Controversy of peptide cyclization from tripeptide

Article abstract of DOI:10.3390/molecules26020389

The present investigation reports an attempt to synthesize naturally occurring α-cyclic tripeptide cyclo(Gly-L-Pro-L-Glu) 1, [cyclo(GPE)], previously isolated from the Ruegeria strain of bacteria with marine sponge Suberites domuncula. Three linear precur

Full text of DOI:10.3390/molecules26020389

molecules
Article
Controversy of Peptide Cyclization from Tripeptide
Chung-Yin Lin 1,2,*, Subrata Chakraborty 3, Chia-Wei Wong 3 and Dar-Fu Tai 4,
*
1
Medical Imaging Research Center, Institute for Radiological Research, Chang Gung Memorial Hospital,
Chang Gung University, Taoyuan 333423, Taiwan
Department of Nephrology and Clinical Poison Center, Chang Gung Memorial Hospital,
Taoyuan 333423, Taiwan
Department of Chemistry, National Dong Hwa University, Hualien 974003, Taiwan;
cpapu2000@yahoo.com (S.C.); linny5782@hotmail.com (C.-W.W.)
Department of Life Science, National Dong Hwa University, Hualien 974003, Taiwan
Correspondence: winwood5782@gmail.com (C.-Y.L.); dftai@gms.ndhu.edu.tw (D.-F.T.);
Tel.: +886-3-211-8800 (ext.3865) (C.-Y.L.)
2
3
4
*
Abstract: The present investigation reports an attempt to synthesize naturally occurring
α-cyclic
tripeptide cyclo(Gly-L-Pro-L-Glu) , [cyclo(GPE)], previously isolated from the Ruegeria strain of
1
bacteria with marine sponge Suberites domuncula. Three linear precursors, Boc-GPE(OBn)2, Boc-
PE(OBn)G and Boc-E(OBn)GP, were synthesized using a solution phase peptide coupling protocol.
Although cyclo(GPE)
with high dilution to form corresponding
then converted to cyclic hexapeptide cyclo(GPE)2
1
was our original target, all precursors were dimerized and cyclized at 0 ◦C
α
-cyclic hexapeptide, cyclo(GPE(OBn))2
7, which was
2. Cyclization at higher temperature induced
racemization and gave cyclic tripeptide cyclo(GPDE(OBn)) 9. Structure characteristics of the newly
synthesized cyclopeptides were determined using 1H-NMR, 13C-NMR and high-resolution mass
spectrometry. The chemical shift values of carbonyls of
the formation of a cyclic hexapeptide.
2 and 7 are larger than 170 ppm, indicating
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Keywords: tripeptide; cyclic hexapeptide; solution phase synthesis; peptide cyclization
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Lin, C.-Y.; Chakraborty, S.;
Wong, C.-W.; Tai, D.-F. Controversy of
Peptide Cyclization from Tripeptide.
Molecules 2021, 26, 389. https://
doi.org/10.3390/molecules26020389
1. Introduction
Marine sponge-derived natural cyclic peptides have generated much interest in recent
years due to their privileged structures and persuasive biological activities (therapeutic po-
tential). The surfaces and internal spaces of sponges provide a specific environmental niche
that contains a high number of bacteria to exceed those of seawater by two or three orders
Academic Editor: Theodore Tselios
Received: 16 November 2020
Accepted: 5 January 2021
of magnitude [
bacteria recently by proving that these bioactive compounds, when isolated from marine
sponges, are symbiotically produced microorganisms [ ]. Among these cyclic peptides, nat-
urally occurring -cyclic tripeptides and tetrapeptides are highly constrained because they
have a small ring size [ ]. Diverse biological activities of natural cyclic peptides, such as
antimicrobial, cytotoxic, anti-HIV, nematicidal and anti-inflammatory characteristics [4–7]
increases their importance as targets for synthetic chemists. However, many biologically
active cyclic peptides have been isolated [ 12], but not fully characterized [13 14]. An-
other hurdle is to meet the supply problem and target identification. Synthesis [15 16] is
1]. Moreover, several studies have increased interest in sponge-associated
Published: 13 January 2021
2
α
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
3
,
8–
,
,
particularly useful for biological applications as well as to confirm the actual structure of
natural products and target identification to overcome the hurdle.
It is extremely challenged for successful drug development of these often complex
molecules. In many cases, total synthesis of natural products revealed structural incor-
rectness of the reported natural products [17–19]. Previously, we reported the synthesis
and X-ray crystallographic characterization of marine cyclic tetrapeptide cyclo(Gly-L-Ser-
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
L-Pro-L-Glu) [20]. Antitumor and antimicrobial activity of some cyclic tetrapeptides and
tripeptides were examined [21]. In addition, some α-cyclic tripeptides also synthesized
Molecules 2021, 26, 389. https://doi.org/10.3390/molecules26020389
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 389
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as the inhibitors for HMG-CoA reductase [22]. As a continuation of natural cyclopeptide
synthesis, we attempted to synthesize a rare example of naturally occurring marine
α-cyclic
tripeptide, cyclo(Gly-L-Pro-L-Glu) ], for applications in vivo as it is more permeable
1 [8
and more resistant to degradation by digestive proteases [23]. When rendered into its
linear form, Gly-L-Pro-L-Glu (GPE) is the N-terminal sequence of insulin-like growth factor
(IGF-1) [24,25]. There is evidence that the peptide has neuroprotective properties and
improves long-term function following brain injury or disease, including hypoxic-ischemic
brain injury, chemical toxins and in animal models of Parkinson’s and Alzheimer’s dis-
ease [26
–
28]. Furthermore, GPE and GPE analogues are thus of interest as promising
28].
neuroprotective agents for the treatment of central nervous system (CNS) diseases [27
,
Despite having therapeutic significance, several challenging features are still encountered in
the synthesis, such as dimerization and racemization of linear natural amino acid sequence,
cyclization only with all 3 amide linkages in cis configuration and less conformation to
raise the accuracy of molecular docking due to its rigidity [22,29–31]. These factors can
produce contamination to influence the in vivo ability of formulation/characterization for
clinical applications.
To address these problems, this paper explores an attempt to synthesize the reported
natural cyclic tripeptide 1 since it possesses a side-chain carbonyl group that may enable
further functionalization after cyclization. Although the literature reported natural cyclic
tripeptide, the characterization data of our synthesized compound turn out to be cyclic
hexapeptide 2. In addition, the cyclization of linear precursor at room temperature (RT)
was involved with racemization to give a cyclic D-tripeptide 9.
