4
Tetrahedron
ACCEPTED MANUSCRIPT
4.2. Collection and Gene Sequencing
4.5. Absolute Configurations of Ahp Unit
Samples of the marine cyanobacterium, Caldora penicillata
Kyanamide (1, 1.0 mg) was dissolved in CH2Cl2 (1.0 mL), and
PCC (4.5 mg) was added. The reaction mixture was allowed to
stand for 13 h at room temperature, after which it was washed
with H2O. The CH2Cl2 portion was dried under argon. The PCC-
oxidized kyanamide was heated with 9 M HCl (1.0 mL) at 110
˚C for 22 h. The hydrolyzed product was evaporated to dryness
and added with 0.1% solution of Nα-(5-fluoro-2, 4-
were collected by hand from the coast of Kyan, Okinawa
Prefecture, Japan in June 2017. Small pieces of collected marine
cyanobacteria were preserved for genetic analysis in RNAlater
(Qiagen). The cyanobacterium was identified by 16S rRNA
sequence analysis (see Supporting Information).
4.3. Extraction and Isolation
dinitrophenyl)-L-alaninamide (L-FDAA, Marfey’s reagent, 200
µL) in acetone and 0.5 M NaHCO3 (300 µL) followed by heating
at 40 ˚C for 90 min. After cooling to room temperature, the
reaction mixture was neutralized with 2M HCl (75 µL) and
diluted with MeOH (1.0 mL). To reveal the absolute
configuration of the Glu unit originated from the Ahp moiety, the
solution was subjected to reversed-phase HPLC [Cosmosil 5C18-
AR-II (4.6 × 250 mm), MeOH/20 mM AcONa = 25:75 (solvent
Approximately 414.8 g (wet weight) of the cyanobacterial
samples were extracted with MeOH (700 mL) at room
temperature for 4 days. The extract was filtered, and the filtrate
was concentrated. The residue was partitioned between H2O (0.2
L) and EtOAc (0.2 L × 3). The material obtained from the
organic layer was further partitioned between 90% aqueous
MeOH (0.1 L) and n-hexane (0.1 L × 3). The aqueous MeOH
fraction (589.0 mg) was separated by column chromatography on
ODS (5.0 g) using 20% aqueous MeOH, 90% aqueous MeOH,
and MeOH. The fraction (143.6 mg) eluted with 90% aqueous
MeOH was subjected to reversed-phase HPLC [Cosmosil 5C18-
AR-II (20 × 250 mm), 70% aqueous MeOH at 5.0 mL/min, UV
detection at 215 nm] to yield kyanamide (1, 9.5 mg, tR = 36.0
min).
E) at 1.0 mL/min, UV detection at 340 nm]. The
L-FDAA
derivatives of standard amino acids were prepared by the same
procedure as that mentioned above. The retention times (min) of
the authentic standards were as follows:
(9.2) in solvent E. The retention time and ESIMS product ion
(m/z [M + Na]+) of the
-FDAA derivative of Glu from the
hydrolysate was 7.7 min (422.1) in solvent E, proving the
configuration of Glu was
L-Glu (7.7) and D-Glu
L
L.
Kyanamide (1): colorless oil; [α]27 – 17.9 (c 0.85, MeOH);
D
1
For H NMR and 13C NMR data, see Table 1; HRESIMS m/z
4.6. Absolute Configurations of Gln and N-Me-Trp Residues in 1.
1022.5324 [M + Na]+ (calcd for C52H73N9O11Na, 1022.5327).
Kyanamide (1, 1.8 mg) was treated with 6 M HCl containing
1% Phenol (500 µL) at 110 ˚C for 16 h. The hydrolysate
subjected to HPLC [Consmosil 5C18-AR-II (10 × 250 mm),
MeOH/H2O = 20:80 with 0.1% TFA at 5.0 mL/min, UV
detection 215 nm] to yield Glu and N-Me-Trp. The Glu was
4.4. Absolute Configurations of Amino Acid Residues except for
N-Me-Trp and Gln Residues
Kyanamide (1, 1.0 mg) was treated with 9 M HCl (500 µL) at
110 ˚C for 18 h. The hydrolysate was subjected to HPLC
[Cosmosil HILIC (4.6 × 250 mm), MeCN/10 mM AcONH4 =
85:15 at 1.0 mL/min, UV detection at 215 nm] to yield Leu, Phe,
Val, and Thr. Each amino acid was added with 0.1% solution of
added with 0.1% solution of α-(5-fluoro-2, 4-dinitrophenyl)-
L-
alaninamide ( -FDAA, Marfey’s reagent, 200 µL) in acetone and
L
0.5 M NaHCO3 (200 µL) followed by heating at 40 ˚C for 90 min.
After cooling to room temperature, the reaction mixture was
neutralized with 2M HCl (50 µL) and diluted with MeOH
(500 µL). The N-Me-Trp was added with 0.1% solution of α-(5-
Nα-(5-fluoro-2,
4-dinitrophenyl)-
L-alaninamide
(L-FDAA,
Marfey’s reagent, 200 µL) in acetone and 0.5 M NaHCO3
(300 µL) followed by heating at 40 ˚C for 90 min. After cooling
to room temperature, the reaction mixture was neutralized with 2
M HCl (75 µL) and diluted with MeOH (1.0 mL). The solution
was subjected to reversed-phase HPLC [Cosmocsil 5C18-AR-II
(4.6 × 250 mm), MeOH/20 mM AcONa = 60:40 (solvent A),
55:45 (solvent B), 50:50 (solvent C), 35:65 (solvent D) at 1.0
mL/min, UV detection at 340 nm]. The L-FDAA derivatives of
standard amino acids were prepared by the same procedure. The
retention times (min) of the authentic standards were as follows:
fluoro-2, 4-dinitrophenyl)-L-leucinamide (L-FDLA, Marfey’s
reagent, 200 µL) in acetone and 0.5 M NaHCO3 (100 µL)
followed by heating at 40 ˚C for 90 min. After cooling to room
temperature, the reaction mixture was neutralized with 2M HCl
(25 µL) and diluted with MeOH (500 µL). The solution was
subjected to reversed-phase HPLC [Cosmocsil 5C18-AR-II (4.6 ×
250 mm), MeOH/20 mM AcONa = 60:40 (solvent A), 25:75
(solvent E) at 1.0 mL/min, UV detection at 340 nm]. The
L-
FDLA derivative, -FDLA derivative, and -FDAA derivatives
D
L
L
-Leu (7.0) and
(19.3) in solvent B,
Thr (7.1) and -Thr (20.0) and
in solvent D. The retention time and ESIMS product ion (m/z [M
+ Na]+) of the
-FDAA derivative of Leu from the hydrolysate
was 7.0 min (406.0) in solvent A, proving the configuration of
Leu was . The retention time and ESIMS product ion (m/z [M +
Na]+) of the
-FDAA derivative of Phe from the hydrolysate was
7.9 min (440.2) in solvent B, proving the configuration of Phe
was . The retention time and ESIMS product ion (m/z [M +
Na]+) of the
-FDAA derivative of Val from the hydrolysate was
8.1 min (392.2) in solvent C, proving the configuration of Val
was . The retention time and ESIMS product ion (m/z [M +
Na]+) of the
-FDAA derivative of Thr from the hydrolysate was
7.1 min (394.1) in solvent D, proving the configuration of Thr
was
D
-Leu (15.9) in solvent A,
-Val (8.1) and -Val (23.5) in solvent C,
-allo-Thr (6.7), -allo-Thr (11.7)
L-Phe (7.9) and D-Phe
of standard amino acids were prepared by the same procedure as
that mentioned above. The retention times (min) of the authentic
L
D
L-
D
L
D
standards were as follows: N-Me-
-Trp- -FDLA (9.5) in solvent A,
Glu- -FDAA (9.2) in solvent E. The retention time and ESIMS
product ion (m/z [M + Na]+) of the
-FDLA derivative of N-Me-
Trp from the hydrolysate was 6.0 min (535.1) in solvent A,
respectively, proving the configurations of N-Me-Trp was . The
retention time and ESIMS product ion (m/z [M + Na]+) of the
L-Trp-
L-FDLA (6.0) and N-Me-
L
D
L-Glu-
L-FDAA (7.7) and
D-
L
L
L
L
L
L
L-
L
FDAA derivative of Glu from the hydrolysate was 7.7 min
(422.1) in solvent E, respectively, proving the configurations of
Gln was L.
L
L
4.7. Cell growth analysis.
L
HeLa S3 cells were cultured at 37 ˚C with 5% CO2 in DMEM
(Sigma) containing 10% heat-inactivated FBS, 100 µg/mL
streptomycin, and 100 units/mL penicillin. HeLa S3 cells were
seeded at 5.0 × 103 cells/well in 96-well plates and cultured
overnight. Various concentrations of compound were added and
L.