Water-soluble, stable and azide-reactive strained dialkynes for biocompatible double strain-promoted click chemistry

Article abstract of DOI:10.1039/c9ob01745c

The Sondheimer dialkyne is extensively used in double strain-promoted azide-alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.

Full text of DOI:10.1039/c9ob01745c

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: K. Sharma, A. V.
Strizhak, E. Fowler, X. Wang, W. Xu, C. Hatt, Y. Wu, H. Sore, Y. H. Lau, M. Hyvönen, L. Itzhaki and D. R.
Spring, Org. Biomol. Chem., 2019, DOI: 10.1039/C9OB01745C.
Volume 15
Number 47
21 December 2017
Pages 9945-10124
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
Accepted Manuscripts are published online shortly after acceptance,
rsc.li/obc
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
shall the Royal Society of Chemistry be held responsible for any errors
single cell transcriptome in vivo analysis (TIVA)
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 5
View Article Online
DOI: 10.1039/C9OB01745C
ARTICLE
Water-soluble, stable and azide-reactive strained dialkynes for
biocompatible double strain-promoted click chemistry
Krishna Sharma,a Alexander V. Strizhak,a Elaine Fowler,a Xuelu Wang,b Wenshu Xu,c Claus Hatt
Jensen,a Yuteng Wu,a Hannah F. Sore,a Yu Heng Lau,*,d Marko Hyvönen,*,b Laura S. Itzhaki,*,c David
R. Spring*,a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The Sondheimer dialkyne is extensively used in double strain-promoted azide-alkyne cycloadditions. This reagent suffers
with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological
systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble,
stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes
to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting
the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and
show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant
advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.
new water-soluble, stable, and azide-reactive strained
cyclooctyne based reagents. Highly reactive strained alkyne
reagents allow probing of fast biological processes and enable
Introduction
Strain-promoted azide-alkyne cycloaddition (SPAAC) reactions
are widely employed for covalent modification of biomolecules
in living systems.1 These utilise a high-energy, strained
cyclooctyne molecule as the reaction driving force, thus
avoiding the use of cytotoxic copper catalysts employed in
copper(I)-catalysed azide-alkyne cycloadditions (CuAAC).2-4
A double SPAAC has been developed using Sondheimer
dialkyne 1, which contains two strained alkyne moieties both
able to undergo SPAAC reactions (Figure 1a).5, 6 The use of a
double-strained dialkyne reagent enables efficient conjugation
of two azide containing molecules in a metal-free environment.
The Sondheimer dialkyne has been used for a range of
applications, including the macrocyclisation of bis-azide
functionalised peptides,7-12 chemical modification of azido-
proteins in cells,5 post-synthetic modification of metal-organic
framework thin films,13 tetramerisation of HIV-related
peptides,14 and for the preparation of fluorescent dyes.15
Notably, these dialkynes are fundamentally limited in their
biological application due to inherently poor water-solubility
and aqueous stability (τ1/2 ∼ 10 min at pH = 7.4).16, 17
reactions in biological systems at low reagent concentrations.
As a result, numerous derivatives of strained cyclooctyne
molecules which exhibit either improved water-solubility,
stability or azide-reactivity have been synthesised and applied
to SPAAC in vitro and in vivo.18-21 However, research has been
less successful towards the development of water-soluble,
stable and azide-reactive dialkyne reagents, despite their
extensive utility.22
(a) Previous work
N3
N
N
N
N
Double SPAAC reagent:
- Low aqueous solubility
- Poor aqueous stability
N
N
N3
1
Sondheimer dialkyne
(b) This work
N+
CF3COO-
Our improved double SPAAC reagent:
- High aqueous solubility
- High aqueous stability
N
22
Following the introduction of SPAAC there has been a
considerable amount of research towards the development of
N
CF3COO-
N
N
N
N+
N
N
CF3COO-
a. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge
CB2 1EW, United Kingdom.
N
N3
N3
N
N+
b. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road,
Cambridge CB2 1GA, United Kingdom.