2. Results and Discussion
To synthesize the cyclic tripeptide, linear precursor Boc-G-P-E(OBn)2
5 (Scheme 1)
was synthesized using standard solution-phase peptide coupling protocols from Fmoc-
L-proline. Subsequently, regioselective enzymatic hydrolysis of the -benzyl ester on
α
glutamate was achieved [32]. The linear precursor was then activated with pentafluo-
rophenol and cyclized in pyridine at high dilution (2 mM, RT). The cyclization ended
up with cyclo(GPE(OBn))2
spectrometry (supplementary Figures S1–S3). Then, deprotection of the benzyl group
furnished the synthesis of cyclo(GPE)2 . The results indicate that GPE sequence is much
easier to produce cyclic products, but others are probably more difficult to be cyclized.
In contrast, Yudin et al. [33] used Boc-protected -amino imide as a model substrate to
demonstrate that medium-sized rings could be constructed through the collapse of cyclol
7. Formation of dimer was confirmed by high-resolution mass
2
β
intermediates derived from the intramolecular cyclization of β-amino imides upon heating
to 50 ◦C for 4 h. We also monitored the cyclization step by using RP-HPLC to analyze the
compounds. Pure cyclo(GPE)2OBn showed peak at retention time 3.78, and the reaction
mixture showed one peak correspond to cyclo(GPE)2OBn and another was salt of TFA.
Analysis of spectroscopic data of reported natural product
1 and synthetic cyclic
hexapeptide
2
, especially 1H and 13C-NMR data, creates a doubt about the correctness
of the natural product structure. Table 1 shows the recorded carbonyl peaks for reported
cyclic peptides in 13C-NMR. All the ring carbonyl peak values of reported cyclic tripeptides
are lower than 170 ppm [34
cyclo(Pro-Pro-BnG) [35], cyclo(L-Pro-L-Pro-D-Pro) [35] and cyclo(L-Pro-L-Pro-L-Pro) [36
showed peaks between 167 and 169 ppm. Glycine carbonyl [36], just like proline, also
appeared in the range around δ 166 ppm.
–36]. Carbonyl peak of proline in cyclo(L-Pro-L-BnG-D-Pro) [34],
]
δ

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Scheme 1. Synthesis of cyclo(GPE)2 and cyclo(GPDE). (
a
) Dibenzyl L-glutamate p-toluenesulphonate,
◦
DCC, HOBt, TEA, DCM, 0 C~rt (
b
) (i) 20% piperidine in DCM, rt (ii) Boc-Gly-OH, DCC, HOBt, TEA,
◦
◦
DCM, 0 C~rt (c) B. licheniformis protease (Sigma type–VIII), pH 7 buffer, acetone, 37 C (
d
) (i) DCC,
◦
pentafluorophenol, DCM (ii) TFA/DCM (1:1), 0 C (iii) pyridine, high dilution, rt (
e) H2, Pd(OH)2/C,
MeOH (f) (i) DCC, pentafluorophenol, DCM (ii) TFA/DCM (1:1), rt (iii) pyridine, high dilution, rt.
Table 1. 13C chemical shift values for carbonyls of reported cyclic tripeptides.
13C Chemical Shift Values of
Cyclic Tripeptides
Carbonyls (ppm)
Solvents
Pro-
Gly-
171.0
166.0
Cyclo(L-Pro-BnG-D-Pro) [34]
Cyclo(L-Pro-L-Pro-BnG) [35]
C6D6
165.0
169.6
168.8
CDCl3
165.2
169.0
168.0
166.0
Cyclo(L-Pro-L-Pro-D-Pro) [35]
CDCl3
Cyclo(L-Pro-L-Pro-L-Pro) [36]
Cyclo (BnG-BnG-BnG) [36]
CDCl3
CDCl3
167.6
166.0
In contrast, in the case of reported natural product
proline and glycine are much higher ( 174.8 and 169.8 ppm) than for other reported
cyclic tripeptides [34 36] and closer to our synthetic cyclic hexapeptide cyclo(GPE)2
173.8 and 169.6 ppm). Table 2 shows complete comparison of 13C chemical shift values of
cyclo(GPE) 1 [8] and cyclo(GPE)2 . To verify the spectra of cyclic monomer and dimer con-
1, ring carbonyl peak values for
δ
–
2 (δ
2
taining same amino acid sequence, we synthesized cyclo (serine-proline-glycine) (SPG) and
cyclo(SPG)2. Compared with natural products, cyclo(SPG) containing L-serine instated L-
glutamic acid in its amino acid sequence. Both the monomer and dimer showed completely
different 1H and 13C-NMR spectra (unpublished work). 13C peak values of cyclo(SPG)2 are
also quite similar to our synthesized hexapeptide cyclo(GPE)2
2 (Figure 1). We used a 3:1
CD3OD/D2O ratio for NMR, which was the cause of little variation of NMR peaks.

Molecules 2021, 26, 389
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Table 2. 13C chemical shift values of cyclo(GPE) 1 [8] and cyclo(GPE)2 2 (ppm) in CD3OD/D2O.
Amino Unit
Reported Data
Cyclo(GPE) 1 [8]
169.8
Synthesized Product
Cyclo(GPE)2 2
169.6
Glycine (carbonyl)
α-
42.2
174.8
43.7
173.8
Proline (carbonyl)
α-
β-
60.0
29.5
63.5
35.4
γ-
δ-
24.9
47.6
26.1
47.7
Glutamate (carbonyl)
Side Chain (carbonyl)
176.2
183.1
175.0
181.6
α-
β-
γ-
57.5
25.8
29.5
54.5
29.0
30.7
Figure 1. 13C-NMR spectra of cyclo(SPG), cyclo(SPG)2 and cyclo(GPE)2 in CD3OD/D2O.
Cyclization of fully deprotected linear G-P-E(OBn)-OH in DMF at various dilution
conditions with different coupling reagent combinations such as PyBOP-HOBt, HATU-
HOAt and BOP-HOBt also gave dimeric compound. After failure to acquire a monomeric
compound, we prepared two other linear precursors, Boc-PE(OBn)G and Boc-E(OBn)GP.
These two compounds were cyclized using the same procedure and producing the same
dimer
2 with lower cyclization yield. We also tried microwave assisted cyclization of linear
precursor with a series of coupling reagents in DMF as well as different activated ester
compounds in pyridine with variation of temperature, power and dilution failed to give
cyclic tripeptide 1.
To identify the specific functional groups in our synthesized compound, an infrared
spectrum was also useful to check the carbonyl position. Amide stretching of small
cyclic peptides was around 1675 cm−1 and usually shifted to 1635 cm−1 for larger rings.
Unfortunately, the reported cyclo(GPE) did not provide this information [
known that cyclic tripeptide can be formed only when all peptide bonds are in crown form
with cis-linkage [37 38]. Small ring size and crown structure will bring all the carbonyls
8]. It is well
,
closer and parallel to each other, which will increase the shielding effect of one carbonyl to
another. As a result, carbonyls on the cyclic tripeptide ring shift upfield (lower than 170
ppm in 13C-NMR). Conversely, carbonyl peaks of cyclic hexapeptide usually appeared in
the downfield region (higher than 170 ppm), because flexible cyclic hexapeptide contains
both cis and trans links [39]. Their cis trans combination keeps ring carbonyls apart from
each other, which results in a less shielding effect. Carbonyl peaks of reported natural
product
1 along with our synthetic cyclic hexapeptide cyclo(GPE)2 2 showed similar peak
patterns to previously synthesized [40] and isolated cyclic hexapeptides [41–43].

