Synthesis of a secretoglobin 3A2 type C (98–139) fragment by Dawson's native chemical ligation

Article abstract of DOI:10.1016/j.tetlet.2017.09.058

A secretoglobin 3A2 type C (98–139) peptide was synthesized by native chemical ligation between ¹¹⁵Ile and ¹¹⁶Cys residues using Dawson's linker. The peptide-N-acyl-benzimidazolinone-glycine amide, a C-terminal thioesters precursor, was provided from 3-amino-4-(methylamino)benzoic acid. In addition, an N-terminal cysteine fragment, the (116–139) peptide, was prepared by ordinary Fmoc-solid phase peptide synthesis. Native chemical ligation of the (98–115) fragment with the Dawson's linker and the (116–139) peptide smoothly proceeded to give SCGB3A2 type C (98–139) peptide.

Full text of DOI:10.1016/j.tetlet.2017.09.058

Tetrahedron Letters xxx (2017) xxx–xxx
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Tetrahedron Letters
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Synthesis of a secretoglobin 3A2 type C (98–139) fragment by Dawson’s
native chemical ligation
⇑
Mariko Kikuchi, Reiko Kurotani, Hiroyuki Konno
Department of Biological Engineering, Graduate School of Science and Engineering, Yamagata University, Yonezawa, Yamagata 992-8510, Japan
a r t i c l e i n f o
a b s t r a c t
A secretoglobin 3A2 type C (98–139) peptide was synthesized by native chemical ligation between 115Ile
and 116Cys residues using Dawson’s linker. The peptide-N-acyl-benzimidazolinone-glycine amide, a C-
terminal thioesters precursor, was provided from 3-amino-4-(methylamino)benzoic acid. In addition,
an N-terminal cysteine fragment, the (116–139) peptide, was prepared by ordinary Fmoc-solid phase
peptide synthesis. Native chemical ligation of the (98–115) fragment with the Dawson’s linker and the
(116–139) peptide smoothly proceeded to give SCGB3A2 type C (98–139) peptide.
Article history:
Received 9 August 2017
Revised 15 September 2017
Accepted 19 September 2017
Available online xxxx
Keywords:
Secretoglobin 3A2
Ó 2017 Elsevier Ltd. All rights reserved.
Native chemical ligation
N-Methyldiaminobenzoyl
N-Methylbenzimidazolinone
Introduction
residues. If there are difficult sequences for SPPS, the scope of
application becomes limited to shorter peptides. Native chemical
Secretoglobin (SCGB) 3A2,1–6 also called UGRP1 (uteroglobin-
related protein 1), is a member of the SCGB gene superfamily,
which is comprised of low molecular weight secretory proteins
of approximately 10 kDa. SCGB3A2 plays a role in lung develop-
ment, as well as anti-inflammatory, and anti-fibrotic activities, in
the lungs of several model mice. There are three types of SCGB3A2,
which have different sequences and peptide lengths on the C-ter-
minus. All types of SCGB3A2, types A, B and C, have a common
amino acid sequence from 1Met to 84Leu. SCGB3A2 type A is the
smallest protein, consisting of 91 amino acids, and 85Glu-91Val of
type A and 106Glu-113Val of type B are comprised of a common
amino acid. Furthermore, type C with 139 amino acids has type B
and a common amino acid from 85Val to 106Phe. In addition, the
expression of SCGB3A2 was reported in human lung carcinomas,
suggesting SCGB3A2 as a potential useful tool for diagnosis of pul-
monary tumors. However, the essential sequences of SCGB3A2
type A, B and C to show their activities are unknown. For this rea-
son, we need preparation methods to analyze a variety of peptide
fragments.
