using various halide ions. The results shown in Fig. 3(b) demonstrate that both chloride and bromide ions allow the formation of nanotubes. However, tube formation does not occur with either fluoride or iodide, even following a month of incubation. The results show that chloride or bromide ions are an essential component of the nanotubes, their negative charges counterbalancing the positive amidinium charges. Given that the nanotube formation is selective for only two of the halide ions, an effect of radius is implied where only chloride and bromide have radii appropriate to chelate the amidinium groups and allow tube formation. It is not unreasonable to consider that the ions could bridge between adjacent amidinium groups and actively hold together the DB921 molecular network in addition to balancing the DB921 positive charges.
SAXS curves, shown in Fig. 2(a) and as measured from nanotubes formed with bromide ions, yield a diameter of 32.0 nm when fitted with a similar model. This diameter is larger than that found for the chloride equivalent (the minimum shifts to smaller Q values), while the wall thickness remains similar. This finding further emphasises the involvement of anions likely forming a diffuse outer shell and influencing the bending energy to form a curved surface. Negative stain TEM images shown in Fig. 3(c) also show this increase in tube diameter. A similar effect of counterions modulating the structure and size has been reported in the case of peptide nanotubes7 and discussed in the context of controlling the chiral architecture of self-assembled amphiphilic systems.28
Nanotube assembly was further examined across a pH range from 4 to 9. Assembly was performed with high chloride concentration (150 mM) with SAXS spectra recorded 2.5 hours after initiation (ESI†). The results show no pH dependence in this pH range.
A time-resolved study was performed by negative stain-EM to image the assembly process. For convenience, nanotubes were assembled in a lower concentration of the DB921 of 4.5 mM, enabling a longer assembly time of several days. Times were measured from the initiation of nanotube formation by mixing the DB921 and sodium chloride solutions. Fig. 4(a)–(c) shows the sequential formation of intermediates before complete nanotube formation. Shortly after (40 minutes) the assembly is initiated, short fibrillar structures of width 2 to 5 nm and lengths of 50 to 100 nm are seen as in Fig. 4(a), left. Additionally, the sample is inter-dispersed with longer fibrils with widths varying between 20 to 30 nm and lengths of 200 to 300 nm (Fig. 4(a), right).
The coexistence of these two morphologies suggests that initial aggregation of the DB921 molecules leads to the formation of short and thin fibrils, which in turn aggregate to form elongated fibrils. Twenty-four hours post-initiation we observe a system of ribbons with widths of approximately 50 nm and lengths of up to several microns (Fig. 4(b)). In particular, thin fibrils that are similar to those displayed in Fig. 4(a) are seen splitting and branching out from the ends of the aggregated fibrils, packed together in a side-by-side manner. Approaching 48 hours, we observe such ribbons helically twisted into mature nanotubes (Fig. 4(c)). This final step of helical winding has been previously identified in many nanotube systems.28–33
A similar time-resolved study was also carried out using SAXS (beamline BM29). The scattering by nanotubes assembled in saturated solution of DB921 (with fixed NaCl concentration at 70 mM and with 4% (v/v) DMSO) was measured at multiple time stamps after initiation varying from a few minutes up to one hour. The resulting SAXS curves are shown in Fig. 4(d). Several salient features are noted. Firstly, there exists an induction period immediately after assembly is initiated, during which no characteristic maxima in the SAXS spectra are observed. Secondly, following this induction time, initially weak maxima emerge that subsequently evolve into the scattering pattern associated with mature nanotubes. At first, the oscillations are weak in amplitude, presumably due to the coexistence of different structural moieties in the solutions. As more nanotubes are formed, their contribution to the total SAXS intensity becomes dominant, characterised by the well-defined maxima. The apparent shift in positions of the fringe maxima to higher Q with time, as shown in Fig. 4(e), is an indication of the maturing of nanotube population. At the same time, the population of other structural moieties such as the ribbons and helices as observed by TEM deplete with time as they are transformed into nanotubes. Such a hierarchical assembly mechanism corroborates the time-resolved TEM data.
The induction period is attributed to the time taken for the assembly to proceed up to the formation of nanotubes. The lack of peaks at early times during the assembly is due to the fact that the characteristic SAXS oscillations strictly pertain to tubular structures. Therefore, all early intermediates have broad scattering features as observed at early times in Fig. 4(d); the onset of oscillations is observed once the ribbons begin to coil.34 Despite not being fully formed nanotubes, their average structure as seen by X-ray scattering would resemble that of a nanotube. Furthermore, the SAXS reveals an apparent decrease in the cross-sectional size over time before reaching a “saturation” limit (Fig. 4(e)). This is consistent with an assembly by helical twisting and packing seen by TEM, corresponding to the tightening of the ribbons as the packing approaches completion. The proposed hierarchical assembly mechanism based on these studies is shown schematically in Fig. 4(f).
Finally, the dependence of assembly rate on NaCl concentration is directly quantified by plotting the induction time as a function of NaCl concentration. Here, the induction time was found by fitting the time dependence of the integrated SAXS intensity over the entire Q range (IInt) with the Avrami equation used to describe polymer crystallisation,35 given by
IInt = a(1 − exp{−[(t − τI)/b]n}). | (1) |
The fitting was performed with parameters a corresponding to the final plateau value of IInt, τI the corresponding induction time, b the growth-rate factor and n is a coefficient fixed to 1 in this case. As shown in Fig. 5(d), it can be seen that at its fastest assembly rate, the nanotube formation completes in approximately 5 minutes.
UV-vis spectra were obtained for the unassembled monomer and assembled nanotubes respectively as shown in Fig. 6(b). The characteristic absorptions of monomeric DB921 at 278 nm and 325 nm are found to be red-shifted to 283 nm and blue-shifted to 310 nm, respectively. Additional peaks are also noted at higher wavelengths of 359 nm and 378 nm. Similar spectral shifts are frequently observed in aggregated organic dye molecules which demonstrate face-to-face (H-type) or tail-to-tail (J-type) aggregation due to their high aromatic nature.37–40 A blue (hypsochromic) shift is attributed to H-type aggregation whilst a red (bathochromic) shift is characteristic of J-type aggregation. For the present system, a simple distinction between H- and J-type aggregation is likely to be insufficient given the complexity of the assembled DB921 molecules in the nanotube. However, the existence of both blue and red-shifted peaks suggests a combination of both H- and J-type configurations in the final nanotube, lending further evidence to the existence of π–π stacking interactions.
The diffraction and spectroscopy measurements suggest that the DB921 molecules stack in a regular way through π–π stacking of the aromatic rings. Together with the π–π stacking and the halide ion stabilising the assembly of DB921 in the nanotubes, each DB921 molecule has a hydrogen bond donor and acceptor symmetrically positioned on either side of the benzimidazole ring. Within the helix of side-to-side DB921 molecules this configuration could form N–H⋯N hydrogen bonds, further knitting together the assembly.41 Molecular curvature has been implicated previously in nanotube formation. A well-known example is the bent-core achiral molecules which form chiral liquid crystals.42,43 As noted earlier DB921 has an inherent curvature to the molecule that may be associated with its specific ability to form helical nanotubes described here from achiral building blocks.
Of particular interest in this series were compounds varying only by a single moiety on the aromatic ring structures. For example, the compound DB985 was examined. This molecule is identical to DB921 but with a hydroxyl group added at the ortho-position on the terminal phenyl ring. This suggests that the additional hydroxyl group sterically distorts the side-by-side alignment of the hydrogen-bonded molecules, preventing regular nanotube formation. Another example is DB1963 where one of the two amidinium groups is modified to be an amide, demonstrating the importance of the dication. A further example is DB1883 which is isostructural to the parent except for the absence of the imidazole nitrogen which is replaced with a carbon atom. This supports the hypothesis that the DB921 molecules are held together in a chain by N–H⋯N hydrogen bonds and that these are crucial in the formation of the regular nanotube structure.
Self-assembly is likely to be driven by a combination of electrostatic interactions, the formation of π–π stacking interactions and inter-molecular N–H⋯H hydrogen bonds, with additional halide ion stabilisation of the charged amidinium groups and controlling the curvature of helical ribbons.
The biophysical characterisation of these nanotubes has allowed a candidate structural model for the nanotubes to be presented. This is based on π–π stacked DB921 molecules, ordered in a helical fashion in the narrow walls of the filaments, with diffuse layers of halide ions screening the charged amidinium groups.
Time-resolved experiments demonstrate an elegant assembly process consisting of the formation of short fibril structures, which in turn associate to large-scale ribbons, in turn winding in a helical manner into the final hollow tubular structure.
It is noted that assembly is triggered by addition of chloride or bromide ions, with a change in size of the tubes according to the halide ion used. In recent years, the remarkable properties of self-assembled systems such as nanotubes have been manipulated for applications such as drug delivery and templating for bottom-up synthesis of nanoscale devices.44 The kinetic studies conducted have demonstrated that various parameters contribute to the assembly rates of the DB921 nanotubes, offering the promising possibility of controlled self-assembly. Adhesion of molecules to intermediate species prior to the completion of assembly may enable their encapsulation in the final tubules, allowing the function of a molecular- or drug-delivery system.
As previously mentioned, π–π interactions between aromatic rings appear to be highly prevalent in the final structure. Pi-conjugated molecules demonstrating extensive π–π stacking have been reported as promising organic semiconductors, in which the large degree of π-overlap enables effective charge transport.45,46 Our findings indicate that DB921 may be explored as a candidate for (opto)electronically active layers in devices for organic electronic applications.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr03875e |
This journal is © The Royal Society of Chemistry 2018 |