Study of (Cyclic Peptide)–Polymer Conjugate Assemblies by Small-Angle Neutron Scattering

Article abstract of DOI:10.1002/chem.201603091

We present a fundamental study into the self-assembly of (cyclic peptide)–polymer conjugates as a versatile supramolecular motif to engineer nanotubes with defined structure and dimensions, as characterised in solution using small-angle neutron scattering (SANS). This work demonstrates the ability of the grafted polymer to stabilise and/or promote the formation of unaggregated nanotubes by the direct comparison to the unconjugated cyclic peptide precursor. This ideal case permitted a further study into the growth mechanism of self-assembling cyclic peptides, allowing an estimation of the cooperativity. Furthermore, we show the dependency of the nanostructure on the polymer and peptide chemical functionality in solvent mixtures that vary in the ability to compete with the intermolecular associations between cyclic peptides and ability to solvate the polymer shell.

Full text of DOI:10.1002/chem.201603091

DOI: 10.1002/chem.201603091
Full Paper
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Supramolecular Structures
Study of (Cyclic Peptide)–Polymer Conjugate Assemblies by
Small-Angle Neutron Scattering
Ming Liang Koh,[a, b] Paul A. FitzGerald,[a] Gregory G. Warr,[a] Katrina A. Jolliffe,*[a] and
Sꢀbastien Perrier*[a, b, c]
Abstract: We present a fundamental study into the self-as-
sembly of (cyclic peptide)–polymer conjugates as a versatile
supramolecular motif to engineer nanotubes with defined
structure and dimensions, as characterised in solution using
small-angle neutron scattering (SANS). This work demon-
strates the ability of the grafted polymer to stabilise and/or
promote the formation of unaggregated nanotubes by the
direct comparison to the unconjugated cyclic peptide pre-
cursor. This ideal case permitted a further study into the
growth mechanism of self-assembling cyclic peptides, allow-
ing an estimation of the cooperativity. Furthermore, we
show the dependency of the nanostructure on the polymer
and peptide chemical functionality in solvent mixtures that
vary in the ability to compete with the intermolecular associ-
ations between cyclic peptides and ability to solvate the
polymer shell.
Self-assembling CPs are a highly versatile design platform[19] in
which alterations to the CPs are reflected in the final cyclic
peptide nanotube assembly; for example, the number of
amino acids in the CP correlates to the diameter of the nano-
tube.[19] Since the earliest examples of self-assembling CPs,
imaging techniques have shown the assemblies as very long
and rigid structures,[17] yet controlling the length of the extend-
ed nanotube structures (i.e. beyond dimerisation) is not
straightforward, requiring the self-assembly process to be
tamed. However, in a recent example, Mizrahi et al. demon-
strated length control through the use of a ‘layer-by-layer’-like
approach, in which the authors sequentially deposited posi-
tively and negatively charged CPs to obtain short nanotubes
of precise lengths.[20] An interesting templating approach was
proposed by Granja et al., stemming from their molecular dy-
namics study of CPs (a and g residues), they suggest that the
use of organochloride molecules in a highly hydrogen bonding
competitive solvent (such as water) would promote the associ-
ation of the CPs around the guest molecule (i.e. enveloping
the molecule in the core of the nanotube) and promote disso-
ciation of the CPs beyond the chain length of the organochlor-
ide molecule.[21]
Introduction
Nanotubes are attractive scaffolds for a variety of applications
such as materials, electronics, sensors, catalysis, drug delivery
and ion channels, owing to their anisotropic geometry, rigid
structure and their internal pore.[1–6] Although there are cova-
lent-based routes to tubular nanostructures, notably, bottle-
brush copolymers[7–9] and carbon nanotubes,[10] the per-
formance of natural systems such as gramicidin A,[11,12] a-he-
molysin,[13] and the tobacco mosaic virus,[14,15] which are capa-
ble of precise manufacturing of nanotubes as well as enabling
dynamic/reversible properties, are highly inspiring incentives
for taking a supramolecular approach.
Self-assembly is a powerful route that has been used to real-
ise synthetic structures with control on the nanometer length
scale.[16] Pioneered in the 1990s, cyclic peptides (CPs) of alter-
nating d and l amino acids adopt a conformation that pro-
motes stacking between CP units by the formation of cylindri-
cal b-sheets.[17,18] Many groups, including our own, have used
self-assembling CPs as a guiding motif for nanotube structures.
[a] Dr. M. L. Koh, Dr. P. A. FitzGerald, Prof. G. G. Warr, Prof. K. A. Jolliffe,
Prof. S. Perrier
The conjugation of synthetic polymers to natural products,
particularly biopolymers, is a simple and effective approach to
combine functionalities which can be engineered for a multi-
tude of applications.[22–24] Likewise the recent developments in
covalent tethering synthetic polymers to self-assembling cyclic
peptides[25–30] have further widened the possibilities by adding
functionality for processing,[31,32] pH[33] or temperature[34,35]
stimuli response, or for drug delivery.[36,37] Additionally, the
tethered polymers allow control over the nanotubular struc-
ture such as the diameter,[38,39] ordering and orientation,[40] pro-
moting formation of macropores,[41] or to stabilise the other-
wise dynamic exchange of CPs.[39,42] In the context of control-
School of Chemistry, The University of Sydney
NSW 2006 (Australia)
E-mail: kate.jolliffe@sydney.edu.au
s.perrier@warwick.ac.uk
[b] Dr. M. L. Koh, Prof. S. Perrier
Department of Chemistry, University of Warwick
Coventry CV4 7AL (UK)
[c] Prof. S. Perrier
Faculty of Pharmacy and Pharmaceutical Sciences
Monash University, VIC 3052 (Australia)
Supporting information including detailed synthetic information for this ar-
ticle is available on the WWW under http://dx.doi.org/10.1002/
chem.201603091.
