Baraphenazines A-G, Divergent Fused Phenazine-Based Metabolites from a Himalayan Streptomyces

Article abstract of DOI:10.1021/acs.jnatprod.9b00289

The structures and bioactivities of three unprecedented fused 5-hydroxyquinoxaline/alpha-keto acid amino acid metabolites (baraphenazines A-C, 1-3), two unique diastaphenazine-type metabolites (baraphenazines D and E, 4 and 5) and two new phenazinolin-type (baraphenazines F and G, 6 and 7) metabolites from the Himalayan isolate Streptomyces sp. PU-10A are reported. This study highlights the first reported bacterial strain capable of producing diastaphenazine-type, phenazinolin-type, and izumiphenazine A-type metabolites and presents a unique opportunity for the future biosynthetic interrogation of late-stage phenazine-based metabolite maturation.

Full text of DOI:10.1021/acs.jnatprod.9b00289

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Baraphenazines A−G, Divergent Fused Phenazine-Based
Metabolites from a Himalayan Streptomyces
Xiachang Wang,†,‡,§ Muhammad Abbas,∥ Yinan Zhang,†,‡,§ Sherif I. Elshahawi,‡,§,⊥
Larissa V. Ponomareva,‡,§ Zheng Cui,§ Steven G. Van Lanen,§ Imran Sajid,∥ S. Randal Voss,#,¶,□
Khaled A. Shaaban,*,‡,§ and Jon S. Thorson*,‡,§
†Jiangsu Key Laboratory for Functional Substances of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023,
People’s Republic of China
‡Center for Pharmaceutical Research and Innovation and §Department of Pharmaceutical Sciences, College of Pharmacy, University
of Kentucky, Lexington, Kentucky 40536, United States
∥Department of Microbiology and Molecular Genetics, University of the Punjab, Quid-i-Azam campus, Lahore 54590, Pakistan
⊥Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, California 92618,
United States
#Department of Neuroscience, ¶Ambystoma Genetic Stock Center, and □Spinal Cord and Brain Injury Research Center, University
of Kentucky, Lexington, Kentucky 40506, United States
S
* Supporting Information
ABSTRACT: The structures and bioactivities of three unprecedented fused 5-hydroxyquinoxaline/alpha-keto acid amino acid
metabolites (baraphenazines A−C, 1−3), two unique diastaphenazine-type metabolites (baraphenazines D and E, 4 and 5) and
two new phenazinolin-type (baraphenazines F and G, 6 and 7) metabolites from the Himalayan isolate Streptomyces sp. PU-10A
are reported. This study highlights the first reported bacterial strain capable of producing diastaphenazine-type, phenazinolin-
type, and izumiphenazine A-type metabolites and presents a unique opportunity for the future biosynthetic interrogation of late-
stage phenazine-based metabolite maturation.
henazine natural products are chorismate-derived diben-
Pzo-annulated pyrazine-based secondary metabolites pri-
marily produced by Streptomyces and Pseudomonas.1−6
Phenazines are known to contribute to Pseudomonas virulence
and to also display a broad range of biological activities
(antihypertensive, antiparasitic, antimalarial, neuroprotective,
radical scavenging, and/or antitumor),1−3 primarily via the
reduction of molecular oxygen to generate reactive oxygen
species (ROS).7 While over 150 naturally occurring phenazines
have been reported,1−3,8 only a small subset are composed of
more than one phenazine-derived unit. These include six
naturally occurring phenazine dimers (connected via a single
covalent bond as exemplified by diphenazithinonin,9 phena-
zostatins A−D,10−12 and izumiphenazine B13) and 11 fused
phenazine-based metabolites (in which the combination of two
phenazine core units affords a new ring system as exemplified
by esmeraldines A and B,14 phenazinolins A−E,15 izumiphe-
nazine A,13 and diastaphenazine,16 Figure 1). Of the latter set,
the recently reported diastaphenazine (isolated from an
endophytic Streptomyces strain) stands out as the first example
in which the new ring formed lacks a heteroatom.16
As part of an effort to characterize novel natural products
from unusual terrestrial environments,17−27 their unique
molecular targets,28 and/or corresponding biocatalysts,29
herein we report the isolation and characterization of seven
new fused phenazine-based metabolites (baraphenazines A−G;
1−7, Figure 1), along with diastaphenazine and izumiphena-
zine A, from the Himalayan isolate Streptomyces sp. PU-10A.
Of the new natural products disclosed herein, baraphenazines
A−C (1−3) represent the first reported examples of fused 5-
hydroxyquinoxaline/alpha-keto acid-based metabolites. In
addition, baraphenazines D and E (4 and 5) embody two
new diastaphenazine-type C−C-fused phenazine-based ana-
logues, while baraphenazines F and G (6 and 7) exemplify two
new phenazinolin-type C−O-fused compounds. This study
highlights the first reported strain capable of producing the
divergent phenazine ring-fused systems of diastaphenazine-
type, izumiphenazine A-type, and phenoazinolin D/E-type
Received: March 29, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00289
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Figure 1. Structures of new compounds (1−7) isolated from Streptomyces sp. PU-10A and related phenazines.
