Total synthesis of five proline-enriched cyclic heptapeptides from the marine sponge Stylissa carteri

Article abstract of DOI:10.1016/j.tetlet.2018.03.083

The total synthesis of five naturally occurring cyclic proline-enriched heptapeptides from the marine sponge Stylissa carteri was reported. The five cyclic heptapeptides were synthesized by applying a two-step solid-phase/solution synthesis strategy. The

Full text of DOI:10.1016/j.tetlet.2018.03.083

Accepted Manuscript
Total synthesis of five proline-enriched cyclic heptapeptides from the marine
sponge Stylissa carteri
Yulei Li, Qi Chang, Minghao Wu, Xia Zhao
PII:
S0040-4039(18)30410-6
https://doi.org/10.1016/j.tetlet.2018.03.083
TETL 49852
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Reference:
Tetrahedron Letters
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27 March 2018
Please cite this article as: Li, Y., Chang, Q., Wu, M., Zhao, X., Total synthesis of five proline-enriched cyclic
heptapeptides from the marine sponge Stylissa carteri, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/
j.tetlet.2018.03.083
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Tetrahedron Letters
Total synthesis of five proline-enriched cyclic heptapeptides from the marine sponge
Stylissa carteri
Yulei Lia,b , Qi Changa,b, Minghao Wua , Xia Zhao a,b 
a Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China
b Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The total synthesis of five naturally occurring cyclic proline-enriched heptapeptides from the
marine sponge Stylissa carteri was reported. The five cyclic heptapeptides were synthesized by
applying a two-step solid-phase/solution synthesis strategy. The linear heptapeptides were
assembled by standard Fmoc chemistry on 2-chlorotrityl chloride resin, cleaved off-resin with
acetic acid/trifluoroethanol/dichloromethane to keep side-chain protecting groups intact, and
subsequently cyclization was achieved by a solution method. The final products were purified
by a preparative RP-HPLC system, and their structures were characterized by HR-QTOF-MS
NMR. The spectral data of synthetic peptides were found to be identical to that reported for the
natural products.
Available online
Keywords:
cyclic heptapeptides
Stylissa carteri
Solid-phase synthesis
Cyclization
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Marine organisms are excellent sources of biologically active
natural products, which have been considered as promising
resources for lead compounds or drug candidates.1 Peptides are
important bioactive natural products which are present in many
marine species and have attracted increasing attention for their
broad spectra of bioactivities, low toxicity and high specificity.2
These marine peptides have high potential medicinal values and
show a hopeful and brilliant prospect for treatment or prevention
on various diseases. In 2010, the global sales of four of 60
peptide drugs on the market reached in excess of US $ 1 billion,
and more than 500 peptides are under clinical development.3,4
Cyclic peptides are one of the underexplored classes of bioactive
peptides with a marine origin that have great promise in
pharmaceutical areas. Unlike linear peptides, they are more stable
in vivo, more lipophilic and membrane permeable, and show
higher specificity for target receptors. These bioactive cyclic
peptides may be considered as attractive targets for drug
discovery and biomedical research.5,6 Especially for the poly-
proline cyclic peptides, proline residue can reduce their backbone
flexibility, thus enhancing the selectivity and affinity for protein
binding.7 Recently, an emerging class poly-proline cyclic
peptides have been mainly isolated from lower sea organisms,
however, their contents are so low that it was not enough for drug
development. What is more, they have been associated with
strong anticancer and antimicrobial activities.8 For further
research of biological activity and structure-activity relationships,
many researchers have focused on the synthesis of cyclic
peptides, especially by solid-phase peptide synthesis method.9
Fig 1. Structures of compound 1-5 (all the amino acids are L-
configuration).
10
heptapeptides from the marine sponge Stylissa carteri. Among
them, Phakellistatin 13 (3) was first reported in 2003 by zhong-
hua wang and his colleagues as a secondary metabolite from the
sponge Phakellia fusca Thiele. This compound was significantly
cytotoxic against the human hepatoma BEL-7404 cell line with
an ED50<10-2 µg/mL. 11 And Hymenamides C (4) and D (5) were
described in 1993 by Kobayashi and his team from the okinawan
marine sponge hymeniacidon sp.12 Carteritins A (1) is a new
cyclic heptapeptide exhibited cytotoxic activity against Hela,
HCT116 and RAW264 cells with IC50 values of 0.70, 1.30 and
1.50 μM, respectively. The five cyclic heptapeptides have been
previously isolated in very low yields (<1%) from the marine
The five cyclic peptides (Fig. 1) are poly-proline cyclic
———
 Corresponding author: Tel & Fax: 86-532- 82031560. E-mail address: zhaoxia@ouc.edu.cn (X. Zhao).