Chem. Eur. J. 2016, 22, 1 – 11
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under reduced pressure. The residue was washed with ice-cold
MeOH (4ꢂ20 mL) then dried under reduced pressure. Boc groups
were removed by treatment with TFA/TIPS/H2O (18:1:1 v/v/v, 5 mL)
for 2 h, then the mixture was triturated with ice-cold diethyl ether
and washed twice with ice-cold diethyl ether to give CP1 as an off-
ling the nanotube length, Biesalski et al. first suggested the
use of increasing the polymer mass and graft density to tailor
the length of the nanotubes by steric repulsion.[25] These au-
thors then investigated the impact of the polymer molecular
mass by using atomic force microscopy to characterize the sur-
face absorbed nanostructure (after drying), which showed an
increase in height and decrease in nanotube length as the
polymer molar mass increased.[38] Recently we reported the
first characterisation of (cyclic peptide)–polymer assemblies in
solution by the use of small-angle neutron scattering (SANS),
confirming their rigid, rod-like structure and providing the di-
mensions of the assembly.[43] An interesting result of this study
showed the impact of the grafted polymer length on the as-
sembly structure in solution, revealing that the nanotube
length depends on both steric hindrance and solvent accessi-
bility to the peptide core, that is, the increase of tethered poly-
mer mass can promote the formation of tubes by shielding
the CP core from a solvent that competes with the intermolec-
ular H-bonding between CPs.[43]
1
white powder: 0.25 g, 71% yield; H NMR (400 MHz, [D1]TFA): d=
8.11 (d, J=8 Hz, 2H, 2ꢂTrp-CHaromatic), 7.66 (d, J=8 Hz, 2H, 2ꢂTrp-
CHaromatic), 7.54 (s, 2H, 2ꢂTrp-CHaromatic), 7.26–7.42 (m, 4H, 4ꢂTrp-
CHaromatic), 5.21 (t, J=8 Hz, 2H, 2ꢂTrp-a-H), 4.60–4.85 (br m, 6H, 4ꢂ
Leu-a-H+2ꢂLys(N3)-a-H), 3.00–3.40 (br m, 8H, 2ꢂLys(N3)-CH2-N3 +
2ꢂTrp-a-CH2), 0.50–2.00 ppm (br m, 48H, 2ꢂLys(N3)-a-CH2-CH2-
CH2 +4ꢂLeu-a-CH2-CH+8ꢂLeu-CH3); MALDI-FTICR MS m/z found:
1156 (M+Na)+; ATR-FTIR: nmax =3270 (NꢀHstr), 2093 (N3), 1626 cmꢀ1
(C=Ostr).
CP2: H2N(-l-Lys(N3)-d-Leu-l-Lys(Boc)-d-Leu)2-OH (0.09 g, 0.08 mmol)
was dissolved in DMF (73 mL) with 1 h of sonication at 408C. The
solution was purged with N2(g). DMTMM·BF4 (0.03 g, 0.09 mmol) in
DMF (3 mL) was purged with N2(g) and added dropwise to the
linear peptide solution. The solution was stirred for 67 h at 408C
under an atmosphere of N2(g). DMF was removed under reduced
pressure. The residue was washed with MeOH precooled to ꢀ788C
(dry ice/acetone) (2ꢂ10 mL) then dried under reduced pressure to
Herein, we directly compare the supramolecular structures
formed by non-conjugated CPs and polymer-conjugated CPs
in solution by SANS, and also examine the influence of the sol-
vating media by varying the strength of competition for H-
bonding sites and polymer solvation. These conjugates present
an ideal system by which to study the self-assembly of cyclic
peptides, providing insight to the growth mechanism includ-
ing an estimation of the degree of cooperativity. Variations to
the cyclic peptide residues with polar and non-polar side
chains also enable a study of the impact of chemical function-
ality at the external periphery of the peptide core.
1
give CP2 as a white powder: 42 mg, 45% yield; H NMR (400 MHz,
[D1]TFA): d=4.60–5.00 (m, 8H, 2ꢂLys(N3)-a-H+2ꢂLys-a-H+4ꢂ
Leu-a-H), 3.41 (t, J=6 Hz, 4H, 2ꢂLys(N3)-CH2-N3), 3.20–3.34 (br m,
4H, Lys-CH2-NH2), 1.25–2.12 (br m, 36H, 2ꢂLys(N3)-a-CH2-CH2-
CH2 +2ꢂLys-a-CH2-CH2-CH2 +4ꢂLeu-a-CH2-CH), 0.91–1.11 ppm (br
m, J=8 Hz, 24H, 8ꢂLeu-CH3); ESI-LCQ MS m/z=1240 (M+Na)+;
ATR-FTIR: nmax =3270 (NꢀHstr), 3037–2800 (CꢀHstr), 2096 (N3),
1624 cmꢀ1 (C=Ostr).
CP3: H2N(-l-Lys(N3)-d-Leu-l-Lys(Boc)-d-Leu)2-OH (0.19 g, 0.15 mmol)
was dissolved in DMF (142 mL) with 75 min of sonication at 408C.
The solution was purged with N2(g)
.
DMTMM·BF4 (0.06 g,
0.18 mmol) in DMF (20 mL) was purged with N2(g) and added drop-
wise to the linear peptide solution. The solution was stirred for
67 h at 408C under an atmosphere of N2(g). DMF was removed
under reduced pressure. Boc groups were removed by treatment
with TFA/thioanisole/TIPS/H2O (88:5:2:5vol, 20 mL) for 2 h, then
the mixture was washed with acetic acid/diethyl ether precooled
to ꢀ788C (dry ice/acetone) (1:1 v/v, 2ꢂ40 mL), washed twice with
diethyl ether pre-cooled to ꢀ788C (dry ice/acetone) (2ꢂ40 mL)
and then dried under reduced pressure to give CP3 as a white
powder: 63 mg, 33% yield; 1H NMR (400 MHz, [D1]TFA): d=4.75–
5.15 (m, 8H, 2ꢂLys(N3)-a-H+2ꢂLys-a-H+4ꢂLeu-a-H), 3.56 (t, J=
8 Hz, 4H, 2ꢂLys(N3)-CH2-N3), 3.33–3.50 (br m, 4H, Lys-CH2-NH2),
1.40–2.26 (br m, 36H, 2ꢂLys(N3)-a-CH2-CH2-CH2 +2ꢂLys-a-CH2-
CH2-CH2 +4ꢂLeu-a-CH2-CH), 1.17 ppm (d, J=8 Hz, 24H, 8ꢂLeu-
CH3); MALDI-FTICR MS: m/z found: 1068 (Mꢀ2N2+Ag)+; ATR-FTIR:
nmax =3273 (NꢀHstr), 3006–2790 (CꢀHstr), 2099 (N3), 1627 cmꢀ1 (C=
Ostr).
Experimental Section
Equipment
Microwave-assisted copper(i)-catalysed azide–alkyne cycloaddition
(CuAAC) reactions were performed using a CEM Discover SP micro-
wave reactor using 5 mL borosilicate microwave vessels.
