Hydrophobic tag-assisted liquid-phase synthesis of a growth hormone-inhibiting peptide somatostatin

Article abstract of DOI:10.1016/j.bmcl.2011.06.004

Hydrophobic tag-assisted liquid-phase peptide synthesis technique and disulfide bond formation have been well-combined, leading to the efficient and practical preparation of a growth hormone-inhibiting peptide somatostatin. Intramolecular disulfide bond f

Full text of DOI:10.1016/j.bmcl.2011.06.004

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4476–4479
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hydrophobic tag-assisted liquid-phase synthesis of a growth
hormone-inhibiting peptide somatostatin
Shingo Kitada a, Shuji Fujita b, Yohei Okada a, Shokaku Kim a, Kazuhiro Chiba a,
⇑
a Department of Applied Life Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
b JITSUBO Co., Ltd, 2-24-16 Naka-cho, Koganei, Tokyo 184-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrophobic tag-assisted liquid-phase peptide synthesis technique and disulfide bond formation have
been well-combined, leading to the efficient and practical preparation of a growth hormone-inhibiting
peptide somatostatin. Intramolecular disulfide bond formation has successfully been carried out even
under relatively high concentrations, enabling the effective peptide modifications in preparative scale.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 6 April 2011
Revised 24 May 2011
Accepted 1 June 2011
Available online 15 June 2011
Keywords:
Hydrophobic tag
Liquid-phase peptide synthesis
Disulfide bond
Cyclic peptide
Somatostatin
Intramolecular cross-linking plays a significant role in both nat-
urally occurring and artificial peptides to preorganize an amino
acid sequence into a rigid conformation, to impart and/or enhance
several biological activities, and to stabilize their structures. A vast
number of cyclic peptides have been synthesized1–17 to investigate
their potential applications as nanomaterials,18 imaging agents,19
and therapeutics.20,21 In particular, disulfide bonds formed be-
tween cysteine residues are essential for many peptides to induce
a wide variety of biological processes. The development of solid-
phase techniques have led to practical and facile peptide synthesis,
into which effective disulfide bond formation can also be intro-
duced.22–31 Alternatively, disulfide bond-containing peptides have
been prepared efficiently through liquid-phase methodologies un-
der high dilution conditions.32–36
Soluble polymer-assisted liquid-phase synthesis has proven to
be promising, especially for multi-step reactions, where the advan-
tages of solid-phase separation and liquid-phase efficiency are
both attained.37–40 Previously, we developed a hydrophobic tag-as-
sisted liquid-phase peptide synthesis method.41–46 In this system, a
simple hydrophobic tag, 3,4,5-trioctadecyloxybenzyl alcohol,
which could be prepared from naturally abundant materials in
one step (Scheme S1 in Supplementary data), served as an effective
protecting group for the peptide C-terminus and as a phase-tag.
The desired hydrophobically tagged peptides, which were soluble
in less polar organic solvents to realize efficient homogeneous li-
quid-phase peptide elongation/deprotection, could be separated
as precipitates from reaction solutions through the addition of po-
lar organic solvents. Most recently, we demonstrated that several
types of hydrophobic tags could be designed simply to incorporate
both Boc- and Fmoc-chemistry, allowing the elaboration of desired
peptide sequences (Scheme S2 in Supplementary data).47 The com-
bination of this technique with disulfide bond formation would be
a great aid for furthering the potential of hydrophobic tag-assisted
peptide synthesis.
The present work began with the evaluation of the stability of the
hydrophobic tag against the conditions used for disulfide bond for-
mation. To this extent, our previous studies also indicated that three
octadecyloxy substituted groups (in total 54 carbons) of the hydro-
phobic tag could be replaced by two docosyloxy substituted groups
(in total 44 carbons) with high precipitability that responded to the
addition of polar organic solvents. Thus, we introduced N-Fmoc-Cy-
s(Acm)-OH 1 to 1.00 mmol of the hydrophobic tag, 2,4-didocosyl-
oxybenzyl alcohol, (HO-TAG) 2 using diisopropylcarbodiimide
(DIC) in the presence of a catalytic amount of dimethylaminopyri-
dine (DMAP), forming N-Fmoc-Cys(Acm)-O-TAG 3 (Scheme 1). In or-
der to integrate disulfide bond formation reactions into the
hydrophobic tag-assisted peptide synthesis, we chose one of the
most common conditions which involved simple iodine oxidation
of cysteine residues, where side chains were protected with N-
(acetyl)aminomethyl (Acm) groups, constructing desired disulfide
bond in one step, including both deprotection and oxidative cou-
pling.48–52 N-Fmoc-Ala-OH 4 and N-Boc-Cys(Acm)-OH 5 were then
introduced into 3 using O-benzotriazole-N,N,N0,N0-tetramethyluro-
nium hexafluorophosphate (HBTU) and 1-hydroxy-1H-benzotria-
⇑
Corresponding author. Tel.: +81 42 367 5667; fax: +81 42 360 7167.
E-mail address: chiba@cc.tuat.ac.jp (K. Chiba).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.004