1
Table 1. H NMR (400 MHz) Spectroscopic Data for Compounds 1−7 in DMSO-d6 (δ in ppm, mult. J in Hz)
no.
1
no.
2
3
no.
4
5
6
7
2
3
7.13, d (7.7)
7.62, t (7.8, 7.9)
2
3
7.14, d (7.7)
7.63, t (7.9, 8.1) 7.57, t (7.6,
7.9)
7.08, d (7.5)
2
3
7.05, d (7.7)
7.48, t (7.7,
8.0)
7.04, d (7.8)
7.47, t (7.8, 8.2)
7.07, d (7.8)
7.59, t (7.8,
8.2)
7.12, d (7.8)
7.65, t (7.8, 8.4)
4
6
7
7.46, d (8.3)
4.92, d (4.0)
4.59, dd (4.3, 8.7)
4
6
7
7.46, d (8.3)
4.98, d (4.1)
4.65, dd (4.1,
8.2)
7.34, d (8.4)
5.09, br s
4.54, t (5.7,
7.1)
4
6α
6β
7.24, d (8.0)
3.14, d (18.0)
3.75, d (18.0)
7.26, d (8.2)
3.03, d (17.8)
3.67, dd (4.5,
17.8)
7.35, d (8.2)
3.34, d (18.0)
3.79
7.32,
3.60, d (18.0)
3.76, dd (5.0,
18.0)
8
3.29, m
8
9α
3.38, m
2.90, m
3.18, m
2.40, m
7
8
4.46, br s
4.95, br s
4.37, br s
4.59, d (4.4)
3.82
4.79, br s
4.91, br s
2.63, 2.86
9α 2.78, dd (3.9,
14.8)
9β 3.32, m
9β
1′
2′
4′
5′
3.39, m
11.00, s
7.35, s
7.37, d (8.3)
6.89, t (7.2, 7.6) 6.98, t (7.5,
7.6)
3.24, m
10.95, s
7.41, s
7.56, d (7.6)
9
4.58, br s
8.08
8.22, d (8.9)
8.52, d (6.6)
8.10
5.04, br s
5.74, br s
2′
3′
5′
6′
6.67, d (8.1)
7.06, d (8.2)
7.06, d (8.2)
6.67, d (8.1)
3′
4′
7′
8′
7.96, d (8.8)
8.22, d (8.8)
8.65, d (7.0)
8.06, t (6.6, 8.4)
8.05, d (9.0)
7.88, d (9.0)
8.59, d (8.2)
7.28, d (8.2)
7.91, s
7.32
7.84, t (7.7, 8.5)
7′
2.89, d (10.7)
6′
7.03, t (7.2, 7.5) 7.08, t (7.5,
7.7)
9′
8.58, d (8.9)
8.49, d (8.7)
7.74, d (8.6)
10.20, s
7′
8′
7.33, d (8.3)
3.30, d (10.6)
7.39, d (8.4)
4.27, d (8.8)
10.11, s
1-OH 10.25, br s
8-OH 5.79, br s
9-OH
10.20, br s
6.19, s
10.57, s
6.16, s
1-OH 10.29, br s
8.28, s
6-OH
5.80, s
6′-OH
10.62, s
9′-OH
11.99, s
congeners and may implicate a broader biosynthetic relation-
ship.
Northern Pakistan (Bara Gali) revealed Streptomyces sp. PU-
10A as capable of novel secondary metabolite production
(based on a comparison to the AntiBase 20178 database).
Scale-up fermentation (10 L) of Streptomyces sp. PU-10A
followed by extraction, fractionation, and standard chromatog-
raphy (Supporting Information, Scheme S1) gave seven new
RESULTS AND DISCUSSION
■
Preliminary LC-MS metabolic profiling of purified actino-
mycete strains isolated from a soil sample collected in
B
DOI: 10.1021/acs.jnatprod.9b00289
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Table 2. 13C NMR (100 MHz) Spectroscopic Data for 1−7, Diastaphenazine, and Izumiphenazine in DMSO-d6 (δ in ppm)
a
a
no.
1
2
3
no.
