Chemical synthesis and biological activity of peptides incorporating an ether bridge as a surrogate for a disulfide bond

Article abstract of DOI:10.1039/d0sc02374d

Disulfide bridges contribute to the definition and rigidity of polypeptides, but they are inherently unstable in reducing environments and in the presence of isomerases and nucleophiles. Strategies to address these deficiencies, ideally without significan

Full text of DOI:10.1039/d0sc02374d

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: R. zhao, P. Shi, J.
Chen, S. sun, J. chen, J. cui, F. Wu, G. Fang, C. Tian, J. Shi, D. Bierer, L. Liu and Y. Li, Chem. Sci., 2020,
DOI: 10.1039/D0SC02374D.
Volume
Number
9
1
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
7
January 2018
Pages 1-268
Chemical
Science
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.
rsc.li/chemical-science
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.
ISSN 2041-6539
EDGE ARTICLE
Xinjing Tang et al.
Caged circular siRNAs for photomodulation of gene
expression in cells and mice
rsc.li/chemical-science

View Article Online
DOI: 10.1039/D0SC02374D
Page 1 of 7
Chemical Science
Journal Name
RSCPublishing
EDGE ARTICAL
Chemical synthesis and biological activity of peptides
incorporating an ether bridge as a surrogate for a
disulfide bond
,
Cite this: DOI:
10.1039/x0xx00000x
Rui Zhao,a,b+ Pan Shi,a+ Junyou Chen, Shuaishuai Sun, Jingnan Chen, Jibin Cui, Fangming
b+
b
b
b
f
e
a
a*
d
c
b*
Wu, Gemin Fang, Changlin Tian, Jing Shi, Donald Bierer, Lei Liu, Yi-Ming Li
Received 00th January 2012,
Accepted 00th January 2012
Disulfide bridges contribute to the definition and rigidity of polypeptides, but they are inherently unstable in
reducing environments and in the presence of isomerases and nucleophiles. Strategies to address these
deficiencies, ideally without significantly perturbing the structure of the polypeptide, would be of great
interest. One possible surrogate for the disulfide bridge is a simple thioether, but these are susceptible to
oxidation. We report the introduction of an ether linkage into the biologically active, disulfide-rich peptides
oxytocin, tachyplesin I, and conotoxin -ImI, using an ether-containing diaminodiacid as the key building
block, obtained by the stereoselective ring-opening addition reaction of an aziridine skeleton with a hydroxy
group. NMR studies indicated that the derivatives with an ether surrogate bridge exhibited very small change
of their three-dimensional structures. The analogs obtained using this novel substitution strategy were found
to be more stable than the original peptide in oxidative and reductive conditions; without a loss of bioactivity.
This strategy is therefore proposed as a practical and versatile solution to the stability problems associated
with cysteine-rich peptides.
DOI: 10.1039/x0xx00000x
www.rsc.org/
of peptides, changing their overall shape, properties, and
activity. For example, some triazole-containing insulin analogs
Introduction
8
were poorly active due to structural distortions introduced into
Disulfide-rich polypeptides have emerged as promising drug
candidates due to their excellent metabolic stability, potent
activity and high target selectivity. These favorable
8
a
their backbone by the triazole moiety. Furthermore, although
thioether and selenoether bridges are similar in structure and
reactivity to the native disulfides, they are also prone to
oxidation, which can be problematic – some thioether-substituted
conotoxin -ImI derivatives have previously been revealed to be
1
characteristics are in part due to the disulfide bonds themselves,
which support the definition of the structure and influence the
topological orientation of the pharmacophore, thereby imparting
4
a
easily oxidized to sulfoxide, leading to a loss of activity.
2
stability and selectivity to the peptides. However, disulfide
Another possible disulfide bridge isostere is an ether bridge,
which in principle has several advantages over those previously
mentioned. For example, ether bridges are inherently more stable
than thioether bridges, are expected to be readily accommodated
by the structure of the peptide, being neither rigid nor planar, and
not susceptible to racemization during solid phase peptide
synthesis (SPPS). However, the formation of ether bonds is
difficult, especially in the context of side chain cyclization, and
usually requires forcing conditions not compatible with delicate
bonds are inherently unstable in reducing environments and in
the presence of isomerases and nucleophiles, resulting in
structural rearrangement of the entire peptide and a complete loss
of biological activity. Accordingly, the replacement of this basic
structural element with a more stable, non-reducing isostere
3
4
5
6
would be highly desirable; and thioether, selenoether, triazole,
7
and hydrocarbon bridge surrogates have all been proposed. In
practice, however, drawbacks are associated with all of these
disulfide bond surrogates. First, the rigidity and planarity of
triazole and dicarba (olefin or hydrocarbon) bridges generally
precludes their proper accommodation into the parent structures
9
peptide functionality.
