One-Pot/Sequential Native Chemical Ligation Using Photocaged Crypto-thioester

Article abstract of DOI:10.1021/acs.orglett.5b03661

A practical and efficient methodology for the chemical synthesis of peptides/proteins using a one-pot/sequential ligation is described. It features the use of photocleavable S-protection on an N-sulfanylethylaniline moiety. Removal of the S-protecting ligated materials under UV irradiation provides a readily usable mixture for subsequent native chemical ligation.

Full text of DOI:10.1021/acs.orglett.5b03661

Letter
pubs.acs.org/OrgLett
One-Pot/Sequential Native Chemical Ligation Using Photocaged
Crypto-thioester
Keisuke Aihara, Kosuke Yamaoka, Naoto Naruse, Tsubasa Inokuma, Akira Shigenaga, and Akira Otaka*
Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima University, 1-78-1 Shomachi,
Tokushima 770-8505, Japan
*
S Supporting Information
ABSTRACT: A practical and efficient methodology for the
chemical synthesis of peptides/proteins using a one-pot/sequential
ligation is described. It features the use of photocleavable S-
protection on an N-sulfanylethylaniline moiety. Removal of the S-
protecting ligated materials under UV irradiation provides a readily
usable mixture for subsequent native chemical ligation.
rotein chemical synthesis has great potential as a chemica1l
P
means for elucidating a wide range of protein functions.
Native chemical ligation (NCL), which has been widely used
for the synthesis of proteins, is among the most powerful
2
methodologies. NCL involves a chemoselective ligation
between N-terminal cysteinyl and C-terminal thioester frag-
ments through an initial transthioesterification, followed by a
rapid intramolecular S → N acyl transfer, to generate a native
peptide bond under mild conditions.
Recent and ever-increasing application of NCL to protein
chemical synthesis has prompted the development of a one-
pot/sequential NCL in a C−to−N- or N−to−C-directive
manner. One-pot synthesis decreases the risk of product loss
that would otherwise have resulted from HPLC purification
3
following every round of NCL. Generally, N-terminal cysteinyl
thioesters, with an N-and/or S-protection on the cysteine,
which are readily removed without affecting the progress of
Figure 1. An outline of one-pot/sequential three-fragment NCL using
SEAlide peptide.
4
,5
NCL, are used in the C−to−N-directive protocol. Alter-
natively, Kent and co-workers developed the kinetically
controlled N−to−C-directive NCL (KCL) protocol which
exploits the difference in reactivity between alkyl thioester and
10
achieved. With the intention of adding the SEAlide peptide to
a multifragment ligation, we planned to use a photocleavable
protecting group on the free thiol group of the SEAlide moiety.
Recently, the 6-nitroveratryl (NV) group was reported to be a
useful protecting group in peptide chemistry, because it can be
6
aryl thioester fragments. This research has encouraged
7
research groups including ours to develop other versions of
the N−to−C protocol. In this context, we previously developed
an N-sulfanylethylanilide (SEAlide) peptide 1 as a crypto-
thioester for the N−to−C-directive one-pot/sequential three-
11
readily removed by UV light under mild conditions. Thus, we
envisioned that the use of a photocleavable protective group on
the SEAlide moiety should contribute the establishment of a
sequential NCL without adding any reagents such as phosphate
salts that modify the reactivity of SEAlide. That is, UV
irradiation after each NCL step would allow sequential NCL to
proceed in a one-pot manner as shown in Figure 2. Herein, we
report a new one-pot/four-fragment sequential ligation method
8
,9
fragment ligation.
In our one-pot protocol, N-terminal
cysteinyl SEAlide peptide initially reacts with a thioester in
the absence of a phosphate salt to afford the first ligation
product; then the resulting SEAlide peptide in the reaction
mixture is converted to a thioester form 2 and allowed to react
with N-terminal cysteinyl peptide only by the addition of a
phosphate salt (Figure 1).
However, use of the SEAlide peptide in a one-pot/sequential
ligation involving more than three fragments has yet to be
Received: December 24, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b03661
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
using a photocaged SEAlide peptide, which enables the
synthesis of protein in the N-to-C direction.
terminal 6 and middle 7 fragments under standard NCL
conditions (3 M guanidine hydrochloride (Gn·HCl)−0.5 M
sodium phosphate buffer (NaPB) (pH 7.0) in the presence of
2
% (v/v) thiophenol at 37 °C) proceeded to give the
corresponding ligated product 9. Next we examined the
removal of the NV group in a one-pot procedure. Direct UV
irradiation of the first ligation mixture gave many byproducts
due to the presence of a thiyl radical derived from thiophenol
during photolysis. We speculated that the generation of the
thiyl radical derived from thiophenol under photolysis
conditions is responsible for generating these byproducts.
