Influence of α-methylation in constructing stapled peptides with olefin metathesis

Article abstract of DOI:10.1016/j.tet.2014.08.004

Ring-closing metathesis is commonly utilized in peptide macro-cyclization. The influence of α-methylation of the amino acids bearing the olefin moieties has never been systematically studied. In this report, controlled reactions unambiguously indicate that α-methylation at the N-terminus of the metathesis sites is crucial for this reaction to occur. Also, we first elucidated that the E-isomers of stapled peptides are significantly more helical than the Z-isomers.

Full text of DOI:10.1016/j.tet.2014.08.004

Tetrahedron 70 (2014) 7621e7626
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Influence of
a
-methylation in constructing stapled peptides with
olefin metathesis
*
Qingzhou Zhang, Xiaodong Shi, Yanhong Jiang, Zigang Li
School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Xili University Town, PKU Campus,
Shenzhen, Guangdong 518055, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 April 2014
Received in revised form 31 July 2014
Accepted 3 August 2014
Available online 7 August 2014
Ring-closing metathesis is commonly utilized in peptide macro-cyclization. The influence of a-methyl-
ation of the amino acids bearing the olefin moieties has never been systematically studied. In this report,
controlled reactions unambiguously indicate that -methylation at the N-terminus of the metathesis
a
sites is crucial for this reaction to occur. Also, we first elucidated that the E-isomers of stapled peptides
are significantly more helical than the Z-isomers.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Macro-cyclic peptide
a
a
-Methylation
-Helix
Ring-closing metathesis
1. Introduction
Recent statistical analysis reveals that
a-helical interfaces are
presented in more than 60% proteineprotein interactions (PPIs)
based on the Protein Data Bank (PDB), approximately 30%t of these
interactions are accomplished by helices composed of 15 amino
acids or less.1 Due to the shallow and large nature of the PPI surface,
small molecule PPI ligands are rare; meanwhile, poor bio-
availability and enzymatic labiality largely limit the application of
peptides.2e4 How to interrupt PPIs remains a formidable task for
the research community. Starting from a few decades ago, re-
searchers noticed that a short peptide with well-defined helical
conformation is more resistant to protease. Numerous methods
have been developed to construct constraint peptides with a fixed
secondary conformation, as shown in Scheme 1.5e11 In general,
most of these methods fix the peptides into cyclic molecules with
constraint conformation by adding a tether to the peptide back-
bone. Salt bridges,12 disulfide bonds,13 amide bonds,14 thioleether
bonds, photo-clicked heterocycle15 formation, olefin metathesis,
etc. have been employed as efficient linking strategy.
Scheme 1. Short summary of a-helix stabilization methods.
serines at the i and iþ4 positions, respectively.16 Shortly after,
Verdine et al. in 2000 reported an all-hydrocarbon linker con-
structed by substituting the amino acids at i, iþ4 positions or i, iþ7
positions with unnatural amino acids bearing an alkenyl side
chain.9 These peptides are named as ‘stapled peptides’ and show
higher helix content, better protease resistance, and cell perme-
ability than their linear analogues. In the past decade, stapled
peptides are utilized to mimic protein binding surfaces in various
Among them, olefin metathesis is the most commonly used
method to construct constraint peptides. Grubbs et al. in 1998 re-
ported that olefin metathesis could be applied in peptide stabili-
zation by adding a tether to the peptides by inserting two O-allyl
biological researches, including Mcl-1,17 hDM2,18 ERa 19
(Scheme 2a). In 2004, Arora et al. constructed a hydrogen bond
,
etc.
* Corresponding author. Tel.: þ86 755 2603 3616; fax: þ86 755 2603 3174; e-mail
addresses: lizg@pkusz.edu.cn, ziganglichembio@gmail.com (Z. Li).
http://dx.doi.org/10.1016/j.tet.2014.08.004
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

7622
Q. Zhang et al. / Tetrahedron 70 (2014) 7621e7626
R'
n
surrogate (HBS) system, which incorporated a 4-pentenoic acid and
N-allyl residue at i, iþ4 positions, respectively (Scheme 2b).11 Their
HBS peptides are also broadly utilized as PPI inhibitors for hDM2,20
Ras-Sos,21 HIF-1,22 etc.
[Ru]
S
[Ru]
S
S
n=2, stable five member ring
intermediate with sulfur chelation
R'
n
n=1, four member ring is too much tension
so double bond chelation favored.
[Ru]
[Ru]
CH
S
S
[Ru]
S
Scheme 3. S-Homoally-cysteine chelates metal catalyst.
form an eight-atom tether, however, only less than 5% of product
was detected (Scheme 4).
Catalysts
No detectable product
standard conditions
Scheme 2. Verdine and Arora utilized olefin metathesis in constructing macro-cyclic
peptides.