Herein, we disclose a new and practical strategy for the
synthesis of cysteine-rich peptides incorporating an ether linkage
a. Hefei National Laboratory of Physical Sciences at Microscale, Department of as a disulfide-bond mimic using an orthogonally protected, ether-
Chemistry and School of Life Sciences, University of Science and Technology of
containing diaminodiacid, obtained by the stereoselective ring-
China, Hefei, Anhui 230009, China, E-mail: shijing@ustc.edu.cn;
b.
10
School of Food and Biological Engineering, Hefei University of Technology, Hefei, opening addition reaction of an aziridine with a hydroxy group.
Anhui 230009, China, E-mail: ymli@hfut.edu.cn;
Department of Chemistry, Tsinghua University, Beijing 100084, China
The utility of this strategy was exemplified in syntheses of the
Department of Medicinal Chemistry, Bayer AG, Aprather Weg 18A, 42096 Wuppertal, ether analogs of oxytocin; the bacteriostatic peptide tachyplesin I
c.
d.
Germany
(TPI-1), which was shown to be more stable than that its wild-
e
School of Life Science, Institute of Physical Science and Information Technology,
Anhui University, Hefei 230601, China
High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, China;
;
type (WT) counterpart under reducing conditions but just as
bioactive; and an ether-substituted conotoxin -ImI analog,
f
+
These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

View Article Online
DOI: 10.1039/D0SC02374D
Chemical Science
Page 2 of 7
ARTICLE
Journal Name
which was significantly more stable in oxidative environments position, we chose the electron-withdrawing p-
1
1
compared to conotoxin -ImI analogs incorporating thioether nitrobenzyloxycarbonyl group (pNZ) for nitrogen protection.
bonds.
After removal of the Trt group, pNZ-Cl was added to obtain
segment 1c. We further obtained Fmoc-homoserine-tBu 2a as
previously reported,4j and then successfully constructed the
key intermediate 3a containing an ether bond by reacting 2a
a. previous work:
O
R'
O
R'
R
O
R
O
Labile:
Reduction&
Nucleophiles
N
H
O
Labile:
oxidation&
isomerization
N
H
O
HN
3 2
and 1c in the presence of catalytic BF •OEt (yield 30%).
HN
HN
HN
O
HN
HN
O
Finally, the ether-containing diaminodiacid product 4a was
obtained by removing the tBu group with TFA (overall yield
3.1%, 7 steps from L-serine) (Scheme 2). Because the ether
bond is slightly shorter than the disulfide bond, we further
prepared a side-chain extended ether-containing diaminodiacid
S
S
S
thioether bond mimic
disulfide-rich peptides
b. this work:
O
R'
O
R'
4b by coupling Fmoc-homohomoserine-tBu (2b) with 1c, to
R
O
R
O
N
H
O
O
Harsh
reaction
N
H
O
O
No Reduction
oxidation &
isomerization
test the influence of linkage length on peptide bio-activity
(Scheme S5).
HN
HN
HN
HN
HN
HN
side chain cyclization
O
X
ONa
R'
HO
ether bond mimic
MS-Cl,Et N
3
TFA
pNZ-Cl
Trt
O
Trt
O
PNZ
O
N
Allyl
SPPS
N
Allyl
N
Allyl
O
H
2
4 h
O
H
O
O
1b
O
R
O
O
R'
PG1
N
N
H
1a
1c
O
O
PG2
R
O
NH2
O
HN
PG removal
1
N
H
O
O
O
cyclization
HN
O
HN
PG1
PNZ
O
O
O
O
diaminodiacid strategy
O
Fmoc
OH
N
Allyl
H
N
H
N
HN
HN
PG2
Allyl
Allyl
N
H
O
O
O
PNZ
O
O
PNZ
OH
PG2
1c
BF OEt
O
TFA
6 h
O
3
2
OH
Scheme 1 Diaminodiacid strategy enabled the development of the first
example of cysteine-rich peptide exploiting the ether-bond as disulfide-bond
mimic. These ether-containing peptide mimics exhibit significant stability to
both reduction and oxidation conditions.