Thus, we examined the extractive removal of thiophenol with
Et O from the ligation mixture. Removal of thiophenol from
2
the ligation mixture, followed by UV irradiation, resulted in the
smooth progress of the reaction to yield the desired uncaged
SEAlide peptide 10. The resulting reaction mixture was directly
brought to the second ligation with C-terminal segment 8
under standard conditions (3 M Gn·HCl−0.5 M NaPB (pH
7.0) in the presence of 2% (v/v) thiophenol at 37 °C) to yield a
reduced form hANP 11 with high purity. Then removal of
thiophenol, followed by dilution and subsequent addition of
DMSO (10% (v/v)), allowed the oxidative folding of 11 to
yield hANP 5 in 26% isolated yield after HPLC purification
(Figure 3).
Figure 2. Strategy of peptide/protein synthesis using photocaged
SEAlide peptide.
First, we synthesized the requisite photocaged SEAlide 3 as
shown in Scheme 1. Deprotection of the trityl (Tr) group of
Scheme 1. Synthesis of the Caged SEAlide
Fmoc-Gly-SEAlide 4 with trifluoroacetic acid (TFA)-triethylsi-
lane in CH Cl , followed by reprotection with 6-nitroveratryl
2
2
bromide (NV-Br)-N,N-diisopropylethylamine (DIPEA), af-
forded the desired photocaged SEAlide 3 in 67% isolated
yield over two steps. Then we attempted to synthesize hANP 5
using this photocaged SEAlide as a crypto-thioester moiety.
The three peptide fragments required for hANP synthesis, N-
terminal 6, middle 7, and C-terminal 8, were synthesized using
a standard Fmoc solid-phase peptide synthesis (SPPS). Then
the resulting fragments were used in a one-pot/sequential
three-fragment ligation involving photoremoval of the NV
group as shown in Scheme 2. The first ligation between N-
Figure 3. HPLC monitoring of one-pot/three-fragment ligation for
the synthesis of hANP. (A) Ligation between 6 and 7 (t < 5 min). (B)
Ligation between 6 and 7 (t = 15 h). (C) Ligation between 8 and 10 (t
‡
< 5 min). (D) Ligation between 8 and 10 (t = 18 h). Released
SEAlide unit. *Nonpeptidic compounds probably derived from
photoremoval of the NV unit. HPLC conditions are described in
the Supporting Information.
Scheme 2. Synthesis of hANP Using Sequential NCL
Next, we applied the photocaged SEAlide peptide to a one-
pot/four-fragment sequential NCL for the synthesis of reduced
2
+
form SNX-482 12. SNX-482, potent inhibitor of R-type Ca
channels isolated from the tarantula Hysterocrates gigas, contains
12
4
1 amino acids and three disulfide bonds. The sequence of
SNX-482 was divided in four fragments consisting of SEAlide
3, photocaged SEAlide 14, 15, and N-terminal cysteinyl
1
peptides 16 as shown in Scheme 3. All peptides were prepared
by Fmoc SPPS where the isopeptide protocol was used for the
13
preparation of 16 due to its insoluble nature. The first NCL
between peptide 13 and 14 under standard NCL conditions (3
M Gn·HCl−0.5 M NaPB (pH 7.0) in the presence of 2% (v/v)
thiophenol at 37 °C) yielded a mixture containing ligated
peptide 17 which was then subjected to UV irradiation to give
peptide 18 after extractive removal of thiophenol. The second
NCL for the synthesis of 19 was initiated by addition of
fragment 16 and thiophenol to the reaction mixture containing
B
DOI: 10.1021/acs.orglett.5b03661
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Scheme 3. Synthesis of Reduced Form SNX-482 Using N-to-
C Directed Sequential NCL
Scheme 4. Convergent Synthesis of Reduced Form SNX-482
6
2
-nitroveratryloxycarbonyl (NVOC)-protected SEAlide peptide
1 was synthesized by standard Fmoc SPPS. NCL between 16
and 21 under standard NCL conditions (6 M Gn·HCl−0.2 M
NaPB (pH 7.0) in the presence of 2% (v/v) thiophenol at 37
°
C) gave a reaction mixture containing NVOC-protected
ligated peptide 22, which was then subjected to the extractive
removal of thiophenol followed by UV irradiation for
deprotection of NVOC group, to afford peptide fragment 23
required for the convergent assembly. The blending of reaction
mixtures containing 23 or 18 in the presence of 80 mM MPAA
and 60 mM TCEP·HCl enabled the convergent ligation to
produce the desired reduced form SNX-482 12 in 30% yield
after HPLC purification (Figure 5).
1
8. After completion of the ligation, removal of thiophenol and
subsequent UV irradiation gave the reaction mixture readily
available for the next NCL step. The third NCL was performed
in the presence of 80 mM (4-carboxymethyl)thiophenol
(
MPAA) and 60 mM tris(2-carboxyethyl)phosphine hydro-
chloride (TCEP·HCl) by the addition of fragment 20 to the
reaction mixture to yield the desired reduced form SNX-482 12
in 24% yield after HPLC purification (Figure 4).
