Supramolecular dimerisation of middle-chain Phe pentapeptides via CB[8] host-guest homoternary complex formation

Article abstract of DOI:10.1039/c3cc45420g

Pentapeptides containing a Phe residue in the middle of the sequence exhibit ternary complex formation in the presence of cucurbit[8]uril, thus opening new perspectives on supramolecular peptide dimerisation studies. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc45420g

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Supramolecular dimerisation of middle-chain Phe
pentapeptides via CB[8] host–guest homoternary
complex formation†
Cite this: Chem. Commun., 2013,
49, 8779
Received 17th July 2013,
Accepted 2nd August 2013
´
Silvia Sonzini, Sean T. J. Ryan and Oren A. Scherman*
DOI: 10.1039/c3cc45420g
www.rsc.org/chemcomm
Pentapeptides containing a Phe residue in the middle of the
sequence exhibit ternary complex formation in the presence of
cucurbit[8]uril, thus opening new perspectives on supramolecular
peptide dimerisation studies.
Supramolecular host–guest chemistry in peptide sequences is a
clever approach to reversibly control not only peptide conforma-
tions, but also their aggregation into dimers and higher order
structures. Peptide dimers can be useful as scaffolds to bind DNA,
as well as proteins, to regulate their activity.1,2 Additionally,
dimers may become important building blocks to further study
hierarchical aggregation pathways of greater complexity. A simple
dimerisation technique exploiting host–guest supramolecular
interactions, which does not require synthetic modification but
only a Phe residue in the sequence, is herein explored.
Fig. 1 (a) Ab 1–42 sequence with highlighted chosen fragments. (b) Schematic
illustrating the homoternary complex formation via host–guest interaction of
pentapeptides and CB[8].
sequences with a general structure H3N+–X1X2F3X4X5–CONH2,
Cucurbit[n]uril, a family of synthetic, macrocyclic host mole- which are capable of exhibiting strong 2 : 1 binding, through the
cules, has already been shown to selectively bind specific amino middle-chain Phe residue, with CB[8] (Fig. 1b). The three chosen
acid side chains.3–5 Such interactions have been exploited for sequences, reported in Fig. 1a, are short fragments adapted from
sensing of protonated and aromatic residues.6,7 One of the larger the wild type human Amyloid Beta 1–40/42 (WT Ab) sequences.
members of this family, cucurbit[8]uril (CB[8]), can simultaneously
The interaction of the Phe residues with CB[8] was investigated
accommodate two guest molecules inside its cavity.8–10 This in an effort to better understand the oligomerisation pathways of
has allowed the dimerisation of large (bio)molecules, which Ab. Sequence 1 (AEFRH) was directly taken from residues 2–6 of
have suitable binding motifs incorporated at their termini. A the WT Ab. Sequences 2 (LVFIA) and 3 (VIFAE) were derived from
previous study on the strength of CB[8] binding with aromatic residues 17–21 and 18–22, respectively, of the WT peptide. In the
amino acids as 1 : 2 homoternary complexes was carried out latter two sequences, however, both residues 19 and 20 are Phe,
by Urbach et al.,11 highlighting the importance of having therefore, one amino acid per fragment has been exchanged
the binding residue at the N-terminal position. Furthermore, for Ile, which maintains the hydrophobic nature of the penta-
complexes between CB[8] and aromatic residues have also been peptides without inserting a second aromatic residue.13 This avoided
reported in bioconjugate systems such as BSA and PEG through potential CB[8] interactions with more than one residue per chain
a heteroternary (1 : 1 : 1) complex.12
None of the reported studies, however, suggest the possibility allowing us to better comprehend mid-chain Phe–CB[8] interactions.
of significant ternary complex formation when the guest residues The pentapeptides were synthesised by solid phase peptide
without dramatically changing the surrounding environment, thus
exist in the middle of the peptide sequence. In fact, the contrary synthesis (SPPS) using standard Fmoc protocols and characterised
was even proposed.11 We report three examples of pentapeptide by HPLC and ESI-MS to ensure their purity (see ESI†). Ternary
complex formation with CB[8] was studied using three different
methods, all of which support strong 2 : 1 homoternary complexa-
tion. The sequences were first analysed by fluorescence titration to
Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of
Cambridge, Lensfield Road, CB2 1EW, Cambridge, UK. E-mail: oas23@cam.ac.uk
evaluate any difference in the presence or absence of CB[8]. When
Phe is irradiated, its emission maximum is 284 nm.14 This is readily
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc45420g
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observed for a solution of 1 in the absence of CB[8], which is a
sequence suitably hydrophilic and well solvated in buffer. On
the other hand, fibres formed from Phe-rich sequences display
a red-shifted emission at about 303 nm, which is attributed to
p–p stacking.15,16 A change in the emission band at 303 nm in
the presence of CB[8] is observed for all of the three sequences
(see Fig. S9–S11, ESI†) and is indicative of Phe dimerisation in
the cavity and variations in the aggregation state, as expected.
Sequence 1 initially shows an intrinsic major peak at 284 nm
and a shoulder at 303 nm; upon increasing CB[8] concentration
a dramatic enhancement of the peak at 303 nm is observed. 2,
the most hydrophobic of the three sequences, exhibits only one
peak at 303 nm, which is quenched upon addition of CB[8]. 3
alone presents two intrinsic peaks at 284 and 303 nm, which
both increase upon CB[8] addition, however, the peak at 284 nm
transitions from being the minor peak to the major one.
We evaluated the change in emission at 284 and 303 nm of 1–3
upon increasing CB[8] concentration (Fig. 2). Notably, analysing the
Fig. 3 1H-NMR in D2O of sequence 3 (0.5 mM) with an increasing amount
ratio between fluorescence upon CB[8] addition (Fi) and the intrinsic of CB[8].
fluorescence (F0) all of the sequences show a change in their
emission behaviour at a 0.5 ratio, supporting the theory of a 2 : 1
binding complex. In addition, 1 presents a second binding event at aromatic signals have been reported previously as evidence of
higher CB[8] concentrations, which might be related to interactions homoternary complex formation between Phe and CB[8].11
with the C-terminal His residue. Further studies, in full agreement Sequence 1 exhibited similar behaviour; in fact, at a 2 : 1 ratio
with the CB[8] data, were also conducted evaluating any changes in of pentapeptide to CB[8] the only peak in the region of 7.3 ppm
the emission spectra upon addition of CB[7] (see Fig. S1, ESI†).
is at 7.36 ppm, which is one of the His side chain peaks,
1H-NMR spectra of host–guest complexes usually exhibit together with the signal at 8.64 ppm (see Fig. S12, ESI†). 2, on
shifted and broadened peaks, therefore 1H-NMR was also used the other hand, exhibits a dramatic broadening of the Phe side
to investigate 2 : 1 complex formation between the pentapeptide chain peaks and the shifted peak is barely visible at a ratio of
sequences and CB[8]. The concentration of the peptides was 2 : 1 of 2 to CB[8] (see Fig. S13, ESI†); this different behaviour is
held constant (either at 0.5 mM or 0.25 mM in the case of 2) and attributed to a fast exchange rate of the ternary complex.
the spectra were recorded at various concentrations of CB[8]. The
titration spectra of 3 are reported in Fig. 3, while the spectra of 1 between the pentapeptide sequences and CB[8] was obtained
and 2 are available in the ESI† (see Fig. S12 and S13). by isothermal titration calorimetry (ITC). The ITC data clearly
A final confirmation of 2 : 1 homoternary complexation
1H-NMR spectra of 3 (Fig. 3) showed a clear upfield shift of Phe indicate that all three pentapeptide sequences exhibit 2 : 1 binding
aromatic peaks from 7.26 and 7.20 ppm to 6.68 and 6.40 ppm, stoichiometry with CB[8]. The binding isotherm obtained for 3 is
respectively. Additionally, the Ha and CH2 protons of Phe exhibit shown in Fig. 4. The 2:1 binding stoichiometry was first checked
an upfield shift as well. The aromatic peaks are completely shifted by fitting the data to a one site binding model; the data points
upfield at a ratio of 2 : 1 peptide : CB[8]. These shifts in the Phe were, then, fitted with a sequential binding site model using the
Origin-Microcal software as reported previously.7 The binding
isotherms of 1 and 2 are reported in the ESI† (see Fig. S15 and
S16). Ka1 and Ka2 are about 105 and 104 MÀ1, respectively, for both
1 and 2. 3 shows higher values of ca. 107 and 105 MÀ1, respectively.
The binding parameters obtained for the homoternary complexes
are all reported in Table 1.
It should be noted that the three sequences exhibit very
different characteristics: sequence 1 (AEFRH) has a negatively
charged (Glu) and a positively charged (Arg) residue flanking the
Phe, sequence 2 (LVFIA) is completely hydrophobic and presents a
challenging steric environment around Phe, and sequence 3 (VIFAE)
possesses a negatively charged (Glu) residue at its C-terminus.
The difference in binding abilities reflects the different
environments of the Phe–CB[8] complexes in the three sequences.
Furthermore, the data obtained imply that the pentapeptide
sequences present different self-assembly in solution, which thus
cause a different strength of the interaction with CB[8]. 1 shows
preferential binding between Phe and CB[8], but fluorescence
Fig. 2 Relative emission at 284 nm or 303 nm vs. the CB[8]/peptide ratio
(peptide sequences 40 mM, CB[8] 0–35 mM).
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8780 Chem. Commun., 2013, 49, 8779--8781
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which form strong homoternary complexes with CB[8]. A binding
stoichiometry of 2 : 1 Phe–CB[8] was confirmed by three indepen-
dent methods. Nevertheless, the sequences present different
binding features strictly related to their amino acid composition
and conformation in solution, confirming that guest environment
plays a significant role in CB[8] binding. This study suggests that
CB[8] can bind Phe (and likely most if not all of the aromatic
residues) not only located at the N-terminus, but also in many
places along a peptide sequence. Homoternary complexation can
be very useful for further Phe-rich peptide dimerisation studies,
such as Ab that is currently under investigation in our laboratory
and other pathologically related peptides.
Both S.S. and S.T.R. are grateful for funding from the ERC
starting investigator grant ASPiRe (240629) and S.T.R. acknowl-
edges the Cambridge Home and European Scholarship Scheme
and Robert Gardiner memorial scholarship.
Fig. 4 ITC titration of sequence 3 into a CB[8] solution.
Table 1 ITC data for CB[8] ternary complexes of 1–3
References
1 B. K. Kim, H. Kang, K. O. Doh, S. H. Lee, J. W. Park, S. J. Lee and
T. J. Lee, Bioorg. Med. Chem. Lett., 2012, 22, 5415–5418.
2 S. Aggarwal, P. Singh, O. Topaloglu, J. T. Isaacs and S. R. Denmeade,
Cancer Res., 2006, 66, 9171–9177.
3 P. Rajgariah and A. Urbach, J. Inclusion Phenom. Macrocyclic. Chem.,
2008, 62, 251–254.
4 D. T. Dang, H. D. Nguyen, M. Merkx and L. Brunsveld, Angew. Chem.,
Int. Ed., 2013, 52, 2915–2919.
5 H. D. Nguyen, D. T. Dang, J. L. J. van Dongen and L. Brunsveld,
Angew. Chem., Int. Ed., 2010, 49, 895–898.
Ka (MÀ2
)
K
a1/Ka2 DH (kcal molÀ1
)
TDS (kcal molÀ1
)
1
2
3
(3.74 Æ 0.7) Â 1010 15.3
(1.58 Æ 0.3) Â 1013 88.3
À15.3 Æ 0.2
À9.02 Æ 0.6
À20.8 Æ 0.2
À0.88 Æ 0.3
À4.49 Æ 0.9
À2.76 Æ 0.3
(7.68 Æ 2.3) Â 109
2.04
6 S. W. Heo, T. S. Choi, K. M. Park, Y. H. Ko, S. B. Kim, K. Kim and
H. I. Kim, Anal. Chem., 2011, 83, 7916–7923.
7 J. M. Chinai, A. B. Taylor, L. M. Ryno, N. D. Hargreaves, C. A. Morris,
P. J. Hart and A. R. Urbach, J. Am. Chem. Soc., 2011, 133, 8810–8813.
8 J. W. Lee, S. Samal, N. Selvapalam, H.-J. Kim and K. Kim, Acc. Chem.
Res., 2003, 36, 621–630.
9 L. Isaacs, Chem. Commun., 2009, 619–629.
10 E. Masson, X. Ling, R. Joseph, L. Kyeremeh-Mensah and X. Lu,
RSC Adv., 2012, 2, 1213–1247.
11 L. M. Heitmann, A. B. Taylor, P. J. Hart and A. R. Urbach, J. Am.
Chem. Soc., 2006, 128, 12574–12581.
12 F. Biedermann, U. Rauwald, J. M. Zayed and O. A. Scherman, Chem.
Sci., 2011, 2, 279–286.
13 O. D. Monera, T. J. Sereda, N. E. Zhou, C. M. Kay and R. S. Hodges,
J. Pept. Sci., 1995, 1, 319–329.
14 H. Du, R. C. A. Fuh, J. Li, L. A. Corkan and J. S. Lindsey, Photochem.
Photobiol., 1998, 68, 141–142.
1
and H-NMR data suggest an additional secondary interaction
with His. 2 is highly prone to fibre formation and likely under-
goes a conformational adjustment prior to binding CB[8]. The
breaking of the strong p–p interactions that are already present
within the peptide bundle causes quenching of the fluorescence
at 303 nm. Moreover, the significantly higher binding constants
measured for 3 are very likely related to the circular conforma-
tion that it adopts in water and its exposed Phe residue. The
cationic N-terminus and the anionic Glu side chain at the
C-terminus are held in close proximity pushing the Phe side
chain far from the backbone and, therefore, allowing better
interaction with CB[8]. This hypothesis is also supported by
computational energy minimisation of 3 (see ESI†).
15 M. J. Krysmann, V. Castelletto, A. Kelarakis, I. W. Hamley, R. A. Hule
and D. J. Pochan, Biochemistry, 2008, 47, 4597–4605.
16 K. E. Marshall, K. L. Morris, D. Charlton, N. O’Reilly, L. Lewis,
H. Walden and L. C. Serpell, Biochemistry, 2011, 50, 2061–2071.
In conclusion, we have reported three pentapeptide sequences,
differing in charge, hydrophobicity and steric hindrance, all of
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 8779--8781 8781