4
5
6
7
diastaphenazine
izumiphenazine
1
2
3
4
4a
5a
6
7
8
153.6, C
153.6, C
111.7, CH
130.3, CH
118.1, CH
142.4, C
151.2, C
71.8, CH
80.5, CH
40.9, CH
32.7, CH2
154.1, C
131.4, C
153.2, C
111.0, CH
130.0, CH
117.9, CH
141.9, C
150.8, C
74.0, CH
84.7, CH
38.6, CH
32.0, CH2
154.6, C
130.6, C
1
2
3
4
4a
5a
6
7
8
153.4, C
111.9, CH
129.8, CH
117.7, CH
142.5, C
152.6, C
35.5, CH2
45.4, CH
80.6, CH
58.1, CH
151.9, C
130.6, C
140.5, C
148.0, C
129.3, CH
128.9, CH
140.8, C
139.3, C
128.4, C
133.8, CH
130.6, CH
133.6, CH
142.5, C
140.5, C
166.3, C
153.3, C
111.7, CH
129.5, CH
117.8, CH
142.3, C
154.0, C
31.9, CH2
40.2, CH
75.0, CH
55.0, CH
152.1, C
131.1, C
140.2, C
146.8, C
128.1, CH
129.6, CH
141.3, C
139.9, C
130.9, C
133.9, CH
130.5, CH
133.1, CH
142.7, C
139.1, C
165.6, C
153.6, C
111.8, CH
131.6, CH
117.8, CH
142.7, C
152.2, C
35.5, CH2
35.9, CH
62.9, CH
75.5, CH
146.6, C
132.6, C
146.6, C
123.1, C
135.8, CH
119.8, CH
139.7, C
140.5, C
114.1, C
138.9, CH
110.3, CH
159.3, C
134.2, C
134.2, C
165.5, C
153.3, C
110.9, CH
132.1, CH
117.6, CH
142.5, C
151.9, C
39.4, CH2
27.0, CH
31.3, CH2
97.2, C
147.4, C
130.6, C
122.2, C
152.7, C
127.9, CH
127.6, CH
136.9, C
135.1, C
153.2, C
111.5, CH
132.4, CH
118.0, CH
139.3, C
139.8, C
165.4, C
153.3, C
111.7, CH
129.8, CH
117.7, CH
142.3, C
153.9, C
31.9, CH2
40.2, CH
75.0, CH
54.9, C
152.0, C
131.5, C
140.4, C
147.7, C
128.6, CH
128.8, CH
140.7, C
139.3, C
129.8, C
133.2, CH
130.7, CH
133.0, CH
142.4, C
139.6, C
166.7, C
118.4, CH
132.3, CH
111.8, CH
153.2, C
135.9, C
149.7, C
71.5, CH
88.7, CH
40.4, CH
33.3, CH2
153.6, C
139.6, C
153.5, C
111.7, CH
130.6, CH
118.0, CH
142.3, C
137.3, C
129.9, C
121.1, CH
158.7, C
124.9, C
138.7, C
131.3, C
165.8, C
111.7, CH
130.4, CH
118.1, CH
142.4, C
151.1, C
71.8, CH
80.5, CH
40.2, CH
32.5, CH2
153.9, C
131.4, C
156.4, C
114.7, CH
130.3, CH
9
9
9a
10a
1′
2′
3′
3a′
4′
5′
6′
7′
7a′
8′
9′
10′
9a
10a
1′
2′
3′
4′
4′a
5′a
6′
7′
8′
9′
9′a
10′a
11′
124.2, CH
109.0, C
127.7, C
118.7, CH
118.2, CH
120.6, CH
111.2, CH
135.9, C
124.6, CH
109.5, C
127.7, C
118.1, CH
118.4, CH
120.9, CH
111.2, CH
135.5, C
125.8, C
130.3, CH
114.7, CH
56.0, CH
101.7, C
170.4, C
48.9, CH
101.4, C
170.8, C
45.2, CH
104.9, C
169.6, C
a
The atom numbers correspond to the atom numbers in compound 1−7 for comparison.
1
Figure 2. H−1H COSY (bold lines) and key HMBC (→) correlations for 1−7.
compounds [baraphenazines A (1, yield: 1.02 mg/L), B (2,
Structure Elucidation. Compound 1 was isolated as a
yield: 1.15 mg/L), C (3, yield: 0.48 mg/L), D (5, yield: 0.94
mg/L), E (4, yield: 0.75 mg/L), F (6, yield: 0.31 mg/L), and
G (7, yield: 0.40 mg/L)] and two previously reported
metabolites [diastaphenazine (yield: 1.02 mg/L) and izumi-
phenazine A (yield: 1.30 mg/L)] (Figure 1).
green, amorphous powder, and its molecular formula was
established by (+)-HR-ESIMS as C21H18N2O7, indicating 14
degrees of unsaturation. The analysis of the 1H/13C and
HSQC NMR data suggested the presence of one methylene,
11 methine (seven aromatic), one hemiketal, seven sp2
C
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Figure 3. Key NOESY correlations for 1−7.
nonprotonated carbons, and one carboxylic acid (Tables 1 and
2). Analysis of the COSY spectrum revealed the presence of
three 1H−1H spin systems, including a 1,2,3-trisubstitued
benzene ring (CH-2/CH-3/CH-4), a 1,4-disubstitued benzene
ring (CH-2′/CH-3′ and CH-4′/CH-5′) and the connectivity
of CH-6/CH-7/CH-8/CH-7′/CH2-9. Key HMBC correla-
tions (Figure 2) established 1 to comprise two key
substructures: a 7,8-disubstitued tetrahydrophenazine-1,6-diol
(supported by HMBC correlations from H-2 to C-4 and C-
10a, from H-3 to C-1 and C-4a, and from H-4 to C-2 and C-
10a) and an 1′,8′,8′-trihydroxy-benzenepropanoic acid (based
on HMBC correlations from H-7′ to C-8′, C-9′, and C-3′;
from H-3′ to C-7′ and C-1′; and from H-2′ to C-4′ and C-6′).