Synthesis
Fmoc-l-Lys(N3)-OH was prepared from Fmoc-l-Lys-OH using a diaz-
otransfer reagent[44,45] in a method described in literature.[27] 4-(4,6-
Dimethoxy-1,3,5-triazin)-4-methylmorpholinium tetrafluoroborate
(DMTMM·BF4) was prepared following literature procedures.[46]
Linear octapeptides were synthesised by fluorenylmethyloxycar-
bonyl (Fmoc) solid phase peptide synthesis[47–49] using: 2-chlorotri-
tyl chloride resin; Fmoc deprotection with piperidine/N,N-dimethyl-
formamide (DMF) (1:4 v/v); amino acid coupling with HBTU/HATU,
N,N-diisopropylethylamine (DIPEA), DMF; and cleaved from the
resin with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP)/dichlorome-
thane (DCM) (1:4 v/v). Cyclic peptides were synthesised by the
head-to-tail coupling of linear peptides as described below.
CP-(NH3·CF3COO)2:
H2N(-l-Lys(Boc)-d-Leu-l-Trp(Boc)-d-Leu)2-OH
(1.00 g, 0.67 mmol) was dissolved in DMF (115 mL) with 10 min of
sonication. The solution was purged with N2(g) at room tempera-
ture, then cooled with an ice/water bath. DMTMM·BF4 (0.26 g,
0.8 mmol) in DMF (15 mL) was purged with N2(g) and added drop-
wise to the linear peptide solution. The solution was brought to
CP1: H2N(-l-Lys(N3)-d-Leu-l-Trp(Boc)-d-Leu)2-OH (0.41 g, 0.31 mmol)
was dissolved in DMF (20 mL) with 5 min of sonication. The solu-
tion was purged with N2(g) at room temperature, then cooled with
an ice/water bath. DMTMM·BF4 (0.12 g, 0.37 mmol) in DMF (5 mL)
was purged with N2(g) and added dropwise to the linear peptide
solution. The solution was brought to room temperature and
stirred for 91 h under an atmosphere of N2(g). DMF was removed
room temperature and stirred for 6 d under an atmosphere of N2(g).
DMF was removed under reduced pressure. The residue was sus-
pended and washed with ice/water-cold MeOH (3ꢂ20 mL) then
dried under reduced pressure. Cyclo[(-l-Lys(Boc)-d-Leu-l-Trp(Boc)-
d-Leu)2] (0.148 g, 0.099 mmol) was treated with TFA/TIPS/H2O
(18:1:1 v/v/v, 5 mL) for 2 h, then the mixture was triturated with di-
ethyl ether precooled to ꢀ788C (dry ice/acetone) and washed
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twice with diethyl ether precooled to ꢀ788C (dry ice/acetone) to
give cyclo[(-l-Lys-d-Leu-l-Trp-d-Leu)2] as an off-white powder:
124 mg, 96% yield; 1H NMR (400 MHz, [D1]TFA): d=8.13 (d, J=
8 Hz, 2H, 2ꢂTrp-CHaromatic), 7.65 (d, J=8 Hz, 2H, 2ꢂTrp-CHaromatic),
7.55 (s, 2H, 2ꢂTrp-CHaromatic), 7.26–7.42 (m, 4H, 4ꢂTrp-CHaromatic),
5.22 (t, J=8 Hz, 2H, 2ꢂTrp-a-H), 4.60–4.87 (m, 6H, 4ꢂLeu-a-H+
2ꢂLys-a-H), 3.04–3.37 (m, 8H, 2ꢂLys-CH2-N+2ꢂTrp-a-CH2), 0.55–
2.00 ppm (br m, 48H, 2ꢂLys-a-CH2-CH2-CH2 +4ꢂLeu-a-CH2-CH+
8ꢂLeu-CH3); ESI-ToF MS: m/z found=1081.0 (M+H)+; ATR-FTIR:
nmax =3273 (N-Hstr), 1627 cmꢀ1 (C=Ostr).
CH2), 1.42 (s, 64H, 32ꢂpBA-CH2), 0.98 (s, 96H, 32ꢂpBA-CH3), 0.73–
0.89 (m, 24H, 8ꢂLeu-CH3), 0.45 (d, J=10.15 Hz, 3H, PPBTCr-CH3),
0.15–0.22 ppm (m, 3H, PPBTCz-CH3); ATR-FTIR nmax =3274 (NꢀHstr),
3045–2750 (CꢀHstr), 1728 (C=Op(BA)), 1625 cmꢀ1 (C=OCPꢀstr).
CP2-(pBA30)2: CP2 (10 mg, 0.008 mmol), pBA30 (0.071 g,
0.017 mmol), sodium ascorbate (0.016 g, 0.081 mmol) and
CuSO4·5H2O (0.009 g, 0.036 mmol) were suspended in DMF (3 mL),
then the mixture was placed in a microwave reactor and irradiated
(dynamic mode) at 1008C for 15 min with a flow of N2(g) and deliv-
ering 200 W in the initial ramp. The DMF was removed under re-
duced pressure. In an attempt to push the reaction to completion,
r-Terminal alkyne RAFT agent (propynyl 2-propanoate)yl butyl tri-
thiocarbonate (PPBTC) was prepared following a similar protocol to
that described previously.[50] r-Terminal N-succinimidyl ester RAFT
agent (NHS-PABTC) was prepared following a previously described
protocol.[35,41] Polymers with a-alkyne and a-NHS functional group
were synthesised by RAFT polymerisation.
a
second microwave irradiation was performed after adding
sodium ascorbate (0.016 g, 0.081 mmol) and CuSO4·5H2O (0.009 g,
0.036 mmol) in DMF (3 mL). The crude product was dissolved in
CHCl3 and washed with an aqueous solution of EDTA (2ꢂ30 mL,
0.055m, pH 8.5), then water (30 mL), and dried over MgSO4. After
concentration in vacuo, CP2-(pBA30)2 was obtained as a dark-
Poly(n-butyl acrylate) with an alkyne end group was synthesised
by RAFT polymerisation: PPBTC (0.25 g, 0.9 mmol), BA (3.9 g,
31.5 mmol) and AIBN (0.015 g, 0.09 mmol) was mixed and diluted
to 40% (w/w) with dioxane, cooled in an ice bath and purged with
N2(g) for 15 min before stirring at 708C under an atmosphere of
N2(g) until 79% conversion determined by 1H NMR. The polymer
was precipitated and washed with ice-cold water/MeOH (1:9) to
brown film: 54 mg, <100% conversion (ATR-FTIR); ATR-FTIR nmax
=
3272 (NꢀHstr), 3040–2800 (CꢀHstr), 2098 (N3), 1729 (C=Op(BA)),
1624 cmꢀ1 (C=OCPꢀstr).
CP2-(pBA30)1.5
.
