Solid Phase Synthesis of Marine Cyclopeptide Phakellistatin 13

Article abstract of DOI:10.1007/s10600-018-2460-6

The synthesis of the naturally occurring marine cyclopeptide phakellistatin 13 has been achieved by solid phase peptide synthesis (SPPS). A general method was described to synthesize the cyclic peptide by a two-step solid-phase/solution synthesis strategy. The linear peptide was assembled by standard Fmoc chemistry on solid-phase and subsequently cyclized in solution phase to yield fully protected cyclic heptapeptide. After removing the protected groups, phakellistatin 13 was obtained and purified by semi-preparative reverse phase high-performance RP-HPLC, and its structure was confirmed by NMR and HR-ESI-MS.

Full text of DOI:10.1007/s10600-018-2460-6

DOI 10.1007/s10600-018-2460-6
Chemistry of Natural Compounds, Vol. 54, No. 4, July, 2018
SOLID PHASE SYNTHESIS OF MARINE
CYCLOPEPTIDE PHAKELLISTATIN 13
1
2
2
Demeng Xia, Chao Liu, Hui Song,
2
2*
Xiang Li, and Na Li
The synthesis of the naturally occurring marine cyclopeptide phakellistatin 13 has been achieved by solid
phase peptide synthesis (SPPS). A general method was described to synthesize the cyclic peptide by a
two-step solid-phase/solution synthesis strategy. The linear peptide was assembled by standard Fmoc chemistry
on solid-phase and subsequently cyclized in solution phase to yield fully protected cyclic heptapeptide. After
removing the protected groups, phakellistatin 13 was obtained and purified by semi-preparative reverse
phase high-performance RP-HPLC, and its structure was confirmed by NMR and HR-ESI-MS.
Keywords: cyclopeptide, solid phase synthesis, phakellistatin 13.
Cyclopeptides have several advantages as potential drug candidates [1, 2]. Structurally their mobility is more restricted
than that of their linear counterparts so that they adopt fewer conformations and, as a result, show higher specificity for the
target receptors [3]. In addition, they are often more stable in vivo. Therefore, they present promising drug candidates. Because
of these advantages, many laboratories have focused on the syntheses of cyclopeptides and cyclic depsipeptides, especially by
solid-phase peptide synthesis methodologies [4].
To date, marine sponges were revealed to be a valuable source for discovering new medicinal agents for the treatment
of cancer and acquired immune deficiency syndrome (AIDS), among others [5]. Several secondary metabolites with unusual
architecture and remarkable biological activity, especially a number of cyclic peptides, such as cyclosquamosin H, dolastatins,
didemnin B, bryostatin, aplidin, and kahalalide F, have undergone clinical trials [6–10].
During the past 30 years, there was a series of phakellistatin (phakellistatins 1–14) isolated from the Western Pacific
Ocean (Federated States of Micronesia) marine sponge Phakellia sp. All of them have been reported to have proline units that
rendered the peptide chain less flexible and showed medium cytotoxicity [11–17]. Phakellistatin 13, isolated by Zhong-Hua Wang in
–
2
2
002, was found to exhibit strong cytotoxicity against the BEL-7404 human hepatoma cell line, with an ED50 < 10 ꢀg/mL,
but was inactive against the HL-60 cell line. However, the yield of phakellistatin 13 isolated from natural products was very
low (0.0006%) [16].
The first total synthesis of phakellistatin 13 was reported by Greenman and coworkers in 2004 using solid-phase
synthesis and liquid cyclization. However, the reagents and conditions used for cyclization requires 4 days, which is
time-consuming. For further pharmaceutical and medicinal chemical investigations, the total synthesis of phakellistatin 13
utilizing a more succinct and efficient cyclization strategy is reported in this paper.
We planned to obtain the cyclopeptide by solid-phase synthesis of the linear precursors followed by cyclization and
deprotection. The synthesis of phakellistatin 13 was carried out by a solid-phase method using the Fmoc/tBu protecting
method and 2-chorotrityl chloride resin as solid support [18].
1
) Department of Emergency Medicine, Changhai Hospital, Second Military Medical University No. 168 Changhai
Road, WujiaochangTown, Yangpu District, 200433, Shanghai, China, e-mail: demengxia@163.com; 2) PharmaceuticalAnalysis
Center, School of Pharmacy, Second Military Medical University No. 325 Guohe Road, Wujiaochang Town, Yangpu District,
2
2
00433, Shanghai, China, e-mail: nana_li1989@yeah.net. Published in Khimiya Prirodnykh Soedinenii, No. 4, July–August,
018, pp. 630–632. Original article submitted August 7, 2017.
0
0
009-3130/18/5404-0745 2018 Springer Science+Business Media, LLC
745