The crucial HMBC correlations from H-8 (δH 3.29) to C-4′
(δC 125.8) and from H-7′ (δH 2.89, d) to C-8 (δC 40.2) and C-
9 (δC 32.5) served as a basis for substructure connectivity to
establish the preliminary 1 scaffold. The relative configuration
of 1 was established by NOESY, which highlighted H-6, H-7,
H-8, and H-9α to adopt the same facial orientation (Figure 3).
Consistent with the putative 1 8′-hemiketal, methylation of 1
in the presence of methyl iodide and silver oxide30 yielded two
per-methyl-1 C-8′ diastereomers (compounds 1a and 1b,
Scheme 1). Thus, the structure of 1 was established as a new
natural product, and 1 was named baraphenazine A to reflect
the producing strain’s point of origin.
Scheme 1. Decomposition of 2
structural divergence deriving from C-7′ substitution (Figure
1). Substitution of the 1 C-7′ phenol side chain with an indole
in both 2 and 3 was established by the presence of
1
characteristic indole H NMR signals [e.g., for 2: δH 7.35 (s,
H-2′), 7.33 (d, H-7′), 7.03 (t, H-6′), 6.89 (t, H-5′), and 7.37
(d, H-4′)]; corresponding 13C NMR signals [e.g., for 2: δC
124.2 (C-2′), 109.0 (C-3′), 127.7 (C-3a′), 118.7 (C-4′), 118.2
(C-5′), 120.6 (C-6′), 111.2 (C-7′), 135.9 (C-7a′)]; and key
HMBC [e.g., for 2: from H-8 (δH 3.38, m) to C-3′ (δC 109.5)
and from H-8′ (δH 3.30, s) to C-2′ (δC 124.2) and C-3a′ (δC
127.7)]; and NOESY correlations (Tables 1 and 2, Figures
1−3). NOESY revealed 2 and 3 to solely differ in C6-OH
orientation (Figure 3) and further established 2 and 3 as
unprecedented fused 5-hydroxyquinoxaline/indolylpyruvic
acid-based metabolites. Compounds 1−3 decomposed in
MeOH, and, as exemplified by 2, the decomposition products
were identified as 1,6-dihydroxyphenazine and the enol form of
indole-3-pyruvic acid (Schemes 1 and S2). As new natural
products, 2 and 3 were designated as baraphenazines B and C.
Compounds 2 and 3 share a common molecular formula
(C23H19N3O6) and also display similar UV and NMR
signatures to that of 1 (Tables 1 and 2) with apparent
D
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The determined molecular formula of 4 (C25H16N4O4) and
2D NMR (Figures 2 and 3) revealed 4 to share a planar
structure identical to the previously reported diastaphena-
zine,16 also a confirmed metabolite of Streptomyces sp. PU-10A.
NOESY of 4 and diastaphenazine (Supporting Information,
Figures S29 and S74) revealed these compounds to stereo-
chemically differ at C7, C8, and C9 with the observed H-6/H-
8 NOE in 4 (Figure 3) and lack thereof in diastaphenazine,
consistent with a 4/diastaphenazine C8 enantiomeric relation-
ship. Consistent with this, the determined optical rotation of 4
transformations/enzymes that contribute to the structural
divergence of major phenazine classes.1−6,32−36
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation was
recorded on a Jasco DIP-370 digital polarimeter (Jasco, Easton,
MD, USA). UV spectra were recorded on an Ultrospec 8000
spectrometer (GE, Pittsburgh, PA, USA). All NMR data were
recorded at 400 MHz for 1H and 100 MHz for 13C with Varian Inova
NMR spectrometers (Agilent, Santa Clara, CA, USA). HPLC-MS was
conducted with an Agilent 6120 Quadrupole MSD mass spectrometer
(Agilent Technologies) equipped with an Agilent 1200 Series
Quaternary LC system and an Eclipse XDB-C18 column (150 × 4.6
mm, 5 μm). HRESIMS spectra were recorded on an AB SCIEX Triple
TOF 5600 System (AB Sciex, Framingham, MA, USA). HPLC
analyses were performed on an Agilent 1260 system equipped with a
photodiode array (PDA) detector and a Phenomenex C18 column
(250 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA). Semipreparative
HPLC separation was performed on a Varian Prostar 210 HPLC
system equipped with a PDA detector using a Supelco DiscoveryBio
wide-pore C18 column (250 × 21.2 mm, 10 μm; flow rate, 8 mL/min;
Sigma-Aldrich, St. Louis, MO, USA). Size exclusion chromatography
was performed on Sephadex LH-20 (25−100 μm; GE Healthcare,
Piscataway, NJ). Amberlite XAD16N resin (20−60 mesh) was
purchased from Sigma-Aldrich. TLC silica gel plates (60 F254) were
purchased from EMD Chemicals Inc. (Darmstadt, Germany).