ligation7,8 has been shown to be the most generally applied
approach for the chemoselective assembly of unprotected peptides
in aqueous solution. Native chemical ligation relies on the selective
reaction of a C-terminal peptide thioester and an N-terminal cys-
teine fragment. Especially, preparation of a C-terminal thioester,
which is labile to basic and nucleophilic conditions, is important
and therefore many methodologies have been reported in the liter-
ature.9–15 These methods were useful to give the designed thioe-
ster precursors. However it is sterically demanding for native
chemical ligation and/or difficult in terms of the preparation of
required linkers. Recently, Dawson et al. reported the N-acyl-ben-
zimidazolinone (Nbz) linker,16 which is converted from an o-ami-
noanilide peptide to give the corresponding thioester by
treatment with 4-mercaptophenylacetic acid (MPAA). Although a
variety of protocols to prepare a C-terminal thioester have been
reported to date, there are few applications. Therefore, it is impor-
tant to show the versatility of the N-acyl-Nbz linker and further
research in this field should be promoted. In the present study,
we report the synthesis of an SCGB3A2 type C (98–139) fragment
(1) as an application of Dawson’s native chemical ligation.
SCGB3A2 type C is one of the difficult expression proteins and its
purification is laborious. Since post-translational modification sim-
ilar to phosphorylation was achieved, the modified proteins are
extremely interesting. By investigation of the native chemical liga-
tion using the peptide fragment (1), it is possible to prepare various
peptides around the C-terminal sequence of SCGB3A2 (Fig. 1).
Peptides are synthesized by solid phase techniques, which has
been an indispensable tool for the construction of libraries for high
throughput screening in biology and drug discovery. However, the
coupling efficiency in solid phase peptide synthesis (SPPS) is radi-
cally suppressed for long peptide sequences that exceed 30–40
⇑
Corresponding author.
E-mail address: konno@yz.yamagata-u.ac.jp (H. Konno).
https://doi.org/10.1016/j.tetlet.2017.09.058
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kikuchi M., et al. Tetrahedron Lett. (2017), https://doi.org/10.1016/j.tetlet.2017.09.058

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M. Kikuchi et al. / Tetrahedron Letters xxx (2017) xxx–xxx
Fig. 1. Sequence of SCGB3A2 type C (98–139) (1).
Result and discussion
Synthetic plan of SCGB3A2 type C (98–139) (1) by native chem-
ical ligation between 115Ile and 116Cys residues is shown in
Scheme 1. To continue the native chemical ligation reaction, 98Thi-
azolidine-4-carboxylic acid (Thz) residue17 is selected as a pro-
tected 98Cys equivalent for 1. To give a C-terminal thioester, the
(98–115) peptide-N-MeNbz-Gly amide (2) can be obtained from
Dawson’s Linker (3). In this case, Rink amide resin is selected as
the solid support. In addition, an N-terminal cysteine fragment
(4), the (116–139) peptide, is prepared by ordinary Fmoc-SPPS
using 2-chlorotritylchloride (2-CTC) resin (Scheme 1).
Synthesis of the N-MeNbz peptide (2) is shown in Scheme 2. At
first, 3-amino-4-(methylamino)benzoic acid (N-MeDbz) (5) was
prepared from 4-fluorobenzoic acid.14 After nitration of the meta
position of 4-fluorobenzoic acid, treatment of 4-fluoro-3-nitroben-
zoic acid with methylamine in MeOH afforded 4-(methylamino)-3-
nitrobenzoic acid followed by hydrogenolysis in the presence of
catalytic Pd/C in an H2 atmosphere to give N-MeDbz (5) in 73%
yield over 3 steps. After Fmoc protection of N-MeDbz (5), Fmoc-
N-MeDbz was coupled with a Gly-Rink amide resin using HATU/
DIPEA to give an Fmoc-N-MeDbz-Gly-Rink amide resin (3). The
Fmoc group of the resin (3) was removed with 20% piperidine/
DMF for 60 min. Due to the difficulty of introducing Fmoc-amino
acids under normal conditions, several coupling conditions were
attempted. For the coupling with N-MeDbz-Gly-resin, Fmoc-Ile-
OH (6 eq.) was introduced using DIPCI (6 eq.)/HOAt (6 eq.)/DIPEA
(4 eq.) in DMF for 20 h. After deprotection of the Fmoc group with
20% piperidine/DMF for 50 min, Fmoc-Met-OH (6 eq.) was coupled
Scheme 2. Solid phase peptide synthesis of the N-MeNbz peptide (2). Reagents and
conditions (a) i) HNO3 (4 eq.), conc. H2SO4, rt, o/n, 75%, ii) MeNH2 (18 eq.), MeOH, rt,
o/n, 98%, iii) Pd/C (cat.), H2, MeOH, rt, o/n, 99%, (b) i) Fmoc-OSu (1 eq.), DIPEA
(1.1 eq.), 50% MeCN/H2O, 61%. ii) Gly-Rink amide resin (1 eq.), HATU (5 eq.), DIPEA
(5.5 eq.), rt, 19 h. (c) SPPS. (d) i) 4-nitrophenyl chloroformate (5 eq.), CH2Cl2, rt, 10 h,
ii) 0.85 M DIPEA/DMF, rt, 2 h. (e) TFA/TIPS/H2O (95:2.5:2.5), rt, 2 h, 5.1% overall
yield from resin loading.
using DIPCI (6 eq.)/HOBt (6 eq.)/DIPEA (4 eq.) for 8 h to give
Fmoc-114Met-115Ile-N-MeDbz-Gly-resin. In these cases, 6 equiva-
lents of each Fmoc-amino acid and coupling reagent and a long agi-
tation time were needed to complete these reactions. Although the
two coupling reactions with the resin were laborious tasks, the
subsequent elongation of the corresponding Fmoc-amino acids
proceeded smoothly to give the desired sequence (see Supporting
information in Table S1). The final coupling reaction with Boc-
Thz-OH, a protected Cys equivalent, occurred in a similar manner
to give the N-MeDbz resin (6) with N-terminus Boc-protection.
All coupling conditions monitored by the Kaiser test18 were opti-
mized to use DIPCI/HOAt or DIPCI/HOBt combinations. The N-
MeDbz resin (6) with 5 equivalents of 4-nitrophenyl chloroformate
in CH2Cl2 for 2 h was treated to afford the thioester precursor and
continuous urea formation by 0.85 M DIPEA/DMF for 1 h at room
temperature was attempted to give the acylated product (7). How-
ever, the major peak on HPLC analysis was the N-MeDbz peptide, a
resin-cleaved product of 6, after cleavage from the resin. By opti-
mization of reaction times to convert from N-MeDbz to N-MeNbz
on solid support, we found 10 h was needed to give the corre-
sponding carbamate and an additional 2 h for acylation, respec-
tively. After cleavage from the resin with TFA/TIPS/H2O
(95:2.5:2.5) for 2 h, crude peptides were purified with preparative
HPLC to give the N-MeNbz peptide (2) in 5.1% yield from resin
loading. The optimization of reaction conditions for the individual
sequences not only increased conversion of the substrates, but also
suppressed the production of by-products, especially hydrolysis
products (Scheme 2).
The HPLC profile and ESI-MS spectra of the purified N-MeNbz
peptide (2) are shown in Fig. 2. The characteristic ion peak, m/
z = 2298.8, was detected by the ESI-TOFMS spectra (positive mode)
(Fig. 2).
For ligation, SCGB3A2 type C (116–139) (4) as a C-terminus
peptide was prepared by ordinary Fmoc-SPPS on 2-CTC resin in
0.6% overall yield from resin loading. In this case, the chain elonga-
tion between 125Gln and 116Cys residues was difficult. For the elon-
gation of 125Gln-123Lys residues and 119Pro-117Ala residues, long
reaction times were needed. We applied microwave-assisted syn-
thesis19,20 to facilitate the coupling reactions from 122Ser residue
to 120Arg residue. In addition, Fmoc-Cys(Trt)-OH to introduce the
116Cys residue was coupled with HATU/HOAt/DIPEA on the resin
Scheme 1. Synthetic plan for SCGB3A2 type C (98–139) (1) by Dawson’s native
chemical ligation.