Ac LTF
N
C
O
EYWAQL
N
C
O
S
NH2
Ac LTF
N
C
O
EYWAQL
N
H
C
O
S NH2
H
H
H
c. Department of Pharmacology, University of Cambridge, Tennis Court Road,
Cambridge CB2 1PD, United Kingdom.
School of Chemistry, Eastern Ave, The University of Sydney, NSW 2006, Australia
PDI-E
28
26
Potent MDM2 inhibiting stapled peptide
Kd = 7 nM
Cellular activity observed
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 5
ARTICLE
Journal Name
Figure 1. a) Previous work: development of the Sondheimer dialkyne for double
strain-promoted azide-alkyne cycloaddition (SPAAC). b) This work: development
of a highly water-soluble and stable, strained dialkyne reagent and an example of
its application in the generation of p53-MDM2 inhibiting stapled peptides.
substituted anilines 12 and 13.24, 25 DIBAL-H reducVtiieownAfrtoicllleoOwnleinde
by Dess-Martin oxidation26 gave the desDirOeId: 1o0.r1t0h3o9-/Ca9nOdB0m17e4t5aC-
dimethylamine substituted substrates 16 and 17.
(a)
NBS (1.05 eq)
BPO (0.1 eq)
PhSO2Na·2H2O
(1.2 eq)
Br
SO2Ph
R1
R1
R1
CO2CH3
CCl4, 80 °C, 6h
CO2CH3
CO2CH3
DMF, 80 °C, 12h
1
1
4.
5.
R = 3-NO2, 57%
2.
3.
R = 3-NO2
1
1
R = 5-NO2, 74%
R = 5-NO2
SO2Ph
CH2OH
DIBAL-H (2.3 eq)
CH2Cl2, -78 °C
DMP (1.1 eq)
CH2Cl2, rt
R1
SO2Ph
CHO
R1
1
1
6.
7.
R = 3-NO2, 84%
8.
9.
R = 3-NO2, 87%
1
1
R = 5-NO2, 91%
R = 5-NO2, 87%
Zn (10 eq)
NH4Cl (15 eq)
(HCHO)n (10 eq)
NaBH4CN (5 eq)
SO2Ph
SO2Ph
SO2Ph
O2N
H2N
N
CO2CH3 acetone:water (5:1)
rt
CO2CH3
27
CO2CH3
glacial acetic acid
rt, 16h
Figure 2. Predicted (clogP) water-solubility values of substituted dialkynes 20-25.
10.
11.
12.
13.
3-NH2, 75%
5-NH2, 70%
3-NMe2, 93%
5-NMe2, 82%
4.
5.
3-NO2
5-NO2
Experimentally determined (logP) values and second-order rate constants (k, in M-1 s-1)
indicated below.
DIBAL-H (2.3 eq)
CH2Cl2, -78 °C
SO2Ph DMP (1.1 eq)
SO2Ph
N
N
CH2Cl2, rt
CH2OH
CHO
Substrates 16 and 17 were then transformed to the
respective cyclic diacetylenes through double elimination.23
These substrates were then subjected to Wittig-Horner
conditions in the presence of LiHMDS and diethyl
chlorophosphate to form the cyclised intermediates 18 and 19
in 15 and 26% respective yields, which were finally treated with
LDA to eliminate the two phenylsulfonyl groups, affording the
desired substituted strained dialkynes 20 and 21 in moderate
yields (Scheme 1b). When this protocol was applied to nitro-
substituted aldehydes 8 and 9 the Wittig-Horner conditions
were unsuccessful, and no product was formed.
14.
15.
3-NMe2, 96%
5-NMe2, 94%
16.
17.
3-NMe2, 82%
5-NMe2, 73%
(b)
ClP(O)(OEt)2
(1.2 eq)
R1
R1
R1
SO2Ph
R2
R2
LDA (5 eq)
LiHMDS (2.0 eq)
SO2Ph
CHO
R2
R2
-78 °C, 2 h
THF
R2
-78 °C, 0.5 h rt,
1.5 h, THF
R1
R1
R = H, R =NMe2, 50%
PhO2S
2
1
2
1
1
2
17.