: Rink Amide MBHA resin
Although olefin metathesis has been proved as an efficient
method to construct constraint peptides, a detailed study of its
scope and limitation is absent. Due to the limit switching sites of
Arora’s HBS peptides, we focus on stapled peptides and systemat-
ically elucidate the correlation between the linker length, double
S
O
3
A
N
A
A
H
Peptide 4 (trace amount
product)
Scheme 4. Linker length has minimal influence on metathesis efficiency. Catalysts
tested: Grubbs first generation, Grubbs second generation, HoveydaeGrubbs second
generation; standard conditions: 20% catalyst agitated for 2 h in DCE.
bond position, a-substitution on the amino acids at the metathesis
sites and the products’ secondary conformations.
2. Result and discussion
a
-Methylation was always present in Verdine’s stapled pep-
For this study, we try to elucidate the efficiency of metathesis
under the standard solid-phase peptide synthesis condition. Pen-
tapeptides with three alanines as the internal residues and the
unnatural amino acids bearing an olefin substitution at the N- and
C-terminus, respectively, were chosen as the model peptides to
avoid any possible sequence perturbation. All metathesis reactions
were carried out in dichloroethane (DCE) on resin at room tem-
perature for 2 h without heating or microwave radiation.
tides; however, Grubbs et al. utilized two O-allyl serines instead of
Verdine’s unnatural amino acid S5, which could undergo metath-
esis efficiently without
et al. successfully used mS5 synthesized stapled BID BH3 peptide,
but there was an internal glycine between the i and iþ4 position.24
Meanwhile, Nomura et al. used mS5 and O-allyl serine synthesized
HIV-1 integrase inhibitors.25 In their study the metathesis reaction
a-methylation requirement. In 2013, Yeo
must be heated. The previous reports hinted the importance of
methylation, however, there was no systematical elucidation.
Peptide Ac-S5-AAA-S-allyl-C-NH2 (5) was synthesized, the me-
tathesis reaction went smoothly to provide a 74% conversion, which
a-
S5 is a common utilized a-methylated unnatural amino acid for
stapled peptides, S indicates the absolute configuration and 5
means there are five atoms in the olefin substitution. Reported by
Verdine et al., if two S5s at i and iþ4 were switched to S6 and S3,
respectively, metathesis efficiency significantly decreased.9 How-
ever, further examination of the tether length was absent. We
highlighted the importance of the a-methylation for the metathesis
reactions. The two isomers had a ratio of 2.2:1. This higher ratio
may be caused by the formation of plausible intermediate 5-2a,
which preferred isomer 5a (Scheme 5a). If these two unnatural
synthesized S5 analogues with and without
a-methyl group and
named them as Sn or mono-Sn (mSn for short), then systematically
amino acids were switched, peptide Ac-mS5-AAA-S-allyl-
methyl-C-NH2 (6) provided only 17% conversion. If N- and C-ter-
minus were both -methylated, peptide Ac-S5-AAA-S-allyl-
methyl-C-NH2 (7) gave only 10% conversion (Scheme 5b). All these
controlled studies showed the importance of incorporating an
methyl substitution on Sn unnatural amino acid while -methyla-
tion of S-allyl-cysteine decreased the reaction efficiency (Scheme
5b).
Further study revealed that peptide Ac-S-allyl-C-AAA-S5-NH2
(8) and Ac-S-allyl-a-methyl-C-AAA-mS5-NH2 (9) were synthesized
and none of them could react. These results indicated that Sn amino
acids must be positioned at the N-terminus to facilitate this re-
action. This finding was further supported by peptide Ac-S4-AAA-S-
allyl-C-NH2 (10), which reacts smoothly to provide 48% conversion
to form a seven-atom tether (Scheme 6).
Then the finding was further tested in the stapled peptide sys-
tem for constructing all-hydrocarbon tether. Peptides Ac-mS5-
AAA-mS4-NH2 (11), Ac-mS4-AAA-mS5-NH2 (12), Ac-mS5-AAA-
a-
tested the tether length and a-methylation influences on the me-
tathesis efficiency.
a
a-
To mimic stapled peptides’ tether length, Ac-S-homoallyl-C-
AAA-mS3-NH2 (1) was first synthesized, which would form
a seven-atom tether. It was comparable with stapled peptides’
eight-atom all-carbon tether since CeS bond length is significantly
a
-
a
ꢀ
ꢀ
longer than CeC bond (1.8 A vs 1.5 A). Interestingly, this reaction
wouldn’t occur with all metathesis catalysts tested. As reported by
Davis et al. in 2008, homoallyl substitution on sulfur may form an
ideal chelating environment for catalyst and the sulfur atom will
compete with the other alkene.23 In contrast, S-allyl-cysteine is too
tight to form sulfureolefin chelating of metal catalysts (Scheme 3).