Staphylococcus aureus, Bacillus subtilis, Salmonella enterica, Mycobacte-
rium aurum, and Saccharomyces cerevisiae strains and PC3 and A549
cells were obtained from ATCC (Manassas, VA, USA); Micrococcus
luteus and Escherichia coli were obtained from NRRL (Peoria, IL,
USA). All solvents used were of ACS grade and purchased from the
Pharmco-AAPER (Brookfield, CT, USA). All other reagents used
were reagent grade and purchased from Sigma-Aldrich.
([α]25 +15.1) differed from that of diastaphenazine
D
(determined [α]25 −278.0; literature [α]20 −1248).16
D
D
NMR also highlighted a close structural relationship between
4 and 5, with divergence at C-6′ (C-6′ carboxylic acid in 4
versus a C-6′ carboxamide in 5). The relative configuration of
5 was also determined by NOESY (Figure 3). In a similar
manner, HRESIMS and 1D/2D NMR (Tables 1 and 2, Figure
2) revealed baraphenazines F and G (6 and 7) as two new
phenazinolin-type15 C−O-fused phenazine dimers, where the
unique dimeric orientation of 6 is unprecedented compared to
previously reported phenazine-based metabolites (Figure 1).
All isolated compounds were evaluated in standard
antibacterial, antifungal, and cancer cell line cytotoxicity assays
(Supporting Information, Table S1). Only 5 displayed
appreciable activity at the concentrations tested [≤50 μM in
cytotoxicity assays and ≤120 μM (49.2−56.2 μg/mL) in
antibacterial/fungal assays]. Specifically, the similar cancer cell
line cytotoxicity [human non-small-cell lung A549, IC50 2.4
μM; human prostate PC3, IC50 4.7 μM] and antimycobacterial
[M. aurum MIC 30 μM (13.1 μg/mL) and anti-Gram-(+) (S.
aureus, MIC 4 μM (1.7 μg/mL); M. luteus, MIC 7.5 μM (3.3
μg/mL); B. subtilis, MIC 7.5 μM (3.3 μg/mL)] potencies of 5
are consistent with those previously reported for the
structurally related diastaphenazine. This bioactivity data may
implicate a general toxicity-based mechanism of action and the
impact of the 5 C-11′-amide on cellular uptake (in contrast to
the corresponding free acid on 1−4, 6, and 7).16 Compounds
1−7 also lacked activity and/or toxicity at the concentrations
tested (≤10 μM) in our recently developed axolotl embryo tail
regeneration assay.25,31
Isolation of Streptomyces sp. PU-10A and Taxonomical
Identification. A soil sample containing PU-10A was collected from
the Himalayan mountain range (Bara Gali, Pakistan; 34°6′0″ N,
73°21′0″ E; altitude 7710 ft). Streptomyces sp. PU-10A was isolated
following previously reported methods.18,37 A full colony isolated on
M2 medium (glucose, 4.0 g; yeast extract, 4.0 g; malt extract, 10.0 g;
CaCO3, 2.0 g; agar, 18.0 g) was used to inoculate M2 broth. After 3
days of incubation at 28 °C the cell pellet was collected and genomic
DNA was prepared using UltraClean microbial DNA isolation kit
(Mo Bio Laboratories, CA, USA). The partial 16S rRNA gene
fragment was amplified using universal primers (27F, 5′-
AGAGTTTGATCMTGGCTCAG-3′; 1492R, 5′-GGTTACCTTGT-
TACGACTT-3′)38 and Advantage GC 2 polymerase (Clontech,
Mountain View, CA, USA). QIAquick gel extraction kit (Qiagen,
Valencia, CA, USA) was used to gel-purify the amplified product,
which was cloned into pGEM T-easy vector (Promega, Madison, WI,
USA) according to the manufacturer’s protocol. The corresponding
ligated plasmid construct was transformed into NEB 5-alpha
Competent E. coli (New England Biolabs, Ipswich, MA, USA)
following manufacturer’s recommendations. The plasmids were
purified using QIAprep Spin Miniprep plasmid purification kit
(Qiagen) and sequenced using plasmid forward and reverse primers.
Three different clones were sequenced for verification. The insert 16S
rRNA sequence (1370 bp) displayed 99% identity (BLAST search) to
the 16S rRNA gene sequence of Streptomyces diastaticus subsp.
ardesiacus strain NBRC 15402 and has been deposited in the NCBI
nucleotide database with the accession number KY484991.
