The stereodynamics of macrocyclic succinimide-thioethers

Article abstract of DOI:10.1002/psc.3075

Maleimide-thiol coupling is a popular bioconjugation strategy, but little is known about the stereoselectivity and the stereodynamics of the succinimide thioether formed in a biopolymer environment. We used thiol 1,4-addition for the macrocyclisation of 5 designed pentapeptides with the ringsize of hexapeptides because they incorporate the succinimide thioether (4-8). Both succinimide diastereomers are observed in the constrained macrocyclic rings in each case. In spite of the low diastereoselectivity of the macrocyclisation reaction, there is a significant influence of the amino acid environment on the epimerization rate of the succinimide. Its half life can be as short as several hours at room temperature when Gly is the amino acid following the succinimide (peptide 8). On the contrary, no epimerization is detectable even after several weeks in the case of d-Phe C-terminal to the succinimide in peptide 4. Already the small selection of examples shows how big the differences in epimerization rates can be and that the local environment has a significant influence. The variation of amino acids in the vicinity of the ligation site points the way towards the synthesis of bioconjugates which are obtained as stable and separable diastereomers.

Full text of DOI:10.1002/psc.3075

Received: 8 December 2017
DOI: 10.1002/psc.3075
Revised: 20 February 2018
Accepted: 21 February 2018
R E S E A R C H A R T I C L E
The stereodynamics of macrocyclic succinimide‐thioethers
|
|
Stefan Lenz Philip Horx Armin Geyer
Institute of Chemistry, Philipps‐University
Marburg, Hans‐Meerwein‐Straße, 35032
Maleimide‐thiol coupling is a popular bioconjugation strategy, but little is known about
Marburg, Germany
the stereoselectivity and the stereodynamics of the succinimide thioether formed in a
biopolymer environment. We used thiol 1,4‐addition for the macrocyclisation of 5
designed pentapeptides with the ringsize of hexapeptides because they incorporate
the succinimide thioether (4‐8). Both succinimide diastereomers are observed in the
constrained macrocyclic rings in each case. In spite of the low diastereoselectivity of
the macrocyclisation reaction, there is a significant influence of the amino acid envi-
ronment on the epimerization rate of the succinimide. Its half life can be as short as
several hours at room temperature when Gly is the amino acid following the
succinimide (peptide 8). On the contrary, no epimerization is detectable even after
several weeks in the case of D‐Phe C‐terminal to the succinimide in peptide 4. Already
the small selection of examples shows how big the differences in epimerization rates
can be and that the local environment has a significant influence. The variation of
amino acids in the vicinity of the ligation site points the way towards the synthesis
of bioconjugates which are obtained as stable and separable diastereomers.
Correspondence
Prof. Dr Armin Geyer, Institute of Chemistry,
Philipps‐University Marburg, Hans‐Meerwein‐
Straße, 35032 Marburg, Germany.
Email: geyer@staff.uni‐marburg.de
|
1
INTRODUCTION
precursor Mal=Gly (1, Figure 2) reacts with Cys‐OMe to the
succinimide containing dipeptide Cys(OMe)‐Suc=Gly (2). 1H NMR
The formation of succinimide thioethers via thiol 1,4‐addition is
frequently used to form bioconjugates or to immobilize bioactive
compounds on surfaces.1-12 It is the preferred method for the
formation of antibody‐drug conjugates.5,13 Yet, only few studies are
dedicated to the construction of homogeneous bioconjugates which
do not exist as mixtures of diastereomers at the ligation site.14,15 Our
study identifies the stereodynamic and conformational properties of
identifies a nearly equimolar mixture of the diastereomeric dipeptides
Cys(OMe)‐(R)‐Suc=Gly and Cys(OMe)‐(S)‐Suc=Gly (3 DS1 and 3 DS2).