Molecules 2021, 26, 389
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The 1H-NMR of synthesized cyclic hexapeptide also showed a similar splitting pattern
to reported natural product
protons was observed for synthetic cyclo(GPE)2. All
showed the same splitting (Gly dd, Pro dd, Glu dd) and coupling constants as reported
natural product . In addition to splitting, chemical shift values of -hydrogens (Gly
4.33, 4.20, Pro 4.51, Glu 4.64) were also identical to the reported data. High-resolution
1
in the same duterum solvent (Table 3). Only one set of GPE
α
-hydrogens of cyclic hexapeptide
1
α
δ
δ
δ
mass spectra show a mass ion peak at 566.2349 corresponding to cyclo(GPE)2 and a peak
28
at 283 corresponding to cyclo(GPE). In addition, our cyclic hexapeptide ([
α
]
−
33 (c 0.1,
].
D
25
MeOH)) also has similar optical rotation as reported ([
α
]
−
27.8 (c 0.0010, MeOH)) [
1 is perhaps a cyclic hexapeptide.
8
D
These results demonstrated that reported natural product
1
Table 3. H-NMR data of cyclo(GPE) 1 [8] and cyclo(GPE)2 2 in CD3OD/D2O.
Amino Unit
Reported Data
Synthesized Product
Cyclo(GPE) 1 [8]
Cyclo(GPE)2 2
4.20 d (17.3)
4.13 d (17.3)
4.33 d (17.3)
4.20 d (17.3)
Glycine
Proline
α-
α-
β-
γ-
δ-
4.49 dd (8.6; 3.5)
2.34 m, 2.08 m
2.08 m
4.51 dd (8.7; 6.1)
2.41 m, 2.13 m
2.13 m
3.67 m
3.96 m
α-
β-
γ-
4.42 dd (9.1; 4.9)
2.58 m, 2.14 m
2.47 m
4.64 dd (10.3; 4.1)
2.48 m, 2.13 m
2.48 m
Glutamate
Coupling constants (hertz) are in parentheses.
As shown in Scheme 1, droping the α-pentafluorophenol ester of G-P-E(OBn)-OH to
pyridine at 50 ◦C resulted in racemization to form cyclo(GPDE(OBn))
(27%). Removal
9
of the benzyl protecting group on glutamate generated a cyclic tripeptide cyclo(Gly-L-Pro-
D-Glu) 10. The analysis of 13C-NMR (Figure 2) and ESIMS data (284.1241 m/z) indicated
that the synthesized cyclic tripeptide 10 was not closely related to reported natural product
1
1
or synthesized cyclic hexapeptide
2
. Their H-NMR data are shown in Table 4. As
compared with the optical rotation, our synthesized cyclic tripeptide 10 has a distinct value
24
25
([
−
α
]
−
150 (c 0.01, MeOH)), which is different to reported natural product
1
([
α
]
D
D
28
27.8 (c 0.0010, MeOH)) or synthesized compound 2 ([
results suggest that this cyclic tripeptide is cyclo(Gly-L-Pro-D-Glu), cyclo(GPDE) 10, not
α
]
−
33 (c 0.1, MeOH)). Our
D
cyclo(Gly-L-Pro-L-Glu) 1. These results also point out that GPE sequence is much easier to
cycle than other precursors.
Figure 2. 13C-NMR spectra of cyclo(GPDE(OBn)) and cyclo(GPDE) in D2O.

Molecules 2021, 26, 389
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1
Table 4. H-NMR data of cyclo(GPE) 1 [8] and cyclo(GPDE) 10 in D2O.
Amino Unit
Reported Data
Synthesized Product
Cyclo(GPE) 1
Cyclo(GPDE) 10
Glycine
α-
4.20 d (17.3)
4.13 d (17.3)
4.24 dd (17.4, 7.6)
4.10 dd (17.1, 3.3)
Proline
α-
β-
γ-
δ-
4.49 dd (8.6; 3.5)
2.34 m, 2.08 m
2.08 m
4.17 t (4.6)
2.67 m, 2.12 m
2.12 m
3.67 m
3.47 m, 3.70 m
Glutamate
α-
β-
γ-
4.42 dd (9.1; 4.9)
2.58 m, 2.14 m
2.47 m
3.79 t (6.4)
2.22 m
2.37 m
The antibiotic activity of natural product 1 was reported [8]. To date, we have
not found any cyclic tripeptide with antibiotic activity [21 22]. We have shown that
,
the structure–activity relationship cannot be neglected. The size of the cyclic peptide
is very critical. Previous studies have disclosed the importance of ring size between 4 and
14 residues on the antimicrobial activity [44,45], such as cyclic hexapeptides [40,46–48].
3. Materials and Methods
All reactions involving air or moisture sensitive reagents were carried out under a
dry argon or nitrogen atmosphere using freshly distilled solvents. Pyridine and dioxane
were distilled from CaH2. The NMR spectra were recorded on Bruker DRX 400 (1H at
400.13 MHz, and 13C at 100.03 MHz). Chemical shifts (
δ
) are reported in ppm relatively to
= 77.0; CD2HOD,
= 3.34, 13CD3OD,
δ = 4.47. MALDI TOF was performed on a Bruker Autoflex MALDI-TOF
the residual solvent peak (CHCl3,
= 49.0; D2O,
δ
= 7.26, 13CDCl3,
δ
δ
δ
mass spectrometer (Bruker Daltonics, Breman, Germany). High-resolution electrospray
ionization mass spectrometry (ESI-MS) was performed on a Shimadzu-LCMS-IT-TOF
mass spectrometer (Shumadzu, Kyoto, Japan). Infrared spectra were recorded on a Perkin
Elmer Spectrum one FT-IR spectrometer (PerkinElmer, Shelton, CT, USA) using KBr pellets
(4000–400 cm−1).