16.
18.
20.
21.
R = H, R =NMe2
R = H, R =NMe2, 15%
1
2
1
2
1
2
19.
R =NMe2, R =H
R =NMe2, R =H, 26%
R =NMe2, R =H, 54%
Scheme 1. a) Synthesis of benzyl sulfone substituted aldehyde substrates. b)
Synthesis of substituted dialkynes.
To further improve their physical properties, dimethylamine
substituted dialkynes 20 and 21 were treated with methyl
trifluoromethanesulfonate in dichloromethane to give
trimethylammonium substituted dialkynes 22 and 24 (see SI 2.1
for details).28 The dialkynes 22 and 24 were further methylated
in acetonitrile to deliver bis-ammonium dialkynes 23 and 25.
Gratifyingly, all of the trimethylammonium substituted
dialkynes 22-25 were found to be readily soluble in water
thereby overcoming this limitation. Water-solubility of meta-
substituted bis-ammonium dialkyne 23 was determined
experimentally by measuring its octanol-water partition
coefficient using the shake-flask method,29 which
demonstrated 23 (logP = -1.50) to be more soluble than the
uncharged dialkyne 20 (logP = 1.11), which was consistent with
predicted solubility trends (Figure 2).
Next, the aqueous stability of these highly water-soluble
trimethylammonium substituted dialkynes 22 and 23 was
investigated. Dialkynes 22 and 23 were subjected to aqueous
buffer conditions (1 mM in PBS, pH 7.4, r.t.) and decomposition
was monitored by UV spectroscopy (see SI 2.3 for details). We
observed no decomposition of 22 nor 23 even after 7 days.
Under the same conditions, Sondheimer dialkyne 1 is known to
have a decomposition half-life of 10 ± 2 minutes.16 Moreover,
23 when stored neat at r.t. did not show any decomposition
after two weeks, whereas the Sondheimer dialkyne completely
In this work, we report the synthesis of water-soluble,
stable, and azide-reactive strained dialkyne reagents which
address
these
limitations.
Meta-trimethylammonium
substituted Sondheimer dialkyne 22 exhibited high water-
solubility, excellent stability and high azide-reactivity (Figure
1b). Finally, these dialkynes were applied to a double strain-
promoted macrocyclisation strategy7 to generate functionalised
p53-based stapled peptides which inhibit the oncogenic p53-
MDM2 interaction (Figure 1b).
Results and discussion
Novel nitro and amine substituted dialkynes were designed to
improve the physical characteristics of these reagents. The
substitutions were made at the ortho- and meta- positions
relative to the alkyne moiety based on Orita’s synthetic route.22,
23
The ortho-nitro and meta-nitro substituted sulfones 8 and 9
were synthesised by subjecting commercially available nitro
substituted arenes 2 and 3 to Wohl-Ziegler bromination,
followed by nucleophilic substitution with benzenesulfinic acid
sodium salt (Scheme 1a). The resulting benzyl sulfones 4 and 5
were subjected to DIBAL-H reduction followed by Dess-Martin
oxidation to deliver the desired benzyl sulfone aldehyde
substrates 8 and 9. Reduction of the nitro-group in sulfones 4
and 5, followed by reductive methylation afforded dimethyl
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 5
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
inhibition. The yields depicted are the isolated yields of the major fraction of the stapled
decomposes within two days.16 We believe that these dialkynes
will exhibit similar stability to thiols as Sondheimer dialkyne.
We also investigated the azide-reactivity of these amine
substituted dialkynes 20-25 in a SPAAC, with benzyl azide as a
model substrate in methanol. We studied the reaction kinetics
by monitoring the consumption of dialkynes 20-25 using UV
spectroscopy and measured their second-order rate constants
(see SI 2.10 for details). The second-order rate constants
determined experimentally are depicted in figure 2. We
observed that the substitution of a single dimethylamine
substituent at the meta-position in 20 with a charged
trimethylammonium substituent (22, 0.097 M-1s-1) led to a 2.5-
fold increase in the rate when compared to 20. Installation of
another trimethylammonium substituent at meta-position in 22
(23, 0.174 M-1s-1) pleasingly led to a further 1.8-fold increase in
View Article Online
peptides obtained after HPLC purification. The major isomer of functionalised stapled
DOI: 10.1039/C9OB01745C
peptides separated by HPLC purification were tested for biological activity.