Three different protected amino acid building blocks of the type
Mal=Xaa, wherein Xaa is either Gly, D‐Ala, or D‐Phe were synthesized
and used in solid phase peptide synthesis according to methods recently
published.19-22 With the intention to minimize the conformational
mobility of the macrocyclic peptides, we introduced the bicyclic β‐turn
mimic Hot=Tap (Hot: Hydroxythreonine, “=” backbone cyclisation,
Tap: Thiaproline)23 oppositely to the ligation site of the macrocyclic
peptides as shown in Figure 3. The oligocyclic environment restricts
the conformational mobility of the cyclopeptide and was expected to
slow down the H/D exchange and the epimerization in solution with
the aim of studying the processes by NMR spectroscopy.
pairs of diastereomeric macrocyclic pentapeptides containing
a
succinimide thioether where the ring closure is achieved by a thiol
1,4‐addition reaction. Figure 1 shows how the ligation site fits the 3‐
atom register of α‐amino acids and thus can be seen as an isosteric
replacement within the peptide backbone. The ligation has structural
analogy to aspartimides formed spontaneously by Asp‐Gly sequences
as a side reaction in peptide synthesis.16 This reaction is also known
from proteins, where in the case of lysozyme the resulting succinimide
kinks and restricts the turn region.17
The linear peptide precursors of the type Mal=Xaa‐Gly‐
Hot=Tap‐Yaa, wherein Yaa is either D‐, L‐Cys, or Tht, were cyclized
in a 1‐pot retro Diels‐Alder, thiol 1,4‐addition reaction yielding 2
diastereomers which were separated by preparative HPLC. The
unequivocal assignment of R/S stereochemistry of the newly formed
stereocenter at the cyclisation site was not possible by NOE and 3J
We use the 3‐letter code “Suc” for succinimide to underline the
similarity of the ligation site with the 3‐atom repeat of the peptide
backbone. The ring‐closing acylation of the amino acid following Suc
is indicated by a double hyphen “=” instead of a hyphen “‐” to show
the restriction by the 5‐membered ring.18 Accordingly, the maleimide
coupling data. Therefore, we distinguished the
2 diastereomers
cyclo[‐(R)‐Suc=Xaa‐Gly‐Hot=Tap‐Yaa] and cyclo[‐(S)‐Suc=Xaa‐Gly‐
J Pep Sci. 2018;e3075.
wileyonlinelibrary.com/journal/psc
Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.
1 of 9
https://doi.org/10.1002/psc.3075

2 of 9
LENZ ET AL.
FIGURE 1 Two stereoisomers are formed by the succinimide‐thioether (Suc) which closely resembles naturally occurring amino succinimides
(Asu) formed by Asp‐Gly sequences for the well‐studied example is lysozyme (PDB‐code: 1AT5). Both succinimides fit the 3‐atom register of
the peptide backbone (right)
FIGURE 2 The addition reaction of Mal=Gly (1) and Cys‐OMe (2) in a thiol 1,4‐addition yields both diastereomeric succinimide dipeptides
Cys(OMe)‐(R)‐Suc=Gly and Cys(OMe)‐(S)‐Suc=Gly (3 DS1 and 3 DS2). The expansion from the 1H NMR shows different chemical shifts for the
high‐field proton of the (R)‐Suc/(S)‐Suc methylene group of 3 in the 1H NMR. The complete 1H NMR is shown in the Supporting Information
FIGURE 3 The proposed mechanism of the
succinimide epimerization is accompanied by
de‐ and reprotonation of Cα (upper left). Three
different scenarios should be distinguishable
by 1H NMR (right). The schematic line spectra
of the dd signal pattern of one of the SucHβ
either remains unchanged in the presence of
D2O, the enolisation simplifies the signal
pattern without epimerization, or the
enolization is accompanied by epimerization
observable as a second signal set with
simplified signal pattern. The general structure
of the succinimide thioether containing
tetracyclic pentapeptides cyclo[‐(R/S)‐
Suc=Xaa‐Gly‐Hot=Tap‐Yaa] and the
nomenclature of the β‐turn mimic Hot=Tap is
shown at lower left
Hot=Tap‐Yaa] as DS1 and DS2 according to their retention times on
the HPLC. The tetracyclic peptides showed sufficient 1H NMR signal
dispersion to allow a complete signal assignment by 2D NMR. In
order to differentiate whether the N‐ or the C‐terminal amino acid
next to the cyclisation site has a bigger influence on the cyclisation
stereochemistry and on the epimerization rate, we varied both sites
and separated the diastereomers. All 5 pairs of cyclic peptides 4 to 8
are shown in Scheme 1.
