Reversibly switching the conformation of short peptide through in-tether chiral sulfonium auxiliary

Article abstract of DOI:10.1016/j.cclet.2017.07.003

A chirality induced helicity method has been developed to modulate the peptide's biophysical and biochemical properties. We report herein a novel approach for reversibly switching the conformation of short constraint α-helical peptides through alkylation of the in-tether thioether and dealkylation of the chiral sulfonium. This traceless redox sensitive tagging strategy broadened our scope of CIH (chirality induced helicity) strategy and provided a valuable approach to functionalize the peptide tether.

Full text of DOI:10.1016/j.cclet.2017.07.003

Accepted Manuscript
Title: Reversibly switching the conformation of short peptide
through in-tether chiral sulfonium auxiliary
Authors: Xiaodong Shi, Yixiang Jiang, Dan Yang, Hui Zhao,
Yuan Tian, Zigang Li
PII:
S1001-8417(17)30239-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.07.003
CCLET 4121
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
16-5-2017
22-5-2017
3-7-2017
Please cite this article as: Xiaodong Shi, Yixiang Jiang, Dan Yang, Hui
Zhao, Yuan Tian, Zigang Li, Reversibly switching the conformation of
short peptide through in-tether chiral sulfonium auxiliary, Chinese Chemical
Lettershttp://dx.doi.org/10.1016/j.cclet.2017.07.003
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Communication
Reversibly switching the conformation of short peptide through in-tether chiral
sulfonium auxiliary
Xiaodong Shi a †, Yixiang Jiang a †, Dan Yang a †, Hui Zhao a, Yuan Tian a, b,*, Zigang Lia,*
a School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China
b School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 611756, China
*Corresponding authors.
E-mail addresses: ty@swjtu.edu.cn (Y. Tian); lizg@pkusz.edu.cn (Z. Li)
† These authors contributed equally to this work.
Graphical Abstract
A novel approach for reversibly switching the conformation of short constraint α-helical peptides through chemselective
alkylation of the in-tether thioether and dealkylation of the chiral sulfonium was developed and it provides a valuable modifiable
site to functionalize the peptides.
ABSTRACT
A chirality induced helicity method has been developed to modulate the peptide’s biophysical and biochemical properties. We report herein a novel
approach for reversibly switching the conformation of short constraint α-helical peptides through alkylation of the in-tether thioether and dealkylation
of the chiral sulfonium. This traceless redox sensitive tagging strategy broadened our scope of CIH (chirality induced helicity) strategy and provided a
valuable approach to functionalize the peptide tether.
Keywords: Cyclic peptide Sulfonium Chiral center α-Helix Reversible Tether modification
Protein-protein interactions (PPIs) play a central role in most biological processes and have been demonstrated to be important
therapeutic targets in the past decades [1]. Synthetic peptides are suitable targeting candidates for PPIs as they are able to precisely
mimic the topological features of target proteins and can be easily synthesized and modified [2]. Chemists have elaborated many efforts
in order to stabilize these targeting epitopes into varies conformational elements, such as α-helix and β-hairpin [3]. Particularly, over
30% of protein secondary structure are helical in nature and over 50% of PPIs involve short α-helices according to a statistical analysis
of protein data bank (PDB) [4]. As α-helix involved in a majority of PPIs, chemical methods, such as disulfide formation [3j], amide
formation [3c], olefin/alkyne methathesis [3a], cysteine alkylation [3k] and perfluoroated arene incorportation [3l] have been utilized to
confine peptides into helical structures.
Our group recently discovered a unique phenomenon [3d] that a precisely positioned chiral center in the tether of short peptides could
dominantly induce helicity of the backbone peptides. This phenomenon could be extended to both a chiral sulfilimine center [5] and a
chiral sulfoxide center [6] (Fig. 1), which provides a valuable modifiable site on the tether [7]. The absolute configuration of the in-
tether chiral center was determined to be R for helicity inducement by peptide crystal structure.
Methionine alkylation is a facile and chemoselective approach for introducing functional groups into peptides [8]. Deming et al.
reported a chemoselective tagging strategy for the modification of peptides in a reversible manner [9]. Met residues could be
chemoselectively alkylated in quantitative yields under acidic condition with satisfying residue tolerances [10]. Inspired by the
versatility of methionine alkylation on especially its great functional group tolerance even for residues like Lys and Cys, we propose a
simple and versatile strategy for reversibly controlling the conformation of short α-helical peptides. A thioether tethered peptide could
be facially synthesized via photo-induced intramolecular thiol-ene reaction but too flexible to induce peptide’s helicity [11]. We could
then functionalize this thioether tether to generate a sulfonium based chiral auxiliary by a facile alkylation of the tether with alkyl
halides under acidic media of trifluoroacetic acid (TFA) [12]. We envision the newly-generated sulfonium chiral center could modulate
the backbone peptides’ secondary structure, similarly as the all-hydrocarbon, sulfilimine and sulfoxide chiral centers. More importantly,