CP2: (5 mg, 0.004 mmol), pBA30 (0.024 g,
0.006 mmol), sodium ascorbate (0.008 g, 0.040 mmol) and
CuSO4.5H2O (0.005 g, 0.020 mmol) were suspended in DMF
(2.5 mL), then the mixture was placed in a microwave reactor and
irradiated (dynamic mode) at 1008C for 15 min with a flow of N2(g)
and delivering 200 W in the initial ramp. The DMF was removed
under reduced pressure, then the crude product was dissolved in
CHCl3 and washed with an aqueous solution of ethylenediaminete-
traacetic acid (EDTA) (2ꢂ30 mL, 0.055m, pH 8.5), then water
(30 mL), and dried over MgSO4. After concentration in vacuo, CP2-
(pBA30)1.5 was obtained as a brown film: 20 mg, ꢂ100% conver-
sion (ATR-FTIR); ATR-FTIR nmax =3270 (NꢀHstr), 3040–2800 (CꢀHstr),
2097 (N3), 1732 (C=Op(BA)), 1625 cmꢀ1 (C=OCPꢀstr).
CP3-(pBA30)2: CP3 (10 mg, 0.008 mmol), pBA30 (0.085 g,
0.021 mmol), sodium ascorbate (0.020 g, 0.098 mmol) and
CuSO4.5H2O (0.010 g, 0.039 mmol) were suspended in DMF (2 mL)
then the mixtrue was placed in a microwave reactor and irradiated
(dynamic mode) at 1008C for 15 min with a flow of N2(g) and deliv-
ering 200 W in the initial ramp. The DMF was removed under re-
duced pressure and the crude product was dissolved in DCM and
washed with an aqueous solution of EDTA (2ꢂ30 mL, 0.055m,
pH 8.5), then water (30 mL), and dried over MgSO4. After concen-
tration in vacuo, CP3-(pBA30)2 was obtained as a dark-brown film:
52 mg, ꢂ100% conversion (ATR-FTIR); ATR-FTIR nmax =3279 (N-Hstr),
3050–2800 (C-Hstr), 1733 (C=Op(BA)), 1631 cmꢀ1 (C=OCPꢀstr).
yield pBA30 as a yellow viscous liquid: Mn (1H NMR)=4200 gmolꢀ1
;
1H NMR (400 MHz, CDCl3): d=4.78–4.88 (m, 1H, pBA-S-CH), 4.58–
4.72 (m, 2H, PPBTCR-COO-CH2), 3.80–4.30 (m, 30ꢂ2H, pBA-COO-
CH2), 3.34 (t, J=6 Hz, 2H, S-CH2), 0.70–2.60 ppm (m, 311H, PPBTCZ-
CH2-CH2-CH3 +30ꢂpBA-CH2-CH2-CH3 +30ꢂpBAbackbone-CH-CH2 +
PPBTCR-CH3 +PPBTCR-CꢁCH);
SEC-DRI(DMF+LiBr):
Mn =
3500 gmolꢀ1, ꢀ=1.16. MALDI-ToF MS: (M+Na)+ observed; repeat-
ing unit m/z=128, end group m/z=276.
Poly(n-butyl acrylate) with an N-succinimidyl ester end group was
synthesised by RAFT polymerisation. NHS-PABTC (0.075 g,
0.22 mmol), BA (1.04 g, 7.8 mmol), ACVA (0.018 g, 0.0065 mmol) in
dioxane (0.94 g) was purged with N2(g) for 15 min before stirring at
708C under an atmosphere of N2(g) until 94% conversion deter-
mined by 1H NMR. The polymer was twice precipitated and
washed with water/MeOH (1:9) precooled to ꢀ788C (dry ice/ace-
tone) to yield pBA33 as a yellow viscous liquid: Mn (1H NMR)=
;
4600 gmolꢀ1 1H NMR (300 MHz, CDCl3): d=4.76–4.89 (br t, 1H,
pBA-S-CH), 3.86–4.20 (m, 33ꢂ2H, pBA-COO-CH2), 3.34 (t, J=6 Hz,
2H, S-CH2), 2.67–2.89 (m, 5H, NHS-CH2-CH2-+R-CH-COO), 0.66–
2.67 ppm (m, 339H, Z-CH2-CH2-CH3 +33ꢂpBA-CH2-CH2-CH3 +33ꢂ
pBAbackbone-CH-CH2 +R-CH3);
4700 gmolꢀ1, ꢀ=1.09.
SEC-DRI(CHCl3+TEA):
Mn =
CP3-(pBA30)1.5
:
CP3 (10 mg, 0.008 mmol), pBA30 (0.062 g,
CP1-(pBA30)2 was synthesised following a previously described
protocol.[29] CP1 (40 mg, 0.035 mmol) was suspended in 2,2,2-tri-
fluoroethanol (TFE) (2 mL) by sonication for 10 min. PBA30 (0.31 g,
0.074 mmol), sodium ascorbate (0.14 g, 0.71 mmol) and
CuSO4·5H2O (0.037 g, 0.14 mmol) in DMF (3 mL) were added to the
cyclic peptide solution, then the mixture was placed in a microwave
reactor and irradiated (dynamic mode) at 1008C for 15 min with
a flow of N2(g) and delivering 200 W in the initial ramp. TFE and
DMF were removed under reduced pressure. The crude product
was dissolved in DCM and washed with an aqueous solution of
EDTA (2ꢂ30 mL, 0.055m, pH 8.5), then water (30 mL), and dried
over MgSO4. After concentration in vacuo, CP1-(pBA30)2 was ob-
tained as a brown film: 145 mg, 100% conversion (ATR-FTIR);
1H NMR (300 MHz, [D1]TFA): d=8.48 (s, 2H, 2ꢂtriazole-CH), 7.60 (s,
2H, 2ꢂTrp-CHaromatic), 7.34 (s, 2H, 2ꢂTrp-CHaromatic), 7.02–7.26 (m,
6H, 6ꢂTrp-CHaromatic), 5.46 (s, 4H, 2ꢂO-CH2-triazole), 5.18 (s, 2H, 2ꢂ
Trp-a-H), 4.52–4.87 (m, 6H, 4ꢂLeu-a-H+2ꢂLys-a-H), 4.24 (s, 64H,
32ꢂpBA-O-CH2), 3.95 (s, 2H, PPBTCz-CH2), 1.71 (s, 64H, 32ꢂpBA-
0.015 mmol), sodium ascorbate (0.021 g, 0.11 mmol) and
CuSO4.5H2O (0.011 g, 0.042 mmol) were suspended in DMF (2 mL),
then the mixture was placed in a microwave reactor and irradiated
(dynamic mode) at 1008C for 15 min with a flow of N2(g) and deliv-
ering 200 W in the initial ramp. The DMF was removed under re-
duced pressure. The crude product was dissolved in DCM and
washed with an aqueous solution of EDTA (2ꢂ30 mL, 0.055m,
pH 8.5), then water (30 mL), and dried over MgSO4. After concen-
tration in vacuo, CP3-(pBA30)1.5 was obtained as a dark brown film:
49 mg, ꢂ100% conversion (ATR-FTIR); ATR-FTIR nmax =3280 (Nꢀ
Hstr), 3060–2800 (CꢀHstr), 1733 (C=Op(BA)), 1631 cmꢀ1 (C=OCPꢀstr).