Then Ac-S-allyl-C-AAA-mS4-NH2 (2) was tested, however, no
product was detectable with LCeMS as well. Then the amino acids
at the N- and C-terminus were switched, Ac-mS4-AAA-S-allyl-C-
NH2 (3) didn’t provide any products as well. Longer CeS bonds may
not be enough to compensate one atom less in the tether. Then Ac-
mS5-AAA-S-allyl-C-NH2 (4) was constructed, which would also

Q. Zhang et al. / Tetrahedron 70 (2014) 7621e7626
7623
Peptide Ac-S3-AAA-S-homoallyl-C-NH2 (17) could not undergo
metathesis while peptide Ac-S3-AAA-S-allyl-homoC-NH2 (18)
reacted smoothly to give 86% conversion. These two controlled
reactions further proved that S-homoallyl-cysteine indeed chelated
the metal catalyst to inhibit the reaction as shown in Scheme 2
(Scheme 8). Interestingly, peptide 18 gave only one isomer from
the metathesis, which suggested plausible intermediate 18-2a,
which provided more space around the sulfur (Scheme 8).
a
[Ru]
S
O
HN
A
N
A
A
O
H
plausive intermediate 17-1
S
2
O
b
A
N
A
A
S
H
S
[Ru]
[Ru]
A
less bolky
O
less bolky
O
O
O
HN
HN
A
N
A
A
N
A
A
H
H
plausive intermediate18-1
Scheme 5. (a) HPLC analysis of the reaction of peptide 5. (b) Peptides 7 and 8 have
plausive intermediate18-2a
lower yield to peptide 5 for their different
a
-methyl pattern.
Scheme 8. Influence of sulfur position.
S
O
2
A
When metathesis is utilized for macro-cyclic peptide construc-
tion, the resulting peptides may exist as E- and Z-mixtures, how-
ever, the conformational differences between the E- and Z-isomers
of stapled peptides have never been explored. We indeed separated
the E- and Z-isomers by HPLC and the isomers with the longer
N
H
A
A
Peptide 10a and 10b
Z/E=1.3/1
Scheme 6. Influence of a-methyl on metathesis efficiency.
retention time showed higher
a-helical contents by circular di-
chroism (CD) measurement (Scheme 9). 1H NMR experiments
showed the isomer with longer retention time was E isomer and CD
measurements showed that the E-isomers had significantly higher
mS5-NH2 (13), and Ac-mS5-AAA-S5-NH2 (14) could not undergo
metathesis reactions smoothly since they were missing -methyl
substitutions. On the contrast, peptide Ac-S5-AAA-mS5-NH2 (15)
and Ac-S5-AAA-S5-NH2 (16) both reacted well with high conver-
sions (Scheme 7).
a
a
-helicity contents (Scheme 9f). This result suggested that for sta-
pled peptides, E-isomers gave better -helicity.
Interestingly, -methyl of the linear peptide could also enhance
-helicity, which could then facilitate the metathesis reactions.
a
a
its
a
Peptide 14 Ac-mS5-AAA-S5-NH2 is less helical than peptide 16 Ac-
S5-AAA-S5-NH2, peptide 16 had an 87% conversion in metathesis
reaction versus 5% conversion for peptide 14 (Scheme 9f).
3. Conclusion
The systematic study of the
a-methylation influence on the
stapled peptides unambiguously indicated that Sn must be placed
at the N-terminus to facilitate the metathesis reactions at mild
conditions. Otherwise, harsher reaction conditions were required,
which was generally not favorable in peptide synthesis. In-
A
terestingly,
effects on metathesis reactions, no matter it was placed at N- or C-
terminus. One possible explanation is that the -methylation of Sn
may help to fix the olefin tether into a preferable torsion angle for
the metathesis reaction to occur, while the bond angle difference
between sp3 C and sp3 S induced the reactivity differences between
a-methylation on the S-allyl-cysteine casted damaging
O
3
a
A
N
A
A
H
Peptide 15a and 15b
Z/E=0.1/1
S5 and
a-methyl-S-allyl-cysteine. Our findings will help the re-
O
NH
1. Grubbs 1 (20%mol)
2. TFA/TIS/H O
87%
3
HN
A
searchers design their peptide sequences with higher successful
rate.
Interestingly, for most reported stapled peptides, they were
drawn in their Z-form; however, our study showed that E-stapled
N
A
A
O
H
Peptide 16a and 16b
Z/E=0.4/1
Scheme 7. Effects of a-methylation on all-hydrocarbon tether construction.
peptides show significantly higher
a-helical contents than its Z-

7624
Q. Zhang et al. / Tetrahedron 70 (2014) 7621e7626
Scheme 10. (a) Synthesis of amino acids mSn and Sn. (b) Synthesis of S-alkenyl
cysteines.
temperature for 1 h. Then 1.05 equiv of alkene bromide was added
under ice bath and the mixture was stirred for 2 h.
Work up: After complete consumption of starting material on
TLC, the reaction mixture was acidified with 5% acetic acid to pH¼5
and left for precipitation overnight. Then, the reaction mixture was
filtered and the solid was dried and used for the nest step without
purification.
