Dmab/ivDde protected diaminodiacids for solid-phase synthesis of peptide disulfide-bond mimics

Article abstract of DOI:10.1016/j.tetlet.2017.03.024

The use of pre-prepared diaminodiacids has been established as an effective approach for the chemical synthesis of peptide disulfide bond mimics. A technical problem often encountered in the implementation of the diaminodiacids strategy is the use of heavy metal reagents to remove the side-chain protecting groups. In the present work, we reported the development of diaminodiacid that carry 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-methylbutyl]amino)benzyl (Dmab) and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde) protecting groups. This pair of protecting groups can be readily removed by mild hydrazinolysis during the solid-phase synthesis on resin. We demonstrated the use of Dmab/ivDde protected diaminodiacids in the successful synthesis of a disulfide surrogate of oxytocin.

Full text of DOI:10.1016/j.tetlet.2017.03.024

Accepted Manuscript
Dmab/ivDde protected diaminodiacids for solid-phase synthesis of peptide di-
sulfide-bond mimics
Yang Xu, Tao Wang, Chao-Jian Guan, Yi-Ming Li, Lei Liu, Jing Shi, Donald
Bierer
PII:
S0040-4039(17)30323-4
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.03.024
TETL 48725
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 January 2017
28 February 2017
8 March 2017
Please cite this article as: Xu, Y., Wang, T., Guan, C-J., Li, Y-M., Liu, L., Shi, J., Bierer, D., Dmab/ivDde protected
diaminodiacids for solid-phase synthesis of peptide disulfide-bond mimics, Tetrahedron Letters (2017), doi: http://
dx.doi.org/10.1016/j.tetlet.2017.03.024
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Dmab/ivDde protected diaminodiacids for
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Yang Xu, Tao Wang, Chao-Jian Guan, Yi-Ming Li, Lei Liu, Jing Shi∗, and Donald Bierer∗

1
Tetrahedron Letters
journal homepage: www.els evier.com
Dmab/ivDde protected diaminodiacids for solid-phase synthesis of peptide
disulfide-bond mimics
a
a
Yang Xu‡ , Tao Wang‡ , Chao-Jian Guana, Yi-Ming Lia, Lei Liua, Jing Shia∗, and Donald Biererb*
a Department of Chemistry, University of Science and Technology of China, Hefei 230026; Tsinghua University, Beijing 100084, P. R. China; School of
Biological and Medical Engineering, Hefei University of Technology, Hefei, 230009, China.
b Bayer AG, Department of Medicinal Chemistry, Aprather Weg 18A, 42096 Wuppertal, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
The use of pre-prepared diaminodiacids has been established as an effective approach for
the chemical synthesis of peptide disulfide bond mimics. A technical problem often
encountered in the implementation of the diaminodiacids strategy is the use of heavy metal
reagents to remove the side-chain protecting groups. In the present work, we reported the
development of diaminodiacid that carry 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclo-
hexylidene)-3-methylbutyl]amino)benzyl (Dmab) and 1-(4,4-dimethyl-2,6-dioxocyclohex-
1-ylidene)-3-methylbutyl (ivDde) protecting groups. This pair of protecting groups can be
readily removed by mild hydrazinolysis during the solid-phase synthesis on resin. We
demonstrated the use of Dmab/ivDde protected diaminodiacids in the successful synthesis
of a disulfide surrogate of oxytocin.
Received in revised form
Accepted
Available online
Keywords:
Diaminodiacid
Disulfide
Peptide
2016 Elsevier Ltd. All rights reserved
Solid phase
Protecting group
a) Orthogonally protected Diaminodiacids
Conformation-rigidified peptidic macrocycles have recently
been shown to exhibit good metabolic stability, strong
bioactivity, and high target selectivity.1 With these advantages
peptidic macrocycles represent promising molecules for
diagnostic and therapeutic applications.2 Most of peptidic
macrocycles contains one or multiple disulfide bridges that
contribute to their thermal and hydrolytic stability, as well as
their structural rigidity.3 However, a potential problem of the
disulfide bonds is their poor stability towards reducing
environment or disulfide isomerases in vivo.4 As a result, various
approaches have been developed to replace the disulfide bonds
with bioisosteric and metabolically stable surrogates such as
thioether,5 diselenide,6 triazole,7 or hydrocarbon bridges.8 One
way to make these disulfide surrogates is to carry out post-chain-
assembly side-chain cyclization reactions such as thiol alkylation
or azide-alkyne cycloaddition. Alternatively, disulfide surrogates
can also be prepared by using pre-prepared diaminodiacids so
that the cyclization step is achieved through peptide coupling
reactions.
O
O
O
H
N
H
N
H
Allyl
pNb
Dmab
N
O
Alloc
OH
O
pNz
OH
O
ivDde
X
Y
X
Y
CH2
S
Fmoc
Fmoc
Fmoc
OH
N
H
N
H
N
H
O
O
O
1
2
3
O
O
ivDde
Dmab
N
H
O
O
b) Diaminodiacid-Based Solid-Phase Synthesis Strategy
O
NH2
HO
X
Y
SPPS
Cyclization
resin
resin
Aa Aa Aa
Aa
Aa Aa Aa
H
N
H
O
O
O
H
In the previous implementation of the diaminodiacid-based
strategy for making peptide disulfide bond mimics, two sets of
orthogonally protected diaminodiacids were usually used that
carried either allyl/allyloxycarbonyl (Alloc) or p-nitrobenzyl
(pNb)/p-nitrobenzyloxycarbonyl (pNz) protecting groups
(compounds 1 and 2). These two pairs of protecting groups are
compatible with Fmoc (9H-fluorenyl-methoxycarbonyl) solid-
phase peptide synthesis (SPPS) and can be selectively removed
NH2
N
Aa
Aa Aa Aa Aa
Aa Aa Aa
Aa
Aa
SPPS and cleavage
Aa
Aa
X
X
Y
Y
Aa
Aa
Aa Aa Aa
Aa
resin
N
H
N
H
O
O
Figure 1 a) Previous (1, 2) and new (3) orthogonally protected
diaminodiacids. b) General strategy for diaminodiacid-based
SPPS
b—y —trea—tment with [Pd(PPh ) ]/PhSiH or SnCl /HCl. Nonetheless,
∗ Corresponding authors. E-mails: shijing@ustc.edu.cn; donald.bierer1@bayer.com; ‡These authors contributed equally to this work.
3 4
3
2

