Structure-activity effects in peptide self-assembly and gelation - Dendritic versus linear architectures

Article abstract of DOI:10.1039/c2cc32921b

We demonstrate that linear-dendritic shape-isomerism can have an impact on the gelation potential of peptides based on lysine, but the architectural effects can be inverted depending on the choice of functional groups, with the nature of these protecting

Full text of DOI:10.1039/c2cc32921b

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Structure–activity effects in peptide self-assembly and
gelation – Dendritic versus linear architecturesw
Cecile A. Lagadec and David. K. Smith*
Received 24th April 2012, Accepted 12th June 2012
DOI: 10.1039/c2cc32921b
We demonstrate that linear-dendritic shape-isomerism can have
an impact on the gelation potential of peptides based on lysine,
but the architectural effects can be inverted depending on the
choice of functional groups, with the nature of these protecting
groups dominating the gelation ability.
a particularly rich source of organo–and hydro-gelators over
the last 10 years,13 as it is both a rich source of hydrogen bond
functionality and also relatively easily functionalised to give a
wide range of different structures. Compounds 1 and 2 are
architectural/structural isomers with Boc protecting groups
and ‘linear’ and ‘dendritic’ structures respectively. Compounds
3 and 4 are equivalent to 1 and 2 but have Z protecting groups
instead of Boc. We reasoned that these compounds would allow
us to deduce the effect of peptide architecture, and the con-
sequent organisation of hydrogen bonding amide groups, on
gelation. The synthesis and characterisation of novel gelators
1–4 is described in full detail in the supporting information.w
Characterisation of the compounds by NMR showed the
difference between linear and dendritic systems in terms of
organising the hydrogen bonding CONH groups. This is
reflected in the resonances of the chiral C–H protons of the
lysine groups (see experimental data in supporting informa-
tionw). For linear compounds 1 and 3, two *C–H are adjacent
to two amide groups (‘chain-like’ and appear more downfield
at ca. 4.2 ppm), and one is between an amide and a carbamate
(‘surface-like’ and appears more upfield at 3.8 ppm). For
dendritic compounds 2 and 4, the situation is reversed with
one ‘chain-like’ *C–H at ca. 4.2 ppm and two ‘surface-like’
groups at ca. 3.8 ppm. These kind of architectural differences,
reflected in the NMR spectra, may underpin different mole-
cular recognition pathways within the self-assembly of these
architectural isomers.
There has recently been intense interest in understanding the
self-assembly of molecular-scale building blocks into fibrillar
materials, a process which can take place in a range of different
solvent environments to give rise to gel-phase behaviour.1
Peptides are particularly versatile building blocks for gelation2
and have been used to create organogels and hydrogels with
high-tech applications ranging from nanoelectronics to tissue
engineering.3 We have been interested in peptide organogels
based on a dumb-bell shaped architecture (Fig. 1) which
assemble as a consequence of intermolecular peptide-peptide
hydrogen bond interactions.4 In particular, we have focussed
on examples where the peptide head groups are dendritically
branched.5
There has been considerable interest in exploring structure–
activity relationships in self-assembling dendritic molecules –
with a number of reports outlining the importance of the
dendritic structure in controlling gelation.6 We have previously
explored structure–activity effects using our dendritic gelators,
and reported the dendritic effects of branched peptide head
groups,7 modifications in the spacer chain,8 variation of amino
acids,9 role of protecting groups10 and solvent effects.11 We
also recently reported that linear peptide oligomers form
effective organogels, even when present as ill-defined mixtures.12
However, there have been no explorations of the way in which
dendritic architectures differ from their linear analogues
in terms of underpinning gels, which would allow the ‘true
dendritic effect’ to be elucidated. This paper therefore explores
a comparison between dendritic and linear peptide gelators
with identical molecular formulae.