Hydrolysis of Ni-complex procedure: 3 M HCl/DCM/MeOH (2:2:1)
cocktail was added to the alkylated NieGly (5 mL cocktail to
1 mmol Ni-complex) and the reaction mixture was heated to reflux
until no Ni-complex was detectable on TLC. DCM and MeOH were
removed by rotvap. The aqueous solution was extracted with CHCl3
for three times. Then the amino acid aqueous solution was directly
used for next step.
Fmoc protection: EDTA (1 equiv) was added to the amino acid
aqueous solution and the reaction pH was adjusted to 7e8 with
NaHCO3. Then equal volume of acetonitrile and 1.1 equiv of
FmoceOSu were added. The mixture was stirred for 3 h, concen-
trated to remove acetonitrile, and acidified with 1 M HCl to pH 2e3,
extracted with ethyl ether, washed with brine, dried over sodium
sulfate, and purified via column chromatograph with elute DCM/
MeOH (50:1).
Scheme 9. (a) CD spectra of 5a/5b. (b) CD spectra of 10a/10b. (c) CD spectra of 15a/15b.
(d) CD spectra of 16a/16b. (e) CD spectra of 18a. (f) CD spectra of linear peptide 5, 8, 14,
15, 16. (g) Calculated helix contents of peptides. The concentration of peptides is
normalized by HPLC. All CDs are measured with peptides in acetonitrile/H2O (1:3).
These procedures have been repeated multiple times for various
amino acids, the yields varied between 30% and 50% from Ni-
complexes.
4.2.1.1.1. mS3. dH (300 MHz, CDCl3) 7.76 (2H, d, J 7.5), 7.59 (2H, d,
J 7.1), 7.40 (2H, t, J 7.4), 7.31 (2H, t, J7.4), 5.71 (1H, dd, J 16.7, 7.5), 5.30
(1H, d, J 7.7), 5.18 (2H, d, J 12.8), 4.46 (3H, dd, J 26.5, 6.3), 4.23 (1H, t, J
6.8), 2.72e2.34 (2H, m).27
isomers. This finding gives a hint we may improve the overall helix
enhancing effect of stapled peptides by developing a more E-se-
lective metathesis catalyst.
4.2.1.1.2. mS4. dH (300 MHz, CDCl3) 7.75 (2H, d, J 7.4), 7.58 (2H, d,
J 6.3), 7.38 (2H, t, J 7.4), 7.30 (2H, t, J 7.3), 5.85e5.69 (1H, m), 5.24
(1H, d, J 7.9), 5.04 (2H, t, J 13.3), 4.52e4.35 (3H, m), 4.21 (1H, t, J 6.6),
2.20e1.93 (3H, m), 1.87e1.73 (1H, m).28
4.2.1.1.3. mS5. dH (400 MHz, CDCl3) 7.76 (2H, d, J 7.4), 7.60 (2H, d,
J 5.2), 7.40 (2H, t, J 7.3), 7.31 (2H, t, J 7.2), 5.84e5.68 (1H, m), 5.24
(1H, d, J 8.1), 5.08e4.93 (2H, m), 4.41 (3H, dd, J 13.4, 7.0), 4.23 (1H, t,
J 6.8), 2.14e1.86 (3H, m), 1.72 (1H, d, J 9.0), 1.54e1.35 (2H, m).
4. Experimental
4.1. General method
The Fmoc-protected natural amino acids were purchased from
GL Biochem (Shanghai). S5 was purchased from Suli Pharmaceuti-
cal Technology Jiangyin Co., Ltd. Homocysteine was purchased from
Xinxiang Zhiyuan Co., Ltd. Peptides were synthesized manually
following standard Fmoc-protected solid phase peptide synthesis
procedure. Peptides were purified with Reverse Phase HPLC (Wa-
ters 600 and Shimadzu Prominence LC-20AT, column: Agilent
4.2.1.2. Sn. Alkylation of NieAla: To a stirred solution of NieAla
(10.9 g, 20 mmol) in THF (100 mL) was added NaH 60% in mineral
oil (3.0 g, 100 mmol) and the mixture was stirred at room tem-
perature for 1 h. Then 5 equiv of alkene bromide was added and the
mixture was stirred for overnight. Then the mixture was filtrated
carefully and the liquid was concentrated with rotvap. The product
was used for the nest step without purification.
The following steps are similar to the synthesis of mSn.
4.2.1.2.1. S3. Yield 1.5 g, 11% overall yield from 20 g Ni-complex.
dH (300 MHz, CDCl3) 7.76 (2H, d, J 7.4), 7.59 (2H, d, J 7.3), 7.40 (2H, t, J
7.2), 7.31 (2H, td, J 7.4, 1.0), 5.67 (1H, s), 5.52 (1H, s), 5.14 (2H, d, J
11.5), 4.49e4.29 (2H, m), 4.22 (1H, t, J 6.6), 2.81 (1H, s), 2.68 (1H, s),
1.61 (3H, s).
