Use of a Removable Backbone Modification Strategy to Prevent Aspartimide Formation in the Synthesis of Asp Lactam Cyclic Peptides?

Article abstract of DOI:10.1002/cjoc.202100272

The synthesis of an Asp lactam derivative of A-183, a selective inhibitor of Factor 7a with good anticoagulant and antithrombotic activity, is described. Our synthesis depends on the use of a removable backbone modification (RBM) strategy to prevent aspar

Full text of DOI:10.1002/cjoc.202100272

Chinese Journal
of Chemistry
CJC
中 国 化 学 - An International Journal
Accepted Article
Title: Use of a removable backbone modification strategy to prevent aspartimide
formation in the synthesis of Asp lactam cyclic peptides
Authors: Tingting Cui, Junyou Chen, Rui Zhao, Yanyan Guo, Jiahui Tang, Yulei Li, Yi-
Ming Li* Donald Bierer, and Lei Liu*
This manuscript has been accepted and appears as an Accepted Article online.
This work may now be cited as: Chin. J. Chem. 2021, 39, 10.1002/cjoc.202100272.
The final Version of Record (VoR) of it with formal page numbers will soon be
published online in Early View: http://dx.doi.org/10.1002/cjoc.202100272.
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Cite this paper: Chin. J. Chem. 2021, 39, XXX—XXX. DOI: 10.1002/cjoc.202100XXX
Use of a removable backbone modification strategy to prevent
aspartimide formation in the synthesis of Asp lactam cyclic pep-
tides
Tingting Cui,‡ Junyou Chen,‡ Rui Zhao,‡,d Yanyan Guo,a Jiahui Tang,a Yulei Li,b Yi-Ming Li*,a Donald Bierer,c
,a
,a
and Lei Liu*,b
a School of Food and Biological Engineering, Engineering Research Center of Bio-process, Ministry of Education, Hefei University of Tech-
nology, Hefei 230009, P. R. China
b Tsinghua-Peking Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biol-
ogy, Center for Synthetic and Systems Biology, Department of Chemistry, Tsinghua University, Beijing 100084 (China)
c Department of Medicinal Chemistry, Bayer AG, Aprather Weg 18A, 42096 Wuppertal, Germany
d Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China
Keywords
Lactam cyclic peptides | Solid-phase peptide synthesis | Aspartimide | Removable backbone modification |
Main observation and conclusion
The synthesis of an Asp lactam derivative of A-183, a selective inhibitor of Factor 7a with good anticoagulant and antithrombotic ac-
tivity, is described. Our synthesis depends on the use of a removable backbone modification (RBM) strategy to prevent aspartimide
formation, which thwarted all attempts to synthesize this target using direct solid-phase peptide synthesis. Validation of the RBM
strategy in the synthesis of a second Asp lactam derivative was also accomplished. The RBM strategy is therefore proposed as a gen-
eral method for the synthesis of Asp lactam cyclic peptides.
Comprehensive Graphic Content
*E-mail: ymli@hfut.edu.cn, lliu@mail.tsinghua.edu.cn
View HTML Article
Supporting Information
‡These authors contribute equally to this work.
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
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through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/cjoc.202100272
This article is protected by copyright. All rights reserved.

First author et al.