2
Tetrahedron
the employment of heavy metal reagents may cause unwanted
facilitate the conduct of related researches towards improved
probe or drug molecules.15 In the present work, the disulfide-
replaced analogue of oxytocin nonapeptide was synthesized via
Dmab/ivDde protected diaminodiacid.
contamination into the target peptides for biological applications.
Furthermore, the heavy metal reagents are not convenient to use
in a fully automatic peptide synthesizer. Therefore, alternative
metal-free protecting group strategies need to be developed for
the diaminodiacid toolbox. In this context we now report the
development of new orthogonally-protected diaminodiacids that
bear 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-
butyl]amino)-benzyl (Dmab) and 1-(4,4-dimethyl-2,6-dioxo-
cyclohex-1-ylidene)-3-methylbutyl (ivDde) protecting groups
(compound 3).11 This pair of protecting groups can be readily
removed by hydrazinolysis (2% NH2NH2 in DMF) under metal-
free conditions and are expected to be compatible with Fmoc-
SPPS.12
The synthesis of oxytocin analogue was carried out by using
the rink amide AM resin. Diaminodiacids 3 was used as the
building block in the synthesis. A linear tripeptide 10 was first
assembled onto the resin by using HCTU (N,N,N′,N′-
tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uranium-hexa-
fluorophosphate) as coupling reagent.9a Then diaminodiacids 3
was attached onto the resin by using HCTU (twice, 30 min and
45 min each). Next, four more amino acids (i.e. Asn, Gln, Ile,
Tyr) were incorporated onto the peptide to give 12. After the N-
terminal Fmoc group and Dmab/ivDde groups were removed by
20% piperidine in DMF and 2% NH2NH2 respectively (20%
piperidine in DMF for 5, 10 min twice; 2% NH2NH2 in DMF for
10 min twice), peptide 14 was obtained by lactamization using
PyBOP ((benzotriazole-1-yloxy) tripyrrolidinophosphonium-
hexafluorophosphate), HOBt (1-hydroxy-1H-benzotriazole), and
NMM (N-methylmorpholine). After acidic cleavage and
deprotection from the resin, crude oxytocin analogue 15 was
obtained.9a
Scheme 1. The synthetic route of Dmab/ivDde protected
diaminodiacid.
Our work started with the synthesis of diaminodiacid 3
(Scheme 1). The synthetic route began with the preparation of
protected amino acid monomers (compounds 6, 8).
Commercially
available
L-homoserine
5
and
S-
(triphenylmethyl)-L-cysteine 7 were used as starting materials.
First, 5 was reacted with N-(9-fluorenylmethoxy carbonyloxy)
succinimide (Fmoc-OSu) and then tert-butylbromide (tBuBr) in
the presence of N,N,N′-triethyl-benzenemethanaminium chloride
(TEBAC). Bromination of the resulting Fmoc- and tert-butyl
protected intermediate afforded in 27% yield over three steps.
Meanwhile 7 was reacted with ivDde-OH and Dmab-OH to
produce the fully-protected amino acid. After that, the
triphenylmethyl protecting group was cleaved with trifluoroacetic
acid (TFA) to yield compound 8 (36%, three steps). Finally, the
fully-protected diaminodiacid 9 was obtained by thioalkylation
reaction between compound 6 and 8 (45% isolated yield). The
tert-butyl group was cleaved with TFA to afford the desired
diaminodiacid 3 (the overall yield of 3 was 12% from 5, or 16%
from 7).
Scheme 3. Synthetic route for the preparation of oxytocin
analogue 15 on solid support using diaminodiacid 3. "P" refers to
protecting groups.
The crude peptide was analyzed by reverse-phase high
performance liquid chromatography (HPLC). We found that the
product contained four major components (Figure 2a, upper). We
speculated that repeated use of piperidine for the deprotection of
Fmoc group may cause decomposition of some Dmab/ivDde
groups, leading to the formation of the side products. To
overcome the problem, we attempted to shorten the time of each
deprotection step (20% piperidine in DMF for 6-8 min). As
shown in Figure 2a (bottom), we found that under the new
conditions, the major peak of the crude peptide became the target
product (peptide 15).
With new protected diaminodiacid building block in hand, we
next examined its use for the solid phase synthesis of peptide
disulfide-bond mimics using oxytocin as a model target
compound. Oxytocin is a multifunctional nonapeptide with N-
terminal cyclic six-residue ring structures and a flexible three-
residue C-terminal tail, all of which was stabilized by an
intramolecular disulfide bond.14 The in vivo receptor of oxytocin
belongs to the G protein-coupled receptor family. Previous works
revealed that oxytocin receptor plays a key role in breast cancer
and autism, and a metabolically stable oxytocin analogue will

3
We now describe development of Dmab/ivDde protected
diaminodiacid that may replace the previously reported
allyl/Alloc or pNb/pNz protected ones. By using 2-
methylpiperidine as reagent for Fmoc deprotection, we
demonstrated that oxytocin disulfide bond mimic 15 could be
synthesized in good efficiency via SPPS. We expect that the new
diaminodiacid would find favorable utilization in the studies on
peptidic macrocycles for diagnostic and therapeutic applications.
Acknowledgements
This work was partly supported by the National Natural Science
Foundation of China (Nos. 21572214, 21572043, 21532004,
81621002, 21521091).
Figure 2 a) HPLC traces of crude oxytocin analogue 15 under
different deprotecting conditions: 20% piperidine in DMF for 5,
10 min twice (upper); 20% piperidine in DMF for 6-8 min once
(bottom). b) ESI-QTOF-MS spectrum of oxytocin analogue 15,
Calcd: 989.2, Found: 988.5.
References and notes
To further improve the synthesis, we paid attention to the
recent finding that the Fmoc group can also be efficiently
removed by 2-methylpiperidine.16 We hypothesized that the
higher steric hindrance of 2-methylpiperidine over piperidine
may lead to higher compatibility to the Dmab/ivDde groups. As a
result, we tested the use of 2-methylpiperidine as the Fmoc
removal reagent for peptide synthesis (20% 2-methylpiperidine
in DMF for 8 min). Indeed we were delighted to obtain the target
product in higher yield from the crude peptide (Fig. 3a, overall
yield = 12%). The circular dichroism (CD) spectrum of 15 in
water showed a negative peak at 196 nm and a positive band at
220-230 nm (Fig. 3c). These observations were consistent with
the structure of native oxytocin.
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