CP-(pBA33)2 by NHS conjugation: Cyclo[(-l-Lys-d-Leu-l-Trp-d-
Leu)2] (0.020 g, 0.015 mmol), pBA33 (0.202 g, 0.044 mmol) and
NMM (0.093 g, 0.092 mmol) were suspended in a mixture DMF/
DMSO (1:3 vol, 4 mL) and stirred for 4 days at room temperature.
Solvents were removed with a stream of N2(g) and the product was
redissolved in DCM, then precipitated into dry ice/acetone-cold
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MeOH/H2O (9:1 vol). The precipitate was redissolved in THF and
purified by preparative size exclusion using a column of Bio-Beads
S-X1. Gravity flow elution permitted isolation of conjugate in the
form of self-assembled structures as detected by laser light scatter-
ing and analysed by SEC(DMF). Concentrated in vacuo, CP-(pBA33)2
was obtained as a yellow film: 9.7 mg, 100% conversion (ATR-
FTIR); Mp (SEC-DRI(DMF+NH4BF4))=6600 gmolꢀ1 (n.b. free poly-
mer Mp 2900 gmolꢀ1); ATR-FTIR nmax cmꢀ1: 3276 (NꢀHstr), 3040–
2800 (CꢀHstr), 1733 (C=Op(BA)), 1624 cmꢀ1 (C=OCPꢀstr).
sample was not filtered) and analysed in a quartz cell at angles
158–1498 taking 3ꢂ10 sec intensity measurements at 28 incre-
ments.
Results and Discussion
A range of self-assembling cyclic peptides (CP1–3) containing
azide moieties were obtained by Fmoc- solid phase peptide
synthesis (SPPS) and poly n-butyl acrylates containing an
alkyne functional group at the a-terminus were prepared
using reversible addition-fragmentation chain transfer (RAFT)
polymerisation. The peptide polymer conjugates were then
prepared by reaction of minimal excess polymer with the ap-
Small-angle neutron scattering (SANS)
SANS measurements were performed on the NG3 beamline at the
National Institute of Standards of Technology Center for Neutron
Research in Gaithersburg, MD, USA. Raw data was corrected for de-
tector sensitivity, background and empty cell scattering, and then
converted to absolute scattered intensity (dS/dW cmꢀ1) from the
absolute neutron flux.[51] Incident neutrons had an average wave-
length of 6 ꢃ and wavelength resolution Dl/l=0.124. Sample-to-
detector distances (SDD) of 1.33 m and 8 m were used and the
data combined for 0.0057 ꢃꢀ1 ꢂqꢂ0.43 ꢃꢀ1. For CP1-(pBA30)2 in
[D8]THF (2 mm), SDD of 1.33 m, 8 m, and 13.17 m were used and
propriate CP in
a microwave-assisted copper(i)-catalysed
azide–alkyne cycloaddition (CuAAC) using a high concentration
of copper catalyst (Scheme 1), following a previously published
procedure.[29] The cyclic peptides (CPs) and the cyclic-peptide
polymer conjugates were then examined using SANS to deter-
mine the structure of the assemblies formed in solution.
the data combined for 0.0030 ꢃꢀ1 ꢂqꢂ0.43 ꢃꢀ1
. Hellma cells
(2 mm path length) were used for data acquisition.
Influence of polymer conjugation
First we examined cyclo[(Lys(N3)-d-Leu-Trp-d-Leu-)2] CP1,
which has two azide containing residues (incorporated to
enable the introduction of two polymer chains with alkyne
end groups). A very limited range of solvents can be used to
obtain macroscopically clear solutions (i.e. submicron sizes) of
unconjugated CP1, and trifluoroacetic acid (TFA) and dimethyl-
sulfoxide (DMSO) are commonly used solvents to dissolve CPs
containing these residues.[28,29,55–57] Small angle neutron scatter-
ing (SANS) enabled direct analysis of the macroscopically dis-
solved CP1 in [D1]TFA and [D6]DMSO (Figure 1).
SANS sample preparation
CP1-pBA30 (1 mm) samples in [D1]TFA/[D8]THF (1:9 v/v and 9:1 v/v)
and [D1]TFA/CDCl3 (1:9 v/v and 9:1 v/v) mixtures were prepared
from mixing appropriate volumes of CP1-pBA30 (1 mm in [D1]TFA),
CP1-pBA30 (1 mm in THF) and CP1-pBA30 (1 mm in CDCl3) then
transferred to Hellma cells for data acquisition. An alternative
sample preparation (as indicated in text), involving the solvent
mixture [D1]TFA/[D8]THF (1:9 v/v), was prepared by dissolving CP1-
pBA30 in [D1]TFA and diluting with [D8]THF (as opposed to mixing
two solutions of CP1-pBA30).
In [D1]TFA, CP1 does not scatter above the background inco-
herent scattering from the solvent. This suggests that any
structure in solution is smaller than 1 nm, corresponding to
the molecular dissolution of CP1. On the other hand, CP1 in
SANS analysis
Models incorporated in the NCNR analysis macro using IGOR Pro
6.34A were used to fit to the collected data.[51] The general proce-
dure followed for fitting was to begin with a uniform, monodis-
perse, hard sphere model, then, wherever necessary, fit complexity
was increased to consider polydisperse spheres, uniform ellipsoids,
uniform cylinders, polydisperse cylinders, and core-shell cylinders
to gain further insight. The parameters: scale (volume fraction), sol-
vent SLD and incoherent background were always fixed and the
other parameters were fit to the data. Repeated fittings with vari-
ous manually input starting parameters were used to assess the
validity of the fit. Solvent SLDs were determined from literature
values of scattering lengths and cross sections[52] using the NCNR
SLD calculator,[53] and SLDs of solvent mixtures were determined
by weighted averages. Where removed, incoherent scattering was
subtracted using the Porod Law (I(q)·q4 vs. q4 plot).[54]
Static light scattering (SLS)
SLS experiments were performed on an ALV-CGS3 goniometer
system. A 633 nm, vertically polarised laser was used, and the
sample was kept at 208C throughout the experiment. Dark count
rate was recorded at 908, distilled and filtered toluene (n=1.491,
Rq =1.35ꢂ10ꢀ5) was used to calibrate the instrument, and filtered
THF (n=1.409) was used as a blank for background subtraction.