Discussion. Given the role of phenazines in Pseudomonas
virulence and quorum-sensing, the biosynthesis of the
phenazine core scaffold (phenazine-1-carboxylic acid, phena-
zine-1,6-dicarboxylic acid, and phenazine) has been well
studied.1−6,32−36 However, the genes/enzymes that contribute
to species-specific structural/functional divergence of phena-
zines remain poorly understood. Previous studies highlighted
Streptomyces sp. IFM 11204, Streptomyces diastaticus subsp.
ardesiacus, and Streptomyces diastaticus YIM DT26 as capable
of producing izumiphenazines, diastaphenazine, and phenazi-
nolins, respectively. In contrast, the current study establishes
Streptomyces sp. PU-10A as the first reported strain to produce
diastaphenazine-type (baraphenazines D and E; 4 and 5,
respectively), phenazinolin-type (baraphenazines F and G; 6
and 7, respectively), and izumiphenazine A-type (baraphena-
zines A−C; 1−3, respectively) metabolites. Among the diverse
set of novel phenazine metabolites identified in this study,
baraphenazines A−C (1−3) stand out as the first reported
examples of fused 5-hydroxyquinoxaline/alpha-keto acid-based
phenazines. Cumulatively, this study implicates Streptomyces
sp. PU-10A as a new genomic and metabolic enabling resource
for future studies to elucidate the key late-stage biosynthetic
Fermentation, Extraction, Isolation, and Purification. The
terrestrial Streptomyces sp. PU-10A was cultivated on M2 agar plates at
28 °C for 7 days. To prepare the seed culture, small pieces of the agar
with the fully grown strain were used to inoculate three 250 mL
Erlenmeyer flasks, each containing 50 mL of medium A (glucose, 10.0
g; soluble starch, 20.0 g; yeast extract, 5.0 g; peptone, 5.0 g; NaCl, 4.0
g; CaCO3, 2 g; K2HPO4, 0.5 g; and MgSO4·7H2O, 0.5 g, dissolved in
1 L of H2O, pH 7.0; sterilized by autoclaving for 30 min at 121 °C)
and grown at 28 °C with shaking (210 rpm) for 3 days. An aliquot of
E
DOI: 10.1021/acs.jnatprod.9b00289
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Article
Scheme 2. Methylation of 1
1
seed culture (1 mL) was subsequently used to inoculate 100 250 mL
baffled flasks, each containing 100 mL of medium A. The
fermentation (10 L total) was continued at 28 °C with shaking
(210 rpm) for 8 days. After 8 days, the culture broth was combined
and centrifuged at 3000g for 30 min (4 °C). The biomass (mycelium)
was extracted with MeOH (3 × 800 mL), and the corresponding
organics were evaporated in vacuo at 40 °C to yield 28.0 g of
mycelium crude extract. The supernatant was mixed with 3% (w/v)
XAD-16 resin and stirred overnight, followed by filtration. The resin
was washed with H2O (3 × 500 mL) and then extracted with MeOH
until the eluent was colorless. The MeOH extract was subsequently
evaporated to afford 6.2 g of XAD crude extract. Both extracts
(obtained from the biomass and supernatant) revealed a similar
metabolite profile based on HPLC and TLC analyses and were
therefore combined (34.2 g).
(0.96) nm; 13C and H NMR data, see Tables 1 and 2; (+)-ESIMS
m/z 434.0 [M + H]+; (−)-ESIMS m/z 431.8 [M − H]−;
(+)-HRESIMS m/z 434.1341 [M + H]+ (calcd for C23H20N3O6,
434.1352).
Baraphenazine D (4): yellow, amorphous powder; [α]25D +15.1 (c
0.57, DMSO); UV (MeOH) λmax (log ε) 226 (9.13), 262 (2.96), 376
1
(0.76) nm; 13C and H NMR data, see Tables 1 and 2; (+)-ESIMS
m/z 437.0 [M + H]+; HRESIMS m/z 437.1252 [M + H]+ (calcd for
C25H17N4O4, 437.1250).
Baraphenazine E (5): yellow, amorphous powder; [α]25D +38.0 (c
0.51, DMSO); UV (MeOH) λmax (log ε) 228 (8.94), 260 (0.71), 372
1
(0.76) nm; 13C and H NMR data, see Tables 1 and 2; (+)-ESIMS
m/z 436.0 [M + H]+; (−)-ESIMS m/z 433.8 [M − H]−;
(+)-HRESIMS m/z 436.1407 [M + H]+ (calcd for C25H18N5O3,
436.1410).
Baraphenazine F (6): yellow, amorphous powder; [α]25D +117.2 (c
0.24, DMSO); UV (MeOH) λmax (log ε) 227 (9.09), 260 (1.80), 280
As highlighted in Scheme S1, the combined crude extract was
subjected to HP-20SS resin column chromatography (8 × 40 cm, 800
g) eluted with a gradient of aqueous CH3CN (10−100%) to yield 10
fractions, A−J. Fraction C (1.2 g) was subjected to a Sephadex LH-20
column (4 × 100 cm, 2 mL/min, MeOH) to obtain three
subfractions, C1−C3. Subfraction C2 (0.3 g) was further purified
by semipreparative HPLC (25−45% aqueous CH3CN over 30 min)
to yield compound 1 (10.2 mg, retention time: 13.4 min) as a green,
amorphous powder. Fraction D (1.7 g) was initially purified by a
Sephadex LH-20 column (4 × 100 cm, 2 mL/min, MeOH) to obtain
four subfractions, D1−D4. Subfraction D2 (0.4 g) was further purified
by semipreparative HPLC (30−35% aqueous CH3CN over 25 min)
to yield compound 4 (9.4 mg, retention time: 17.2 min) as yellow,
amorphous powders. Subfraction D3 (0.5 g) was further purified by
semipreparative HPLC (30−35% aqueous CH3CN over 22 min) to
yield compounds 2 (11.5 mg, retention time: 15.7 min), 3 (4.8 mg,
retention time: 17.0 min), and diastaphenazine (10.2 mg, retention
time: 14.5 min) as green, amorphous powders. Fraction g (2.2 g) was
purified by a Sephadex LH-20 column (4 × 100 cm, 2 mL/min,
MeOH) to obtain three subfractions, G1−G3. Subfraction G2 (0.4 g)
was further purified by semipreparative HPLC (30−55% aqueous
CH3CN over 30 min) to yield compounds 6 (3.1 mg, retention time:
19.0 min), 7 (4.0 mg, retention time: 20.6 min), and izumiphenazine
A (13.0 mg, retention time: 19.9 min) as yellow, amorphous powders.