CHCl , 40 °C
3
Fmoc
Fmoc
N
tBu
N
H
Fmoc
O
N
H
tBu
H
O
O
O
2
a
3a (30% yield)
4a (95% yield)
Scheme 2 Chemical synthesis of ether-containing diaminodiacids 4a
Results and Discussion
With ether-containing diaminodiacids in hand, we first tested
their compatibility with Fmoc-based SPPS by synthesizing ether-
containing analogs of oxytocin, a peptide hormone with a
plethora of both physiological and psychological biological
functions. The synthesis of the ether-containing analog of
oxytocin was attempted first. Gly, Leu and Pro were
Although ether bridge possibly has advantages over
previously mentioned surrogates, the replacement of a disulfide
moiety with an ether is not straightforward. The major reason
for this is that whereas a disulfide bridge can be readily
formed by the gentle oxidation of two proximal thiols, the
reaction conditions used to construct ether bonds are
relatively harsh; for example, the Williamson ether synthesis
requires high temperatures, strongly basic sodium alkoxides,
and prolonged reaction times.9 To avoid having to expose a
fully-formed peptide to such forcing conditions, we
envisioned pre-preparation of a diaminodiacid unit containing
an ether bond, followed by its incorporation into a polypeptide
by conventional SPPS. We expected that under Lewis acid
catalysis, selective β-position ring-opening addition of
azacyclopropane derivatives through the side chain hydroxyl
groups of homoserine derivatives will construct the key
12
sequentially coupled to Rink Amide AM resin to give 5. Then,
4a was condensed to the ɑ-amino of the pendant Pro of 5 to
yield 6. Finally, the Tyr-Ile-Gln-Asn tetrapeptide was added
on to the 4a moiety of 6. After removal of the allyl protecting
group, the side-chain carboxyl group of 4a and N-terminal
amino group of Tyr were efficiently cyclized using
PyAOP/NMM. Then, the pNZ group was removed and finally
the oxytocin analog 9 was obtained after cleavage from the
resin using TFA (Fig. 1a). HPLC analysis of the purified
products gave rise to a single peak, and 10 was also identified
by LC-MS as an oxytocin derivative containing an ether bond
10
skeleton of the ether-containing diaminodiacid. Vederas et al.
used a similar reaction to a synthesize a natural product DAP
analog which was crucial for bacterial cell wall layer
(Fig. 1b), HPLC analysis of purified 10 showed a single
product peak, and it was identified by LC-MS as an oxytocin
derivative containing an ether bond (Fig. 1b), proving that the
new diaminodiacid can be readily used in SPPS. The circular
dichroism (CD) spectrum confirmed that 10 had a secondary
1
0d
formation
.
First, we synthesized aziridine derivative 1b by cyclization
of 1a using methanesulfonyl chloride and triethylamine. To
activate the aziridine for regioselective ring opening at the β-
structure, very similar to that of its native counterpart (Fig.
4e
1
c). To verify the three-dimensional structure of diaminodiacid-
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/D0SC02374D
Page 3 of 7
Journal Name
Chemical Science
ARTICLE
replaced analogue, solution NMR experiments including DQF- are sensitive to the nature of the disulfide bond surrogates.
COSY, TOCSY, NOESY and ROESY were carried out to For example, when the Cys7-Cys12 disulfide bond was
determine the structure of 10. With a total of 52 NOE distance replaced with a thioether bond, the bio-activity of the
restraints (including 3 long-range NOEs) and 6 dihedral resulting TPI-1 analog was significantly reduced compared to
4
b
restraints, 20 lowest energy structures selected from 100 TPI-1. Our synthesis of a TPI-1 derivative incorporating an
calculated structures were observed with an all heavy atom ether bond (TPI-2) in place of the Cys7-Cys12 disulfide bond
RMSD (residue 1-8) of 1.476 Å (Fig. 1d and Supplementary is depicted in Fig. 2a. A linear peptide 11 containing 4a was
Table S1). The ROESY cross peaks and NMR structure first assembled on the resin. Removal of the allyl and Fmoc
showed that 10 incorporated the correct Cys1-Cys6 ether groups of 11 gave peptide 12. Cyclization using PyAOP and
bond connectivity (Fig. 1d). The overall solution NMR NMM followed by removal of the pNZ protecting group gave
structure of 10 is a turning loop structure, almost identical to 13. After condensation of the hexapeptide Lys-Trp-Cys-Phe-
the native oxytocin. In our proton assignment, similar Arg-Val with 13, the resulting peptide was cleaved from the
chemical shifts for Cα-H atoms were observed for native resin and purified by HPLC to give the full-length peptide 14,
4
h
Oxytocin and 10, except for the replaced Cys residues (Fig. which was refolded under neutral conditions (33% acetonitrile,
S11), thus indicating that replacement of the Cys1-Cys6 1:1 GSSG/GSH, pH 7.5). HPLC and ESI-MS analysis
disulfide bond with an ether bond had little influence on the demonstrated the purity and correct molecular weight of the
tertiary structure of Oxytocin.
ether bond substituted TPI-2 (Fig. 2b). For comparison,
wildtype (WT) TPI-1 and TPI-3 incorporating a side-chain
containing an extra methylene group were obtained under the
same conditions. Using previous reported thioether-containing
diaminodiacids, we further synthesized two other TPI-1
derivatives incorporating thioether linkages of different
lengths (TPI-4 and TPI-5) (Fig. 2d). The CD spectra of TPI-
O
H
O
NH2
H
N
a.
