Ahmpatinin iBu, a new HIV-1 protease inhibitor, from Streptomyces sp. CPCC 202950?

Article abstract of DOI:10.1039/c7ra13241g

Ahmpatinin ⁱBu (1) and statinin ⁱBu (2), two new linear peptides, a novel pyrrolidine derivative, ()-(S)-2-[3-(6-methylheptanamido)-2-oxopyrrolidin-1-yl] acetic acid (3), and three known pepstatin derivatives (4–6) along with their corresponding methanolysis artifacts (7–9) were isolated from Streptomyces sp. CPCC 202950. Their structures were elucidated on the basis of extensive spectroscopic data using Marfey's analysis, chiral-phase HPLC, and ECD and OR calculation to determine the absolute configurations. Compound 1 contains an unusual amino acid, 4-amino-3-hydroxy-5-(4-methoxyphenyl)pentanoic acid (Ahmppa), and 3 is the first natural product with a 2-(3-amino-2-oxopyrrolidin-1-yl)acetic acid system. Compounds 1, 2, and 4–9 are HIV-1 protease inhibitors. In particular, ahmpatinin ⁱBu (1) exhibits significant inhibitory activity against HIV-1 protease with an IC₅₀ value of 1.79 nM. A preliminary structure–activity relationship is discussed.

Full text of DOI:10.1039/c7ra13241g

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i
Ahmpatinin Bu, a new HIV-1 protease inhibitor,
from Streptomyces sp. CPCC 202950†
Cite this: RSC Adv., 2018, 8, 5138
ac
Ming-Hua Chen,
‡
Shan-Shan Chang,‡ab Biao Dong,a Li-Yan Yu,a Ye-Xiang Wu,a
a
a
b
a
*
*
Ren-Zhong Wang, Wei Jiang, Zeng-Ping Gao and Shu-Yi Si
Ahmpatinin iBu (1) and statinin iBu (2), two new linear peptides, a novel pyrrolidine derivative, (ꢀ)-(S)-2-[3-
(6-methylheptanamido)-2-oxopyrrolidin-1-yl] acetic acid (3), and three known pepstatin derivatives (4–6)
along with their corresponding methanolysis artifacts (7–9) were isolated from Streptomyces sp. CPCC
202950. Their structures were elucidated on the basis of extensive spectroscopic data using Marfey's
analysis, chiral-phase HPLC, and ECD and OR calculation to determine the absolute configurations.
Compound 1 contains an unusual amino acid, 4-amino-3-hydroxy-5-(4-methoxyphenyl)pentanoic acid
(Ahmppa), and 3 is the first natural product with a 2-(3-amino-2-oxopyrrolidin-1-yl)acetic acid system.
Compounds 1, 2, and 4–9 are HIV-1 protease inhibitors. In particular, ahmpatinin iBu (1) exhibits
significant inhibitory activity against HIV-1 protease with an IC50 value of 1.79 nM. A preliminary
structure–activity relationship is discussed.
Received 11th December 2017
Accepted 4th January 2018
DOI: 10.1039/c7ra13241g
rsc.li/rsc-advances
In our previous report, a new HIV-1 protease inhibitor named
4862F15 and 11 known compounds16 were isolated from the
Introduction
liquid fermentation of the strain Streptomyces sp. CPCC 202950.
Unexpectedly, the crude extract of the rice culture of Strepto-
myces sp. CPCC 202950 also signicantly inhibited HIV-1
protease activity with an IC50 value of 20.0 ng mlꢀ1. Extensive
investigation of the secondary metabolites of the rice culture
extract of the strain resulted in the isolation of two new linear
peptides, ahmpatinin iBu (1) and statinin iBu (2), a new
pyrrolidine derivative, (ꢀ)-(S)-2-[3-(6-methylheptanamido)-2-
oxopyrrolidin-1-yl]acetic acid (3), and three known pepstatin
derivatives along with their corresponding methanolysis arti-
facts (4–9) (Fig. 1). Among them, compound 1 contains an
unusual amino acid, 4-amino-3-hydroxy-5-(4-methoxyphenyl)
pentanoic acid. Compound 3 represents the rst natural
The most effective treatment for HIV infection, HAART (Highly
Active AntiRetroviral Therapy) that including several HIV-1
protease inhibitors combination with reverse transcriptase
inhibitors, and other inhibitors with integrase, membrane
fusion, and viral attachment, have signicantly reduced the
mortality of AIDS patients and improved the quality of life of
those infected with HIV.1–4 However, according to World
Health Organization and Joint United Nations Program on
HIV/AIDS data, in 2016, there were still 36.7 million people
living with HIV, 1.8 million newly infected and 1.0 million
HIV-related deaths.5,6 Therefore, AIDS is still a substantial
threat to global public health. HIV-1 protease (HIV-1 PR) is an
essential enzyme in the life cycle of HIV and has been used as
a promising target for AIDS therapy.7–9 Although 11 HIV-1 PR
inhibitors such as amprenavir, indinavir, nelnavir, ritonavir,
saquinavir, and tipranavir have been approved by the FDA and
applied extensively in clinical evaluations,10,11 drug toxicity,
resistance, and drug–drug interactions remain serious prob-
lems for HIV/AIDS treatments,1,12–14 prompting us to discover
new anti-HIV drugs.
aInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Peking
Union Medical College, Beijing 100050, China. E-mail: sisyimb@hotmail.com
bSchool of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing
100102, China. E-mail: gaozp@bucm.edu.cn
cKey Laboratory for Uighur Medicine, Institute of Materia Medica of Xinjiang, Urumqi
830004, China
† Electronic supplementary information (ESI) available: 1D, 2D NMR, HRESIMS
and IR spectra for compounds 1–3. See DOI: 10.1039/c7ra13241g
‡ These authors have contributed equally to this work.
Fig. 1 Chemical structures of compounds 1–9.
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product with
a
2-(3-amino-2-oxopyrrolidin-1-yl)acetic acid spectrum of 1 (Table 1) in DMSO-d6 exhibited characteristics of
system, which was synthesized in a previous study.17 Herein, we a typical peptide, with ve NH protons (dH 7.4–7.9) and ve
report the isolation, structural elucidation, and biological amino acid a-protons (dH 3.8–4.3). Furthermore, para-disubsti-
activities of these compounds.
tuted aromatic ring protons [dH 6.80 (2H, d, H-3 and H-5) and
7.12 (2H, d, H-2 and H-6)], two oxygenated methine protons (dH
3.82 and 3.85), four methylene protons, four methine protons,
and a methoxyl singlet [dH (3.70, s, 3H)] along with nine addi-
tional methyl doublets (dH 0.7–1.2) were also observed in the 1H
NMR spectrum. Six carbonyl signals (dC 170–176) and ve a-
amino acid carbon signals (dC 48–58) were observed in the 13C
Results and discussion
i
Ahmpatinin Bu (1) was isolated as a white powder and was
determined to be C37H61N5O10 on the basis of positive HRE-
SIMS, indicating 10 degrees of unsaturation. The 1H NMR
Table 1 NMR spectroscopic data of 1 and 2a
1
2
No.
dH
dC, type
dH
dC, type
Val1
Val2
Sta
CO
a
171.1, C
171.0, C
4.14, dd (7.8, 8.4)
1.96, m
0.81, d(6.6)
0.81, d(6.6)
7.79, d(8.4)
57.7, CH
30.1, CH
18.4, CH3
19.8, CH3
4.14, ddb
1.94, m
0.81, d(6.6)
0.81, d(6.6)
7.78, d(9)
57.7, CH
30.1, CH
18.2, CH3
19.8, CH3
b
g
g0
NH
CO
a
170.6, C
170.6, C
4.14, dd(7.8, 8.4)
1.96, m
0.81, d(6.6)
0.83, d(6.6)
7.71, d(9)
57.8, CH
30.4, CH
18.2, CH3
19.4, CH3
4.14, ddb
1.94, m
0.81, d(6.6)
0.83, d(6.6)
7.68, d(9)
57.8, CH
30.5, CH
18.1, CH3
19.4, CH3
b
g
g0
NH
CO
a
170.7, C
170.8, C
2.11, m
3.82, m
3.82, m
1.34, m
39.2, CH2
69.0, CH
50.7, CH
38.7, CH2
2.10, m
3.81, m
3.85, m
1.32, m
39.5, CH2
69.0, CH
50.7, CH
38.5, CH2
b
g
d
1.25, m
1.25, m
3
1.52, m
24.2, CH
21.6, CH3
23.4, CH3
1.52, m
24.2, CH
21.6, CH3
23.5, CH3
z
z0
0.78, d(6.6)
0.83, d(6.6)
7.47, d(9)
0.78, d(6.6)
0.83, d(6.6)
7.50, d(9)
NH
CO
a
Ala
172.1, C
48.2, CH
18.0, CH3
174.2, C
47.4, CH
17.2, CH3
4.22, m
1.12, d(7.2)
7.85, d(7.2)
4.17, dqb
1.23, d(7.8)
8.07, d(7.2)
b
NH
CO
a
Ahmppa
172.8, C
38.8, CH2
2.23, dd(15.6, 3.6)
2.16, dd(15.6, 8.4)
3.85, m
3.83, m
2.75, dd(13.8, 6)
2.56, dd(13.8, 8.4)
b
g
d
67.2, CH
54.4, CH
35.4, CH2
1
130.9, C
2
3
4
7.12, d(8.4)
6.80, d(8.4)
130.0, CH
113.5, CH
157.5, C
5
6
6.80, d(8.4)
7.12, d(8.4)
7.53, d(9)
3.70, s
113.5, CH
130.0, CH
NH
OMe-4
CO
a
54.9, CH3
176.2, C
33.6, CH
19.2, CH3
19.3, CH3
Acyl
176.2, C
2.51, m
0.95, d(6.6)
0.98, d(6.6)
2.52, m
0.95, d(6.6)
0.98, d(6.6)
33.6, CH
19.2, CH3
19.3, CH3
b
b0
NMR data (d) were measured at 600 MHz for 1H and at 150 MHz for 13C in DMSO-d6. Proton coupling constants (J) in Hz are given in parentheses.
