Synthesis of an O-acyl isopeptide by using native chemical ligation to efficiently construct a hydrophobic polypeptide

Article abstract of DOI:10.1016/j.tetlet.2011.10.116

By using dimethylformamide to suppress the O-to-N acyl migration, we efficiently synthesized an O-acyl isopeptide by native chemical ligation of a peptide-thioester and a Cys-O-acyl isopeptide. The reaction mixture was then loaded onto an octadecylsilane

Full text of DOI:10.1016/j.tetlet.2011.10.116

Tetrahedron Letters 52 (2011) 7146–7148
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of an O-acyl isopeptide by using native chemical ligation
to efficiently construct a hydrophobic polypeptide
Youhei Sohma a, , Hitomi Kitamura a, Hiroyuki Kawashima b, Hironobu Hojo c, Masayuki Yamashita b,
⇑
Kenichi Akaji a, Yoshiaki Kiso d,
⇑
a Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
b Department of Pharmaceutical Manufacturing Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
c Department of Applied Biochemistry, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
d Laboratory of Peptide Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
By using dimethylformamide to suppress the O-to-N acyl migration, we efficiently synthesized an O-acyl
isopeptide by native chemical ligation of a peptide-thioester and a Cys-O-acyl isopeptide. The reaction
mixture was then loaded onto an octadecylsilane reverse-phase HPLC column, and the isopeptide was
purified by using a linear gradient of CH3CN in 0.1% aqueous trifluoroacetic acid. The recovery rate of
the O-acyl isopeptide was considerably higher than that of the corresponding native polypeptide. Synthe-
sis of O-acyl isopeptides via native chemical ligation, with O-to-N acyl migration as the final step to give
the native form, has potential as an efficient method of constructing hydrophobic polypeptides.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 26 September 2011
Revised 15 October 2011
Accepted 19 October 2011
Available online 25 October 2011
Keywords:
O-Acyl isopeptide
HPLC
Hydrophobic peptide
Native chemical ligation
Purification
In ligation chemistry, the synthesis of hydrophobic polypep-
tides is problematic because of the purification step via reverse-
phase HPLC using conventional mixed aqueous/organic solvent
systems.1–6 Polypeptides derived from membrane proteins and
amyloid proteins are representatives of such hydrophobic pep-
tides. The solubility of these polypeptides in the solvents that are
commonly used for HPLC is very low. Additionally, because the
interaction between the hydrophobic peptides and the stationary
phase (e.g., C18, C8, and C4 reverse phase) is extremely strong, pos-
sibly causing aggregation, the elution of the polypeptides from the
column is highly obstructed.
of the peptide-a-thioester by increasing the solubility, and thereby
facilitates the use of the peptide-thioester in the ligation. An alter-
native approach is the use of the polar aprotic organic solvent
dimethylformamide (DMF), instead of a conventional guanidine-
containing aqueous solution, for the ligation,6 since hydrophobic
peptides are more soluble in DMF than in aqueous solution during
ligation. However, in these approaches, the problem associated with
the reverse-phase HPLC purification of the resulting polypeptide
after the ligation is not overcome, because the solubilizing effects
of the Arg-tag and DMF are no longer available at the purification
step.
Chemical protein synthesis, which enables us to introduce non-
standard amino acids into proteins, provides an efficient and versa-
tile tool for uniquely elucidating the molecular basis of protein
function.7 Native chemical ligation,8 the thioester-mediated cova-
lent joining of unprotected peptide segments at a cysteine residue,
substantially facilitates the total chemical synthesis of proteins. The
When the N-acyl linkage on the hydroxyamino acid residue is
replaced with an O-acyl linkage, the resulting species (referred to
as an O-acyl isopeptide) shows suppressed aggregation relative
to the native peptide, and due to the presence of the protonated
amino group, O-acyl isopeptides are water-soluble.9–12 Further-
more, O-acyl isopeptides can be converted into the corresponding
native polypeptides via an O-to-N acyl migration reaction under
neutral pH conditions.
Here, we report a new strategy for the efficient construction of a
hydrophobic polypeptide via the synthesis of an O-acyl isopeptide,
prepared by native chemical ligation followed by high-recovery
HPLC purification (Fig. 1).13 We were inspired by a report that
native chemical ligation efficiently takes place in DMF in the
presence of base.6 Since, in the synthesis and handling of O-acyl
isopeptide,9–11 we experienced a remarkably slower O-to-N acyl
peptide-a-thioester-(Arg)n species, which contains a solubilizing
Arg-tag in the C-terminal thioester leaving group, has been reported
to improve the efficacy of native chemical ligation of hydrophobic
polypeptides.4,5 The Arg-tag improves the preparation and handling
⇑
Corresponding authors. Tel.: +81 75 595 4636; fax: +81 75 595 4787 (Y.S.); tel.:
+81 749 64 8113; fax: +81 749 64 8140 (Y.K.).
E-mail addresses: ysohma@mb.kyoto-phu.ac.jp (Y. Sohma), y_kiso@nagahama
-i-bio.ac.jp (Y. Kiso).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.10.116