Functionalised stapled peptides 27-29 were then tested in a
competitive fluorescence polarisation (FP)35, 36 assay for MDM2
binding affinity (see SI 2.8 for details). Stapled peptide 28 which
contained one trimethylammonium substituent, was found to
exhibit the highest binding affinity (7.0  3.9 nM). Installation of
a second trimethylammonium group on the dialkyne caused a
slight reduction in the binding affinity (29, 17.0  6.2 nM).
A crystal structure of MDM2 complexed with meta-
trimethylammonium functionalised peptide 28 was obtained to
help determine whether the charged trimethylammonium
group interacts with the protein (17-108, E69A/K70A;37 Figure
3). Previous studies have shown that the staple moiety of the
peptide can interact with the protein.7, 38-41 The MDM2-bound
stapled peptide 28 was shown to be in an α -helical
conformation as expected, with the binding triad (F3, W7, L10)
in the correct orientation to engage the respective binding
hotspots on MDM2 (Figure 3: Top view).7 Two MDM2-bound
peptide molecules were observed in one asymmetric unit, with
the staple moiety shifted away from the MDM2 surface due to
the charged trimethylammonium group (see SI Figures
S2.7.1.3.1a and S2.7.1.3.1b). The bis(triazolyl) staple of the
bound peptide 28 exists as the anti regioisomer with the
charged trimethylammonium group pointing away from MDM2
surface (Figure 3: Side view).
Next, we tested the ability of functionalised stapled
peptides 27-29 to inhibit the p53-MDM2 interaction in an
established p53 reporter cell-based assay.36 We treated p53
reporter cells with linear peptide 26 and dialkynes 1, 20, 22, and
23 in a 96-well format (Figure 4). In situ parallel stapling with
multiple substituted dialkynes provides a faster way of
screening resultant stapled peptides directly in the cellular
medium, without performing individual stapling reactions and
purifications for each dialkyne variant.7,42 p53 activation values
are reported as fold activation over the activation value
obtained when cells were treated with 1% DMSO. This provides
an indirect comparative measure of the cellular p53 levels
where higher values are indicative of increased cellular activity.
We also tested the corresponding purified stapled peptides 27-
29 in this assay which confirmed the potencies (see SI 2.9 for
details). Peptide 29 gave a negligible response in this assay,
most likely due to cell permeability issues with the doubly
charged dialkyne. Peptide 28 was able to permeate the cell and
gave 1.7-fold activation of p53. We feel that the advantages in
usability of our water-soluble, stable and azide-reactive
dialkynes make these reagents a substantial improvement over
the Sondheimer dialkyne.
the rate over dialkyne 22. However, presence of
a
dimethylamine or trimethylammonium substituent at the
ortho-position (21, 24, and 25) led to a significant decrease in
the rate of reaction, making them essentially unreactive
towards benzyl azide at rt (see SI Figure S2.10). Overall,
trimethylammonium substituted 22 and 23 were found to be
the most reactive dialkynes. We believe that the high azide-
reactivity of 22 and 23 stems from the electronic effect of the
charged trimethylammonium substituent which potentially
lowers the transition state barrier required for SPAAC with
azide, as has been reported previously with BARAC in the
presence of a fluoro substituent.30 The poor reactivity of ortho-
substituted 21, 24, and 25 is attributed to the steric effects of
bulky dimethylamine/trimethylammonium substituent.