Technologies XDB-C18 250ꢀ9.4 nm, 5
mm). All compounds were
characterized with LCeMS (Shimadzu LCMS2020 using an UHPLC
column, Agilent Technologies ‘Poroshell 120 SB-C18’ 75ꢀ3.0 mm,
2.7 mm) and/or nuclear magnetic resonance (NMR) spectroscopy
(AVANCE-III 300M, 400, 500M). HRMS was taken on ABI Elite.
4.2. Unnatural amino acid synthesis
4.2.1. Synthesis of mSn and Sn (Scheme 10a).26
4.2.1.1. mSn. Alkylation of Ni-Gly: KOH powder (11.2 g,
200 mmol) was added to a stirred solution of NieGly (10.6 g,
20 mmol) in DMF (100 mL) and the mixture was stirred at room
4.2.1.2.2. S4. Yield 0.5 g, 3.9% overall yield from 20 g Ni-com-
plex. dH (400 MHz, CDCl3) 7.77 (2H, d, J 7.5), 7.59 (2H, d, J 7.4), 7.40
(2H, t, J 7.4), 7.32 (2H, td, J 7.4, 0.9), 5.76 (1H, s), 5.57 (1H, s), 4.99

Q. Zhang et al. / Tetrahedron 70 (2014) 7621e7626
7625
(2H, dd, J 27.6, 13.4), 4.40 (2H, s), 4.22 (1H, t, J 6.5), 2.32e2.17 (1H,
m), 2.00 (3H, d, J 35.3), 1.62 (3H, s).
(1H, s), 5.30 (1H, s), 4.34 (1H, s), 4.22 (1H, s), 4.09 (2H, s), 3.08 (2H,
d, J 6.6), 1.91 (3H, s), 1.82 (3H, s), 1.79e1.73 (2H, m), 1.55 (3H, s), 1.35
(3H, s), 1.28e1.15 (12H, m).
4.2.2. Synthesis of S-allyl-cysteine and relative derivatives (Scheme
10b).29
L
-Cysteine hydrochloride (2.90 g, 16 mmol) was mixed with
4.3.1.2. Compound 5b. dH (500 MHz, DMSO) 8.46 (1H, s), 8.15
(1H, s), 7.85 (1H, s), 7.80 (1H, s), 7.54 (1H, s), 7.14 (1H, s), 7.08 (1H, s),
5.43 (1H, s), 5.35 (1H, s), 4.24 (1H, dd, J 9.7, 8.1), 4.12 (1H, s),
4.03e3.96 (2H, m), 2.93 (2H, s), 2.81 (1H, s), 2.73 (1H, s), 2.00 (3H, d,
J 6.8), 1.89 (4H, s), 1.78e1.69 (2H, m), 1.58 (2H, s), 1.40 (2H, s),
1.38e1.19 (13H, m).
allyl bromide (3.0 g, 2.2 mL, 25 mmol) in 50 mL 2 M NH4OH/EtOH
(5:4) and stirred at room temperature for 20 h. The reaction mixture
was concentrated and S-allyl-C precipitated out as white solid. The
solid was filtered, washed with ethanol (50 mL twice), dried under
reduced pressure, and used without further purification. Then Fmoc
protection was taken in H2O/acetonitrile with 1 equiv of FmoceOSu
and 3 equiv of NaHCO3. The product was purified with column with
DCM, DCM/MeOH (20:1) to get 4.30 g Fmoc-protected amino acid,
4.3.2. cyclo-[S4-A-A-A-S-allyl-C]-NH2 (10a/10b)
4.3.2.1. Compound 10a. dH (400 MHz, DMSO) 8.12 (1H, d, J 6.1),
7.93 (1H, s), 7.80 (1H, d, J 7.1), 7.75e7.68 (2H, m), 7.29 (1H, s), 7.17
(1H, s), 5.45e5.36 (2H, m), 4.26 (1H, d, J 6.0), 4.20e4.15 (1H, m),
4.12e4.08 (1H, m), 4.03 (1H, d, J 7.1), 3.06 (2H, dd, J 11.5, 6.2),
2.67e2.65 (1H, m), 2.61 (1H, s), 1.96 (3H, s), 1.86 (3H, s), 1.81e1.74
(1H, m), 1.37 (3H, s), 1.32e1.15 (9H, m).
70% overall yield from L-cysteine hydrochloride.
This protocol was used for other cysteine derivative synthesis.
4.2.2.1. Fmoc-S-allyl-C. Yield 4.3 g, 70%. dH (400 MHz, CDCl3)
7.76 (2H, d, J 7.5), 7.61 (2H, d, J 4.2), 7.40 (2H, t, J 7.4), 7.31 (2H, t, J
7.4), 5.82e5.65 (2H, m), 5.16e5.05 (2H, m), 4.40 (2H, d, J 7.1), 4.24
(1H, t, J 7.1), 3.16 (2H, d, J 7.1), 2.98 (2H, ddd, J 34.3, 14.1, 5.0).