of 6 and D‐Cys in the case of 7. Fmoc‐L‐allo‐trityl‐thiothreonine
(Fmoc‐Tht(Trt)‐OH, 9) was synthesized in 7 steps from Thr in an over-
all yield of 20%. Detailed reaction conditions can be found in the
Supporting Information.
|
2
MATERIALS AND METHODS
Reagents
Peptides 4, 5, and 8 differ in the amino acids of the Mal=Xaa
building block. Mal=D‐Phe in the case of 4, Mal=D‐Ala in 5, and
Mal=Gly in the case of 8. Peptides 6 and 7 differ in the amino acids
bearing the thiol group which is L‐allo‐thiothreonine (Tht) in the case
|
2.1
All chemicals and solvents used for the synthesis of peptides were
purchased from Iris Biotech and were not purified further.

LENZ ET AL.
3 of 9
SCHEME 1 The 18‐membered macrocyclic
ring is a common feature of all cyclic peptide
analogs 4 to 8 which differ only at the amino
acids N‐ or C‐terminal to the succinimide
cyclisation site. Each peptide exists as a pair of
diastereomers DS1 and DS2
|
|
2.2
Resin loading
2.6
HPLC
2‐Chlorotritylchloride resin (1.60 mmol/g) was loaded with Fmoc‐
Xaa(Trt)‐OH (Xaa = Cys, D‐Cys, Thiothreonine) by adding the protected
amino acid (1.50 eq) and DIPEA (6.00 eq) in DMF (10 mL/g resin) and
stirring for 4 hours. After washing the resin with DMF, methanol, and
DCM several times, the resin was treated with a mixture of DCM/
methanol/DIPEA (80:15:5) 2 times for 30 minutes and washed several
times with DMF, methanol, and DCM before it was dried under
vacuum. The loading of the used resins was estimated to be between
0.30 and 0.40 mmol/g by UV‐Vis spectroscopy at 289 and 300 nm
after cleaving the Fmoc protecting group with 20% piperidine in
DMF for 20 minutes.
Analytical HPLC was done on a Thermo Fisher Ultimate 3000 LC
System. The cyclic peptides were purified by semi‐preparative
reversed‐phase HPLC on a Thermo Fisher Ultimate 3000 LC System.
|
2.7
Mass spectrometry
Mass spectra (ESI+) were acquired on a Thermo Fisher Scientific LTQ‐
FT.
|
2.8
NMR measurement
NMR spectra (1H, TOCSY, COSY, ROESY, 1H‐13C‐HSQC) were
acquired on a 500‐MHz spectrometer, and the kinetic measurements
were performed on a 300‐MHz spectrometer, both Bruker AV series.
|
2.3
Solid phase peptide synthesis
Linear precursor peptides were synthesized on a microwave assisted
peptide synthesizer. Fmoc‐strategy was applied. The 2CTC‐resins
loaded with Fmoc‐Xaa(Trt)‐OH (Xaa = Cys, D‐Cys, Thiothreonine;
scheduled quantity: 0.1 mmol, 1.00 eq) ran through the following
cycles of Fmoc‐deprotection and amino acid coupling:
|
2.9
Molecular modeling
Molecular modeling and molecular dynamics calculations were
performed using Molecular Operating Environment software version
2014.0901 (Chemical Computing Group, Montreal, Canada). The
structure was assembled using the Protein Builder program; later, the
Suc=Gly building block was modeled starting from cysteine using the
builder interface of the Molecular Operating Environment program
and energy minimized using the Amber12EHT force field. The peptide
was afterwards analyzed through multiple simulated annealing
processes in implicit solvent using Born as the dielectric constant with
extended cutoff. Several iterations were performed, and the structure
with the lowest energy was further minimized.24
•
•
Fmoc‐deprotection: T = 50°C, Pmicrowave = 30 W, t = 210 seconds
with piperidine (20 w% in DMF, 3.00 mL/deprotection)
Amino acid coupling: T = 50°C, Pmicrowave = 30 W, t = 600 seconds
with Fmoc‐protected amino acid and maleimde building blocks
(0.2 M in DMF, 5.00 eq, 2.5 mL/coupling), DIC (0.5 M in DMF,
5.00 eq, 1 mL/coupling), and Oxyma (1 M in DMF, 5.00 eq, 0.5 mL).