Y. Sohma et al. / Tetrahedron Letters 52 (2011) 7146–7148
7147
O
H2N
C2-peptide
HS
O
O
+
N-peptide
S
R
O
C1-peptide
H2N
native chemical ligation
(in DMF)
HPLC purification
O
H2N
C2-peptide
SH
C1-peptide
O-acyl isopeptide
O
O
N-peptide
O
N
H
O-to-N acyl shift
SH
C1-peptide
OH
C2-peptide
O
O
N-peptide
N
N
H
H
hydrophobic polypeptide
Figure 1. Synthetic scheme for constructing a hydrophobic polypeptide via the
efficient purification of O-acyl isopeptide prepared by use of native chemical
ligation.
migration in organic solvents (e.g., MeOH, DMF, and DMSO) com-
pared with aqueous solutions we anticipated that the use of DMF
would enable us to synthesize the O-acyl isopeptide by native
chemical ligation without losing the O-acyl moiety. As a proof-of-
concept study, we conducted the native chemical ligation of H-
Ser(Ala-Val-Gln-Glu-Ala-Cys-Ala-Val)-Ala-Ile-Leu (1), an O-acyl
isopeptide of a transmembrane domain derived from the protein
NpHtrll.6 The peptide-
a-aryl thioester H-Ala-Val-Gln-Glu-Ala-S-
Figure 2. Native chemical ligation of peptide-
a-aryl thioester 2 with Cys-O-acyl
C6H4-CH2-COOH (2, 3.0 mg) and the Cys-O-acyl isopeptide H-
Ser(Cys-Ala-Val)-Ala-Ile-Leu-OH (3, 3.0 mg) were dissolved in
DMF containing 20 mM 4-mercaptophenylacetic acid (MPAA),14
40 mM triethylamine, and 20 mM tris(2-carboxyethyl)phosphine
hydrochloride (TCEPÁHCl), at a concentration of 3.5 mM for each
peptide (pH of the reaction mixture was approximately 5), and
the solution was allowed to stand at 37 °C. The native chemical
ligation reaction was approximately 70% complete after 1 h, yield-
ing ligated O-acyl isopeptide 1 (Fig. 2). Importantly, only a small
amount of the corresponding N-acyl peptide (i.e., 5) was detected
on the HPLC trace at t = 1 h, indicating that the O-to-N acyl migra-
tion in 1 was mostly suppressed during the reaction (Fig. 2). Addi-
tionally, we confirmed that the epimerization at the C-terminal Ala
residue of 2 during the reaction was below detectable limits
(<0.5%, Fig. S1). Moreover, 1 remained soluble in DMF during the
reaction, with no precipitation. After the 1-h reaction, the reaction
mixture was loaded onto a C18 reverse-phase HPLC column
(250 Â 20 mm) at a time, and 1 was purified by using a linear gra-
dient of CH3CN in 0.1% aqueous TFA. The isolation yield of 1 in this
reaction was 54%. Purified 1 was convertible to the parent N-acyl
peptide (i.e., 5) in 0.1 M phosphate buffer (pH 7.5, including 10%
DMF) via the O-to-N acyl migration, nearly quantitatively after
an overnight reaction at 37 °C.
isopeptide 3, to yield O-acyl isopeptide 1, in DMF was monitored by use of
analytical HPLC (UV profiles at 230 nm are shown). (A) t = 0 h; (B) t = 1 h. The
chromatographic separations were performed by using a linear gradient (0–100%)
of buffer
B in buffer A over 40 min (buffer A = 0.1% TFA in water; buffer
B = acetonitrile). Compound 5 = Ala-Val-Gln-Glu-Ala-Cys-Ala-Val-Ser-Ala-Ile-Leu,
#non-peptidic.
peptide) in the presence of 20 mM MPAA, 40 mM triethylamine,
and 20 mM TCEPÁHCl, a gel-like precipitate was observed after
the 1-h reaction. After dissolution of the precipitate in hexa-
fluoro-2-propanol, HPLC analysis indicated that the major compo-
nent was the ligated product, Ala-Val-Gln-Glu-Ala-Cys-Ala-Val-
Ser-Ala-Ile-Leu (5, Fig. S2). In contrast, 5 was barely detected on
analysis of the solution layer of the ligation mixture. These results
indicate that the native chemical ligation of 2 and 4 proceeded well
and that the resulting 5 precipitated due to the low solubility in
DMF. The crude precipitate containing 5 was then dissolved in
hexafluoro-2-propanol, and the solution was loaded onto a re-
verse-phase HPLC column (250 Â 20 mm) at a time and eluted
with a binary solvent system (a linear gradient of CH3CN in 0.1%
aqueous TFA) to afford the purified 5. Although 5 was predomi-
nantly obtained after the ligation reaction, as was the case with
O-acyl isopeptide 1, the isolation yield of 5 after the HPLC purifica-
tion was 18%, approximately one-third that of 1. This result sug-
gests that the recovery rate of 5 in the purification step was
In a control ligation of peptide-a-aryl thioester 2 (3.0 mg) with
Cys-Ala-Val-Ser-Ala-Ile-Leu (4, 3.0 mg) in DMF (3.5 mM each

7148
Y. Sohma et al. / Tetrahedron Letters 52 (2011) 7146–7148
isopeptide. Upon subsequent HPLC purification with the CH3CN–
0.1% aq TFA mixed solvent system, the recovery rate of O-acyl iso-
peptide 1 from the reverse-phase column was considerably higher
than that of the corresponding native peptide 5. The difference in
recovery yield between 1 and 5 tended to be greater with increas-
ing amounts of loaded peptides. Currently, the problematic step in
the chemical synthesis of hydrophobic polypeptides is that of puri-
fication by reverse-phase HPLC with conventional mixed aqueous/
organic solvent systems. The strong interaction of the hydrophobic
peptide with the stationary phase greatly hinders the elution of the
peptide from the column. Therefore, this synthetic scheme that in-
cludes the efficient purification of an O-acyl isopeptide, prepared
by the use of ligation chemistry, and the O-to-N acyl migration
as the final step to give the native form, has potential as an efficient
method for constructing hydrophobic polypeptides.
Figure 3. A graph of loaded crude peptide versus recovered pure peptide. The crude
Acknowledgments
reaction mixture was loaded onto
a reverse-phase HPLC column (ODS,
250 Â 20 mm) at a time and eluted by using a linear gradient of CH3CN in 0.1%
aqueous TFA. Values on the vertical axes at the filled circles (for 1): 0.36 mg (at
[1+1] mg scale), 2.8 mg (at [3+3] mg scale), and 4.5 mg (at [6+6] mg scale); open
circles (for 5): 0.47 mg (at [1+1] mg scale), 0.94 mg (at [3+3] mg scale), and 1.1 mg
(at [6+6] mg scale).
This research was supported, in part, by the ‘Academic Frontier’
Project for Private Universities: matching fund subsidy from MEXT
(Ministry of Education, Culture, Sports, Science, and Technology) of
the Japanese Government, a Grant-in-Aid for Scientific Research
(KAKENHI), the Kyoto Pharmaceutical University Fund for the Pro-
motion of Scientific Research, and the Takeda Science Foundation.
We thank Mr. W. Yamanashi for the technical assistance.
lower than that of 1, most probably because more 5 than 1 was re-
tained on the ODS phase of the column due to its greater
hydrophobicity.
In the synthesis of 1 from 3 mg of each peptide ([3+3] mg scale
ligation), the isolation yield of 1 (2.8 mg) was approximately three
times that of the corresponding N-acyl peptide (5) (0.94 mg), ow-
ing to improvement of the recovery rate upon reverse-phase HPLC
purification. At the [6+6] mg scale, the difference in recovery rate
between 1 and 5 after purification was even more pronounced
(yield of 1: 4.5 mg, yield of 5: 1.1 mg) (Fig. 3). In contrast, there
was no noteworthy difference between the yields of 1 and 5 at
the [1+1] mg scale (1: 0.36 mg, 5: 0.47 mg). Thus, the amount of
5, derived from a ligation at a scale greater than [3+3] mg, needed
to maintain a reasonable recovery rate for the loaded peptide
would exceed the capacity of a C18 column of 250 Â 20 mm. For
O-acyl isopeptide 1, the recovery rate was considerably improved
compared with that of 5 at the [3+3] mg and [6+6] mg scales, sug-
gesting that the O-acyl isopeptide overcame the retention problem
of the parent peptide as a result of a decrease in its hydrophobicity.
In conclusion, here we report the synthesis of an O-acyl isopep-
tide using native chemical ligation to efficiently construct a hydro-
phobic polypeptide. By using DMF to suppress the O-to-N acyl
migration, we efficiently synthesized O-acyl isopeptide 1, a deriva-
tive of the transmembrane domain of NpHtrll, by native chemical
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.10.116.
References and notes
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ligation of
a peptide-thioester with a Cys-O-acyl isopeptide.
Ligated O-acyl isopeptide 1 was soluble in DMF during the reaction,
whereas the corresponding N-acyl peptide 5 predominantly pre-
cipitated, reaffirming the advantage of the solubility of the O-acyl