a
The assignments were based on H–1H COSY, HSQC, and HMBC experiments. The J-value was not determined due to overlapping signals.
1
b
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NMR spectrum, further indicating that 1 was a peptide. Analysis
of the DEPT and 13C NMR spectra revealed additional 1,4-dis-
ubstitued benzene ring groups (dC 113–158), ten methyl groups,
four methylenes, and six sp3 methines.
A comparison of the NMR spectra of compounds 1 and
i
ahpatinin Bu18 revealed that the mono-substituted benzene
moiety in ahpatinin iBu was replaced with a para-disubstituted
aromatic ring group in 1. This was conrmed by 2D NMR
(Fig. S6–S9†). HMBC correlations (Fig. 2) of H-4-OMe and C-4, d-
H-Ahmppa/C-1, C-2, C-6, C-b-Ahmppa and C-g-Ahmppa,
a-H-Ahmppa/C-b-Ahmppa, C-g-Ahmppa and C-CO-Ahmppa,
b-H-Ahmppa and C-CO-Ahmppa, and NH-Ahmppa and C-g-
Ahmppa indicated the presence of a 4-amino-3-hydroxy-5-(4-
methoxyphenyl)pentanoic acid (Ahmppa) residue. Integrated
2D NMR (1H NMR, COSY, HSQC, and HMBC) data were used to
assign ve partial structures consisting of the amino acids
alanine, two valines, statine (Sta), and an isobutyl (iBu) group.
HMBC correlations from a-H-Val1 (dH 4.14) and NH-Val1 (dH
7.79) to iBu-C]O (dC 176.2), a-H-Val2 (dH 4.14) to Val1-C]O (dC
171.1), g-H-Sta (dH 3.82) to Val2-C]O (dC 170.6), a-H-Ala (dH
4.22) to Sta-C]O (dC 170.7), and g-H-Ahmppa (dH 3.83) to Ala-
Fig. 3 The key NOESY correlations of 1 and 2.
S18†) revealed the sequence of iBu–Val1–Val2–Sta–Ala. The
absolute congurations of the amino acid residues and the
statine unit were also conrmed to be L-Ala, L-Val and (3S,4S)-
statine using the same protocol as 1. Hence, compound 2 was
deduced to be iBu-L-Val1-L-Val2-(3S,4S)-Sta-L-Ala and was named
i
statinin Bu.
Compound 3 was isolated as a white powder. Its molecular
formula C14H24N2O4, requiring four degrees of unsaturation,
was established from the HRESIMS data (m/z 285.1802 [M + H]+,
calcd for C14H25N2O4, 285.1808). The IR spectrum of 3 displayed
the presence of hydroxy and/or amino (3287 cmꢀ1), carboxyl
(1700 cmꢀ1) and amide (1645 cmꢀ1) functionalities. Analysis of
the 1H NMR, 13C NMR and HSQC spectra (Table 2) of 3 revealed
two doublet methyls, seven sp3 methylenes, two sp3 methines,
and three carbonyls (dC 170.0, 172.3, and 172.4). The 1H–1H
COSY cross-peaks of H2-200/H2-300/H2-400/H2-500/H-600/H3-700(H3-800)
and HMBC correlations of H3-700(H3-800)/C-600 and C-500; H-600/C-
400, C-500, C-700 and C-800; H2-500/C-300, C-400, C-600, C-700 and C-800; H2-
400/C-200, C-300, C-500 and C-600; H2-300/C-100, C-200, C-400 and C-500; and
H2-200/C-100, C-300 and C-400 indicated the presence of a 6-meth-
ylheptanoyl moiety in 3. Meanwhile, in conjunction with the
degrees of unsaturation and the molecular composition, 1H–1H
i
C]O (dC 172.1) established the sequence Bu–Val1–Val2–Sta–
Ala–Ahmppa, which was veried by NOESY correlations (Fig. 3
and S9†) from NH-Val1 (dH 7.79) to a-H-iBu (dH 2.51), NH-Val2
(dH 7.71) to a-H-Val1 (dH 4.14), NH-Sta (dH 7.47) to a-H-Val2
(dH 4.14), NH-Ala (dH 7.85) to a-H-Sta (dH 2.11), and NH-Ahmppa
(dH 7.53) and a-H-Ala (dH 4.22).
