An in-tether sulfilimine chiral center induces helicity in short peptides

Article abstract of DOI:10.1039/c6cc04508a

A precisely positioned sulfilimine chiral center in the tether of a stabilized peptide would determine the peptide's secondary structure. Peptide sulfilimines could be prepared by a facile chloramine T oxidation and the two resulting peptide diastereomers showed significant differences in their secondary structures, which were supported by circular dichroism spectroscopy and NMR.

Full text of DOI:10.1039/c6cc04508a

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An in-tether sulfilimine chiral center induces
helicity in short peptides†
Cite this:DOI: 10.1039/c6cc04508a
Huacan Lin, Yixiang Jiang, Qingzhou Zhang, Kuan Hu and Zigang Li*
Received 29th May 2016,
Accepted 26th July 2016
DOI: 10.1039/c6cc04508a
www.rsc.org/chemcomm
A precisely positioned sulfilimine chiral center in the tether of a stabilized configuration of the chiral center should be R as shown in Fig. 1,10
peptide would determine the peptide’s secondary structure. Peptide and we propose that the sulfilimine center will also have an
sulfilimines could be prepared by a facile chloramine T oxidation and R configuration.
the two resulting peptide diastereomers showed significant differences
Pentapeptides were prepared to mimic a single-turn helix, to
in their secondary structures, which were supported by circular avoid any sequence perturbation. Different methods for sulfilimine
dichroism spectroscopy and NMR.
preparation were tested and chloramine T stood out as the best
nitrenoid transfer reagent (see Tables S1–S3 for detailed conditions
Protein–protein interactions (PPIs) mediate various kinds of screened, ESI†).11 Compared with other reported reactions such as
biological processes including transcription regulation and signal RCM and azide–alkyne click reactions to construct a tether, the
transduction.1 Short helices in the length of 4–15 amino acids chloramine T oxidation method was free of metal catalysts and
count for about 30% interaction subdomains of all known PPIs.2 compatible with aqueous solution. Then the resulting peptide
Thus, chemically induced helical conformation of short peptides is diastereomers were separated by reverse phase HPLC and their
a focused research area of peptide sciences. Side chain–side chain helicity contents were examined by circular dichroism (CD)
ligation is the most important stabilization strategy, including spectroscopy and are summarized in Fig. 2. Peptides 1–7 with
disulfide,3 lactam bridges,4 all hydrocarbon linkers,5 oxime,6 different tether lengths (peptides 1–4) or different linking
perfluorobenzyl thioether7 and click reactions.8 Recently, Moore et al. patterns (peptides 5–7) were prepared and converted into the
reported that an in-tether chiral center would influence the corresponding sulfilimine easily by chloramine-T oxidation as
secondary structures of stapled peptides.9 Meanwhile, we reported shown in Fig. 2A. Sulfilimines were obtained in satisfying yields
that an in-tether chiral center will dominate the secondary structure varying from 53% to 85% as summarized in Fig. 2. The peptide
of peptides with single-bonded tethers.10 In this report, we further diastereomers were separated by HPLC and the separation
demonstrate that the chirality-induced helicity concept could be spectrum of peptide 3 is presented in Fig. 2C. The peptide
translated into a sulfur-based chiral center of sulfilimine (Fig. 1). diastereomers were called peptides A and B based on their
Structural elucidation of a helical peptide containing an in-tether HPLC retention time respectively. The CD spectra of peptides
carbon chiral center unambiguously showed that the absolute 1A to 7A and peptides 1B to 7B are summarized in Fig. 2D. The
results clearly indicated that only peptide 3B showed characteristic
features of a helical structure in water. When trifluoroethanol (TFE)
was added to aqueous 3B solution, the CD spectrum of 3B showed
some 310 helix features (Fig. S6, ESI†), indicated by the increased
intensity at 204 nm and reduced intensity at 190 nm. Notably, for a
seven-atom tether, when the S = NTs chiral center was switched to
positions other than peptide 3, the helicity greatly diminished in
peptides 5, 6 and 7 (Fig. 2D). Thus, 20-membered ring size with the
S = NTs chiral center positioned at the second site from the
C terminus was determined as the optimal linking strategy, similar
to the carbon chiral center case.10
Analogous cyclic pentapeptides 8–14 with different substituted
amino acid residues were prepared and tested for their secondary
structure in H2O as shown in Fig. 3. Yields comparable to
Fig. 1 An in-tether chiral center will influence the backbone peptides’
secondary structure. Abbreviation: Ts, p-tolylsulfonyl group.