To further explore the potential utility of the photocaged
SEAlide peptide, we attempted to synthesize reduced form
SNX-482 12 via a convergent route (Scheme 4). N-Terminally
Figure 5. HPLC profiles for the convergent synthesis of reduced form
SNX-482. (A) Ligation between 16 and 21 (t < 5 min). (B) Ligation
between 16 and 21 (t = 43 h). (C) Ligation between 18 and 23 (t < 5
‡
min). (D) Ligation between 18 and 23 (t = 43 h). Released SEAlide
§
¶
unit. MPAA. Thiophenol. *Nonpeptidic compounds probably
derived from photoremoval of the NV unit. HPLC conditions are
described in the Supporting Information.
In summary, we successfully extended the usefulness of the
SEAlide peptide by caging of the sulfanyl moiety with NV
protection, which allowed the 41-residue SNX-482 to be
synthesized by one-pot/sequential four-fragment ligation in an
N−to−C-directive manner. Alternatively, the combination of
the photocaged SEAlide and NVOC-protected N-terminal
cysteine peptides enabled the convergent assembly of the four
fragments in a one-pot synthesis. These successful results
indicate that the photocaged SEAlide peptide can be employed
in sequential and convergent approaches to peptide/protein
synthesis.
Figure 4. HPLC profiles of sequential ligation for the synthesis of
reduced form SNX-482. (A) Ligation between 13 and 14 (t < 5 min).
(
B) Ligation between 13 and 14 (t = 23 h). (C) Ligation between 15
and 18 (t < 5 min). (D) Ligation between 15 and 18 (t = 20 h). (E)
Ligation between 16 and 20 (t < 5 min). (F) Ligation between 16 and
‡
§
¶
2
0 (t = 25 h). Released SEAlide unit. MPAA. Thiophenol.
*
Nonpeptidic compounds probably derived from photoremoval of the
NV unit. HPLC conditions are described in the Supporting
Information.
C
DOI: 10.1021/acs.orglett.5b03661
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(
11) (a) Karas, J. A.; Scanlon, D. B.; Forbes, B. E.; Vetter, I.; Lewis,
ASSOCIATED CONTENT
Supporting Information
■
R. J.; Gardiner, J.; Separovic, F.; Wade, J. D.; Hossain, M. A. Chem. -
Eur. J. 2014, 20, 9549. (b) Mukherjee, S.; Van Der Donk, W. A. J. Am.
Chem. Soc. 2014, 136, 10450. (c) Ren, W.; Ji, A.; Ai, H. J. Am. Chem.
Soc. 2015, 137, 2155. (d) Miyajima, R.; Tsuda, Y.; Inokuma, T.;
Shigenaga, A.; Imanishi, M.; Futaki, S.; Otaka, A. Biopolymers, in press
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b03661.
(
DOI: 10.1002/bip.22757). (e) Tang, S.; Wan, Z.; Gao, Y.; Zheng, J.-
S.; Wang, J.; Si, Y.-Y.; Chen, X.; Qi, H.; Liu, L.; Liu, W. Chem. Sci.
016, in press (DOI: 10.1039/C5SC03404C).
12) (a) Newcomb, R.; Szoke, B.; Palma, A.; Wang, G.; Chen, X. H.;
General procedures and additional HPLC, NMR, and
MS data (PDF)
2
(
Hopkins, W.; Cong, R.; Miller, J.; Urge, L.; Tarczy-Hornoch, K.; Loo,
J. A.; Dooley, D. J.; Nadasdi, L.; Tsien, R. W.; Lemos, J.; Miljanich, G.
Biochemistry 1998, 37, 15353. (b) Newcomb, R.; Chen, X.; Dean, R.;
Dayanithi, G.; Cong, R.; Szoke, B.; Lemos, J.; Bowersox, S.; Miljanich,
G. CNS Drug Rev. 2000, 6, 153.
AUTHOR INFORMATION
Corresponding Author
■
*
E-mail: aotaka@tokushima-u.ac.jp.
Notes
(13) For a general review of an O-acyl isopeptide, see: (a) Cheng, Q.;
The authors declare no competing financial interest.
Benson, D. R.; Rivera, M.; Kuczera, K. Biopolymers 2006, 83, 297.
Examples for the synthesis of O-acyl building blocks: (b) Yoshiya, T.;
Taniguchi, A.; Sohma, Y.; Fukao, F.; Nakamura, S.; Abe, N.; Ito, N.;
Skwarczynski, M.; Kimura, T.; Hayashi, Y.; Kiso, Y. Org. Biomol. Chem.
ACKNOWLEDGMENTS
■
This research was supported in part by a Grant-in-Aid for
Scientific Research (KAKENHI). K.A. is grateful for a JSPS
fellowship (14J07568). This paper is dedicated to Professor
Nobutaka Fujii on the occasion of his retirement from Kyoto
University.
2
007, 5, 1720.
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D
DOI: 10.1021/acs.orglett.5b03661
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