2
Tetrahedron
Scheme 2. Total synthesis of Carteritins A (a)Fmoc-Pro-OH, DMF, DCM, DIPEA,1h; (b) 20% piperidine in DMF, (2×5min); (c)Fmoc-
Tyr(tBu)-OH, HCTU, DIPEA, DMF, 1h; (d) Fmoc-Glu(Trt)-OH, HCTU, DIPEA, DMF, 1h; (e)Fmoc-Pro-OH, HCTU, DIPEA, DMF,
1h; (f) Fmoc-Pro-OH, HCTU, DIPEA, DMF, 1h; (g)Fmoc-Ile-OH, HCTU, DIPEA, DMF, 1h; (h)Fmoc-Phe-OH, HCTU, DIPEA, DMF,
1h; (i) AcOH/TFE/DCM (1:2:16), 3 h; (j) PyBop, HOBt, DIPEA, NMP, DCM, 0 oC-r.t. overnight; (k) TFA/TIPS/ phenol/H2O(88/5/5/2,
v/v) for 1h with shaking
sponge Stylissa carteri.10 It is obvious that the extraction
procedure is very difficult and has an extremely low yield, which
greatly hinders further biological activity and development, so
chemical synthesis becomes of great importance. As a result,
these cyclic heptapeptides were obtained by combining solid-
phase method with conventional solution method for its further
biological studies in this paper.
synthesizing natural cyclic heptapeptides stylissatin A,16 we
found that the residues of Pro and the beta location of carbon
which is not replaced are the favourable points of cyclization to
get the target compounds. So the point of cyclization Carteritins
A , Carteritins B, Phakellistatin 13, hymenamides C and D is Pro
- Phe, Pro - Tyr, Pro - Phe, Pro - phe, and Pro – Leu, respectively,
to obtain the linear precursor with Pro as the C terminal amino
acid, and another residue as the N-terminal amino acid. The
linear precursors were synthesized directly by standard solid-
Results and discussion
Retrosynthetic strategy
phase
peptide
synthesis
procedures,
using
a
9-
fluorenylmethyloxycarbonyl/tert-
butoxycarbonylation(Fmoc/Boc) protecting scheme and 2-
chlorotrityl chloride resin (CLTR-Cl) as a solid support. The
cyclization of the linear precursors was prepared in solution. The
final crude cyclic heptapeptides were purified by a preparative
RP-HPLC system to give the target compounds.
Representative total synthesis of Carteritins A
Scheme 2 shows the total synthetic process of Carteritins A,
including synthesis of the linear heptapeptide, cyclization and
side-chain deprotection. The synthesis of the linear heptapeptide
13 was performed in a peptide synthesizer under anhydrous
condition. The first amino acid Fmoc-Pro-OH was loaded on the
2-Cl-trityl-Cl resin (1 mmol/g) after treatment by N, N-
Diisopropylethylamine (DIPEA) (540.0 μL) in a mixed solution
of N, N-Dimethylformamide (DMF) (5 mL) and dichloromethane
(DCM) (5 mL) for 1 h, then the solvent was drained and the resin
was washed sequentially with DMF, DCM and MeOH (3-5mL).
The Pro-linked resin was then used for construction of the full
length peptide. The protocols employed were as follows:
deprotection of Fmoc with 20% piperidine in DMF (15 min) and
peptide coupling using 5 eq. of amino acid, 4.5 eq. of O-(7-
azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium-hexafluor-
ophosphate (HCTU) and 10 eq. of DIPEA. All coupling reactions
were performed 1 h at room temperature. In the synthesis of 8-
13, amino acids Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-
Pro-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Phe-OH were
sequentially installed to construct the linear peptide. To cleave
the linear peptide from the solid phase with intact side chain
protecting groups, the resin was treated with AcOH/TFE/DCM
Scheme1. Retrosynthetic analysis of Carteritins A
Scheme 1 describes a retrosynthetic method to obtain
Carteritins A, and the method can be applied to the retrosynthetic
analysis of Carteritins B, Phakellistatin 13, hymenamides C and
D. It is well known that the head-to-tail connected cyclic peptides
can be easily synthesized in good purity using standard
procedures and orthogonally protected amino acid residues.
However, the cyclization step is critical and essentially depended
on the peptide sequence, structural constraint, and the resulting
ring size.13,14,15 These peptides are poly-proline cyclic
heptapeptides. According to our previous experience in
o
(1:2:16, v/v) at 35 C with shaking for 3 h. The synthesis of the