Report
H
O
H
O
O
N
N
strategy to
Use RBM
prevent
formation in the
Pg
Pg
Tbe
aa
aa aa
Tbe
N
Ac
aa
aa
aa
O
O
H
H
H
N
aspartimide
( )n
SPPS
N
SPPS
aa
Fmoc
aa
aa
HN
aa aa
Resin
N
H
N
H
Asp
of
lactam
aa
synthesis
peptides
cyclic
aa
Asp(OTbe)
coupling
O
O
O
Pg
Pg
Pg
Pg
H
Pg
Pg
aa
TFA
aa
N
N
aa
aa
aa
Resin
Pg
CONH2
Resin
Pg
H
H
O
H
Pg
O
Pg
N
Pg
N
HO
O
Reductive
amination
O
aa aa
O
Lactam
peptide
N
Ac
cyclic
aa
H
Pg
( )n
HO
NO2
N
aa
OHC
HN
aa
aa
N
H
Ac
O
O
N
H
H
O
Pg
aa
NH2
N
HO
N
O
N
HO
RBM group
O
aa aa
O
N
N
aa
aa
Pg
HO
O
aa
O
H
N
H
Tbe
H
O
NO2
Pg
( )n
Pg
O
NO2
Tbe
aa
HO
O
HN
SnCl 2
Tbeoc ( )n
Pg
NO2 Tbe,Tbeoc
aa
O
H
aa
Fmoc
HN
SPPS
SPPS
N
H
Fmoc
N
O
removal
Pg
N
O
aa
Resin
aa
NO2
aa
Pg
aa
N
H
aa
N
Ac 2
O
aa
aa
Pg
H
O
N
O
N
aa
aa
Pg
Pg
Cyclization
aa
Pg
Pg
aa
Pg
H
Deacetylation
aa
O
Pg
Resin
Pg
SPPS
Pg
Resin
Resin
Pg
Pg
aa
Pg
Resin
O
HO
NH
O
HO
O
O
NH
O
N
N
H
NH2
O
O
H
N
NH
NH
HN
O
H
HN
O
NH
HN
HN
OH
O
N
NH
O
NH
OH
H
HN
HN
HN
O
O
H
N
OH
N
H
2N
N
O
O
O
H
OH
O
HN
O
O
NH2
O
N
H
HN
O
Ac
O
N
O
O
NH
Ac
H
N
H
HN
O
OH
NH
NH2
O
NH
O
HN
H
N
H
H
2N
HO
N
HN
NH
NH2
N
O
H
O
O
O
NH
KIIIA7-14
A-183
Cyclo
[Asp9,Lys13]
lactam
peptide
cyclic
www.cjc.wiley-vch.de
© 2018 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2018, XX, 1－X

Running title
Chin. J. Chem.
We first attempted to synthesize A-183 lactam derivative by
standard Fmoc-SPPS. This target incorporates Asp and Dap resi-
dues, and we planned to protect their side chains using our pre-
viously developed tert-butyl disulfide-based strategy for which the
deprotection conditions are mild and metal-free.[13] Accordingly,
2-(tert-butyldisulfanyl)ethanol (Tbe) and 2-(tert-butyldisulfa-
nyl)ethoxycarbonyl (Tbeoc) were installed onto the Asp and Dap
side chains to give Fmoc-Asp(OTbe) and Fmoc-Dap(Tbeoc), re-
spectively, which were then submitted to standard Fmoc SPPS
using Rink amide resin (Figure 1). A linear peptide incorporating
both Asp(OTbe) and Dap(Tbeoc) was expected after cleavage
from the resin using trifluoroacetic acid (TFA), but no product of
the expected molecular weight was detected by HPLC and ESI-
MS even after repeated trials using several different SPPS condi-
tions.
Background and Originality Content
Lactam cyclic peptides have attracted widespread attention
recently due to their structural rigidity, metabolic stability, and
interesting biological activity;[1-7] and the Asp-based lactam cyclic
peptide drug bremelanotide (Scheme 1), a highly potent syn-
thetic analogue of α-melanocyte-stimulating hormone, was re-
cently approved by FDA.[8] Asp-based lactam cyclic peptides are
usually synthesized by Fmoc solid-phase synthesis (SPPS),[9-15]
which generally entails removal of the temporary protecting
groups on the side chains of Asp and Lys (or their derivatives)
prior to on-resin Asp-Lys lactam cyclization.
A-183 is a 15-residue peptide-based selective inhibitor of Fac-
tor 7a reported to exhibit good anticoagulant and antithrom-
botic effects,[16-18] but its metabolic stability is poor due to the
presence of a disulfide bond which is prone to undergo reduction
under physiological conditions. We sought to synthesize a deriv-
ative of A-183 incorporating an Asp-Dap (a Lys derivative) amide
bond in place of this disulfide bond, a modification anticipated to
enhance its metabolic stability, but our efforts to synthesize it
through direct SPPS repeatedly failed.
