Multifunctional polyurethanes synthesized from different triarylamine units with electrochromic, photogeneration, memory storage and sensing properties

Article abstract of DOI:10.1039/c8nj05159c

In this work, a series of multifunctional polyurethane (PU) materials containing three kinds of different triarylamine (TAA) units was prepared. These polymers show good solubility in common organic solvents and excellent thermal stability under a N₂ atmosphere. The PU films show reversible color changes with high contrast ratios and electrochromic stability. Moreover, they have good memory storage performance. The ON/OFF current ratio is about two orders of magnitude. Meanwhile, they can be used as detectors for 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenol (TNP). The polymer solutions rapidly exhibit fluorescence-quenching phenomena when exposed to trace amounts of TNT and TNP. The PU films also have mechanical properties that make them suitable for use in stretchable devices.

Full text of DOI:10.1039/c8nj05159c

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RESEARCH ARTICLE
Naphthomycin-derived macrolactams with two
new carbon skeletons from endophytic
Streptomyces†
Cite this: Org. Chem. Front., 2019, 6,
177
Zhouxin Zhang,‡a,b Pei Cao,‡a Ning-Ning Shang,‡a,b Jing Yang,a Li Wang,a,b
a
Yijun Yana and Sheng-Xiong Huang
*
Chemical investigation of an endophytic Streptomyces strain led to the isolation of naphthomycin
congeners 1–4. Compound 1 contains a unique phenalene scaffold with a bridged oxetane ring, which
was confirmed to be a result of an intramolecular [2 + 2] cycloaddition between the olefin and the ketone
in the known compound 4 by chemical conversion. 2 features an unprecedented acenaphthene moiety
in the ansamycin class of polyketides. Possible biosynthetic pathways are proposed for 1–3. Compounds
1–3 were tested for cytotoxicity against selected human cancer cell lines and they showed moderate
activity, with IC50 values ranging from 10.6 μM to 32.5 μM.
Received 15th October 2018,
Accepted 21st November 2018
DOI: 10.1039/c8qo01107a
rsc.li/frontiers-organic
exploration of related natural products, have emerged as
alternative strategies. Recently, dozens of novel ansamycins
Introduction
Ansamycins generally comprise a rigid aromatic chromophore with remarkable bioactive properties, such as divergolides,4a
(naphthalene/benzene or the reduced quinone) connected to a natalamycin,4b ansalactam,4c aminorifamycin,4d spora-
polyketide chain, usually featuring as flexible macrocyclic lactam,4d mccrearamycin4e and so forth, have been obtained
lactam.1 Based on the relatively conserved skeleton frame- from various terrestrial or marine natural resources.
works, structurally diversified ansamycins with a 3-amino-5-
hydroxybenzoic acid (AHBA) moiety, which exhibit intriguing similar endophytes (mainly actinomycetes)5 chemically and
biological and pharmaceutical activities to serve as valuable biologically highlighted an extract of strain named
Continued efforts on the screening of extract libraries from
a
antibiotics and anticancer agents, are predominantly gener- Streptomyces sp. KIB-H2054 which was isolated from the plant
ated from microorganisms.1 Greatly inspired by the potential Campylotropis polyantha, which had antibacterial activity
prospects of clinical applications exemplified by rifamycin- against Bacillus subtilis ATCC 6633 and Escherichia coli ATCC
derived drugs, the knowledge to disclose the biosynthetic 8099. As
a result, three new naphthomycin congeners,
mechanisms within ansamycins has been continuously naphthomycins O–Q (1–3), which possess two new carbon
updated, relying on popular and extensive studies.2 skeletons, were obtained from this strain. Herein, the iso-
Undoubtedly, the extraordinary architectural complexity of lation, structure elucidation, and biological evaluation,
ansamycins makes it quite challenging to afford diversity- together with the suggested biosynthetic origins of compounds
oriented libraries through either semi-synthesis or total syn- 1–3, are described.
thesis efforts.3a–d However, combinational biosynthesis origi-
nating from bioengineering3e,f and site-specific chemical
modification and functionalization,3g whose targeting interfer-
ences are significantly and substantially depended on the deep
aState Key Laboratory of Phytochemistry and Plant Resources in West China,
CAS Center for Excellence in Molecular Plant Sciences, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204, China.
