Journal of Natural Products
Article
under a 12:12 h light/dark regimen, in ASM1 growth medium. The
cultured cells were harvested by centrifugation after a period of 4
weeks of incubation, lyophilized, and kept at −20 °C until extraction.
Extraction and Isolation. The lyophilized material (6 g) was
extracted three times with 70% aqueous MeOH (1 g/30 mL) by probe
sonication (amplitude of 30%, 2 min, Soni Omni Disruptor) and
centrifuged (7000g, 4 °C, 10 min, Eppendorf 5804R), and the
supernatants were combined. The MeOH was removed in a rotatory
evaporator system, and the aqueous suspension split in three aliquots.
These aliquots were subjected to SPE extraction (Waters Sep-Pak Vac
35 cm3 10 g C18 cartridge) and eluted with 20−100% aqueous MeOH
to yield five fractions (70 mL). The fractions were concentrated under
vacuum, and the residue dissolved in 20% aqueous MeOH and
analyzed by LCHRMS. The 40% MeOH fraction was further purified
by semipreparative HPLC (column, Phenomenex Luna RP-C18 250 ×
10 mm, 5 μm; mobile phases A (H2O) and B (MeCN), both
containing 0.1% formic acid; flow rate 4.5 mL/min) to yield
compounds 1 (2.4 mg, 0.040% yield of dry cells weight) and 2 (0.4
mg, 0.007% yield of dry cells weight). The 80% MeOH fraction
containing the compounds 3−6 was concentrated under a stream of
nitrogen (TE-concentrator, Technal), reduced with NaBH4, and
separated in the same way as 1 and 2 with slight gradient modifications
to yield 3a (1.0 mg, 0.017% yield of dry cells weight), 4a (0.8 mg,
0.014% yield of dry cells weight), 5a (0.8 mg, 0.013% yield of dry cells
weight), and 6a (0.9 mg, 0.015% yield of dry cells weight).
the L- and D-amino acid standards-FDAA derivatives were as follows
(m/z, L-, D-): Arg (427, 16.2 min, 17.2 min); Hty (448, 35.8 min, 39.2
min); Ile (384, 40.4 min, 46.1 min); Leu (384, 41.4 min, 46.7 min);
allo-Ile (384, 39.4 min, 45.0 min); Lys (399, 15.6 min, 16.5 min); Pro
(368, 29.8 min, 31.5 min); Tyr (434, 32.6 min, 35.5 min).
Compounds(3a−6a were oxidized using Jones’ reagent as
previously described.31 After evaporation the products were hydro-
lyzed and subjected to Marfey’s analyses as described above.
2-Hydroxy-4-(4-hydroxyphenyl)butanoic acid. The acid hy-
drolysates (1 mL) of compounds 3a−6a were extracted with ethyl
ether (1 mL × 3) to separate the hydroxy acid from the amino acids
mixture. The ether was removed and the residue dissolved in MeOH
(1 mL). The hydroxy acid from the authentic samples and a standard
of Hhpba (AnalytiCon Discovery) were subjected to LCMS analysis to
confirm their identity using the same chromatographic conditions as
described for Marfey’s analyses.
Protease Inhibition Assays. Chymotrypsin, thrombin, carbox-
ypeptidase A, and their respective colorimetric substrates were all
purchased from Sigma Chemical Co. The thrombin and chymotrypsin
inhibitory activities were measured using the methods reported by
Zafrir-Ilan and Carmeli,31 Anas et al.,12 and Shin et al.35 with minor
modifications. Thrombin was dissolved in a Tris-imidazole buffer (pH
8.2) containing NaCl (30 mM) to prepare a 5.79 U/mL solution.
Chymotrypsin was dissolved in 50 mM Tris-HCl (pH 7.6), 1 mM of
CaCl2, and 100 mM NaCl to prepare a 50 U/mL solution. Substrates
were prepared as follows: N-benzoyl-Phe-Val-Arg-p-nitroanilide hydro-
chloride/DMSO (6 mg/600 μL)/20-fold volume of Tris-imidazole
buffer for thrombin and N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide/50
mM Tris-HC1 (pH 7.6, 1 mg/mL) for chymotrypsin. For the
thrombin protease inhibitory activity assay, 90 μL of enzyme solution
and 20 μL of sample solution previously dissolved in DMSO were
added to each microtiter plate well and preincubated at 37 °C for 5
min. Then, 90 μL of substrate solution was added to start the reaction,
and the absorbance was read at 405 nm before and after 30 min of
incubation at 37 °C. For chymotrypsin, 30 μL of Tris-HCl buffer, 50
μL of enzyme, and 20 μL of test sample were added to each microtiter
plate well and preincubated at 37 °C for 5 min. Then, the substrate
solution (100 μL) was added and the absorbance was read at 405 nm
before and after 30 min. In both assays the 4-(2-aminomethyl)-
benzenesulfonyl fluoride hydrochloride was used as a positive control.
Carboxypeptidase A inhibition activity was measured according to the
manufacturer’s instructions. Briefly, 2 μL of enzyme, 1 μL of test
substances, and 97 μL of ultrapure H2O were added to each microtiter
plate well. Then, an aliquot of 100 μL of the substrate solution (N-4-
(methoxyphenylazoformyl)-Phe-OH) was added to start the reaction.
After 5 min of incubation at 25 °C, 100 μL of the sodium carbonate
solution was added to stop the reaction and the absorbance was read at
350 nm. A carboxypeptidase inhibitor from potato tuber was used as
positive control.
Namalide B (1): white, amorphous powder; [α]20 −8 (c 0.75,
D
1
MeOH); UV (MeOH) λmax (log ε) 277 (3.06) nm; H, 13C, and 15N
NMR data (800 MHz for 1H, 201 MHz for 13C, and 81 MHz for 15N,
DMSO-d6) Table 1; HRMS (ESI-QTOF) m/z 576.3400 [M + H]+
(calcd for C29H46N5O7, 576.3392; Δ −1.4 ppm).
Namalide C (2): white, amorphous powder; [α]20 −34 (c 0.1,
D
1
MeOH); UV (MeOH) λmax (log ε) 277 (2.94) nm; H, 13C, and 15N
NMR data (800 MHz for 1H, 201 MHz for 13C, and 81 MHz for 15N,
DMSO-d6) Table 1; HRMS (ESI-QTOF) m/z 562.3227 [M + H]+
(calcd for C28H44N5O7, 562.3235; Δ 3.8 ppm).
Dihydrospumigin K (3a): colorless, amorphous powder; [α]20
D
1
−15 (c 0.25, MeOH); UV (MeOH) λma1x (log ε) 277 (2.85) nm; H,
13C, and 15N NMR data (800 MHz for H, 201 MHz for 13C, and 81
MHz for 15N, DMSO-d6) Table 2; HRMS (ESI-QTOF) m/z 613.3360
[M + H]+ (calcd for C31H45N6O7, 613.3344; Δ 2.5 ppm).
Dihydrospumigin L (4a): colorless, amorphous powder; [α]20D −10
1
(c 0.31, MeOH); UV (MeOH) λmax (log ε) 277 (2.85) nm; H, 13C,
and 15N NMR data (800 MHz for 1H, 201 MHz for 13C, and 81 MHz
for 15N, DMSO-d6) Table 2; HRMS (ESI-QTOF) m/z 613.3369 [M +
H]+ (calcd for C31H45N6O7, 613.3344; Δ 4.1 ppm).
Dihydrospumigin M (5a): colorless, amorphous powder; [α]20
D
1
−19 (c 0.25, MeOH); UV (MeOH) λma1x (log ε) 277 (2.85) nm; H,
13C, and 15N NMR data (800 MHz for H, 201 MHz for 13C, and 81
MHz for 15N, DMSO-d6) Table 3; HRMS (ESI-QTOF) m/z 599.3205
[M + H]+ (calcd for C30H43N6O7, 599.3188; Δ 3.9 ppm).
Dihydrospumigin N (6a): colorless, amorphous powder; [α]20
D
1
−10 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 277 (2.85) nm; H,
13C, and 15N NMR data (800 MHz, DMSO-d6) Table 3; HRMS (ESI-
ASSOCIATED CONTENT
■
QTOF) m/z 599.3207 [M + H]+ (calcd for C30H43N6O7 599.3188; Δ
3.4 ppm).
S
* Supporting Information
The Supporting Information is available free of charge on the
Hydrolysis, Oxidation, and Marfey’s Analysis for Determi-
nation of the Absolute Configuration of the Amino Acids.
Hydrolysis (6 N HCl, 105 °C, for 16 h) was performed using 0.1 mg
of compounds 1−6. The hydrolysates were dried under a nitrogen
stream and resuspended in H2O (100 μL). FDAA solutions (Sigma-
Aldrich) in acetone (0.05 M, 50 μL) and NaHCO3 (1 M, 100 μL)
were added to each aqueous solution, and the reaction mixture was
heated in a dry block (40 °C, 1 h) (Eppendorf Thermomixer R). The
mixture was cooled to room temperature, and HCl (2 M, 40 μL) was
added to stop the reaction. The solution was dried under a nitrogen
stream, and the resulting residues were reconstituted in MeOH/H2O
(1:1, 100 μL) prior to LC/MS analysis (Phenomenex Luna C18, 150
× 2.1 mm, 0.19 mL/min, 50 °C, A (0.1% aqueous formic acid
solution), B (MeCN/MeOH, 90/10) also containing 0.1% formic acid,
linear gradient elution of 5−60% B). The masses and retention time of
MS/MS spectra and assignment of the corresponding
major product ions for the protonated molecules of 1−6
and 3a−6a; base peak LC-MS chromatogram and
selected LC-MS extracted ion chromatograms for
original (3−6) and reduced (3a−6a) spumigin-contain-
ing fractions. 1H NMR, 1H−13C HSQC, HMBC,
1
TOCSY, NOESY, COSY, and H−15N HSQC spectra
for 1, 2, and 3a−6a; LC/MS data of Marfey derivatives
of the hydrolysates of 1, 2, and 3a−6a; CPA inhibition
curves and estimation of IC50 values for 1 and 2 (PDF)
I
J. Nat. Prod. XXXX, XXX, XXX−XXX
Sanz, Miriam