the detachment and recovery of the parent thioether tethered stapled peptides could possibly enable the conformation switching between
random coil and α-helix (Fig. 1).
Model peptide Ac-Y-(cyclo-1,5)-mS5AAAC-NH2 was synthesized through intramolecular thiol-ene reaction (Fig. 2A) [11a]. Then
the cleaved peptide was let react with a panel of different alkylation reagents (Fig. 2B) in TFA at room temperature. We chose
alkylating reagents in terms of their sizes and functional groups. Propinyl and allyl group were successfully attached to the peptide by
alkyaltion reagents (II) and (III) respectively, which might enable a further bioorthogonal modifications [13]. Methyl group of (IV) and
the aromatic groups benzyl of (V), 4-fluorobenzyl of (VI) and 4-acetoxylbenzyl of (VII) were also tested as alkylation groups by there
corresponding alkylation reagents [14]. The alkylation reaction occurred smoothly over 48 h in quantitive conversions except for
aliphatic group of allyl and methyl. Two products of identical molecular weight but different HPLC retention time were identified,
which represented two sulfonium epimers, similar as in our previous reports [3d]. The two epimers of peptides 2a/2b to 7a/7b (Table 1)
could be readily separated by reverse-phase HPLC (Fig. 2C, Figs. S3-S8 in Supporting information). The ratio of the two epimers was
approximately 1:1 according to HPLC traces in Figs. S3-S8. The electron withdrawing vinyl and benzyl groups tended to accelerate the
alkylation process. The retention time differences of the peptide epimers indicated a significant conformational difference between the
two epimers.
The peptide epimers’ conformational preferences were tested and relative helicity of 2b-7b epimers summarized in Table 1. Details
of molar elipticities at λ = 215, 206 and 190 nm were summarized in Table S2 in Supporting information. Consistent with our previous
report, the b-epimers with longer retention time showed helical features in circular dichroism (CD) spectroscopy study while the a-
epimers was mainly random coil. As shown in Fig. 3A, epimer 2b displayed featured helical conformation with double minima at 206
nm and 215 nm [15]. Epimer 2a and its parent thioether tethered peptide 1 showed typical CD spectra of random coils. Sulfonium
peptides with different alkylation groups showed similar pattern as peptide 2a and 2b. The CD spectra were summarized in Fig. 3A and
3B. CD spectra of 3a-7a epimers were shown in Fig. S1 in Supporting information.
2D 1H NMR spectroscopy was conducted in H2O:D2O (9:1) at 298 K. We used peptide 10b (peptide 5b without concentration
determining residue Tyr) as model peptide. The low amide coupling constants (3JNH-Hα ＜6 Hz of three alanines) suggested a helical
structure (Table S3) [3c, 16, 17]. However, due to the signal overlapping of protons, we were unable to unambiguously determine the
key ROEs from NH(i)–Hα (i+4). Then the thermal stability of 2b-7b epimers was tested using CD spectroscopy [18]. We observed
gradual decreases of molar ellipticity as temperature increases from 25 oC to 50 oC (Fig. 4A, Fig. S2 in Supporting information). To
further understand the cause of decrease in helicity of the sulfonium functionalized peptides, we incubated the peptide 8b (Ac-(cyclo-
1,5)-[mS5AYAC(4-F-Bn)]-NH2) under 50 oC overnight. We detected about 36% racemization of peptide 8b which indicated the
decrease of helical content is attributed to the racemization of the chiral auxiliary (Fig. S12 in Supporting information). Meanwhile, the
in vitro serum stability assay (Fig. 4B) showed that cyclic peptide 9 and the sulfonium peptides 10a and 10b remained more than 60
percent intact after 12 h, while the linear analogue was degraded within a few hours.
Furthermore, to explore the detachment of the sulfonium auxiliary, peptide 5b was chosen for model study (Fig. 5A). 2-
Mercaptopyridine (PyS) was used as dealkylation reagent as reported previously [9]. PyS stock (100 μL, 1 mg/mL, 3 equiv.) was added
to the aqueous solution of peptide 5b (0.6 mmol/L, 500 μL) at room temperature. HPLC traces were recorded at different time intervals
as shown in Fig 5B. The regenerated thioether-tethered peptide 1 was observed over time. Peptide 1 ca. 62% was regenerated during 48
h according to integration of the HPLC traces (Fig. 5B and 5C). Thus, the sulfonium chiral center could be successfully dealkylated to
regenerate the parent
thioether tethered peptides. The time dependency of dealkylation process was also studied by CD. The CD spectra of peptide 5b
displayed α-helix at the beginning then gradually switched to a random coil conformation as shown in Fig. S13 in Supporting
information.
To test whether this concept could be translated into longer peptides, model peptide FITC-βA-(cyclo-5,9)-HKILmS5RLLCDS-NH2
(11) targeting estrogen receptor (ER-α) [19] and its 4-bromomethyl-benzyl octanoate alkylated analogue (12) were synthesized [20],
and peptide 12 shows enhanced helical content and cell permeability compared with peptide 11 (Fig. S14 in Supporting).
In summary, we have developed an in-tether chiral sulfonium strategy for reversibly switching peptide conformation by alkylation
and dealkylation of the sulfonium auxiliary under mild conditions. We couldn’t get the crystal structure of sulfonium peptides to
determine the absolute configuration of the sulfonium center. However, we previously reported a carbon or a sulfoxide chiral center at
the same position both need to be R for helicity inducement [3d, 6a], thus, we believe the sulfonium center will prefer the same absolute
configuration. This strategy broadened our scope of CIH strategy and provided a valuable approach to functionalize the peptide tether.
The release of the parent unmodified peptides is unique and not seen in previously reported on-tether modification of constraint
peptides [21]. This traceless redox sensitive tagging strategy could be utilized for various biological application, such as fabrication of
detachable cell penetrating peptides for cargo delivery [22]. Further investigations and biological applications of this sulfonium
auxiliary is under current investigation in the laboratory and will be reported in due course.