E-mail: sxhuang@mail.kib.ac.cn
bUniversity of the Chinese Academy of Sciences, Beijing 100049, China
†Electronic supplementary information (ESI) available: Spectroscopic data, as
well as the NMR and MS spectra of 1–3 and corresponding isomers 1a–3a. See
DOI: 10.1039/c8qo01107a
‡These authors contributed equally to this work.
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Results and discussion
The molecular formula of naphthomycin O (1) was deduced
from its HRESIMS data and was assigned as C40H47NO9
(m/z 708.3142 [M + Na]+, calcd 708.3143), corresponding to an
unsaturation index of 18. The IR absorption bands of 1 at 3439
and 1634 cm−1 suggested the presence of hydroxyl and conju-
1
gated carbonyl groups, respectively. The H and 13C NMR data
of 1 (Table S2†) showed 40 carbon resonances due to four Fig. 2 Key ROESY correlations of naphthomycins O (1) (A) and P (2) (B).
carbonyl groups, nine olefinic groups, five oxygenated carbons
including two quaternary carbons, and four tertiary and three
secondary methyl groups. Careful analysis of the NMR and MS naphthomycin E (4) on the basis of a common biosynthetic
spectra showed that 1 seemed to be an analogue of naphtho- origin. The ROESY correlations between H-20 and 22-Me,
mycin E (4) which is a known compound and was isolated in together with the cross peak of H-20 and H-30, indicated that
this study too.
H-20 was approaching the benzocyclohexenone plane (Fig. 2A).
The gross structure of 1 was elucidated by the analysis of Furthermore, the H–1H coupling constant between H-20 and
2D NMR data and by comparison with the NMR data of 4. It H-21 (11.8 Hz) suggested that they were oppositely oriented.
was obvious that 1 shared the same side chain from C-1 to An additional ROESY correlation between 20-Me and 22-Me
C-19 as 4, however a different aromatic chromophore existed required the ether linkage between C-22 and C-31 to be located
in 1, instead of the naphthoquinone core in 4. The HMBC on the opposite side of the benzocyclohexenone ring. These
correlations from H-27 (δH 7.99, s) to C-25, 26-Me, C-27a, C-28 data limited the relative configurations of C-21, C-22 and C-31
and C-31a, from H-30 (δH 8.16, s) to C-23, C-28, C-29, C-31 and to being 21S*, 22S*, and 31R*.
1
C-31a, from 26-Me (δH 2.34, s) to C-25, C-26 and C-27, from
The elemental composition of naphthomycin P (2) was
H-21 (δH 3.32, d) to C-22, C-23, C-31, and C-31a, as well as determined to be C40H49NO9 by HRESIMS (m/z 710.3302
22-Me (δH 1.82, s) to C-21, C-22 and C-23 allowed the aromatic [M + Na]+, calcd for 710.3300). The 1H and 13C NMR spectra of
chromophore in 1 to be assigned as an oxygen-bearing phena- 2 had some similarities with those of 4. A careful comparison
lene core (Fig. 1, left). In addition, the HMBC correlations of of the NMR data between 2 and 4 reached a conclusion that 2
NH (δH 8.59, s) with C-1, C-2, C-28 and C-30, and of H-20 also possesses the same side chain from C-1 to C-19, except for
(δH 2.88, m) with C-21, C-22 and C-31 (Fig. 1A and Fig. S2†), a different conjugated aromatic moiety. A series of HMBC
verified that the side chain of 1 was connected to the oxyge- correlations from 26-Me, H-27 and H-30 to the carbons in the
nated phenalene subunit at C-29 and C-21, respectively, in the naphthalene ring defined the naphthalene ring moiety
form of a macrocyclic lactam. To sum up, only one oxygen (Fig. S6†). The HMBC correlations (Fig. 1B) from H-22 to C-23,
atom and one unsaturation were not accounted for in 1. The C-24, C-30, C-31 and C-31a, and from H-30 to C-22, C-31 and
chemical shifts at C-22 (δC 88.0) and C-31 (δC 77.8) suggested C-31a established the acenaphthene moiety in 2. Furthermore,
that an ether linkage, which could balance the molecular the HMBC correlations from H-20 to C-21 and C-22, from
weight of one oxygen atom and one unsaturation precisely, 21-Me to C-20, C-21 and C-22, and from H-22 to C-20, C-21 and
existed between C-22 and C-31.
