Journal of Natural Products
Article
death. Comparison of the minimum bactericidal concen-
trations (MBCs) on days 7 and 14 revealed the time-
dependent bactericidal activity of the RUFs. Importantly, the
RUFs also showed strong activity against M. abscessus, the
cause of many drug-resistant nontuberculous mycobacterial
infections.
were extracted with ethyl acetate and dried. The Streptomyces strain
MJM3502 was identified as being identical with Streptomyces atratus
(
NRRL B-16927) through classification using the 16S rDNA
12
sequence.
Extraction and Isolation. The whole broth of a 500 L culture of
MJM3502 was extracted with a 2 times volume of methanol and then
concentrated under vacuum at 40 °C to 1/10th volume. After adding
deionized water to the concentrated solution to achieve 90%
methanol, the mixture was defatted by partitioning with ethyl acetate.
The methanol−water layer was extracted with one volume of ethyl
acetate twice, and this was cleaned by shaking with one volume of
deionized water (pH 3.0). The ethyl acetate layer was evaporated to
dryness to yield a yellowish solid. A RUF-enriched fraction (28 g;
aliquot of 41 g extracted from a total of 350 L of culture broth) was
obtained from 3502 EtOAc extract by silica gel CC with a hexane/
EtOAc (5:5) eluent. The RUF-enriched fraction was further
chromatographed on silica gel using a gradient elution of hexane/
acetone (7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, and 1:5) to give six
fractions (A−F). From fraction B (3 g), 300 mg of rufomyazine (9)
was purified by recrystallization. The remainder was separated by
Concluding Remarks. With the isolation and identifica-
tion of eight new compounds (1−8), this study expands the
chemical space of RUFs to 12 natural congeners. This has
enabled SAR studies aimed at optimizing ClpC1 binding
affinities in the context of the ClpC1-NTD-RUF complex
11
(
PDB entry code 6cn8). The newly characterized RUFs,
targeting the NTD of ClpC1, exhibited variable in vitro anti-
TB activity and selectivity indices. While some of the new
compounds exhibited similarly high potencies to 12, no new
analogues with even higher potency were encountered.
Considering that this study mined the minor constituents of
the S. atratus strain MJU3502 metabolome, the outcomes
indicate that 12 might be the most potent of the naturally
produced RUFs. Semisynthetic modifications of RUFs are one
logical next step and are ongoing in our laboratory. Notably,
the anti-M. abscessus activities of the RUFs highlight the
potential of RUFs to serve as more universal antimycobacterial
drug leads. In addition to structural modification, other
ongoing work includes microsome stability and PK/PD studies
of this class of potential anti-TB leads.
silica gel CC using a gradient elution of CHCl /MeOH (200:1, 100:1,
3
50:1, 30:1, 20:1, and 10:1), then further purified by semipreparative
HPLC (60% CH CN in H O, 2.5 mL/min) to give compounds 4 (20
3
2
mg) and 10 (50 mg). Similarly, ∼6 g of RUF I and II mixtures (12)
was purified from fraction C (10 g) by a series of columns packed
with silica gel (CHCl /MeOH) and Sephadex LH-20 (MeOH or
3
EtOH). Compounds 6 (3.5 mg), 11 (2.1 mg), and 13 (8.1 mg) were
then purified by semipreparative HPLC (60% CH CN in H O, 2.5
3
2
mL/min) from the remaining material of fraction C. Fractions D (1.0
g), E (1.7 g), and F (1.7 g) were each chromatographed on a silica gel
EXPERIMENTAL SECTION
General Experimental Procedures. Fourier transform infrared
FTIR) spectra were recorded on a Nicolet 6700 FTIR spectropho-
■
(
column using a gradient elution of CHCl /MeOH (100:1, 50:1, 30:1,
3
2
0:1, and 10:1), then purified by semipreparative HPLC (45−60%
CH CN in H O, 2.5 mL/min) to give 1 (100 mg), 2 (10 mg), 3 (10
3
2
tometer. Optical rotation values were measured on a PerkinElmer 241
polarimeter at room temperature; concentrations are reported in g/
mg), 5 (12 mg), 7 (3 mg), and 8 (10 mg).
Rufomycin NBZ1 (1): pale yellow crystals; [α]
25
D
−82 (c 1.2,
100 mL. All 1D/2D NMR spectra were acquired on a JEOL (JEOL
MeOH); IR (KBr) νmax 3275, 2955, 2872, 1628, 1539, 1512, 1456,
Resonance Inc., Peabody, MA, USA) ECZ 400S spectrometer. For
UHPLC analysis, a Shimadzu (Kyoto, Japan) Nexera UHPLC-UV
system equipped with a DAD was used, employing a Kinetex 1.7 μm
XB-C18 100 Å column (50 mm × 2.1 mm) and LabSolutions software
for data analysis. The column oven, detector cell, and autosampler
temperature were maintained at 40, 40, and 4 °C, respectively,
throughout the analysis. UHPLC-HRESIMS and ESIMS/MS spectra
were carried out by using a Bruker Impact II, quadrupole time-of-
flight (qTOF) equipped with a Shimadzu UHPLC (Kyoto, Japan).
The ion source was operated in the positive electrospray ionization
mode using a capillary voltage at 4.0 kV, nebulizer and drying gas
−1
1
13
1
327, 1244, 1084, 966, 758 cm ; H and C NMR (CD OD), see
3
+
Table 1; (+)-HRESIMS m/z 1044.5760 [M + H] (calcd for
C H N O , 1044.5764).
5
4
78
9
12
25
Rufomycin NBZ2 (2): pale yellow, amorphous solid; [α] −41 (c
D
0
1
.6, MeOH); IR (KBr) ν 3275, 2955, 1628, 1539, 1456, 1317,
max
−1
1
13
245, 1207, 968, 750 cm ; H and C NMR (CD OD), see Table 1;
3
+
(+)-HRESIMS m/z 1044.5782 [M + H] (calcd for C H N O ,
54 78 9 12
1
044.5764).
Rufomycin NBZ3 (3): pale yellow, amorphous solid; [α]25 −118
D
(
c 0.3, MeOH); IR (KBr) νmax 3271, 2927, 2856, 1630, 1537, 1456,
−
1
1
13
1317, 1205, 1084, 968, 750 cm ; H and C NMR (CD OD), see
3
(
N ) at 0.4 bar and 4.0 L/min, respectively, dry temperature of 225
2
+
Table 1; (+)-HRESIMS m/z 1028.5853 [M + H] (calcd for
°
C, and mass scan range set from m/z 50 to 2000. The MS/MS
spectra were acquired using varied collision energy ranging from 20 to
C H N O , 1028.5815).
5
4
78
9
11
25
D
Rufomycin NBZ4 (4): white, amorphous solid; [α] −70 (c 0.2,
70.0 eV. A 10 mM sodium formate solution was introduced to the ion
MeOH); IR (KBr) ν 3270, 2956, 2864, 1629, 1516, 1456, 1234,
source as the internal calibration. The separation was performed on a
CORTECS C18 (100 × 3.0 mm, 2.7 μm) UHPLC column. Data were
collected and processed by the Data Analysis 4.4 software (Bruker
Daltonik GmbH, Germany). Sephadex LH-20 (Pharmacia, Uppsala,
Sweden) and silica gel (ICN EcoChrom 32−63, 60 Å) was used for
column chromatography (CC). Semipreparative HPLC was per-
formed on a Shimadzu HPLC (Kyoto, Japan) connected to a PDA
detector (Shimadzu, model SPD-20A) and equipped with a Kinetex
EVOC18 (250 × 10 mm, S-5, 100 Å) column. TLC was analyzed by
UV detector and vanillin-sulfuric acid spray (3 g vanillin, 95 mL
ethanol, and 1.5 mL sulfuric acid). All solvents used were obtained
from Fisher Scientific (Fair Lawn, NJ, USA) or Sigma-Aldrich (St.
Louis, MO, USA). L-FDLA and D-FDLA were purchased from
Arctom Chemicals. Amino acid standards were purchased from Sigma
and AmBeed.
max
−
1
1
13
968, 922, 750 cm ; H and C NMR (CD OD), see Table 1;
3
+
(+)-HRESIMS m/z 967.5990 [M + H] (calcd for C H N O ,
54 79 8 8
9
67.6015).
Rufomycin NBZ5 (5): pale yellow, amorphous solid; [α]25 −70 (c
D
1.1, MeOH); IR (KBr) νmax 3271, 2956, 1628, 1537, 1456, 1315,
−
1
1
13
1
244, 1207, 1182, 1072, 966, 748 cm ; H and C NMR (CD OD),
3
+
see Table 2; (+)-HRESIMS m/z 1088.6021 [M + H] (calcd for
C H N O , 1088.6027).
56 82
9
13
2
5
Rufomycin NBZ6 (6): pale yellow, amorphous solid; [α] −81 (c
D
0
.1, MeOH); IR (KBr) ν 3286, 1628, 1522, 1433, 1327, 1234,
max
−
1
1
13
1171, 1089, 752 cm ; H and C NMR (CD OD), see Table 2;
3
+
(+)-HRESIMS m/z 1014.5695 [M + H] (calcd for C53H N O ,
76 9 11
1014.5659).
Rufomycin NBZ7 (7): pale yellow, amorphous solid; [α]25
−57 (c
D
Strain Material. The strain MJM3502 was obtained from the
Extract Collection of Useful Microorganisms (ECUM) at Myongji
University, Republic of Korea, and was fermented in glucose-soybean
starch medium (rich medium). The culture medium supernatants
0.1, MeOH); IR (KBr) νmax 3304, 2929, 2872, 1539, 1456, 1323,
1257, 1082, 956, 744 cm ; H and C NMR (CD
(+)-HRESIMS m/z 1060.5703 [M + H] (calcd for C H N O ,
−
1
1
13
OD), see Table 2;
3
+
5
4
78
9
13
1060.5714).
I
J. Nat. Prod. XXXX, XXX, XXX−XXX