Allyl
N
Allyl
O
PNZ
O
PNZ
Tyr
SPPS Ile
Gln
Allyl
H2N
O
O
Pro
Leu
removal
4
a
H
N
H
N
Fmoc
N
Pro
Asn
N
H
Pro
Leu
Resin Gly
H
O
O
5
Leu
Resin Gly
Resin Gly
1
and its ether bond analogs were measured; WT TPI-1 was
6
7
O
O
O
NH2
H
N
found to give rise to a positive peak at 196 nm and a negative
peak at 218 nm, while the spectra of its ether bond analogs
showed a weak negative absorption at 218 nm (Fig. 2c). This
suggests the β-sheet structure of TPI-2 and TPI-3 to be less
ordered than that of TPI-1, consistent with the previous
NH2
NH2
HO
PNZ
N
N
Tyr
Tyr
Tyr
Ile
Gln
cyclization
H
H
O
O
TFA
Ile
O
Ile
PNZ removal
H
N
H
N
H
N
Gln
Asn
Gln
Asn
N
Pro
N
Pro
Leu
Resin Gly
Asn
N
Pro
Leu
H
H
H
O
O
O
Leu
Resin Gly
4
b
Gly
results of thioether derivatives. We further conducted solution
NMR spectroscopy (including DQF-COSY, TOCSY, NOESY,
H2NCO
8
9
10
1
3
15
c.
C-HSQC and N-HSQC) for TPI-2. The determined solution
b.
5
0
structures revealed that a total of 136 NOE distance restraints
(including 8 long-range NOEs) and 22 dihedral restraints, 20
lowest energy structures selected from 100 calculated structures
were observed with an all heavy atom RMSD (residue 5-14) of
Obs: 973.5Da
Calc: 973.7 Da
-
5
1
.317 Å (Supplementary Table S2 and Fig. S15). The NOESY
cross peaks and NMR structure showed that TPI-2 incorporated
the correct Cys7-Cys12 ether bond connectivity (Fig. 2d)
-
10
Oxytocin analog 10
Native Oxytocin
.
-15
2
1
22
23
24
210
230
250
Furthermore, we also observed the similar chemical shifts for
Retention time (min)
wavelength/nm
Cα-H of TPI-2 (The residue Lys1 subject to fast protein
exchange) compared with native TPI-1 (Fig. S13), except for
d.
4b
those corresponding to the two replaced Cys residues, thus
confirming that TPI-2 maintained a three dimensional structure
similar to the TPI-1.
1O
6CH
2
Antimicrobial assays were then used to ascertain the
biological activity of the TPI analogs. Three bacterial strains,
including two Gram-positive bacteria (Bacillus subtilis and
Staphylococcus epidermidis) and one Gram-negative bacteria
Figure 1. a. Synthetic route for the preparation of oxytocin analog using
diaminodiacid 4a. b. HPLC traces and ESI-MS analysis of purified oxytocin
analog. c. CD spectra of oxytocin analogue in water. d. Backbone ensembles (Escherichia coli) were used. The three strains were cultured
of 20 lowest-energy NMR structures of oxytocin analog 10 (left) and a ribbon
representation of 10 (right). The C-O bridge is shown in red.
8
o
to 10 CFU at 37 C using LB medium; and the minimal
inhibitory concentration (MIC) of the TPI derivatives tested
1
4
using the standard two-fold dilution method.
Each
Next, our attention turned to tachyplesin I (TPI-1). TPI-1
adopts a β-sheet conformation stabilized by two cross-linked
experiment was repeated three times. The ether-containing
TPI-2 and TPI-3 had identical antibacterial activity to WT
TPI-1, exerting significant antibacterial activity against both
1
3
disulfide bonds, and the folding and activity of its analogs
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

View Article Online
DOI: 10.1039/D0SC02374D
Chemical Science
Page 4 of 7
ARTICLE
Journal Name
Gram-positive and Gram-negative bacteria at low Gram-positive and Gram-negative bacteria (Table 1).
concentrations. TPI-4 had a slightly inferior MIC value for Collectively, these results indicate that ether bond substitution
Bacillus subtilis compared to TPI-1, whereas the antibacterial may exert less of an effect on the activity of disulfide-rich
activity of TPI-5 incorporating a side-chain containing an polypeptides than thioether bonds, especially in peptides
extended thioether bond was dramatically decreased, for both sensitive to structural alterations such as TPI-1.
a.