With substituted Sondheimer dialkynes 20-25 in hand, we
investigated their application in stapling phage display peptide
PDI-E 26 (Ac-LTFXEYWAQLXS-NH2, X=Orn(N3)) for targeting the
32
oncogenic p53-MDM2 interaction.31, The p53 protein is a
transcription factor and tumour suppressor. Its activity is
inhibited by interaction with the E3 ligase MDM2, which is
overexpressed in many cancers and targets p53 for
proteasome-mediated degradation.33 The stapling of peptide
26 with Sondheimer dialkyne 1 has been previously shown by
our group to be a potent inhibitor of the p53-MDM2
interaction.7 Stapling reactions between peptide 26 and
dialkynes 20, 22, and 23 successfully gave the stapled peptides
27-29 in 25-36% yields within 24 h (Scheme 2).34 Ortho-
substituted dialkynes 21, 24, and 25 were found to be
unreactive under the reaction conditions.
Top View
Side View
Scheme 2. Application of substituted Sondheimer dialkynes in double strain-promoted
macrocyclisation strategy to generate functionalised stapled peptides for p53-MDM2
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 5
ARTICLE
Journal Name
Acknowledgements
View Article Online
DOI: 10.1039/C9OB01745C
Figure 3. Crystal structure of 28 bound to MDM2 at 2.0 Å resolution (PDB ID: 6H22). Top
and side views depicting the bound stapled peptide 28 in -helical conformation and the
staple as anti regioisomer (only the staple and side chains of the three binding residues
are shown for clarity). The 2Fo-Fc electron density map is contoured at 1.
D.R.S. acknowledges support from the Engineering and Physical
Sciences Research Council (EP/P020291/1) and Royal Society
(Wolfson Research Merit Award). K.S. would like to thank Trinity
College, Cambridge Trust, Cambridge Nehru Trust and the
Cambridge Philosophical Society for providing fellowships. We
would like to thank X-ray crystallographic facility at the
Department of Biochemistry for access to crystallisation
instrumentation and the PNAC service (Department of
Biochemistry, University of Cambridge). We are grateful for the
Diamond Light Source for access to beamline I04 (proposal
mx14043) and for the data that contributed to these results.
N3
N3
R2
DMEM
R1
Ac LTF
N
C
O
EYWAQL
N
C
O
S NH2
H
H
in situ stapling
p53 reporter cells
in 96-well plate
Substituted dialkynes
20, 22, 23, 1 (0.5 mM)
PDI-E 26 (50 M)
27, 28, 29, 30
Notes and references
1.
2.
3.
4.
5.
N. J. Agard, J. A. Prescher and C. R. Bertozzi, J. Am. Chem.
Soc., 2004, 126, 15046-15047.
V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chem. Int. Ed., 2002, 41, 2596-2599.
C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem.,
2002, 67, 3057-3064.
L. M. Gaetke and C. K. Chow, Toxicology, 2003, 189, 147-
163.
I. Kii, A. Shiraishi, T. Hiramatsu, T. Matsushita, H. Uekusa, S.
Yoshida, M. Yamamoto, A. Kudo, M. Hagiwara and T.
Hosoya, Org. Biomol. Chem., 2010, 8, 4051-4055.
H. N. C. Wong, P. J. Garratt and F. Sondheimer, J. Am.
Chem. Soc., 1974, 96, 5604-5605.
Figure 4. p53 activation in a cellular reporter assay for in situ stapling of peptide 26 (50
M) with substituted dialkynes 20, 22, 23 and dialkyne 1 (0.5 mM) in parallel, giving
stapled peptides 27, 28, 29, and 30 respectively (performed as technical triplicate). Data
reported as fold activation over 1% DMSO. DMEM- Dulbecco’s Modified Eagle’s Medium.
6.
7.
Y. H. Lau, Y. Wu, M. Rossmann, B. X. Tan, P. deꢀAndrade, Y.
S. Tan, C. Verma, G. J. McKenzie, A. R. Venkitaraman, M.
Hyvönen and D. R. Spring, Angew. Chem. Int. Ed., 2015, 54,
15410-15413.
Conclusions
In conclusion, substituted strained dialkyne reagents have been
synthesised for biocompatible double strain-promoted click
chemistry. These highly azide-reactive novel dialkyne reagents
were shown to be both water-soluble and stable under aqueous
conditions. To demonstrate their extensive utility, we applied
these dialkynes to an in situ double-strain promoted
macrocyclisation strategy to generate functionalised stapled
peptide inhibitors of the oncogenic p53-MDM2 interaction.
These stapled peptides were all shown to bind MDM2 with low
nanomolar affinity by FP assay. Furthermore, the structure of
our MDM2-bound functionalised stapled peptide 28 was
elucidated through X-ray crystallography, which confirmed the
α-helical conformation of the peptidic backbone, and indicated
the staple moiety was shifted away from the MDM2 surface.
Peptide 28 was found to permeate the cell and gave
comparable biological data to the unsubstituted dialkyne. These
water-soluble, stable and azide-reactive strained dialkyne
reagents could be successfully applied to numerous
therapeutically relevant protein-protein interactions and
conjugation applications.
8.
9.
Y. H. Lau, P. de Andrade, Y. Wu and D. R. Spring, Chem. Soc.
Rev., 2015, 44, 91-102.
K. Sharma, D. L. Kunciw, W. Xu, M. M. Wiedmann, Y. Wu,
H. F. Sore, W. R. J. D. Galloway, Y. H. Lau, L. S. Itzhaki and
D. R. Spring, in Cyclic Peptides: From Bioorganic Synthesis
to Applications, RSC, 2018, pp. 164-187.
O. Demeter, E. A. Fodor, M. Kállay, G. Mező, K. Németh, P.
T. Szabó and P. Kele, Chem. Eur. J., 2016, 22, 6382-6388.
O. Demeter, A. Kormos, C. Koehler, G. Mező, K. Németh, E.
Kozma, L. B. Takács, E. A. Lemke and P. Kele, Bioconjugate
Chemistry, 2017, 28, 1552-1559.
A. Kormos, C. Koehler, E. A. Fodor, Z. R. Rutkai, M. E.
Martin, G. Mező, E. A. Lemke and P. Kele, Chem. Eur. J.,
2018, 24, 8841-8847.
Z. Wang, J. Liu, H. K. Arslan, S. Grosjean, T. Hagendorn, H.
Gliemann, S. Bräse and C. Wöll, Langmuir, 2013, 29, 15958-
15964.
C. Hashimoto, W. Nomura, T. Narumi, M. Fujino, T.
Nakahara, N. Yamamoto, T. Murakami and H. Tamamura,
Bioorg. Med. Chem., 2013, 21, 6878-6885.
A. Horner, D. Volz, T. Hagendorn, D. Furniss, L. Greb, F.
Ronicke, M. Nieger, U. Schepers and S. Brase, RSC Adv.,
2014, 4, 11528-11534.
10.
11.
12.
13.
14.
15.
16.
17.
D. A. Sutton, S.-H. Yu, R. Steet and V. V. Popik, Chem.
Comm., 2016, 52, 553-556.
M. F. Debets, J. C. M. van Hest and F. P. J. T. Rutjes, Org.
Biomol. Chem., 2013, 11, 6439-6455.
Conflicts of interest
There are no conflicts to declare.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
18.
19.
E. M. Sletten and C. R. Bertozzi, Acc. Chem. Res., 2011, 44, 41.
666-676.
J. Dommerholt, S. Schmidt, R. Temming, L. J. A. Hendriks, F. 42.
P. J. T. Rutjes, J. C. M. vanꢀHest, D. J. Lefeber, P. Friedl and
F. L. vanꢀDelft, Angew. Chem. Int. Ed., 2010, 49, 9422-9425.
J. C. Jewett, E. M. Sletten and C. R. Bertozzi, J. Am. Chem.
Soc., 2010, 132, 3688-3690.
B. R. Varga, M. Kállay, K. Hegyi, S. Béni and P. Kele, Chem.
Eur. J., 2012, 18, 822-828.
F. Xu, L. Peng, K. Shinohara, T. Morita, S. Yoshida, T.
Hosoya, A. Orita and J. Otera, J. Org. Chem., 2014, 79,
11592-11608.