|
3
RESULTS AND DISCUSSION
|
2.4
Resin cleavage
|
3.1
Synthesis of the Mal=Gly and Mal=D‐Xaa
Resin cleavage was performed with a mixture of TFA/TIPS (96:4) for
240 minutes. Peptides were precipitated from cold diethylether
(40 mL), washed 2 to 3 times with diethylether and lyophilised from
water.
building blocks
The maleimide Mal=Gly 1 was obtained by condensation of malic
anhydride (10) with Gly in refluxing acetic acid.25,26 Chiral amino acids
are expected to show significant racemization under these reaction
conditions. Therefore, the chiral maleimide building blocks Mal=D‐
Phe 12a and Mal=D‐Ala 12b were synthesized in a 2‐step process
according to Toru et al27 (Scheme 2) although alternative synthesis at
room temperature or with microwave irradiation exist.28-30 To prevent
side reactions of the maleimide double bond in peptide synthesis, a
dihydrofuran protecting group was used to obtain the building blocks
13a to 13c which are compatible to the reaction conditions used for
|
2.5
Cyclization of peptides
The crude linear precursor peptides were used without further
purification and dissolved in 100 mL of n‐butanol to reach a peptide
concentration of c = 1 mM. After refluxing for 4 hours, the solvent
was evaporated, and the resulting diastereomeric mixture of the
peptide was purified by preparative HPLC.

4 of 9
LENZ ET AL.
SCHEME 2 Synthesis of maleimide building
blocks compatible for SPPS. Reaction
conditions for Gly: (A) HOAc, 120°C, 8 hours;
(B) reaction conditions for chiral amino acids:
1) HOAc, RT, 12 hours; 2) ZnBr2, HMDS,
MeCN, 80°C, 3 hours; (C) furan, CHCl3, 36°C,
4 days
solid phase peptide synthesis. This method has been implied by
Grandas et al.31,32 The retro Diels‐Alder deprotection was described
to be complete at 120°C.
chromatogram. The more polar peptide with shorter retention time is
diasteromer 1 (DS1) and the second diastereomer is DS2 (Figure 4).
The diastereomers were separated by preparative reversed‐phase
HPLC and analyzed by NMR spectroscopy. The 1H NMR spectra of
DS1 are shown in Figure 5 and the spectra of DS2 in Figure 6. In spite
of the significant signal dispersions shown by both sets of spectra,
there are no characteristic chemical shifts, coupling, or NOE patterns
which would allow an unequivocal stereochemical assignment of the
(R)‐Suc/(S)‐Suc stereochemistry.
|
3.2
Synthesis of cyclic peptides
The linear precursor peptides were synthesized on a semi‐automatic
microwave‐assisted peptide synthesizer in a 0.1 millimolar scale. The
first amino acid loaded on a 2‐CTC resin was the selectively protected
thiol amino acid to obtain resins 14 to 16. The solid‐phase peptide
synthesis (SPPS) was performed according to standard protocols for
the coupling with DIC/Oxyma and the Fmoc deprotection with 20%
piperidine in DCM. Cyclisation of these peptides takes place in a 1‐
pot retro DA reaction with an additional thiol 1,4‐addition. To prevent
oligomerisation, the cyclisation was carried out at dilute conditions
with a peptide concentration of 1 mM. The solvent of choice for the
cyclisation was n‐butanol because its boiling point at 118°C fits the
temperature for the retro DA reaction. Its relatively low vapor pressure
simplified workup (Scheme 3). All cyclization experiments yielded 2
diastereomers in a close to equimolar mixture of both diastereomers
(Scheme 3). There were no directing effects observed of the other
stereo centers within the linear precursor peptide. The cyclic peptide
was the only observed product without dimer formation.
|
3.3
Stereodynamic behavior of the cyclic
succinimide thioether containing pentapeptides
The pure peptide 8 showed the slow formation of a second signal set
in the course of the NMR experiments which was identified as the sec-
ond diastereomer. The same was observed for cyclopeptide
(Figure 7).
