Potent Elastase Inhibitors from Lyngbya spp.
Journal of Natural Products, 2007, Vol. 70, No. 10 1599
MeOH (60–90% for 25 min, then 90–100% for 10 min, and finally
100% MeOH for 10 min) to afford lyngbyastatin 5 (1), tR 13.7 min
(0.47 mg), and lyngbyastatin 6 (2), tR 15.0 min (0.17 mg), along with
known lyngbyastatin 4, tR 12.2 min (9.6 mg), as the most potent elastase
inhibitors in the sample.
which allowed detection of D-glyceric acid (tR 13.8 min) but not
L-glyceric acid (tR of standard, 10.6 min).
CrO3 oxidations of 1–4 followed by acid hydrolysis were carried
out as described.4 The resulting hydrolyzates were derivatized with
L-FDLA and aliquots subjected to reversed-phase HPLC as above.
When compared to the Marfey profiles without prior oxidation, the
HPLC profiles for derivatives resulting from compounds 1 and 2 showed
one new peak for L-Glu (tR 16.2 min) and one peak with increased
intensity for L-Phe (tR 28.5 min). For compounds 3 and 4, both peaks
were already present in the original profile; however, they appeared to
be larger after oxidation, while the corresponding D-amino acid
derivatives were not detected.
Protease Inhibition Assays. The test samples for 1–4 were prepared
in DMSO by (log/2)-fold dilutions ranging from 1 mM to 100 pM. All
assays were performed in triplicate. Phenylmethylsulfonyl fluoride
(PMSF) was used as a positive control in the enzyme assays.
To test the inhibition of porcine pancreatic elastase (Elastase-high
purity; EPC, EC134), 75 µg/mL solution of elastase was prepared using
Tris-HCl (pH 8.0). The Km for elastase was determined to be 1.5 mM
for N-succinyl-Ala-Ala-Ala-p-nitroanilide, a concentration that was used
subsequently for the inhibitor dose–response experiments. After pre-
incubation of 165 µL of Tris-HCl (pH 8.0), 10 µL of elastase solution,
and 10 µL of test samples in DMSO (5% final concentration) in a
microtiter plate at 30 °C for 20 min, 15 µL of substrate solution (1.5
mM final concentration) was added to the mixture. The increase in
absorbance was measured for 30 min at intervals of 5 min at 405 nm.
Competitive binding was determined by plotting enzyme activity against
substrate concentrations in the presence of different inhibitor concentra-
tions (Lineweaver–Burk plot).
Inhibitory activity against R-chymotrypsin (bovine pancreas; Sigma,
C4129) was determined as follows. A 1 mg/mL solution of chymo-
trypsin was prepared in assay buffer (50 mM Tris-HCl/100 mM NaCl/1
mM CaCl2, pH 7.8). After preincubation of 80 µL of assay buffer
solution, 10 µL of enzyme solution, and 10 µL of test solution in DMSO
in a microtiter plate at 37 °C for 10 min, 50 µL of substrate solution
(N-succinyl-Gly-Gly-Phe-p-nitroanilide, 0.75 mM final concentration
corresponding to Km) was added to the mixture. The increase in
absorbance was measured for 30 min at intervals of 5 min at 405 nm.
Inhibitory activity against trypsin was assayed as described above
for chymotrypsin, using trypsin from porcine pancreas (Sigma, T0303)
and NR-benzoyl-DL-arginine-4-nitroanilide hydrochloride as the sub-
strate solution.
Lyngbya sp. was collected from a mangrove channel, Kemp Channel,
at the northern end of Summerland Key, Florida Keys (24°39.730′ N,
81° 27.791′ W) in May 2006. We suspect it is a L. majuscula of gray-
black color, yet it appears thinner than generally described (cell width:
17.3 µm; sheath: 0.9 µm; length: 3.8 µm). A voucher specimen is
retained at the Smithsonian Marine Station. The freeze-dried sample
was extracted with CH2Cl2–MeOH (1:1). The resulting lipophilic extract
(24.1 g) was partitioned between hexanes and 20% aqueous MeOH,
the methanolic phase was evaporated to dryness, and the residue was
further partitioned between n-BuOH and H2O. The n-BuOH layer was
concentrated and subjected to chromatography over Si gel using CH2Cl2
and increasing gradients of i-PrOH. Consecutive fractions that eluted
with 50 and 75% i-PrOH were individually applied to C18 SPE
cartridges, and elution was initiated with H2O followed by aqueous
solutions containing 25, 50, 75, and 100% MeOH. Both times, the
fractions eluting with 75% aqueous MeOH were then purified by
semipreparative RP HPLC (YMC-Pack ODS-AQ, 250 × 10 mm, 2.0
mL/min; UV detection at 220 and 254 nm) using a MeOH–H2O linear
gradient (50–100% for 60 min and then 100% MeOH for 10 min).