S. Kitada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4476–4479
4477
S(Acm)
O
OC22H45
S(Acm)
OH
DIC
TAG
(Fmoc)HN
HO
DMAP
(Fmoc)HN
O
+
CH2Cl2
OC22H45
2 (HO-TAG)
O
3
1
quant.
Scheme 1. Introduction of N-Fmoc-Cys(Acm)-OH 1 to the hydrophobic tag, 2,4-didocosyloxybenzyl alcohol, (HO-TAG) 2.
OH
(Fmoc)HN
O
S(Acm)
S(Acm)
O
O
HBTU
HOBt
S(Acm)
O
H
4
N
N
H
TAG
(Boc)HN
CH2Cl2
then
TAG
(Fmoc)HN
O
+
O
DBU
Piperidine
CH2Cl2
O
S(Acm)
OH
6
3
93% (over 4 steps)
(Boc)HN
O
5
Scheme 2. Construction of the model peptide for the evaluation of the stability of the hydrophobic tag against the conditions used for disulfide bond formation.
(Boc)HN
S
S
O
S(Acm)
S(Acm)
O
HN
O
O
H
TAG
NH
N
I2
N
H
TAG
(Boc)HN
O
O
CH2Cl2/DMF
O
O
7
6
95% (1.0 mM)
98% (10 mM)
Scheme 3. Iodine oxidation of N-Boc-Cys(Acm)-Ala-Cys(Acm)-O-TAG 6.
S(Acm)
S(Acm)
S(Acm)
O
O
O
O
TAG
(Fmoc)HN
H
N
H
HBTU
HOBt
N
O
N
H
N
TAG
(Boc)HN
H
CH2Cl2
3
O
O
O
R
11
DBU
Piperidine
CH2Cl2
+
8
R
OH
72% (over 27 steps)
(Fmoc)HN
O
Scheme 4. Synthesis of hydrophobically tagged peptide 8.
zole (HOBt) in the presence of N,N-diisopropylethylamine (DIPEA) to
afford N-Boc-Cys(Acm)-Ala-Cys(Acm)-O-TAG 6 in 93% isolated yield
over four steps (Scheme 2). All reaction steps, including both elonga-
tion and deprotection, were efficiently carried out homogeneously
in the liquid-phase, realizing both high reaction rate and rapid reac-
tion monitoring. The desired hydrophobically tagged peptides could
be separated as precipitates from the reaction solutions after the
completion of the reactions. Iodine oxidation was then attempted
with 6 at 1.0 mM concentration to give the desired disulfide bond-
containing peptide 7 in 95% isolated yield (Scheme 3). Notably, the
iodine oxidation of 6 also gave 7 in 98% isolated yield even at a
10 mM concentration of 6. These results suggested that disulfide
bond formation was effectively combined with the hydrophobic
tag-assisted peptide synthesis, which was successfully carried out
even under relatively high concentrations.
With these results in hand, we next turned our attention to the
synthesis of a growth hormone-inhibiting peptide, somatostatin, as
a model of bioactive disulfide bond-containing peptides. The
receptor subtypes of somatostatin are well-established targets for
several therapeutic studies. To date, several synthetic strategies
that address the disulfide bond formation have been proposed to
elaborate somatostatin based on both solid-phase and liquid-phase
techniques.53–55 In order to remove all protective groups under
acidic conditions at the final step of the synthesis, we chose the

4478
S. Kitada et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4476–4479
(Boc)HN
O
NH
O
S(Acm)
S(Acm)
O
O
O
HN
H
N
H
S
N
S
I2
O
N
H
N
H
11
TAG
(Boc)HN
O
O
O
R
CH2Cl2/DMF
HN
R
O
TAG
NH
8
O
O
11
9
73% (1.0 mM)
64% (10 mM)
Scheme 5. Iodine oxidation of hydrophobically tagged peptide 8.
Boc
Ala
Ala
Trt
Asn
Gly
Boc
Lys
Asn
Lys
Gly
Phe
Cys
Phe
Cys
Phe
Trp
Phe
Trp
Lys
S
S
2.5% TIS
S
S
2.5% H2O
TFA
Lys
Thr
Cys
Boc
Cys
O
TAG
Ser
Thr
tBu
Ser
tBu
Phe
O
Thr
10
Phe
Thr
tBu
95%
9
Scheme 6. Deprotection of hydrophobically tagged disulfide bond-containing peptide 9.
Boc and t-Bu groups for the protection of both the N-terminus and
side chains. Several N-Fmoc amino acids (AAs) (except the last, N-
Boc-Ala-OH) were repeatedly introduced to 3 using HBTU and
HOBt in the presence of DIPEA to afford the desired hydropho-
bically tagged peptide 8 in 72% isolated yield over 27 steps
(Scheme 4). When 8 at a concentration of 1.0 mM was oxidized
using iodine, the desired disulfide bond-containing peptide 9 was
formed in 73% isolated yield (Scheme 5). Furthermore, even at a
concentration of 10 mM, 8 could give 9 in 64% isolated yield
through iodine oxidation. Finally, deprotection of 9, including the
N-terminal Boc group, side chain protective groups, and C-terminal
TAG, was achieved in a single step under acidic conditions to give
deprotected compound, somatostatin, 10 in 95% isolated yield
(Scheme 6).
In conclusion, disulfide bond formation has effectively been
combined with hydrophobic tag-assisted peptide synthesis, lead-
ing to the efficient and practical preparation of a growth hor-
mone-inhibiting peptide somatostatin. Disulfide bond formation
has successfully been carried out even under high concentrations.
This approach should find applications not only for the synthesis of
naturally occurring disulfide bond-containing peptides, but also for
the creation of artificial cyclic peptides.
Supplementary data
Supplementary data (general information, experimental details,
characterization data, and copies of 1H NMR and 13C NMR spectra
of new compound) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2011.06.004.
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