G. Adrian, B. David, H. Gernot, R. Christoph, O. Christian
View Article Online
and G. T. N., Angew. Chem. Int. EdD.,O2I0:1104.,150339, /2C498O9B-204179435.C
Gradual product formation over 18h and losses due to the
formation of syn/anti byproducts in an in situ stapling assay
could result in slight variation in the cellular response levels
observed.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
A. Orita, D. Hasegawa, T. Nakano and J. Otera, Chem. Eur.
J., 2002, 8, 2000-2004.
K. S. MacMillan, T. Nguyen, I. Hwang and D. L. Boger, J. Am.
Chem. Soc., 2009, 131, 1187-1194.
M. Chandrasekharam, B. Chiranjeevi, K. S. V. Gupta and B.
Sridhar, J. Org. Chem., 2011, 76, 10229-10235.
D. B. Dess and J. C. Martin, J. Org. Chem., 1983, 48, 4155-
4156.
Calculated using ACD/Labs software, I-Lab 2.0
ilab.acdlabs.com.
-
J. T. Reeves, D. R. Fandrick, Z. Tan, J. J. Song, H. Lee, N. K.
Yee and C. H. Senanayake, Org. Lett., 2010, 12, 4388-4391.
OECD, Paris, 1981, Test Guideline 107, Decision of the
Council C(81) 30 final.
C. G. Gordon, J. L. Mackey, J. C. Jewett, E. M. Sletten, K. N.
Houk and C. R. Bertozzi, J. Am. Chem. Soc., 2012, 134, 9199-
9208.
31.
32.
B. Hu, D. M. Gilkes and J. Chen, Cancer Res., 2007, 67, 8810.
J. Phan, Z. Li, A. Kasprzak, B. Li, S. Sebti, W. Guida, E.
Schönbrunn and J. Chen, J. Biol. Chem., 2010, 285, 2174-
2183.
33.
34.
P. Chène, Nat. Rev. Cancer, 2003, 3, 102.
Stapling reactions with water-soluble reagents 22-25 were
carried out in 1:1 H2O:MeCN due to the partial solubility of
starting linear peptide 26 in water.
35.
Y. H. Lau, P. de Andrade, S.-T. Quah, M. Rossmann, L.
Laraia, N. Skold, T. J. Sum, P. J. E. Rowling, T. L. Joseph, C.
Verma, M. Hyvonen, L. S. Itzhaki, A. R. Venkitaraman, C. J.
Brown, D. P. Lane and D. R. Spring, Chem. Sci., 2014, 5,
1804-1809.
36.
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 and D. P. Lane, ACS Chem. Biol., 2013,
8, 506-512.
37.
38.
B. Anil, C. Riedinger, J. A. Endicott and M. E. M. Noble, Acta
Cryst. D, 2013, 69, 1358-1366.
Y. S. Chang, B. Graves, V. Guerlavais, C. Tovar, K. Packman,
K.-H. To, K. A. Olson, K. Kesavan, P. Gangurde, A.
Mukherjee, T. Baker, K. Darlak, C. Elkin, Z. Filipovic, F. Z.
Qureshi, H. Cai, P. Berry, E. Feyfant, X. E. Shi, J. Horstick, D.
A. Annis, A. M. Manning, N. Fotouhi, H. Nash, L. T. Vassilev
and T. K. Sawyer, Proc. Natl. Acad. Sci., 2013, 110, E3445-
E3454.
39.
40.
M. L. Stewart, E. Fire, A. E. Keating and L. D. Walensky, Nat.
Chem. Biol., 2010, 6, 595.
C. Phillips, L. R. Roberts, M. Schade, R. Bazin, A. Bent, N. L.
Davies, R. Moore, A. D. Pannifer, A. R. Pickford, S. H. Prior,
C. M. Read, A. Scott, D. G. Brown, B. Xu and S. L. Irving, J.
Am. Chem. Soc., 2011, 133, 9696-9699.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins