Converting disulfide bridges in native peptides to stable methylene thioacetals

Article abstract of DOI:10.1039/C6SC02285E

Disulfide bridges play a crucial role in defining and rigidifying the three-dimensional structure of peptides. However, disulfides are inherently unstable in reducing environments. Consequently, the development of strategies aiming to circumvent these deficiencies-ideally with little structural disturbance-are highly sought after. Herein, we report a simple protocol converting the disulfide bond of peptides into highly stable methylene thioacetal. The transformation occurs under mild, biocompatible conditions, enabling the conversion of unprotected native peptides into analogues with enhanced stability. The developed protocol is applicable to a range of peptides and selective in the presence of a multitude of potentially reactive functional groups. The thioacetal modification annihilates the reductive lability and increases the serum, pH and temperature stability of the important peptide hormone oxytocin. Moreover, it is shown that the biological activities for oxytocin are retained.

Full text of DOI:10.1039/C6SC02285E

View Article Online
View Journal
Chemical
Science
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C. M. B. K. Kourra
and N. Cramer, Chem. Sci., 2016, DOI: 10.1039/C6SC02285E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, 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 shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemicalscience

Please do not adjust margins
Chemical Science
Page 1 of 6
View Article Online
DOI: 10.1039/C6SC02285E
Journal Name
ARTICLE
Converting Disulfide Bridges in Native Peptides to Stable
Methylene Thioacetals
C. M. B.K. Kourraa and N. Cramera
Received 00th January 20xx,
Accepted 00th January 20xx
Disulfide bridges play a crucial role in defining and rigidifying the three‐dimensional structure of peptides. However,
disulfides are inherently unstable in reducing environments. Consequently, the development of strategies aiming to
circumvent these deficiencies ‐ ideally with little structural disturbance ‐ are highly sought after. Herein, we report a
simple protocol converting the disulfide bond of peptides into highly stable methylene thioacetal. The transformation
occurs under mild, biocompatible conditions, enabling the conversion of unprotected native peptides into analogues with
enhanced stability. The developed protocol is applicable to a range of peptides and selective in the presence of a
multitude of potentially reactive functional groups. The thioacetal modification annihilates the reductive lability and
increases the serum, pH and temperature stability of the important peptide hormone oxytocin. Moreover, it is shown that
the biological activities for Oxytocin are retained.
DOI: 10.1039/x0xx00000x
www.rsc.org/
activity. For instance, replacements of the disulfide group with a
lactam,10 thioether,11 selenium12 or dicarba13 analogues have been
Introduction
reported.5a Many of these methods require significant modification
Peptides have recently been enjoying a renewed interest in their
application as therapeutic agents.1 They provide a large chemical
space with a diverse array of molecular frameworks for the
development of novel therapeutics for a plethora of biomedical
applications. However, peptides exhibit inherent drawbacks as drug
candidates, due to their conformational flexibility and low
stability.1a,2 Several strategies have evolved to address these
unfavourable pharmacokinetics by increasing stability through local
or global constraints. This involves the incorporation of non‐natural
amino acids3 or amide bond surrogates,2,4 as well as various
cyclisation modes,5 side chain functionalisations6 and other
modifications.7,8 These approaches stabilise the secondary and
tertiary structure of the peptide by constraining it to a more
defined geometry. This increases protease resistance as the
vulnerable bonds are less easily accessible.
Disulfide bridges play a crucial role in defining and rigidifying
the three‐dimensional structure of natural peptides and proteins.
However, disulfides are inherently unstable in reducing
environments and towards nucleophiles. Reduction or scrambling
of the disulfide bonds is caused by transhydrogenases, thiol
oxidoreductases, disulfide isomerases and other thiol containing
agents, such as serum albumin or glutathione.9 All of them possess
the ability to decrease the lifespan of therapeutic disulfide
containing agents. Therefore, disulfide bond engineering emerged
as an important strategy to improve the metabolic stability of
disulfide‐containing peptides, whilst maintaining their biological
of the synthetic building blocks, at the initiation of the synthesis
towards the final peptide product, which can be problematic.11b,14 In
addition to the synthetic efforts of a de novo approach, these
modifications may introduce steric distortions that can negatively
impact activity and selectivity.15
Concept
Hence, we envisioned a disulfide stapling procedure performed
under mild, biocompatible reaction conditions (aqueous media, low
to ambient temperature and mild pH) that can be used directly with
unprotected native peptides. A key aim of our strategy was to insert
a minimal linker into the disulfide bond and maintain the structural
integrity necessary for the biological activity of the peptides. We
proposed to achieve this by the insertion of a methylene group in
between the two sulfur atoms, giving a thioacetal functional group
(Scheme 1). Thioacetals are utilised in organic synthesis as a rugged
protecting group for carbonyl groups. Chemically, they are inert
towards nucleophiles and most reductive conditions.