The absolute congurations of Ala and Val in 1 were deter-
mined to be L using Marfey's method (Fig. S27† and Experi-
mental section),19,20 whereas the Ahmppa residue was established
to be 3S, 4S by chiral-phase HPLC aer acid hydrolysis in
comparison to synthetic standards (Fig. S28† and Experimental
section). In addition, the acid hydrolysis of 1 followed by HPLC
1
purication gave statine, and its H NMR data agreed with the
Table 2 NMR spectroscopic data of 3a
synthetic standard of (3S,4S)-statine. Meanwhile, the specic
rotation {[a]2D0 ¼ ꢀ19.5 (c 0.05, H2O)} of the statine from the
hydrolysis of 1 was consistent with that of (3S,4S)-statine {[a]D20
¼
3
ꢀ20.4 (c 0.502, H2O)}.21 Therefore, the nal structure of 1 was
determined to be iBu-L-Val1-L-Val2-(3S,4S)-Sta-L-Ala-(3S,4S)-
Ahmppa and was named ahmpatinin iBu.
Compound 2 was obtained as a white amorphous powder,
and the molecular formula was assigned as C25H46N4O7 by
HRESIMS based on a [M ꢀ H]ꢀ ion at 513.3275 (calc. 513.3283).
The MS data for 2 indicated the absence of the Ahmppa moiety
relative to compound 1. Extensive analysis of 2D NMR (1H–1H
COSY, HSQC, HMBC, and NOESY) spectroscopic data (Fig. S15–
No.
dH
dC, type
1
2
170.0, C
44.1, CH2
3.99, d(17.4)
3.89, d(17.4)
20
30
40
172.4, C
49.5, CH
26.2, CH2
4.41, m
2.29, m
1.76, m
3.33, m
5000
100
2
43.8, CH2
172.3, C
2.10, t(7.2)
1.47, m
1.25, m
1.14, m
1.51, m
0.84, d(6.6)
0.84, d(6.6)
8.13, d(8.4)
35.3, CH2
25.5, CH2
26.4, CH2
38.2, CH2
27.3, CH
22.5, CH3
22.5, CH3
300
4000
500
600
7
800
NH
a
1
NMR data (d) were measured at 600 MHz for H and at 150 MHz for
13C in DMSO-d6. Proton coupling constants (J) in Hz are given in
parentheses. The assignments were based on 1H–1H COSY, HSQC,
and HMBC experiments.
Fig. 2 The key 1H–1H COSY and HMBC correlations of 1–3.
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COSY correlations of NH/H-30/H-40/H-50 along with HMBC
correlations of NH/C-20, C-30 and C-40; H-30/C-20 and C-40; H2-40/
C-20, C-30 and C-50; H2-50/C-20, C-30 and C-40; and H2-2/C-1, C-20
Table 3 Inhibitory activities of 1–9 against HIV-1 protease
Compd
IC50 (nM)
Compd
IC50 (nM)
and C-50 demonstrated the presence of
a 2-(3-amino-2-
1
2
3
4
5
1.79
6
7
8
9
9.90
203.30
138.60
782.10
1.82
oxopyrrolidin-1-yl)acetic acid moiety.17 Finally, the HMBC
correlation from H-30 and NH to C-100 suggested that the
two moieties were connected via an amide bond. Thus,
compound 3 was conrmed to be 2-(3-(6-methylheptanamido)-
2-oxopyrrolidin-1-yl)acetic acid. The absolute conguration at
C-30 was determined by comparing the experimental circular
dichroism (CD) spectrum and optical rotation (OR) data with
the computed ECD and OR data. A comparison of the experi-
mental CD spectrum with the calculated ECD spectrum of 3
(Fig. 4) demonstrated that compound 3 possessed an S cong-
uration, which was veried by the computed OR, which was
ꢀ41.5 for (S)-3. The experimental OR value was ꢀ100.5 in
acetonitrile. Therefore, compound 3 was elucidated to be
(ꢀ)-(S)-2-[3-(6-methylheptanamido)-2-oxopyrrolidin-1-yl]acetic
acid.
3190.0
>10 000
92.10
22.27
Indinavir
showed weaker inhibitory ability than 1 and 4–9, suggesting
that the presence of the statine or Ahmppa unit could enhance
the HIV-1 protease inhibitory activity. In addition, all of the
compounds were not active at toward Hela, HepG2, and U2OS
tumor cells at 100 mM in in vitro tests.
Experimental
General experimental methods
ORs were measured using a Rudolph Research Autopol III
automatic polarimeter. UV, CD, and IR spectra were recorded
using a Cary 300 spectrometer, a JASCO J-815 CD spectrometer,
and a Nicolet 5700 FT-IR spectrometer (FT-IR microscope
In addition to compounds 1–3, the known pepstatin Ac
(4),18,22 pepstatin Pr (5),18,22 pepsinostreptin (6),18 pepstatin Ac
methyl ester (7), pepstatin Pr methyl ester (8), and pepsin-
ostreptin methyl ester (9) were also isolated and identied from
the strain Streptomyces sp. CPCC 202950. Compounds 4–6
incubated with methanol showed the presence of correspond-
ing methyl esters 7–9, respectively. These results indicated that
7–9 were indeed artifacts derived from methanolysis during the
isolation process. Furthermore, a new benzamide analog and
seven known compounds were isolated from the inactive frac-
tions of the crude extract in our previous report.23
Pepstatin and ahpatinin derivatives have been shown to
exhibit signicant inhibitory activity against aspartic
protease.18,24–28 HIV-1 PR is a 22 kDa dimeric aspartyl protease.
As expected, compounds 1 and 2 along with pepstatin deriva-
tives 4–9 from the active fractions showed signicant inhibitory
activity against HIV-1 protease with IC50 values of 1.79 nM to
3.19 mM, while compound 3 was inactive (the positive control
indinavir gave an IC50 value of 1.82 nM) in preliminary in vitro
assays (Table 3). A comparison of the activities of 4–6 and 7–9
demonstrated that the exposed terminal carboxyl group was
very important for the HIV-1 protease inhibitory activity. Anal-
ysis of the inhibitory abilities of 1 and 4–9 indicated that the
1
transmission), respectively. H and 13C NMR spectra were ob-
tained at 600 and 150 MHz, respectively, using a Bruker-
AVIIIHD-600 spectrometer with solvent peaks used as refer-
ences. HR-ESIMS data were measured using a Micromass
Autospec-Ultima ETOF spectrometer and Thermo LTQ Orbitrap
XL mass spectrometer. Column chromatography was per-
formed using silica gel (200–300 mesh, Qingdao Marine
Chemical Inc., China) and MCI gel CHP20P (Mitsubishi
Chemical Corporation, Tokyo, Japan). HPLC separation was
performed using an Agilent 1200 series (quaternary pump,
autosampler, diode array detector) with a Shiseido Capcell-Pak
C18 MGII column (5 mm, 250 ꢁ 10 mm). TLC was performed
using glass-precoated silica gel GF254 plates (Qingdao Marine
Chemical Inc., Qingdao, China). Cervical cancer (Hela), hepa-
tocellular carcinoma (HepG2), and human osteosarcoma
(U2OS) cell lines were obtained from the National Infrastructure
of Cell Line Resource (Beijing, China).
Microorganism and fermentation
Ahmppa unit was better than the statine moiety. Compound 2 Strain CPCC 202950 was identied as a member of the genus
Streptomyces on the basis of 16S rRNA sequence analysis and
deposited at the China Pharmaceutical Culture Collection
(Institute of Medicinal Biotechnology, Chinese Academy of
Medical Sciences and Peking Union Medical College, no. CPCC
202950).
The strain was cultured on slants of YM (0.4% yeast extract,
1% malt extract, 0.4% glucose, and 1.2% agar) at 28 ꢂC for 7 d.
Agar plugs were cut into small pieces (about 0.5 ꢁ 0.5 ꢁ
0.5 cm3) under aseptic conditions and 5 of these pieces were
used to inoculate in three Erlenmeyer asks (500 ml), each
containing 100 ml of media (glucose 0.5%, yeast extract 0.5%,
soluble starch 2.0%, soybean meal 1%, peptone 0.5%, beef
extract 0.5%, corn steep liquor 0.4%, CaCO3 0.4%, and CoCl2-
$6H2O 0.002%), and the nal pH was adjusted to 7.2. Aer
Fig. 4 Experimental ECD spectrum of 3 (blue) and the calculated ECD
spectra of (R)-3 (red) and (S)-3 (black).
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Statine.18 The (3S,4S)- and (3R,4S)-isomers of statine were
sterilization, three asks of the inoculated media were incu-
bated at 28 ꢂC on a rotary shaker at 220 rpm for two days to
prepare the seed culture. Spore inoculum was prepared by
suspending the seed culture in sterile, distilled H2O to give
a nal spore/cell suspension of 1 ꢁ 106/ml. Fermentation was
carried out in 30 Fernbach asks (500 ml), each containing 80 g
of rice. Distilled H2O (120 ml) was added to each ask, and the
contents were soaked overnight before autoclaving at 15 psi for
30 min. Aer cooling to room temperature, each ask was
inoculated with 10.0 ml of the spore inoculum and incubated at
prepared from Boc-L-Leu following the published procedure to
1
prepare the mixture of statine: H NMR (DMSO-d6, 600 MHz)
(3S,4S)-isomer (major product) d 0.87 (6H, d, J ¼ 6.6 Hz, z-CH3
and z0-CH3), 1.31 (1H, m, d-Ha), 1.45 (1H, m, d-Hb), 1.69 (1H, m,
3-H), 2.43 (1H, dd, J ¼ 8.4, 15.6 Hz, a-Ha), 2.55 (1H, dd, J ¼ 4.8,
15.6 Hz, a-Hb), 3.09 (1H, m, b-H), 3.93 (1H, m, g-H), 5.70 (1H,
br s, b-OH), 7.62 (2H, br s, g-NH2), 12.32 (1H, br s, –COOH);
(3R,4S)-isomer (minor product) d 0.84 (3H, d, J ¼ 6.6 Hz, z-CH3),
0.89 (3H, d, J ¼ 6.6 Hz, z0-CH3), 1.27 (1H, m, d-Ha), 1.40 (1H, m,
d-Hb), 1.64 (1H, m, 3-H), 2.29 (1H, dd, J ¼ 8.4, 15.6 Hz, a-Ha),
2.43 (1H, dd, J ¼ 4.8, 15.6 Hz, a-Hb), 3.13 (1H, m, b-H), 4.11 (1H,
m, g-H), 5.50 (1H, br s, b-OH), 7.74 (2H, br s, g-NH2), 12.32 (1H,
br s, –COOH).
ꢂ
28 C for 30 day.
Extraction and isolation
Ahmppa.18 The (3S,4S)- and (3R,4S)-isomers along with the
(3R,4R)- and (3S,4R)-isomers of Ahmppa were prepared from N-
Boc-4-methoxy-L-Tyr and N-Boc-4-methoxy-D-Tyr following the
procedure to prepare the mixture of statine isomers, respec-
tively. The (3S,4S)- and (3R,4S)-isomers and (3R,4R)- and (3S,4R)-
isomers of the Ahmppa mixture were subjected to reversed-
phase semipreparative HPLC (Capcell PAK C18-MGII 5 mm,
10 mm ꢁ 250 mm, 1.5 ml minꢀ1, 20% MeCN in 0.1% tri-
uoroacetic acid) to yield (3S,4S)- and (3R,4S)-Ahmppa and
The fermented material was ultrasonicated with 95% EtOH
(2 ꢁ 8.0 l ꢁ 40 min), and the EtOH extracts were combined and
evaporated under reduced pressure to yield an aqueous
suspension (2.0 l). The suspension was partitioned with EtOAc
(4 ꢁ 2.0 l). The EtOAc extract (15.0 g) was chromatographed over
a silica column using a gradient elution of increasing MeOH
(0–100%) in CH2Cl2 to give 10 fractions (F1–F10). The fraction of
F6 was also subjected to silica gel CC using a gradient elution of
increasing MeOH (0–100%) in CH2Cl2 to give six parts (F6-1
–
1
F6-6). Further purication of F6-1 and F6-3 with reversed-phase
(3R,4R)- and (3S,4R)-Ahmppa, respectively. H NMR (DMSO-d6,
semipreparative HPLC (Capcell PAK C18-MGII 5 mm, 10 mm ꢁ
250 mm, 1.5 ml minꢀ1, 37% MeCN in 0.1% triuoroacetic acid)
yielded 2 (13.5 mg) and 1 (8.6 mg), respectively. The aqueous
phase was chromatographed over MCI gel (CHP20P, 1 l) with
successive elution using H2O, 10% EtOH, 20% EtOH, 35%
EtOH, 50% EtOH, and 70% EtOH to afford fractions M1–M5. M4
was further separated by reversed-phase semipreparative HPLC
600 MHz) (3S,4S) and (3R,4R)-isomer (major product) d 2.46 (2H,
br d, J ¼ 6.0 Hz, a-H2), 2.81 (2H, br d, J ¼ 7.2 Hz, d-H2), 3.26 (1H,
m, b-H), 3.73 (3H, s, 4-OCH3), 3.87 (1H, m, g-H), 6.89 (2H, d, J ¼
8.4 Hz, H-2 and H-6), 7.20 (2H, d, J ¼ 8.4 Hz, H-3 and H-5), 5.78
(1H, br s, b-OH), 7.92 (2H, br s, g-NH2), 12.23 (1H, br s, –COOH);
(3R,4S) and (3S,4R)-isomer (minor product) d 2.29 (1H, dd, J ¼
9.0, 15.6 Hz, a-Ha), 2.54 (1H, dd, J ¼ 3.6, 15.6 Hz, a-Hb), 2.70
(1H, dd, J ¼ 7.8, 14.4 Hz, d-Ha), 2.80 (1H, dd, J ¼ 6.6, 14.4 Hz, d-
Hb), 3.38 (1H, m, b-H), 3.73 (3H, s, 4-OCH3), 4.10 (1H, m, g-H),
6.89 (2H, d, J ¼ 8.4 Hz, H-2 and H-6), 7.19 (2H, d, J ¼ 8.4 Hz, H-3
and H-5), 5.56 (1H, br s, b-OH), 7.80 (2H, br s, g-NH2), 12.31 (1H,
br s, –COOH).
(Capcell PAK C18-MGII 5 mm, 10 mm ꢁ 250 mm, 1.5 ml minꢀ1
,
35% MeCN in 0.1% triuoroacetic acid) to afford 3 (102.0 mg).
Physical–chemical properties of 1–3
Ahmpatinin iBu (1). White amorphous powder; [a]2D0 ꢀ75.0 (c
0.5, CH3CN : H2O 1 : 1); UV (MeOH) lmax (log 3) 223 (4.44), 275
(3.56) nm; IR nmax 3285, 2961, 2925, 2873, 2851, 1635, 1547,
(3S,4S)-Statine from acid hydrolysates of 1. Approximately
ꢂ
5.0 mg of 1 was hydrolyzed with 2 ml of 6 M HCl at 110 C for
16 h. The hydrolysate was then evaporated to dryness and
redissolved in H2O. The solution was chromatographed by
reversed-phase semipreparative HPLC (Capcell PAK C18-AQ 5
mm, 10 mm ꢁ 250 mm, 1.5 ml minꢀ1, 14% MeCN in 0.1% tri-
uoroacetic acid) to yield (3S,4S)-statine (0.5 mg): [a]2D0 ¼ ꢀ19.5
1
1515, 1468, 1390, 1249, 1183, 1148, 1099, 1038, 722 cmꢀ1; H
NMR (DMSO-d6, 600 MHz) data and 13C NMR (DMSO-d6, 150
MHz) data, see Table 1. (+)-HR-ESIMS m/z 736.4474 [M + H]+
(calcd for C37H62N5O10, 736.4439).
Statinin iBu (2). White amorphous powder; [a]2D0 ꢀ85.5 (c 0.6,
CH3CN : H2O 1 : 1); IR nmax 3285, 3080, 2964, 2874, 1727, 1636,
1
(c 0.05, H2O); H NMR (DMSO-d6, 600 MHz) d 0.87 (6H, d, J ¼
6.6 Hz, z-CH3 and z0-CH3), 1.32 (1H, m, d-Ha), 1.45 (1H, m, d-Hb),
1.69 (1H, m, 3-H), 2.43 (1H, dd, J ¼ 7.8, 15.6 Hz, a-Ha), 2.56 (1H,
dd, J ¼ 4.8, 15.6 Hz, a-Hb), 3.09 (1H, m, b-H), 3.93 (1H, m, g-H),
5.70 (1H, br s, b-OH), 7.63 (2H, br s, g-NH2), 12.33 (1H, br s,
–COOH).
1
1545, 1466, 1389, 1284, 1229, 1150, 1098, 1053, 721 cmꢀ1; H
NMR (DMSO-d6, 600 MHz) data and 13C NMR (DMSO-d6, 150
MHz) data, see Table 1. (ꢀ)-HR-ESIMS m/z 513.3275 [M ꢀ H]ꢀ
(calcd for C25H45N4O7, 513.3283).
Chiral-phase HPLC analysis of the acid hydrolysate of 1.29,30
Ahmpatinin iBu (1, 0.5 mg) was dissolved in 6 N HCl (1 ml) and
heated at 110 ꢂC for 16 h. Aer cooling to room temperature (rt),
the solvent was evaporated, and traces of HCl were removed by
repeated drying under vacuum with distilled H2O. The dried
hydrolysate was dissolved in 200 ml of 2 mM CuSO4/H2O solu-
tion. The hydrolysate of 1 and authentic (3S,4S)-, (3R,4S)-,
(ꢀ)-(S)-2-[3-(6-Methylheptanamido)-2-oxopyrrolidin-1-yl]
acetic acid (3). White amorphous powder; [a]2D0 ꢀ100.5 (c 1.0,
MeCN); CD (MeCN) 216 (D3 ꢀ15.88) nm; IR nmax 3287, 3066,
2947, 2871, 1700, 1644, 1551, 1465, 1439, 1397, 1300, 1263,
1
1188, 1148, 926, 721, 636 cmꢀ1; H NMR (DMSO-d6, 600 MHz)
data and 13C NMR (DMSO-d6, 150 MHz) data, see Table 1.
(+)-HR-ESIMS m/z 285.1802 [M + H]+ (calcd for C14H25N2O4,
285.1808).
5142 | RSC Adv., 2018, 8, 5138–5144
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(3R,4R)-, and (3S,4R)-Ahmppa were analyzed with a chiral-phase hepatocellular carcinoma (HepG2), and human osteosarcoma
column (MCI GEL CRS10W, 4.6 ꢁ 50 mm, Mitsubishi Chemical (U2OS) cell lines with IC50 values of 1.42, 1.56, and 2.78 mM,
Corporation) using 2 mM CuSO4/H2O solution as the mobile respectively.
phase with ow rate at 0.8 ml minꢀ1 and UV detection at 254 nm
on a Shimadzu LC-20A HPLC instrument. Ahmppa residue (tR ¼
Conclusions
12.07 min) in ahmpatinin iBu (1) was found to correspond to the
(3S,4S)-conguration by comparison of the retention times
(tR, min) with those of standard Ahmppa units: (3S,4S)-Ahmppa
(11.96), (3R,4S)-Ahmppa (10.63), (3R,4R)-Ahmppa (10.06), and
(3S,4R)-Ahmppa (10.36).
In summary, we have isolated two new linear peptides, ahm-
i
i
patinin Bu (1) and statinin Bu (2), and a novel pyrrolidine
anolog, (ꢀ)-(S)-2-[3-(6-methylheptanamido)-2-oxopyrrolidin-1-
yl]acetic acid (3), from a Streptomyces sp. strain. The unusual
amino acid, 4-amino-3-hydroxy-5-(4-methoxyphenyl)pentanoic
HPLC analysis of the acid hydrolysates of 1 and 2 using
Marfey's method. Approximately 0.5 mg of 1 and 2 were
hydrolyzed with 1 ml of 6 N HCl at 110 ꢂC for 16 h. The
hydrolysate was evaporated to dryness and redissolved in H2O
(200 ml). To one portion (100 ml), 100 ml of a 1% solution of 1-
uoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) in acetone
and 20 ml of 1 M NaHCO3 were added. The reaction mixture was
heated at 40 ꢂC for 1 h, cooled to room temperature, neutralized
with 2 M HCl (10 ml), and diluted with MeCN (100 ml). Similarly,
the standard D-Ala, L-Ala, D-Leu, and L-Leu were derivatized
separately. The Marfey's derivatives of the hydrolysate and
standards were analyzed by HPLC using the following condi-
tions. Column, Agilent ZORBAX SB-Aq C18 column (5 mm,
4.6 mm ꢁ 150 mm); ow rate, 1.0 ml minꢀ1; solvent A, 0.1%
triuoroacetic acid in an aqueous solution; solvent B, MeCN;
elution, 20–50% B in A over 60 min; UV detection at 340 nm;
i
acid (Ahmppa) of ahmpatinin Bu was identied for the rst
time from a natural source, and 3 represents the rst reported
instance of a natural pyrrolidine possessing a 2-(3-amino-2-
oxopyrrolidin-1-yl)acetic acid system. In addition, ahmpatinin
iBu (1) displayed signicant inhibitory activity against HIV-1
protease.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Financial support from the National Natural Science Founda-
tion of China (NNSFC, 81302675), CAMS Innovation Fund for
Medical Sciences (CIFMS, 2016-I2M-2-002), PUMC Youth Fund
and the Fundamental Research Funds for the Central Univer-
sities (3332015133), Natural Science Foundation of Beijing
Municipality (7144224), and National Infrastructure of Micro-
bial Resources (NIMR 2017-3) is acknowledged.
ꢂ
column temperature, 30 C. The retention times for the FDAA
derivatives of the standards D-Ala, L-Ala, D-Leu, and L-Leu were
39.93, 36.26, 49.43, and 44.10 min, respectively. The FDAA
derivatives of the amino acids from the hydrolysate of 1 and 2
showed peaks at 36.30 (L-Ala) and 44.16 (L-Leu) min, respec-
tively, and the amino acids were assigned to be L-Ala and L-Leu
in 1 and 2, respectively.
ECD and OR calculations of compound 3. Conformational
analysis of the S-enantiomer was carried out via Monte Carlo
searching with the MMFF94s molecular mechanics force eld
using the Spartan 10 soware.31 Seventy-two geometries
having relative energies within 6 kcal molꢀ1 were optimized
using DFT at the B3LYP/6-31G (d) level in vacuum with the
Gaussian 09 program.32 The 52 B3LYP/6-31G(d)-optimized
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5144 | RSC Adv., 2018, 8, 5138–5144
This journal is © The Royal Society of Chemistry 2018