7
In order to determine the mechanism behind this epimerization
process, the epimerization was followed in the presence of D2O with
the aim to estimate the enolization rate of the succinimde (Figure 8).
Peptide 7 was dissolved in D2O where H/D exchange of the slightly
acidic stereocenter of the succinimide ring (green) ceased the corre-
sponding 3J coupling to the neighboring diastereotopic methylene
group (CH2, blue, Hh). This observation connects the epimerization
process between DS1 and DS2 and enolization of the succinimide ring.
The 2 possible diastereomers of the cyclic peptides were differen-
tiated according to their retention times on the reversed‐phase HPLC
SCHEME 3 Synthesis of the cyclic peptides 4 to 8, starting from preloaded CTC‐resin. SPPS was carried out on a microwave‐assisted peptide
synthesizer. For complete deprotection of the Trt‐protecting group, the TFA cleavage time was extended to 4 hours. The linear precursor
peptide was used without further purification. The retro DA reaction of the dihydrofuryl protected N‐terminal maleimide at 120°C under high
dilution conditions in n‐butanol is followed by the macrocyclisation of the peptide

LENZ ET AL.
5 of 9
FIGURE 4 The HPLC chromatogram of the
cyclisation reaction shows the crude cyclic
pentapeptide 6 containing a succinimide
thioether neighbouring L‐allo‐thiothreonine as
a nearly 1:1 ratio of both diastereomers DS1
and DS2. Similar chromatograms were
observed for all investigated cyclic peptides
The epimerization rate was investigated by NMR spectroscopy for
all cyclic peptides (4‐8). The well‐separated Hot = Tap‐3H or 8aH
proton served as reference signal for the integration. The cyclic
pentapeptides which contained the Suc=Gly motif (6‐8) showed a ratio
of approximately 3:1 DS1/DS2 after reaching the thermodynamic
equilibrium.
resistance to epimerization. The NMR kinetics of the separated
diastereomers of peptide 4 in DMSO‐d6 in the presence of small
amounts of D2O showed no epimerization for more than 60 days
(Figure 10). Remarkably, DS2 shows complete H/D exchange in this
measuring window due to enolisation, while H/D exchange is
completely absent for DS1. This is the first time that stable succinimide
epimers could be separated and analyzed.
The epimerization equilibrium and velocity strongly depend on the
configuration of the stereogenic centers of the peptide backbone close
to the succinimide. The epimerization velocity of peptide 8 is 4 times
faster than peptide 7 (Figure 9). The additional methyl group of L‐
allo‐thiothreonine (6) halves the epimerization velocity relative to pep-
tide 8 (see Table 1).
The wide 1H signal dispersion allows a complete signal assignment
and
a closer look at the conformational properties of the
cyclopeptides. The temperature dependence of the secondary amide
NH protons differentiated the internal or external (solvent accessible)
orientation of the amide protons. Sequential NOEs characterize the
peptide backbone and identify the dominating rotamer about the
thiol‐succinimide linkage was identified from a combination of 3J and
NOE data.
Altogether, there is a strong conformational influence of the mac-
rocyclic on the acidity of the succinimide ring and its tendency for
enolization.
Cyclopeptide 4 and 5 with the D‐configurated amino acid at the C‐
terminal position relative to the succinimide ring showed significant
In spite of the significant chemical shift differences, the relevant
NOE and coupling data were surprisingly similar for both diastereomers,
FIGURE 5 1H NMR (500 MHz, 300 K, DMSO‐d6) of the aliphatic region of the DS1 of all cyclic peptides. The diastereotopic proton pairs are
colored and show characteristic signal dispersions.

6 of 9
LENZ ET AL.
FIGURE 6 1H NMR (500 MHz, 300 K, DMSO‐d6) of the aliphatic region of the DS2 of all cyclopeptides. The diastereotopic proton pairs are
colored and show characteristic signal dispersions.
FIGURE 7 Epimerization (1H NMR,
500 MHz, 300 K, DMSO‐d6) of the cyclic
peptides 7 DS‐2 (top) and DS‐1 (below) leads
to the identical mixture of both diastereomers
as before the separation by preparative HPLC
FIGURE 8 1H NMR (600 MHz, 300 K, D2O) of peptide 7 (DS1), time‐dependent H/D exchange of the acidic proton of the succinimide ring
(green) is accompanied by the loss of 3J coupling of the methylene group (CH2, blue)

LENZ ET AL.
7 of 9
FIGURE 9 The equilibration of DS1 and DS2 was studied by NMR for 4 different peptides at room temperature. The 1H NMR integrals of well‐
separated signals served as reference. Epimerization was the only process which explained the changes in signal intensity and multiplicity. A retro
Michael‐type ring opening was not observed. Cyclopeptide 5 behaves similar to 4 (Supporting Information)
TABLE 1 Comparison of the epimerization time needed for each diastereomer to reach a 50% extent
t1/2 DS1,
h
t1/2 DS2,
h
Peptide
cyclo[‐(R/S)‐Suc=Gly‐Gly‐Hot=Tap‐L‐Cys] (8)
cyclo[‐(R/S)‐Suc=Gly‐Gly‐Hot=Tap‐Tht] (6)
cyclo[‐(R/S)‐Suc=Gly‐Gly‐Hot=Tap‐D‐Cys] (7)
cyclo[‐(R/S)‐Suc=D‐Xaa‐Gly‐Hot=Tap‐L‐Cys] (4, 5)
50
15
100
30
200
140
>>1000
>>1000
FIGURE 10 1H NMR (first: 300 MHz, 300 K, DMSO‐d6; above: 300 MHz, 300 K, D2O/DMSO‐d6, 1:12), time‐dependent H/D exchange for the
stable diastereomers of cyclic peptide 4. After the addition of D2O to the NMR sample, only the second diastereomer (DS2, right) undergoes
enolization at the succinimide ring, which is determined by loss of the 3J coupling of the methylene group. For the 2 diastereomers of the Suc=D‐
Phe containing cyclopeptide 4 no epimerization is detected over a time period of 60 days

8 of 9
LENZ ET AL.
FIGURE 11 Simulated annealing calculations
for both diastereomers of peptide 8 show that
the main difference between the 2
diastereomers is the alignment of the
succinimide ring. Left: Suc‐CH2 above the
macrocyclic plane; right: Suc‐CH2 under the
plane. This leads to the conclusion that
enolization of the succinimide‐ring facilitates
the rotation around the C‐S‐ and C‐N bond,
and therefore epimerization is accompanied
by a ring flip (180° rotation)
molecular size. Cancer Res. 2010;70:1595‐1605. https://doi.org/
10.1158/0008‐5472.CAN‐09‐2724
and therefore it was not possible to assign the succinimide stereochem-
istry unequivocally. Molecular modeling of the 2 diastereomers of
peptide 8 shows that the main difference between the 2 diastereomers
is the alignment of the succinimide ring (Figure 11).
8. Anderson CJ, Ferdani R. Copper‐64 radiopharmaceuticals for PET
imaging of cancer: advances in preclinical and clinical research. Cancer
Biother Radiopharm. 2009;24:379‐393. https://doi.org/10.1089/
cbr.2009.0674
9. Danhier F, Le BA, Préat V. RGD‐based strategies to target alpha(v)
beta(3) integrin in cancer therapy and diagnosis. Mol Pharm.
2012;9:2961‐2973. https://doi.org/10.1021/mp3002733
|
4
CONCLUSION
It was shown that the synthesis of cyclic peptides with a native
backbone register is possible by ring closure in a thiol 1,4 addition of a
Cys side chain with a maleimide. NMR was used to quantify the
stereodynamic properties of the obtained succinimide thioether.
Epimerization of the cyclic peptides occurs by enolization of the
succinimide ring whereby the configuration of the stereocenter at the
succinimide ring and the conformation of the macro cyclic ring has a
major influence on the ring acidity and epimerization velocity of the
corresponding diastereomers. The epimerization velocity is strongly
influenced by neighbouring stereo centers of the peptide backbone. In
the case studied, introducing a D‐Xaa C‐terminal to the succinimide
suppresses enolization and epimerization of the peptide backbone.
10. Tsai M‐Y, Lin C‐Y, Huang C‐H, et al. Vapor‐based synthesis of maleimide‐
functionalized coating for biointerface engineering. Chem Commun.
2012;48:10969‐10971. https://doi.org/10.1039/c2cc35892a
11. Kotagiri N, Li Z, Xu X, Mondal S, Nehorai A, Achilefu S. Antibody quan-
tum dot conjugates developed via copper‐free click chemistry for rapid
analysis of biological samples using a microfluidic microsphere array
system. Bioconjug Chem. 2014;25:1272‐1281. https://doi.org/
10.1021/bc500139u
12. Park EJ, Gevrek TN, Sanyal R, Sanyal A. Indispensable platforms for
bioimmobilization: maleimide‐based thiol reactive hydrogels. Bioconjug
Chem. 2014;25:2004‐2011. https://doi.org/10.1021/bc500375r
13. Gunnoo SB, Madder A. Chemical protein modification through cyste-
ine. Chembiochem. 2016;17:529‐553. https://doi.org/10.1002/
cbic.201500667
14. Akkapeddi P, Azizi S‐A, Freedy AM, Cal PMSD, Gois PMP, Bernardes
GJL. Construction of homogeneous antibody–drug conjugates using
site‐selective protein chemistry. Chem Sci. 2016;7:2954‐2963.
https://doi.org/10.1039/C6SC00170J
ORCID
Armin Geyer http://orcid.org/0000-0001-8096-2266
15. Loidl G, Groll M, Musiol HJ, Ditzel L, Huber R, Moroder L.
Bifunctional inhibitors of the trypsin‐like activity of eukaryotic
proteasomes. Chem Biol. 1999;6:197‐204. https://doi.org/10.1016/
S1074‐5521(99)80036‐2
REFERENCES
1. Tsao TC, Bailey K. The extraction, purification and some chemical
properties of actin. Biochim Biophys Acta. 1953;11:102‐113. https://
doi.org/10.1016/0006‐3002(53)90013‐4
16. Mergler M, Dick F. The aspartimide problem in Fmoc‐based SPPS. Part
III. J Pept Sci. 2005;11:650‐657. https://doi.org/10.1002/psc.668
2. Moore JE, Ward WH. Cross‐linking of bovine plasma albumin and wool
keratin. J Am Chem Soc. 1956;78:2414‐2418. https://doi.org/10.1021/
ja01592a020
17. Noguchi S, Miyawaki K, Satow Y. Succinimide and isoaspartate residues
in the crystal structures of hen egg‐white lysozyme complexed with tri‐
N‐acetylchitotriose. J Mol Biol. 1998;278:231‐238. https://doi.org/
10.1006/jmbi.1998.1674
3. Wu CW, Yarbrough LR. N‐(1‐pyrene)maleimide:
cross‐linking reagent. Biochemistry. 1976;15:2863‐2868. https://doi.
a fluorescent
18. Haack M, Enck S, Seger H, Geyer A, Beck‐Sickinger AG. Pyridone
dipeptide backbone scan to elucidate structural properties of a flexible
peptide segment. J Am Chem Soc. 2008;130:8326‐8336. https://doi.
org/10.1021/ja8004495
org/10.1021/bi00658a025
4. Ourailidou ME, Van Der Meer JY, Baas BJ, et al. Aqueous oxidative
heck reaction as
a
protein‐labeling strategy. ChemBioChem.
2014;15:209‐212. https://doi.org/10.1002/cbic.201300714
19. Keller O, Rudinger J, Acta C. Preparation and some properties of
maleimido acids and maleoyl derivatives of peptides. Helv Chim Acta.
1975;58:531‐541. https://doi.org/10.1002/hlca.19750580224
5. Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the
next generation of therapeutic antibodies. Nat Rev Immunol.
2010;10:345‐352. https://doi.org/10.1038/nri2747
20. Schrimpf A, Geyer A. Two opposing D‐amino acids give zigzag hairpin
epitopes an additional kink to create antibody‐selective peptide anti-
gens. Chembiochem. 2016;17:2129‐2132. https://doi.org/10.1002/
cbic.201600479
6. Merten H, Brandl F, Plückthun A, Zangemeister‐Wittke U. Antibody‐drug
conjugates for tumor targeting—novel conjugation chemistries and the
promise of non‐IgG binding proteins. Bioconjug Chem. 2015;26:2176‐
2185. https://doi.org/10.1021/acs.bioconjchem.5b00260
21. Schrimpf A, Linne U, Geyer A. Eight at one stroke—a synthetic tetra‐
disulfide peptide epitope. Org Biomol Chem. 2017;15:2512‐2521.
https://doi.org/10.1039/C6OB02746F
7. Zahnd C, Kawe M, Stumpp MT, et al. Efficient tumor targeting with
high‐affinity designed ankyrin repeat proteins: effects of affinity and

LENZ ET AL.
9 of 9
22. Elduque X, Sanchez A, Sharma K, Pedroso E, Grandas A. Protected
maleimide building blocks for the decoration of peptides, peptoids,
and peptide nucleic acids. Bioconjug Chem. 2013;24:832‐839. https://
doi.org/10.1021/bc4000614
agents on Bacillus spp. strains: docking affinity and in vitro inhibition
of glutamate‐racemase. J Enzyme Inhib Med Chem. 2013;28:1026‐
1033. https://doi.org/10.3109/14756366.2012.705837
30. Chandrasekhar S, Padmaja MB, Raza A. Solid phase‐solid state synthe-
sis of N‐alkyl imides from anhydrides {#}. Synlett New York.
1999;7:1597‐1599. DOI:ISSN 0936–5214
23. Eckhardt B, Grosse W, Essen L‐O, Geyer A. Structural characterization
of a‐turn mimic within a protein‐protein interface. Proc Natl Acad Sci.
2010;107:18336‐18341. https://doi.org/10.1073/pnas.1004187107
31. Elduque X, Pedroso E, Grandas A. Orthogonal protection of peptides
and peptoids for cyclization by the thiol‐ene reaction and conjugation.
J Org Chem. 2014;79:2843‐2853. https://doi.org/10.1021/jo500427c
24. Molecular Operating Environment (MOE), 2013.08; Chemical Comput-
ing Group ULC, 1010 Sherbooke St. West,Suite #910, Montreal, QC,
Canada, H3A 2R7, 2018.
32. Sanchez A, Pedroso E, Grandas A. Maleimide‐dimethylfuran exo
adducts: effective maleimide protection in the synthesis of oligonucle-
otide conjugates. Org Lett. 2011;13:4364‐4367. https://doi.org/
10.1021/ol201690b
25. Sinclair AJ, del Amo V, Philp D. Structure‐reactivity relationships in a
recognition mediated [3+2] dipolar cycloaddition reaction. Org Biomol
Chem. 2009;7:3308‐3318. https://doi.org/10.1039/b908072d
26. De Figueiredo RM, Oczipka P, Fröhlich R, Christmann M. Synthesis of
4‐maleimidobutyric acid and related maleimides. Synthesis (Stuttg).
2008;8:1316‐1318. https://doi.org/10.1055/s‐2008‐1032016
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
27. Reddy P, Kondo S, Toru T, Ueno Y. Lewis acid and
hexamethyldisilazane‐promoted efficient synthesis of N‐alkyl‐and N‐
arylimide derivatives. J Org Chem. 1997;62:2652‐2654. https://doi.
org/10.1021/jo962202c
28. Romano R, Bayerl TM, Moroder L. Lipophilic derivatization and its
effect on the interaction of cholecystokinin (CCK) nonapeptide with
phospholipids. BBA‐Biomembranes. 1993;1151:111‐119. https://doi.
org/10.1016/0005‐2736(93)90093‐F
How to cite this article: Lenz S, Horx P, Geyer A. The
stereodynamics of macrocyclic succinimide‐thioethers. J Pep
Sci. 2018;e3075. https://doi.org/10.1002/psc.3075
29. Tamay‐Cach F, Correa‐Basurto J, Villa‐Tanaca L, Mancilla‐Percino T,
Juárez‐Montiel M, Trujillo‐Ferrara JG. Evaluation of new antimicrobial