3.1. Fmoc-L-Prolyl-L-Glutamyl Dibenzyl Ester (3)
To a solution of dibenzyl L-glutamyl p-toluenesulphonate (8.88 g, 17.79 mmol) and
triethylamine (3 g, 29.65 mmol) in dichloromethane (20 mL) in 50 mL round bottom
flask Fmoc-L-proline (4 g, 11.86 mmol), DCC (2447 mg, 11.86 mmol) and HOBt (1601 mg,
◦
◦
11.86 mmol) was added at 0 C. The reaction mixture was stirred for 2 h at 0 C and 17 h at
room temperature. The reaction was monitored by TLC and the resultant white mixture
was filtered to remove 1,3-dicyclohexylurea (DCU). After evaporation of the filtrate, the
resultant mass was dissolved in ethyl acetate and washed successively with 5% citric
acid solution, water, 5% sodium bicarbonate solution and water. The organic extract was
concentrated and purified by column chromatography using hexane/ethyl acetate (70:30)
22
as eluent to obtain compound
1H-NMR (CDCl3, 400 MHz)
4.24–4.34 (m, 3H), 4.41–4.46 (m, 2H), 4.61–4.69 (m, 1H), 5.04–5.17 (m, 4H), 7.30–7.39 (m,
14H), 7.59 (s, 2H), 7.75–7.77 (d, 2H, J = 7.16 Hz). 13C-NMR (CDCl3, 100 MHz)
27.2, 28.5,
3
as a white solid (6 g, 78%). [
α
]
=
−
56 (c 0.1, CHCl3).
D
δ
1.88–1.99 (m, 4H), 2.25–2.40 (m, 4H), 3.46–3.56 (m, 2H),
δ
30.1, 34.0, 47.1, 47.3, 51.8, 60.4, 66.5, 67.3, 67.7, 120.0, 125.1, 125.2, 127.1, 127.7, 128.2, 128.3,
128.5, 128.5, 128.6, 135.3, 135.8, 143.8, 144.0, 171.4, 171.7, 172.5. IR (KBr) 3326, 3065, 2952,
1736, 1704, 1532, 1451, 1418, 1353, 1262, 1169, 1119, 1089, 988, 758, 740, 698, 621 cm−1
.
MALDI TOF: m/z calcd. for C39H38N2O7Na [M + Na]+ 669.257; found 669.170.

Molecules 2021, 26, 389
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3.2. L-Prolyl-L-Glutamyl Dibenzyl Ester (4)
To a solution of Fmoc-L-prolyl-L-glutamyl dibenzyl ester
3
(6 g, 9.28 mmol) in dichlorome-
thane (16 mL) in a round bottom flask piperidine was added (4 mL). The mixture was stirred for
2 h. The reaction was monitored by TLC which showed the complete disappearance of starting
material. The reaction mixture was concentrated and purified by column chromatography
using DCM/MeOH (97:3) as eluent to obtain compound
1H-NMR (CDCl3, 400 MHz)
1.65–1.70 (m, 4H), 1.85–1.88 (m, 1H), 2.00–2.10 (m, 2H), 2.29–2.41
(m, 3H), 2.87–2.99 (m, 2H), 3.69–3.73 (m, 1H), 4.62–4.64 (m, 1H), 5.00–5.14 (m, 4H), 7.32 (s, 10H),
8.13–8.15 (d, 1H, J = 8.48 Hz). 13C-NMR (CDCl3, 100 MHz)
26.2, 27.6, 30.3, 30.9, 47.3, 51.1,
4 as a pale yellow liquid (3.7 g, 94%).
δ
δ
60.5, 66.5, 67.2, 128.2, 128.3, 128.4, 128.6, 128.6, 135.3, 135.8, 171.7, 172.3, 175.5. MALDI TOF:
m/z calcd. for C24H29N2O5 [M + H]+ 425.207; found 425.267.
3.3. Boc-Glycyl-L-Prolyl-L-Glutamyl Dibenzyl Ester (5)
Dichloromethane (20 mL) and triethylamine (953 mg, 9.43 mmol) were taken in a
round bottom flask and cooled at 0 ◦C. To this, L-prolyl-L-glutamyl dibenzyl ester
4 (2 g,
4.71 mmol) was added and stirred for 5 min. Boc-glycine (825 mg, 4.71 mmol), DCC
(972 mg, 4.71 mmol) and HOBt (636 mg, 4.71 mmol) were added. The reaction mixture was
stirred 2 h at 0 ◦C and 17 h at rt. The reaction was monitored by TLC which showed the
complete disappearance of both starting materials. The resultant white mixture was filtered
to remove 1,3-dicyclohexyleurea (DCU). Filtrate was dried, and residue was dissolved
in ethyl acetate. The organic layer was washed with 5% aq. citric acid solution followed
by water and then with 5% aq. NaHCO3 solution followed by water. The organic layer
was dried over Na2SO4, and the mixture was purified by column chromatography using
hexane/ethyl acetate (65:35) as eluent to obtain compound
5
as colorless liquid. (2.25 g,
δ
22
1
82%). [
α
]
=
−
66 (c 0.1, CHCl3). H-NMR (CDCl3, 400 MHz) 1.4 (s, 9H), 1.95–2.06 (m,
D
4H), 1.97–2.04 (m, 2H), 2.17–2.24 (m, 2H), 2.36–2.41 (m, 2H), 3.31–3.37 (m, 1H), 3.44–3.49 (m,
1H), 3.82 (dd, 1H, J = 3.92 Hz, J = 17.2 Hz), 3.96 (dd, 1H, J = 4.5 Hz, J = 17.4 Hz), 4.49–4.53
(m, 1H), 4.54–4.58 (m, 1H), 5.06–5.16 (m, 4H), 5.25–5.26 (d, 1H, J = 1.76 Hz), 5.47 (s, 1H),
7.26–7.38 (m, 10H). 13C-NMR (CDCl3, 100 MHz)
δ 24.8, 26.9, 27.9, 28.4, 30.2, 43.1, 46.3,
51.9, 60.1, 66.5, 67.2, 79.7, 135.3, 135.8, 155.8, 168.5, 171.2, 171.4, 172.7. IR (KBr) 3316, 3034,
−1
2977, 1733, 1653, 1528, 1455, 1391, 1367, 1251, 1168, 1057, 1029, 971, 866, 751, 699, 581 cm
.
HRMS (ESI): m/z calcd. for C31H40N3O8 [M + H]+ 582.2810; found 582.2814.
3.4. Boc-Glycyl-L-Prolyl-L-Glutamic Acid γ-Benzyl Ester (6)
To a solution of 0.1 M, pH 7 phosphate buffer (32 mL) containing B. subtilis protease
(Sigma type–VIII) (10 mg) in a 100 mL round bottom flask a solution of Boc-glycyl-L-prolyl-
L-glutamyl dibenzyl ester
5
(1 g, 1.72 mmol) in acetone was added drop by drop (8 mL). The
◦
reaction mixture was stirred at 35 C overnight. The reaction was monitored by TLC which
showed the complete disappearance of the starting material. Then, the pH of the solution
was basified to 8 and unchanged ester was extracted with ethyl acetate. The aqueous layer
was acidified to pH 2, centrifuged to remove enzyme and extracted with ethyl acetate.
