Extended Solution-phase Peptide Synthesis Strategy Using Isostearyl-Mixed Anhydride Coupling and a New C-Terminal Silyl Ester-Protecting Group forN-Methylated Cyclic Peptide Production

Article abstract of DOI:10.1021/acs.oprd.1c00078

Herein, we present a new and efficient convergent solution-phase synthetic strategy for producing peptides containingN-methyl amino acids. Specifically, we have synthesized a model cyclic octapeptide with twoN-methyl amino acids, utilizing an isostearyl-m

Full text of DOI:10.1021/acs.oprd.1c00078

pubs.acs.org/OPRD
Article
Extended Solution-phase Peptide Synthesis Strategy Using
Isostearyl-Mixed Anhydride Coupling and a New C‑Terminal Silyl
Ester-Protecting Group for N‑Methylated Cyclic Peptide Production
Akihiro Nagaya, Shota Murase, Yuji Mimori, Kazuya Wakui, Madoka Yoshino, Ayumu Matsuda,
Yutaka Kobayashi, Haruaki Kurasaki, Douglas R. Cary, Keiichi Masuya, Michiharu Handa,
and Naoki Nishizawa*
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ABSTRACT: Herein, we present a new and efficient convergent solution-phase synthetic strategy for producing peptides containing
N-methyl amino acids. Specifically, we have synthesized a model cyclic octapeptide with two N-methyl amino acids, utilizing an
isostearyl-mixed anhydride coupling methodology and a novel silyl ester-protecting group, cyclohexyl di-tert-butyl silyl (cHBS). This
newly developed method uses an isostearic acid chloride (ISTA-Cl) and silylation reagent that allows coupling between N- and C-
terminally unprotected amino acids with sterically hindered N-methyl amino acids. High yields of four dipeptide fragments are
efficiently synthesized by omitting the traditional C-terminal deprotection step. The silyl ester-protecting group at the C-terminus is
stable during general peptide synthesis, and is selectively cleaved by fluoride ions. This group further suppresses diketopiperazine
formation during the deprotection of the Nα-amino-protecting group. The linear octapeptide precursor is convergently synthesized
utilizing protection and selective deprotection of the silyl ester-protecting group, and the cyclic octapeptide can be obtained with
high purity using this novel methodology, via a route shorter than conventional solution-phase peptide synthesis strategies.
KEYWORDS: liquid-phase peptide synthesis, solution-phase peptide synthesis, silyl, isostearyl-mixed anhydrides, cHBS
requirements can be minimized.25 As such, we have developed
INTRODUCTION
■
a new coupling method using isostearic acid halide (ISTA-X)
Discovering candidate peptides using in vitro display
and silylation agents,26 by which N- and C-terminal
libraries,1−3 multiple peptide loop cyclization,4,5 or cell-
unprotected amino acids can be introduced without double
penetrating lipopeptides6,7 in addition to conventional peptide
insertion by a temporary protecting effect of the carboxyl
drug discovery approaches, such as using endogenous
functionality by the silyl group (Scheme 1).
peptides8−10 or phage display,11,12 has accelerated the pace
Generally, prolonged reaction times caused by the low
of peptide drug discovery. The new methodologies for
reactivity of N-methyl amino acids increases the risk of
identifying novel peptides composed of non-canonical amino
epimerization; however, this method allows for the rapid
acids allow for the creation of cyclic peptide libraries with
coupling of N-methyl amino acids without epimerization. It
significantly greater structural diversity, compared to those
also enables N-to-C reverse elongation, which is in the
previously studied.13,14 In particular, the strategy of incorpo-
opposite direction of the conventional C-to-N elongation
ration of N-methyl amino acids can be utilized to improve cell
method. Reverse elongation permits the rapid and environ-
membrane permeability and protease resistance.15 However,
mentally friendly synthesis of peptide fragments for convergent
the synthesis of peptides containing N-methyl amino acids is
LPPS in fewer steps due to elimination of the deprotection
difficult due to steric hindrance, which represents a significant
step.25 In a previous study, we showed that tetrapeptides
obstacle in peptide drug research and development.16−18 Solid-
containing N-methyl amino acids could be efficiently
phase peptide synthesis (SPPS) has been widely used for long-
synthesized without epimerization using this method.26 One
chain peptide manufacturing and, more recently, for the
of the limitations of peptide fragment synthesis by convergent
synthesis of short-chain peptides.19−21 Although SPPS is
LPPS is C-terminal carboxy protection during peptide
straightforward and rapid, it is not considered economically
elongation. The protective groups must be selectively
or environmentally favorable (green chemistry) due to its
excessive use of reaction reagents and washing solvents.22−24
However, convergent solution-phase (liquid-phase) peptide
Received: March 1, 2021
synthesis (LPPS) is suitable for producing short to moderate
length peptide-active pharmaceutical ingredients (APIs) as it
allows for the purification of intermediates by crystallization
and facilitates scale-up production. LPPS also has a better
green chemistry profile than SPPS as the reagent and solvent
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A

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Scheme 1. Coupling Between N-Protected Amino Acids and N-, C-Terminal Unprotected Amino Acids With ISTA-X and
a
Silylating Agents
a
BSA, N,O-bis(trimethylsilyl)acetamide; DIPEA, N,N-diisopropylethylamine; ISTA-X, isostearic acid halide; MeCN, acetonitrile; PG, protecting
group; and TMS, trimethylsilyl.
a
Scheme 2. Peptide Elongation on the C-Terminal Silyl Ester-Protecting Group
a
DKP, diketopiperazine; PG, protecting group
deprotected over Nα- and side chain-protecting groups. The
most frequently used methyl and ethyl esters are stable under
all deprotection conditions used in standard Boc, Fmoc, and
Cbz chemistry; however, the amino acids that can be
incorporated at the C-terminus are limited as deprotection
requires basic hydrolysis, which has a high risk of
epimerization.27 Although the development of new coupling
reagents has helped to suppress epimerization during fragment
condensation,28 the epimerization risk during the basic
deprotection step limits the fragmentation sites that can be
used in synthetic strategies for peptide synthesis by convergent
LPPS. Furthermore, there is a high risk of peptide cleavage by
diketopiperazine (DKP) formation when the C-terminal amino
acid is proline (Pro) or an N-methyl amino acid.29,30 DKP
structures can form under Fmoc deprotection conditions, even
with natural amino acids other than Pro. Additionally,
solubility issues can be limiting during solution-phase synthesis
due to decreasing solubility of the protected peptide during
long peptide chain construction. The decrease in solubility
induces incomplete coupling/deprotection reactions and
complicates separation and purification in postreaction work-
up. Although SPPS and tag-assisted LPPS have been utilized to
significantly improve the solubility of the protected pep-
tides,31,32 these efforts have not resulted in enhanced purity of
the peptidic intermediate in the workup process. To address
this issue, we evaluated the use of silyl ester-type carboxyl-
protecting groups as a C-terminal-protecting group.
Previously, the trimethylsilyl group has been used in peptide
synthesis as an aqueous labile temporary protecting group of
the carboxylic acid.33,34 However, there are no examples of C-
terminal carboxylic acid protection with a silyl ester during
peptide elongation, due to instability under the peptide
synthesis conditions. We hypothesized that a silyl ester with
bulky hydrocarbon substituents would allow for enhanced
stability under peptide synthesis conditions. Additionally, we
expected that the steric bulk would prevent DKP formation
and the high hydrophobicity of the bulky silyl group would
result in improved overall solubility for the protected peptide
fragment. The silyl ester could be cleaved by a fluorine source,
such as potassium fluoride without affecting other protecting
groups on the peptide (Scheme 2). These effects were
expected to overcome the limitations of conventional C-to-N
solution-phase synthesis, and in combination with N-to-C
reverse elongation could be expected to greatly improve the
diversity of synthetic strategies available for peptide synthesis.
B
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a
Table 1. Tests of BIBS Methodology Applied to Fmoc-, Boc-, and Cbz-Based Chemistry for the Synthesis of Dipeptide (3)
deprotection
condensation
yield
(%)
yield
(%)
entry PG-Phe-OBIBS (1)
condition
condition
Fmoc-Phe-OH (1.2 eq.) EDCI (1.3 eq.)/CH2Cl2, 0 °C, 3 h
1
2
PG = Fmoc (1a) Et2NH/CH2Cl2, room temperature, 6 h
98
100
95
PG = Boc (1b)
PG = Cbz (1c)
4 N HCl-dioxane/CH2Cl2, room temperature, 7
h
Pd−C, H2/TFE, room temperature, 24 h
3
100
a
Bn, benzyl; EDCl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; PG, protecting group; and TFE, 2,2,2-trifluoroethanol.
a
Scheme 3. Evaluation of DKP Suppression During Fmoc Deprotection and Selective Deprotection of BIBS
a
Bn, benzyl; Bzl, benzyl; COMU, 1-[(1-(cyano-2-ethoxy-2-oxoethylidene aminooxy) dimethylamino morpholino)] uronium hexafluorophosphate;
and DIPEA, N,N-diisopropylethylamine.
In this study, we reported synthetic studies of a model cyclic
octapeptide using a combination of our new coupling method
with silyl ester carboxylate protection. This model peptide
derived from an in vitro display library had a challenging
synthetic target due to the presence of both a thioether linkage
from the N-terminus to a cysteine (Cys) residue, and the
coupling of a sterically hindered N-methylleucine (MeLeu)
and sarcosine (Sar) amino acid. It also contained a Pro residue
that made the structure prone to DKP formation.
Obtained Fmoc-Phe-Phe-OBIBS (3) showed high solubility in
organic solvents due to the high hydrophobicity of BIBS group.
The solubility of Fmoc-Phe-Phe-OBzl (4) in cyclopentyl
methyl ether (CPME), toluene, and ethyl acetate was
approximately 2−7 mg/mL, while that of (3) was 300−400
mg/mL. It is expected to improve solubility during peptide
elongation by using silyl-protecting groups. Another type of
highly bulky “super silyl” protecting group, tris(trimethylsilyl)-
silyl, which was expected to be superior at preventing DKP
production, was cleaved under the conditions used for
deprotection of the Fmoc group (data not shown).36 Hence,
we concluded that the super silyl-protecting group was not
suitable for peptide elongation syntheses.
To evaluate the preventative effect of DKP formation by
bulky silyl groups, the Fmoc-deprotection reactions of (3) and
(4) were both conducted. Deprotection was complete in 3 h
with diethylamine treatment, and DKP formation was not
observed for the C-terminal BIBS-protected dipeptide (5),
while 89% of the Bzl-protected peptide (6) was converted to
DKP product (7) (Figure S1). Peptide (5) was coupled with
Cbz-Ser(tBu)-OH to produce high-yield Cbz-Ser(tBu)-Phe-
Phe-OBIBS (8). Treatment with potassium fluoride resulted in
selective removal of the C-terminal BIBS group without
cleavage of the Cbz or tBu groups. The deprotected BIBS
group was easily removed by washing with n-heptane, and the
RESULTS AND DISCUSSION
■
Evaluation of Silyl-Protecting Groups in Peptide
Synthesis. To evaluate the applicability of bulky silyl
carbonyl-protecting groups in peptide synthesis, the stability
of the di-tert-butyl-isobutylsilyl (BIBS) group was examined.35
The BIBS-protecting group was introduced into the carboxyl
group by employing the reagent di-tert-butylisobutylsilyl
trifluoromethanesulfonate (BIBS-OTf) under basic conditions.
The resultant BIBS-protected, Nα-protected phenylalanine was
treated under conditions typically employed for the depro-
tection of Fmoc, Boc, and Cbz groups in standard peptide
chemistry (Table 1). Each of the deprotection reactions
proceeded quickly without cleavage of the silyl group, and the
resulting compound H-Phe-OBIBS was coupled with Fmoc-
Phe-OH to produce the corresponding dipeptide in high yield.
C
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a
Scheme 4. Synthesis of cHBS-H
a
nBuLi, n-butyllithium; PhBr, bromobenzene; and Bzl, Ru−Al, ruthenium on alumina
Scheme 5. Overall Synthetic Scheme for the Synthesis of Cyclic Octapeptide (24)
Table 2. Synthesis of Dipeptide Fragments With Isostearyl-Mixed Anhydrides
time
b
yield
c
diastereomer ratio
(% d.r.)
a
entry
PG-AA1-OH
H-AA2-R
PG-AA1-AA2-R
method
(h)
(%)
1
2
3
4
Fmoc-Phe-OH (13a)
Cbz-Lys(Boc)-OH (13b)
Cbz-Thr(tBu)-OH (13c)
Fmoc-Pro-OH (13d)
H-Ala-OH (14a)
H-MeLeu-OH (14b)
H-Sar-OH (14c)
Fmoc-Phe-Ala-OH (15a)
Cbz-Lys(Boc)-MeLeu-OH (15b)
Cbz-Thr(tBu)-Sar-OH (15c)
A
A
A
B
2
15
3
91
100
99
>99.5 : 0.5
>99.5 : 0.5
99.4 : 0.6
d
H-Cys(Trt)-NH2 (14d)
Fmoc-Pro-Cys(Trt)-NH2 (15d)
2
101
>99.5 : 0.5
a
Method: (A) reaction equivalent ratios are 13a−c (1.0 equiv) and 14a−c (1.2 equiv). (B) Reaction equivalent ratios are 13d (1.2 equiv) and 14d
b
c
d
(1.0 equiv). Reaction time after adding silylated 14a−d. Isolated yield without column chromatography. The Fmoc group of 15d was
deprotected with piperidine in methylene chloride to yield H-Pro-Cys(Trt)-NH2 (15e).
corresponding C-terminal free tripeptide (9) was obtained in
high purity without column purification (Scheme 3).
We formulated a synthetic plan for the model cyclic
octapeptide (Scheme 5) comprising the following steps: (1)
four dipeptides (15a−15d), including N-methyl amino acid
dipeptides, were synthesized using isostearyl-mixed anhy-
drides; (2) the Sar carboxy group of the third dipeptide
(15c) was protected with cHBS; (3) hexapeptide (19) was
constructed by the sequential condensation of (15b) and
(15a) with the cHBS-protected dipeptide (16), and (15e) was
introduced after removal of the cHBS group; (4) full-length
protected peptide (22) was constructed by introducing a
chloroacetyl group into the N-terminus; and (5) deprotection
of the side chain-protecting group and cyclization produced
the target peptide (24).
