An intramolecular tryptophan-condensation approach for peptide stapling

Article abstract of DOI:10.1039/c7ob02667f

Stapled peptides are gaining tremendous interest as next-generation therapeutic agents to target protein-protein interactions. Herein, we report an intramolecular peptide stapling method which links two tryptophan residues at the C2 position of the indole

Full text of DOI:10.1039/c7ob02667f

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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An Intramolecular Tryptophan-Condensation Approach for
Peptide Stapling†
Eunice Y.-L. Hui,*a Bhimsen Rout,b Yaw Sing Tan,c Chandra S. Verma,c,d,e Kok-Ping Chana and
Charles W. Johannes*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
was achieved by incubating peptides in trifluoroacetic acid (TFA) in
the absence of an inert environment, limiting this approach to
peptides containing aliphatic side groups. In this report, we present
a new peptide-stapling methodology that links two Trp residues via
a concomitant condensation reaction with an aldehyde.
Stapled peptides are gaining tremendous interest as next-
generation therapeutic agents to target protein-protein
interactions. Herein, we report an intramolecular peptide stapling
method which links two tryptophan residues at C2 position of the
indole moieties via acid-mediated condensation with an aldehyde.
We extend the strategy used to synthesise 4,4-difluoro-4-bora-
3a,4a-diaza-s-indacene, also known as boron dipyrromethene
(BODIPY),8 to fuse indoles into a C2-symmetric structure with an
arylaldehyde. Using skatole as a model to mimic tryptophan
Intramolecular stapling of linear peptides is used as an
approach to confer enhanced α-helicity, protease resistance
and target binding affinity relative to their linear
counterparts.1-3 With the emerging trend of stapled peptides residues, condensation reactions were carried out using skatole and
as a new class of medicine,4 there is an urgent need to develop
4-bromobenzaldehyde in the presence of acids. A variety of acids
a feasible synthetic stapling route using natural amino acids
and mild table-top chemical reagents; as an alternative to
classical stapling,5 which involves the use of expensive and
synthetically challenging unnatural amino acids5a-d and
reagents.5e
and methods were screened (Table 1). Desired condensation
product 3 was obtained in good yield (72%) using (1S)-(+)-camphor-
10-sulphonic acid (CSA, Table 1, entry 2). The formation of various
possible side products (ESI,† Fig. S1) might have aꢀributed to low
With the rare occurrence of tryptophan (Trp) residues in
protein structures,6 these residues are ideal for controlled
single-site modification, functionalisation and derivatisation.
However, it is not easy to activate the aromatic scaffold of
tryptophan under mild conditions, thus posing significant
challenges to site-specific functionalisation. Utilising Pd-
catalysed C-H activation, Lavilla and colleagues successfully
linked Trp to iodo-phenylalanine (Phe) or iodo-tyrosine (Tyr).6
This method involves the use of transition metals which is not
cost effective in large scale manufacturing and requires
complex purifications to remove traces of toxic metals that
may bind to the peptide. Makwana and Mahalakshmi recently
reported a Trp - Trp cross linking strategy to form hyperstable
peptides with β-hairpin and α-helical structures.7 Crosslinking
Table 1 Optimisation of the conditions for skatole condensation
Br
Br
Acid catalyst
+
N
H
CHO
NH HN
1
2
3
Entry
Acids
Methoda
Yield
1
2
3
4
5
6
CH3SO3H
C9H15C(O)SO3Hc
CH3C6H4SO3H
CF3COOH
HCl
A
A
A
A
B
C
0b
72
62
38
4
H2SO4
85
aMethod A: 1 (0.32 mmol), 2 (0.16 mmol) and acid (5 equiv) were
dissolved in CH2Cl2 and the mixture was heated at 40 °C for 6 h in a
sealed tube. Method B: 1 (0.32 mmol), 2 (0.16 mmol) were dissolved
in acid and the mixture was heated at 100 °C for 3 h in a sealed tube.
Method C: 1 (0.32 mmol), 2 (0.16 mmol) and acid (10 µL) were
dissolved in EtOH and the mixture was stirred at room temperature
c
for 18 hours. b3 was not obtained. (1S)-(+)-Camphor-10-sulfonic acid
(CSA).