The resultant organic layer was dried over Na2SO4, concentrated and purified by column
chromatography using DCM/MeOH (97:3) as eluent to obtain compound
6
as a white
δ
22
1
solid. (727 mg, 86%). [
α
]
=
−
64 (c 0.1, CHCl3). H-NMR (CDCl3, 400 MHz) 1.41 (s,
D
9H), 1.88–1.93 (m, 2H), 2.16–2.25 (m, 2H), 2.37–2.50 (m, 2H), 3.39–3.41 (m, 1H), 3.51–3.55
(m, 1H), 3.84 (dd, 1H, J = 3.84 Hz, J = 17.28 Hz), 4.00 (dd, 1H, J = 5.64 Hz, J = 17.48 Hz),
4.48–4.53 (m, 2H), 5.07 (s, 2H), 5.65 (s, 1H), 7.28–7.33 (m, 5H), 7.46–7.48 (d, 1H, J = 7.56 Hz),
8.20 (br s, 1H). 13C-NMR (CDCl3, 100 MHz)
δ 24.8, 26.8, 28.3, 30.3, 43.0, 46.5, 51.9, 53.5,
60.4, 66.5, 79.9, 128.2, 128.3, 128.6, 135.6, 135.8, 156.1, 169.2, 171.6, 173.2, 173.6. IR (KBr)
3327, 2978, 1731, 1652, 1531, 1454, 1392, 1367, 1252, 1167, 1057, 865, 749, 699 cm−1. HRMS
(ESI): m/z calcd. for C24H33N3O8 [M]+ 491.2268; found 491.2268.

Molecules 2021, 26, 389
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3.5. Cyclo(Glycyl-L-Prolyl-L-Glutamyl(OBn))2 (7)
Boc-glycyl-L-prolyl-L-glutamyl -benzyl ester
to a 10 (mL) round bottom flask containing DCM (2 mL) and cooled at 0 C. Pentafluorophe-
nol (202 mg, 1.1 mmol) and DCC (206 mg, 1 mmol) were added. The reaction mixture was
stirred for 1 h at 0 ◦C and 22 h at rt. The solvent was evaporated, and the residue was dis-
solved in ethyl acetate and filtered to remove 1,3 dicyclohexyleurea (DCU). The filtrate was
γ
α
-acid
6
(491.53 mg, 1 mmol) was added
◦
evaporated to obtain Boc-glycyl-L-prolyl-L-glutamyl γ-benzyl ester α-pentafluorophenol
ester. Boc-glycyl-L-prolyl-L-glutamyl γ-benzyl ester α-pentafluorophenol ester (658 mg,
1 mmol) was taken in a (25 mL) round bottom flask and cooled to 0 ◦C. TFA/DCM (1:1)
◦
(
4 mL) was added and stirred 1 h at 0 C. The solvents were evaporated and triturated with
ether followed by decanting to remove free pentafluorophenol. The residue was dried in
vacuum, which was used directly in the ensuing cyclization procedure. Glycyl-L-prolyl-
L-glutamyl γ-benzyl ester α-pentafluorophenol ester (492 mg, 1 mmol) was dissolved in
dioxane (20 mL). This solution was added dropwise with efficient stirring to a 1 L round
bottom flask containing 500 mL pyridine. Addition was completed after 6 h. After stir-
ring over 36 h at rt, the solvent was distilled off. Residue was dissolved in DCM. The
organic layer was washed with 1 N HCl solution, followed by water and then with 5%
sodium bicarbonate solution, followed by water. The organic layer was dried over sodium
sulphate, and the mixture was purified by column chromatography using DCM/MeOH
22
(95:5) as eluent to obtain compound
CHCl3). H-NMR (CDCl3, 400 MHz)
7
as a white solid. (110 mg, 30%). [
δ
α
]
=
−
46 (c 0.5,
D
1
1.93–1.96 (m, 8H), 2.22–2.29 (m, 4H), 2.38–2.45 (m,
4H), 3.44–3.46 (m, 2H), 3.75–3.81 (m, 4H), 4.20–4.21 (d, 1H, J = 4.72 Hz), 4.24–4.25 (d, 1H,
J = 4.96 Hz), 4.33 (dd, 2H, J = 4.04 Hz), 4.44–4.49 (m, 2H), 5.07 (s, 4H), 7.13–7.15 (d, 2H,
J = 8.24 Hz), 7.29–7.34 (m, 10H), 7.73 (s, 2H). 13C-NMR (CDCl3, 100 MHz)
δ 24.9, 26.9, 29.7,
30.6, 42.9, 46.9, 52.1, 61.4, 66.5, 128.2, 128.3, 128.4, 128.6, 135.8, 168.2, 171.5, 171.8, 173.2.
IR (KBr) 3329, 3065, 2951, 1732, 1647, 1516, 1454, 1386, 1325, 1260, 1165, 1004, 738, 699,
597 cm−1. ESI: m/z calcd. for C38H47N6O10 [M + H]+ 747.33; found 747.27.
3.6. Cyclo(Glycyl-L-Prolyl-L-Glutamyl)2 (2)
Cyclo(glycyl-L-prolyl-L-glutamyl(OBn))2
7 (200 mg, 0.27 mmol) was dissolved in 4 mL
of MeOH in a 10 mL round bottom flask and 20% palladium hydroxide on carbon was
added under nitrogen. The vessel was puged three times with nitrogen and three times with
hydrogen, and the reaction mixture was then stirred for 2 h under hydrogen at atmospheric
pressure. The catalyst was removed by filtration, and the filtrate was evaporated to obtain
28
compound
D2O, 400 MHz)
2
as a white solid. (144 mg, 95%). [
α
]
=
−
33 (c 0.1, MeOH). 1H-NMR (CD3OD,
D
δ
2.13–2.21 (m, 6H), 2.27–2.38 (m, 6H), 2.41–2.53 (m, 4H), 3.71–3.77 (m,
2H), 3.94–3.99 (m, 2H), 4.2 (d, 2H, J = 17.32 Hz), 4.33 (d, 2H, J = 17.56 Hz), 4.51 (dd, 2H,
J = 8.72 Hz, J = 6.12 Hz), 4.64 (dd, 2H, J = 10.28 Hz, J = 4.08 Hz). 13C-NMR (CD3OD, D2O,
400 MHz)
δ 26.1, 29.0, 30.7, 35.4, 43.7, 47.7, 54.5, 63.5, 169.6, 173.8, 175.0, 181.6. IR (KBr)
3405, 2944, 1635, 1564, 1404, 1328, 1189, 1111, 1050, 919, 599 cm−1. HRMS (ESI): m/z calcd.
for C24H34N6O10 [M]+ 566.2336; found 566.2349.