We initially explored the gelation ability of these com-
pounds in 1,2-dichlorobenzene (DCB), and where appropriate
determined gel–sol transition temperatures (Tgel values) using
the reproducible tube inversion methodology.14 Dendritic
compound 2 was a more effective gelator than the linear
analogue, compound 1, which might suggest that the dendritic
architecture is preferable for self-assembly and gelation
(Table 1). However, linear gelator 3 was a much more effective
We synthesised compounds 1–4 (Fig. 2) using stepwise
solution-phase peptide chemistry with appropriate protecting
group methodologies. Gelators derived from lysine have been
Department of Chemistry, University of York, Heslington, York,
YO10 5DD, UK. E-mail: dks3@york.ac.uk
w Electronic supplementary information (ESI) available: Synthesis
and characterisation, IR spectra, Job plots and titration curves. See
DOI: 10.1039/c2cc32921b
Fig. 1 Schematic diagram of the self-assembly of a dumbbell-shaped
peptide into a fibrillar gel-phase material.
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studies were in agreement with the Tgel values reported above,
with NMR peaks emerging from the baseline at around the
Tgel value, as the gelator molecules become mobile.
To probe this further, we investigated gelation in a wider
range of solvents. As we have observed before, gelation was
best supported in solvents with effectively no hydrogen bonding
ability, such as chlorobenzene, dibromoethane and toluene.
Limited gelation was observed in phenetole, which has some
ability to accept hydrogen bonds with its oxygen atom. In each
case, the trends observed in dichlorobenzene were replicated
i.e., Z-protected compounds are more effective than Boc-protected
analogues, with Boc protection the branched architecture is
preferred, whereas with Z protection the linear architecture is
better. In more polar, hydrogen bond acceptor solvents, such
as acetonitrile and dimethoxyethane, gelation was not observed.
Gels were also not observed in hydrogen bond donor solvents
such as methanol. In decane, there was no gelation – as a very
low polarity solvent, it is unable to sufficiently solubilise the
compounds and self-assembly is therefore unable to take place.
These solvent effects demonstrate how gelation occurs at a
borderline of solubility – compounds must be soluble enough
to allow self-assembly, but also must not interact too strongly
with the solvent, or self-assembly is disrupted.
Fig. 2 Structures of linear and branched gelators derived from lysine
investigated in this paper.
Table 1 Visual observations of gelation for compounds 1–4 (and Tgel
values at 1% wt/vol), in a range of different solvents. G = gel, PG =
partial gel, S = solution, O = opaque
The solvent effects indicate that, as expected, peptide–peptide
hydrogen bond interactions are primarily responsible for gelation
in this case. However, the data also indicate that the choice of
protecting groups has a profound impact on the self-assembly
of these peptides, with Z groups encouraging hierarchical self-
assembly and gelation in comparison with the the Boc groups. We
suggest that this is a consequence of the ability of these groups to
participate in p–p interactions. The better ability of Z-protected
compounds to aggregate is supported by their lower observed
solubility – which will also help drive the gelation process.10 In
addition, changing the protecting groups inverts the dendritic/
linear architectural preferences of the overall assembly process.
We suggest that the linear compound is better able to organise the
aromatic rings of the Z groups for mutual interaction than the
branched isomer can. Indeed, for the branched isomer, there is
little difference between Z and Boc derivatives, suggesting ineffec-
tive organisation of the p-stacking groups in this morphology. For
the Boc protected system, we suggest that steric hindrance
between the bulky Boc groups plays a role in limiting the assembly
of the linear isomer, but has less impact on the dendritic system
where all of these groups are on the periphery of the molecule.
We attempted to probe this hypothesis further using circular
dichroism spectroscopy, but unfortunately, the aromatic/halo-
genated solvents which support gelation obscured the bands
associated with peptide self-assembly.
1
Boc-protected
2
3
Z-protected
4
Linear
Branch
Linear
Branch
Solvent
Chlorobenzene
1,2-Dichlorobenzene
1,2-Dibromoethane
Phenetole
OS
PG
OS
OS
OG (29)
OG (30)
OG (26)
PG
G (72)
G (120)
G (110)
G (85)
OG (27)
G (31)
G (41)
OG (24)
gelator than dendritic compound 4, which would suggest that
a linear architecture was more effective for underpinning
gel-phase materials (Table 1). Fig. 3 illustrates the dependence
of Tgel on gelator concentration for compounds 3 and 4.