Me-21 (Fig. 1B) demonstrated the fragment of C-19–C-20–
Next, the relative configuration of 1 was discussed (Fig. 2A C-21–C-22 in the macrocyclic ring, which was linked to the ace-
and Fig. S3†). The ROESY correlations of 2-Me/H-3, H-3/H-6, naphthene moiety at C-22. Just like 1, the stereochemistry of 2
H-6/8-Me and 12-Me/H-14 indicated the geometries of olefins at C-8, C-9, C-15, C-18, C-19 and C-20 was also suggested to be
to be 2Z, 4Z, 6E and 12E, respectively. The 1H–1H coupling con- the same as that of 4, and the geometries of the olefins of 2
stant between H-16 and H-17 (15.4 Hz) indicated a trans were resolved as 2Z, 4Z, 6E, 12E, and 16E, respectively, based
double bond at C-16. The stereochemistry at C-8, C-9, C-15, on the relevant ROESY correlations (Fig. S6†) and the corres-
C-18, C-19 and C-20 in 1 is suggested to be the same as that of ponding proton coupling constant. Moreover, the ROESY
correlations of H-22 with H-20 and H-30 together with the ROE
effect of H-22 and 21-Me indicated that H-22 and 21-OH were
in opposite configurations. Another ROESY correlation
between H-20 and H-30 limited the two possible configur-
ations of C-21 and C-22 to be 21S*, 22R* or 21R*, 22S*
(Fig. 2B). Ultimately, the ROESY correlation between H-19 and
H-22 indicated that the configurations at C-21 and C-22 could
only be 21S* and 22R* on the basis of the energy-minimized
modeling of the simplified structure 2 (Fig. S8†).
Naphthomycin Q (3) was found to possess the molecular
formula C40H49NO10 from the HRESIMS data (m/z 726.3247
Fig. 1 Key 2D NMR correlations of naphthomycins O (1) (A) and P (2)
(B).
1
[M + Na]+, calcd for 726.3249). The H NMR spectra of 3 were
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similar to those of 4 except for the presence of one more
singlet aromatic proton at δH 7.63 (H-24). The 13C NMR spec-
trum of 3 revealed one significant up-shifted carbon C-23
(δC 172.2) when compared to that of 4 (δC 202.4). Taking the
above NMR variations into consideration, it could be inferred
that the carbon bond between C-23 and C-24 in 4 was cleaved
to access a free carboxylic acid moiety at C-23 in 3. In this way,
the 18 Dalton molecular weight variation between 4 (MW: 685)
and 3 (MW: 703) could be accounted for coincidently. This
deduction was also supported by the HMBC correlations of
H-24 with C-26, C-28, and C-31a and of 22-Me with C-21, C-22,
and C-23 (Fig. S11†).
Fig. 3 HPLC analysis (detected at 236 nm) of the photocatalytic
(254 nm UV-light) cycloaddition product of 4. (I) The reaction mixture of
4; (II) standard 1; (III) standard 4.
Small amounts of compounds 1a, 2a, and 3a (Fig. S1†) were
also isolated, which are double bond geometry isomers of 1, 2, clearly detect the presence of 1. In addition, we found that
and 3, respectively. Careful analyses of the 1D and 2D NMR visible light could not convert 4 to compound 1 in the reactor
data of 1a–3a (Tables S3, S5 and S7†) showed that the geome- and only ultraviolet light could do this pericyclic reaction.
tries of the triene system were 2E, 4E, and 6E, determined by
Naphthomycin P (2) has a unique naphtho-cyclopentone
ROESY experiments (Fig. S5, S10 and S14†). Although we have moiety. Additionally, a methyl group, which is supposed to be
not yet provided direct evidence for the absolute configuration at C-22 according to the biosynthetic route of naphthomycins,6
of all of the new compounds, we have pictured the molecules was found to attach to C-21. It was recently reported that
with the same stereochemistry as that of the known compound quinone moieties can be aromatized into phenols under redu-
4 due to co-isolation from the same strain and the shared bio- cing conditions in the biosynthesis of rifamycin SV.2c Hence, it
synthetic machinery.