3.7. Cyclo(Glycyl-L-Prolyl-D-Glutamyl) (10)
Boc-glycyl-L-prolyl-L-glutamyl γ-benzyl ester α-acid 6 (100 mg, 0.2 mmol) was added
to a 10 (mL) round bottom flask containing DCM (2 mL), pentafluorophenol (41.5 mg,
0.22 mmol) and DCC (41.3 mg, 0.3 mmol). Reaction mixture was stirred for 1.5 h at
rt. The solvent was evaporated, and the residue was dissolved in ethyl acetate and fil-
tered to remove 1,3 dicyclohexyleurea (DCU). The filtrate was evaporated to obtain Boc-
glycyl-L-prolyl-L-glutamyl
L-glutamyl -benzyl ester
TFA/DCM (1:1) (2 mL) and stirred for 1 h at 0 C. The solvents were evaporated to remove
TFA. The residue was dried in vacuum, which was used directly in the ensuing cyclization
γ-benzyl ester α-pentafluorophenol ester. Boc-glycyl-L-prolyl-
γ
α
-pentafluorophenol ester (131.5 mg, 0.2 mmol) was taken in
◦
procedure. The TFA salt of glycyl-L-prolyl-L-glutamyl
γ-benzyl ester α-pentafluorophenol
ester (111.5 mg, 0.2 mmol) was dissolved in 1,4-dioxane. This solution was added dropwise

Molecules 2021, 26, 389
9 of 11
◦
with efficient stirring to a 1 L round bottom flask containing 500 mL pyridine at 50 C. After
dropping was completed, the temperature was lowered to rt and stirring over 60 h at rt, the
solvent was distilled off. The residue mass was dissolved in DCM, hexane, ethyl ester and
washed with ethyl acetate to obtain cyclo(glycyl-L-prolyl-D-glutamyl(OBn)) (20 mg, 27%).
Cyclo(glycyl-L-prolyl-D-glutamyl(OBn)) (20 mg, 0.053 mmol) was dissolved in 2 mL
MeOH and DMF in a 10 mL round bottom flask, and palladium hydroxide on carbon was
added under nitrogen. The reaction mixture was then stirred for 2 h. The catalyst was
removed by filtration, and the filtrate was evaporated to obtain a white solid compound
24
(10.5 mg, 70%). [
α
]
=
−
150 (c 0.01, MeOH). 1H-NMR (D2O, 400 MHz)
δ 2.06–2.18 (m,
D
3H), 2.19–2.24 (m, 2H), 2.32–2.42 (m, 2H), 2.64–2.70 (m, 1H), 3.35–3.59 (m, 1H), 3.67–3.72 (m,
1H), 3.79 (t, 1H, J = 6.4 Hz), 4.10 (dd, 1H, J = 17.1 Hz, 3.3 Hz), 4.17 (t, dd, 1H, J = 4.6 Hz),
4.24 (dd, 1H, J = 17.4 Hz, 7.6 Hz). 13C-NMR (D2O, 100 MHz)
δ 22.1, 26.4, 27.8, 32.0, 45.0,
45.6, 57.2, 57.7, 161.4, 164.9, 174.4. IR (KBr) 3375, 2927, 2328, 2858, 1652, 1430, 1314, 1151,
1097, 985, 874, 751, 631 cm−1. HRMS(ESI): m/z calcd. for C12H18N3O5 [M + H]+ 284.1242;
found 284.1242.
4. Conclusions
In summary, an α-cyclic hexapeptide cyclo(GPE)2 2 was synthesized via cyclization of
fully deprotected linear precursor with a series of coupling reagents in DMF. Additionally,
we were able to obtain cyclo(GPDE) with much synthetic effort. Even though the character-
ization data of the reported natural product and our synthetic cyclo(GPE)2 indicate that
the reported compound may be a dimer of cyclo(GPE), this strategy still provided a way to
synthesize cyclic peptide of varying ring size. In light of the cyclic peptide synthesis using
a solution phase method, it can also provide opportunities to explore the synthesis of cyclic
products with various functionalities. The unusual antibiotic activity of natural product 1
raised our suspicion as a cyclic tripeptide. The characterization data of the reported natural
product and our synthetic cyclo(GPE)2 also indicate that the reported compound may be a
dimer of cyclo(GPE).
Supplementary Materials: The following are available online, Figure S1: 1H NMR spectrum of
cyclo(Gly-L-Pro-L-Glu(OBn))2
Glu(OBn))2 in CDCl3, Figure S3: HRMS(ESI) spectrum of cyclo(Gly-L-Pro-L-Glu(OBn))2
S4: 1H NMR spectrum of cyclo(Gly-L-Pro-L-Glu)2
in CD3OD/D2O, Figure S5: 13C NMR spectrum
of cyclo(Gly-L-Pro-L-Glu)2 in CD3OD/D2O, Figure S6: IR spectrum of cyclo(Gly-L-Pro-L-Glu)2
, Figure S7: HRMS(ESI) spectrum of cyclo(Gly-L-Pro-L-Glu)2 , Figure S8: 1H NMR spectrum of
cyclo(Gly-L-Pro-D-Glu(Bn))
in CDCl3, Figure S9: 13C NMR spectrum of cyclo(Gly-L-Pro-D-Glu(Bn))
in CDCl3, Figure S10: IR spectrum of cyclo(Gly-L-Pro-D-Glu(Bn)) , Figure S11: HRMS(ESI) spectrum
7
in CDCl3, Figure S2: 13C NMR spectrum of cyclo(Gly-L-Pro-L-
7
7, Figure
2
2
2
2
9
9
9
of cyclo(Gly-L-Pro-D-Glu(Bn)) 9, Figure S12: 1H NMR spectrum of cyclo(Gly-L-Pro-D-Glu) 10 in D2O,
Figure S13: 13C NMR spectrum of cyclo(Gly-L-Pro-D-Glu) 10 in D2O, Figure S14: IR spectrum of
cyclo(Gly-L-Pro-D-Glu) 10, Figure S15: HRMS(ESI) spectrum of cyclo(Gly-L-Pro-D-Glu) 10.
Author Contributions: The listed authors contributed to this work as described in the following:
conceived and designed the experiments, D.-F.T.; performed the experiments, S.C. and C.-W.W.;
analyzed the data, S.C., C.-W.W. and C.-Y.L.; wrote the paper, S.C., C.-Y.L. and D.-F.T. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Ministry of Science and Technology, grant number 92-2751-
B-259-001-Y, 104-2113-M-259-002, 107-2221-E-182-022, 109-2221-E-182-008-MY3 and Chang Gung
Memorial Hospital, Linkou, Taiwan, grant number CMRPD1K0101.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are availiable on request from corre-
sponding authors. The data are not publicly available due to confidentiality due diligence.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2021, 26, 389
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Sample Availability: Samples of the compound are not available from the authors.
References
1.
2.
3.
4.
5.
6.
Friedrich, A.B.; Fischer, I.; Proksch, P.; Hacker, J.; Hentschel, U. Temporal variation of the microbial community associated with
the mediterranean sponge Aplysina aerophoba. FEMS Microbiol. Ecol. 2001, 38, 105–113. [CrossRef]
Proksch, P.; Edrada, R.A.; Ebel, R. Drugs from the seas–current status and microbiological implications. Appl. Microbiol. Biotechnol.