Fraction I (2.3 g) was subjected to a Sephadex LH-20 column (4 ×
100 cm, 2 mL/min, MeOH) to obtain five subfractions, I1−I5.
Subfraction I3 (0.4 g) was further purified by semipreparative HPLC
(20−45% aqueous CH3CN over 30 min) to yield compound 5 (7.5
mg, retention time: 15.1 min) as a yellow, amorphous powder.
1
(0.21) nm; 13C and H NMR data, see Tables 1 and 2; (+)-ESIMS
m/z 469.0 [M + H]+; HRESIMS m/z 469.1176 [M + H]+ (calcd for
C25H17N4O6, 469.1148).
Baraphenazine G (7): yellow, amorphous powder; [α]25D −36.3 (c
0.18, DMSO); UV (MeOH) λmax (log ε) 227 (8.18), 272 (0.34) nm;
13C and 1H NMR data, see Tables 1 and 2; (+)-ESIMS m/z 469.0 [M
+ H]+; HRESIMS m/z 469.1176 [M + H]+ (calcd for C25H17N4O6,
469.1148).
Diastaphenazine: yellow, amorphous powder; [α]25 −278.0 (c
D
0.65, DMSO) [lit., [α]20D −1248 (c 0.11, DMSO)];16 13C NMR data,
see Table 2; (+)-ESIMS m/z 437.0 [M + H]+; HRESIMS m/z
437.1249 [M + H]+ (calcd for C25H17N4O6, 437.1250).
Izumiphenazine A: yellow, amorphous powder; [α]25 −360.3 (c
D
0.28, DMSO) [lit., [α]20 −480 (c 0.14, MeOH)];13 13C NMR data,
D
see Table 2; (+)-ESIMS m/z 469.0 [M + H]+; HRESIMS m/z
469.1127 [M + H]+ (calcd for C25H17N4O6, 469.1148).
Methylation of Compound 1.30 CH3I (0.1 mL) and Ag2O
(23.17 mg, 0.1 mmol) were added to 1 (4.1 mg, 0.01 mmol) dissolved
in tert-butyl ether (0.1 mL), and the mixture was stirred at room
temperature for 72 h. The reaction was subsequently evaporated to
dryness under vacuum, dissolved in 0.5 mL of MeOH, and purified via
semipreparative HPLC (35−70% aqueous CH3CN over 30 min) to
yield 1a (1.9 mg, 47.5% yield) and 1b (1.5 mg, 37.5% yield) (Scheme
2).
O-Permethyl-(8′S)-baraphenazine A (1a): green, amorphous
powder; 13C and 1H NMR data, see Table 3; (+)-ESIMS m/z
481.1 [M + H]+; (+)-HRESIMS m/z 481.2004 [M + H]+ (calcd for
C26H29N2O7, 481.1975); m/z 503.1823 [M + Na]+ (calcd for
C26H28N2O7Na, 503.1794).
O-Permethyl-(8′R)-baraphenazine A (1b): green, amorphous
powder; 13C and 1H NMR data, see Table 3; (+)-ESIMS m/z
481.1 [M + H]+; (+)-HRESIMS m/z 481.2002 [M + H]+ (calcd for
C26H29N2O7, 481.1975); m/z 503.1825 [M + Na]+ (calcd for
C26H28N2O7Na, 503.1794).
Decomposition of 2. Compound 2 (5.0 mg) was dissolved in 0.5
mL of MeOH with a trace of trifluoroacetic acid at room temperature
for 48 h, and the decomposition products were purified via
semipreparative HPLC (35−45% aqueous CH3CN over 25 min) to
yield two compounds [1,6-dihydroxyphenazine (2a, 2.2 mg, 44.0%
Baraphenazine A (1): green, amorphous powder; [α]25 −66.7 (c
D
0.45, DMSO); UV (MeOH) λmax (log ε) 228 (8.69), 274 (0.64) nm;
13C and 1H NMR data, see Tables 1 and 2; (+)-ESIMS m/z 411.0 [M
+ H]+; (−)-ESIMS m/z 408.9 [M − H]−; (+)-HRESIMS m/z
411.1190 [M + H]+ (calcd for C21H19N2O7, 411.1192).