Linear compound 3 forms a room temperature gel above
ca. 2 mg mlÀ1 (0.2% wt/vol), while dendritic compound 4
forms an effective gel above ca. 4 mg mlÀ1 (4% wt/vol)
indicating they are both efficient gelators. Compound 3 has
a maximum Tgel value of 125 1C, while for compound 4 it is
only 40 1C. Furthermore, compounds 3 and 4 are both more
effective gelators than compounds 1 and 2 – clearly the nature
of the protecting groups (i.e., the molecular formula of the
gelator) plays a more important role than whether the archi-
tecture is linear or dendritic. Variable temperature NMR
We employed scanning electron microscopy (SEM) to gain
further insight into the self-assembly process. Initially, the
xerogels formed by 3 and 4 were imaged after drying from
DCB and phenetole under ambient conditions. In DCB rela-
tively similar looking fibres were observed (Fig. 4A and B),
whereas in phenetole it appeared that the linear compound
(Fig. 4C) formed a somewhat better defined gel fibre network
than the dendritic analogue (Fig. 4D) – consistent with its
higher Tgel value. We were unable to image the Boc-protected
gelators by SEM, as they did not form such effective extended
gel-fibre networks as their Z-protected analogues.
Fig. 3 Effect of weighed mass of gelator (in 0.5 mL of DCB), on the
Tgel value as determined by the inverted vial method.
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organisation of hydrogen bonding groups to underpin gelation.
Furthermore, this relationship is not a simple one – the nature
of functional groups present (Boc or Z) inverts the archi-
tectural effect – indeed these protecting groups play a domi-
nating role in controlling gelation. This indicates the subtlety
of molecular recognition pathways within self-assembled
peptide nanomaterials, and demonstrates the importance of
carefully optimising molecular structures to obtain the desired
materials behaviour.
We acknowledge EPSRC and Givaudan for funding
(Industrial CASE award).
Notes and references
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Fig. 4 SEM images of xerogels formed by Z-protected gelators. A:
Linear gelator 3 dried from DCB, B: gelator 4 dried from DCB, C:
gelator 3 dried from phenetole, D: gelator 4 dried from phenetole.
Scale bars: 100 nm (A,B,D) and 200 nm (C).
5 D. K. Smith, Adv. Mater., 2006, 18, 2773–2778.
6 For selected classic and recent examples see: (a) W.-D. Jang,
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Fig. 5 SEM images of A: compound 3, and B: compound 4, after
drying from DCB under low temperature conditions to create an
aerogel. Scale bar: 100 nm. Insets show larger area images.
We also applied a new approach to sample preparation in
order to image the gels formed by compounds 3 and 4 under
more representative conditions. We used critical point drying
to remove solvent under low temperature supercritical condi-
tions from samples of the gel under high vacuum, on the SEM
stub. We were able to do this in our laboratory and then
transfer the samples into the SEM machine. Remarkably, the
expanded aerogels formed during this drying process were
highly stable. Because the samples are dried at liquid nitrogen
temperatures, they are less susceptible to thermal collapse
during drying, and should be more representative of the three
dimensional solvated structure of the gel.15 This was indeed
the case, as can be seen in Fig. 5. There were some differences
in the gel networks, with linear compound 3 appearing to form
larger fibres (ca. 40 nm) than those of dendritic compound 4
(ca. 20 nm). We suggest that these larger, more aggregated
fibres may reflect greater fibril aggregation, perhaps driven by
p-stacking of the Z groups.
12 C. A. Lagadec and D. K. Smith, Chem. Commun., 2011, 47, 340–342.
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In summary, we have demonstrated that by careful synthesis
it is possible to make linear and dendritic gelators with
identical molecular formulae, but which exhibit significantly
different organogelation characteristics, and thus elucidate the
‘true dendritic effect’ on gelation. This demonstrates that
molecular architecture plays a role in enabling the effective
14 S. R. Raghavan and B. H. Cipriano, in Gel Formation Phase
Diagrams using Tabletop Rheology and Calorimetry, Chapter 8 in
Molecular Gels, Materials with Self-Assembled Fibrillar Networks,
ed. R. G. Weiss and P. Terech, Springer, Dordrecht, Netherlands,
2006.
15 R. H. Wade, P. Terech, E. A. Hewat, R. Ramasseul and F. Volino,
J. Colloid Interface Sci., 1986, 114, 442–451.
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