The thioacetal group has been sporadically previously used to
functionally rebridge disulfide bonds within peptide structures.16
However, these reports suffer from significant shortcomings by
using either strongly basic conditions, employing the linker reagent
as the solvent or organic and non‐biocompatible solvents.
Furthermore, these methods were not applied to unprotected
native peptides. Moreover, this method would also allow the
convenient introduction of radiolabeled methylene groups for
tracing or imaging purposes. In line with our aims for minimising
structural disturbance with the disulfide modification, CH2X2 (X= Cl,
Br or I) was selected as the smallest possible electrophilic linker.
This is complementary to the highly reactive multi‐bromo benzylic
linkers employed to invoke the cyclisation of prior non‐cyclised
a. Laboratory of Asymmetric Catalysis and Synthesis, Institute of Chemical Sciences
and Engineering, Ecole Polytechnique Fédérale de Lausanne, EPFL SB ISIC LCSA,
BCH 4305, CH‐1015 Lausanne, Switzerland.
Electronic Supplementary Information (ESI) available: Experimental procedures
and characterisation of all new compounds. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1‐3 | 1
Please do not adjust margins

Please do not adjust margins
Chemical Science
Page 2 of 6
ARTICLE
Journal Name
peptide segments due to the sterically larger size of this linker.17 For
our approach, one end of the reduced disulfide bridge will be
alkylated with the electrophilic reagent. A facile ionisation provides
a thiocarbenium ion18 of superior electrophilicity, leading to a rapid
cyclisation to the desired methylene thioacetal before any
additional reaction with another linker molecule would occur.
View Article Online
DOI: 10.1039/C6SC02285E
Scheme 2. Performing the mild one‐pot thioacetal transformation on
glutathione.
Application of the thioacetal modification to a scope of bioactive
peptides
With this simple and effective protocol we aimed to apply this
method to bioactive peptides and drugs, containing a plethora of
proteinogenic amino acids, to rigorously test the compatibility of
our approach (Scheme 3). The selected cyclic peptides have a broad
variety of relevant biological activities, which would allow a
comparative study of the native parent peptide to the modified
analogue. Firstly, we focused on cyclic octapeptide Octreotide as
more complex prototype substrate. Octreotide is pharmacologically
related to somatostatin and is a potent growth hormone inhibitor.21
It is an important drug marketed for the treatment of a variety of
hormone producing tumors.22 Application of the one‐pot methylene
insertion protocol provided the desired modified SCS‐Octreotide (1)
in a good yield of 78%. Importantly, the transformation remained
efficient under dilute conditions (5 mM) as well as a largely reduced
amount of the organic co‐solvent (5:1 ratio water/THF). Exposure of
octreotide to CH2I2 and NEt3 without prior disulfide reduction led to
full recovery. No chemical modifications occurred at any other sites,
highlighting the high chemoselectivity of the linker protocol. The
pharmacologically related natural tetradecapeptide hormone
Somatostatin (4),23 performed efficiently under the one‐pot
transformation to provide the thioacetal bridged analogue in 72%
yield. Next, neurohypophysial nonapeptide hormones Oxytocin
(OXT)24 and related Arginine Vasopressin (AVP),25 were targeted.
Oxytocin is marketed as a drug to instigate uterine contractions as
well to prevent post‐partum hemorrhage. Vasopressin constricts
the blood vessels to increase arterial blood pressure and regulates
the body’s retention of water. Both OXT and AVP smoothly
underwent the cut‐and‐sew methylene insertion procedure,
affording the desired SCS‐analogues 2 and 3 in high yields, 78% and
85% respectively. The antibacterial peptide Bactenecin26 was
chosen as a challenging substrate, due to the presence of multiple
nucleophilic guanidine groups of the arginine residues, that
potentially could react with the electrophilic linker reagent.
Nonetheless, SCS‐Bactenecin 4 was isolated in 53% yield without
the formation, of detectable side‐products.