CP-(pBA33)2 in THF (2 mm) was prepared with filtered THF (but the
Figure 1. SANS data of CP1 in [D1]TFA and [D6]DMSO. CP1 is molecularly dis-
solved in [D1]TFA but forms 1–15 nm structures in [D6]DMSO. The Figure
shows the fit of a uniform ellipsoid model (disk-like) to CP1 in [D6]DMSO
(4 mm). This has been interpreted as bundles of short tubes in [D6]DMSO.
See Figures S1–S3 for model fitting.
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Scheme 1. Synthetic overview of cyclic peptides and (cyclic peptide)–polymer conjugates.
[D6]DMSO shows significant small-angle scattering over the
probed q-range, which increases with concentration, indicating
the presence of small aggregates. These scattering functions
were inconsistent with spherical aggregates, but could be ade-
quately fit by models with disk-like geometry such as homoge-
neous oblate spheroids or cylinders. Best fit parameters are
similar for the two models, yielding short cylinders with radii
of 12ꢃ0.5 nm and lengths of 4.0 nm, or spheroids with large
radii of 13ꢃ0.5 nm and short (axial) radii of 2.3 nm, very close
to half the best-fit cylinder length. Including polydispersity into
the cylinder radius improves the fit slightly but does not affect
cylinder length (see Figure S1–S3 for further details).[51] These
dimensions are larger than expected for a single CP1 nanotube
(which would have a radius of around 0.5 nm), but in line with
earlier observations by Ghadiri et al. who observed electron
(and optical) microscopy images of unconjugated CPs in bun-
dles (microcrystals).[17,56,58] We interpret the SANS patterns to
mean that CP1 exists as bundles of short tubes in [D6]DMSO,
a concept that has also been proposed in the study of CP ion
channel mechanism.[59–61] Indeed, although early work in the
field presented the mechanism of ion transport through the in-
ternal cavities of cyclic peptide nanotubes,[62,63] assembly into
‘barrel and stave’ structures has also been suggested.[64] Fur-
thermore, the work of Ramesh et al. and Kodama et al. have
shown that the transport of ions is possible with cyclic pep-
tides with only 4 or 6 residues, for which the internal pore is
too small to allow ion transport, thus suggesting a barrel and
stave assembly.[59–61]
(pBA30; Mn =4100 g.molꢀ1, ꢀ=1.16) yielding CP1-(pBA30)2
(Scheme 1). We probed the effect of the solvent environment
on the assembly of the (cyclic peptide)–polymer conjugates,
using a variety of solvents and mixtures to empirically correlate
the influence of the solvent H-bonding strength in competition
with H-bonding between CPs, and the solvent quality towards
the peptide and polymer.
Remarkably, unlike CP1, the peptide–polymer conjugate
CP1-(pBA30)2 in [D1]TFA shows an assembly of appreciable size
by SANS (Figure 2). This is likely due to the polymer shielding
the peptide from the [D1]TFA molecules, a phenomenon ob-
served in a previous study.[43] This scattering pattern can also
be fit to short cylinders. Here the best fit cylinder radius is
1.4 nm and length 6.5 nm (note, a fit to prolate ellipsoids gives
comparable radii of 1.5 nm and 4.2 nm, respectively, see Fig-
ures S4 and S5). This cylinder radius is greater than expected
for the cyclopeptide alone, but is consistent with expectations
for an isolated CP nanotube surrounded by a polymer sheath
swollen by solvent. More complex models, to account for a sol-
vent-penetrable polymer shell, were not necessary to fit the
data; however the obtained radii and scattering length densi-
ties (SLDs) must be interpreted as an average of the peptide
core and solvated polymer shell. The fact that the obtained
radius is only slightly larger than expected for a single, uncon-
jugated cyclic peptide nanotube and does not account for the
full length of the polymer chain, shows not only the low con-
trast of the polymer but is also evidence of isolated cyclic pep-
tide nanotubes that are sterically stabilised by the polymer
chains. These findings match previous SANS analyses per-
formed on these systems.[43]
Using microwave-assisted CuAAC, we conjugated CP1 with
two chains of alkyne-functionalised poly(n-butyl acrylate)
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Figure 2. SANS data of CP1-(pBA30)2 in [D1]TFA and [D6]DMSO. CP1-(pBA30)2
exists as short individual tubes. Precipitation is observed for the conjugate
in [D6]DMSO. See Figures S4 and S5 for model fitting.
Figure 3. SANS data of CP1-(pBA30)2 in CDCl3 and [D8]THF. Conjugate exists
as long and individual tubes. See Figure S6 for model fitting.
CP1-(pBA30)2 was difficult to dissolve in [D6]DMSO, and this
is clearly seen in the qꢀ4 scattering behaviour in Figure 2 at
low q (<0.03 ꢃꢀ1) corresponding to Porod scattering by large
particles (>100 nm).[65,66] This is consistent with the poor solu-
bility of pBA in DMSO being carried over to the conjugate.
However SANS reveals a coexistence of precipitate with a pop-
ulation of small, low aspect ratio aggregates in [D6]DMSO, de-
spite the incompatibility between [D6]DMSO and the pBA
chains. These aggregates are comparable in their dimensions
with those seen in TFA. A cylinder fit yields a radius of 1 nm
and a length of 6 nm, (or radii of 1 nm and 4 nm for a prolate
spheroid). Here also the assemblies are short, and consistent
with isolated nanotubes sterically stabilised by the polymer
conjugate, and very different from the cyclopeptide CP1 alone.
Figure 4. Uniform cylinder model fits to SANS data of (a) CP1-(pBA30)2 in
[D8]THF (2 mm); and (b) CP-(pBA33)2 in THF (2 mm). Incoherent background
scattering has been subtracted. SLS data has been offset to match the SANS
data. See also Table 2.