was proposed that 2 results from a 1,4-naphthalenediol inter-
Naphthomycin O (1) was characterized with the occurrence mediate, and 22-Me is migrated through a cyclopropane inter-
of an oxetane ring, which has rarely been encountered in the mediate 4b because some ansamycins, such as sporalactam B
ansamycin class.4 Naphthomycin O (1) is proposed to be and tolypomycin Y,4d,7 were reported to encompass a cyclopro-
derived from the major metabolite 4 through an intra- pane unit previously. Cyclopropanation could be realized via a
molecular [2 + 2] cycloaddition of the C-21–C-22 olefin to the radical pathway initiated by radical related enzymes.8 When
C-31 ketone (Scheme 1A). To confirm this conjecture, a solu- the cyclopropane ring suffers in acidic conditions, a ring
tion of naphthomycin E (4) was treated with UV irradiation opening process is initiated, and subsequent aldol conden-
(254 nm), and the presence of 1 was detected by HPLC analysis sation and dehydration would generate the cyclopentone ring
(Fig. 3). This transformation not only confirmed the biosyn- (Scheme 2).
thetic origin of the unique oxetane ring, but also supported
Naphthomycin Q (3) features an open polyketide chain.
the structure identification of 1. To prove whether compound 1 Since C-23 and C-24 are derived from one acetate unit on the
was an artificial product during the isolation process or not, basis of the biosynthetic pathway of naphthomycin,1e it is
we conducted the HPLC analysis of fresh extract. We could suggested that 3 is derived from 4 by a cleavage reaction
between C-23 and C-24. This type of cleavage reaction was pro-
posed to be a Baeyer–Villiger-style oxidation in the literature,
just as reported in some ansamycins with an opening poly-
Scheme 1 (A) Chemical conversion of 1 from 4 via a photochemical [2
+ 2] cycloaddition between the double bond and the ketone; (B) pro-
posed biosynthetic pathways of 3.
Scheme 2 Proposed biosynthetic pathway of 2.
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ketide chain, such as proansamycin B-M1,9a protorifamycin was collected in Hailuogou, Sichuan Province, China, in 2015.
I-M1,9a streptovaricin U9b and ansalactam C.4c However, a phe- It was identified as Streptomyces sp. by a 16S rRNA gene
nolic hydroxyl group must be generated at C-24 after the sequence (GenBank accession no. MH542663) and showed a
Baeyer–Villiger-style oxidation process, rather than an aromatic 99.0% identity to the Streptomyces nitrosporeus strain NRRL
proton in compounds 3 and 3a. Therefore, we prefer retro- B-1316 (GenBank accession no. NR044140.1). This strain was
Friedel–Crafts acylation at C-23 and C-24 in this case, which grown on ISP2 agar plates (glucose 4 g, malt extract 10 g, yeast
could construct a free carboxylic acid at C-23 and an aromatic extract 4 g, and agar 20 g in 1 L of water, pH 7.2) for five days
hydrogen at C-24 (Scheme 1B). As mentioned above, a bio- at 30 °C. Then, it was inoculated into 250 mL baffled
mimetic chemical transformation from 4 to 3 was planned via Erlenmeyer flasks containing 50 mL of sterile seed medium
the key retro-Friedel–Crafts acylation reaction, with the aid of (tryptone soy broth, 30 g L−1) and cultivated for two days at
the Lewis acid SnCl4 as the literature reported.10 30 °C on a rotary shaker (220 rpm). After that, aliquots
Unfortunately, neither 3 nor 3a was detected (Fig. S15†). (12.5 mL) of the culture were transferred into 1000 mL baffled
Consequently, the retro-Friedel–Crafts acylation of 4 is more Erlenmeyer flasks filled with 250 mL of production medium
likely to be enzymatic (Scheme 1B).
(pH 7.0) consisting of 0.1% tryptone (w/v), 3% glucose (w/v),
Previous studies demonstrated that ansamycins have 0.5% beef extract (w/v), 0.25% CaCO3 (w/v), 0.5% NaCl (w/v),
diverse bioactivities such as antitumor11 and antibacterial activi- and 0.1% minor elements concentrate (v/v) (ferrous sulphate
ties.12 All isolates were evaluated for their cytotoxicity against heptahydrate (1.0 g), copper sulphate pentahydrate (0.45 g),
the HL-60, SMMC-7721, A-549, MCF-7, and SW480 human zinc sulphate heptahydrate (1.0 g), manganese sulphate tetra-
tumor cell lines, as well as their antibacterial activity. hydrate (0.1 g), and potassium molybdate (0.1 g), made up to
Compounds 1 and 2 showed broad cytotoxicities towards all five 1 L with distilled water and clarified by the addition of a few
cell lines (Table S1†). However, 3 only showed inhibitory activity drops of concentrated hydrochloric acid), and cultured on a
against the HL-60 and A549 cells (Table S1†). Besides, com- rotary shaker (220 rpm) at 30 °C for five days.