Scheme 1. Thioacetal linkages by methylene insertion into disulfides for
enhanced stability.
Results and discussion
Investigation into a one‐pot biocompatible disulfide bond
modification
The initial assessment and optimisation of the disulfide to
thioacetal conversion, was conducted on N,N’‐Bis‐Boc L‐cystine
dimethyl ester as a simple model substrate (see SI). A completely
chemoselective thiol alkylation of the sulfhydryl group forming the
desired methylene thioacetal was observed, with no detectable side
products. Potentially interfering amino acids were added to the
reaction mixture and were innocent bystanders. Diiodomethane
was the most efficient linker reagent, although dibromomethane
and even dichloromethane reacted. Despite its higher reactivity, it
did not result in any undesired side products. A mild base is
required for the reaction to proceed as thiols themselves do not
react. The reaction performs well at ambient temperature. The
addition of an organic co‐solvent helps solubilise the linker reagent
and THF was favoured due to its miscibility with water and low
boiling point. The protocol was completed by a prior disulfide bond
reduction using water soluble tris‐(2‐carboxyethyl)phosphine (TCEP)
(Scheme 2).19 Pleasingly, CH2I2 was found to be compatible with
TCEP under the reaction conditions, even though it had been
reported to impede bioconjugations with maleimides and
α‐haloacyl groups.20 For instance, after the reduction of glutathione
disulfide (GSSG) to glutathione (GSH), addition of diiodomethane
and triethylamine provided the thioacetal modified version GSCSG
in 53% yield.
2 | J. Name., 2012, 00, 1‐3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 6
Chemical Science
Please do not adjust margins
Journal Name
ARTICLE
View Article Online
DOI: 10.1039/C6SC02285E
Scheme 4.Synthesis of double thioacetal modified SCSα‐Conotoxin MII (9)
Conditions: a) 1.5 eq. TCEP∙HCl, 3.0 eq. K2CO3, 10:1 H2O/THF, 23°C, 2 h, then 6.0
eq. CH2I2, 10 eq. NEt3, 23°C, 9 h
Stability enhancing thioacetal modification
One key goal of this thioacetal stapling technology was to
improve the stability of the modified peptides to various adverse
storage and metabolic conditions. The native disulfide bridge is
particularly labile under reductive environments and suffers from
scrambling reactions in the presence of free thiols. To verify the
anticipated reductive stability of the thioacetal modified peptides,
OXT and SCS‐OXT were incubated at 37°C in pH 7.0 PBS buffer
containing blood levels of L‐glutathione (1.0 mM).29 Under these
conditions, the half‐life of oxytocin is 8 hours (Fig. 1). In contrast,
SCS‐OXT is, as expected, completely stable and even after 5 days no
degradation was observed.
Scheme 3. Employing the thioacetal modification on a variety of cyclic bioactive
peptides. Conditions: a) 1.5 eq. TCEP∙HCl, 3.0 eq. K2CO3, 5:1 H2O/THF, 23°C, 2 h,
then 4.0 eq. CH2I2, 5.0 eq. NEt3, 23°C, 9 h; [b] 2.5 eq. CH2I2 used; [c] 1.5 eq.
TCEP∙HCl, 3.0 eq. K2CO3, 10:1 H2O/THF, 23°C, 2 h, then 10 eq. CH2I2, 15 eq. NEt3,
23°C, 12 h; [d] 8.0 eq. CH2I2, 12 eq. NEt3.
Besides the illustrated modification of unprotected native
peptides, the thioacetal introduction can also be embedded in
classical peptide synthesis. For instance, the neurotoxic bicyclic
hexadecapeptide α‐Conotoxin MII containing two critical disulfide
bridges is a potent and specific competitive antagonist of neuronal
nicotinic acetylcholine receptors.27 To prevent disulfide scrambling
and to ensure correct oxidative folding of α‐Conotoxin MII following
SPPS, an orthogonal 4‐monomethoxytrityl/acetamidomethyl
(Mmt/Acm) protecting group strategy was pursued (Scheme 4).28
The thioacetal group proved to be stable towards the acetic
acid/iodine deprotection‐cyclisation conditions in the formation of
8. The application of our thioacetal stapling protocol was
successfully performed twice, yielding the double modified
SCS2‐α‐Conotoxin MII in a good overall yield.