Acknowledgements
We acknowledge financial support from the National Natural Science Foundation of China (No. 21372023 and 81572198); Ministry
of Science and Technology of the People’s Republic of China (No. 2015DFA31590); the Shenzhen Science and Technology Innovation
Committee (No. JSGG20140519105550503, JCYJ20150331100849958, JCYJ20150403101146313, JCYJ20160301111338144,
JCYJ20160331115853521, JSGG20160301095829250 and ZDSYS201504301539161); the Shenzhen Peacock Program (No.
KQTD201103).

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Fig. 1. Schematic illustration of reversibly switching of peptide conformation through an in-tether chiral sulfonium.
Fig. 2. (A) Alkylation of thioether tethered peptides in TFA at room temperature (1 mg/mL, alkylation reagent: 10.0 equiv.). (B) Alkylation reagents used in this
study and alkylation conversions. (C) HPLC separation spectrum of alkylated peptide epimers 5a and 5b.
4
3
2
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B
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W
a v e l e n g t h ( n m )
W
a v e l e n g t h ( n m )
Fig. 3. (A) CD spectra of thioether tethered peptide 1 and sulfonium functionalized peptide 2a/2b in double distilled water. (B) CD spectra of chiral sulfonium
induced α-helical peptides 3b-7b in 20% TFE.
2
3
3
5
0
5
 C
 C
 C
A
3
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i m e ( h )
W
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g
t h ( n
m
)
Fig. 4. (A) CD spectra of 5b at elevated temperatures from 25 oC to 50 oC. (B) In vitro serum digestion assay of peptides at 37 oC for 0-12h. Percentage intact,
mean ± s. d. and n = 3. * represent peptides without the residue Tyr.

Fig. 5. (A) Dealkylation of chiral sulfonium α-helical peptides in the presence of PyS in double distilled water. (B) HPLC traces of dealkylation process of 5b at
different time intervals. (C) Plots of percent regenerated peptides versus time.

Table 1
Peptide sequences used in this study and relative helicity.
Peptide
Relative
helicitya
0.81
Ac-Y-(cyclo-1,5)-[mS5AAAC(propinyl)]-NH2 (2b)
Ac-Y-(cyclo-1,5)-[mS5AAAC(allyl)]-NH2 (3b)
Ac-Y-(cyclo-1,5)-[mS5AAAC(Me)]-NH2 (4b)
Ac-Y-(cyclo-1,5)-[mS5AAAC(Bn)]-NH2 (5b)
Ac-Y-(cyclo-1,5)-[mS5AAAC(4-F-Bn)]-NH2 (6b)
Ac-Y-(cyclo-1,5)-[mS5AAAC(4-acetoxyl-Bn)]-NH2 (7b)
0.69
0.68
1.00
0.88
0.57
a The relative helicity was calculated based on reported method in Ref. 3c with peptide 5b set as 100% helical.