OH
HO
O
Cl
Cl
O
O
a
b, c
+
Cl
O
NH
NH
2
NHFmoc
O
HN
1
O
2-Chlorotrityl chloride Thr7
resin
OR
Fmoc-Gly-OH
N
1
O
Pro1
1d
d
O
7
d
H
N
1
c
7c
O
C
O
t
O Bn
7
b
1a
1e
NH
1
b
6
6
e
N
7
a
O
NH
N
HN
C
d
2a
Boc
Leu
6
2b CH
2
Gly2
HN
O
6
b
6
c
NH
O
C6a
O
5
3a
C
HN
e
HN
3b
2
R
2
O
N
C
5
b
5
d
5
a
5
f
4a
C
3c
3d
Trp5
NH
C
4b
5
k
5c
N
O
4e
O 3e
3f
5
h
4
c
Phe3
4
d
Pro4
3
g
3
, 4
= tBu, R
= R = H
3
4
: R
: R
1
1
2
= Boc
e
2
a. i. Fmoc-Gly-OH, DIPEA and DCM, r.t., 1 h; ii. 20% piperidine–DMF, r.t., 10 min; b. i. Fmoc-AA-OH, DCC, HOBt and NMP, r.t.,
4
h; ii. 20% piperidine–DMF, r.t., 10 min; c. AcOH–TFE–DCM (1:2:16), r.t., 1 h, 73.2%; d. PyAOP, HOAt, DIPEA and DCM, r.t.,
1
2 h, 87.6%; e. TFA–Et SiH–DCM (1:1:8), r.t., 3 h, 48.4%
3
Scheme 1
As shown in Scheme 1, the target peptide is obtained by solid-phase synthesis. The starting Fmoc-protected amino
acid (Fmoc–Gly–OH) was loaded on 2-chorotrityl chloride resin by treatment with N,N-diisopropylethylamine (DIPEA) to
obtain 1. For the remaining amino acids, the standard Fmoc SPPS was followed to build the on-resin linear peptide as the
precursor for phakellistatin 13. All the Fmoc protected amino acids were activated by 1-hydroxybenzotriazole (HOBt) and
N,Nꢁ-dicyclohexylcarbodiimide (DCC) in the presence of N-methyl-2-pyrrolidone (NMP) before the next coupling was carried
out. The progress of the amino acid coupling was checked through the ESI-MS test. Fmoc deprotection before each coupling
step was achieved by treatment of the resin-bound peptide with 20% piperidine/DMF solution. The free linear peptide 2 was
obtained by treatment of the cleavage cocktail of AcOH–tetrafluoroethylene (TFE)–DCM (1:2:16) for 1 h at room temperature
with stirring (Scheme 1) [19]. Subsequently, the cyclization was accomplished using (3-hydroxy-3H-1,2,3-triazolo[4,5-b]
pyridinato-O)tri-1-pyrrolidinylphosphorus hexafluorophosphate (PyAOP)–1-hydroxy-7-azabenzotriazole (HOAt)–DIPEA in
DCM at room temperature for 12 h to give the protected cyclopeptide 3. In order to reduce side reactions, cyclization was
performed under highly diluted conditions with a cyclic peptide concentration of 0.5 mg/mL. PyAOP was chosen as the
coupling reagent on account of the higher coupling efficiency. The side chain deprotection was realized by treatment with
TFA–Et SiH–DCM (1:1:8) for 3 h with stirring to provide crude phakellistatin 13 (4).
3
Finally, the crude compound was analyzed and purified by analytic and preparative HPLC using water and acetonitrile
1
13
as mobile phase. Further confirmation was carried out by HR-QTOF-MS and H NMR and C NMR.
In summary, the total synthesis of phakellistatin 13 was accomplished in good chemical yield via standard Fmoc
SPPS utilizing DCC and HOBt as coupling reagents and solution phase cyclization by PyAOP–HOAt–DIPEA. A general
synthesis method was developed to prepare cyclic peptide by combining solid-phase and conventional solution methods with
good yield and high purity. This strategy is an effective method to synthesize cyclopeptides especially when there is no
suitable anchoring side chain to be linked to the resin. The synthesis of phakellistatin 13 laid the foundation for further studies
on structural modification and pharmacological activity.
746

