Synthesis of Peptide Disulfide-Bond Mimics by Using Fully Orthogonally Protected Diaminodiacids

Article abstract of DOI:10.1021/acs.orglett.8b02459

A new strategy was developed for the synthesis of peptide disulfide-bond mimics using fully orthogonally protected diaminodiacids. This method overcomes the previous problems of heavy-metal contamination and poor compatibility with Fmoc chemistry and provides a practical avenue for the efficient preparation of peptide disulfide-bond mimics.

Full text of DOI:10.1021/acs.orglett.8b02459

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Synthesis of Peptide Disulfide-Bond Mimics by Using Fully
Orthogonally Protected Diaminodiacids
Tao Wang,†,§,# Jian Fan,‡,# Xiao-Xu Chen,† Rui Zhao,‡ Yang Xu,‡ Donald Bierer,⊥ Lei Liu,∥
Yi-Ming Li,§ Jing Shi,*,‡ and Ge-Min Fang*,†
†School of Life Science, Institute of Physical Science and Information Technology, Anhui University, Hefei 230601, P. R. China
‡Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China
§School of Biological and Medical Engineering, Hefei University of Technology, Hefei 230009, P. R. China
∥Tsinghua University, Beijing 100084, P. R. China
⊥Department of Medicinal Chemistry, Bayer AG, Aprather Weg 18A, 42096 Wuppertal, Germany
S
* Supporting Information
ABSTRACT: A new strategy was developed for the synthesis of peptide disulfide-bond mimics using fully orthogonally
protected diaminodiacids. This method overcomes the previous problems of heavy-metal contamination and poor compatibility
with Fmoc chemistry and provides a practical avenue for the efficient preparation of peptide disulfide-bond mimics.
isulfide-rich polypeptide scaffolds, which are frequently
Dpresent in the structure of FDA-approved peptide drugs
such as insulin, linaclotide, and plecanatide, have attracted a
great deal of attention from peptide-based drug discovery.1
Although disulfide-rich scaffolds confer peptides with
enhanced enzymatic stability and structural diversity, the
disulfide bridge can be unstable toward thiol and disulfide
isomerases in vivo.2 A promising and effective strategy to
improve the metabolic integrity of disulfide-containing
polypeptides is to replace reducible disulfide bridges with
nonreducible bonds such as thioether, selenoether, triazole, or
hydrocarbon bridges.3 This strategy, proposed as early as the
1960s, has been widely used by many groups to engineer a
variety of bioactive peptides4 and displayed an attractive
prospect in peptide drug discovery, as showcased by an
uterotonic and antihemorrhagic drug, carbetocin, a recently
Figure 1. Orthogonally protected diaminodiacids used for the
approved oxytocin analogue containing a thioether bond.5
preparation of disulfide-bond surrogates. Fmoc, Tbe, and Mtt are
A number of methods have been developed for the
fully orthogonal to each other.
preparation of disulfide peptide mimics. Among them, one
efficient and versatile method is diaminodiacid-based solid-
phase peptide synthesis (SPPS) (Figure 1).6,7 This approach
molecular structures of disulfide mimics through the same
synthetic chemistry strategy.3a The diaminodiacids used in this
avoids the use of intermolecular reactions such as thiol
alkylation, azide−alkyne cycloaddition, and olefin metathesis
strategy contain two amino groups and one carboxyl group that
needs to be temporarily protected. More specifically, one
amino protecting group is Fmoc, and the remaining protecting
groups can be Alloc, pNz, Dmab, or ivDde groups, which are
that are commonly used to construct disulfide-bonded mimic
scaffolds on peptide substrates, thereby reducing the number
of synthetic steps and increasing the overall yield of the final
product. More importantly, many different diaminodiacids can
be readily incorporated into the peptides using the same
synthetic strategy, thus enabling the synthesis of diverse
Received: August 2, 2018
© XXXX American Chemical Society
A
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orthogonal to Fmoc.7,9 When using Alloc- and pNz-protected
diaminodiacids, we encountered an annoying issue, namely,
metal contamination of target peptides,8 probably as a result of
the high affinity of the remaining palladium to thiol-rich
peptides.