School of Chemical Biology and Biotechnology, Peking University Shenzhen
Graduate School, Shenzhen, 518055, China. E-mail: lizg@pkusz.edu.cn
† Electronic supplementary information (ESI) available: Experimental procedures,
supporting tables and figures. See DOI: 10.1039/c6cc04508a
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Fig. 2 (A) Structures of cyclic sulfilimine pentapeptides 1–4. (B) Conversion of
thioether peptides into sulfilimine peptides 1–7. Abbreviation: homoC,
homocysteine. (C) Representative HPLC separation of peptide diastereomers.
(D) CD spectroscopy of peptides 1A–7A and 1B–7B in H2O at 25 1C. a Reaction
conditions: thioether peptide (1.0 equiv.), chloramine-T (1.2 equiv.) in
CH3CN at r.t. for 24 h. b Yield after HPLC purification and calculated using
1,3,5-tribromobenzene as the internal standard.
Fig. 3 (A) Molar ellipticities [y]215, [y]207, [y]190, [y]215/[y]207 and relative helicity
of 3B and 8B–14B. (B) CD measurements of 8A–14A and 8B–14B in H2O at
25 1C. (C) Two dimensional NMR summary results of 12B performed in 90%
peptides 1–7 were achieved, indicating that the preparation of
sulfilimines using chloramine-T was compatible with different
functional groups (Table S4, ESI†). CD spectra of peptides 8A–14A H2O:10% D2O at 25 1C. Distance restraints in ROEs and their intensities were
reflected in the bar thickness. (D) The dependence of amide NH chemical shifts
showed a single deep minimum at 200–205 nm, supporting the
adoption of a random coil structure in H2O, while their B
diastereomers 8B–14B displayed good characteristic features
on temperature for each residue at 288 K, 293 K, 298 K, 303 K, 308 K and 313 K.
aRelative helicity refers to [y]215(X)/[y]215(10B).
of an a-helix, indicated by a deep minimum at 215/207 nm and
a high maximum at 190 nm (Fig. 3B). The significant difference of their involvement in the a-helix. The temperature coefficient
between A and B diastereomers of peptides 8–14 in CD spectra Dd/T of each residue could be determined by line slopes intuitively
confirmed the decisive stabilization effect of the S = NTs chiral (Fig. 3D). Among the five residues, the first alanine and fifth
center. Details of molar ellipticities at l = 215, 207 and 190 nm cysteine had the minimum line slopes. The observation of strong
are summarized in Fig. 3. The high [y]215/[y]207 ratio of peptides signals at daN(i,i + 3) and dab(i,i + 3) of 12B suggested the adoption
3B and 8B–14B demonstrated a well-defined a-helix in H2O. of a-helical conformation in H2O.
The highest ratio 0.94 was found in peptide 3B, while peptide
To verify whether our a-helix stabilizing strategy was applied to
10B with isoleucine had the lowest value of 0.78. Even in the induce left-handed a-helicity in D-pentapeptides, we prepared 15B
case of substitution with an a-helix breaking residue glycine, composed of all D amino acid residues, termed D-Ac-(cyclo-1,5)-
peptide 8B still showed satisfactory a-helicity with [y]215/[y]207 [S5AIAC(NTs)]-NH2, which was the mirror image of 10B composed
of 0.93. These results illustrated our strategy’s good residue of all L-residues. The CD spectrum of 15B showed opposite
tolerance. As for the absolute configuration of the in-tether chiral absorption characteristics at 215 nm, 207 nm and 190 nm,
center, although we didn’t have direct evidence, it was very reason- suggesting a left-handed a-helix in 15B (Fig. 4A). The neutralization
able to propose an R absolute configuration for the sulfilimine titration by adding 15B to 10B solution showed that the right-
center because the current sulfilimine system was translated from handed a-helicity characteristic in the CD spectrum exhibited by
our previous report of an in-tether chiral carbon center.10
10B was gradually offset by the addition of 15B. The absorption
2D 1H NMR spectroscopy in H2O : D2O (9 : 1) at different curve returned to the baseline when the quantity ratio of 15B/10B
temperatures was also conducted with peptide 12B. The low amide was 1 : 1. Then excess addition of 15B to the L/D mixture exhibited
coupling constants 3JNH-CHa o 6 Hz of S5 and two alanine residues left-handed a-helicity characteristic in the CD spectrum, which
suggested their involvement in the helical structure (Fig. 3C). In certified the feasibility of left-handed a-helicity induced by the
addition, the temperature coefficient Dd/T o 4.5 ppb KÀ1 of the sulfilimine chiral center with all D-residues (Fig. 4B).
