Total Synthesis of Cyclic Lipodepsipeptide Ophiotine

Article abstract of DOI:10.1007/s10600-020-03175-z

The first total synthesis of the natural nematicidal cyclic lipodepsipeptide ophiotine via a convergent strategy that involved both solid-phase peptide synthesis and liquid phase chemistry is reported. The pre-made dipeptide building block was synthesized

Full text of DOI:10.1007/s10600-020-03175-z

DOI 10.1007/s10600-020-03175-z
Chemistry of Natural Compounds, Vol. 56, No. 5, September, 2020
TOTAL SYNTHESIS OF CYCLIC LIPODEPSIPEPTIDE OPHIOTINE
1,2
3
3
3
Jing Wang, Chao Liu, Hong-Gang Hu, Yan Zou,
3
1,2*
Qin-Jie Zhao, and Guang-Ming Ye
The first total synthesis of the natural nematicidal cyclic lipodepsipeptide ophiotine via a convergent strategy
that involved both solid-phase peptide synthesis and liquid phase chemistry is reported. The pre-made
dipeptide building block was synthesized in the liquid phase, which was then assembled into the peptide
backbone through standard Fmoc chemistry on solid support. After cleavage from the resin, the linear
peptide was cyclized by the liquid phase macrocyclization and the side chain deprotection successively,
which led to the crude ophiotine. Finally, the crude product was purified by preparative reverse-phase high-
performance liquid chromatography, and its structure was confirmed by NMR and HR-ESI-MS.
Keywords: ophiotine, cyclic lipodepsipeptide, solid-phase peptide synthesis.
Cyclic peptides have attracted a great deal of attention during recent years because of their intriguing chemical
structures and potent biological activities. Owing to their better structural stability in vivo compared with their linear counterparts,
they are considered as promising drug candidates [1]. Among normal cyclic peptides, natural cyclic lipopeptides, which are
found in natural organisms such as fungus bacteria, marine organisms containing ester bonds are regarded as potential leading
compounds for novel antimicrobial agents [2–5]. It was reported that many cyclic lipopeptides such as polymyxin [6], daptomycin
[7], paenibacterin [8], pseudofactin [9], surfactin [10], iturin [11], and fengycin [12] showed great antimicrobial activity.
Ophiotine is a newly discovered natural nematicidal cyclic lipodepsipeptide isolated from Phaeosphariaceous fungus
that parasitize eggs of the plant parasitic cyst nematode Heterodera filipjevi. Ophiotine displays obvious nematicidal activity
at 100 μg/mL [13], which makes it a leading compound for novel nematicidal therapy. However, the content of ophiotine in
Phaeosphariaceous fungus was quite small; only 20.9 mg ophiotine can be obtained from 1 L of the seed culture by biological
extraction [13]. This extremely low extraction yield and time-consuming procedure can hardly meet the requirements of
further biological activity and structure–activity relationship studies. Therefore, the chemical total synthesis of ophiotine is
urgently needed.
In previous studies, the total synthesis of lipopeptide was achieved through total liquid phase chemistry [14, 15];
however, the synthetic procedures were complicated and time-consuming due to the repeated and tedious purification process,
which led to unsatisfactory chemical yields. Herein we planned to obtain the cyclic lipodepsipeptide via a more efficient
solid-phase synthesis/liquid-phase macrocyclization strategy.
Our work began with the retrosynthetic analysis. The macrocyclization point was identified between the non-chiral
β-Ala and Thr residue according to the reported article [16]. Considering the racemization problem during the ester bond
formation with a strong coupling reagent, the dipeptide building block 1 was identified by disconnecting the linear peptide by
a liquid method [17].
At the beginning of our study, the dipeptide building block 1 was synthesized as shown in Scheme 1. Initially, the free
amino acid D-phenylalanine (2) was protected with allyl chloroformate (Alloc-Cl) to afford 3 with a good yield of 74%.
Then, Boc-L-Thr(tBu)-OH (4) was readily coupled with 3 through an esterification reaction promoted by
N,N-dicyclohexylcarbodiimide (DCC) and catalytic 4-dimethylaminopyridine (DMAP) to provide 5 in 84% yield.
1) Clinical College of No. 98 Hospital of PLA, Anhui Medical University, 313000, HuXhou, P. R. China, e-mail:
shygm98@163.com; wangjing_000_jyxk@163.com; 2) WuXi Branch of Ruijin Hospital, 214000, WuXi, P. R. China;
3) School of Pharmacy, Second Military Medical University, 200433, Shanghai, P. R. China. Published in Khimiya Prirodnykh
Soedinenii, No. 5, September–October, 2020, pp. 756–759. Original article submitted August 26, 2019.
0009-3130/20/5605-0883 ©2020 Springer Science+Business Media, LLC
883

9
9
12
a
12
7
O
7
O
O
3
1
O
O
1
O
5
O
3
5
O
N
H
O
N
H
O
N
H
H N
2
23
15
15
O
OH
26
23
c
O
OH
O
O
25
24
14
3
21
b
HO
17
O
18
25
N
O
N
H
O
20
32
31
2
HO
17
18
H
O
27
O
O
21
O
N
H
O
1
5
29
O
4
a. Alloc-Cl, Na CO , H O–MeCN, 0°C to r.t., overnight; b. DMAP, DCC, DCM, r.t., 4 h; c. i) TFA–DCM 1:3, Pd(PPh ) –PhSiH , r.t.,
2
3
2
3 4
3
2 h; ii) FmocOSu, NaHCO , H O–1,4-dioxane, 0°C to r.t., overnight.
3
2
Scheme 1. Synthesis route of dipeptide building block 1.
O
O
R
AllocHN
d
b
a
O
O
O
O
Cl
O
O
SPPS
SPPS
N
H
NHTrt
NHFmoc
HN
O
7, 8
6
c
7
8
t
O Bu
7
3
R
O
O
27
O
O
29
1
N
H
N
9
O
R
Boc
H
1
f
O
NH
NH
O
O
O
NH
N
N
19
O
O
O
O
26
H
N
20
33
11
R
35
31
N
H
NHTrt
NH
N
H
NHTrt
13
25
17
36
45
HN
HN
O
15
(CH ) CH
2 7
3
O
O
23
O
O
H N
2
9, 10
11, 12
g
e
9: R =
12
9
10
11
7: R = AllocHN; 8: R = NH ; 10: R = OH; 11: R = OtBu, R = Boc; 12: R = OH, R = H
2
1
1
a. Dipeptide building block 1, DIPEA, DCM–DMF, r.t., 2 h; b. Fmoc-D-Gln(Trt)-OH, decanoic acid, 20% piperidine–DMF, HOBT, DCC,
DMF, DCM, r.t., 2 h; c. Pd(PPh ) , PhSiH , DCM, DMF, r.t., 0.5 h; d. Fmoc-L-Ser(tBu)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-β-Ala-OH,
3 4
3
HOBT, DCC, DMF, DCM, 20% piperidine–DMF, r.t., 2 h; e. TFE–DCM 1:4, r.t., 2 h; f. PyAOP, HOAt, NMM, DCM, DMF, r.t., overnight;
g. TFA–DCM 1:3, r.t., 2 h.
Scheme 2. Synthesis route of ophiotine (12).
Subsequently, the t-butyloxy carbonyl (Boc) and allyloxycarbonyl (Alloc) groups of 5 were removed using trifluoroacetic
acid (TFA)–dichloromethane (DCM) 1:3 and Pd(PPh ) /PhSiH , respectively; then the Fmoc group was capped on the
3 4
3
amino terminal to provide 1 via N-(9-fluorenylmethoxycarbonyloxy)succinimide (FmocOSu) and NaHCO with 70% yield.
3
With the dipeptide building block 1 in hand, we synthesized ophiotine through a solid-phase synthesis/liquid-phase
macrocyclization strategy as shown in Scheme 2. First of all, the dipeptide building block 1 was coupled with the
2-chlorotritylchloride resin in the treatment of DIPEA for 2 h to afford compound 6. The peptide backbone was elongated
with Fmoc-D-Gln(Trt)-OH and decanoic acid via standard Fmoc SPPS to provide compound 7. Then the Alloc protective
group was successfully cleaved by Pd (PPh ) /PhSiH in DCM/DMF solution on the solid phase to obtain compound 8.
3 4
3
The on-resin linear hexapeptide 9 was obtained after the coupling of Fmoc-L-Ser(tBu)-OH, Fmoc-D-Trp(Boc)-OH, and
Fmoc-β-Ala-OH via the standard Fmoc SPPS. The side chain protected linear lipodepsipeptide 10 was finally
884

obtained from the cleaved 1:4 cocktail tetrafluoroethylene (TFE)–DCM. Lastly, the liquid-phase macrocyclization
with (7-azabenzotriazol-1-yloxy)tripyrrolidino-phosphonium-hexafluorophosphate (PyAOP)/3H-[1,2,3]-triazolo[4,
5-b]pyridin-3-ol (HOAt)/N-methylmorpholine (NMM) and the subsequent side chain deprotection by 1:3 TFA–DCM afford
crude ophiotine (12).
Next, the crude compound was analyzed and purified by analytic and preparative RP-HPLC using water and
acetonitrile as the mobile phase. The crude product can be easily purified to 93.4% purity, and the resulting total yield was
39% (based on initial resin load). The molecular weight was confirmed using HR-ESI-MS and found to be identical to the
1
13
theoretical molecular mass. Furthermore, the purified product was also confirmed by H NMR and C NMR.
In summary, we have successfully achieved the first total synthesis of the natural cyclic lipodepsipeptide ophiotine,
which may serve as a promising compound in the development of antibiotics, via a solid/liquid-phase synthesis strategy with
satisfactory chemical yield and purity. Our effort can not only promote pharmacological activity studies of the lipodepsipeptide
ophiotine, but also provide a generic method for the synthesis of other lipodepsipeptides both in laboratory research and
industrial production.
EXPERIMENTAL
1
13
General. H NMR and C NMR spectra were obtained on a Bruker Avance 600 MHz NMR spectrometer.
The chemical shifts of protons are given on the δ scale, ppm, with tetramethylsilane (TMS) as internal standard. HR-ESI-MS
was measured on an Agilent 6538 UHD Accurate Mass Q-TOF LC/MS mass spectrometer. Purification of crude peptides was
obtained using preparative RP-HPLC (LC-20AD, Shimadzu) with dual wavelength (214 and 254 nm). All chemical reagents
were purchased from Adamas-Beta and Sinopharm Chemical Reagent Co., Ltd.
((Allyloxy)carbonyl)-D-phenylalanine (3). Compound 2 (5 g, 0.03 mol) and Na CO (3.18 g, 0.03 mol) were
2
3
dissolved in water (25 mL) and acetonitrile (15 mL); then Alloc-Cl (3.04 mL, 0.03 mol) was added at 0°C. The mixture was
stirred overnight at room temperature. After the reaction was completed, the acetonitrile was removed by rotary evaporation.
The mixture was acidified with HCl to pH and extracted with ethyl acetate twice. The organic phase was collected, dried over
anhydrous sodium sulfate, concentrated, and purified through column chromatography over silica gel. Compound 3 was
1
obtained as a viscous oil (5.5 g, 74%). H NMR (600 MHz, CDCl , δ, ppm, J/Hz): 9.49 (1H, s), 7.35–7.32 (2H, m), 7.30–7.27
3
(1H, m), 7.24–7.23 (2H, m), 5.92–5.82 (1H, m), 5.55 (1H, d, J = 6), 5.32–5.23 (2H, m), 4.75–4.71 (1H, m), 4.59–4.56 (2H, m),
13
3.27–3.24 (1H, m), 3.15–2.98 (1H, m). C NMR (150 MHz, CDCl , δ, ppm): 175.62 (C, C-6), 156.10 (C, C-4), 135.81 (C, C-8),
3
132.47 (CH, C-2), 129.40 (CH, C-10, 12), 128.66 (CH, C-9, 13), 127.18 (CH, C-11), 118.14 (CH , C-1), 66.10 (CH , C-3), 54.70
2
2
+
(CH, C-5), 37.63 (CH , C-7). HR-ESI-MS m/z 250.1097 [M + H] (calcd for C H NO , 249.1001).
2
13 15
4
Tert-Butyl-O-(((allyloxy)carbonyl)-D-phenylalanyl)-N-(tert-butoxycarbonyl)-L-threoninate (5). Compound 3
(1 g, 4 mmol), Boc-Thr(tBu)-OH (4, 1.1 g, 4 mmol), DCC (827 mg, 4 mmol), and DMAP (49 mg, 0.4 mmol) were dissolved
in DCM (20 mL). The mixture was stirred for 4 h at room temperature. After the reaction was completed, the mixture was
washed with 1 M HCl solution. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and
1
purified through column chromatography over silica gel. Compound 5 was obtained as a viscous oil (1.7 g, 84%). H NMR
(600 MHz, CDCl , δ, ppm): 7.21–7.19 (1H, m), 7.15–7.11 (3H, m), 5.58–5.75 (1H, m), 5.59–5.58 (1H, m), 5.35–5.23 (2H, m),
3
5.17–5.09 (1H, m), 5.09–5.07 (1H, m), 4.52–4.51 (1H, m), 4.43–4.42 (2H, m), 4.28–4.27 (1H, m), 1.39 (9H, s), 1.38 (9H, s),
13
1.18–1.17 (3H, m). C NMR (150 MHz, CDCl , δ, ppm): 170.48 (C, C-6), 168.84 (C, C-17), 155.81 (C, C-4), 155.62 (C,
3
C-22), 135.90 (C, C-8), 132.49 (CH, C-2), 129.16 (CH, C-10, 12), 128.46 (CH, C-9, 13), 126.92 (CH, C-11), 117.57 (CH ,
2
C-1), 82.40 (C, C-18), 79.81 (C, C-23), 71.97 (CH, C-14), 65.60 (CH , C-3), 57.49 (CH, C-16), 55.01 (CH, C-5), 37.96
2
(CH , C-7), 28.20 (CH , C-19, C-20, 21), 27.81 (CH , C-24, 25, 26), 16.55 (CH , C-15). HR-ESI-MS m/z 507.2520
2
3
3
3
+
[M + H] (calcd for C H N O , 506.2628).
26 38
2 8
N-(((9H-Fluoren-9-yl)methoxy)carbonyl)-O-(((allyloxy)carbonyl)-D-phenylalanyl)-L-threonine (1). Compound 5
(1.7 g, 3.36 mmol) was dissolved in DCM–TFA solution (20 mL, 3:1) and stirred at room temperature for 2 h. After the
reaction was completed, the mixture was removed by rotary evaporation to obtained a crude mixture, which was used directly
in the next step without purification. The crude mixture (1.1 g, 2.76 mmol) and NaHCO (465 mg, 5.53 mmol) were stirred in
3
water (20 mL) at room temperature. FmocOSu (1.86 g, 5.53 mmol) in 1,4-dioxane solution (10 mL) was added at 0°C. The
reaction mixture was stirred overnight at room temperature. After the reaction was completed, the 1,4-dioxane was removed
by rotary evaporation, and the mixture was acidified with HCl to pH 2 and extracted with ethyl acetate. The organic phase was
885

washed with brine, dried over anhydrous sodium sulfate, concentrated, and purified through column chromatography over
1
silica gel. Compound 1 was obtained as white solid powder (1.1 g, 70% in two steps). H NMR (600 MHz, DMSO-d , δ, ppm,
6
J/Hz): 7.92 (2H, d, J = 6), 7.81–7.77 (3H, m), 7.71–7.70 (1H, m), 7.45–7.43 (2H, m), 7.36–7.32 (3H, m), 7.31–7.29 (4H, m),
7.24–7.22 (1H, m), 5.88–5.83 (1H, m), 5.36–5.34 (1H, m), 5.25–5.22 (1H, m), 5.16–5.14 (1H, m), 4.44–4.42 (2H, m),
13
4.41–4.35 (3H, m), 4.32–4.27 (2H, m), 3.15–3.12 (3H, m), 2.79–2.75 (1H, m), 1.23–1.22 (3H, m). C NMR (150 MHz,
DMSO-d , δ, ppm): 171.49 (C, C-17), 171.38 (C, C-6), 157.21 (C, C-4), 156.32 (C, C-18), 144.31 (C, C-21, 32), 144.19
6
(C, C-26), 141.23 (C, C-27), 138.11 (C, C-8), 133.87 (CH, C-2), 129.56 (CH, C-10, 12), 128.64 (CH, C-9, 13), 128.14 (CH,
C-11, 25, 28), 127.56 (CH, C-30), 126.94 (CH, C-23), 125.75 (CH, C-22, 24), 120.58 (CH, C-31, 29), 117.51 (CH , C-1),
2
71.39 (CH, C-14), 66.39 (CH , C-19), 64.96 (CH , C-3), 57.96 (CH, C-16), 56.02 (CH, C-5), 47.20 (CH, C-20), 36.86 (CH ,
2
2
2
+
C-7), 17.08 (CH , C-15). HR-ESI-MS m/z 573.2229 [M + H] (calcd for C H N O , 572.2159).
3
32 32 2 8
General Procedures for the Fmoc Solid Phase Peptide Synthesis. The amino acid residues were attached to the
resin via a single coupling procedure (120 min at 35°C). All peptides were synthesized with a scale of 0.1 mmol.
a) The 2-chlorotritylchloride resin (loading capacity 0.93 mmol/g) was swollen in DCM–DMF solvent mixture for
10 min.
b) After treatment with 20% piperidine–DMF for 15 min, the resin was washed (5 × DMF, 5 × DCM, 5 × DMF).
c) After pre-activation of 3.0 equiv of Fmoc-protected amino acid/dipeptide building block–decanoic acid in DMF
for 15 min using 3.0 equiv. of BtOH and 9 equiv of DCC, the solution was added to the resin. After 120 min, the resin was
washed (5 × DMF, 5 × DCM, 5 × DMF). The coupling reaction was monitored using the ninhydrin test.
d) To the peptide resin was added a solution of PhSiH (24 equiv.) in 2 mL of DCM in the presence of argon, and the
3
resin was manually stirred for 2 min. Subsequently, a solution of Pd(PPh ) (0.25 equiv.) in 6 mL of DCM was added.
3 4
The reaction was mechanically stirred for 30 min under argon. Then the resin was washed (5 × DMF, 5 × DCM, 5 × DMF).
e) The cleavage cocktail TFE–DCM (1:4) was added to the resin at room temperature. After stirring for 2 h, the
cleavage cocktail was collected and concentrated.
f) A solution of PyAop (5 equiv.), HOAt (5 equiv.), and NMM (10 equiv.) in DCM–DMF solution was added to the
peptide DMF solution. After overnight reaction, the solution was concentrated.
g) The concentrated mixture was dissolved in MeOH and purified on a Sephadex LH-20 gel column. After concentration,
the resulting white residue was dissolved in CH CN–water and analyzed by HPLC and HR-ESI-MS.
3
1
(R)-N -((3R,6S,9R,16S,17R)-9-((1H-Indol-3-yl)methyl)-3-benzyl-6-(hydroxymethyl)-17-methyl-2,5,8,11,15-
pentaoxo-1-oxa-4,7,10,14-tetraazacycloheptadecan-16-yl)-2-decanamidopentanediamide, Ophiotine (12). White
1
lyophilized powder (34.1 mg, 39% yield, 93.4% purity). H NMR (600 MHz, DMSO-d , δ, ppm, J/Hz): 10.76 (1H, s),
6
8.15–8.12 (2H, m), 7.98 (1H, d, J = 6), 7.94 (1H, d, J = 6), 7.17–7.65 (1H, m), 7.61 (1H, d, J = 6), 7.32 (1H, d, J = 6), 7.27–7.25
(2H, m), 7.24–7.23 (2H, m), 7.16–7.11 (3H, m), 7.05–7.04 (1H, m), 6.96–6.94 (1H, m), 5.58 (3H, m), 4.62–4.57 (2H, m),
4.33–4.19 (3H, m), 4.05–4.03 (1H, m), 3.62–3.52 (2H, m), 3.16–3.09 (4H, m), 2.92–2.89 (2H, m), 2.71–2.67 (1H, m),
2.52–2.51 (1H, m), 2.27–2.17 (4H, m), 2.00–1.98 (3H, m), 1.81–1.78 (1H, m), 1.45–1.43 (2H, m), 1.23 (8H, s), 1.06–1.02
13
(5H, m), 0.87–0.85 (4H, m). C NMR (150 MHz, DMSO-d , δ, ppm): 175.13 (C, C-31), 173.68 (C, C-9), 172.61 (C, C-36),
6
172.29 (C, C-15), 171.53 (C, C-35), 171.02 (C, C-26), 170.55 (C, C-29), 170.12 (C, C-12), 138.17 (C, C-3, 20), 136.51 (CH,
C-5), 129.69 (CH, C-7), 128.46 (CH, C-4), 127.82 (CH, C-8), 126.64 (C, C-6, 25), 123.99 (CH, C-19), 121.23 (CH, C-22),
118.95 (CH, C-23), 118.60 (CH, C-24), 111.68 (CH, C-21), 110.71 (C, C-18), 67.01 (CH, C-10), 62.19 (CH, C-16), 58.72
(CH, C-28), 55.64 (CH , C-27), 54.68 (CH, C-11), 53.91 (CH, C-32), 52.63 (CH, C-1), 45.96 (CH , C-2), 35.88 (CH , C-13),
2
2
2
35.80 (CH , C-37), 35.60 (CH , C-14), 31.73 (CH , C-34), 29.36 (CH , C-43), 29.27 (CH , C-41, 42), 29.14 (CH , C-39, 40),
2
2
2
2
2
2
27.84 (CH , C-17), 25.68 (CH , C-33), 22.55 (CH , C-38), 19.98 (CH , C-44), 14.40 (CH , C-30), 18.81 (CH , C-45).
2
2
2
2
3
3
+
HR-ESI-MS m/z 875.4687 [M + H] (calcd for C H N O , 874.4589).
45 62
8 10
ACKNOWLEDGMENT
We are grateful to the National Natural Science Foundation of China (21402235) and the Instrumental Analysis
Center of our university for NMR spectroscopic and mass spectrometric analysis.
886

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