Fig. 1. Reductive stability of SCS‐OXT vs OXT (1.0 mM glutathione in pH 7.0 PBS
buffer at 37°C). Data points are displayed as the mean value ± SEM of three
independent experiments. Curves for the stability assays were fitted by
non‐linear, least squares regression analysis using Prism (GraphPad software
version 6.07, La Jolla, CA).
This confirmed the inherent chemical stability of a thioacetal
moiety against reduction. In further tests, we exposed the SCS
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
Chemical Science
Page 4 of 6
ARTICLE
Journal Name
analogues of OXT and AVP to human serum at 37°C (Table 1). For (neurotransmitter/ neuromodulator)31 were assessed. Notably,
View Article Online
both analogues, we found a significant improvement in the serum SCS‐AVP has a modulated selectivity prDoOfilIe: 10a.1t03t9h/Ce6SdCif0f2e2r8e5nEt
half‐lives. For SCS‐OXT the half‐life was more than doubled, receptors compared to AVP. SCS‐AVP displays similar binding
equating to an additional 40 hours. In addition, SCS‐AVP, displayed affinity (Ki = 0.77 nM) as the benchmark standard for the V1A
1
an approximately 1.5‐fold enhancement in the serum half‐life in receptor, [d(CH2)5 ,Tyr(Me)2]‐AVP (Ki = 0.74 nM).32 For the V2
relation to its parent peptide. OXT is included in the WHO’s list of receptor, SCS‐AVP exhibited
a slight decrease in affinity
essential medicines. A major concern is its thermal lability, (Ki = 4.52 nM vs AVP’s Ki = 1.22 nM). Concerning the V1B receptor,
particularly in countries where it is difficult to ensure proper SCS‐AVP binds weaker than the parent AVP (Ki = 0.87 nM vs AVP’s
refrigerated handling and storage, as stability studies indicate that Ki = 0.096 nM).
liquid oxytocin formulations rapidly degrade at temperatures
Besides binding affinities, the functional activity of the
greater than 30°C.30 Thus, OXT analogues with an enhanced thermal SCS‐analogues was also investigated. An in‐vitro uterine contraction
stability profile would be of significant value. Consequently, we assay yielded an EC50 = 4.90 nM for SCS‐OXT, thus remaining a full
undertook a comparative study to compile thermal and pH stability agonist with
a
potency of similar magnitude as OXT
data between SCS‐OXT and its unmodified parent peptide (Table 1). (EC50 = 1.20 nM).33 Prior investigations into the effect of changing
The rate of degradation of the peptides were compared by the ring size in native OXT demonstrated a significant decrease in
subjecting them to two different temperatures (40°C and 55°C) the agonist potency of the ring‐adjusted analogue by a factor of up
under four pH values (2.0, 4.5, 7.0 and 9.0). Native oxytocin is most to 500.34 The macrocyclic ring size modification in Oxytocin
stable under slightly acidic or neutral conditions.30a Overall, we previously involved the carbon framework of the cysteine residues
observed sizeable improvements in stability across the majority of (homocysteine) and not within the disulfide motif. However, our
temperature and pH conditions. The most significant gains in data suggests that increasing the size of Oxytocin’s 20‐membered
stability of the thioacetal modified analogue were obtained at the ring by insertion of the extra methylene group in between the two
most medically useful, physiological pH (7.0) increasing the half‐life sulfur atoms does not significantly reduce its agonist potency.
approximately 4 to 5‐fold. Even at the best storage pH 4.5, the Assaying the activation of the V1A receptor by SCS‐AVP resulted in
SCS‐OXT displays superior stability at 55°C. Harsher conditions (high an EC50 of 290 nM (EC50 = 0.71 nM for AVP) with both compounds
temperature and extremes of pH) were more destructive to both having essentially the same binding affinity (Ki values for SCS‐AVP:
peptides due to the relative ease of peptide bond cleavage under 0.77 nM, AVP 0.74 nM). Consequently, the thioacetal modification
such conditions. Nevertheless, at 55°C and pH 9.0, our SCS‐OXT transforms SCS‐AVP to a partial agonist at the V1A receptor. Overall,
showed a 3.3‐fold increase in half‐life.
the modified compounds SCS‐OXT and SCS‐AVP remain biologically
active. The thioacetal bridge can alter the selectivity profile of the
parent peptides at the different receptors and has the potential to
modulate their ligand efficacy.