H
tBu
tBu Boc tBu
N
Boc
(a)
Tbeoc
H
N
Trp
Leu Val Glu Trp Glu Glu
NH2
tBu
N
H
SPPS
Thr
O
Tbe
Resin
Boc
O
Trp
linear intermediate
Glu
O
H
N
tBu
Thr
Resin
Glu
Arg
N
H
tBu
O
tBu
pbf
unknown
by-products
H
N
Tbeoc
Leu Val Glu Trp Glu Glu
H
N
Trp
NH2
N
H
Thr
TFA
O
Tbe
O
Trp
Glu
O
H
N
Thr
Resin
Glu
N
H
Arg
5
11
17
23
O
Retention
time
(min)
(b)
O
O
S
O
O
O
S
S
S
(Tbeoc)
S
(Tbeoc)
NH
( )4
O
S
NH
OH
(Tbe)
OH
Fmoc
Fmoc
OH
N
Fmoc
N
N
H
H
O
H
O
O
Fmoc-Lys(Tbeoc)-OH
Fmoc-Asp(OTbe)-OH
Fmoc-Dap(Tbeoc)-OH
Figure 1. (a) Direct SPPS of the linear peptide of A-183 derivative. (b)
Chemical structure of 2-(tert-butyldisulfanyl)ethanol protected amino ac-
ids.
To identify the problem, we carefully monitored the peptide
chain elongation process. As shown in Figure 2a, sequential cou-
pling of Arg(Pbf), Glu(OtBu), and Asp(OTbe) onto the Rink amide
resin gave intermediate 1, which gave rise to a single peak with
the correct molecular weight by HPLC / ESI-MS analysis after
cleavage using TFA. Next, we coupled Fmoc-Thr(OtBu) onto 1
and again cleaved the product from the resin. HPLC / ESI-MS
analysis showed that target product 2 was obtained, as well as a
by-product with a molecular weight of 166 units less than that of
2 (Figure 2a). Further coupling of Glu(OtBu), Trp(Boc) and
Thr(OtBu) onto 2 followed by cleavage and analysis showed that
no target product 3 had been formed, but a by-product having a
molecular weight 166 units less than 3 had been synthesized in-
stead. Noting that the difference in molecular weight between 3
and by-product could be accounted for by loss of 2-(tert-butyldi-
sulfanyl)ethanol (MW 166), we hypothesized that the Asp(OTbe)
had converted to the corresponding aspartimide under the basic
(piperidine) conditions used for Fmoc deprotection.
Scheme 1 Structures of bremelanotide, A-183, and its lactam derivative
More careful analysis revealed that the problem stemmed
from the nucleophilic attack of the β-carboxyl of the protected
Asp by the amide nitrogen of the preceding residue of Asp, re-
sulting in the formation of aspartimide by-product.[19-20] To over-
come this problem, we report the use of our recently developed
removable backbone modification (RBM) strategy to synthesize
Asp-based lactam cyclic peptides. Both the Asp lactam derivative
of A-183 and another Asp lactam cyclic peptide cy-
clo[Asp9,Lys13]KIIIA7-14 were successfully synthesized by the
RBM strategy.
To confirm this hypothesis, we synthesized the tripeptide
Asp(OTbe)-Pro-Arg(Pbf) 4, which is similar to the sequence in 2,
but incorporating a Pro residue in place of the Glu(OtBu) and
then added Thr(OtBu) to 4 to generate 5. After cleavage of 5 by
TFA, HPLC and ESI-MS analysis showed the formation of a single
product with the expected mass. Sequential addition of
Results and Discussion
Aspartimide formation during the synthesis of an Asp-
based A-183 lactam cyclic peptide derivative
Glu(OtBu), Trp(Boc) and Thr(OtBu) to 5 yielded 6, which also
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de

First author et al.