H2N Lys Trp
Cys
O
O
O
H
N
H
N
O
Phe
Allyl
NH2
NH2
H
N
O
PNZ
HO
PNZ
Tyr
N
Tyr
Arg
Tyr
Arg
H
Tyr
Arg
Gly
N
H
Val Arg
cyclization
SPPS
O
Allyl removal
Fmoc removal
O
Arg
Gly
O
O
H
N
H
N
PNZ removal
H
N
TFA cleavage
Gly
Gly
H
Ile
N
H
Tyr Arg
Ile
N
H
Tyr Arg
Arg
Cys
Ile
N
H
Tyr Arg
Arg
Cys
N
Ile
N
H
Tyr Arg
Arg
Cys
O
O
O
Arg
O
Cys
Resin Arg
Resin Arg
Resin Arg
2
HNCO Arg
1
1
12
13
14
c.
12
b.
O
Obs: 2229.071 Da TPI-2
Calc: 2229.754 Da
H
10
8
N
Val Arg Phe Cys Trp Lys NH2
N
Tyr
H
folding
Arg
Gly
O
S
S
6
4
2
0
H
N
Ile
N
H
Tyr
Arg Arg
Cys
Arg CONH2
O
2
10
230
250
270
-
2
4
TPI-2
TPI-1
TPI-2
TPI-3
-
2
1
23
25
wavelength/nm
Retention time (min)
-6
e.
d.
O
O
H
H
N
N
Val Arg Phe Cys Trp Lys NH2
Val Arg Phe Cys Trp Lys NH2
N
N
H
Tyr
H
Tyr
7O
12S
S
S
S
S
Arg
Gly
S
S
S
Arg
Gly
H
H
12CH
7S
N
N
N
H
Cys
Arg CONH2
2
Ile
N
H
Cys
Arg CONH2
Ile
Tyr
Arg Arg
Tyr
Arg Arg
O
O
TPI-1
TPI-4
O
H
N
O
Val Arg Phe Cys Trp Lys NH2
H
N
H
N
Val Arg Phe Cys Trp Lys NH2
Tyr
Arg
Gly
Ile
N
Tyr
H
O
CH2
S
S
Arg
Gly
Ile
S
S
S
H
N
H
N
N
H
Tyr Arg Arg Cys Arg CONH2
N
H
Tyr Arg Arg Cys Arg CONH2
O
O
TPI-2
TPI-1
TPI-3
TPI-5
Figure 2. a. Solid-phase synthetic route for the preparation of ether-containing TPI-2 using diaminodiacid 4a. b. HPLC traces and ESI-MS analysis of purified
refolded TPI-2. c. CD spectra of TPI-1, TPI-2 and TPI-3 in water. d. NMR structure of TPI-1 (right) and ether-containing analogue TPI-2 (left). Disulfides
are shown in yellow and the ether bond in red. e. The structure of side-chain extended ether-containing TPI-3 and different lengths of thioether linkage
substituted TPI-1 derivatives TPI-4 and TPI-5.
Table 1. Structures of different TPI-1 analogs and their minimal inhibitory
concentrations (MIC) against several bacterial strains. MIC values were
environment. After one hour in aqueous dithiothreitol (DTT,
obtained by using the standard two-fold dilution protocol
We also studied the stability of these peptides in a reducing
2
50 μm) solution, 80% TPI-1 had been completely reduced to
S
S
linear peptide, whereas TPI-2 exhibited less than 40%
reduction (Fig. S9). Thus, the replacement of the disulfide
functionality with an ether improves the stability of the parent
peptide, as expected.