pound 3 was found to have weak antibacterial activity against
Extraction and isolation of compounds. The fermentation
E. coli ATCC 8099 with an inhibition zone of 11 mm, while the broth (20 L) was centrifuged (4000 rpm, 15 min), and the super-
positive control kanamycin showed an inhibition zone of natant was extracted with EtOAc (10 L × 3). The EtOAc extract
18 mm. However, 1 and 2 exhibited no antibacterial activity, pre- was subsequently evaporated in vacuo to afford 5.0 g of oily
sumably due to the loss of the naphthalene unit.
crude extract. The mycelium was extracted with acetone (1 L × 2)
and was then concentrated in vacuo to remove the acetone to
yield the aqueous concentrate. This aqueous concentrate was
finally extracted with EtOAc (1 L × 3) to give 1.0 g of solid crude
extract after removing the EtOAc. Both extracts revealed an iden-
tical set of metabolites based on HPLC and TLC analyses and
Experimental section
General experimental procedures
Optical rotations were recorded in MeOH using a JASCO P1020 therefore both extracts were combined for further purification.
digital polarimeter. UV spectra were acquired in MeOH with a The crude extract (6.0 g) was fractionated by a silica gel
Shimadzu UV-2401PC UV-VIS spectrophotometer. IR spectra using a PE/EA gradient (20 : 1 to 0 : 1) as the eluent to afford
were measured on a Bruker Tensor 27 FTIR spectrometer with nine fractions (F1–F9). F5 was sequentially subjected to
KBr disks. NMR spectra were recorded in CDCl3 or CD3OD Sephadex LH-20 column elution with MeOH to collect four
using a Bruker AV 600 MHz spectrometer with TMS as an fractions (F5A–F5D). F5B was further purified by semiprepara-
internal standard. ESIMS spectra were recorded using a Waters tive HPLC (YMC-Triart C18 column, 250 × 10 mm i.d., 5 μm,
Xevo TQ-S Ultrahigh Pressure Liquid Chromatography Triple 3.0 mL min−1, CH3CN : H2O = 52 : 48, 0.1% acetic acid) to
Quadrupole Mass Spectrometer. HRESIMS data were obtained afford 1 (tR = 22.5, 6.7 mg), 1a (tR = 23.5 min, 4.5 mg), 2 (tR
using an Agilent 1290 UPLC/6540 Q-TOF and a UPLC-IT-TOF 21.4 min, 11.4 mg), 2a (tR = 20.3 min, 3.1 mg), and 4 (tR
=
=
mass instrument. Silica gel (200–300 mesh and 300–400 mesh, 15.3 min, 60 mg). F6 was subjected to Sephadex LH-20 column
Qingdao Marine Chemical Inc., China), and Sephadex LH-20 elution with MeOH to collect two fractions (F6A and F6D). F6C
(25–100 µm, Pharmacia Biotech Ltd, Sweden) were used for was purified by semipreparative HPLC (YMC-Triart C18
the chromatograph column. Semipreparative HPLC was con- column, 250 × 10 mm i.d., 5 μm, 3.0 mL min−1, CH3CN : H2O =
ducted on a HITACHI Chromaster system, equipped with a 50 : 50, 0.1% acetic acid) to afford 3 (tR = 18.0 min, 24.2 mg)
DAD detector and a YMC-Triart C18 column (250 × 10 mm i.d., and 3a (tR = 18.7 min, 13.7 mg).
5 μm), and had a flow rate of 3.0 mL min−1. The HPLC analysis
Photocatalytic reaction of naphthomycin E (4). Compound 4
conditions were as follows: a HITACHI Chromaster system (5 mg, 7.3 × 10−3 mmol) and methanol (0.02 mL) were dis-
equipped with a DAD detector, a YMC-Triart C18 column (250 solved in methylbenzene (1.0 mL). This reaction mixture was
× 10 mm i.d., 5 μm) with a mobile phase of CH3OH–H2O, placed in a UV photochemical reactor for 30 min then the
0–20 min: 10%–100%, 20–24 min: 100%, and 24–28 min: 10%, mixture was concentrated and the crude product was analyzed
and flow rate of 1 mL min−1
.
by HPLC (A: water; B: methanol; 0 min 10% B; 20 min 100% B;
Strain isolation and cultivation. The strain designated 24 min 100% B; 24.1 min 10% B; 28 min 10% B; flow rate was
KIB-H2054 was isolated from Campylotropis polyantha, which 1.0 mL min−1).