Report
gave rise to a single peak with the expected molecular weight by
HPLC / ESI-MS after cleavage. Therefore, replacement of the
Glu(OtBu) with Pro successfully disabled aspartimide formation,
presumably because the Pro residue does not contain an amide
nitrogen capable of nucleophilic attack on the β-carboxyl moiety
of the Asp.[21-23]
(a)
(c)
O
*
O
*
O
O
Tbe
Tbe
Tbe
O
Tbe
O
H
N
H
O
O
H
t
Bu
H
N
Fmoc
N
pbf
N
pbf
Arg
TFA
TFA
Fmoc
N
Fmoc
N
N
H
Fmoc
N
H
Glu Arg
N
N
H
Arg
Glu Arg
H4
O
O
CONH2
O
O
O
O
1
Resin
Resin
H2NOC
24
28
32
35
13
19
25
*
31
SPPS
SPPS
*
O
O
O
O
Byproduct
-166]
Glu
Arg
Tbe
O
Tbe
Tbe
O
H
N
Tbe
O
H
H
t
Bu
O
H2N
pbf
Arg
N
H
N
N
N
t
[M2
Bu
N
N
Thr
Thr
t
N
H
Bu
H2N
pbf
N
Thr
H2N
Thr
TFA
TFA
H
N
TFA
TFA
Arg
H2NOC
H2N
O
H
O
Glu
O
O
H
Arg
2
O
Resin
O
5
Resin
H2NOC
SPPS
SPPS
12
17
22
27
13
19
Byproduct
25
31
O
O
O
Tbe
*
O
Tbe
O
Tbe
O
H
H
Tbe
O
t
Bu
N
N
O
H
N
pbf
H
t
t
Bu
N
O
Thr
Bu
N
H
t
-166]
Bu
N
[M3
N
Thr
H
pbf
Glu
Arg
Arg
Resin
t
N
Thr
Glu
Bu
N
H
O
O
Glu
Trp
Thr
NH2
N
H
Thr
Glu
Trp
Glu
Arg
Glu
Boc Trp
Arg
O
O
3
O6
H2NOC
Resin
Boc
H2NOC
Trp
t
Bu
Thr
NH2
Thr
t
Bu
Thr
NH2
14
19
24
29
13
19
25
31
NH2
NO2
(b)
(d)
O
Tbe
Tbe
Tbe
O
,
HNO3 AcOH
0oC
O
O
N
O
O
Base
CHO
O
O
O
H
Base
CHO
Base
H
H
N
N
N
OH
N
N
OH
molecular
N
H
N
H
N
H
RBM
AspartOimide
H
O
O
O
O
2-hydroxy-4-methoxy-5-nitrobenzaldehyde
Figure 2. Aspartimide formation during the synthesis of the A-183 derivative. (a) The aspartimide-forming side reaction occurred during the synthesis of A-183.
(b) Base can promote aspartimide formation and Asp-Pro sequence can prevent aspartimide formation. (c) Asp-Pro sequence can prevent aspartimide for-
mation. (d) Synthesis route of the RBM molecule.
Synthesis of Asp-based A-183 lactam cyclic peptides deriva-
tive through an RBM strategy
7. Coupling of Asp(OTbe) to 7 led to the formation of a phenol es-
ter intermediate which underwent simultaneous O-to-N acyl
transfer to generate the desired amide 8. Sequential condensation
of Dap(Tbeoc), Trp(Boc), Thr(OtBu), Trp(Boc), Glu(OtBu), and
Thr(OtBu) with 8 afforded 9. HPLC and ESI-MS analysis of the
product obtained by cleavage of 9 from the resin with TFA showed
a single peak with the expected molecular weight and no detecta-
ble aspartimide by-products.
To definitively implicate the backbone amide nitrogen in the as-
partimide formation side reaction, we decided to protect it, to
prevent its nucleophilic attack.[19] However, due to the steric bulk
of the Asp and Glu residues flanking it on either side, the direct in-
troduction of such a protecting group was anticipated to be chal-
lenging. Recently we developed a method to disrupt the for-
mation of secondary structure by nascent peptides during SPPS
and protein ligation by protecting selected amide nitrogen atoms
with the RBM building block 2-hydroxy-4-methoxy-5-nitrobenzal-
dehyde.[24-31] This strategy is applicable to any secondary amide ni-
trogen regardless of its steric encumbrance because the building
block is attached by reductive amination when the amide nitrogen
is still a free amine. The amino acid that is next in the peptide se-
quence is attached to the phenolic group of the building block,
whereupon the protected peptide is obtained by para-nitrophenol
induced intramolecular O-to-N acyl transfer that is a highly effi-
cient process applicable to bulky amino acids.