2002, 59, 125–134. [PubMed]
Li, L.; Zhan, H.; Duan, P.; Liao, J.; Quan, J.; Hu, Y.; Chen, Z.; Zhu, J.; Liu, M.; Wu, Y.-D.; et al. Self-assembling nanotubes consisting
of rigid cyclic γ–peptides. Adv. Funct. Mater. 2012, 22, 3051–3056. [CrossRef]
Zainuddin, E.N.; Jansen, R.; Nimtz, M.; Wray, V.; Preisitsch, M.; Lalk, M.; Mundt, S.; Lyngbyazothrins, A.-D. antimicrobial cyclic
undecapeptides from the cultured cyanobacterium Lyngbya sp. J. Nat. Prod. 2009, 72, 1373–1378. [CrossRef] [PubMed]
Williams, D.E.; Yu, K.; Behrisch, H.W.; Soest, R.V.; Andersen, R.J. Rolloamides A and B, cytotoxic cyclic heptapeptides isolated
from the caribbean marine Sponge Eurypon laughlini. J. Nat. Prod. 2009, 72, 1253–1257. [CrossRef]
Ford, P.W.; Gustafson, K.R.; McKee, T.C.; Shigematsu, N.; Maurizi, L.K.; Pannell, L.K.; Williams, D.E.; Silva, E.D.; Lassota, P.;
Allen, T.M.; et al. HIV-inhibitory and cytotoxic depsipeptides from the Sponges Theonella mirabilis and Theonella swinhoei Collected
in Papua New Guinea. J. Am. Chem. Soc. 1999, 121, 5899–5909. [CrossRef]
7.
8.
9.
Capon, R.J.; Ford, J.; Lacey, E.; Gill, J.H.; Heiland, K.; Friedel, T. Phoriospongin A and B: Two new nematocidal depsipeptides
from the australian marine sponges Phoriospongia sp. and Callyspongiabilamell- ata. J. Nat. Prod. 2002, 65, 358–363. [CrossRef]
Mitova, M.; Popov, S.; De Rosa, S. Cyclic peptides from a Ruegeria strain of bacteria associated with the sponge Suberites domuncula.
J. Nat. Prod. 2004, 67, 1178–1181. [CrossRef]
Aracil, J.M.; Badre, A.; Fadli, M.; Jeanty, G.; Banaigs, B.; Francisco, C.; Lafargue, F.; Heitz, A.; Aumelas, A. Nouveaux cy-
clotétrapeptides isolés de l’ascidie cystodytes delle chiajei. Tetrahedron Lett. 1991, 32, 2609–2612. [CrossRef]
10. Kawagishi, H.; Somoto, A.; Kuranari, J.; Kimura, A.; Chiba, S. A novel cyclotetrapeptide produced by Lactobacillus helveticus as a
tyrosinase inhibitor. Tetrahedron Lett. 1993, 34, 3439–3440. [CrossRef]
11. Yang, L.; Tan, R.X.; Wang, Q.; Huang, W.Y.; Yin, Y.X. Antifungal cyclopeptides from Halobacillus litoralis YS3106 of marine origin.
Tetrahedron Lett. 2002, 43, 6545–6548. [CrossRef]
12. Shin, J.; Seo, Y.; Lee, H.S.; Rho, J.R.; Mo, S.J. A new cyclic peptide from a marine-derived bacterium of the genus Nocardiopsis. J.
Nat. Prod. 2003, 66, 883–884. [CrossRef] [PubMed]
13. Xie, W.; Zou, B.; Pei, D.; Ma, D. Total synthesis of cyclic tetrapeptide FR235222, a potent immunosuppressant that inhibits
mammalian histone deacetylases. Org. Lett. 2005, 7, 2775–2777. [CrossRef] [PubMed]
14. Kulkarni, S.S.; Ross, N.C.; McLaughlin, J.P.; Aldrich, J.V. Synthesis of Cyclic Tetrapeptide CJ 15,208: A Novel Kappa Opioid
Receptor Antagonist. Adv. Exp. Med. Biol. 2009, 611, 269. [PubMed]
15. Davies, J.S. The cyclization of peptides and depsipeptides. J. Peptide Sci. 2003, 9, 471–501. [CrossRef] [PubMed]
16. Hamada, Y.; Shioiri, T. Recent progress of the synthetic studies of biologically active marine cyclic peptides and depsipeptides.
Chem. Rev. 2005, 105, 4441–4482. [CrossRef]
17. Schmidt, U.; Langner, J. Cyclotetrapeptides and cyclopentapeptides: Occurrence and synthesis. J. Peptide Res. 1997, 49, 67–73. [CrossRef]
18. Tajima, H.; Wakimoto, T.; Takada, K.; Ise, Y.; Abe, I. Revised structure of cyclolithistide A, a cyclic depsipeptide from the marine
sponge Discodermia japonica. J. Nat. Prod. 2014, 77, 154–158. [CrossRef]
19. Usami, Y. Recent synthetic studies leading to structural revisions of marine natural products. Mar. Drugs 2009, 7, 314–330. [CrossRef]
20. Chakraborty, S.; Tyagi, P.; Tai, D.F.; Lee, G.H.; Peng, S.M. A lead (II) 3D coordination polymer based on a marine cyclic peptide
motif. Molecules 2013, 18, 4972–4985. [CrossRef]
21. Chakraborty, S.; Tai, D.F.; Lin, Y.C.; Chiou, T.W. Antitumor and antimicrobial activity of some cyclic tetrapeptides and tripeptides
derived from marine bacteria. Mar. Drugs 2015, 13, 3029–3045. [CrossRef] [PubMed]
22. Chakraborty, S.; Lin, S.H.; Shiuan, D.; Tai, D.F. Syntheses of some α-cyclic tripeptides as potential inhibitors for HMG-CoA
reductase. Amino Acids 2015, 47, 1495–1505. [CrossRef] [PubMed]
23. New, R.; Bogus, M.; Bansal, G.S.; Dryjska, M.; Zajkowska, K.; Burnet, M. Efficacy of bioactive cyclic peptides in Rheumatoid
Arthritis: Translation from in vitro to in vivo models. Molecules 2017, 22, 1613. [CrossRef] [PubMed]
24. Sara, V.R.; Carlsson-Skwirut, C.; Bergman, T.; Jornvall, H.; Roberts, P.J.; Crawford, M.; Hakansson, L.N.; Civalero, I.; Nordberg, A.
