Head-to-tail macrocyclization of cysteine-free peptides using an o-aminoanilide linker

Article abstract of DOI:10.1016/j.bmcl.2018.03.027

A head-to-tail macrocyclization protocol for the preparation of cysteine-free cyclic peptides was investigated. The o-aminoanilide linker constructed in the peptide sequence by a standard Fmoc-based peptide synthesis procedure was subjected to nitrite-med

Full text of DOI:10.1016/j.bmcl.2018.03.027

Accepted Manuscript
Head-to-tail macrocyclization of cysteine-free peptides using an o-aminoanilide
linker
Takumi Ohara, Masato Kaneda, Tomo Saito, Nobutaka Fujii, Hiroaki Ohno,
Shinya Oishi
PII:
S0960-894X(18)30206-3
https://doi.org/10.1016/j.bmcl.2018.03.027
BMCL 25681
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
20 February 2018
6 March 2018
11 March 2018
Please cite this article as: Ohara, T., Kaneda, M., Saito, T., Fujii, N., Ohno, H., Oishi, S., Head-to-tail
macrocyclization of cysteine-free peptides using an o-aminoanilide linker, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.027
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Head-to-tail macrocyclization of cysteine-free peptides using an o-aminoanilide linker
Takumi Ohara, Masato Kaneda, Tomo Saito, Nobutaka Fujii, Hiroaki Ohno, Shinya Oishi*
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
*Corresponding Author:
Shinya Oishi, Ph.D.
Graduate School of Pharmaceutical Sciences
Kyoto University
Sakyo-ku, Kyoto, 606-8501, Japan
Tel: +81-75-753-4561; Fax: +81-75-753-4570,
E-mail: soishi@pharm.kyoto-u.ac.jp
1

ABSTRACT. A head-to-tail macrocyclization protocol for the preparation of cysteine-free cyclic
peptides was investigated. The o-aminoanilide linker constructed in the peptide sequence by a
standard Fmoc-based peptide synthesis procedure was subjected to nitrite-mediated activation under
acidic conditions toward N-acyl benzotriazole as the active ester species. The subsequent cyclization
smoothly proceeded by neutralization in the presence of additives such as 1-hydroxybenzotriazole
(
HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) to afford the expected cyclic pentapeptide, a
CXCR4 antagonist. The cyclization efficiencies were dependent on the precursor open-chain
sequence. The application of this step-wise activation-cyclization protocol to microflow reaction
systems is also described.
Keywords: chemokine; CXCR4 antagonist; cyclic peptide; macrocyclization: microflow reaction
Abbreviations: CXCR4, CXC chemokine receptor 4; Dbz, 3,4-diaminobenzoic acid; DPPA,
diphenylphosphoryl azide; Nal, 3-(2-naphthyl)-L-alanine; NCL, native chemical ligation; Pbf,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; SDF-1, stromal cell-derived factor 1; SPPS,
solid-phase peptide synthesis
2

Cyclic peptides are attractive scaffolds that provide great chemical diversity arising from their
1
,2
various macrocyclic structures and the combination of their component amino acids. A number of
approved drugs as well as promising clinical candidates with head-to-tail cyclic peptide structures
3
have been developed through medicinal chemistry efforts. For the facile preparation of cyclic
peptides by solid-phase approaches, several characteristic linkers have been reported, which are
converted into active ester species at the C-terminus of the precursor open-chain peptides. For
example, Kaiser oxime resin was employed for the synthesis of side-chain bridged and head-to-tail
4
cyclic peptides via a Boc strategy of solid-phase peptide synthesis (SPPS). Sulfamylbutyryl linker,
compatible with the basic conditions of Fmoc-SPPS, is subjected to activation by treatment with
5
iodoacetonitrile or (trimethylsilyl)diazomethane. The subsequent nucleophilic addition of the N-
6
terminal amino group led to formation of the head-to-tail cyclic peptides. This safety-catch linker
7
,8
has also been employed for the preparation of peptide thioester segments for native chemical
9
,10
ligation (NCL) in protein synthesis.
The 3,4-diaminobenzoic acid (Dbz) linker (o-aminoanilide linker) is another useful active ester
1
1
precursor (Scheme 1). The Dbz linker is activated by treatment with p-nitrophenyl chloroformate
to form an N-acyl benzimidazolone intermediate, which can be converted into the peptide thioester
in the presence of an appropriate thiol. By taking advantage of similar activation principles, a
12,13
number of protocols for peptide thioester preparations have also been developed.
Recently, an
alternative activation protocol of Dbz linker 1 was reported, in which NaNO -mediated triazole
2
1
4
formation provides a reactive N-acyl benzotriazole 2. Treatment of 2 with a thiol affords a peptide
thioester 3 as an active species for NCL. A poly(arginine) solubilizing tag can be conjugated at the
C-terminus of peptide segments via the Dbz linker to improve the solubility during protein synthesis
1
5
and then be converted into the C-terminal acid by hydrolysis. We envisioned that the cyclization
would proceed even without a Cys group to yield cyclic peptide 4 when the N-terminal amino group
is located in close proximity to the C-terminal N-acyl benzotriazole intermediate 2. In this study, we
3

investigated a Cys-free macrocyclization protocol using a C-terminal Dbz linker for cyclic peptide
synthesis.
Scheme 1. Application of a C-terminal Dbz linker for the preparation of thioesters and cyclic
peptides.
As a model peptide, we chose the cyclic pentapeptide, FC131 (5, Figure 1), which exhibits highly
potent antagonistic activity against stromal cell-derived factor 1 (SDF-1) binding to CXC chemokine
16
receptor 4 (CXCR4). FC131 and its derivatives also inhibit CXCR4-mediated HIV-1 infection to
1
6,17
show potent anti-HIV activity.
Initially, we synthesized two linear peptide substrates 9a,b, in
which Gly carboxamide was subjected to activation by nitrite-mediated benzotriazole formation of
the Dbz linker. The cyclization at the Gly-D-Tyr peptide bond in FC131 is advantageous in that no
epimers are generated and steric hindrance derived from the side-chain can be avoided during the
activation–cyclization process (Scheme 2). For the preparation of peptide 9a without employing
2
protecting groups for the side-chain functional groups [H-D-Tyr-Arg-Arg-Nal-Gly-Dbz-Gly-NH ;
Nal: 3-(2-naphthyl)-L-alanine], the Dbz linker was loaded on the Rink amide resin 6a via a Gly
linker. The peptide sequence was constructed using standard Fmoc-based solid-phase synthesis
(
2
SPPS). The final deprotection and cleavage from resin 8a with a cocktail of TFA/H O/m-
4

cresol/thioanisole provided peptide 9a. The side-chain protected peptide 9b having a Dbz linker [H-
D-Tyr(t-Bu)-Arg(Pbf)-Arg(Pbf)-Nal-Gly-Dbz-Gly-OH] was also synthesized by Fmoc-based SPPS
on the (2-Cl)Trt resin 6b as well as cleavage from resin 8b by treatment with 1,1,1,3,3,3-hexafluoro-
2
2 2
-propanol (HFIP)/CH Cl (2:8) at room temperature.
Figure 1. Structure of FC131 (5).
Scheme 2. Syntheses of unprotected and protected FC131 (5 and 10).
H2N
Rink amide resin (6a)
Cl
(2-Cl)Trt resin (6b)
1
) Fmoc-Gly-OH, HBTU,
HOBt, (i-Pr)2NEt, DMF
) 20% piperidine/DMF
) Fmoc-Dbz-OH, HBTU,
HOBt, (i-Pr)2NEt, DMF
) 20% piperidine/DMF
1) Fmoc-Gly-OH, (i-Pr)2NEt,
CH2Cl2
2) 20% piperidine/DMF
3) Fmoc-Dbz-OH, HBTU,
HOBt, (i-Pr)2NEt, DMF
4) 20% piperidine/DMF
2
3
4
H-Dbz-Gly-NH
7a
H-Dbz-Gly-O
7b
1) Fmoc-Xaa-OH,
1) Fmoc-Xaa-OH,
Fmoc-based SPPS
t-Bu Pbf Pbf
HOBt, DIPCI, DMF
) 20% piperidine/DMF
Fmoc-based SPPS
t-Bu Pbf Pbf
HOBt, DIPCI, DMF
2) 20% piperidine/DMF
2
H-D-Tyr-Arg-Arg-Nal-Gly-Dbz-Gly-NH
8a H-D-Tyr-Arg-Arg-Nal-Gly-Dbz-Gly-O
8b
Cleavage
Purification
TFA/H2O/m-cresol/
thioanisole (80:10:5:5)
Cleavage
& Purification
20% HFIP/CH2Cl2
&
NH2
H
t-Bu Pbf Pbf
NH2
H
H-D-Tyr-Arg-Arg-Nal-Gly
N
H-D-Tyr-Arg-Arg-Nal-Gly
N
9a
9b
NH
NH
O
CONH2
O
CO2H
Activation
Activation
Cyclization
Table 1
Cyclization
Table 1
t-Bu Pbf Pbf
ref. 16
cyclo(D-Tyr-Arg-Arg-Nal-Gly)
5
cyclo(D-Tyr-Arg-Arg-Nal-Gly) 10
5

Next, the key activation–cyclization processes of 9a,b were investigated (Table 1). When linear
unprotected peptide 9a was treated with NaNO in diluted aq. HCl by the standard protocol for Dbz
2
1
4
activation followed by cyclization of the intermediate 11a under basic conditions, only hydrolyzed
product was obtained (data not shown). The same reaction using isoamyl nitrite in DMF gave the
expected cyclic peptide 5 in 27% yield (entry 1). To improve the cyclization efficiency, we
attempted the conversion of N-acyl benzotriazole into an alternative active ester form by addition of
auxiliary reagents. Using a catalytic quantity of 1-hydroxybenzotriazole (HOBt, 10 mol%), the
product yield of 5 was significantly improved (entry 2). The use of stoichiometric amounts of HOBt
was more effective (entry 3). The best result for the preparation of unprotected cyclic peptide 5 was
obtained with 1-hydroxy-7-azabenzotriazole (HOAt, entry 4). In the case of the protected peptide
substrate 9b, activation and the macrocyclization process resulted in satisfactory yield of 10 even in
the absence of auxiliary reagents (entry 5). Because the choice of base for the cyclization of 11b had
a significant effect on the product yield (Supplementary Table S2), N,N-diisopropylethylamine ((i-
2
Pr) NEt) was employed for further experiments. The cyclization efficiency was not improved in the
presence of catalytic and stoichiometric amounts of HOBt (entries 6 and 7), while the addition of
HOAt slightly increased the product yield (entry 8). These results suggested that the ability of the N-
acyl benzotriazole to be converted to an active ester species was similar to that of the HOBt ester
and slightly less compared with that of the HOAt ester.
6

Table 1. Cyclization of the unprotected and protected peptides 9a,b with a C-terminal Dbz linker.
a
entry
compound
additive (eq.)
-
yield (%)
1
2
3
4
5
6
7
8
9a
9a
9a
9a
9b
9b
9b
9b
27
49
57
71
77
79
77
91
HOBt (0.1)
HOBt (1.5)
HOAt (1.5)
-
HOBt (0.1)
HOBt (1.5)
HOAt (1.5)
a
HPLC yield.
We next investigated the nitrite-mediated activation–cyclization process at the different
cyclization sites for the preparation of protected cyclic peptide 10 (Table 2). Favorable
conformation(s) of the linear precursor for macrocyclization may improve the product yield, while
the efficiency of acylation might be decreased due to the presence of the side-chain of the C-
terminal amino acid. We synthesized four additional protected pentapeptides 9c–f by the identical
procedure for the synthesis of 9b on (2-Cl)Trt resin. Cyclization of peptide 9c at the Nal–Gly
peptide bond smoothly proceeded in the presence or absence of auxiliary reagents to provide the
cyclic peptide 10 in high yields (83–98%) with low epimerization. In contrast, no or significantly
less product 10 was obtained by the activation–cyclization processes from 9d–f in the absence of
any auxiliary reagents (10% yield from 9d; no product from 9e; 39% yield from 9f). In the case of
cyclization from 9d at the Arg(Pbf)–Nal peptide bond, epimerized product 12d was preferentially
obtained over 10. These results suggested that the cyclization efficiencies were highly dependent on
7

the peptide sequence and therefore, its conformations. For the linear substrates 9d–f, the auxiliary
reagents were effective at improving the cyclization yield. In the Dbz linker-mediated cyclization
from 9d and 9f, even catalytic amounts of HOBt (10 mol%) significantly increased the yield of 10.
In particular, addition of stoichiometric amounts of HOAt facilitated the efficiency of
macrocyclization from all linear substrates 9b–f (68% yield from 9d; 42% yield from 9e; 80% yield
from 9f) with minimal levels of byproduct epimer formation. The yields exceeded those from
diphenyl phosphoryl azide (DPPA)-mediated macrocyclization of linear pentapeptide substrates,
1
8
which is one of the conventional protocols for cyclic peptide synthesis (Supplementary Table S3).
Table 2. Cyclization of protected peptides at alternative peptide bonds.
a
yield (10 / 12c-f; %)
sequence
Xaa1-Xaa2-Xaa3-Xaa4-Xaa5]
additive (eq.)
compound
[
HOBt
0.1)
77 / - 79 / -
HOBt
(1.5)
HOAt
(1.5)
none
(
9
b
D-Tyr(t-Bu)-Arg(Pbf)-Arg(Pbf)-Nal-Gly
Gly-D-Tyr(t-Bu)-Arg(Pbf)-Arg(Pbf)-Nal
Nal-Gly-D-Tyr(t-Bu)-Arg(Pbf)-Arg(Pbf)
Arg(Pbf)-Nal-Gly-D-Tyr(t-Bu)-Arg(Pbf)
Arg(Pbf)-Arg(Pbf)-Nal-Gly-D-Tyr(t-Bu)
77 / -
91 / -
98 / 1
68 / 5
42 / 0
80 / 0
9
c
83 / 2 90 / 1 96 / 1
10 / 14 47 / 6 59 / 7
9
d
9
e
f
-
-
37 / 0
9
39 / 0 67 / 0 76 / 0
a
HPLC yield.
8

During the course of our investigation, the on-resin cyclization of a protected peptide with a C-
1
9
terminal Dbz linker was reported. The 14-residue sunflower trypsin inhibitor-1 (SFTI-1) was
obtained in 42% yield via on-resin cyclization, final deprotection and intramolecular disulfide bond
1
9
formation. With reference to this approach, we assessed the synthesis of the cyclic pentapeptide via
on-resin cyclization of the linear protected peptide on polyethylene glycol–polystyrene (PEG–PS)
resin. However, FC131 (5) was obtained in only 11% yield with concomitant formation of a cyclic
decapeptide (6% yield, cyclic dimer peptide) (Supplementary Scheme S1). This indicated that lower
loading rate of the N-acyl benzotriazole intermediate on solid support or high dilution in solution-
phase before the macrocyclization step under basic conditions would be needed to obtain the
expected monomeric cyclic pentapeptide in good yield.
On the basis of these insights, we designed a microflow reactor using DMF as the solvent for the
synthesis of cyclic pentapeptide 10 via the activation–cyclization process using the Dbz linker-
containing substrate 9b (Figure 2). Two Y-shape mixers were connected with tubing. To the first
mixer, substrate 9b and isoamyl nitrite in DMF were introduced, after which the activation of the
Dbz-linker occurred within 2.5 min to form N-acyl benzotriazole intermediate 11b (step 1). In the
second mixer, a mixture of 11b was diluted with a 100-fold volume of DMF containing excess (i-
2
Pr) NEt and catalytic HOBt (10 mol%). The cyclization process continued in the tubing for two
hours (step 2) and the mixture was poured into an HCl solution to quench the reaction. The protected
cyclic peptide 10 was obtained in satisfactory yield (80%). Recently, there have been a number of
2
0
synthetic processes for peptide materials using microflow reactors. This microflow-based approach
using a Dbz-linker-containing linear peptide should be applicable to the synthesis of a variety of
cyclic peptides even on a large scale.
9

Figure 2. Dbz linker-mediated macrocyclization using a microflow reaction system.
In summary, we established a Dbz activation-cyclization protocol for the synthesis of the cyclic
peptide, FC131, a CXCR4 antagonist that does not contain a Cys residue. Both side chain-protected
and unprotected linear substrates were subjected to macrocyclization via nitrite-mediated acyl
benzotriazole formation in the presence of HOAt and HOBt as additives to afford the expected
cyclic peptide in moderate to good yields. This nitrite-mediated protocol has an advantage over a
safety-catch sulfonamide linker in that the peptide C-terminus can be activated under mild acidic
conditions without protection of the N-terminal amino group. The investigations using a series of
linear substrates demonstrated that the efficiency primarily depended on the macrocyclization site.
The best result involved the macrocyclization at the Nal-Gly peptide bond with minimal
epimerization. The stepwise activation–cyclization protocol using a continuous microflow reaction
system process also gave a satisfactory result comparable with that obtained from the batch-wise
process under the corresponding conditions. The established protocol should be applicable to the
synthesis of a variety of cyclic pentapeptides including natural products and receptor ligands and be
adaptable to large scale preparations.
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research (15KT0061 and 15H04654) from
JSPS, Japan; the Platform Project for Supporting Drug Discovery and Life Science Research funded
by the Japan Agency for Medical Research and Development (AMED), Japan.
1
0

Supplementary data
Supplementary data associated with this article can be found, in the online version, at #####.
1
1

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