Table 1. Serum, temperature and pH‐stability of OXT and SCS‐OXT. [a]
OXT half-life [d]
SCS-OXT half-life [d]
Table 2. Binding Affinity and Functional Activity of OXT, SCS‐OXT, AVP and SCS AVP. [a,b]
Entry
pH
40°C
55°C
-
40°C
55°C
-
Entry Receptor
SCS-OXT
1.41 ±0.3
OXT
0.24 ±0.04
1.2033
SCS-AVP
AVP
1631
-
1
2
3
4
Serum
2.0
1.2 ±0.2[b]
14 ±0.9
3.0 ±0.1[b]
27 ±0.4
133 ±7.0
34 ±1.2
8.6 ±0.8
1
2
OXTR Ki [nM]
41 ±10
-
3.2 ±0.4
18 ±1.3
2.0 ±0.2
0.9 ±0.15
3.6 ±0.4
32 ±1.7
8.0 ±0.4
3.0 ±0.2
OXTR EC50 [nM]
V1AR Ki [nM]
V1AR EC50 [nM]
V2R Ki [nM]
4.90
±0.34
4.5
121 ±6.2
7.2 ±0.5
3.9 ±0.5
7.0
3
4
5
6
15 ±3.5
6431
0.77
±0.23
0.74
±0.14
5
9.0
[a]
All data are displayed as the mean value ± SEM (calculated by Prism: GraphPad
software version 6.07, La Jolla, CA), of three independent experiments for each pH
and temperature variable, and five for the Oxytocin serum half‐life assay. [b] at 37°C.
-
-
-
-
-
-
290 ±20
0.71
±0.10
4.52
±0.52
1.22
±0.31
Evaluation of the biological activities of the thioacetal modified
peptides
Although the introduction of the methylene bridge is the
smallest possible reinforcement of a disulfide, it involves a minor
structural disturbance. The length and the directionality of the
disulfide and the methylene thioacetal are slightly different. The
impact of such change might level out with the increasing size of
the peptide and the length of the loop. We have therefore assessed
the binding affinities of the modified OXT and AVP peptides (Table
2). For the Oxytocin receptor (OXTR), SCS‐OXT demonstrated a
slight decrease in binding affinity, (Ki = 1.41 nM) compared to OXT
(Ki = 0.24 nM). For SCS‐AVP, the vasopressin receptors V1AR
(vasopressor activity), V2R (water retention) and V1BR
V1BR Ki [nM]
0.87
±0.24
0.10
±0.04
[a]
[b]
For each assay, five concentrations were performed in duplicate. IC50 (or
EC50) values were determined by a non‐linear, least squares regression analysis
using MathIQTM (ID Business Solutions Ltd., UK) by Eurofins Pharma Discovery
Services. Inhibition constants (Ki) were calculated with the Cheng and Prusoff
equation using the observed IC50 of the tested compound, the concentration of
radioligand employed in the assay, and the historical values for the KD of the
ligand.
Conclusions
4 | J. Name., 2012, 00, 1‐3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 6
Chemical Science
Please do not adjust margins
Journal Name
ARTICLE
4. a) C. Proulx, D. Sabatino, R. Hopewell, J. Spiegel, Y. García‐
In conclusion, we have developed an experimentally facile, one‐pot
transformation reinforcing disulfide linkages in peptides as
thioacetals. No synthetic incorporation of engineered amino acids is
needed for this stapling process. The protocol operates under mild,
bio‐compatible conditions (aqueous solution, mild pH and at
ambient temperature) and is tolerant towards unprotected amino
acids possessing a variety of functional groups. Furthermore, the
high site specificity for the insertion into disulfide bonds further
enhanced the reactions utility and versatility. The methylene
thioacetal bridge completely eliminates the reductive liability of the
disulfide group, contributing to an enhanced structural stability. We
have exemplarily shown for Oxytocin that the thioacetal analogue
exhibits superior stability while maintaining binding affinities and
functional activities of the same magnitude. This methodology is an
additional tool for stapling peptides using natural linking points with
potential impact for the medicinal application of bioactive peptides.
View Article Online
Ramos and W. D. Lubell, Future Med. Chem. 2011, 3, 1139‐1164;
DOI: 10.1039/C6SC02285E
b) J. Vagner, H. Qu and V. J. Hruby, Curr. Opin. Chem. Biol. 2008,
12, 292‐296; c) I. E. Valverde, A. Bauman, C. A. Kluba, S.
