The CuAAC reactions were performed in aq DMF, in the
presence of CuSO O and sodium ascorbate (NaAsc)
Á5H
M
triple helix assembly (accounting for the higher T ) and an
4
2
unexpected intermolecular triple helix complex (accounting
(
Table S1, ESIw). The use of an equivalent of CuSO Á5H O
for the lower TM = 28.4 and 31.2 1C for CP3 and CP4,
4
2
À1
and NaAsc (5 equiv.) gave almost undetectable levels of a
product, presumably due to the sequestration of Cu(I) by the
peptide substrates. In contrast, CuSO Á5H O (5 equiv.)–
respectively). Moreover, multiple DSC cycles (at 6 1C h
established that both transitions were completely reversible
)
(Fig. 3, inset) in a manner similar to those previously reported
1
4
2
3
NaAsc (25 equiv.) when mixed with alkyne-bearing SS3
by Henkel et al. We are currently investigating the exact
nature of these melt transitions for CP3 and CP4.
(
2 equiv.) and azido-bearing DS1 (1 equiv.) afforded
quantitatively the desired product CP1 within 5 min. Moreover,
the CuAAC reaction rate was found to be dependent on
substrates concentration. Notably, extended exposure to the
click conditions (412 h) resulted in partial degradation of the
peptides, and the addition of protecting ligands such as
In conclusion, using a rational approach we have developed
a facile synthetic strategy to covalently tethered homo and
heterotriple helices. Our strategy is modular, in which ‘dual
stranded’ peptides are clicked with either a similar or unique
linear peptide. Significantly, our designed end-stapled
polypeptides displayed characteristic triple helical features
with enhanced thermal stability. These discrete biomolecules
with the capacity to form stable triple helices will enable
comprehensive SAR study of specific collagen–integrin inter-
actions and have implications in nanostructure design.
We thank Prof. Pat Guiry, Dr Jens Neilsen and Fergal O’
Meara at University College Dublin, Ireland, for access
and assistance with CD spectroscopy. We would like to
acknowledge financial support from Cancer Research, UK.
8c
THTPA or TBTA, or the exclusion of O , had no effect on
2
reaction efficiency. Thus, the required biomolecules CP1–4
were produced using our optimised CuAAC condition, i.e.
1
mM DS1, CuSO Á5H O (5 equiv.)–NaAsc (25 equiv.) in
4
2
B1% aq DMF, and purified by direct injection of the reaction
mixture onto an RP-HPLC column (see Fig. S12–S15, ESIw).
Notes and references
1
2
(a) R. O. Hynes, Cell (Cambridge, Mass.), 1992, 69, 11–25;
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(
2
Res., 2010, 339, 247–257.
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Circular dichroism spectroscopic analysis of the end-
stapled/clicked peptides CP1–4 showed triple-helical character,
3
4
J. Emsley, C. G. Knight, R. Farndale, M. J. Barnes and
R. Liddington, Cell (Cambridge, Mass.), 2000, 101, 47–56.
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9
,12
as well as an anticipated
Fig. 3). For example, compared to the ‘non-tethered’
homotriple helix SS1 (T = 13.1 1C, Table 1), our
end-stapled homotriple helix CP1 showed T = 30.0 1C.
Gratifyingly, our novel end-stapled hetero-polypeptide CP2
displayed triple helical character with T = 27.5 1C.
and noticeable shift to higher TM
(
M
M
5
For example: (a) B. Grab, A. J. Miles, L. T. Furcht and
G. B. Fields, J. Biol. Chem., 1996, 271, 12234–12240;
(
b) A. V. Persikov, J. A. Ramshaw, A. Kirkpatrick and
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M
The longer clicked polypeptides CP3 and CP4, with a GPO
(
extension, showed broadly two main melt transitions, in which
C. S. Smerling, N. Pugh, A. Konitsiotis, B. Leitinger, P. G. de
Groot, G. E. Jarvis and N. Raynal, Biochem. Soc. Trans., 2008, 36,
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`
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M
the higher T were at an impressive 42.4 and 41.1 1C,
respectively. Closer investigation using heating/cooling cycles
on a differential scanning calorimeter (DSC) confirmed the
6
7
8
occurrence of two T s. We speculate that these dual melt
M
transitions are due to the formation of both intramolecular
1
32, 3242–3243; (b) V. Gauba and J. D. Hartgerink, J. Am. Chem.
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(a) V. V. Rostovtsev, L. G. Green, V. V. Fokin and
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(
b) C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem.,
2
002, 67, 3057–3064; (c) M. Meldal and C. W. Tornøe, Chem. Rev.,
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2
J.-C. Horng, A. J. Hawk, Q. Zhao, E. S. Benedict, S. D. Burke and
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9
1
1
1
1
0 Fmoc Solid Phase Synthesis: A Practical Approach, ed. W. C. Chan
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Fig. 3 (top) Melt transition curves and (bottom) first derivative
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This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 2589–2591 2591