4.3.2.2. Compound 10b. dH (400 MHz, DMSO) 8.30 (1H, s), 8.04
(1H, s), 8.00 (1H, d, J 5.6), 7.82 (1H, d, J 7.8), 7.69 (1H, d, J 7.7), 7.23
(1H, s), 7.09 (1H, s), 5.39 (1H, s), 5.32 (1H, s), 4.17 (2H, d, J 7.2), 4.03
(1H, s), 3.97 (1H, d, J 7.5), 3.07 (2H, s), 2.87e2.82 (1H, m), 2.72 (1H,
dd, J 12.7, 7.7), 2.05 (3H, d, J 22.6), 1.89 (3H, d, J 7.2), 1.79 (1H, s),
1.42e1.15 (12H, m).
4.2.2.2. Fmoc-
a-methyl S-allyl-C.
a-Methyl cysteine was syn-
thesized as previous reported.30 Yield 0.3 g, 45% from
a-methyl
cysteine hydrochloride. dH (400 MHz, CDCl3) 7.77 (2H, d, J 7.5),
7.63e7.56 (2H, m), 7.40 (2H, t, J 7.3), 7.31 (2H, td, J 7.4,1.1), 5.90e5.78
(1H, m), 5.22e5.09 (2H, m), 4.84 (1H, s), 4.43 (2H, d, J 6.9), 4.23 (1H,
t, J 6.7), 3.83 (2H, dd, J 9.3, 3.8). dC (101 MHz, CDCl3) 177.69, 154.82,
143.75, 143.71, 141.27, 133.93, 127.70, 127.09, 125.06, 119.96, 117.68,
66.84, 60.29, 47.07, 35.86, 30.90, 23.38. ESI HRMS m/z found
420.1242 (MþNa)þ, calcd for C22H23NNaO4S exact mass: 420.1245.
4.3.3. cyclo-[S5-A-A-A-mS5]-NH2 (15a/15b)
4.3.3.1. Compound 15a. dH (500 MHz, DMSO) 7.98 (1H, s), 7.91
(1H, s), 7.86e7.83 (1H, m), 7.64 (2H, s), 7.14 (1H, s), 7.03 (1H, s), 5.32
(2H, d, J 4.7), 4.27 (1H, s), 4.20e4.14 (1H, m), 4.06 (2H, d, J 6.5),
2.02e1.95 (2H, m), 1.86 (7H, s), 1.62 (4H, s), 1.52e1.43 (2H, m),
1.42e1.17 (12H, m).
4.2.2.3. Fmoc-S-allyl-homoC. Yield 0.15 g, 23% from homo-
cysteine hydrochloride. dH (400 MHz, CDCl3) 7.76 (2H, d, J 7.5), 7.58
(2H, d, J 3.3), 7.40 (2H, t, J 7.4), 7.31 (2H, t, J 7.4), 5.76 (1H, dt, J 16.5,
7.1), 5.38 (1H, d, J 8.1), 5.08 (2H, dd, J 10.9, 4.5), 4.47 (2H, dd, J 35.5,
5.7), 4.22 (1H, dd, J 8.6, 5.0), 3.13 (2H, d, J 7.1), 2.52 (2H, t, J 7.3),
2.23e2.13 (1H, m), 1.98 (1H, dd, J 14.2, 7.2). dC (75 MHz, CDCl3)
176.59, 156.25, 143.82, 143.62, 141.34, 134.04, 127.82, 127.15, 125.11,
120.07, 117.44, 67.21, 53.07, 47.11, 34.48, 31.70, 26.13. ESI HRMS m/z
found 398.1421 (MþH)þ, calcd for C22H24NO4S exact mass:
398.1426.
4.3.3.2. Compound 15b. dH (500 MHz, DMSO) 8.84 (1H, d, J 3.3),
8.40 (1H, s), 7.97 (1H, d, J 6.3), 7.43 (1H, d, J 8.6), 7.10 (1H, s), 6.98
(1H, d, J 8.2), 6.61 (1H, s), 5.39e5.32 (1H, m), 5.27 (1H, dd, J 10.0,
4.2), 4.23e4.16 (1H, m), 3.98e3.88 (2H, m), 3.82 (1H, dd, J 7.4, 3.4),
2.00e1.83 (6H, m), 1.76e1.48 (7H, m), 1.44e1.20 (15H, m), 1.16 (2H,
d, J 12.4).
4.3.4. cyclo-[S5-A-A-A-S5]-NH2 (16a/16b)
4.3.4.1. Compound 16a. dH (500 MHz, DMSO) 8.10 (1H, s), 7.79
(2H, d, J 7.7), 7.57 (1H, s), 7.25 (1H, d, J 3.6), 7.18 (1H, s), 7.06 (1H, s),
5.32 (2H, s), 4.19e4.08 (3H, m), 1.87 (7H, s), 1.63 (2H, s), 1.27 (15H,
ddd, J 37.8, 23.5, 11.7).
4.2.2.4. Fmoc-S-homoallyl-C. Yield 0.53 g, 81% from cysteine
hydrochloride. dH (300 MHz, CDCl3) 7.76 (2H, d, J 7.4), 7.60 (1H, d, J
7.0), 7.40 (1H, t, J 7.4), 7.31 (1H, t, J 7.3), 5.75 (1H, ddd, J 20.7,17.1, 9.0),
5.05 (1H, t, J 12.6), 4.71e4.52 (1H, m), 4.41 (1H, d, J 7.0), 4.23 (1H, t, J
6.9), 3.05 (1H, d, J 4.7), 2.68e2.53 (1H, m), 2.32 (1H, d, J 7.0). dC
(75 MHz, CDCl3) 175.32, 155.97, 143.67, 143.62, 141.32, 136.19,
127.80, 127.13, 125.12, 120.05, 116.47, 77.48, 77.06, 76.64, 67.42,
53.48, 47.06, 34.12, 33.71, 32.16. ESI HRMS m/z found 420.1241
(MþNa)þ, calcd for C22H23NNaO4S exact mass: 420.1245.
4.3.4.2. Compound 16b. dH (500 MHz, DMSO) 8.86 (1H, s), 8.41
(1H, s), 7.84 (1H, d, J 6.6), 7.20 (1H, d, J 8.5), 7.07 (1H, s), 6.88 (1H, s),
6.55 (1H, s), 5.42e5.34 (1H, m), 5.34e5.24 (1H, m), 4.10e4.01 (1H,
m), 3.96e3.85 (2H, m), 1.93 (4H, s), 1.90e1.83 (1H, m), 1.70 (3H, dd, J
15.5, 9.1), 1.55 (1H, d, J 7.5), 1.39e1.20 (15H, m).
4.3.5. cyclo-[S3-A-A-A-S-allyl-homoC]-NH2 (18a). dH (500 MHz,
DMSO) 8.81 (1H, s), 8.61 (1H, s), 7.67 (1H, d, J 6.4), 7.42 (1H, d, J 9.3),
7.21 (1H, d, J 7.9), 7.09 (1H, s), 6.64(1H, s), 5.30 (2H, s), 4.30e4.23
(1H, m), 4.06e4.01 (1H, m), 3.97e3.91 (1H, m), 3.77 (1H, d, J 10.1),
3.02 (1H, d, J 13.7), 2.93 (1H, d, J 5.7), 2.80 (1H, d, J 7.7), 2.42 (1H, d, J
8.9), 2.37 (1H, d, J 12.7), 2.23 (1H, d, J 5.9), 1.94 (3H, s), 1.76 (2H, d, J
9.7), 1.37e1.24 (12H, m).
4.3. Olefin metathesis procedure
Ac-X-A-A-A-Y-Resin (50 mg) was swelled in 1 mL DCM for 5 min
then washed with DCE (1 mL) twice. Catalyst of 20% was dissolved in
1 mL DCE and added to the resin. The mixture was agitated with N2
for 2 h. The resin was then cleaved with cocktail of TFA/TIS/H2O
(95:2.5:2.5) for 30 min, dried with nitrogen, precipitated with cold
ether for three times, and the precipitatewas collected and dissolved
in 0.5 mL acetonitrile/H2O (1:1) for purification after filtration.
4.4. CD measurement procedure
4.3.1. cyclo-[S5-A-A-A-S-allyl-C]-NH2 (5a/5b)
Peptides were dissolved in 25% acetonitrile. CD spectra were
obtained on a Chirascan Circular Dichroism Spectrometer at 25 ꢁC
using the standard measurement parameters: wavelength,
4.3.1.1. Compound 5a. dH (400 MHz, DMSO) 7.97 (1H, d, J 8.1),
7.88 (1H, s), 7.84 (1H, s), 7.74 (2H, s), 7.39 (1H, s), 7.16 (1H, s), 5.45

7626
Q. Zhang et al. / Tetrahedron 70 (2014) 7621e7626
8. Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y.-S.; Pentelute, B. L. J. Am. Chem.
Soc. 2013, 135, 5946e5949.
9. Schafmeister, C. E.; Po, J.; Verdine, G. L. J. Am. Chem. Soc. 2000, 122, 5891e5892.
10. Cabezas, E.; Satterthwait, A. C. J. Am. Chem. Soc. 1999, 121, 3862e3875.
11. Chapman, R. N.; Dimartino, G.; Arora, P. S. J. Am. Chem. Soc. 2004, 126,
12252e12253.
190e250 nm; step resolution, 0.5 nm; speed, 20 nm/s; accumula-
tions, 10; response, 1 s; bandwidth, 1 nm; path length, 0.1 cm. Every
sample was scanned twice and the final CD spectra were smoothed
to reduce noise.
12. Marqusee, S.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8898e8902.
13. Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz, P. G. J. Am. Chem.
Soc. 1991, 113, 9391e9392.