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M. Kikuchi et al. / Tetrahedron Letters xxx (2017) xxx–xxx
3
Fig. 2. (a) HPLC profile of the N-MeNbz peptide (2). Condition: flow 1 mL/min,
tR = 22.2 min (0–30 min gradient from 10 to 70% MeCN/H2O). (b) ESI-MS spectra of
the N-MeNbz peptide (2), [M+H]+ m/z found 2298.8, calcd. for 2298.2.
for 28 h. The results affected deprotection of the Fmoc groups. For
this reason, it was important to treat with the deprotection condi-
tion for a long time and/or use irradiation with microwaves. This
suggests a limitation of SPPS based on linear synthesis and these
peptides are frequently called difficult sequences, resulting from
the intermolecular aggregation with hydrogen bonding networks
between protected peptide chains on the resin. Finally, cleavage
from the resin with TFA/TIPS/H2O (95:2.5:2.5) followed by purifi-
cation with preparative HPLC gave SCGB3A2 type C (116–139)
(4) as a C-terminus peptide. The HPLC profile and ESI-MS spectra
of the purified SCGB3A2 type C (116–139) (4) are shown in
Fig. 3. The ESI-TOFMS spectra (positive mode) showed observed
m/z = 1446.3 as a bivalent ion peak (see Supporting information
in Table S2) (Fig. 3).
Fig. 3. (a) HPLC profile of SCGB3A2 type C (116–139) (4). Condition: flow 1 mL/min,
tR = 15.4 min (0–30 min gradient from 10 to 70% MeCN/H2O). (b) ESI-MS spectra of
SCGB3A2 type C (116–139) (4), [M+2H]2+ m/z found 1446.3, calcd. for 1446.3.
Thus, application of native chemical ligation between 115Ile and
116Cys residues was a practical strategy. The native chemical liga-
tion reaction between the N-MeNbz peptide (2) and SCGB3A2 type
C (116–139) (4) using GnÁHCl (6 M)/Na2HPO4 (200 mM)/TCEPÁHCl
(20 mM)/MPAA (200 mM)/pH 7.0 as a ligation buffer at room tem-
perature for 20 h was successfully conducted to give SCGB3A2 type
C (98–139) (1). This result suggests the following: (i) the N-MeNbz
peptide (2) was quickly converted to a C-terminal thioester inter-
mediate (8), and subsequently (ii) trans-thioesterification between
a C-terminal thioester intermediate (8) and SCGB3A2 type C (116–
139) (4) gave the coupled product with the thioester (9), and
finally (iii) the S to N acylshift between 115Ile and 116Cys residues
proceeded to yield SCGB3A2 type C (98–139) (1) (Scheme 3).
The HPLC profile of the native chemical ligation reaction for
SCGB3A2 type C (98–139) (1) at 20 h is shown in Fig. 4a. Interest-
ingly, the hydrolyzed product of the N-MeNbz peptide (2) was not
detected to yield SCGB3A2 type C (98–139) (1) in satisfactory yield,
although the C-terminus peptide (4) was verified at about 10% on
the HPLC profile. The MALDI-TOFMS spectra (positive mode) of
SCGB3A2 type C (98–139) (1) derived from native chemical ligation
were depicted as a trivalent ion peak of 1648.2. Native chemical
ligation could be a powerful tool as it is difficult to prepare short
peptides with difficult sequences (Fig. 4).
Scheme 3. Synthesis of SCGB3A2 type C (98–139) (1). Reagents and conditions (a)
SCGB3A2 type C (116–139) (4), 6 M GnÁHCl, 200 mM Na2HPO4, 20 mM TCEPÁHCl,
200 mM MPAA, pH 7.0, rt, 20 h.
for the preparation of peptide/protein was demonstrated. Synthe-
sis of the full sequence of SCGB 3A2 is now underway.
In conclusion, we synthesized SCGB type C (98–139) (1) by
native chemical ligation using Dawson’s linker. A powerful tool
Please cite this article in press as: Kikuchi M., et al. Tetrahedron Lett. (2017), https://doi.org/10.1016/j.tetlet.2017.09.058

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M. Kikuchi et al. / Tetrahedron Letters xxx (2017) xxx–xxx
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2017.09.
058.
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