X1 X2 X3
Lys Trp Cys Phe Arg Val Aa Tyr Arg Gly Ile Aa Tyr Arg Arg Cys Arg CONH2
Compound X1
X2
X3
B. subtilis S.aureus E.coli
Although thioether crosslinks are among the most popular
disulfide surrogates, they remain susceptible to sulfoxide
formation under oxidative conditions, which may influence
biological activity. Conotoxin -ImI was selected as the basis
for a study to examine whether ether-bond substitutions are more
TPI-1
TPI-2
S
S
4
4
8
8
4
4
O
CH2
TPI-3
TPI-4
O
S
CH2 CH2
CH2
4
8
8
4
4
4
a
stable than thioethers in an oxidative environment. Conotoxin
-ImI analogs -ImI-1 and -ImI-2 incorporating different
8

TPI-5
CH2
S
CH2
16
32
16
surrogates for the Cys2-Cys7 disulfide bond (Fig. 3) were
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

View Article Online
DOI: 10.1039/D0SC02374D
Page 5 of 7
Journal Name
Chemical Science
ARTICLE
readily prepared using 4a and thioether-containing suggested that these ether-contaning derivatives exhibited very
diaminodiacids, respectively. HPLC and MS analysis found that small change of their tertiary structures. The ether analog of the
about 30% -ImI-2 was oxidized to sulfoxide after 4 hours bacteriostatic peptide TPI-2 was found to be more stable under
under 0.1% hydrogen peroxide. When the oxidation process was reducing conditions than wildtype TPI-1, but equally bioactive.
extended to 12 hours, almost all the -ImI-2 was oxidized. Also, the ether-containing conotoxin analog was more stable in
Meanwhile, no oxidation by-products of -ImI-1 were observed an oxidative environment compared to an analog incorporating a
after 12 hours under the same oxidation conditions (Fig. 3). thioether. Further studies to explore the scope of this strategy for
These results indicate that the ether-substituted peptide is more the improvement of the properties of cysteine-rich peptides are
stable under oxidative conditions than the thioether-containing underway.
analog. Therefore, the ether-bond substitution strategy developed
herein is expected to replace the thioether bond strategy for
disulfide-rich peptides that are sensitive to oxidative conditions.
Acknowledgements
This work was supported by the National Key R&D Program of
China (2017YFA0505400 and 2017YFA0505200), the National
X
Natural Science Foundation of China (No. 91753205, 21877024,
1977089, 81621002, 31971152 and 21621003), the
Fundamental Research Funds for the Central Universities
JZ2019HGPB0105). Bayer AG would like to acknowledge
Professor Dr. Ulrich Koert from the Phillipps University of
Marburg for useful scientific discussions
H2N Gly Cys Aa Ser Asp Pro Arg Aa Ala Trp Arg Cys CONH2
2
S
S

-ImI-1; X=O
(
*

-ImI-2; X=S
t=12h
t=4h
Notes and references
1
a) W. J. Wedemeyer, E. Welker, M. Narayan, Biochemistry, 2000, 39, 15,
207-4216. b) A. Henninot, J. C. Collins, J. M. Nuss, J. Med. Chem. 2018,
4

-ImI-2; X=S
61, 1382-1414. c) M. Góngora-Benítez, J. Tulla-Puche, and F. Albericio,
Chem. Rev. 2014, 114, 901-926.
2
a) J. Gehrmann, P. F. Alewood, D. J. Craik, J. Mol. Biol. 1998, 278, 401-
t=0h
4
2
15. b) V. Lavergne, R. J. Taft, P. F. Alewood, Curr. Top. Med. Chem.
012, 12, 1514-1533.
2
0
21
22
23
24
20
21
22
23
Retention time (min)
Retention time (min)
3 a) C. J. Armishaw, N. L. Daly, S. T. Nevin, D. J. Adams, D. J. Craik, P. F.
Alewood, J. Biol. Chem. 2006, 281, 14136-14143. b) J.-Y. Chang, B.-Y.
Lu, L. Li, Anal. Biochem. 2005, 342, 78-85. c) K. Hu, W. Xiong, C. J. Sun,
C. Wang, J. X. Li, F. Yin, Y. X. Jiang, M.-R. Zhang, Z. Li, X. W. Wang, Z.
G. Li, CCS Chem. 2020, 2, 42-51.
1
.0
0
.8
0
0
.6
.4
4
a) Z. Dekan, I. Vetter, N. L. Daly, D. J. Craik, R. J. Lewis, P. F. Alewood,
J. Am. Chem. Soc. 2011, 133, 15866-15869. b) H. K. Cui, Y. Guo, F. L.
Feng, H. N. Chang, Y. J. Wang, F. M. Wu, C. Tian, L. Liu, Angew. Chem.
Int. Ed. 2013, 52, 9558-9562. c) Y. Guo, D. M. Sun, F. L. Wang, Y. He, L.
Liu, C. L. Tian, Angew. Chem. Int. Ed. 2015, 54, 14276-14281. d) C. M. B.
K. Kourra, N. Cramer, Chem. Sci. 2016, 7, 7007. e) S. S. Sun, J. Y. Chen,
R. Zhao, D. Bierer. J. Wang. G. M. Fang, Y. M. Li, Tetrahedron Lett.,
-ImI-1

-ImI-2
0
.2
0
4
8
Time h
12
Figure 3. Oxidative stability studies on -ImI-1 and -ImI-2 under 0.1%
hydrogen peroxide. After 12 hours, almost all -ImI-2 is oxidized, whereas
no oxidation by-products of -ImI-1 were observed. * refers to -ImI-2 in
which thioether is oxidized to sulfoxide.