Vomstein, M. A. Walter and T. L. Mindt, Angew. Chem. Int. Ed.
2013, 52, 8957‐8960.
5. a) Y. H. Lau, P. de Andrade, Y. Wu and D. R. Spring, Chem.
Soc. Rev. 2015, 44, 91‐102; b) C. J. White and A. K. Yudin, Nature
Chem. 2011, 3, 509‐524.
6. a) H. Jo, N. Meinhardt, Y. Wu, S. Kulkarni, X. Hu, K. E. Low, P.
L. Davies, W. F. DeGrado and D. C. Greenbaum, J. Am. Chem.
Soc. 2012, 134, 17704‐17713; b) A. M. Spokoyny, Y. Zou, J. J.
Ling, H. Yu, Y.‐S. Lin and B. L. Pentelute, J. Am. Chem. Soc. 2013,
135, 5946‐5949.
7. S. Shaunak, A. Godwin, J.‐W. Choi, S. Balan, E. Pedone, D.
Vijayarangam, S. Heidelberger, I. Teo, M. Zloh and S. Brocchini,
Nat. Chem. Biol. 2006, 2, 312‐313.
8. a) C. E. Schafmeister, J. Po and G. L. Verdine, J. Am. Chem.
Soc. 2000, 122, 5891‐5892; b) G. L. Verdine and G. J. Hilinksi,
Methods Enzymol. 2012, 503, 3‐33; c) L. D. Walensky, A. L. Kung,
I. Escher, T. J. Malia, S. Barbuto, R. D. Wright, G. Wagner, G. L.
Verdine and S. J. Korsmeyer, Science 2004, 305, 1466‐1470.
9. a) A. Holmgren and M. Bjornstedt, in Methods Enzymol.,
Academic Press, 1995, vol. 252, pp. 199‐208; b) G. Powis and W.
R. Montfort, Annu. Rev. Pharmacol. Toxicol. 2001, 41, 261‐295.
10. C. W. Smith, R. Walter, S. Moore, R. C. Makofske and J.
Meienhofer, J. Med. Chem. 1978, 21, 117‐120.
Acknowledgements
This work is supported by the Swiss National Centre of
Competence in Research Chemical Biology. We gratefully
acknowledge C. Heinis for the use of the peptide synthesiser in
his laboratory and K. Johnsson for helpful discussions.
11. a) N. Assem, D. J. Ferreira, D. W. Wolan and P. E. Dawson,
Angew. Chem. Int. Ed. 2015, 54, 8665‐8668; b) Z. Dekan, I.
Vetter, N. L. Daly, D. J. Craik, R. J. Lewis and P. F. Alewood, J. Am.
Chem. Soc. 2011, 133, 15866‐15869; c) G. Ösapay, L. Prokai, H.‐S.
Kim, K. F. Medzihradszky, D. H. Coy, G. Liapakis, T. Reisine, G.
Melacini, Q. Zhu, S. H. H. Wang, R.‐H. Mattern and M. Goodman,
J. Med. Chem. 1997, 40, 2241‐2251.
Notes and references
General procedure for the synthesis of SCS‐OXT. To a round
bottom flask containing a stirrer bar was added 1.00 eq. of Oxytocin
acetate powder (3.20 mg, 3.20 µmol) and H2O (3.60 mM). A
solution of TCEP∙HCl (1.50 eq.) and K2CO3 (3.00 eq.) in H2O
(29.0 mM) was added via syringe. The reaction was stirred at room
temperature for 1.25 hours. The progress of the reaction was
monitored by HPLC‐MS. Upon completion of this first step of the
process, triethylamine (5.00 eq.) in THF (0.30 M) and then
diiodomethane (4.00 eq.) in THF (0.25 M) were added sequentially
to the reaction mixture via micro‐syringe. The reaction mixture was
stirred at room temperature for 6.5 hours. The progress of the
reaction was monitored by HPLC‐MS. Once adjudged complete, the
reaction mixture was diluted with H2O (5.00 mL) and lyophilised.
Then, the crude peptide was purified by RP preparative HPLC, using
a linear gradient of 5% B to 50% B over 30 minutes at a flow rate of
25.0 mL/min with UV detection at 220 nm, 254 nm and 280 nm.