Recently developed Dmab/ivDde-protected diaminodiacids
avoid the use of heavy-metal reagents; however, neither Dmab
nor ivDde is fully stable toward piperidine,9 rendering them
unsuitable for Fmoc-based peptide synthesis of long sequences
and for use in an automatic synthesizer. In addition, removing
the side-chain carboxyl protecting group of the diaminodiacid
also liberates its side-chain amino functional group, which
during the subsequent cyclization reaction tends to react with
the coupling reagent, thereby lowering the yield of the peptide
synthesis.10 Therefore, there is a strong need to develop new
diaminodiacids whose protecting groups are fully compatible
with Fmoc SPPS and can be removed under metal-free
conditions.
Tbe ester toward piperidine was confirmed by the high stability
of Boc-Phe-Tbe in 25% piperidine over 24 h (Figure S2). Then
we investigated the conditions for Tbe deprotection (Table
S2). Boc-Phe-Tbe was nearly quantitatively converted to Boc-
Phe-OH after reduction by 2-mercaptoethanol in the presence
of an organic base (Et3N, DIPEA, or DBU). We used 2-
mercaptoethanol/DIPEA solution as the deprotection con-
ditions for the subsequent peptide synthesis. In view of the
widespread use of the thioether linkage in the synthesis of
disulfide-bond mimics,15 the Fmoc/Mtt/Tbe-protected C−S-
bridged or S−C-bridged diaminodiacids were synthesized
according to the previously reported procedure, as shown in
Scheme 1 (also see Schemes S1 and S3). These two
Scheme 1. Chemical Synthesis of Fmoc/Mtt/Tbe-Protected
Diaminodiacids
Herein we report a new, practical strategy using thioether-
containing diaminodiacids bearing three completely orthogo-
nal protecting groups (Fmoc, Mtt, and Tbe) as the key
building blocks to synthesize cysteine-rich disulfide-bond
mimics. This method becomes possible on the foundation of
the following findings: (1) hexafluoroisopropanol (HFIP) can
chemoselectively release the Mtt protecting group in the
presence of multiple Trt protecting groups of cysteine residues
(of note, 1% TFA in DCM, which is widely used for Mtt
deprotection, can result in partial removal of Trt from the Cys
residue) and (2) the thiol-sensitive carboxyl protecting group,
Tbe, is fully compatible with Fmoc chemistry. The efficiency
and practicality of this method are illustrated by the successful
synthesis of thioether-containing oxytocin and hepcidin
analogues. The user-friendly diaminodiacid bearing three
fully orthogonal protecting groups makes solid-phase synthesis
of Cys-rich disulfide-bond mimics much simpler and more
efficient.
The acid-sensitive Mtt protecting group was used to protect
the side-chain amino group of the diaminodiacid, but very few
high-quality protecting groups match our requirement for the
side-chain carboxyl group.11 The widely used allyl-based
protecting groups are unacceptable because of the use of
palladium-based reagents.12 We turned our attention to our
recently developed 2-(tert-butyldisulfanyl)ethyloxycarbonyl
(Tbeoc) group for N-terminal cysteine protection in chemical
protein synthesis.13 The metal-free conditions for Tbeoc
deprotection may become completely orthogonal to both
Fmoc and Mtt, so we envisioned the possibility of developing a
practically useful, Fmoc-chemistry-compatible carboxylic pro-
tecting group by coupling 2-(tert-butyldisulfanyl)ethanol
(Tbe) to the side-chain carboxylic acid of the diaminodiacid.
Our interest in the Tbe ester was further stimulated by the
recent finding from Kent’s group that the peptide α-ester
having 2-mercaptoethanol at the C-terminus can be converted
quantitatively into the peptide α-carboxylate via an intra-
molecular displacement mechanism under weakly alkaline
conditions.14
diaminodiacids can provide two peptide disulfide mimics
with subtle differences in the topological structure (∼CH2−S∼
vs ∼S−CH2∼), which could enable in-depth studies of the
disulfide-bond function of bioactive peptides.
With thioether-containing diaminodiacids in hand, we next
tested their compatibility with Fmoc-based SPPS by synthesiz-
ing a model peptide, namely, a cystathionine analogue of
oxytocin. The purity of crude oxytocin prepared from previous
Dmab/ivDde-protected diaminodiacids was less than 50%.9
Therefore, it was tested here whether Mtt/Tbe is more
compatible with Fmoc chemistry than Dmab/ivDde. The
synthesis of the oxytocin analogue started with the Rink amide
AM resin, as shown in Figure 2. After assembly of the
tripeptide, Pro-Leu-Gly, on the resin, the C−S-bridged
diaminodiacid was coupled to the α-N-terminal amino group
of Pro via a double coupling method. Subsequently, the linear
precursor of the oxytocin mimic was finished by assembly of
the tetrapeptide Tyr-Ile-Gln-Asn onto the N-terminal amino
group of the diaminodiacid. After removal of the Tbe
protecting group by 2-mercaptoethanol in DIPEA-containing
NMP, the side-chain carboxyl group of the diaminodiacid was
efficiently cyclized with the N-terminal amino group of Tyr
using PyAOP/NMM. It is noteworthy that although the side-
chain protecting group Mtt is very bulky, it does not hinder the
intramolecular cyclization between the side-chain carboxylic
acid group of the diaminodiacid and the N-terminal amino
group of the peptide. Finally, the intact oxytocin analogue was
cleaved from the resin in TFA. The purity of the crude
oxytocin analogue, as shown in Figure 2, reached 70%, which is
significantly higher than that synthesized using the previous
In order to study the compatibility of the Tbe protecting
group with Fmoc-based SPPS, the following two issues needed
to be addressed: (1) during peptide chain elongation, the Tbe
ester should be completely stable toward 25% piperidine in
DMF, and (2) the Tbe protecting group must be able to be
efficiently removed while the other side-chain protecting
groups of the polypeptide are preserved. The stability of the
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Figure 2. Synthetic route for the preparation of oxytocin and hepcidin disulfide mimics and analytical RP-HPLC traces of the crude synthetic
peptide intermediates (methionine was replaced by norleucine to avoid side reactions). Conditions for Tbe removal: 2-mercaptoethanol (5 M),
DIEA (1.25 M) in NMP. Conditions for Mtt removal: HOBt (5.0 equiv), HFIP (50 vol %) in 1,2-dichloroethane (DCE). Conditions for hepcidin
folding: GSSG (5.0 equiv), GSH (5.0 equiv), 2:1 water/acetonitrile, pH 7.5, rt, 14 h.
Dmab/ivDde-based diaminodiacid,7 demonstrating the high
compatibility of the Tbe/Mtt-protected diaminodiacid with
Fmoc-based peptide synthesis.
strategy to access thioether-containing hepcidin by using the
Mtt/Tbe-based diaminodiacid.
The S−C-bridged diaminodiacid (Figure 1) was selected to
synthesize a hepcidin derivative in which the Cys11−Cys19
disulfide bond was replaced by a thioether bond. After the S−
C diaminodiacid coupling, we analyzed the purity of the
peptide intermediate by HPLC. As expected, the predominant
peak in the HPLC corresponded to the desired peptide. After
assembly of the tetrapeptide and the heptapeptide onto the N-
terminus of the diaminodiacid, we confirmed the high
efficiency of peptide synthesis and the high stability of the
Tbe group toward piperidine by HPLC. Prior to the
intramolecular cyclization reaction, the Tbe group was
quantitatively removed with high chemoselectivity by 2-
mercaptoethanol/DIPEA/NMP, as shown in Figure 2. The
following steps were intramolecular cyclization, Mtt depro-
tection, and assembly of the remaining amino acid residues.