EXPERIMENTAL
General Methods. H NMR and 13C NMR spectrum was obtained on a Bruker Avance 600 MHz NMR spectrometer.
The chemical shifts of protons are given on the ꢂ scale, ppm, with tetramethylsilane (TMS) as internal standard. HR-QTOF-MS
was measured on an Agilent 6538 UHD Accurate Mass Q-TOF LC/MS mass spectrometer. Purification of crude peptides was
carried out using HPLC (LC-20AD, Shimadzu) with dual wavelength (220 and 254 nm).
1
Synthesis of the Linear Precursor of Phakellistatin 13 (4). The first amino acid was loaded on the resin by treatment
of DIPEA for 1 h, and then the Fmoc protection was subsequently removed by treatment with 20% piperidine–DMF solution
for 10 min. After the rest amino acid had been loaded, all protected amino acids were coupled using HOBt and DCC. Typically,
the amino acids (1.5 equiv.) were treated for 2 min with HOBt (2 equiv.) and DCC (2 equiv.) in NMP. The solution was added
to the resin, which was then agitated for 4 h at room temperature. After coupling and Fmoc deprotection, the resin was washed
three times with NMP and DCM, respectively. Then the resin was treated withAcOH–TFE–DCM (1:2:16) at room temperature
for 1 h. The protected free linear peptide 2 was obtained with a yield of 73.2%.
Cyclization of the Linear Precursor 2 of Phakellistatin 13 (4). The linear peptide 2 was cyclized by treatment with
PyAOP (5 equiv.), HOAt (5 equiv.), and DIPEA (6 equiv.) in DCM. Generally, PyAOP, HOAt, and DIPEA were dissolved in
DCM in a round-bottom flask. The protected linear peptide was dissolved in DCM (0.5 mg/mL) and dropped into the flask at
0
ꢃC. The mixture was stirred for 12 h at room temperature, and the reaction was monitored by ESI-MS. After the reaction was
completed, DCM was removed by rotary evaporation, and the resulting oil was purified by Sephadex LH-20 to give the
protected cyclopeptide 3 as a white solid powder with a yield of 87.6%.
Removal of the Side Chain Protecting Group. The protected cyclopeptide 3 was treated with TFA–Et SiH–DCM
3
(
1:1:8) at room temperature for 3 h. The reaction was monitored by ESI-MS. After the reaction was completed, toluene was
added, and the mixture was concentrated in vacuum. The residue was purified by RP-HPLC to give the cyclic peptide as a white
solid with an isolated yield of 48.4% and HPLC purity of over 95.0%. Its structure was confirmed by NMR and HR-QTOF-MS
and agrees with the literature.
1
Phakellistatin 13 (4). H NMR (600 MHz, CD OD, ꢂ, ppm, J/Hz): 9.32 (1H, d, J = 3.2), 8.47 (1H, d, J = 7.6), 7.76
3
(
1H, s), 7.48 (1H, d, J = 9.6), 7.05–7.40 (11H, m), 6.94 (1H, t, J = 7.2), 4.60–4.35 (5H, m), 4.06–4.29 (3H, m), 3.71–3.77 (1H,
m), 3.45–3.64 (2H, m), 3.38 (1H, d, J = 7.2), 3.18–3.25 (4H, m), 3.12 (1H, s), 3.05 (1H, d, J = 12.8), 2.90–2.99 (1H, m), 2.57
(
(
1H, t, J = 10.4), 2.39–2.47 (1H, m), 2.06–2.12 (2H, m), 1.95–2.03 (1H, m), 1.83–1.93 (1H, m), 1.68–1.76 (1H, m), 1.55–1.61
1H, m), 1.46 (1H, d, J = 12.8), 1.28–1.38 (2H, m), 1.18 (1H, d, J = 6.4), 1.09 (1H, t, J = 6.4), 0.99–1.04 (1H, m), 0.95 (1H, d,
1
3
J = 6.4), 0.87–0.91 (1H, m), 0.35–0.36 (1H, m), –0.02–0.08 (1H, m). C NMR (150 MHz, CD OD, ꢂ, ppm): 172.59, 172.49,
3
1
6
2
72.13, 171.81, 169.88, 169.68, 168.11, 136.73, 135.15, 128.36, 127.71, 125.95, 123.24, 120.71, 118.20, 117.30, 110.62,
6.46, 60.25, 59.72, 58.48, 55.53, 54.82, 53.31, 51.57, 50.85, 45.60, 41.47, 40.23, 39.64, 35.74, 29.19, 28.64, 27.20, 26.57,
–
+
3.96, 23.81, 23.05, 21.65, 20.34, 20.87, 18.25. HR-QTOF-MS m/z 797.33 [M – H] (C H N O ); m/z 821.43 [M + Na]
4
2 53 8 8
+
(
C H N O Na); m/z 1619.90 [2M + Na] (C H N O Na).
42 54 8 8 84 108 16 16
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