(Solvent A = 99.9% v/v H2O and 0.1% v/v formic acid; Solvent B =
95% v/v MeCN: 5% v/v H2O and 0.1% v/v formic acid). The pure
product was isolated in 78% yield (2.50 mg) as a fluffy white
powder.
12. a) A. D. de Araujo, M. Mobli, J. Castro, A. M. Harrington, I.
Vetter, Z. Dekan, M. Muttenthaler, J. Wan, R. J. Lewis, G. F. King,
S. M. Brierley and P. F. Alewood, Nat. Commun. 2014,
5; b) H.
Vogt and S. Brase, Org. Biomol. Chem. 2007, , 406‐430; c) M.
5
Muttenthaler, A. Andersson, A. D. de Araujo, Z. Dekan, R. J.
Lewis and P. F. Alewood, J. Med. Chem. 2010, 53, 8585‐8596; d)
R. Walter and V. du Vigneaud, J. Am. Chem. Soc. 1966, 88, 1331‐
1332.
13. a) R. F. Nutt, D. F. Veber and R. Saperstein, J. Am. Chem. Soc.
1980, 102, 6539‐6545; b) J. L. Stymiest, B. F. Mitchell, S. Wong
and J. C. Vederas, Org. Lett. 2003,
14. S. Brocchini, S. Balan, A. Godwin, J.‐W. Choi, M. Zloh and S.
Shaunak, Nat. Protoc. 2006, , 2241‐2252.
5, 47‐49.
1
15. a) R. J. Clark, H. Fischer, L. Dempster, N. L. Daly, K. J.
Rosengren, S. T. Nevin, F. A. Meunier, D. J. Adams and D. J. Craik,
Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 13767‐13772; b) J. A.
Ember, S. D. Sanderson, S. M. Taylor, M. Kawahara and T. E.
Hugli, J. Immunol. 1992, 148, 3165‐3173.
16. a) S. Lindman, G. Lindeberg, A. Gogoll, F. Nyberg, A. Karlén
and A. Hallberg, Bioorg. Med. Chem. 2001, 9, 763‐772; b) H. I.
Mosberg and J. R. Omnaas, J. Am. Chem. Soc. 1985, 107, 2986‐
2987; c) H. I. Mosberg, J. R. Omnaas and A. Goldstein, Mol.
Pharmacol. 1987, 31, 599‐602; d) M. Ueki, T. Ikeo, K. Hokari, K.
Nakamura, A. Saeki and H. Komatsu, Bull. Chem. Soc. Jpn. 1999,
72, 829‐838; e) M. Ueki, T. Ikeo, M. Iwadate, T. Asakura, M. P.
1. a) A. K. Sato, M. Viswanathan, R. B. Kent and C. R. Wood,
Curr. Opin. Biotechnol. 2006, 17, 638‐642; b) D. Goodwin, P.
Simerska and I. Toth, Curr. Med. Chem. 2012, 19, 4451‐4461; c)
P. Vlieghe, V. Lisowski, J. Martinez and M. Khrestchatisky, Drug
Discov. Today 2010, 15, 40‐56.
2. M. Góngora‐Benítez, J. Tulla‐Puche and F. Albericio, Chem.
Rev. 2014, 114, 901‐926.
3. a) R. P. Cheng, S. H. Gellman and W. F. DeGrado, Chem. Rev.
2001, 101, 3219‐3232; b) S. Y. Hong, J. E. Oh and K.‐H. Lee,
Biochem. Pharmacol. 1999, 58, 1775‐1780; c) M. Tanaka, Chem.
Pharm. Bull. 2007, 55, 349‐358; d) M. T. Dohm, R. Kapoor and A.
E. Barron, Curr. Pharm. Des. 2011, 17, 2732‐2747; e) R.
Lemmens‐Gruber, M. R. Kamyar and R. Dornetshuber, Curr.
Med. Chem. 2009, 16, 1122‐1137.
Williamson and J. Slaninova, Bioorg. Med. Chem. Lett. 1999,
1767‐1772.
9,
17. a) S. Chen, J. Morales‐Sanfrutos, A. Angelini, B. Cutting and C.
Heinis, ChemBioChem 2012, 13, 1032‐1038; b) C. Heinis, T.
Rutherford, S. Freund and G. Winter, Nat. Chem. Biol. 2009, 5,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1‐3 | 5
Please do not adjust margins

Please do not adjust margins
Chemical Science
Page 6 of 6
ARTICLE
Journal Name
502‐507; c) P. Timmerman, J. Beld, W. C. Puijk and R. H. Meloen,
ChemBioChem 2005, , 821‐824.