Unexpectedly, when 1% TFA was used to remove Mtt, we
encountered the problem of Trt deprotection of the Cys side
We further tested the practical applicability of the method
by synthesizing the mimics of a more challenging but
pharmaceutically interesting peptide, hepcidin, containing
four pairs of disulfide bonds. This 25 amino acid peptide
hormone has been used as a key element in regulating iron
entry into the circulation, and diseases associated with
hepcidin dysregulation can be treated by intravenous injection
of synthetic hepcidin.16 The folding yield of synthetic linear
hepcidin is less than 12% because of misfolding of the disulfide
bonds.17 Replacement of one disulfide bond with a C−S bridge
significantly increases the yield of hepcidin folding by up to
60%, and therefore, a diaminodiacid-based approach appears to
be a promising hepcidin engineering method.5b However, the
previous method for synthesizing thioether-containing hepci-
din encountered the issue of palladium contamination. In this
context, we attempted to establish a practical, metal-free
C
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Notes
chain, which resulted in multiple side products after the
subsequent amino acid coupling (Figure S16).
The authors declare no competing financial interest.
To solve this problem, we used HFIP/HOBt/DCE to
deprotect the Mtt group.18 To our delight, the HFIP
conditions could selectively remove Mtt without interfering
with the Trt protecting group of Cys residues. Of note, HFIP is
not the first choice for Mtt deprotection because of its
potential toxicity. After completing the linear thioether-
containing hepcidin, we obtained a crude product with an
HPLC purity of over 80%. The subsequent peptide folding was
carried out under common neutral conditions: 33% acetoni-
trile, 1:1 GSSG/GSH, pH 7.5. As analyzed by HPLC, the
folding yield of the hepcidin derivative was 70%, which is
significantly higher than that of wild-type hepcidin. The IC50
value for the hepcidin disulfide thioether mimic was measured
to be 241 nM, which is similar to that of disulfide-linked
hepcidin (224 nM). Taken together, these results show that
the described Mtt/Tbe-protected diaminodiacid is fully
compatible with Fmoc SPPS and avoids the use of heavy
metals, thus providing a user-friendly and practically useful
method of synthesizing bioactive cysteine-rich disulfide-bond
mimics.
To conclude, we have developed user-friendly diaminodia-
cids for Fmoc-based SPPS of cysteine-rich disulfide-bond
mimics. The diaminodiacids reported here contain three
orthogonal protecting groups (Fmoc, Mtt, and Tbe).
Compared with the previously developed pNz/pNb, Alloc/
Allyl, and Dmab/ivDde-protected diaminodiacids, the present
Mtt/Tbe type avoids the use of cytotoxic heavy-metal reagents
and is fully compatible with Fmoc SPPS. The practicability of
this method has been exemplified by the efficient synthesis of
oxytocin and hepcidin analogues. We anticipate that this work
will provide a highly efficient and practically useful strategy for
the efficient preparation and optimization of cysteine-rich
disulfide-bond mimics for diagnostic and therapeutic applica-
tions.
ACKNOWLEDGMENTS
■
This work was supported by the National Key R&D Program
of China (2017YFA0505200), the National Natural Science
Foundation of China (21572214, 21532004, 21807001, and
91753205), and the Fundamental Research Funds for the
Central Universities (PA2017GDQT0021).
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: fanggm@ahu.edu.cn.
*E-mail: shijing@ustc.edu.cn
ORCID
Lei Liu: 0000-0001-6290-8602
Yi-Ming Li: 0000-0001-6716-8199
Jing Shi: 0000-0003-0180-4265
Ge-Min Fang: 0000-0003-4149-4062
Author Contributions
#T.W. and J. F. contributed equally.
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