3
2.93(1H, t, J = 15.0Hz), 2.45(1H, d, J = 15.0Hz), 2.11(3H, m),
linear heptapeptide 14 yielded a side-chain protected linear
precursor in 70% of overall yield. By HPLC and ESI-MS
analysis, the linear hexapeptide 14 was found to be obtained in
high purity and used directly for subsequent cyclization without
further purification. Cyclization was performed in dry DCM with
the liner peptide concentration of 0.5 mg/mL at -5oC.
Benzotriazol-1-yl-oxytripyrrolidinophosphonium-hexafluorophos
phate(PyBOP) and 1-hyd-roxy-benzotriazole (HOBT) were
chosen as the coupling reagents. The crude peptide was initially
purified by a Sephadex LH-20 column. The deprotection of 15
was performed by using TFA/TIPS/Phenol/H2O (88/5/5/2, v/v)
with shaking for 3h, and then the crude cyclic peptide was
precipitated at 0 °C with cool diethyl ether. The final crude cyclic
peptide was purified by a preparative reversed-phase HPLC to
give the target compound as a white solid powder, with a total
yield of 21% and HPLC purity over 98%. The HR-QTOF-MS,
1H and 13C NMR spectroscopic data (Supporting Information) of
synthetic 1 were in agreement with those of the natural product.
2.03-1.97(4H, m), 1.88(2H, m ), 1.84-1.71(4H, m), 1.60(2H, m),
1.49(2H, m), 1.23(1H, s), 1.03(1H, m), 0.90(3H, d, J = 5.0Hz),
0.76(3H, t, J = 5.0Hz)
13C NMR (125MHz, DMSO-d6, δ, ppm): 174.24, 172.31,
172.09, 171.91, 171.52, 170.77, 170.55, 168.20, 156.33, 138.99,
130.56, 129.08, 128.56, 127.58, 126.71, 115.42, 63.13, 60.76,
59.38, 56.18, 55.84, 54.40, 51.94, 47.32, 47.24, 47.22, 37.47,
36.53, 36.41, 32.32, 30.91, 28.89, 28.58, 27.32, 25.39, 24.69,
24.19, 22.29, 17.28, 12.33.
HR-QTOF-MS: C44H58N7O10 Calcd. for, (M+H)+, 844.4200,
+
,
found, 844.4237, C44H57N7NaO10 Calcd. for, (M+Na)+,
+
866.4200, found, 866.4048.
1H NMR, 13C NMR, HR-QTOF-MS Data of Carteritins B:
Yield: 19.5%.1H NMR (500 MHz, DMSO-d6, δ, ppm, J/Hz):
9.14(1H, s), 7.79(1H, d, J = 10.0Hz), 7.58(1H, d, J = 10.0Hz),
7.47(1H, d, J = 5.0Hz), 7.17(1H, d, J = 10.0Hz), 7.09(1H, d, J =
10.0Hz), 6.97(1H, d, J = 10.0Hz), 6.82(1H, d, J = 5.0Hz),
6.70(1H, d, J = 10.0Hz), 6.62(3H, m), 4.92(1H,m), 4.43(1H,m),
4.21(1H,m), 4.17(2H,m), 3.97(2H,m), 3.82(2H, m), 3.78(3H, m),
3.49(1H, d, J = 10.0Hz), 3.24(2H, m), 3.06(2H, m), 2.87(1H, dd,
J = 10.0,15.0Hz), 2.81(1H, m), 2.74(1H, dd, J = 10.0,15.0Hz ),
2.57(2H, m), 2.25(1H, m), 2.06(1H, m), 1.97(2H, m), 1.86(2H,
m), 1.75(2H, m), 1.52(3H, m),1.34(2H, m), 1.26(1H, m),
Total synthesis of Carteritins B, Phakellistatin 13,
hymenamides C and D
As a result, the naturally occurring cyclic peptide Carteritins
A (1) was successfully synthesized by a two-step solid-
phase/solution synthesis strategy. Then the method was also
applied to the synthesis of its close structural analogues,
Carteritins B(2), Phakellistatin 13(3), hymenamides C(4) and
D(5). All the peptides were obtained about total yields of 20%,
1.03(1H, m), 0.94(3H, d,
5.0Hz),0.84(1H, d, J = 10.0Hz), 0.43(1H, m).
J = 5Hz), 0.90(3H, d, J =
13C NMR (125MHz, DMSO-d6, δ, ppm): 172.15, 171.59,
171.32, 170.72, 170.62, 170.41, 169.52, 156.59,156.50, 156.39,
131.09, 130.23, 129.78, 127.97, 127.92, 126.81, 115.89, 115.59,
115.29, 63.26, 61.46, 60.53, 57.53, 55.55, 54.15, 52.26, 52.01,
47.54, 46.45, 40.73, 37.65, 35.83, 30.86, 29.23, 25.61, 25.11,
1
and their HR-QTOF-MS, H and 13C NMR spectroscopic data ,
(Supporting Information) were in agreement with those of the
natural products. The optical rotation value of the final
products ： Carteritins A (1): [α]20 -120o ( c 0.33, MeOH);
D
Carteritins B (2): [α]20 -98o ( c 0.30, MeOH); Phakellistatin
23.97, 23.15, 22.49, 20.97.
D
HR-QTOF-MS:C46H58N7O10 Calcd. for, (M+H)+, 884.4100,
+
13(3): [α]20D -126o ( c 0.09, MeOH); Hymenamides C (4): [α]20
-
,
D
+
138o ( c 0.40, MeOH); Hymenamides D (5): [α]20 -87o ( c 0.15,
found,
884.4171,
C46H57N7NaO10
Calcd.
for,
D
(M+Na)+,906.4100, found, 906.3940.
MeOH);
Cytotoxicity assays
Conclusions
The synthesized cyclic heptapeptides 1–5 were evaluated for
cytotoxicities against human cervical cancer cell line (HeLa),
human colon cancer cell line (HCT116), and murine macrophage
line (RAW264.7), human hepatoma(BEL-7402), human lung
carcinoma cell (A-549). All the products showed cytotoxicities
against the five cell lines with IC50 values of above 25μM, in
An efficient solid-phase/solution strategy was developed to
the total synthesis of the natural products Carteritins A,
Carteritins B, Phakellistatin 13, hymenamides C and D from the
marine sponge Stylissa carteri. The spectral data of synthetic
peptides were found to be identical to that reported for the natural
products. However, a difference in biological activity was noted,
the synthetic peptides were found to be weakly cytotoxic against
five cell lines (A-549, Hela, BEL-7402, HCT116 and
RAW264.7). It may be attributed to conformational difference or
the presence of biologically active impurity in the naturally
isolated material, or configuration was changed in the synthesis.
1
despite of the HR-QTOF-MS, H and 13C NMR spectroscopic
data (Supporting Information) of these targeted compounds were
identical with those of the natural products. Several research
groups have reported that although the synthetic proline-
containing cyclic peptides are chemically equivalent to their
natural counterparts, their biological activities are deviant in
some cases.1 It may be attributed to the conformational difference
or the presence of biologically active impurity in the naturally
isolated material, and/or the configuration was changed in the
synthesis. The same reasons may explain the variation in
biological activity of the synthetic and the natural compounds in
our experiments.
Acknowledgments
This research was supported by NSFC-Shandong Joint Fund
(U1606403) and Innovation Project of Qingdao National
Laboratory
for
Marine
Science
and
Technology
(No.2015ASKJ02). We are grateful to the Instrumental Analysis
Center of Ocean University of China for NMR spectroscopic and
mass spectrometric analysis.
Spectroscopic data of Carteritins A and B
1H NMR, 13C NMR, HR-QTOF-MS Data of Carteritins A:
Yield:21%. 1H NMR (500 MHz, DMSO-d6, δ, ppm,
J/Hz):8.01(1H, d, J = 5.0Hz), 7.82(1H, d, J = 10.0Hz), 7.49(1H,
d, J = 10.0Hz), 7.27(2H, t, J = 5.0Hz), 7.22(2H, t, J = 5.0Hz),
7.18(1H, t, J = 5.0Hz), 7.08(2H, t, J = 5.0Hz), 6.90(1H, t, J =
10.0Hz), 6.66(2H, t, J = 5.0Hz), 4.85(1H, t, J = 10.0Hz),
4.47(2H, td, J = 12.5,5.0Hz), 4.36(2H,q, J = 5.0Hz), 4.29(2H,d, J
= 10.0Hz), 4.25(2H,t, J = 10.0Hz), 3.93(1H,t, J = 10.0Hz),
3.85(1H,m),3.76(1H, m), 3.47(1H, m), 3.35-3.25(4H, m),
Supplementary Material
Supplementary data associated with this article can be found
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4
Tetrahedron
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Total synthesis of five proline-enriched cyclic heptapeptides
from the marine sponge Stylissa carteri
Yulei Lia,b , Qi Changa,b, Minghao Wua , Xia Zhao a,b
*

6
Tetrahedron
Highlights
• Five proline-enriched cyclic heptapeptides from
marine sponge were synthesized totally.
• A two-step solid-phase/solution strategy was
effective to cyclic peptides synthesis.
•Their structures were identical to those of natural
products .