Identification of Gly-Pro-Glu (GPE), the aminoterminal tripeptide of insulin-like growth factor 1 which is truncated in brain, as a
novel neuroactive peptide. Biochem. Biophys. Res. Commun. 1989, 165, 766–771. [CrossRef]
25. Gorecki, D.C.; Beresewicz, M.; Zabłocka, B. Neuroprotective effects of short peptides derived from the Insulin-like growth factor
1. Neurochemistry International. Neurochem. Int. 2007, 51, 451–458. [CrossRef] [PubMed]
26. Guan, J.; Gluckman, P.D. IGF-1 derived small neuropeptides and analogues: A novel strategy for the development of pharmaceu-
ticals for neurological conditions. Br. J. Pharmacol. 2009, 157, 881–898. [CrossRef]
27. Cacciatore, I.; Cornacchia, C.; Baldassarre, L.; Fornasari, E.; Mollica, A.; Stefanucci, A.; Pinnen, F. GPE and GPE analogues as
promising neuroprotective agents. Mini Rev. Med. Chem. 2012, 1, 13–23. [CrossRef]
28. Sizonenko, S.V.; Sirimanne, E.S.; Williams, C.E.; Gluckman, P.D. Neuroprotective effects of the N-terminal tripeptide of IGF-1,
glycine-proline-glutamate, in the immature rat brain after hypoxic-ischemic injury. Brain Res. 2001, 22, 42–50.
29. White, C.J.; Yudin, A.K. Contemporary strategies for peptide macrocyclization. Nat. Chem. 2011, 3, 509–524. [CrossRef]

Molecules 2021, 26, 389
11 of 11
30. Kartha, G.; Ambady, G.; Shankar, P.V. Structure and conformation of a cyclic tripeptide. Nature 1974, 247, 204–205. [CrossRef]
31. Meng, X.Y.; Zhang, H.X.; Mezei, M.; Cui, M. Molecular docking: A powerful approach for structure-based drug discovery. Curr.
Comput. Aided Drug Des. 2011, 7, 146–157. [CrossRef] [PubMed]
32. Miyazawa, T.; Ogura, M.; Nakajo, S.; Yamada, T. Synthesis of monoesters of N-protected α-aminodicarboxylic acids via the
microbial protease-catalyzed regioselective hydrolysis of their diesters. Biotechnol. Tech. 1998, 12, 431–434. [CrossRef]
33. Mendoza-Sanchez, R.; Corless, V.B.; Nguyen, Q.N.; Bergeron-Brlek, M.; Frost, J.; Adachi, S.; Tantillo, D.J.; Yudin, A.K. Cyclols
revisited: Facile synthesis of medium-sized cyclic peptides. Chem. Eur. J. 2017, 23, 13319–13322. [PubMed]
34. Kessler, H.; Bermel, W.; Krack, G.; Bats, J.W.; Fuess, H.; Fuess, H.; Hull, W.E. Peptidkonformationen, 27. cyclo-[L-Pro-(Bzl)Gly-D-
Pro]. Synthese und Konformation im Kristall und in Lösung. Chem. Ber. 1983, 116, 3164–3181.
35. Kessler, H.; Schuck, R.; Siegmeier, R.; Bats, J.W.; Fuess, H.; Forster, H. Peptidkonformationen, 22. Die Konformation von cyclo-
[Pro2-(NB)Gly] im Kristall und in Lösung durch Röntgenstrukturanalyse und ein- und zweidimensionale NMR-Spektroskopie.
Liebigs Ann. Chem. 1983, 2, 231–247. [CrossRef]
36. Kessler, H.; Kondor, P.; Krack, G.; Kramer, P. Conformational equilibrium in the backbone of cyclic tripeptides. J. Am. Chem. Soc.
1978, 100, 2548–2550.
37. Venkatachalam, C.M. Stereochemical studies of cyclic peptides. II. molecular structure of cyclotriprolyl. Biochim. Biophys. Acta
1968, 168, 397–401. [CrossRef]
38. Tosso, R.D.; Zamora, M.A.; Suvire, F.D.; Enriz, R.D. Ab Initio and DFT study of the conformational energy hypersurface of cyclic
Gly-Gly-Gly. J. Phys. Chem. A 2009, 113, 10818–10825. [CrossRef]
39. Kartha, G.; Varughese, K.I.; Aimoto, S. Conformation of cyclo(-L-Pro-Gly-)3 and its Ca2+ and Mg2+ complexes. Proc. Natl. Acad.
Sci. USA 1982, 79, 4519–4522.
40. Dahiya, R.; Gautam, H. Total synthesis and antimicrobial activity of a natural cyclohexapeptide of marine origin. Mar. Drugs
2010, 8, 2384–2394.
41. Vera, B.; Vicente, J.; Rodriguez, A.D. Isolation and structural elucidation of euryjanicins B D, proline-containing cycloheptapep-
−
tides from the Caribbean marine sponge Prosuberites laughlini. J. Nat. Prod. 2009, 72, 1555–1562. [CrossRef] [PubMed]
42. Mitova, M.I.; Stuart, B.G.; Cao, G.H.; Blunt, J.W.; Cole, A.L.J.; Munro, M.H.; Chrysosporide, G.A. Cyclic pentapeptide from a New
Zealand sample of the fungus Sepedonium chrysospermum. J. Nat. Prod. 2006, 69, 1481–1484. [CrossRef] [PubMed]
43. Hsieh, P.W.; Chang, F.R.; Wu, C.C.; Wu, K.Y.; Li, C.M.; Chen, S.L.; Wu, Y.C. New cytotoxic cyclic peptides and dianthramide from
Dianthus superbus. J. Nat. Prod. 2004, 67, 1522–1527. [CrossRef] [PubMed]
44. Lee, D.L.; Hodges, R.S. Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S.
Biopolymers 2003, 71, 28–48. [CrossRef] [PubMed]
45. Falanga, A.; Nigro, E.; De Biasi, M.G.; Daniele, A.; Morelli, G.; Galdiero, S.; Scudiero, O. Cyclic peptides as novel therapeutic
microbicides: Engineering of human defensin mimetics. Molecules 2017, 22, 1217. [CrossRef] [PubMed]
46. Dartois, V.; Sanchez-Quesada, J.; Cabezas, E.; Chi, E.; Dubbelde, C.; Dunn, C.; Granja, J.; Gritzen, C.; Weinberger, D.; Reza Ghadiri,
M.; et al. Systemic antibacterial activity of novel synthetic cyclic peptides. Antimicrob. Agents Chemother. 2005, 49, 3302–3310.
[CrossRef] [PubMed]
47. Scheinpflug, K.; Krylova, O.; Nikolenko, H.; Thurm, C.; Dathe, M. Evidence for a novel mechanism of antimicrobial action of a
cyclic R-, W-rich hexapeptide. PLoS ONE 2015, 10, e0125056. [CrossRef]
48. Wiese, J.; Abdelmohsen, U.R.; Motiei, A.; Humeida, U.H.; Imhoff, J.F. Bacicyclin, a new antibacterial cyclic hexapeptide from
Bacillus sp. strain BC028 isolated from Mytilus edulis. Bioorg. Med. Chem. Lett. 2018, 28, 558–561. [CrossRef]