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-phuric acid for condensation, crude cyclised peptide with lower
purity was obtained.
yields (Table 1). The condensation was also performed using
mineral acids9,10 (Table 1, entry 5-6), of which sulphuric acid (Table
1, entry 6) resulted in the highest yield (85%) of product 3. After the
successful linkage of skatole at the C2 position, it was envisioned
that this method can be adopted to link Trp residues present in
various positions of the linear peptides to produce cyclic stapled
peptides as an alternative to using unnatural amino acids in
conventional stapling.
1
The H NMR spectrum of 5d shows a singlet peak for the newly
formed C-H at δ 6.13, which indicates the successful cyclisation of
1
linear counterpart 4d (ESI,† Fig. S7). The H NMR singlet peaks for
the newly formed aliphatic C-H (highlighted in red in the figure in
Table 2) for stapled peptides 5a, 5b and 5d (ESI,† Fig. S8) have
different chemical shifts. The slight changes in chemical shifts are
due to the different number of amino acid residues present
between the two Trp residues that result in different ring sizes and
ring strains upon peptide stapling.
One of the key benefits of stapled peptides is their enhanced
proteolytic stability2 over the linear counterparts. Hence,
tryptophan-linked stapled peptide 5d (Fig. 1A) and its linear
counterpart 4d were treated with chymotrypsin to compare their
proteolytic resistance. HPLC profiles revealed that stapled peptide
5d was resistant towards enzymatic degradation, whereas its linear
precursor 4d degraded rapidly. This suggests that peptide stapling
through tryptophan condensation could bring about enhanced
stability against proteolytic cleavage and may be useful for the
development of systemic peptide-drugs resistant to proteolytic
degradation.
Encouraged by our findings above, a further step was taken to
investigate the effect of tryptophan stapling technique on peptide
structure. Circular dichroism (CD) studies on peptide 5d and its
linear precursor 4d (ESI,† Fig. S4 and S5) were carried out. It was
observed that the cyclisation resulted in very marginal
conformational change in the peptide (Fig. 1B). Although
tryptophan condensation did not impart any α-helicity to the i, i+4
peptide, we postulate that the tryptophan linkage resulted in a
macrocyclic structure that is highly resistant to proteolytic
degradation.
With these preliminary results, sMTide-02, a stapled peptide that
binds to MDM2 and activates p53,11 was selected as a model
system to demonstrate the applicability of our new stapling
approach. The sequence was truncated to encompass key residues
thus enabling us to establish a minimum-residual-spacing between
two tryptophan residues that is required for cyclisation.
Although conversion using sulphuric acid is higher than when
using CSA for the model cyclisation (Table 1), CSA was chosen over
harsh sulphuric acid for peptide stapling considering the sensitive
nature of various amino acids. Peptide 4, 4 bromobenzaldehyde12
and CSA in 1, 2-dichloroethane (DCE) were heated and mixed at an
elevated temperature of 60 °C for three hours (Table 2, entries 1-9).
Good conversions were observed for Trp residues located at i, i+n
(n=1 to 4) positions (Table 2, entries 1-4). Moreover, this stapling
strategy is used to cyclise peptides with various side chains (Table 2,
entries 5-7). This methodology was also successfully used for the
synthesis of cyclic peptides conta-ining Asn-Gly-Arg (-NGR-)13 and
Arg-Gly-Asp(-RGD-)14 which are important fragments of biologically
active compounds (Table 2, entries 8, and 9). When CSA-mediated
tryptophan condensation was carried out in solution phase, the
conversion was lower as compared to the solid phase for the
formation of stapled peptide 5d. In addition, with the usage of sul-
Table 2 Synthesis of various stapled peptides using CSA
AA
NH2
Ac
AA
Trp
Trp
AA
NH2
Ac
AA
(AA)n
H
Trp
Trp
CSA
DCE
(AA)n
H H
N
H
N
H
N
H
N
H
Br
4
5
Entry
(%)a
i, i+n
Linear Peptide (4)
Cyclic Peptide (5)
Conv.
1
2
3
4
5
6
7
8
9
i, i+1
i, i+2
i, i+3
i, i+4
i, i+4
i, i+4
i, i+4
i, i+4
i, i+4
Ac-WWALL-NH2 (4a)
Ac-WAWLL-NH2 (4b)
Ac-WALWL-NH2 (4c)
Ac-WALLW-NH2 (4d)
5a
5b
5c
5d
5e
5f
100
98
93
93
Ac-EYWALLW-NH2 (4e)
Ac-YWALLWS-NH2 (4f)
Ac-ALWALLW-NH2 (4g)
Ac-WNGRW-NH2 (4h)
Ac-WRGDW-NH2 (4i)
95
95
5g
5h
5i
100
81
94
Coupling conditions: Peptide 4 (1 equiv), CSA (6 equiv) and 4-bromobenzaldehyde
(10 equiv) in 1, 2-dichloroethane (DCE) were heated at 60 °C for 3 h in a sealed
tube. The resin was agitated with a stream of nitrogen gas. Isolated yields of 1%
are obtained for 5a, 5b and 5d after solid phase peptide synthesis and CSA
mediated condensation. aPercent conversion was determined by estimating the
consumption of linear peptide 4 using HPLC.15 AA– amino acid, HPLC- high-
Fig. 1 (A) Chymotrypsin proteolysis and B) circular dichroism study of linear peptide 4d
(dark red) and corresponding cyclic peptide 5d (blue).
performance liquid chromatography, conv.- conversion.
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Fig. 3 Emission spectra of the 10 µM solution of the linear peptide 4d (orange) and
cyclized peptide 5d (blue) in methanol. Each spectrum represents average of three
different measurements with standard deviations. λexc. = 230 nm.
study provides a strong complement to existing methods for the
synthesis of stapled peptides using natural amino acids.
Fig. 2 Conformations of 4d and 5d observed in MD simulations. (A) Superimposition of
representative conformations of 4d. Side chain atoms are not shown. (B)
Representative conformations of 5d. Percentage populations of the respective
conformations are indicated. The tryptophan linker is in yellow while the rest of the
peptide is in grey.
Conflicts of interest
There is no conflict of interest.
To understand the conformation of linear and cyclic peptides,
insilico studies on 4d and 5d were performed. Molecular dynamics
(MD) simulations of the peptides in water followed by root-mean-
square-deviation-based clustering of the sampled structures
revealed that 4d was highly flexible and adopted a variety of
extended and folded conformations (See Supplementary
Information for computational details). In contrast, the stapled
peptide 5d assumed two main conformations (Fig. 2). The dominant
conformation of 5d (98%) was non-α-helical and characterised by
the absence of an i, i + 4 backbone hydrogen bond between the
stapled residues. There is a small cluster of α-helical conformations
(2%). The paucity of helical conformations obtained from the
simulations explained the non-helical conformation of 5d in
solution, as indicated by its circular dichroism (CD) spectrum.
We observed that the two indole rings of 5d are nearly
perpendicular to each other in the MD simulations (Fig. 2B). Hence,
emission spectra of the tryptophan on the linear 4d and cyclized 5d
were obtained (Fig. 3). The high intensity of emission for 4d in
solution probably results from π-π stacking16 between the two
aromatic indole rings in the tryptophan residues. Fluorescence
intensity reduced drastically upon cyclisation indicating that the
indole rings are structurally restricted17,18 and are unable to align
properly to form π-π stacking interactions. This photophysical
observation is in agreement with the MD simulations (Fig. 2).
Acknowledgement
The authors thank A*STAR Joint Council Office (JCO)
[1231BFG036] for funding. The authors wish to thank Mr Simon
Choo for his assistance in CD measurement.
References
1
2
3
Y. H. Lau, Y. Wu, M. Rossmann, B. X. Tan, P. de Andrade, Y. S.
Tan, C. Verma, G. J. McKenzie, A. R. Venkitaraman, M.
Hyvçnen and D. R. Spring, Angew. Chem. Int. Ed., 2015, 54
15410–15413.
G. H. Bird, N. Madani, A. F. Perry, A. M. Princiotto, J. G.
Supko, X. He, E. Gavathiotis, J. G. Sodroski and L. D.
Walensky, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 14093-
14098.
(a) H. E. Blackwell, R. H. Grubbs, Angew. Chem. Int. Ed., 1998,
37, 3281–3284. (b) S. Baek, P. S. Kutchukian, G. L. Verdine, R.
Huber, T. A. Holak, K. W. Lee and G. M. Popowicz, J. Am.
Chem. Soc., 2012, 134, 103–106.
,
4
5
A. Burgess, K.M. Chia, S. Haupt, D. Thomas, Y. Haupt and E.
Lim Front. Oncol., 2016, 6, 7.
(a) K. V. Lawson, T.E. Rose, P.G. Harran, Proc. Natl. Acad. Sci.
U. S. A. 2013, 110, E3753. (b) P. M. Cromm, S. Schaubach, J.
Spiegel, A. Fürstner, T. N. Grossmann, H. Waldmann, Nat.
Commun. 2016, 7, 11300. (c) V. Martí-Centelles, M. D.
Pandey, M. I. Burguete, S. V. Luis, Chem. Rev. 2015, 115,
Conclusions
8736. (d) K. Cui, Y. Guo, Y. He, F.-L. Wang, H.-N. Chang, Y.-J.
Wang, F.-M. Wu, C.-L. Tian, L. Liu, Angew. Chem. Int. Ed.
2013, 52, 9558. (e) H. E. Blackwell, J. D. Sadowsky, R. J.
Howard, J. N. Sampson, J. A. Chao, W. E. Steinmetz, D. J.
O'Leary, R. H. Grubbs, J. Org. Chem., 2001, 66 (16), 5291.
In summary, a new intramolecular tryptophan stapling strategy
using an aldehyde under mild acidic condition for peptides is
reported. Several stapled peptides were synthesized, of which two,
peptides 4d vs. 5d were further investigated for helicity, proteolytic
stability, fluorescence measurements and their conformational
landscape explored using MD simulations. The use of natural amino
acid i.e. tryptophan as a cyclization fragment and the resulting
enhanced proteolytic stability upon condensation are key new
developments we report. Further investigations could result in
improved yields, making this method more synthetically viable. This
6
7
L. Mendive-Tapia, S. Preciado, J. Garcia, R. Ramon, N.
Kielland, F. Albericio and R. Lavilla, Nat. Commun., 2015, 6,
7160.
K. M. Makwana and R. Mahalakshmi, Org. Lett., 2015, 17
,
2498-2501.
This journal is © The Royal Society of Chemistry 20xx
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8
9
Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu, R.
Banerjee, J. Zhao, C. He, Nat. Commun. 2011, , 495.
P. Dydio, D. Lichosyt, T. Zieliński and J. Jurczak, Chem. Eur. J.,
2012, 18, 13686-13701.
2
10 M.R. Mason, T.S. Barnard, M.F. Segla, B.H. Xie, K.
Kirschbaum, J. Chem. Crystallogr., 2003, 33, 531–540.
11 C.J. Brown, S.T. Quah, J. Jong, A.M. Goh, P.C. Chiam, K.H.
Khoo, M.L. Choong, M.A. Lee, L. Yurlova, K. Zolghadr, T.L.
Joseph, C.S. Verma, D.P. Lane, ACS Chem. Biol., 2013, 8, 506-
512.
12 Condensation was carried out using 4-bromobenzaldehyde
instead of benzaldehyde due to higher yield.
13 R. E. Wang, Y. Niu, H. Wu, M. N. Amin, J. Cai, Am. J. Nucl.
Med. Mol. Imaging, 2011, 1, 36-46.
14 S. Jackson, W. DeGrado, A. Dwivedi, A. Parthasarathy, A.
Higley, J. Krywko, A. Rockwell, J. Markwalder, G. Wells, R.
Wexler, S. Mousa, and R. Harlow J. Am. Chem. Soc., 1994,
116, 3220-3230.
15 High conversions and low isolated yields indicate the
formation of many side products and further process
optimization for synthetic application is required.
16 (a) B. Rout, L. Unger, G. Armony, M. A. Iron, D. Margulies,
Angew. Chem. Int. Ed. 2012, 21, 12477. (b) B. Rout, P. Milko,
M. A. Iron, L. Motiei, D. Margulies, J. Am. Chem. Soc. 2013,
135, 15330.
17 L. Unger-Angel, B. Rout, T. Ilani, M. Eisenstein, L. Motiei, D.
Margulies, Chem. Sci., 2015, 6, 5419.
18 Y. Nissinkorn, N. Lahav-Mankovski, A. Rabinkov, S. Albeck, L.
Motiei, D. Margulies, Chem. Eur. J. 2015, 21, 15981.
4 | J. Name., 2012, 00, 1-3
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