Baraphenazine B (2): green, amorphous powder; [α]25D −144.9 (c
0.44, DMSO); UV (MeOH) λmax (log ε) 228 (8.72), 260 (1.46), 280
1
(0.23) nm; 13C and H NMR data, see Tables 1 and 2; (+)-ESIMS
m/z 434.0 [M + H]+; (−)-ESIMS m/z 431.9 [M − H]−;
(+)-HRESIMS m/z 434.1358 [M + H]+ (calcd for C23H20N3O6,
434.1352).
Baraphenazine C (3): green, amorphous powder; [α]25 +35.0 (c
D
0.13, DMSO); UV (MeOH) λmax (log ε) 227 (8.50), 263 (0.83), 281
F
DOI: 10.1021/acs.jnatprod.9b00289
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Journal of Natural Products
Article
1
Table 3. H and 13C NMR (400 and 100 MHz)
ASSOCIATED CONTENT
■
Spectroscopic Data for Compounds 1a and 1b in DMSO-d6
(δ in ppm)
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00289.
compound 1a
δH, mult. (J in
compound 1b
δH, mult. (J in
no.
δC, type
Hz)
δC, type
Hz)
Workup isolation scheme; 1D/2D NMR and HRMS
spectra of isolated compounds (PDF)
1
155.1, C
155.2, C
2
3
4
4a
5a
6
7
8
108.3, CH 7.26, d (7.8)
130.1, CH 7.74, t (7.8, 8.6)
119.7, CH 7.58, d (8.4)
142.6, C
108.5, CH 7.29, d (7.9)
130.6, CH 7.77, t (7.7, 8.4)
119.8, CH 7.60, d (8.5)
142.6, C
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: khaled_shaaban@uky.edu.
*E-mail: jsthorson@uky.edu.
149.3, C
149.0, C
81.3, CH
81.7, CH
41.7, CH
4.66, d (3.7)
5.14, dd (3.3, 8.2) 78.7, CH
3.23, m 40.4, CH
80.4, CH
4.65, br s
4.82, d (8.8)
3.44, m
ORCID
Xiachang Wang: 0000-0002-1106-1904
Yinan Zhang: 0000-0002-0362-1473
Sherif I. Elshahawi: 0000-0003-1879-1552
Steven G. Van Lanen: 0000-0003-1539-2732
Imran Sajid: 0000-0002-4781-1455
Khaled A. Shaaban: 0000-0001-7638-4942
Jon S. Thorson: 0000-0002-7148-0721
9α
9β
9a
34.0, CH2 3.06, m
3.40, m
153.9, C
31.2, CH2 2.68, d (14.6)
3.37, m
153.5, C
10a
1′
2′
3′
4′
5′
6′
7′
8′
9′
132.5, C
158.3, C
132.7, C
158.6, C
113.5, CH 6.85, d (8.2)
129.1, CH 7.08, d (8.4)
129.5, C
129.1, CH 7.08, d (8.4)
113.5, CH 6.85, d (8.2)
59.6, CH
109.8, C
167.3, C
113.3, CH 6.85, d (6.9)
130.6, CH 7.09, d (7.2)
126.0, C
130.6, CH 7.09, d (7.2)
113.3, CH 6.85, d (6.9)
57.0, CH
104.6, C
167.4, C
Notes
The authors declare the following competing financial
interest(s): J.S.T. is a co-founder of Centrose (Madison, WI,
USA).
3.05, d (5.1)
2.49, d
ACKNOWLEDGMENTS
■
1-OCH3 55.9, CH3 4.02, s
6-OCH3 57.2, CH3 3.37, s
1′-OCH3 54.9, CH3 3.72, s
8′-OCH3 50.8, CH3 2.60, s
9′-OCH3 51.5, CH3 3.23, s
55.9, CH3 4.02, s
56.7, CH3 3.26, s
54.9, CH3 3.74, s
50.8, CH3 3.22, s
52.0, CH3 3.38, s
This work was supported by National Institutes of Health
grants R24 OD21479 (J.S.T.) and T32 DA016176 (Y.Z.), the
University of Kentucky College of Pharmacy, the University of
Kentucky Markey Cancer Center and the National Center for
Advancing Translational Sciences (UL1TR000117 and
UL1TR001998), and the Higher Education Commission
(HEC) Pakistan grant (HEC-NRPU Project 2121).
yield) and the enol form of indole-3-pyruvic acid (2b, 1.7 mg, 34.0%
yield)].
REFERENCES
■
1,6-Dihydroxyphenazine (2a): green, amorphous powder; 1H
NMR (400 MHz, DMSO-d6) δ 7.20 (2H), 7.70 (2H), 7.83
(2H),10.35 (2H, br s); 13C NMR (100 MHz, DMSO-d6) δ 109.1
(C-2,7), 118.6 (C-4,9), 132.60 (C-3,8), 132.67 (C-5a, 10a), 143.7 (C-
4a, 9a), 153.2 (C-1,6); (+)-ESIMS m/z 213.1 [M + H]+; (−)-ESIMS
m/z 210.9 [M − H]−.
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G
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