2
019, 60, 1197-1201. f) Q. Qu, S. Gao, F. Wu, M. Zhang, Y. Li, L. Zhang,
D. Bierer, C. Tian, J. Zheng, L. Liu, Angew. Chem. Int. Ed. 2020, 59, 6037.
g) H. Lin, Y. Jiang, Q. Zhang, K. Hu, Z. G. Li, Chem. Commun. 2016, 52,
1
0389-10391. h) K. Hu, H. Geng, Q. Z. Zhang, Q. S. Liu, M. S. Xie, C. J.
Sun, W. J. Li, H. C. Lin, F. Jiang, T. Wang, Y. D. Wu, Z. G. Li, Angew.
Chem. Int. Ed. 2016, 55, 8013-8017. i) Y. Tian, J. X. Li, H. Zhao, X. Z.
Zeng, D. Y. Wang, Q. S. Liu, X. G. Niu, X. H. Huang, N. H. Xu, Z. G. Li,
Chem. Sci. 2016, 7, 3325-3330. j) T. Wang, J. Fan, X. X. Chen, R. Zhao, Y.
Xu, D. Bierer, L. Liu, Y. M. Li, J. Shi, G. M. Fang, Org. Lett. 2018, 20, 19,
6074-6078. h) J. Koehbach, M. O'Brien, M. Muttenthaler, M. Miazzo, M.
Akcan, A. G. Elliott, N. L. Daly, P. J. Harvey, S. Arrowsmith, S.
Gunasekera, T. J. Smith, S. Wray, U. Goransson, P. E. Dawson, D. J. Craik,
Conclusions
A selection of cysteine-rich peptides incorporating an ether-
bond as a disulfide surrogate have been efficiently synthesized
using a strategy based on diaminodiacid building blocks,
obtained by the stereoselective ring-opening addition reaction of
an aziridine skeleton with a hydroxy group. NMR studies
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

View Article Online
DOI: 10.1039/D0SC02374D
Chemical Science
Page 6 of 7
ARTICLE
Journal Name
M. Freissmuth, C. W. Gruber, Proc. Natl. Acad. Sci. USA 2013, 110
1183-21188.
2
5
a) K. Arai, T. Takei, M. Okumura, S. Watanabe, Y. Amagai, Y. Asahina, L.
Moroder, H. Hojo, K. Inaba, M. Iwaoka, Angew. Chem. Int. Ed. 2017, 56,
5
522-5526. b) K. Medini, P. W. R. Harris, A. Menorca, K. Hards, G. M.
Cook and M. A. Brimble, Chem. Sci. 2016, 7, 2005-2010. c) M.
Muttenthaler, S. T. Nevin, A. A. Grishin, S. T. Ngo, P. T. Choy, N. L.
Daly, S.-H. Hu, C. J. Armishaw, C.-I. Wang, R. J. Lewis, J. L. Martin, P.
G. Noakes, D. J. Craik, D. J. Adams, P. F. Alewood, J. Am. Chem. Soc.
2
010, 132, 3514-3522. d) S. Fiori, S. Pegoraro, S. Rudolph-Bçhner, J.
Cramer, L. Moroder, Biopolymers 2000, 53, 550-564. e) S. Pegoraro, S.
Fiori, S. Rudolph-Bohner, T. X. Watanabe, L. Moroder, J. Mol. Biol. 1998,
2
84, 779-792. f) Y. Z. Yin, Q. R. Fei, W. D. Liu, Z. R. Li, H. Suga, and C.
L. Wu, Angew. Chem. Int. Ed. 2019, 58, 4880-4885. g) C. Zuo, W.-W. Shi,
X.-X. Chen, M. Glatz, B. Riedl, I. Flamme, E. Pook, J. Wang, G.-M. Fang,
D. Bierer, L. Liu, Sci. China Chem., 2019, 62, 1371-1378.
6
7
a) K. Holland-Nell, M. Meldal, Angew. Chem. Int. Ed. 2011, 50, 5204-5206.
b) M. Empting, O. Avrutina, R. Meusinger, S. Fabritz, M. Reinwarth, M.
Biesalski, S. Voigt, G. Buntkowsky, and H. Kolmar, Angew. Chem. Int. Ed.
