Journal of Natural Products
Article
H-6b), 1.33 (1H, m, H-15a), 1.25 (3H, s, CH -20), 1.24 (1H, m, H-
amino acid in the hydrolysate under the same HPLC conditions was
25.4, indicating the presence of D-Leu in the hydrolysate. The
stereochemistry of the α-hydroxy acid was determined using a
different chiral column for the HPLC analysis [CHIRALPAK MA (+)
(4.6 × 50 mm), Diacel Chemical industries, Ltd.; solvent, 2.0 mM
3
1
2
2
5b), 0.96 (3H, d, J = 6.2 Hz, CH -27), 0.95 (3H, d, J = 7.5 Hz, CH -
3
3
1), 0.91 (3H, t, J = 6.3 Hz, CH -16), 0.85 (3H, d, J = 6.1 Hz, CH -
3
3
1
3
6), 0.84 (3H, d, J = 6.8 Hz, CH -18); C NMR (CD OD) δ 174.6
3
3
(
(
(
C, C-28), 173.6 (C, C-1), 172.5 (C, C-22), 102.1 (C, C-9), 81.2
CH, C-13), 80.1 (CH, C-7), 77.7 (CH, C-3), 74.7 (C, C-8), 74.2
CuSO −MeCN (95:5); flow rate, 1.0 mL/min; detection at 254 nm].
4
CH, C-29), 70.8 (CH, C-5), 66.7 (CH, C-11), 57.7 (OCH , C-19),
The t min for authentic standards were 7.0 for R-Hba and 10.0 for S-
3
R
5
7.4 (OCH , C-17), 50.5 (CH, C-23), 42.8 (CH, C-12), 38.8 (CH ,
Hba. The retention time of the α-hydroxy acid in the hydrolysate
under these conditions was 10.0, indicating the presence of S-Hba in
the hydrolysate.
3
2
C-24), 38.7 (CH, C-4), 37.3 (CH , C-10), 35.4 (CH , C-14), 33.6
2
2
(
CH , C-2), 32.5 (CH , C-30), 31.4 (CH , C-6), 30.5 (CH , C-31),
2 2 2 2
2
6.0 (CH, C-25), 23.6 (CH , C-27), 21.1 (CH , C-26), 20.3 (CH ,
Single-Crystal X-ray Structure Determination of des-
Thiomethyllooekeyolide A (3). A selected colorless crystal of 3
was used in this study. X-ray intensity data were collected at 100 K on
a Bruker DUO diffractometer using Cu Kα radiation (λ = 1.541 78
Å), from an ImuS power source, and an APEXII CCD area detector.
Raw data frames were read by the program SAINT and integrated
using 3D profiling algorithms. The resulting data were reduced to
produce hkl reflections and their intensities and estimated standard
deviations. The data were corrected for Lorentz and polarization
effects, and numerical absorption corrections were applied based on
indexed and measured faces. The structure was solved and refined in
SHELXTL2013, using full-matrix least-squares refinement. The non-
H atoms were refined with anisotropic thermal parameters, and all of
the H atoms were calculated in idealized positions and refined riding
on their parent atoms. The structure refined with this data confirms
the absolute configuration to be correct as presented here based on
3
3
2
C-15), 19.6 (CH , C-20), 15.2 (CH , C-32), 14.3 (CH , C-16), 12.0
3
3
3
(
CH , C-21), 9.7 (CH , C-18); HRESI/APCIMS m/z 686.3528 [M +
3
3
+
Na] (calcd for C H NO SNa, 686.3545).
32
57
11
2
5
D
Looekeyolide B (2): white, amorphous powder; [α]
+36.1 (c
0
.22, MeOH); UV (MeOH) λmax (log ε) 220 (3.44); IR (solid smear)
νmax 3400, 2957, 2934, 1736, 1679, 1555, 1151, 1095, 1052, 1032,
−
1
1
13
1
010, 752 cm ; H NMR, C NMR, DQF COSY, HMBC, and
NOESY data, see Table 1; HRESI/APCIMS m/z 702.3528 [M +
+
Na] (calcd for C H NO SNa, 702.3493).
32
57
12
Desulfurization. Looekeyolide B (2, 4.5 mg) was dissolved in
EtOH (0.5 mL) and treated with an excess of fresh Raney-Ni (2400)
as a slurry in H O (0.1 mL) and refluxed for 0.5 h. The product was
filtered and concentrated to give a white solid. This solid was further
purified by reversed-phase HPLC (semipreparative, 5 μm, RP-C )
using MeOH−H O (8.5−1.5) to give 3.7 mg of the desulfurized
2
1
8
2
compound des-thiomethylooekeyolide A (3). This material on
crystallization in a mixture of benzene and hexanes (1:95) yielded
colorless crystals of des-thiomethylooekeyolide A (3). A selected
crystal from this batch was used in X-ray crystallography studies.
des-Thiomethylooekeyolide A (3): colorless crystals; mp 196−197
the value of the Flack x parameter of 0.02(16). All H O and hydroxy
2
protons were obtained from a difference Fourier map and refined
freely. All of those protons are involved in an extensive network of
hydrogen bonding. In the final cycle of refinement, 6020 reflections
(of which 5945 are observed with I > 2σ(I)) were used to refine 419
°
(
1
C; [α]25 +29.3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 220
3.27); IR (solid smear) νmax 3303, 2929, 2934, 1737, 1667, 1644,
parameters, and the resulting R , wR , and S (goodness of fit) were
1 2
D
3.22%, 8.91%, and 1.076, respectively. The refinement was carried out
−
1
1
2
551, 1458, 1277, 1152, 1095, 1012, 969, 935, 752 cm ; H NMR
by minimizing the wR function using F rather than F values. R is
2 1
(
(
600 MHz, CD OD) δ 4.96 (1H, dd, J = 9.6, 6.1 Hz, H-13), 4.72
1H, dd, J = 11.0, 1.8 Hz, H-11), 4.71 (1H, dd, J = 8.4, 4.8 Hz, H-29),
calculated to provide a reference to the conventional R value, but its
function is not minimized.
3
4
3
4
.55 (1H, dd, 10.8, 4.2 Hz, H-23), 4.38 (1H, dd, J = 10.9, 4.8 Hz, H-
), 3.62 (1H, ddd, J = 10.2, 10.2, 3.0 Hz, H-5), 3.44 (1H, dd, J = 12.0,
.8 Hz, H-7), 3.39 (3H, s, OCH -19), 3.33 (3H, s, OCH -17), 2.56
Identifying the Biosynthetic Gene Cluster. DNA was
extracted from the BBD layer or from the unicyanobacterial culture
with a PowerSoil DNA isolation kit (MoBio) or Qiagen AllPrep
3
3
11
(
1H, d, J = 17.8, 10.9 Hz, H-2a), 2.36 (1H, dd, J = 17.8, 10.9 Hz, H-
DNA/RNA Micro kit, as previously described. Metagenomic
libraries were constructed with a TruSeq DNA sample preparation
kit (Illumina, San Diego, CA, USA) and sequenced at the University
of Maryland Institute for Bioscience and Biotechnology Research on
an Illumina HiSeq with a 100 bp paired-end protocol. The
unassembled, quality-filtered reads are publicly available through
NCBI’s Sequence Read Archive (SRA) under the BioProject ID
PRJNA269585. Quality-filtering, metagenomic assembly, and the
recovery of metagenome-assembled genomes were previously
2
4
b), 2.22 (1H, m, H-4), 2.16 (1H, m, H-14a), 2.09 (1H, ddd, J = 11.7,
.8, 2.7 Hz, H-6a), 1.89 (1H, dd, J = 14.4, 11.6 Hz, H-10a), 1.80 (1H,
m, Ha-30), 1.76 (1H, m, Hb-30), 1.74 (1H, m, H-24a), 1.68 (1H, dd,
J = 14.4, 2.4 Hz, H-10b), 1.64 (1H, m, H-25), 1.62 (1H, m, H-24b),
1
=
.53 (1H, m, H-14b), 1.49 (1H, q, J = 7.5 Hz, H-12), 1.42 (1H, ddd, J
11.7, 11.7, 11.7 Hz, H-6b), 1.33 (1H, m, H-15a), 1.25 (3H, s, CH3-
0), 1.24 (1H, m, H-15b), 1.02 (3H, t, J = 7.5 Hz, CH -31), 0.96
2
3
(
(
(
1
8
7
3H, d, J = 6.2 Hz, CH -27), 0.95 (3H, d, J = 7.5 Hz, CH -21), 0.91
3H, t, J = 6.3 Hz, CH -16), 0.85 (3H, d, J = 6.1 Hz, CH -26), 0.84
3H, d, J = 6.8 Hz, CH -18); C NMR (CD OD) δ 174.9 (C, C-28),
3
3
11
described. Biosynthetic gene clusters in the Roseofilum MAGs
were identified with antiSMASH v. 3.0 to predict open reading frames
3
3
1
3
3
3
26
73.6 (C, C-1), 172.5 (C, C-22), 102.1 (C, C-9), 81.2 (CH, C-13),
0.1 (CH, C-7), 77.7 (CH, C-3), 76.8 (CH, C-29), 74.7 (C, C-8),
0.8 (CH, C-5), 66.7 (CH, C-11), 57.7 (OCH , C-19), 57.4 (OCH ,
(ORFs). The predicted ORFs were annotated by performing
BLASTp against the NCBI database. Sequence motifs of KR, AT, and
MT domains were determined after protein sequence alignment by
ClustalW, and the corresponding figures were prepared with Jalview
3
3
C-17), 50.5 (CH, C-23), 42.7 (CH, C-12), 38.8 (CH , C-24), 38.7
2
27
(
CH, C-4), 37.3 (CH , C-10), 35.4 (CH , C-14), 33.6 (CH , C-2),
2. The specificity codes of A domains were predicted with
2
2
2
28
3
2
1.4 (CH , C-6), 26.1 (CH , C-30), 25.8 (CH, C-25), 23.6 (CH , C-
NRPSpredictor2.
H O Activity of Looekeyolide A (1) and Looekeyolide B (2).
2
2
3
7), 21.1 (CH , C-26), 20.3 (CH , C-15), 19.7 (CH , C-20), 14.3
3
2
3
2
2
(
CH , C-16), 12.0 (CH , C-21), 10.1 (CH , C-31), 9.7 (CH , C-18);
Serial concentrations of stock solutions of looekeyolide mixture
(1:2:lyngbic acid 1:1:1, estimated by NMR), 2 alone, and lyngbic acid
alone dissolved in DMSO (0.5 μL each) were incubated with 49.5 μL
of 10 μM H O (prepared using the buffer in the assay kit) at 37 °C in
3
3
3
3
+
HRESI/APCIMS m/z 640.3685 [M + Na] (calcd for
C H NO Na, 640.3667).
31
55
11
Acid Hydrolysis and Chiral HPLC Analysis. des-Thiomethy-
2
2
looekeyolide A (3, 0.2 mg) was suspended in 6 N HCl (0.3 mL) and
heated at 115 °C for 18 h in a sealed tube. The hydrolysate was
concentrated to dryness. The residue was reconstituted in 0.2 mL of
a 96-well plate. Equivalent amounts of DMSO were used as controls.
After a 24 h treatment, the H O level was measured using the
2
2
fluorimetric H O assay kit (Sigma-Aldrich) according to the
2
2
H O and analyzed by chiral HPLC, comparing the retention times
with those of authentic standards [Phenomenex Chirex (D)
manufacturer’s instructions (n = 2). Briefly, the master mix was first
prepared by mixing the red peroxidase substrate and the horseradish
peroxidase with the assay buffer. Master mix (50 μL) was added to
each well, and the plate was incubated for 20 min at room
temperature in the dark. The fluorescence intensity (λex = 540/λem
= 590 nm) was then recorded on a SpectraMax M5 (Molecular
2
Penicillamine, 4.6 × 250 mm, 5 μm]; detection at 254 nm. Using
the solvent mixture of 2.0 mM CuSO −MeCN (90:10), with a flow
4
rate of 1.0 mL/min, the retention times (tR min) for authentic
standards were 22.2 for L-Leu and 25.4 for D-Leu. The t min of the
R
I
J. Nat. Prod. XXXX, XXX, XXX−XXX