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Retro-Friedel–Crafts acylation reaction of naphthomycin nm; IR (KBr) νmax 3441, 2973, 2927, 1632, 1570, 1503,
E (4), with the aid of a Lewis acid, SnCl4. Compound 4 (5 mg, 1384 cm−1; for 1H, 13C, and 2D NMR data, see Table S3;†
7.2 × 10−3 mmol) was dissolved in THF (1.0 mL). This solution ESIMS [M + Na]+ m/z 708; HRESIMS [M + Na]+ m/z 708.3125
was degassed with a stream of nitrogen. The reaction was then (C40H47NO9, calcd [M + Na]+ 708.3143).
placed in an ice bath, and SnCl4 was added in a dropwise
Naphthomycin P (2). Pale yellow solid; [α]2D3:2 = +278.6
manner. The ice bath was removed and the solution was refluxed (c 0.05, methanol); UV (methanol) λmax (log ε) 308.0 (1.99),
at 58 °C. A small amount of the reaction solution was taken at 226.5 (2.16), 194.5 (1.97) nm; IR (KBr) νmax 3439, 3030, 2965,
10, 20, 35, 50, 80 and 110 minutes. The solution was hydrolyzed 2926, 2857, 1633, 1531, 1439, 1373, 1318, 1274 cm−1; for 1H,
by adding water, dried and evaporated to remove water and THF, 13C, and 2D NMR data, see Table S4;† ESIMS [M + Na]+ m/z
and the crude product was analyzed by HPLC (A: water; B: metha- 710; HRESIMS [M + Na]+ m/z 710.3302 (C40H49NO10, calcd
nol; 0 min 10% B; 20 min 100% B; 24 min 100% B; 24.1 min [M + Na]+ 710.3300).
10% B; 28 min 10% B; flow rate was 1.0 mL min−1).
2a: Pale yellow solid; [α]2D3:2 = +16.4 (c 0.11, methanol); UV
Cytotoxicity assay. Five human tumor cell lines, HL-60, (methanol) λmax (log ε) 293.0 (1.67), 228.0 (1.88), 194.0 (1.71)
SMMC-7721, A-549, MCF-7, and SW480 were used in this cyto- nm; IR (KBr) νmax 3431, 3030, 2969, 2928, 2882, 1638, 1529,
1
toxicity assay, all of which were obtained from ATCC 1439, 1378, 1318, 1278 cm−1; for H, 13C, and 2D NMR data,
(Manassas, VA, USA). All of the cells were cultured in see Table S5;† ESIMS [M + Na]+ m/z 710; HRESIMS [M + Na]+
RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA) sup- m/z 710.3296 (C40H49NO10, calcd [M + Na]+ 710.3300).
plemented with 10% fetal bovine serum (Hyclone) at 37 °C in
Naphthomycin Q (3). Pale yellow solid; [α]2D3:2 = −72.5 (c 0.12,
a humidified atmosphere with 5% CO2. The cytotoxicity assay methanol); UV (methanol) λmax (log ε) 348.0 (1.25), 307.5
was conducted by a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxy- (1.59), 278.5 (1.61), 215.0 (1.69) nm; IR (KBr) νmax 3428, 3036,
menthoxyphenyl)-2-(4-sulfopheny)-2H–tetrazolium (MTS) assay. 2966, 2928, 2876, 1656, 1587, 1501, 1330 cm−1; for 1H, 13C,
Firstly, cells were seeded into each well of a 96-well cell culture and 2D NMR data, see Table S6;† ESIMS [M + Na]+ m/z 726;
plate. After cell attachment overnight, the test compound HRESIMS [M + Na]+ m/z 726.3247 (C40H47NO10, calcd [M + Na]+
(40 µM) was added. After incubating for two days, the cells 726.3249).