Synthesis Strategy. For our synthetic studies of cyclic
octapeptides, the cyclohexyl di-tert-butyl silyl (cHBS) group
showed similar stability under acidic and basic conditions to
the BIBS group and was chosen as a selectively cleavable C-
terminal-protecting group. cHBS was more suitable for large-
scale manufacturing than BIBS as the necessary starting
materials are more readily accessible. cHBS-H (12) was
synthesized by a two-step reaction with phenyllithium addition
to di-tert-butylsilane (10) and hydrogenation of the phenyl
group (Scheme 4) because one-pot synthetic procedure with
di-tert-butyldichlorosilane, lithium, and cyclohexene was
reported in low yield and Grignard reaction or hydrosilylation
did not proceed in preliminary studies.37
In this process, (15e) was introduced after the construction
of the hexapeptide (20) because epimerization during the
D
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a
Scheme 6. Synthesis of H-Thr(tBu)-Sar-OcHBS (17)
a
Reagents and conditions: (a) trifluoromethanesulfonic acid (1.2 equiv)/PhMe, 0 °C; (b) DIPEA (1.9 equiv)/CPME, 0 °C; and (c) 10% Pd−C
(0.2 wt), H2/TFE, 40 °C. *16 was used for the next reaction without isolation. The yield including the residue of cHBS was 132.9%, and the HPLC
purity at 210 nm was 96.9%. **17 was used for the next reaction without isolation. The yield from 15C was 130.4%, and the HPLC purity at 210
nm was 95.1% (Figure S2).
a
Scheme 7. Construction of the Linear Octapeptide (22) Using Dipeptide Fragment Condensation
a
Reagents and conditions: (a) 15b (1.3 equiv), DIPEA (6.0 equiv), oxyma (3.0 equiv), COMU (1.5 equiv)/CH2Cl2, −20 °C; (b) 10% Pd−C (0.2
wt), H2/TFE, 40 °C; (c) 15a (1.3 equiv), NMM (1.4 equiv), IBCF (1.2 equiv)/THF, −10 °C; (d) KF (2.0 equiv)/THF−MeOH, 0 °C; (e) 15e
(1.1 equiv), ISTA−Cl (1.2 equiv), DIPEA (1.2 equiv)/THF−MeCN, 0 °C; and (f) Et2NH (30 equiv)/CH2Cl2, r.t.; (g) DIC (1.2 equiv), ClAcOH
(2.4 equiv)/CH2Cl2, r.t.
condensation between fragments (20) and (15e) could be
avoided by the presence of an achiral Sar at the C-terminus of
(20). The use of H-Thr(tBu)-Sar-OH was not considered an
appropriate fragment for condensation by our isostearyl-mixed
anhydride conditions because rapid conversion to DKP was
observed during removal of the Cbz group of (15c) in
preliminary experiments (see Supporting Information). The
progress of the reaction and byproduct formation, including
epimerization during dipeptide formation and fragment
condensation, were analyzed by HPLC, LC−MS, and SFC.
Authentic samples incorporating the corresponding D-amino
acid enantiomer at each position were produced using solid-
phase synthesis to determine the extent of epimerization. All
epimerized standard samples could be separated from the
target peptide standard during HPLC analysis.
E
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a
Scheme 8. Synthesis of a Cyclic Octapeptide (24)
a
Reagents and conditions: (a) TFA: m-cresol: thioanisole: H2O/TIS/DTT = 80:5:5:5:2.5:2.5 v/v/v/v/v/w, r.t.; (b) 100 mM NH3HCO3 aq/
MeCN (1:1), peptide concentration: 5 g/L, r.t.
Synthesis of Dipeptide Units. Four dipeptide fragments
were synthesized using the isostearyl-mixed anhydride method-
ology (Table 2). ISTA-Cl was reacted with (13a−13d) at 0 °C
for 2−3 h, followed by coupling with the corresponding
silylated amino acid (14a−14d). The silylation showed not
only a temporary protective effect but also enhanced the
solubility of the amino acids in organic solvents. This property
was also effective at improving the solubility of the C-terminal
amidated cysteine derivative (14d) in which the reaction did
not proceed without silylation due to its low solubility. All
reactions were completed within 2−15 h and quenched by
adding 10% KHSO4. Impurities were removed during the
washing and extraction steps of this method. The rate of
epimerization was evaluated by comparison with authentic
samples of the corresponding diastereomers synthesized
separately, using supercritical fluid chromatography (SFC)
with a chiral column. We found that all target dipeptides were
produced in high yield and in high purity without column
purification of the final products.
Fragment Condensation. Dipeptide Cbz-Thr(tBu)-Sar-
OcHBS (16) was obtained through the introduction of cHBS-
H (12) into Cbz-Thr(tBu)-Sar-OH (15c) following the
method described by Yamamoto et al. (Scheme 6).36 Although
rapid conversion of H-Thr(tBu)-Sar-OH to DKP was observed
in our preliminary study, no DKP formation was observed
when the Cbz group was removed from (16) (Figure S2). The
significant suppression of DKP formation for Sar, which is
prone to DKP products, indicated the superior DKP-
suppressive effect of the cHBS group.
The N-terminal chloroacetylated linear octapeptide (22)
was constructed by fragment condensation of four dipeptides
and chloroacetic acid (Scheme 7). Fragment condensation of
(15b) and (17) yielded Cbz-Lys(Boc)-MeLeu-Thr(tBu)-Sar-
OcHBS (18). Slow condensation rates due to the high steric
hindrance of MeLeu and Thr(tBu) tended to cause
epimerization, which was suppressed to below 0.5% by using
1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethyla-
minomorpholino)] uronium hexafluorophosphate (COMU)
as a condensing agent and the addition of ethyl cyano-
(hydroxyimino)acetate (Oxyma). After Cbz deprotection, the
corresponding tetrapeptide was coupled with Fmoc-Phe-Ala-
OH (15a). We used conventional mixed anhydrides with IBCF
as a coupling reagent due to the fast activation and a short
reaction time. The coupling between the dipeptide (15a),
which has Ala at the C-terminus and the tetrapeptide, which
has the Lys at N-terminus proceeded quickly with good
enantiopurity. Next, the cHBS group of the hexapeptide (19)
was selectively removed by potassium fluoride treatment and
the resulting compound (20) was coupled with H-Pro-
Cys(Trt)-NH2 (15e) using ISTA-Cl. The condensation was
completed quickly within 2 h. The Oxyma-based condensation
agent has higher thermal stability than triazole-based reagents;
however, concerns remain about HCN gas generation.38
Unlike Oxyma and chloroformate, ISTA-Cl is a less-hazardous
coupling agent with high thermal stability that does not
generate HCN or CO2 gas.26 The introduction of the
chloroacetyl group was performed by adding chloroacetic
anhydride, which was activated by N,N′-diisopropylcarbodii-
mide (DIC) in situ. This afforded the full length linear
octapeptide (22) with side chains protected in good HPLC
purity (83.2%).
The linear precursor peptide (23) was produced by
removing the side chain protection of (22) with a TFA-
scavenger cocktail (Scheme 8). Cyclization by thioether bond
formation was performed in aqueous solution, which produced
a crude product (24). Major impurities included cyclic
tetrapeptide (25) and cyclic decapeptide (26), derived from
the remaining dipeptide (15a) produced during the synthesis
of (21).39 These impurities were easily removed by HPLC
purification to yield a purified peptide with a purity higher than
99% (Figure S3) in 73.5% yield from dipeptide (15c) to the
final purified product (24) (TFA salt).
CONCLUSIONS
■
Convergent solution-phase peptide synthesis is an excellent
strategy for manufacturing short- to medium-length peptides;
however, conventional methodologies possess many limita-
tions, including unidirectional elongation from only the C-
terminus to N-terminus, which requires care in avoiding the
risk of C-terminal epimerization. Frequently used C-terminal-
protecting groups including methyl and ethyl esters have a high
risk of side reactions during deprotection and limit the options
for C-terminal amino acids that can be employed. Repeated
coupling in the N-terminus to the C-terminus direction
without an intermediate deprotection step using isostearyl-
mixed anhydrides allows for the efficient synthesis of C-
terminus-free peptide fragments. The silyl ester carboxy-
protecting group was suitable for this methodology and
orthogonal to typical peptide synthesis methods, enabling the
synthesis of N-protected peptide fragments through selective
deprotection without side reactions. In this study, we have
reported the efficient synthesis of a highly challenging cyclic
octapeptide containing two N-methyl amino acids, using a
combination of two new methods for the synthesis of peptide
F
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fragments without the requirement of column purification in
the intermediate steps. We believe that these methods could
greatly enhance the flexibility of solution-phase peptide
synthesis strategies and help accelerate future research in
peptide manufacturing.
used in the next reaction and a portion was purified by column
chromatography to confirm the structure.
1
Cyclohexyl di-tert-butylsilane (12). H NMR (CDCl3 with
0.05% v/v TMS, 600 MHz): δ 3.19 (s, 1H), 1.82−1.85 (m,
2H), 1.71−1.75 (m, 3H), 1.43−1.50 (m, 2H), 1.20−1.26 (m,
3H), 1.05 (s, 18H), 0.97−1.02 (m, 1H); 13C NMR (600 MHz,
CDCl3): δ 30.7, 29.9, 29.1, 27.2, 25.2, 20.2; IR (ATR, cm−1):
2963, 2925, 2855, 2082, 1471, 1446, 1388, 1364, 1102, 1040,
1013, 1005, 934, 884, 848, 823, 807, 799, 783.
EXPERIMENTAL SECTION
■
General Information. All reactions for substrate prepara-
tion were performed in standard, dry glassware fitted with
rubber septa. Reactions were monitored by HPLC or LC−MS.
1H and 13C NMR spectra were collected with a 600 MHz
spectrometer using the deuterated solvent as an internal
deuterium reference. High-resolution mass spectrometry
(HRMS) was performed on an electrospray ionization-mass
spectrometer. Solid-phase syntheses of reference peptides were
conducted by Fmoc SPPS that produced Fmoc cleavage with
20% piperidine/NMP and an amino acid condensation
reaction using DIC/oxyma on an automated peptide
synthesizer. Protected peptide resins were treated with
HFIP−DCM (3:7) to obtain side chain-protected peptides
and a TFA scavenger cocktail to produce deprotected peptides,
which were then analyzed by HPLC without purification and
used as reference compounds. Amino acids were purchased
from Watanabe Chemical Industries, Tokyo Chemical
Industry, GL Biochem, and Merck. ISTA-Cl was synthesized
as previously described.26 Di-tert-butylsilane and Di-tert-
butylisobutyl trifluoromethanesulfonate were purchased from
Gelest. Other reagents and solvents were purchased from
Kanto Chemical, FUJIFILM Wako Pure Chemical, Tokyo
Chemical Industry, and Watanabe Chemical Industries.
ClAc-Phe-Ala-Lys(Boc)-MeLeu-Thr(tBu)-Sar-Pro-Cys(Trt)-
NH2 (22). To a mixture of 6.8 g unpurified 12 (quantitated
purity: 43%, quantified based on the purified 12, 13.0 mmol)
and TfOH (2.3 g, 15.3 mmol) at 0 °C in PhMe, 15c (3.9 g,
10.3 mmol) in CPME and DIPEA (2.6 g, 20.1 mmol) were
added. The mixture was stirred for 2 h at 0 °C, and washed
with 5% NaHCO3, 10% KHSO4, and 10% NaCl. The organic
layer was concentrated and dissolved in n-heptane, washed
with 50% MeCN−H2O and 10% NaCl, and concentrated to
yield crude 16 (8.0 g), to which 10% Pd−C (1.2 g) was added
in TFE and stirred under a H2 gas atmosphere at 40 °C for 1.5
h. Pd−C was removed and concentrated to yield crude 17 (6.1
g). Next, 15b (6.6 g, 13 mmol), COMU (6.4 g, 14.9 mmol),
Oxyma (4.3 g, 30.3 mmol), and DIPEA (7.8 g, 60.4 mmol)
were added to the crude 17 (6.1 g) in CH2Cl2 at −20 °C. After
stirring for 3 h at −20 °C, TFE was added and stirred for an
additional 1 h at −20 °C before the solution was washed with
10% KHSO4, 5% NH3, and H2O and concentrated to obtain
the brown oil of crude 18 (13.9 g). Next, 10% Pd−C (1.9 g)
was suspended in the TFE solution of crude 18 (13.9 g) under
a H2 gas atmosphere at 40 °C for 2.5 h. Pd−C was removed
and concentrated to yield H-Lys(Boc)-MeLeu-Thr(tBu)-Sar-
OcHBS (18b, 12.7 g). Furthermore, 15a (6.0 g, 13.1 mmol)
was stirred with NMM (1.4 g, 13.8 mmol) and IBCF (1.6 g,
11.7 mmol) in THF at −10 °C for 5 min and the mixture was
added to a 18b (12.7 g) solution in THF and stirred for 1 h at
−10 °C. AcOiPr was added to the mixture and stirred for 1 h
at −10 °C, after which the organic layer was washed with 10%
KHSO4, 5% NaHCO3, and 10% NaCl and concentrated. IPE
was added to the concentrated solution to form precipitated
crude 19 (15.3 g). Next, crude 19 (15.1 g, set as 10.2 mmol)
was dissolved in a mixture of THF−MeOH (2:1) and KF (1.2
g, 20.7 mmol) was added; the mixture was stirred for 2 h at 0
°C. Subsequently, CHCl3 was added and the mixture was
washed with 10% KHSO4 and 10% NaCl before IPE was
added to obtain the precipitated crude 20 (10.2 g) product.
Next, crude 20 (9.4 g, assumed as 9.4 mmol) in MeCN was
mixed with DIPEA (1.4 g, 10.8 mmol) and 50% ISTA-Cl in
PhMe (6.4 mL, 10.4 mmol) at 0 °C and stirred for 6 h before
15e (4.7 g, 10.2 mmol) in THF was added to the solution and
stirred for an additional 2 h at 0 °C. The mixture was diluted in
AcOiPr and washed with 10% KHSO4, 5% NaHCO3, and 10%
NaCl before it was concentrated. IPE was added to the
remaining residue and the precipitated solid 21 (12.7 g) was
collected. A mixture of 21 (12.0 g, assumed as 8.9 mmol) in
CH2Cl2 was prepared with Et2NH (17.8 g, 243 mmol) at room
temperature and stirred for 3 h. The mixture was washed with
H2O, 10% NH4Cl, and 5% NaHCO3. Separately, ClAcOH (1.8
g, 19.0 mmol) was mixed with DIC (1.2 g, 9.5 mmol) in
CHCl2 for 10 min at room temperature before being added to
the solution. The mixture was then stirred for 0.5 h at room
temperature, followed by washing with 10% KHSO4 and H2O.
The organic layer was concentrated, IPE was added to
precipitate the product, and the resulting pale red solid was
General Procedure for Synthesizing Dipeptide Frag-
ments. A solution of PG-AA-OH (1 equiv for 13a−c, 1.2
equiv for 13d) in MeCN was stirred at 0 °C and DIPEA (2.0
equiv, relative to 13a−d), 50% ISTA-Cl in PhMe (1.1 equiv,
relative to 13a−d) was added. The mixture was stirred at 0 °C.
Separately, H-AA-OH (14a−c) (1.2 equiv) or H-AA-NH2
(14d) (1 equiv) and N,O-bis(trimethylsilyl)acetamide (BSA, 2
equiv for 14a and 14d, 1.3 equiv for 14b and 14c, relative to
14a−d) was mixed in MeCN at 60 °C, and the mixture was
stirred at 60 °C until the solution became clear. A solution of
silylated 14a−d was added to an activated 13a−d at 0 °C, and
the mixture was stirred for 2−15 h at 0 °C. Next, 10% KHSO4
was added to the reaction solution, washed thrice with n-
heptane, and extracted with AcOiPr. The obtained organic
layer was washed with 10% NaCl and concentrated to produce
the desired dipeptide (15a−d). Characterization data for 15a−
d are listed in Supporting Information.
Cyclohexyl di-tert-butylsilane (12). Bromobenzene (5.7 g,
36 mmol) in THF was added to 1.55 M n-BuLi (35 mL, 54
mmol) in n-hexane and stirred for 2 min at 0 °C, followed by
removal from the ice bath. The mixture was stirred for 6 h at
room temperature. Subsequently, di-tert-butyl silane (2.0 g, 14
mmol) in THF was added and incubated with stirring for 15 h
at room temperature. The reaction mixture was washed with 2
M HCl and 5% NaCl before the organic layer was
concentrated and filtered with the addition of n-hexane and
silica gel. The filtrate was concentrated and crude 11 was
dissolved in 25 mL n-hexane, followed by the addition of Ru−
Al (5%, 0.61 g). This mixture was then stirred under 0.8 MPa
H2 gas for 19 h at 30 °C. The solution was filtered through
silica gel and concentrated to produce crude 12 (5.9 g, 81%
yield over 2 steps) as a colorless liquid. The unpurified 12 was
G
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Article
collected to obtain crude 22 (12.2 g, quantitative yield from
15c).
ClAc-Phe-Ala-Lys(Boc)-MeLeu-Thr(tBu)-Sar-Pro-Cys(Trt)-
NH2 (22). ESI-MS (monoisotopic): [M + H]+ 1337.6777
(calculated for C70H98Cl1N10O12S1, 1337.6775).
Kazuya Wakui − Chemical Research Laboratories, Nissan
Chemical Corporation, Funabashi 274-8507 Chiba, Japan
Madoka Yoshino − Chemical Research Laboratories, Nissan
Chemical Corporation, Funabashi 274-8507 Chiba, Japan
Ayumu Matsuda − PeptiDream, Inc., Kawasaki, Kanagawa
210-0821, Japan
(&)CH2CO-Phe-Ala-Lys-MeLeu-Thr-Sar-Pro-Cys(&)-NH2
(24). To a crude sample of 22 (10.0 g, set as 7.3 mmol), a
mixture of TFA (80.0 mL), m-cresol (5.0 mL), thioanisole (5.0
mL), H2O (5.0 mL), TIPS (2.5 mL), and DTT (2.5 g) at 0 °C
was added, and the resulting mixture was stirred for 90 min at
room temperature. IPE was added to the reaction solution and
the resulting precipitate was collected by filtration to obtain
crude 23 (7.4 g, quantitative yield). A crude sample of 23 (502
mg) was dissolved in 100 mL 100 mM aqueous NH4HCO3−
MeCN (1:1) and stirred for 1 h at room temperature. The
mixture was concentrated and lyophilized to produce crude 24
(522 mg) as a white amorphous solid. Next, 20.0 mg of crude
24 sample was applied to the preparative HPLC, and a linear
density gradient elution (60 min) was conducted with eluent
ratios A/B: 80:20−78:22 using a Triart Prep C18−S (10 μm,
10.0 × 250 mm). Eluent A: 0.1% TFA in water; eluent B: 0.1%
TFA containing MeCN. The fractions containing the product
were collected and lyophilized to produce 11.7 mg white
powder 24 (70.4% yield from crude 22).
(&)CH2CO-Phe-Ala-Lys-MeLeu-Thr-Sar-Pro-Cys(&)-NH2
(24). ESI-MS (monoisotopic): [M + H]+ 903.4744 (calculated
for C42H67N10O10S1, 903.4762), elution time on RP-HPLC:
19.6 min. Elution conditions: an XBridge C18 column (2.5
μm, 100 × 2.1 mm), linear density-gradient elution with
eluents A/B: 90/10 (0−2 min), 70/30−5/95 (2−15 min), and
5/95 (15−25 min), 0.1% TFA in water as eluent A and 0.1%
TFA containing MeCN as eluent B, flow rate: 1 mL/min, and
column oven temperature: 40 °C.
Yutaka Kobayashi − PeptiDream, Inc., Kawasaki, Kanagawa
210-0821, Japan
Haruaki Kurasaki − PeptiDream, Inc., Kawasaki, Kanagawa
210-0821, Japan; orcid.org/0000-0003-4276-0545
Douglas R. Cary − PeptiDream, Inc., Kawasaki, Kanagawa
210-0821, Japan; orcid.org/0000-0003-3940-6078
Keiichi Masuya − PeptiDream, Inc., Kawasaki, Kanagawa
210-0821, Japan
Michiharu Handa − Chemical Research Laboratories, Nissan
Chemical Corporation, Funabashi 274-8507 Chiba, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.1c00078
Notes
The authors declare no competing financial interest.
Abbreviations used for amino acids and designation of peptides
follow the rules of the IUPAC-IUB Commission of
Biochemical Nomenclature in IUPAC-IUB Commission on
Biochemical Nomenclature.40 Amino acid symbols denote the
L-configuration unless indicated otherwise. Abbreviated
nomenclature for cyclic peptides followed the rules of the
application by Albericio et al.41 “&” indicated the bridging
points in the cyclic peptide.
ACKNOWLEDGMENTS
■
The authors thank Masumi Nakamura for measurement of
optical purity by SFC; Kei Nagae for precise measurement of
1
optical rotation, H NMR, 13C NMR, and IR spectra; Yutaka
ASSOCIATED CONTENT
■
Hirokawa and Takayuki Nagatsuka for kindly supporting the
publication; Hiroyuki Kousaka and Norio Tanaka for their
valuable discussions; and Editage (www.editage.jp) for English
language editing.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.1c00078.
Synthetic procedure and characterization data of the
BIBS group;, characterization data of dipeptide frag-
ments (15a−d); the results and experimental procedures
of deprotection of 15c and additional figures; HPLC
profile of Fmoc deprotection reaction of 3; HPLC
profile of Cbz deprotection reaction of 16; HPLC profile
ABBREVIATIONS
■
APIs, active pharmaceutical ingredients; BIBS, di-tert-butyl-
isobutylsilyl; BIBS-OTf, di-tert-butylisobutylsilyl trifluorome-
thanesulfonate; Bn, benzyl; Boc, tert-butoxycarbonyl; BSA,
N,O-bis(trimethylsilyl)acetamide; Bzl, benzyl; Cbz, benzylox-
ycarbonyl; cHBS, cyclohexyl di-tert-butylsilyl; cHBS-H, cyclo-
hexyl di-tert-butylsilane; ClAcOH, chloroacetic acid; COMU,
1-[(1-(cyano-2-ethoxy-2-oxoethyl idene aminooxy) dimethyla-
mino morpholino)] uronium hexafluorophosphate; CPME,
cyclopentyl methyl ether; DIC, N,N′-diisopropylcarbodiimide;
DIPEA, N,N-diisopropylethylamine; DKP, diketopiperazine;
DTT, DL-dithiothreitol; EDCl, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; Et2NH, diethylamine; Fmoc,
9-fluorenylmethyloxycarbonyl; HPLC, high performance liquid
chromatography; IBCF, isobutyl chloroformate; ISTA-Br,
isostearic acid bromide; ISTA-Cl, isostearic acid chloride;
ISTA-X, isostearic acid halide; LC−MS, liquid chromatog-
raphy-mass spectrometry; LPPS, solution-phase (liquid-phase)
peptide synthesis; MeCN, acetonitrile; MeLeu, N-methylleu-
cine; MeOH, methanol; nBuLi, n-butyllithium; NMM, N-
methylmorpholine; Oxyma, ethyl cyanohydroxyiminoacetate;
Pd−C, palladium on carbon; PG, protecting group; PhBr,
bromobenzene; PhBS-H, di-tert-butyl(phenyl)silane; PhMe,
1
of crude and purified 24; and copies of the H and 13C
NMR spectra (PDF).
AUTHOR INFORMATION
■
Corresponding Author
Naoki Nishizawa − Chemical Research Laboratories, Nissan
Chemical Corporation, Funabashi 274-8507 Chiba, Japan;
orcid.org/0000-0002-5530-2511; Email: nishizawan@
nissanchem.co.jp
Authors
Akihiro Nagaya − Chemical Research Laboratories, Nissan
Chemical Corporation, Funabashi 274-8507 Chiba, Japan
Shota Murase − Chemical Research Laboratories, Nissan
Chemical Corporation, Funabashi 274-8507 Chiba, Japan
Yuji Mimori − Chemical Research Laboratories, Nissan
Chemical Corporation, Funabashi 274-8507 Chiba, Japan
H
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(19) Rasmussen, J. H. Synthetic peptide API manufacturing: A Mini
Review of Current Perspectives for Peptide Manufacturing. Bioorg.
Med. Chem. 2018, 26, 2914−2918.
toluene; Ru−Al, ruthenium on alumina; r.t, room temperature;
Sar, sarcosine; SFC, supercritical fluid chromatography; SPPS,
solid-phase peptide synthesis; tBu, tert-butyl; tBu2SiH2, di-tert-
butylsilane; TFA, trifluoroacetic acid; TFE, 2,2,2-Trifluoroe-
thanol; TfOH, trifluoromethanesulfonic acid; THF, tetrahy-
drofuran; TIS, triisopropylsilane; TMS, trimethylsilyl; Trt,
triphenylmethyl
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