2
011, 50, 5207-5211. c) A. White, S. de Veer, G. Wu, P. Harvey, K. Yap,
G. J. King, D. Craik, Angew. Chem. Int. Ed. 2020,
doi.org/10.1002/anie.202003435.
a) M. A. Hossain, K. J. Rosengren, S. Zhang, R. A. D. Bathgate, G. W.
Tregear, B. J. van Lierop, A. J. Robinson, J. D. Wade, Org. Biomol. Chem.
2
009, 7, 1547-1553. b) C. A. Macraild, J. Illesinghe, B. J. Van Lierop, L.
Amanda, M. Chebib, B. G. Livett, A. J. Robinson, R. S. Norton, J. Med.
Chem. 2009, 52, 755. c) T. Wang, Y. F. Kong, Y. Xu, J. Fan, H. J. Xu, D.
Bierer, J. Wang, J. Shi, Y. M. Li, Tetrahedron Lett., 2017, 58, 3970. d) F.
L. Feng, Y. Guo, S. J. Li, Y. M. Li, Org. Biomol. Chem. 2015, 13, 6286-
6
290. e) Y. Wu, Y. H. Li, X. Li, Y. Zou, H. L. Liao, L. Liu, Y. G. Chen, D.
Bierer, H. G. Hu, Chem. Sci. 2017, 8, 7368. f) M. Pan, Q. Y. Zheng, S.
Gao, Q. Qu, Y. Y. Yu, M. Wu, H. Lan, Y. L. Li, S. L. Liu, J. B. Li, D. M.
Sun, L. N. Lu, T. Wang, W. H. Zhang, J. W. Wang, Y. M. Li, H.-G. Hu, C.
L. Tian, L. Liu, CCS Chem. 2019, 1, 476-489.
8
a) G. M. Williams, K. Lee, X. Li, G. J. Cooper, M. A. Brimble, Org.
Biomol. Chem. 2015, 13, 4059 b) M. A. Hossain, L. M. Haugaard-
Kedstrom, K. J. Rosengren, R. A. D. Bathgate, J. D. Wade, Org. Biomol.
Chem. 2015, 13, 10895.
9
1
a) A. Williamson, Justus Liebigs Ann. Chem. 1851, 77, 37. b) S. Mandal,
S. Mandal, S. K. Ghosh, P. Sar, P. Ghosh, R. Saha, B. Saha, RSC Adv.
2
016, 6, 69605.
0 a) X. E. Hu, Tetrahedron 2004, 60, 2701-2743. b) D. Tanner, Angew.
Chem. Int. Ed. Engl. 1994, 33, 599-619. c) B. A. BhanuPrasad, G. Sekar,
V. K. Singh, Tetrahedron Lett. 2000, 41, 4677-4679. d) H. Q. Liu, V. R.
Pattabiraman, J. C. Vederas, Org. Lett. 2007, 9, 4211-4214. e) C. C. Chen,
S. Gao, H. S. Ai, Q. Qu, C. Tian, Y. M. Li, Sci. China: Chem., 2018, 61,
7
02-707.
1
1
1 B. McKeever, G. Pattenden, Tetrahedron 2003, 59, 2071-2712.
2 M. Muttenthaler, A. Andersson, A. D. de Araujo, Z. Dekan, R. J. Lewis, P.
F. Alewood, J. Med. Chem. 2010, 53, 8585-8596.
1
3 a) T. Nakamura, H. Furunaka, T. Miyata, F. Tokunaga, T. Muta, S.
Iwanaga, M. Niwa, T. Takao, Y. Shimonishi, J. Biol. Chem. 1988, 263,
1
6709. b) H. Tamamura, R. Ioma, M. Niwa, S. Funakoshi, T. Murakami, N.
Fujii, Chem. Pharm. Bull. 1993, 41, 978.
1
4 C. Ramalingan, I.-S. Lee, Y.-W. Kwak, Chem. Pharm. Bull. 2009, 57, 591.
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 7 of 7
Chemical Science
O
R'
View Article Online
DOI: 10.1039/D0SC02374D
R
O
O
R'
N
O
R'
H
O
R
O
R
O
O
NH2
O
HN
N
H
O
O
N
H
O
No Reduction
oxidation &
isomerization
PG1 removal
cyclization
PG2 removal
SPPS
HN
O
HN
HN
HN
HN
PG1
O
O
O
HN
diaminodiacid strategy HN
PG2
PG2
ether bond
mimic
O
H
PG1
N
7O
O
PG2
12CH2
1O
O
6CH2
Fmoc
OH
3S
N
H
O
16S
Oxytocin mimic
Tachyplesin I mimic
We report the first introduction of an ether linkage as surrogate into the disulfide-rich peptides using ether-containing
diaminodiacid