were submitted to the MTS-based assay. Formazan crystals
3a: Pale yellow solid; [α]2D3:2 = −76.9 (c 0.12, methanol); UV
that were formed lastly in the viable cells were measured at (methanol) λmax (log ε) 347.0 (1.33), 308.5 (1.59), 278.5 (1.51),
490 nm using a microplate reader (BioTek Instruments, Inc. 215.0 (1.62), nm; IR (KBr) νmax 3429, 3034, 2964, 2927, 2875,
1
USA). Compounds with a growth inhibition rate of 50% were 1656, 1587, 1502, 1330 cm−1; for H, 13C, and 2D NMR data,
further evaluated at the concentrations of 0.064, 0.32, 1.6, 8.0, see Table S7;† ESIMS [M + Na]+ m/z 726; HRESIMS [M + Na]+
and 40 µM, using cisplatin as the positive controls. All of the m/z 726.3242 (C40H47NO10, calcd [M + Na]+ 726.3249).
experiments were carried out in triplicate.
Antibacterial assay. Bacillus subtilis ATCC 6633 and
Escherichia coli ATCC 8099 from the stock were inoculated into
a sterile LB broth of 5 mL, each incubated for 18–24 h at
Conclusions
37 °C. From the overnight culture, 0.1 mL of each organism In summary, our discovery of naphthomycins O–Q (1–3) has
was taken and put into 9.9 mL of sterile distilled water to enriched the members of ansamycins with two different skel-
obtain a 10−2 inoculum concentration of the organism. From etons. Naphthomycin O (1) contains a unique phenalene
the diluted inoculum, 0.2 mL was put into the prepared sterile scaffold with a bridged oxetane ring, which is proposed to be
LB agar and cooled to about 40–45 °C, and was then poured derived from the intermolecular [2 + 2] cycloaddition of the
into a sterile dish and allowed to solidify for about 45–60 min. olefin to the ketone of 4. Our successfully biomimetic syn-
Six filter papers of 6 mm diameter were placed on the LB agar thesis of
1 from 4 verified this biosynthetic origin.
evenly. 10 µL of the compounds with a concentration of 2 mg Naphthomycin P (2) has a unique naphtho-cyclopentone,
mL−1 was added to the filter paper, including the controls. which is hypothesized to undergo an enzyme involved process
The studies were done in triplicate to confirm the results with
a
1,4-naphthalenediol-cyclopropane intermediate.
obtained. The plates were left on the bench for about 2 h to Naphthomycin Q (3), possessing an opening polyketide side
allow the extract to diffuse properly into the LB agar. The chain, indicates the possibility of the enzymatic retro-Friedel–
plates were incubated for 18–24 h at 37 °C.
Crafts acylation of 4. The identification of structurally cyclized (1),
Naphthomycin O (1). Pale yellow solid; [α]2D3:2 = −317.4 rearranged (2), and cleaved (3) naphthomycins highlights
(c 0.06, methanol); UV (methanol) λmax (log ε) 195.5 (1.75), the distinguishing feature of biosynthetic versatility in the
231.0 (1.85), 277.0 (1.81), 316.5 (1.59), 374.5 (0.90) nm; IR ansamycin class of polyketides, which also reflects their
(KBr) νmax 3439, 2968, 2928, 1634, 1518, 1459, 1383 cm−1; for inherent fascination and attraction in nature.
1H, 13C, and 2D NMR data, see Table S2;† ESIMS [M + Na]+ m/z
708; HRESIMS [M + Na]+ m/z 708.3142 (C40H47NO9, calcd [M +
Na]+ 708.3143).
Conflicts of interest
1a: Pale yellow solid; [α]2D3:2 = −102.0 (c 0.10, methanol); UV
(methanol) λmax (log ε) 195.0 (1.62), 229.0 (1.78), 300.0 (1.58) There are no conflicts to declare.
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D. Siegel, S. M. Roe, C. Prodromou, D. Ross and
C. J. Moody, Nat. Chem., 2013, 5, 307–314.
Acknowledgements
This research was financially supported by the National
Natural Science Foundation of China (No. U1702285 and
81522044), the Applied Basic Research Foundation of Yunnan
Province (No. 2013HA022), the Key Research Program of
Frontier Sciences, CAS (No. QYZDB-SSW-SMC051), and the
Yunnan Innovative Research Team for Discovery and
Biosynthesis of Bioactive Natural Products (2018HC012).
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