residues was also consistent with the helical structure. The low
Stabilities of a-helicity induced by the sulfilimine chiral center
temperature coefficient Dd/T of alanine and cysteine was indicative were explored by thermal denaturation and guanidineÁHCl
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stabilization effects. Furthermore, we have proven that the
absolute configuration of the S = NTs chiral center dominates
the backbone peptides’ secondary structure. Namely, the B
diastereomers with longer retention time in HPLC spectra
showed better helical contents in H2O while A diastereomers
appeared as random coils. CD and 2D NMR experiments
provided strong evidence to support the secondary structure
determination. This new stapling methodology presented good
tolerance to different residues, including glycine. Notably, this
novel helicity-induced method showed good thermal stability,
in guanidineÁHCl denaturation as well as serum digestion. This
discovery enriches the toolbox of peptide stabilization and
helps in further understanding the correlation between the
in-tether chiral center and the backbone peptides’ secondary
structures, and it also provides one valuable modification site
for further applications.
We acknowledge financial support from the National Natural
Science Foundation of China (Grant 21102007, 31300600 and
21372023), MOST 2015DFA31590, MOST 2013CB911500, the
Shenzhen Science and Technology Innovation Committee
(KQCX20130627103353535, SGLH20120928095602764, ZDSY2013-
0331145112855 and JSGG20140519105550503) and the Shenzhen
Peacock Program (KQTD201103). We thank the Beijing NMR
Center at Peking University for their help.
Fig. 4 (A) CD spectrum comparison between L-peptide 10B and D-analog
15B. (B) The absorption change trend of the CD spectrum in the neutralization
titration experiment. Peptide 15B solution was titrated into 10B solution slowly
until 15B was excessive. (C) Full CD spectra of 3B at different temperatures
from 25 1C to 70 1C. (D) Molar ellipticity at 215 nm of 3B at different
temperatures from 25 1C to 70 1C. (E) Molar ellipticity at 215 nm of 12B under
the condition of increasing guanidineÁHCl at 25 1C. (F) In vitro serum digestion
assay of peptide 11B and its linear analog.
Notes and references
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denaturants (Fig. 4). CD measurements of 3B in H2O from 25 1C
to 70 1C showed that 3B maintained a well-defined a-helix even
at 70 1C (Fig. 4C). A gradual increase in molar ellipticity was
observed at 215 nm when the temperature was increased from
25 1C to 70 1C, which indicated that the a-helix content
decreased in 3B (Fig. 4D). Despite some a-helix unwinding,
over 60% a-helicity was preserved even at 70 1C, which suggested
that the sulfilimine chiral center-induced a-helicity was thermally
stable. Pentapeptide 12B was used for chemical denaturation under
the condition of increasing guanidineÁHCl from 0 to 7 M. The
molar ellipticity at 215 nm of 12B remained almost unchanged with
increasing guanidineÁHCl, suggesting that the a-helical conforma-
tion of 12B was stable even in the presence of high concentration of
guanidineÁHCl (Fig. 4E). Besides, the in vitro serum stability assay
showed that more than 90% of the helical peptide 11B with the
sulfilimine chiral center remained intact after 10 hours while over
50% degradation of its linear analog, termed Ac-S5ALAC(SH)-NH2,
was observed (Fig. 4F). Thus, peptide 11B with enhanced helicity
induced by the sulfilimine chiral center showed better proteolytic
resistance.
In summary, we have demonstrated a novel and effective
helix-stabilizing strategy by introducing a precisely positioned
S = NTs chiral center via chloramine-T oxidation. The tether
length and S = NTs positions were screened for the best helix
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