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Organic &BiomolecularChemistry

1477-0520(2010)8:3;1-H

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http://dx.doi.org/10.1039/c3ob27347d

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1

Low-molecular-weight gelators consisting of hybrid

cyclobutane-based peptides

Sergi Celis,a Pau Nolis,

b Ona Illa,

a Vicenç Branchadell

a and Rosa M.

Ortuño*a

a Departament de Química, Universitat Autònoma de Barcelona, 08193 Cerdanyola del

Vallès, Barcelona, Spain

b Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona,

08193 Cerdanyola del Vallès, Barcelona, Spain

Electronic supplementary information (ESI) available: Appearance of gels in the

different solvents, IR data, computational calculations on an alternative aggregation

pattern for 1, and NMR details on the study of gelation of 1.

Page 1 of 24 Organic & Biomolecular Chemistry

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Abstract

Some hybrid tetrapeptides consisting of (1R,2S)-2-aminocyclobutane-1-carboxylic acid

and glycine, β-alanine, or γ-aminobutyric acid (GABA) joined in alternation,

compounds 1-3 respectively, have been investigated to gain information on the non-

covalent interactions responsible for their self-assembly to form ordered aggregates, as

well as on parameters such as their morphology and size. All three peptides formed nice

gels in many organic solvents and significant difference in their behaviour was not

observed. Scanning electron microscopy (SEM) and circular dichroism (CD) pointed

out that peptide 1, which contains the shortest C2 linear residue, presented the most

defined fibril network and afforded nanoscale helical aggregates. Tetrapeptide 3, with

C4 linear residues in its structure, also showed bundles of fibres whereas a

homogeneous spherulitic network was observed for tetrapeptide 2, with a C3 spacer

between cyclobutane residues. Computational calculations for 1 allowed to model the

self-assembly of the molecules and suggested a head-to-head arrangement to give

helical structures corresponding to hydrogen-bonded single chains. These features were

corroborated by a high-resolution NMR spectroscopy study of the dynamics of the

gelation process in toluene-d8 which evidenced that molecules self-assemble to afford

ordered aggregates with a supramolecular chirality.

Page 2 of 24Organic & Biomolecular Chemistry

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Introduction

In the last few years there has been a growing interest in the design of molecules that

are able to self-assemble in a predictable manner. These systems offer the perspective of

modulating the properties of the resulting materials by a precise design of their

molecular structures. The progress in this area has been directed from the fundamental

scientific curiosity but also from the numerous applications that can be attributed to

these hierarchically assembled aggregates. Particular examples are gels, which are

materials that nowadays have multiple applications in the fields of cosmetics, lubricants,

and food industry, amongst many others.1 Recently, in the field of biomedicine,

molecular gels that respond to different stimuli have been developed to generate

modular materials1a that can be used, for example, in controlled drug delivery2 or in

tissue regeneration3 and that present better biocompatibility indexes than classical

materials. In addition, nanometric fibrillar conductor gels have been developed and

these are of special importance in molecular electronics.4 Furthermore, these materials

can derive in new catalysts able to carry out processes under milder energetic conditions

and in a faster and more selective manner.5 Another important aspect is the introduction

of chirality in the structure of the molecules that self-assemble and generate gels,

conferring these materials with an extra value.6 Of special interest are peptide-based

gels.7 Many of them are classified as low-molecular-weight gelators (LMWGs) and

consist of short peptides or peptidomimetics.8 Many are formed by oligomers generated

from natural amino acids, especially dipeptides, which mainly form hydrogels.9 These

materials have found main applications for the production of functional biological

materials.10 In the recent years, efforts to develop LMWGs based on peptidomimetics


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have been investigated to accomplish the design and preparation of new organogelators

with tuned properties. For instance, bolaamphiphilic gelators have been prepared and,

among them, some ambidextrous gelators are capable of forming gels in organic

solvents and in water.11

Our group has a broad experience in the synthesis and structural studies of

designed peptides based on optically active 2-aminocyclobutane-1-carboxylic acid

derivatives.12-14 Their conformational bias in solution has been investigated pointing out

the relationship between the preferred folding-type and the stereochemistry of the

monomeric units.15 Most of these molecules are prone to self-assemble producing nano-

sized fibres and some of them gelate in various organic solvents (Fig. 1).12,14,16