View Article Online
DOI: 10.1039/C6SC02285E
6
18. Y. Apeloig and M. Karni, J. Chem. Soc., Perkin Trans. 2 1988,
625‐636.
19. J. A. Burns, J. C. Butler, J. Moran and G. M. Whitesides, J.
Org. Chem. 1991, 56, 2648‐2650.
20. D. E. Shafer, J. K. Inman and A. Lees, Anal. Biochem. 2000,
282, 161‐164.
21. W. Bauer, U. Briner, W. Doepfner, R. Haller, R. Huguenin, P.
Marbach, T. J. Petcher and J. Pless, Life Sci. 1982, 31, 1133‐1140.
22. a) S. W. J. Lamberts, E. P. Krenning and J.‐C. Reubi, Endocr.
Rev. 1991, 12, 450‐482; b) G. Ösapay and K. Ösapay, Expert Opin.
Ther. Pat. 1998,
8, 855‐870; c) L. Degen and C. Beglinger,
Digestion 1999, 60(suppl 2), 9‐14.
23. P. Brazeau, W. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier
and R. Guillemin, Science 1973, 179, 77‐79.
24. a) H.‐J. Lee, A. H. Macbeth, J. H. Pagani and W. S. Young 3rd,
Prog. Neurobiol. 2009, 88, 127‐151; b) V. du Vigneaud, C.
Ressler, C. J. M. Swan, C. W. Roberts, P. G. Katsoyannis and S.
Gordon, J. Am. Chem. Soc. 1953, 75, 4879‐4880.
25. a) Z. R. Donaldson and L. J. Young, Science 2008, 322, 900‐
904; b) V. du Vigneaud, H. C. Lawler and E. A. Popenoe, J. Am.
Chem. Soc. 1953, 75, 4880‐4881.
26. D. Romeo, B. Skerlavaj, M. Bolognesi and R. Gennaro, J. Biol.
Chem. 1988, 263, 9573‐9575.
27. a) G. E. Cartier, D. Yoshikami, W. R. Gray, S. Luo, B. M.
Olivera and J. M. McIntosh, J. Biol. Chem. 1996, 271, 7522‐7528;
b) S. C. Harvey, J. M. Mcintosh, G. E. Cartier, F. N. Maddox and C.
W. Luetje, Mol. Pharmacol. 1997, 51, 336‐342.
28. a) K. B. Akondi, M. Muttenthaler, S. Dutertre, Q. Kaas, D. J.
Craik, R. J. Lewis and P. F. Alewood, Chem. Rev. 2014, 114, 5815‐
5847; b) M. C. Munson and G. Barany, J. Am. Chem. Soc. 1993,
115, 10203‐10210.
29. F. Michelet, R. Gueguen, P. Leroy, M. Wellman, A. Nicolas
and G. Siest, Clin. Chem. 1995, 41, 1509‐1517.
30. a) A. Hawe, R. Poole, S. Romeijn, P. Kasper, R. van der
Heijden and W. Jiskoot, Pharm. Res. 2009, 26, 1679‐1688; b) H.
V. Hogerzeil, G. J. A. Walker and M. J. de Goeje, WHO Action
Programme on Essential Drugs and Vaccines 1993,
WHO/DAP/93.6
.
31. M. Thibonnier, D. M. Conarty, J. A. Preston, P. L. Wilkins, L.
N. Berti‐Mattera and R. Mattera, in Oxytocin and Vasopressin:
Molecular, Cellular, and Clinical Advances, ed. H. H. Zingg,
Bourque, Charles W., Bichet, Daniel G., Plenum Press, New York,
1998, vol. 449, ch. 34, pp. 251‐276.
32. M. Kruszynski, B. Lammek, M. Manning, J. Seto, J. Haldar and
W. H. Sawyer, J. Med. Chem. 1980, 23, 364‐368.
33. Standard reference value for this assay as determined by
Eurofins Pharma Discovery Services
34. a) D. Jarvis, M. Bodanszky and V. Du Vigneaud, J. Am. Chem.
Soc. 1961, 83, 4780‐4784; b) C. W. Smith and M. F. Ferger, J.
Med. Chem. 1976, 19, 250‐254.
6 | J. Name., 2012, 00, 1‐3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins