ISSN 1359-7345

Chemical Communications

www.rsc.org/chemcomm Volume 49 | Number 41 | 21 May 2013 | Pages 4575–4704

1359-7345(2013)49:38;1-#

COMMUNICATIONSourav Saha et al.Controllable self-assembly of amphiphilic macrocycles into closed-shell and open-shell vesicles, nanotubes, and fi bers

This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 4601--4603 4601

Cite this: Chem. Commun.,2013,49, 4601

Controllable self-assembly of amphiphilic macrocyclesinto closed-shell and open-shell vesicles, nanotubes,and fibers†

Atanu Mitra,z Dillip K. Panda,z Lucas J. Corson and Sourav Saha*

Depending on functional groups, amphiphilic hexaamide macro-

cycles self-assemble into closed-shell and open-shell vesicles in

polar solvents. In the presence of water, open-shell vesicles morph

into closed-shell vesicles, whereas acidification of the medium

transforms vesicles into nanotubes and fibers.

In Nature, amphiphilic phospholipids self-assemble into bilayermembranes that compartmentalize complex biological reactions,regulate transport of ions and nutrients, and safeguard geneticinformation.1 Similarly, amphiphilic molecules,2–4 macrocycles,5,6

dendrimers,7–9 polymers,10,11 and supramolecular assemblies12

self-assemble into micelles,2,7 vesicles,3,5,8,10,12 nanotubes andfibers.4,6,9,11 Morphologies of different nanostructures depend ongeometry, functional groups, and concentration of building blocks,as well as polarity and pH of the medium. These nanostructuresshow remarkable potential as drug and gene delivery systems(DDS),13 nanoreactors,14 artificial ion channels,6a and in tissueregeneration4c and other applications.15

To serve as controlled DDS,13 vesicles must be able to encapsulatedrug molecules and release them under the influence of externalstimuli, e.g., solvent, pH, ions, light, heat, and chemicals.16 However,if guest molecules are present during the vesicle formation, theycould interrupt the vesicle formation17 or become embedded withinthe membranes instead of being encapsulated within the vesicles, ascenario that could hinder efficient release of drug molecules. Thesepotential drawbacks can be averted by first constructing open-shellvesicles in a guest-free medium, then loading them with guestmolecules, and finally closing the guest-containing open vesiclesunder appropriate external stimuli. Although myriad closed-shellvesicles have been constructed from different amphiphilic buildingblocks, open-shell vesicles with a single pore are extremely rare.18

Furthermore, stimuli-driven transformation of preformed open-shellvesicles into closed ones remains a formidable challenge.

Herein, we report design, synthesis, and self-assembly of twoamphiphilic macrocycles, MC1 and MC2 (Fig. 1), into closed andopen vesicles and their H+-driven transformation into nanotubesand fibers, which have been analyzed using scanning electronmicroscopy (SEM), transmission electron microscopy (TEM), atomicforce microscopy (AFM), and dynamic light scattering (DLS). MC1and MC2 macrocycles are composed of three isophthalic acid units(A) and three m-phenylenediamine units (B) connected alternatelyvia six amide bonds (Fig. 1). Two isophthalic acid units (A) carry anoctyl chain at the 5-position, while the third one carries a 5-nitro-group in MC1 and a 5-amino-group in MC2, rendering thesemolecules amphiphilic.

MC1 was synthesized (Fig. 1) by coupling an ABA moiety bearingtwo terminal carboxylic acid groups with a BAB moiety carrying twoterminal amine groups in the presence of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT). In this reaction, CDMT not only serves as anefficient amide-coupling reagent by activating ABA diacid into anactivated diester, but also liberates 2,4-dimethoxy-6-hydroxy-1,3,5-triazine (DMHT), which serves as a templating agent (Scheme S1,ESI†) to facilitate the formation of the 30-membered macrocycleMC1 and prevents the formation of linear polymers and largermacrocycles. The first amide coupling between the BAB diamineand CDMT-activated diester of ABA diacid moieties releases DMHT,

Fig. 1 Synthesis of MC1 and MC2 and DFT energy minimized structures of threepossible conformations of MC1. Conformation I is more stable than II and III byca. 15 and 28 kcal mol�1, respectively.

Department of Chemistry and Biochemistry and Integrative NanoScience Institute,

Florida State University, 95 Chieftan Way, Tallahassee, FL 32306-4390, USA.

E-mail: saha@chem.fsu.edu; Tel: +1 850 645 8616

† Electronic supplementary information (ESI) available: Experimental section:synthesis, characterization, NMR, MS, and additional DLS, AFM, SEM, and TEMdata. See DOI: 10.1039/c3cc40535d‡ These authors made equal contributions.

Received 21st January 2013,Accepted 26th February 2013

DOI: 10.1039/c3cc40535d

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4602 Chem. Commun., 2013, 49, 4601--4603 This journal is c The Royal Society of Chemistry 2013

which can potentially form a H-bonded 1 : 1 complex with theresulting acyclic (AB)3 chain and preorganize it into a semicyclicconformation. The proximity of amine and activated ester ends ofthe DMHT-bound folded conformation of (AB)3 chain facilitates thesubsequent intramolecular amide coupling to form MC1 in anexcellent yield. Attempts to couple ABA and BAB moieties with othercoupling agents, e.g., SOCl2 and dicyclohexylcarbodiimide, yieldedmostly polymeric materials instead of the desired MC1 macrocycle,suggesting that DMHT indeed acted as a templating agent tofacilitate macrocyclization. The templating effect of DMHT wasapparent from the ESIMS of the crude reaction mixture (Fig. S1,ESI†), which in addition to MC1 (m/z = 1016.4) showed a 1 : 1MC1�DMHT complex (1173.4), but no polymer or larger macrocycles.After washing the crude product with MeOH, the complex dis-appeared and HR-MALDI-TOF analysis revealed (Fig. S2, ESI†)MC1 (m/z = 1016.915) and its self-assembled dimer (2032.957), trimer(3048.215), and tetramer (4064.010). MC1 was reduced to MC2 withhydrazine and Pd/C (Scheme S1, ESI†). MC2 (m/z = 986.972) and itsself-assembled dimer (1972.982) and trimer (2957.979) were alsofound by HR-MALDI-TOF analysis (Fig. S2, ESI†).

Unlike an analogous hexaamide macrocycle6c that projects all sixamide-CQO groups inside its cavity because of intramolecularH-bonding between amide-NH groups and heteroatoms at the outerrim, MC1 and MC2 do not have any provision for intramolecularH-bond formation. Therefore, these macrocycles are free to adopt oneof the three possible conformations (Fig. 1): (I) one that wouldalternately project amide-NH and CQO groups (three each) insidethe cavity, (II) the second that would project all six –NH bonds inwardand all six CQO bonds outward, and (III) the third that would carryall CQO bonds projecting inward and all –NH bonds projectingoutward. In silico (B3LYP/6-31G) structural analysis suggests that thefirst scenario (I) is significantly more stable than the second and thirdconformations by ca. 15 and 28 kcal mol�1, respectively (Fig. 1). Polarhexaamide rings of MC1 and MC2 adopt a twisted planar geometry,which allows them to self-assemble into bilayer membranes in a tail-to-tail fashion through arene–arene19 and hydrophobic interactions.

MC1 and MC2 structures were determined using 1H NMRspectroscopy (Fig. S3, ESI†). Each macrocycle displays six amide-NH signals, indicating that three sets of constitutionally equivalent–NH groups are located in two different spatial environments: oneinside and the other outside the macrocycle cavity. If all six amide-NH groups were located either inside or outside the macrocycle, theyshould have displayed only three different signals, the lack of whichruled out the latter two scenarios. Variable-temperature 1H NMRstudies did not display any change in the MC1 spectrum withtemperature (Fig. S4, ESI†), ruling out the interconversion of differ-ent conformations via rotation about amide bonds at highertemperatures. The rigidity of the twisted planar conformationrenders these macrocycles inherently chiral.

SEM and TEM studies show that (Fig. 2a–d) in THF the NO2-functionalized MC1 self-assembles into closed-shell vesicles,whereas the NH2-functionalized MC2 forms open-shell vesicles,demonstrating that different functional groups influence the self-assembly of two similar macrocycles such that they producesignificantly different vesicular architectures under identical con-ditions. It is plausible that to avoid electrostatic repulsions betweenN-lone-pair electrons, NH2-functionalized MC2 stacks differently

from the MC1 macrocycle in bilayer membranes, which leads to theformation of open-shell vs. closed-shell vesicles.

DLS measurements (Fig. S5, ESI†) show that the average diametersof closed and open vesicles obtained from 1 mM solutions of MC1and MC2 in THF are 90 and 300 nm, respectively, which are inexcellent agreement with those observed from SEM. While SEM andTEM images (Fig. 2b and d) clearly revealed the hollow interiors ofopen vesicles of MC2, the hollowness of closed-shell MC1 vesicles wasconfirmed by AFM cross-sectional analysis (Fig. S6a, ESI†).5b,e Thewidth (125 nm) of a typical MC1 vesicle (obtained from a 1 mM THFsolution) on the mica surface is ca. 5 times larger than its height(25 nm), indicating that surface-adhered MC1 vesicles becomeflattened upon evaporation of solvents from their hollow cavities.5b,e

Less polar solvents, such as 10% toluene in THF prevented theformation of vesicles (Fig. 2e) by disrupting arene–arene and hydro-phobic interactions between amphiphilic macrocycles. Conversely,in the presence of 20% H2O in THF, significantly larger (ca. 4 times)MC1 vesicles were formed than those formed in pure THF (Fig. S7,ESI†) and MC2 formed closed-shell vesicles (Fig. 2f) instead of openvesicles that were formed in THF. AFM cross-sectional analysisconfirmed the hollowness of closed MC2 vesicles obtained fromaqueous THF (Fig. S6b, ESI†),5b,e as the surface-adhered dry vesiclesdisplayed a width/height ratio of ca. 10. Furthermore, larger vesiclesare formed at higher concentrations of macrocycles (Fig. S8, ESI†).

The toluene-induced disruption and H2O-induced enlarge-ment of vesicles suggest that amphiphilic macrocycles areoriented in a tail-to-tail fashion in bilayer membranes (Fig. 3),

Fig. 2 Field-emission SEM (FE-SEM) images of (a) closed MC1 vesicles and (b) openMC2 vesicles obtained from 1 mM solutions in THF. TEM images of (c) closed MC1vesicles and (d) open MC2 vesicles obtained from 1 mM solutions in THF. SEMimages of (e) disruption of MC1 vesicles in the presence of 10% toluene in THF and(f) closed vesicle formation by MC2 in the presence of 20% H2O in THF.

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projecting p-stacked polar macrocycles in contact with polarsolvents while octyl chains enjoy hydrophobic interactions insidemembranes. Stronger H-bonding interactions between H2O andthe NH2-group of MC2 can dissipate electrostatic repulsionsbetween –NH2 groups, allowing MC2 macrocycles to reorganizein bilayer membranes such that only closed vesicles are formed.

Finally, we have investigated the effect of H+ on the self-assembly of MC1 and MC2 macrocycles. Upon addition of 10%AcOH into a 1 mM solution of MC1 in THF, vesicles began totransform into nanotubes, as evident from the coexistence of bothvesicle and nanotube structures in the medium (Fig. 4a). In thepresence of a stronger acid, i.e., 10% trifluoroacetic acid (TFA) in1 mM solutions of MC1 and MC2 in 9 : 1 THF–H2O, vesicles werecompletely transformed into nanotubes and fibers (Fig. 4b and c,FE-SEM and Fig. S9, AFM, ESI†). The H+-driven morphologicalchange is attributed to protonation of polar macrocyclic head-groups, particularly N and O atoms, which disrupts the stackingof protonated macrocycles in bilayer membranes due to electrostaticrepulsions. To minimize electrostatic repulsion between cationiccenters, protonated macrocycles could rearrange in a circular stair-case fashion (Fig. 3),5e an orientation that would still allow parallel-displaced macrocycles to participate in arene–arene interactions andform nanotubes and fibers.

In conclusion, we have demonstrated that depending on thenature of functional groups (nitro vs. amino), amphiphilic macro-cycles MC1 and MC2 self-assemble into closed- and open-shellvesicles in THF, respectively. While the presence of H2O leads tothe formation of larger MC1 vesicles and converts open-shell MC2vesicles into closed ones, toluene disrupts the formation of normalvesicles. Such an excellent control over the formation of open andclosed vesicles could be exploited for controlled loading of guestmolecules. On the other hand, acidification of the medium trans-forms these vesicles into nanotubes and fibers, demonstrating thepossibility of pH-driven delivery of encapsulated guest molecules.

Inspired by solvents and stimuli-driven morphological changes ofthese nanostructures, we have begun to investigate their guestloading and release properties, which will be reported in due course.

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Fig. 3 Graphical representation of self-assembly of amphiphilic macrocyclesinto bilayer membranes that form vesicles in neutral polar solvents, their H+-driven transformation into nanotubes.

Fig. 4 FE-SEM images of (a) partial transformation of MC1 vesicles into nano-tubes in the presence of 10% AcOH in a 1 mM THF solution, (b) MC1 fibers in thepresence of 10% TFA in a 1 mM 9 : 1 THF–H2O solution, and (c) MC2 fibers in thepresence of 10% TFA in a 1 mM 9 : 1 THF–H2O solution.

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