Fig. 1 Some polycylobutane β-peptides forming organogels

Self-assembly of chiral trans-cyclobutane-containing β-dipeptides into ordered

aggregates was investigated by using several experimental techniques and

computational calculations. The obtained results allowed us to characterize the

assemblies and to present a model for self-assembling that suggested molecular


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arrangements to give helical structures corresponding to hydrogen-bonded single-

chains. These, in turn, interacted with one another to afford bundles. Their significant

geometry obtained computationally fitted well to the main peaks observed in wide-angle

X-ray diffraction spectra of the aggregates in the solid state.16

Very recently, the synthesis and conformational study in solution of a family of

hybrid peptides was achieved. These compounds present cyclobutane β-amino acids and

linear residues such as glycine, β-alanine and γ–amino butyric acid (GABA), 1-3

respectively, joined in alternation (Fig. 2). Results from that study account for the

spacer length effect on folding and show that conformational preference for these hybrid

peptides can be tuned from β-sheet-like folding for those containing a C2 or C4 linear

segment to a helical folding for those with a C3 spacer between cyclobutane units.17

Compound 4 was also prepared and studied to verify the influence of the terminal ester

group in the observed folding with peptide 3.

Fig. 2 Structure of cyclobutane-based hybrid peptides subjected to study

In this paper we describe the gelation behaviour of molecules 1-3 in different

organic solvents. Compound 4 has also been considered to investigate the possible

effect of the lipophilic alkyl chain in the gelation capability of this type of products. For

all of them, their properties such as morphology, size and type of supramolecular


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arrangement in gels was analyzed by using several techniques including scanning

electron microscopy (SEM), circular dichroism (CD) and IR spectroscopy. For gels

produced from α,β-tetrapeptide 1 in toluene, the self-assembly of the molecules has

been modelled suggesting a helical arrangement of the single chains produced. All these

results are in good accordance with high-resolution NMR experiments that, in addition

to studying the dynamics of the sol-gel process, pointed out the chiral recognition of the

prochiral linear segments during aggregation.

Results and discussion

The synthesis, full characterization and secondary structure in solution of cyclobutane-

based hybrid peptides 1-4 (Fig. 2) has been described in reference 17.

1. Gelation studies

Gelation capability was tested for compounds 1-4 in several solvents with

different dielectric constants. Results are summarized in Table 1. Very nice gels were

obtained in toluene in a 7-12 mM minimum-gel-concentration (mgc) range; at lower

concentration as shorter is the linear spacer for 1-3. This concentration compares well

with the mgc values reported for other tetrapeptides with good gelation properties.18

Higher concentration, but in a similar range in most cases, was required to gelate ethyl

acetate, acetonitrile and acetone. For these solvents, compound 4 afforded gels at lower

concentration than tetrapeptides 1–3, which were more soluble, showing thus the

influence of the lipophilic C6-alkyl chain. None of these compounds neither was soluble

in water nor was able to form hydrogels. In contrast, nice gels were obtained from iso-

propanol but they disappeared when some water was added. Tetrapeptides 1-3 formed


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gels in methanol and ethanol at rather high concentrations whereas compound 4 was

very soluble. In the other solvents, behaviour of compounds 1-4 is quite similar

although compound 4 requires lower mgc in most cases pointing out, again, the role of

the alkyl chain. They are insoluble in pentane and diethyl ether and very soluble in

chloroform, tetrapeptide 1 being the only able to form a gel in this solvent at mgc = 26

mM. (See the ESI for appearance of these gels in the different solvents).

Table 1. Gelation behaviour of compounds 1-4 in common solvents.a

Solvent and minimum gel concentrationb,c

Pentane

Toluene

1,4-dioxane

Et 2O

CHCl 3

EtOAc

THF

CH2Cl 2

i PrOH

Acetone

Ethanol

Methanol

MeCN

H2O

1 I 3

(7)

33

(70) I

122

(26)

8

(18)

100

(211)

60

(132)

50

(105)

50

(105)

100

(211)

100

(211)

17

(35) I

2 I 4

(8)

100

(199) I S

10

(20)

100

(199)

100

(199)

50

(99)

20

(40)

100

(199)

200

(398)

10

(20) I

3 I 6

(12)

100

(188) I S

8

(16)

50

(94)

200

(377)

100

(188)

14

(27)

200

(377)

200

(377)

11

(21) I

4 I 6

(12)

33

(65) I S

5

(10)

25

(49)

25

(49)

50

(97)

7

(14) S S

5

(10) I

a Dielectric constant increases from left to right. b mgc in mg/mL and, in parenthesis, mM. c I: insoluble.

S: soluble.

All gels were stable at room temperature for weeks. To check their thermoreversibility,

the best gels at concentrations equal or higher than mgc (Table 2) were heated to just

below the boiling temperature and then they were allowed to cool down to room

temperature. The heating-cooling cycle was repeated twice, thus providing evidence for

the gel formation as the physical bonds could be disrupted upon heating and reformed

during cooling. Gels from the four compounds were thermoreversible in toluene,


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acetonitrile, dichloromethane and, in the case of compound 1, also in chloroform. They

were not in alcohols and ethers.

Table 2. Thermoreversible behaviour of compounds 1-4 in some solvents.

Compound

Solvent and concentrationa Toluene

CHCl 3

CH2Cl 2

MeCN

c1 c2 c1 c1 c1 c2

1 7 15 26 132 35 50

2 8 20 S 199 20 43

3 12 22 S 377 21 35

4 12 21 S 49 10 20

a c1 (mgc) and c2 in mM. S: soluble

2. Scanning Electron Microscopy

SEM experiments were carried out to investigate the morphology of the gels obtained

from 40 mM solutions in toluene. This concentration allowed us to obtain the best

defined structures to be analyzed. SEM micrographs of xerogels from 7, 15 and 40 mM

solutions of 1 in toluene, with different magnification each, are provided in the ESI.

Toluene is a solvent appropriate to perform NMR experiments conducted to gain insight

into the sol-gel process. SEM micrographs of the corresponding xerogels were obtained

at a pressure of 70 Pa for all four tetrapeptides (Fig. 3). Tetrapeptide 1 exhibited the

most defined structures, giving bundles of fibres with variable width (100-600 nm).

Tetrapeptide 2 gave homogeneous and non defined structures with the appearance of


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rough spheres and more poorly defined and wider bundles were observed for

tetrapeptide 3. Compound 4 gave wide and undefined structures although it is possible

to appreciate very small fibres that are intertwined with each other. This fits well with

the higher molecular flexibility of 4 due to the absence of the ester group at the C-

terminus, thus avoiding hydrogen-bond interactions. This fact accounts for the less

ordered molecular and supramolecular structures observed. The different behaviour of

tetrapeptide 2, affording a spherulitic instead of a fibrillar network in the xerogel, was

also evidenced in solution. In that case, a helical folding was observed while 1 and 3

presented β-sheet-type folding.17

Furthermore, the definition of the supramolecular structures for 1–3 correlate

well with the level of conformational order observed for the same molecules in

solution.17 These properties emphasize the influence of the linear segment length

connecting the cyclobutane moieties.

10 µµµµm 40 µµµµm

(a) (b)

20 µµµµm 10 µµµµm

(c) (d)

Fig. 3 SEM images of samples of 1 (a), 2 (b), 3 (c) and 4 (d) as xerogels (from toluene) on graphite at 70

Pa.


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3. IR spectroscopy

The N-H and C=O regions of the vibrational spectra of 1-4 in the solid state were

investigated by IR spectroscopy (ATR mode), as were the corresponding bands for the

xerogels from toluene (30 mM in KBr) and solution (5 mM in chloroform). Note that

toluene was not used due to aggregation problems. Results are summarized in Table S1

in the ESI.

No significant differences were observed between the spectra of the four

xerogels. In chloroform solution, tetrapeptides 1-3 showed one band at about 3420 -

3440 cm-1 due to non-associated N-H stretching. Bands corresponding to associate N-H

stretching were not observed in any case. In contrast, compound 4 showed two bands of

comparable intensity and about 3440 and 3310 cm-1 (toluene) corresponding to free and

associate N-H stretching, respectively. The N-H stretching bands were shifted to lower

wave number and their intensity was increased when passing from liquid solution to

solid, in accordance with the formation of aggregates.

Relevant features were not observed in the C=O region.

4. Circular Dichroism Spectroscopy

CD spectroscopy was used to obtain more information about the self assembly

of the four compounds. The solid-state CD spectra of the xerogels (30 mM in KBr)

from toluene displayed bisignate Cotton effects (Fig. 4). They showed a negative Cotton

effect at 224-226 nm and a positive one at 242-251 nm. This CD signature differs from

the ones obtained for 0.5 mM solutions of these compounds in methanol that showed

only one negative peak at 217-219 nm for 1 and 3 in agreement with β-sheet-like

structures, and one peak at 223 nm attributable to a 14-helical folding in solution for 2.17


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Fig. 4. CD spectra from xerogels from toluene in KBr, c = 30 mM.

Therefore, comparison of the CD spectra indicates that the supramolecular

arrangement in the xerogel is different from the molecular conformational structures in

solution. Moreover, the CD signature observed for the xerogels obtained from toluene

suggests that the molecules are organised in helical structures in the supramolecular

aggregates, as observed for related systems previously studied in our laboratory and

shown in Fig. 1.16 Similar CD spectroscopic features were observed for the gels formed

from other systems also described as arranged in supramolecular helical structures.19

5. Computational calculations for tetrapeptide 1.

To establish the main interactions responsible for the supramolecular arrangement of

these compounds, computational calculations were carried out on tetrapeptide 1 (Fig. 5).


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HN

O

NH

O

NH

O

NHO

MeO

O

O Ph10

2527

18 1916

15

24

9

Fig. 5 Structure of hybrid α,β-tetrapeptide 1 showing some selected atom numeration.

A conformational search was accomplished for a tetrameric aggregate of 1. In

the most stable structure, intramolecular hydrogen bonds prevail in the terminal units,

whereas the central tetrapeptide molecules interact with each other forming

intermolecular hydrogen bonds with their neighbours (CO9-NH10, CO15-NH16, CO18-

NH19 and CO24-NH25). This is in accordance to a head-to-head-type orientation.

From the interaction pattern between the central molecules, the structures of

dimeric, D, tetrameric, T, and hexameric, H, aggregates were optimized using the M06-

2X density functional method with the 6-31G(d) basis set (Fig. 6). These structures

show a tendency of tetrapeptide 1 to form helical aggregates.

An alternative interaction pattern has also been explored for the dimeric and

tetrameric aggregates (see ESI), but the corresponding structures are energetically less

favourable than those shown in Fig. 6.


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D T

H

Fig. 6 Structures of dimeric (D), tetrameric (T) and hexameric (H) aggregates of peptide 1 optimized

at the M06-2X/6-31G(d) level of calculation. Selected interatomic distances are in Å. Non polar

hydrogen atoms were omitted for clarity

If we focus our attention in the hydrogen bond distances between the central

molecules, we can observe a tendency to strengthen the interaction as the number of

molecules increases. This fact can be confirmed when the aggregation energies are

considered (see Table 3). As a matter of fact, the aggregation energy per molecule

(∆E/n) linearly increases (in absolute value) with the number of molecules, in such a

way that there is a cooperative effect in the aggregation process.


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Table 3. Aggregation energiesa computed for tetrapeptide 1

In vacuob In toluenec

∆E ∆E/n ∆E ∆E/n

D (n=2) −22.1 −11.0 −15.8 −7.9

T (n=4) −76.9 −19.2 −50.6 −12.6

H (n=6) −149.7 −25.0 −105.8 −17.6

a All values in kcal mol-1. ∆E corresponds to the n 1 → (1)n process. b M06-2X/6-31G(d) level of

calculation. c SMD-M06-2X/6-311+G(d,p)//M06-2X/6-31G(d) level of calculation.

From the central units of the hexameric aggregate H, idealized helical octameric and

hexadecameric aggregates were built and their structures were optimized at the MMFF

level of calculation in chloroform solution. The structure obtained for the

hexadecameric aggregate is shown in Fig. 7.

Side view

Top view

Fig. 7. Structure of the hexadecameric aggregate of peptide 1 optimized at the MMFF level of calculation

in chloroform. Non polar hydrogen atoms have been omitted for clarity.


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6. High-resolution NMR spectroscopy studies for tetrapeptide 1

Gelation induces reduced mobility of the molecules. Therefore, if gelation is slow

compared to NMR time scale measurement, this dynamic process can be monitored by

variable temperature 1H-NMR experiments. By decreasing temperature a continuous

signal line broadening is observed and finally a complete signal loss is produced due to

the increasing “solid-like” part of the gel, which is NMR invisible.20 Furthermore, if

gelation process is monitored in sufficiently slow motion, a differential behaviour of

signals can be observed which helps to better understand the gel-formation dynamic

process. Adequate conditions of sample concentration and appropriate cooling down

gradient temperature are crucial in these experiments because gelation process strongly

depends on both interdependent factors. In this specific study, a controlled cooling

down regime was applied to a 30 mM sample in toluene-d8. Solutions with other

concentrations (ca. 15 ad 45 mM) were also used affording results similar to those

obtained with the 30 mM sample, which gave the most suitable signals for the NMR

studies undertaken regarding intensity and resolution. 1H-NMR spectra were acquired in

5 K steps, starting from 375 K and lowering to 275 K (Fig. 8). An equilibration period

of three minutes was used as sample thermal equilibration period.

For all protons, two well separated stages are observed during the cooling

regime. In the former, just prior to gelation, the intensity increases and signals become

sharper. This is due to a molecule fixation positioning with a restricted conformational

motion. In a second stage, signal intensity starts to decay and this is due to gelation

process itself. It is noticeable that this occurs at slightly different temperatures for each

NH proton (Fig. 8a).


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Unfortunately, NH19 proton is overlapped by the toluene signal and its complete

behaviour cannot be evidenced. Compared with NH10 and NH25, the fastest intensity

attenuation corresponds to NH16, which starts its signal decay at 350 K vs. 340 K for the

other protons. This observation correlates satisfactorily with the performed calculations

in the sense that NH16 is positioned in a centred point of the gel structure which is

expected to be a more rigid region compared to the gel sides (Fig. 7). (Graphical plots

for each proton are shown in the ESI).

NH19 NH16 NH25 NH10

Fig. 8 Gelation process monitored by variable-temperature 1H NMR spectroscopy experiments in 30 mM

solutions of 1 in toluene-d8. A 400 MHz Bruker Avance III spectrometer equipped with a cooling unit

BCU-Xtreme was used. (a) NH region from 5.6 ppm to 7.3 ppm. (b) Aliphatic region from 2.8 ppm to 5.2

ppm.

Another interesting feature about gel formation was observed in the aliphatic

region (Fig 8 b). Here a quite noticeable differential behaviour of diastereotopic protons


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was observed during gelation process, both in terms of chemical displacement direction

and gelation point. It should be highlighted that, to the best of our knowledge, this

behaviour has not been reported before. In order to better visualize the differences,

graphical plots are presented in the ESI for the three pairs of diastereotopic protons H7,

H17 and H26. For instance, it is observed that while H7a proton starts to attenuate at 330

K, H7b attenuates at 350 K. This indicates that gelation is being oriented in a direction

that restrains the mobility of one of the diastereotopic protons. A more pronounced

signal decay for both protons starts at 310 K, which is attributed to the global network

gelation. Moreover, a remarkable perturbation in the chemical shift displacement

tendency of H7b is observed at this point due to a conformational change to

accommodate gelation. Finally, once the gel is totally formed below 290 K, the same

very low intensity is observed for both protons. Similar differential behaviour is

observed for H17 and H26 diastereotopic pairs of protons, although in a minor degree for

the later pair. This can be also explained with the help of computational calculations

that suggest a structure for aggregates where it is clearly observed that proR H17 is

parallel to hydrogen bonding direction, while proS H17 is in a perpendicular position

(Fig. 9).


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Fig. 9 Three consecutive peptide molecules from the hexadecameric aggregate of peptide 1 optimized at the MMFF level of calculation in chloroform. proR and proS hydrogen atoms of the central molecule are represented by yellow and brown spheres, respectively, and they correspond to

H7, H17 and H26 methylene protons from left to right.

On the other hand, both proR and proS H26 protons are situated approximately at

the same angle regarding hydrogen bonding direction, therefore justifying their more

similar behaviour.

Furthermore, cyclobutane H11/H20 protons also show a remarkable differential

behaviour since H11 starts gelation much earlier than H20 (365 K vs. 335 K). This is

consistent with the different structural surroundings for both protons and their distinct

alignment with respect to hydrogen bonding direction.

Conclusions

The three hybrid α,β-, β,β- and γ,β-tetrapeptides investigated have shown their

capability to gelate several organic solvents, being toluene that requiring the lowest

mgc. SEM revealed that, while 1, 3 and 4 present fibrillar networks in the xerogel, a

spherulitic netwok is evidenced for 2. The most defined and regular morphology was

observed for 1, fact that correlates the flexibility of tetrapeptides 1–3 with their ability

to self assemble into ordered aggregates, as previously observed in solution.

Computational calculations performed on 1 suggest a helical arrangement of the

self-assemblies in excellent agreement with CD and NMR spectroscopies and suggest

that in the aggregates there is a more rigid central region and two more flexible ones

corresponding to the N- and C-terminus at the sides of the aggregate structure. This

agrees nicely with NMR results and allowed us to conclude that the structure of 1

around the gelation point has two differentiated regions concerning mobility. In the

more rigid central one, NH16 is the hydrogen-bonding promoter thus acting as the


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gelation driving-force. Furthermore, NMR experiments pointed out that gelation occurs

in such a way that a chiral supramolecular structure is produced, which strongly agrees

with a certain degree of chiral helicity in accordance with CD studies and also with

computational calculations, as shown in Fig. 7.

Experimental

Procedure for gel preparation

A small amount (5.0 ± 0.1 mg) of peptide is weighted in a 2 mL transparent-glass vial

with septum screw-on cap. When the 5 mg amount of peptide is soluble in a specific

solvent, a new vial containing 10 ± 0.1 mg is prepared and the solubility-gelification

checked again. In a second step, a certain volume of solvent to be tested is added and

the vial closed. The minimum volume added is 0.05 mL. Then the mixture is heated

under the boiling point of the solvent using a balloon system in order to avoid solvent

pressure and, once a solution is obtained the mixture is sonicated for 1 to 5 minutes. For

high concentrations and also in some solvents, previous sonication is needed for a good

solubilization during heating and sonication time is usually shorter than for diluted gels.

Then, the mixture is left to stabilize and to reach room temperature. To state that the

mixture is a gel the tube inversion test is done just by turning the vial upside down (see

ESI). If the sample is a gel it does not drop and if it drops a little it can be classified as

gel-like mixture. We can also state the mixtures as solutions or insoluble systems. In

order to determine the mgc, a new volume of solvent is added to the gel and the process

is repeated until no gel is formed: the last volume added determines the mgc.

SEM measurements


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Wet gels were disposed on a carbon-film-coated copper grid and dried by standing for

30 minutes on the grid. The resulting xerogels (dry gels) were then introduced into the

microscope working at 70 Pa and 5 kV.

Computational details

A Montecarlo conformational search21 using the MMFF22 force field implemented in

the Macromodel program23 was carried out for a tetrameric aggregate of 1. The solvent

effect has been included through the GB/SA method 24 using chloroform as solvent.

The structures of dimeric, tetrameric and hexameric aggregates were optimized

using the M06-2X25 density functional method with the 6-31G(d) basis set. Harmonic

vibrational frequencies were computed for the structures of dimeric and tetrameric

aggregates to ensure that they correspond to energy minima (all frequencies are real).

The energies of the obtained structures were recalculated at the same level of

calculation with the 6-311+G(d,p) basis set in toluene solution using the SMD

method.26 These calculations were done using the Gaussian-09 program.27

Acknowledgements

The authors thank financial support from Spanish Ministerio de Ciencia e Innovación

(grant CTQ2010-15408/BQU) and Generalitat de Catalunya (grant 2009SGR-733 and

XRQTC). They are also grateful to European Union for COST Action CM0803. Time

allocated in the Servei de Ressonància Magnètica Nuclear (UAB) and CESCA is

gratefully acknowledged

References


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