Influence of solvent
Since the conjugation of a polymer provides solvent solubility
to the nanotubes, the conjugate CP1-(pBA30)2 was dissolved in
tetrahydrofuran-[D] and chloroform-d (CDCl3). In these less
competitive solvents, H-bonding between CPs is promoted,
and the SANS patterns exhibit a qꢀ1 power law of over a wide
q-range (Figure 3), characteristic of long rigid rods. This obser-
vation is consistent with previously reported electron micros-
copy imaging of chemically locked-in CP-polymer conju-
gates.[39,43] With no Guinier regime in the available q range, fit-
ting the data gave cylinders with very high aspect ratios, with
a lower bound on the average length>110 nm, and a cylinder
radius of ~1 nm in both solvents (Figure S6). Even at very low
q (0.001 ꢃꢀ1) the qꢀ1 behaviour persists in the scattering data
(Figure 4 and Table 1) showing the rods to be extremely long
and rigid, raising the minimum observed length of the assem-
bly to 1700 nm. Such long assemblies support the notion of
a cooperative assembly mechanism,[67] which has been sug-
gested to originate from the stabilisation of multiple bonds
and peptide backbone conformation.[56,68] The number- and
weight- average degree of polymerisation, hDPin and hDPiw, re-
Table 1.
a) SANS only
b) SANS and SLS overlayed
Scale
Radius [ꢃ]
0.019[a]
8.87
>2100
2.70
6.35[a]
0.01[a]
9.10
>17000
2.80
6.35[a]
Length [ꢃ]
SLD cylinder (ꢂ10ꢀ6 ꢃꢀ2
]
]
SLD solvent (ꢂ10ꢀ6 ꢃꢀ2
[a] Held parameter value.
spectively, of a supramolecular assembly with a single nuclea-
tion step can be described by:[69]
2
s þ ð1 ꢀ sÞð1 ꢀ K½AꢄÞ
ð1Þ
ð2Þ
hDPin¼
hDPiw¼
2
sð1 ꢀ K½AꢄÞ þ ð1 ꢀ sÞð1 ꢀ K½AꢄÞ
3
3
sð1 þ K½AꢄÞ þ ð1 ꢀ sÞð1 ꢀ K½AꢄÞ
sð1 ꢀ K½AꢄÞ þ ð1 ꢀ sÞð1 ꢀ K½AꢄÞ
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Where s=K2/K; [A] is the concentration of unassembled mo-
nomer, K2 is the equilibrium constant for the dimerization of
two monomers, and K is the equilibrium constant for the addi-
tion of one monomer to an assembly with two units or more.
Based on thermodynamic studies of partially N-methylated CPs
(monotopic species), the highest equilibrium constants for the
formation of dimers have been found to be in the order of
2000mꢀ1 in CDCl3.[70] For an isodesmic supramolecular poly-
merisation (i.e. K2 =K) with association constant K~2000mꢀ1 at
1 mm, we calculate a weight average degree of polymerisation
hDPiw around 3 (length ca. 1.5 nm)1.[69] This is significantly
shorter than the assembly observed by SANS; in fact, K~
107 mꢀ1 is required to explain the observed minimum length
through an isodesmic growth mechanism. If we however
assume a cooperative growth mechanism (in the simplest case
of a nucleation step at two units, that is, K2 <K) and an associa-
tion constant for the first two units of the assembly K2
~2000mꢀ1, then to obtain a hDPiw around 220 (length
~110 nm, see footnote) we can estimate the equilibrium con-
stant for subsequent growth K to be greater than 200000mꢀ1
in CDCl3 (Figure 5). If we consider longer assemblies (length~
1700 nm, hDPiw around 3400, see footnote) such as those ob-
served in THF, and with the continued assumption of K2 ~
2000mꢀ1, then we estimate K~1 200000mꢀ1 (Figure 5).
Table 2. Length parameter from fitting uniform cylinder model to scatter-
ing profiles of CP1-(pBA30)2 at 1 mm in various solvent mixtures. See Fig-
ures S4–9 for scattering profiles and model fitting.
Composition of [D1]TFA [vol%]
100
90
10
0
Length in [D1]TFA/[D8]THF (nm)
Length in [D1]TFA/CDCl3 (nm)
10
10
10
10
30
10
>110
>110
closer look at the fitted parameters for radius and contrast of
CP1-(pBA30)2 in the pure solvents suggests that the polymer
chains are better solvated in CDCl3 (larger radius and lower
contrast against solvent) than in [D8]THF (smaller radius and
higher contrast). Note that this comparison was made by fixing
the scale, for the fitting, according to the volume fraction of
each sample. As a cooperative system with the observed
lengths, it is reasonable to assume that the entire sample is
contributing to the scattering (as the unassembled species is
at least 3 orders of magnitude lower than the total concentra-
tion). The difference in solvation means that TFA can more
easily access the CP and disrupt its stacking when mixed with
CDCl3 than [D8]THF, which explains the observed differences in
assembly lengths between [D1]TFA/[D8]THF (1:9 v/v) and
[D1]TFA/CDCl3 (1:9 v/v). To probe this, CP1-(pBA30)2 was dis-
solved first into [D1]TFA, then diluted with [D8]THF to a final
concentration and solvent mixture of [D1]TFA/[D8]THF (10
vol%). In this case even shorter tubes were observed, leading
to the conclusion that a non-thermodynamic product is
formed in one or other addition sequence (Figure S9). The de-
tails of this require further study, but do suggest the potential
for obtaining kinetic products in order to access a wider range
of structures.[71,72]
Influence of cyclic peptide side-chain functionality
A key feature of using self-assembling cyclic peptides is their
versatility in chemical functionality by the inclusion of residues
with various functional groups. Here we use CP2, which substi-
tutes (from CP1) the two Trp residues for two Lys(Boc) resi-
dues, as a model compound that might be potentially used to
functionalise the immediate shell of a tube (i.e. conjugated to
the peptide and not the polymer). Attempts to conjugate
pBA30 to CP2 by microwave-assisted CuAAC resulted in con-
version <100% as judged by ATR-FTIR (Figure S10). We attri-
bute this difference in conjugation efficiency to the bulky Boc
protecting groups which reduce the accessibility of the sites
(based on our findings of attempting to conjugate 4 polymers
to a CP with 4 azide sites).[29] Conjugation efficiency of pBA30
to Boc-deprotected CP2, that is, CP3, was difficult to ascertain
due to the relative insolubility of CP3 which biased the obser-
vation of the conjugate. Our uncertainty with conversion led
us to synthesise samples in which the cyclic peptide was in
excess over the polymer during conjugation, to determine
whether a higher number of unconjugated sites would impact
the assembly. Conjugates CP2-(pBA30)2, CP2-(pBA30)1.5 (2.1 eq
and 1.5 eq of polymer used, respectively) and CP3-(pBA30)2,
CP3-(pBA30)1.5 (2.1 eq and 1.5 eq of polymer used, respective-
Figure 5. Calculated number- and weight-average degree of polymerisation
hDPin and hDPiw versus total concentration of CP units for isodesmic (blue
lines) and cooperative growth mechanisms, with s=0.01 (red lines) and
s=0.0016 (black lines).[69] A cooperative growth mechanism is used to ex-
plain the long assembly.