Removal of the side chain protecting groups of Asp(OTbe) and
Dap(Tbeoc) in 9 was accomplished using 2-mercaptoethanol/DI-
PEA in 40% H2O/DMF. The exposed carboxyl and amino groups of
the Asp and Dap side chains were then cyclized on resin using
PyAOP/HOAT/N-methylmorpholine (NMM) to give a lactam 10.
Sequential addition of the remaining amino acids by standard
Fmoc-SPPS gave the full-length product 11. The RBM group be-
came an acid-sensitive group by reduction of its NO2 group to NH2
using SnCl2 on the solid phase, followed by acetylation of the NH2
group using acetic anhydride on the solid phase. Finally, TFA cleav-
age was carried out to cleave the peptide from the resin to yield
Asp lactam cyclic peptide 13. HPLC and ESI-MS analysis demon-
strated the purity and correct molecular weight of the peptide 13
(total yield 25.7%). Collectively the above observations demon-
strated that the RBM strategy could effectively prevent aspar-
timide formation, and enable the efficient synthesis of the Asp-
based A-183 amide cyclic peptide derivative.
To implement the above RBM strategy to protect the amide
backbone between Asp(OTbe) and Glu(OtBu), we first synthesized
the RBM building block 2-hydroxy-4-methoxy-5-nitrobenzalde-
hyde from commercially available materials (Figure 2d).[24] The
RBM group was then installed onto the amino group of Glu(OtBu)
on the solid-phase by reductive amination to obtain intermediate
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© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
Chin. J. Chem. 2021, 39, XXX－XXX

Running title
Chin. J. Chem.
Synthesis of Cyclo [Asp9,Lys13] KIIIA7-14 derivative
obtained 14, without observable aspartimide formation (Figure 4).
Further Asp-Lys cyclization was conducted and all the protecting
groups including RBM were removed. HPLC and ESI-MS analysis
demonstrated the purity and correct molecular weight of the final
peptide 17 (total yield 35.4%). Thus the RBM strategy could con-
stitute a generally practicable approach to prevent aspartimide
formation during synthesis of the Asp-based lactam cyclic peptide
derivatives.
To assess the versatility of our RBM strategy for the synthesis
of other Asp-based lactam cyclic peptides, we next synthesized cy-
clo [Asp9,Lys13] KIIIA7-14, an Asp lactam derivative of the potent
analgesic KIIIA.[31] We first attempted its synthesis using direct
Fmoc SPPS under standard conditions, but again observed signifi-
cant aspartimide formation (Figure S11). However, by installing
the RBM group between Asp(OTbe) and Arg(Pbf) (Figure 4), we
tBu
tBu
pbf
pbf
O
tBu
O
pbf
Activated
O-to-N
Transfer
Acyl
HO
O
Tbe
H
O
Tbe
(a)
O
H
N
O
Tbe
Glu Arg Resin
+
N
Glu Arg Resin
H2N
NO2
Fmoc
Glu Arg Resin
Reductive
OH
O
Fmoc
O
N
H
N
amination HO
N
Fmoc
H
O
N
H
NO2
O
Glu Arg Resin
O
OH
NO2
O
O
O
O2N
CHO
8
tBu
pbf
7
H
N
Boc
O
Boc
tBu
tBu
tBu
Boc
Boc
Tbeoc
Fmoc
O
H
N
H
N
NH2
HO
Trp
Trp
tBu
Trp
N
N
H
tBu
Leu Val Glu Trp Glu Glu
NH2
tBu
Boc
N
H
Thr
Trp
Glu
Thr
H
Thr
Trp
Glu
O
SPPS
HN
Tbe
removal
HN
O
to
O
NO2 NH2
Trp
Tbe,Tbeoc
SPPS
Boc
HO
N
O
Boc
HO
O
O
NO2
Cyclization
O
Ac2O
Piperdine
Glu
O
NO2
NO2
N
O
10 tBu
20%
Thr
tBu
N
Resin
Thr
Glu Arg
N
tBu
Thr
N
Resin
tBu
Glu
Arg
Resin
Glu
Arg
N
H
H
H
11
tBu
O
tBu
O
9
tBu
pbf
tBu
pbf
tBu
pbf
(b)
tBu
tBu Boc tBu
Boc
O
O
H
N
H
N
H
Trp
tBu
Trp
N
Leu Val Glu Trp Glu Glu
NH2
N
H
Leu Val Glu
Trp Glu Glu
N
Ac
Crude
Thr
Trp
Glu
H
Thr
Trp
Glu
HN
HN
TFA
HO
N
O
Boc
[M+2H+]
1009.4
Ac
O
N
H
O
H
N
Thr
Thr
tBu
N
Resin
Glu
Arg
N
Glu
Arg CONH2
Purified
14
H
800 1000 1200
19
H
O
tBu
O
tBu
pbf
24
12
13
Retention
time
(min)
Figure 3. (a) Synthesis of A-183 derivative using RBM strategy. Specially, when hydroxyl group was acylated, RBM group could not be cleaved by TFA. After the
acetylated hydroxyl group on the RBM group was deacetylated with 20% piperidine /DMF, the RBM group could be completely removed. (b) RP-HPLC traces of
the crude and purified 13 and ESI-MS of purified 13.
O
HO
N
O
(a)
Tbe
O
Reductive Amination
Fmoc-Asp(OTbe)-OH
NO2
SPPS
O
N
OH
+
Fmoc
Arg Resin
H2N
Resin
Arg
N
H
The RBM strategy can also prevent aspartimide formation
associated with commercially available Asp(OAllyl)
O2
CHO
1O4
pbf
pbf
HO
O
HO
O
O
H
O
N
Tbeoc
Tbe
O
SPPS
Finally, we tested whether aspartimide formation was due to the
use of 2-(tert-butyldisulfanyl)ethanol as the Asp protecting group
by attempting the direct SPPS of A-183 using commercially availa-
ble Fmoc-Asp(OAllyl) instead of Fmoc-Asp(OTbe). As shown in Fig-
ure 5, we first synthesized 18 by direct Fmoc-SPPS. As expected,
coupling of Thr(OtBu) to 18 to obtain 19 was accompanied by sig-
nificant formation of a by-product with a molecular weight of 58
mass units less than 19, presumably corresponding the conversion
of Asp(OAllyl) to aspartimide. However, by using the RBM group
to protect the Glu(OtBu) before the Asp(OAllyl), we were able to
obtain 21, which was coupled with Thr(OtBu) to give 22 without
observable formation of an aspartimide-containing byproduct.
NO2
Tbe
O
( )4
NO2
H
N
N
His
Resin
Fmoc
N
Arg
Fmoc
N
Arg Asp His
Resin
Arg
N
H
N
H
O
H
O
O
Trt
pbf
15
tBu
Trt
pbf
pbf
O
H
HO
O
N
removal
( )4
NO2
Tbeoc, Tbe
H
Trp
H2N Lys
N
N
Arg Asp His
Resin
Arg
cyclization
N
N
SPPS
H
H
O
O16
Boc Boc
tBu
Trt
pbf
pbf
O
H
N
to
Ac2O
piperidine
TFA
NO2 NH2
( )4
H
H
Trp
Ac
Lys
N
N
Arg Asp His
Arg
N
N
CONH2
H
20%
H
O
O
17
(b)
(c)
Crude
[M+2H+]
582.5
During the peptide chain extension process to give 23, HPLC
and ESI-MS analysis demonstrated the purity and correct molecu-
lar weight of target products (Figure 5b). Next, the nitro group of
RBM was reduced to an amino group, which was subsequently
acetylated by acetic anhydride. The resulting peptide was cleaved
from resin, and HPLC and ESI-MS analysis demonstrated the purity
and correct molecular weight of 24. Thus, we concluded that the
aspartimide formation side reaction was not limited to 2-(tert-bu-
tyldisulfanyl)ethanol protection, but also possible with other ester
Purified
700
14
19
Retention
24
(min)
600
500
time
Figure 4. (a) Synthesis of Cyclo [Asp9,Lys13] KIIIA7-14 using the RBM strat-
egy. (b) RP-HPLC traces of crude and purified 17. (c) ESI-MS of purified 17.
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de

First author et al.
Report
protection groups, and that our RBM strategy could be used to
prevent the aspartimide side reaction when using Asp(OAllyl).
before Asp(OTbe), to prevent aspartimide formation.[32-36] This
RBM strategy may constitute a simple, efficient, and general
method for the synthesis of Asp-based lactam cyclic peptides,
which we established to be inaccessible by direct Fmoc SPPS.
Conclusions
Asp-based lactam cyclic peptide A-183 and cyclo [Asp9,Lys13]
KIIIA7-14 lactam cyclic peptides have been successfully synthe-
sized using an RBM strategy, wherein a 2-hydroxy-4-methoxy-5-ni-
trobenzaldehyde group is appended to the residue immediately
Supporting Information
The supporting information for this article is available on the
WWW under https://doi.org/10.1002/cjoc.2021xxxxx.
(a)
*
(b)
Allyl
HO
O
HO
O
*
O
O
O
O
Allyl
O
Fmoc
NO2
Allyl
O
Fmoc
Allyl
NO2
O
t
Bu
H
O
N
pbf
Glu Arg
TFA
t
Bu
H
N
pbf
N
H
Fmoc
N
TFA
N
H
N
Fmoc
N
H
Glu Arg
N
H
Glu Arg
CONH2
O
Glu Arg
O
CONH2
O
Resin
O
21
Resin
18
SPPS
10
15
25
20
30
SPPS
10
15
25
20
30
HO
O
*
*
HO
O
O
O
O
Allyl
O
Byproduct
[M19
Allyl
Allyl
O
NO2
Allyl
O
O
NO2
H2N
H
O
N
t
Bu
-58]
H2N
H
N
t
Thr
N
H
Bu
t
N
Bu
TFA
pbf
Arg
Thr
t
N
N
Bu
Glu
Thr
N
H2N
pbf
Arg
Glu
Arg
Thr
TFA
TFA
N
H2N
H
Glu
O
H
Glu
O
22
H
H
O
Arg
19
H2NOC
O
Resin
Resin
1)SPPS
2) Acetylation
2NOC
5
10
20
1)SPPS
Reduction of
3) Acetylation
15
25
5
10
20
15
25
NO2
2)
O
Allyl
AcO
O
O
O
O
N
*
H
AcO
O
Allyl
Byproduct
Allyl
O
N
O
*
O
H
N
Thr
t
N
Bu
NNAc
-58]
Allyl
t
[M20
Bu
Glu
H20O
tBu
pbf
t
O
N
Thr
Glu
NHAc
Bu
N
Glu
Thr
Glu
Arg
TFA
H
Arg
t
Bu
Glu
Arg
N
H23O
pbf
Arg
O
t
Thr
Bu
N
Glu
Trp
Glu
Resin
H
Boc
H2NOC
O
Trp
Glu
Thr
Trp
Resin
Boc
t
H2NOC
t
Bu
Thr
Trp
Thr
HN
Ac
Bu
Thr
10
15
25
20
30
10
15
25
HN
20
30
HN
Ac
Ac
HN
Deacetylation
Ac
(c)
O
Allyl
Allyl
*
O
O
N
O
Allyl
HO
O
O
O
H
N
O
O
Allyl
Base
H
Base
H
Thr
N
O
NHAc
N
N
H
Glu
Arg
TFA
N
H24O
t
Bu
N
t
N
H
Bu
N
Glu
pbf
Arg
AspartOimide
H
t
Bu
Thr
N
O
Glu
O
H
Trp
O
Glu
H2NOC
RBM Strategy:
Resin
O
Boc
Thr
Allyl
HO
N
O
Trp
O
Base
10
15
25
20
30
t
Bu
HN
N
Thr
Ac
O
NO2
group
N
RBM
H
HN
AspartOimide
Ac
N
H
O
Figure 5. (a) Aspartimide formation occurred during the synthesis of the A-183 derivative 13 using Asp (OAllyl). (b) The RBM strategy also prevented aspar-
timide formation when using Asp(OAllyl) in place of Asp(OTbe). (c) Proposed mechanism of RBM-prevented aspartimide formation during SPPS.