Acknowledgements
14. Shepherd, N. E.; Hoang, H. N.; Abbenante, G.; Fairlie, D. P. J. Am. Chem. Soc. 2005,
We thank the National Natural Science Foundation of China
Grant 21102007, the Shenzhen Science and Technology Innovation
Committee SW201110018, ZDSY20130331145112855, the Shenzhen
Peacock Program (KQTD201103) to Z.L.
127, 2974e2983.
15. Madden, M. M.; Vera, C. I. R.; Song, W.; Lin, Q. Chem. Commun. 2009,
5588e5590.
16. Blackwell, H. E.; Grubbs, R. H. Angew. Chem., Int. Ed. 1998, 37, 3281e3284.
17. Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. Nat. Chem. Biol. 2010, 6,
595e601.
Supplementary data
18. Bernal, F.; Wade, M.; Godes, M.; Davis, T. N.; Whitehead, D. G.; Kung, A. L.; Wahl,
G. M.; Walensky, L. D. Cancer Cell 2010, 18, 411e422.
19. Phillips, C.; Roberts, L. R.; Schade, M.; Bazin, R.; Bent, A.; Davies, N. L.; Moore, R.;
Pannifer, A. D.; Pickford, A. R.; Prior, S. H. J. Am. Chem. Soc. 2011, 133,
9696e9699.
20. Henchey, L. K.; Porter, J. R.; Ghosh, I.; Arora, P. S. ChemBioChem 2010, 11,
2104e2107.
21. Patgiri, A.; Yadav, K. K.; Arora, P. S.; Bar-Sagi, D. Nat. Chem. Biol. 2011, 7, 585e587.
22. Henchey, L. K.; Kushal, S.; Dubey, R.; Chapman, R. N.; Olenyuk, B. Z.; Arora, P. S.
J. Am. Chem. Soc. 2009, 132, 941e943.
H NMR and C NMR of amino acids and cyclic peptides, LCeMS
spectra of peptides. Supplementary data associated with this article
can be found in the online version, at http://dx.doi.org/10.1016/
j.tet.2014.08.004.
References and notes
23. Lin, Y. A.; Chalker, J. M.; Floyd, N.; Bernardes, G. J.; Davis, B. G. J. Am. Chem. Soc.
2008, 130, 9642e9643.
24. Yeo, D. J.; Warriner, S. L.; Wilson, A. J. Chem. Commun. 2013, 9131e9133.
25. Nomura, W.; Aikawa, H.; Ohashi, N.; Urano, E.; Metifiot, M.; Fujino, M.; Maddali,
1. Jochim, A. L.; Arora, P. S. ACS Chem. Biol. 2010, 5, 919e923.
2. Berg, T. Proteineprotein Complexes: Analysis, Modeling and Drug Design; Impe-
rial: London, UK, 2010; pp 318e339.
3. Hajduk, P. J.; Greer, J. Nat. Rev. Drug Discov. 2007, 6, 211e219.
4. Conte, L. L.; Chothia, C.; Janin, J. J. Mol. Biol. 1999, 285, 2177e2198.
5. Chorev, M.; Roubini, E.; McKee, R. L.; Gibbons, S. W.; Goldman, M. E.; Caulfield,
M. P.; Rosenblatt, M. Biochemistry 1991, 30, 5968e5974.
6. Cantel, S.; Le Chevalier Isaad, A.; Scrima, M.; Levy, J. J.; DiMarchi, R. D.; Rovero,
P.; Halperin, J. A.; D’Ursi, A. M.; Papini, A. M.; Chorev, M. J. Org. Chem. 2008, 73,
5663e5674.
7. Jo, H.; Meinhardt, N.; Wu, Y.; Kulkarni, S.; Hu, X.; Low, K. E.; Davies, P. L.; De-
Grado, W. F.; Greenbaum, D. C. J. Am. Chem. Soc. 2012, 134, 17704e17713.
ꢁ
K.; Ozaki, T.; Nozue, A.; Narumi, T. ACS Chem. Biol. 2013, 8, 2235e2244.
26. Saghiyan, A. S.; Dadayan, S. A.; Petrosyan, S. G.; Manasyan, L. L.; Geolchanyan,
A. V.; Djamgaryan, S. M.; Andreasyan, S. A.; Maleev, V. I.; Khrustalev, V. N.
Tetrahedron: Asymmetry 2006, 17, 455e467.
27. Oppolzer, W.; Lienard, P. Helv. Chim. Acta 1992, 75, 2572e2582.
28. Kaul, R.; Surprenant, S.; Lubell, W. D. J. Org. Chem. 2005, 70, 3838e3844.
29. Bautista, A. D.; Appelbaum, J. S.; Craig, C. J.; Michel, J.; Schepartz, A. J. Am. Chem.
Soc. 2010, 132, 2904e2906.
30. Armstrong, M. D.; Lewis, J. D. J. Org. Chem. 1951, 16, 749e753.