To probe solvent conditions with properties between those
of TFA and THF or CDCl3, solvent mixtures of [D1]TFA/[D8]THF
and [D1]TFA/CDCl3 were used. Table 2 shows the results of
these experiments (see Figure S4–9 for scattering profiles and
model fitting). With only 10 vol% [D1]TFA, the assemblies are
found to be significantly shorter than those observed in either
neat [D8]THF or CDCl3 and yield comparable dimensions to our
previous findings with the same solvent composition.[43]
A
1
A length of 5 ꢁ per unit[17] has been used to estimate the length of assembly
from DP.
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The scattering patterns of CP3 conjugates at both grafting
densities are similar to CP1-(pBA30)2 in the intermediate q
range, which indicates the presence of isolated cylinders. How-
ever a transition to Porod scattering is seen at lower q, indica-
tive of large particles or precipitates.[65,66] This could be the
result of the use and incompatibility of CDCl3 as the solvent
and the polar nature of the lysine residues in the CP3 conju-
gates. Also of note, in both CP2 and CP3 conjugates, the dif-
ference between using 2.1 and 1.5 equivalents of polymer per
CP during the conjugation reaction has a minimal impact on
the final assembly. For conjugations involving CP2, this sug-
gests a maximum grafting efficiency of 1.5 polymers per CP.
Conclusion
Figure 6. SANS data of CP1-(pBA30)2, CP2-(pBA30)2, CP2-(pBA30)1.5, CP3-
(pBA30)2, CP3-(pBA30)1.5, in CDCl3. Variation of peptide and variation of poly-
mer equivalents used during conjugation. See Figures S11 and S12 for
model fitting.
We have shown the direct characterisation of nanotube form-
ing (cyclic peptide)–polymer conjugates in a variety of condi-
tions. These conditions and self-assembly products have been
summarised in Scheme 2. Fitting the form factor with cylindri-
cal models suggests the attachment of pBA can sterically stabi-
lise these assemblies, preventing the formation of bundles as
otherwise observed in the assembly of the unconjugated cyclic
peptide in [D6]DMSO. Interestingly, in the case of the conjugate
in [D6]DMSO, large structures were also observed to coexist
with isolated nanotubes; this complex behaviour is likely to be
related to the incompatibility (i.e. low solubility) of pBA with
DMSO. In [D1]TFA, in which there was no assembly of the un-
conjugated cyclic peptide, the polymer promoted the forma-
tion of (short) nanotubes in the conjugate, a phenomenon we
have attributed to the polymer chains shielding the solvent
from the CP hydrogen bond donors and acceptors. This notion
was further explored by using solvent mixtures varying in their
ability to compete with the H-bonding between CPs. It was
found that 10 vol% [D1]TFA was enough to significantly short-
en the length of the assembly from a non-polar solvent. CP-
pBA conjugates in THF were found to exist as individual and
long (>1700 nm) nanotubes; which, by extension of the ob-
served length and concentration, suggests a strongly coopera-
tive system in which we estimate for nucleation K2 =2000mꢀ1,
ly) in CDCl3 were examined by SANS to assess the impact of
changing residue function on the CP assembly (Figure 6). It is
clear from the scattering profiles that the structures of these
two conjugates differ from the CP1 based conjugates. The
scattering patterns are all consistent with extended, anisotrop-
ic structures, and can all be fit satisfactorily by rigid rod
models (Figure 6). At wavevectors q<0.03 ꢃꢀ1, CP2-based con-
jugates show a trend towards the expected qꢀ1 power law of
rigid rods (see Figure S11 for model fitting), but the steep
region indicates much larger cylinder radii than CP1-derived
aggregates. This has been modelled by including polydispers-
ity in the radial component via a Schultz distribution with
a fitted polydispersity of ca. 1.0 (see Figure S12 for model fit-
ting). We interpret this to be a result of the incomplete conju-
gation and the consequent tendency of the tubes to partially
bundle. This value of radial polydispersity is reasonable as the
aggregation of even two tubes will be statistically significant
when compared to an isolated nanotube.
Scheme 2. Simplified summary depicting: (a) self-assembling units, (b) key assembly conditions, and (c) products of self-assembly with short descriptions
below. Length scales and concentrations have not been correctly represented for the purpose of illustration.
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and for elongation K>1200000mꢀ1 as a minimum for cooper-
ative growth to explain the observations. We explored the var-
iation of residues of the CP from Trp to Lys(Boc) or Lys, for
which an observable difference was found between the result-
ing self-assembled structure for each of these species. Specifi-
cally, the incomplete conjugation due to Lys(Boc) residues led
to partial lateral aggregation; in the case of Lys residues, al-
though unbundled nanotubes were observed, aggregates
were also present in CDCl3 which have been attributed to the
polar nature of the Lys residues.
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The authors acknowledge R. Chapman (Sydney) for providing
pBA30; P. G. Young (Sydney) for providing DMTMM·BF4; A. Sere-
lis (DuluxGroup, Australia) for providing PABTC; and S. Larnau-
die (Warwick) for providing DMTMM·BF4 and NHS-PABTC. The
authors thank P. Butler (NIST) for support with the SANS experi-
ments. The Australian Research Council Discovery
(DP140100241; K.A.J. and S.P.) Programme is acknowledged
for funding. M.L.K. acknowledges the Australian Government
for the provision of an Australian Postgraduate Award research
scholarship. The Royal Society Wolfson Merit Award
(WM130055; SP) and the Monash–Warwick Alliance (MLK) are
acknowledged for financial support. We acknowledge the sup-
port of the National Institute of Standards and Technology, U.S.
Department of Commerce, in providing the neutron research
facilities used in this work. This work utilized facilities support-
ed in part by the National Science Foundation under Agree-
ment No. DMR-0944772.
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Keywords: cyclic peptide · nanotubes · polymer conjugates ·
raft polymerization · self-assembly · supramolecular
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Received: June 28, 2016
Published online on && &&, 0000
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ÝÝ These are not the final page numbers!

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FULL PAPER
Supramolecular nanotubes based on
(cyclic peptide)-polymer conjugates
were studied by small-angle neutron
scattering. Direct characterisation of
nanostructures in solution shows that
self-assembled products formation is
controlled by the grafted polymer, the
functional groups of the peptide, and
the various solvent conditions. In addi-
tion, this investigation examines the co-
operative nature of the growth mecha-
nism of self-assembling cyclic peptides.
&
Supramolecular Structures
M. L. Koh, P. A. FitzGerald, G. G. Warr,
K. A. Jolliffe,* S. Perrier*
&& – &&
Study of (Cyclic Peptide)–Polymer
Conjugate Assemblies by Small-Angle
Neutron Scattering
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
11
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