Maas, M. N.; Hintzen, J. C. J.; Löffler, P. M. G.; Mecinović, J. Targeting
SARS-CoV-2 spike protein by stapled hACE2 peptides. Chem. Commun.
Acknowledgement
2021, 57, 3283-3286.
This work was supported by the National Key R&D Program of
China (2017YFA0505200), the National Natural Science Founda-
tion of China (No. 91753205, 21877024, 21621003, 81621002),
the Fundamental Re-search Funds for the Central Universities
(JZ2019HGPB0105).
Wang, J.-X.; Fang, G.-M.; He, Y.; Qu, D.-L.; Yu, M.; Hong, Z.-Y.; Liu, L. Pep-
tide o-aminoanilides as crypto-thioesters for protein chemical synthesis.
Angew. Chem. Int. Ed. 2015, 54, 2194-2198.
Zuo, C.; Shi, W. W.; Chen, X. X.; Glatz, M.; Riedl, B.; Flamme, I.; Pook, E.;
Wang, J. W.; Fang, G. M.; Bierer, D.; Liu, L. Chimeric protein probes for
C5a receptors through fusion of the anaphylatoxin C5a core region with a
small-molecule antagonist. Sci. China Chem. 2019, 62, 1371-1378.
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Manuscript accepted: XXXX, 2021
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Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de

Entry for the Table of Contents
Use of a removable backbone modification strategy to prevent aspartimide formation in the synthesis of Asp lactam cyclic peptides
Tingting Cui,‡ Junyou Chen,‡ Rui Zhao,‡,d Yanyan Guo,a Jiahui Tang,a Yulei Li,b Yi-Ming Li*,a Donald Bierer,c and Lei Liu*,b
,a
,a
Chin. J. Chem. 2021, 39, XXX—XXX. DOI: 10.1002/cjoc.202100XXX
RBM
Strategy:
H
O
Pg
NH2
N
HO
O
O
aa aa
O
N
H
aa
O
H
HO
N
O
Tbeoc
H
N
O
N
Tbe
NH
aa
Pg
( )n
HO
Pg
NO2
Tbe,Tbeoc
removal
aa aa
O
Ac
N
aa
aa
Tbe
aa
HN
RBM group
aa
NO2
H
O
SnCl2
Ac2O
Fmoc
N
( )
n
N
SPPS
O
...
HN
NO2
aa
H
N
H
O
aa
Pg
Cyclization
SPPS
aa
N
O
O
N
aa
Deacetylation
Pg
aa
Pg
H
aa
aa
Pg
H
N
O
TFA
aa
Resin
Pg
Pg
N
aa
aa
aa
CONH2
Pg
Resin
Pg
H
aa
O
Pg
Resin
Lactam
peptide
cyclic
R
R
O
O
O
N
Allyl
HO
O
O
Base
Base
Base
O
O
H
H
O
NO2
group
N
N
N
RBM
N
H
N
H
N
R=Tbe; Allyl
AspartOimide
H
N
H
O
O
O
Our synthesis depends on the use of an RBM strategy to prevent aspartimide formation, which thwarted all at-
tempts to synthesize the Asp-based lactam cyclic peptide A-183 and cyclo [Asp9,Lys13] KIIIA7-14 lactam cyclic
peptides using direct SPPS.
Chin. J. Chem. 2018, template
© 2018 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim