Efficient synthesis of trypsin inhibitor SFTI-1 via intramolecular ligation of peptide hydrazide

Article abstract of DOI:10.1016/j.tetlet.2014.03.093

Cyclic peptide trypsin inhibitor SFTI-1 was synthesized via intramolecular ligation of a linear peptide hydrazide with high yield. This cyclization strategy did not cause epimerization at the C-terminal Arg residue. CD spectrum and NMR spectroscopy analysis demonstrated that well-folded SFTI-1 could be obtained via standard oxidative folding process. Thus, we present a simple and cost-efficient strategy for the synthesis of SFTI-1.

Full text of DOI:10.1016/j.tetlet.2014.03.093

Tetrahedron Letters 55 (2014) 2883–2886
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Efficient synthesis of trypsin inhibitor SFTI-1 via intramolecular
ligation of peptide hydrazide
Yi-Qun Chen a,b, Chen-Chen Chen b, Yao He c, Mu Yu c, Lin Xu d, Chang-lin Tian c, Qing-Xiang Guo d,
Jing Shi d, , Min Zhang a, , Yi-Ming Li b,
⇑
⇑
⇑
a School of Life Science, Anhui University, Hefei, Anhui 230601, China
b School of Medical Engineering, School of Chemical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China
c China High Magnetic Field Laboratory, Chinese Academy of Sciences, School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
d Department of Chemistry, University of Science and Technology of China, Hefei 230026, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclic peptide trypsin inhibitor SFTI-1 was synthesized via intramolecular ligation of a linear peptide
hydrazide with high yield. This cyclization strategy did not cause epimerization at the C-terminal Arg res-
idue. CD spectrum and NMR spectroscopy analysis demonstrated that well-folded SFTI-1 could be
obtained via standard oxidative folding process. Thus, we present a simple and cost-efficient strategy
for the synthesis of SFTI-1.
Received 27 February 2014
Revised 15 March 2014
Accepted 20 March 2014
Available online 27 March 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Cyclic peptides
Protein chemical synthesis
SFTI-1
Peptide hydrazides
Cyclic peptides, which are generally referred to peptides with an
end-to-end or head-to-tail linkage of the peptide backbone through
an amide bond, have been considered as useful tools in pharmaceu-
tical science and chemical biology.1 Compared to their linear
counterparts, the cyclization of peptide has generated conforma-
tional constrained analogues with improved bioactivity, selectivity,
bioavailability, and higher receptor-binding affinities.2,3 For the
preparation of cyclic peptides, a classical approach relies on pro-
tected linear precursors which are lactamized in organic solvents,
either at high dilution or using pseudo-dilution on a solid support.4
A major drawback of this approach is the necessity of high enthalpic
activation for the acylating moiety during the lactamization pro-
cess, which often results in epimerization of the C-terminal amino
acid residue and oligomerization to dimers and trimers.5
To overcome aforementioned limitations, many groups devel-
oped alternative methods to cyclize fully unprotected peptides
and proteins in aqueous solution mainly based on the native chem-
ical ligation (NCL).6 Moreover, Staudinger ligation, thiazolidine-
forming ligation, and an imine-induced ring-closing/contraction
strategy used for peptide cyclization have also been reported with
own advantages such as the moderately high concentrations for
cyclization.7 Recently, peptide hydrazides were reported to be a
thioester equivalent reagent in NCL.8 The hydrazide based ligation
has been used for protein total and semi-synthesis, protein modi-
fication, and the synthesis of cyclic peptides and cyclic proteins.9
One of the main advantages is that peptide hydrazides can be
easily prepared by using Fmoc solid-phase peptide synthesis, and
therefore various cyclic peptides could be synthesized with high
efficiency in an epimerization-free manner.10 Another advantage
is that good yield can be obtained for moderately high concentra-
tions of linear reactants.
Sunflower trypsin inhibitor (SFTI-1) is a naturally occurring
cyclic peptide with only 14 residues, which is regarded as one of
the smallest and most potent trypsin inhibitor as known.11 Since
its discovery, SFTI-1 has attracted much interest because of its
small size and high potency, which make it an ideal candidate as
a platform for the design of novel small proteinase inhibitors.12
However, the biosynthesis and peptide cyclization mechanisms
for SFTI-1 are still beyond complete understanding, which has re-
stricted their production by genetic approaches.13 Based on this,
many groups have reported the synthesis of SFTI-1 using solid-
phase Fmoc chemistry with the latter cyclization of SFTI-1 in the
solution or an on-resin cyclization approach.13 Recently, Goransson
et al. first synthesized SFTI-1 under microwave-accelerated
solid phase peptide synthesis and native chemical ligation with
improved efficiency (45% isolated yield).14 Moreover, Tam and
co-workers reported the synthesis of SFTI-1 using a C-terminal
⇑
Corresponding authors. Tel.: +86 551 62877080.
E-mail address: lym2007@mail.ustc.edu.cn (Y.-M. Li).
http://dx.doi.org/10.1016/j.tetlet.2014.03.093
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

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Y.-Q. Chen et al. / Tetrahedron Letters 55 (2014) 2883–2886
thioethylamido moiety (TEA) containing thioester surrogate
(17% isolated yield).15 In spite of these improvements, efforts are
needed to overcome current constraints, for example, relatively
long time (16–24 h), high temperature, and time-consuming steps
for the synthesis of thioester and its surrogate for cyclization.
Herein, we report a highly efficient and time-efficient method of
the synthesis of SFTI-1 via the ligation of peptide hydrazide. Take
the advantage of this hydrazide based native chemical ligation,
we found that SFTI-1 can be synthesized with increased simplicity
and lower costs (Scheme 1).
Our study began with the synthesis of linear peptide precursor
1a by using the commercially available 2-Cl-(Trt)-Cl resin
(Scheme 1). As previously reported, a hydrazine-Trt(2-Cl) resin
was readily prepared as the key intermediate, and all the peptides
were synthesized by the solid-phase method using standard Fmoc
chemistry. After completing the synthesis, the peptides were
cleaved from the resin simultaneously with the side chain depro-
tection in a one-step procedure, using a mixture of TFA/phenol/
water/triisopropylsilane (88:5:5:2, v/v/v/v). The crude peptides
were dissolved in CH3CN/H2O (1:1, v/v) and purified using RP-
HPLC. The purity of linear peptide 1a was confirmed by analytical
HPLC and ESI-MS.
Then, we carried out the peptide cyclization in a one-pot fash-
ion. Firstly, the linear peptides 1a (1 mM) were dissolved in
800 lL of an aqueous phosphate buffer (100 mM) with 6.0 M guan-
idinium chloride and 10 equiv of oxidation solution NaNO2 was
added drop-wise to the reaction system. The reaction mixtures
were held at À10 °C, pH 3.0, and stirred for 20 min. After that,
we added 40 equiv of thiol additive MPAA (4-mercaptophenylace-
tic acid) to the reaction tubes. Solutions were then adjusted to pH
7.0, stirred for 2–4 h at room temperature. The reaction was
monitored using HPLC and the cyclic peptide (1b) was identified
by ESI-MS. The high yield (61% isolated yield) was obtained
through aforementioned hydrazide based ligation. We next exam-
ined the correlation between the peptide concentration and the
yield. We found that relatively high yield (57% isolated yield) can
be obtained in low concentration (e.g., 60 lM) of the reactant
while moderately high concentrations (e.g., 10 mM) also resulted
in good yield (53% isolated yield). To further perform the
intra-molecular disulfide bridge formation of SFTI-1, the oxidative
folding was conducted using the solution of CH3CN/H2O (1:1, v/v).
It has been reported that the disulfide bond is likely to reduce its
conformational flexibility significantly in natural analogue.11b In
detail, 1 mg SFTI-1 was dissolved in 800 lL of 50% CH3CN with
Scheme 1. (a) The concise scheme for linear peptide synthesis (SPPS); (b) The synthesis of SFTI-1 via the ligation of peptide hydrazides.

Y.-Q. Chen et al. / Tetrahedron Letters 55 (2014) 2883–2886
2885
200 lL DMSO (Dimethyl sulfoxide) and the pH was adjusted to 6.0.
After stirred for 2 h at room temperature, the oxidized SFTI-1 (1c)
was acquired with nearly 55% isolated yield (Fig. 1).
Another important issue in the cyclic peptide synthesis was epi-
merization at the cyclization site. Then, we test the possibility of
racemization of the C-terminal residue under ligation conditions
because the epimerization of Arg at the C-terminal has not been
reported in previous studies.8a We compared the cyclization of
H-Cys-Thr-Lys-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro-Asp-Gly-(L)Arg-
NHNH2 and H-Cys-Thr-Lys-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro-Asp-
Gly-(D)Arg-NHNH2 to test whether the peptide hydrazide
cyclization caused any epimerization at the C-terminal amino acid.
The peptide ligation process was performed as mentioned
above. Through co-injection analysis with either L- or D-Arg at
the C-terminus (Fig. 2), we confirmed that hydrazide-based liga-
tion at Arg residue was epimerization-free.
With the compounds in hand, we next explored the three-
dimensional structure of synthetic SFTI-1. The folding of this syn-
thetic peptide was found to occur automatically in PBS (6.0 M
guanidinium chloride, 0.2 M NaH2PO4) buffer at pH 7.4. The circu-
lar dichroism (CD) spectrum of SFTI-1 in water indicated three
peaks, including a positive peak at 198 nm, a negative minimum
at 208 nm, and a strong positive band at 228–230 nm (Fig. 3).
These observations were consistent with those previously reported
that SFTI-1 contained well defined b-sheet structure.16
Figure 2. HPLC analysis of the crude cyclization mixture of linear peptides. No
epimerization at the cyclization site was observed.
To further corroborate the above conclusions, we conducted
solution NMR spectroscopy (including DQF-COSY, TOCSY, and
Figure 3. CD spectrum analysis of the structure of peptide 1c.
Table 1
Structural statistics for the final 20 conformers of SFTI-1
Structural restraints
NOE distance restraints
Intraresidue
239
92
Sequential
69
Medium-range (2 6 |iÀj| 6 4)
51
27
Long-range (|iÀj| P 5)
Statistics for 20 structures
Rmsd for experimental restraints
NOE distance constraints
Rmsd from idealized covalent geometry
Bonds (Å)
Angles (°)
Impropers (°)
0.0388 0.0008
0.0052 0.0001
0.8983 0.0063
0.7198 0.0117
Average RMSD of Atomic Coordinates (Å)
Backbone atoms
All heavy atoms
0.078
0.676
Ramachandran analysis (%)
Most favored
Additionally allowed
Generously allowed
Disallowed
70.6
29.4
0
0
NOESY) for synthetic SFTI-1. The three-dimensional structure of
SFTI-1 was determined using solution NMR methods (Table 1). Pro-
tons of all residues in the sequence were assigned using the data
from DQF-COSY, TOCSY, and NOESY spectra. For cyclic SFTI-1 pep-
tide, a set of 200 structures were calculated using a total of 239 dis-
tance restrains comprising 92 intra-residue, 69 sequential, 51
Figure 1. Analytical HPLC trace and ESI-MS for linear peptide (obs: 1547.4; calcd:
1547.8 Da); cyclic peptide (obs: 1514.8; calcd: 1515.8 Da), and oxidized peptide
SFTI-1 (obs: 1513.5; calcd: 1513.8 Da).

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Y.-Q. Chen et al. / Tetrahedron Letters 55 (2014) 2883–2886
medium-range, and 27 long-range, which were derived from the
NMR data. 20 low overall energy and low constraint violation struc-
tures were selected for further analysis. The root-mean-square devi-
ation (RMSD) from the structure ensembles of SFTI-1 is 0.078 Å for
the backbone atoms and 0.676 Å for all heavy atoms. The deter-
mined solution structures revealed that SFTI-1 adopted an ordered
b-hairpin structure, consistent with the CD data. The two b-strands,
comprising residues 2–5 and 9–12, are joined by one b-turn and one
loop, identically to the solution NMR structure reported before. A
strong NOE signal between Hd(P9) and Ha(P8) suggested that P9
is in the trans-conformation, at the same time P8 is in the cis-confor-
mation since there exists a strong cross signal between Ha(P8) and
Ha(I7). In the SFTI-1 structure, distance between CO(R2) and
HN(F12) is 1.75 Å and distance between CO(I10) and HN(T4) is
2.75 Å, close enough to generate hydrogen bonds to maintain the
overall b-hairpin structure. The electrostatic surface shows that
SFTI-1 have a hydrophobic surface (I7, I10, and F12) in one end
and a charged surface on the other end (positive residues R2, K5
on the side, and negative residue D14 on the other side) (Fig. 4).
In summary, we reported a highly efficient and facile strategy
for the synthesis of cyclic peptide trypsin inhibited SFTI-1 via
hydrazide based native chemical ligation. Our experiments con-
firmed that hydrazide-based ligation at fully unprotected Arg res-
idue was in an epimerization-free manner. The cyclization can be
easily performed with high yield (61% isolated yield). Another
advantage of this strategy is that peptide hydrazides can be easily
prepared by using the routine use of Fmoc solid-phase peptide syn-
thesis. CD spectrum and NMR spectroscopy demonstrated that the
well three-dimensional structure of SFTI-1 could be obtained via
standard oxidative folding process. Moreover, chemically synthe-
sized SFTI-1 has been demonstrated that own actual inhibition
activity as natural analogue,16 which should greatly expand its
application. Finally, we considered that the chemoselectivity is
another advantage of ligation of peptide hydrazide for the synthe-
sis of cyclic peptide.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21372058 to Y.M.L.; 31340044 to M. Zhang;
21272223 to Q.X. Guo).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.03.093.
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Figure 4. Structure analysis of SFTI-1. (a) Backbone alignment of 20 lowest-energy
structures of cyclic SFTI-1; (b) the ribbon representation of the SFTI-1 peptide; (c)
the surface diagram of SFTI-1. Positively charged areas are colored in blue,
negatively charged areas in red and uncharged areas in white.