The fraction that had eluted with 50% i-PrOH from Si gel yielded
compound 3, tR 35.2 min (7.4 mg), while the 75% i-PrOH fraction
furnished additional amounts of 3 (3.1 mg) and somamide B (4), tR
26.2 min (1.2 mg). Both compounds accounted for most of the elastase-
inhibitory activity of the extract.
Lyngbyastatin 5 (1): colorless, amorphous powder;21 UV (MeOH)
λ
max (log ε) 210 (4.57), 280 (sh) (3.79) nm; 1H NMR, 13C NMR, COSY,
HMBC, and ROESY data, see Table 1; HRESI/APCIMS m/z [M +
Na]+ 1079.4711 (calcd for C53H68N8O15Na 1079.4702).
Lyngbyastatin 6 (2): colorless, amorphous powder;21 UV (MeOH)
λ
max (log ε) 210 (4.48), 280 (sh) (3.65) nm; 1H NMR, 13C NMR, COSY,
and ROESY data, see Table 1; HRESI/APCIMS m/z [M + Na]+
1195.4257 (calcd for C54H69N8O18SNa2 1195.4246).
Lyngbyastatin 7 (3): colorless, amorphous powder; [R]20D –7.4 (c
0.27, MeOH); UV (MeOH) λmax (log ε) 230 (3.80), 280 (sh) (3.12); IR
(film) νmax 3373 (br), 2961, 1733, 1645 (br), 1539, 1446, 1203, 1026
cm-1; 1H NMR, 13C NMR, COSY, HMBC, and ROESY data, see Table
2; HRESI/APCIMS m/z [M + Na]+ 969.4710 (calcd for C48H66N8O12Na
969.4698).
Somamide B (4): colorless, amorphous powder,21 UV (MeOH)
λmax (log ε) 230 (3.74), 280 (sh) (3.10) nm; NMR data, see ref 5;
HRESI/APCIMS m/z [M + Na]+ 941.4407 (calcd for C46H62N8O12Na
941.4385).
Acknowledgment. This article was developed under the auspices
of the Florida Sea Grant College Program with support from NOAA,
Office of Sea Grant, U.S Department of Commerce, Grant No.
NA06OAR4170014. The authors gratefully acknowledge NSF for
funding through the External User Program of the National High
Magnetic Field Laboratory (NHMFL), which supported our NMR
studies at the Advanced Magnetic Resonance Imaging and Spectroscopy
(AMRIS) facility in the McKnight Brain Institute of the University of
Florida on samples originating from V.J.P. The 600 MHz 1 mm triple-
resonance HTS cryogenic probe was developed through collaboration
between the University of Florida, NHMFL, and Bruker Biospin.9 We
thank L. Fisher and K. Banks (Broward County Environmental
Protection Department) and C. Ross, A. Capper, and R. Ritson-Williams
(Smithsonian Marine Station) for assistance with collections. We thank
K. Arthur and D. Littler for identifying the Lyngbya sp. collected in
Summerland Key. Mass spectral analyses were performed at the UCR
Mass Spectrometry Facility, Department of Chemistry, University of
California at Riverside. This is contribution #705 from the Smithsonian
Marine Station at Fort Pierce.
Amino Acid Analysis by Modified Marfey’s Method.11 Samples
(∼50 µg each) of compounds 1–4 were subjected to acid hydrolysis (6
N HCl) at 110 °C for 24 h. The hydrolyzates were evaporated to
dryness, dissolved in H2O (100 µL), and divided into two equal portions.
To one portion were added 1 M NaHCO3 (50 µL) and a 1% v/v solution
of 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) in acetone, and
the mixture was heated at 80 °C for 3 min. The reaction mixture was
then cooled, acidified with 2 N HCl (100 µL), dried, and dissolved in
H2O–MeCN (1:1). Aliquots were subjected to RP HPLC (Alltech
Alltima HP C18 HL 5 µm, 250 × 4.6 mm, UV detection at 340 nm)
using a linear gradient of MeCN in 0.1% (v/v) aqueous TFA (30–70%
MeCN over 50 min). The retention times (tR, min) of the derivatized
amino acids in the corresponding hydrolyzates of compounds 1–4
matched with those of L-Thr (13.8), L-Val (23.6), L-Phe (28.5), and
N-Me-L-Tyr (40.6). HPLC profiles derived from compounds 1 and 2
additionally revealed peaks for derivatives of L-Ala (19.8) and L-Htyr
(44.8), while the profiles of compounds 3 and 4 additionally gave peaks
for L-Glu (16.2). Here glutamic acid must have derived from glutamine
present in 3 and 4. For comparison, the L-FDLA derivatives of the
other standard amino acids not detected in the hydrolyzates had the
following retention times (tR in min): L-allo-Thr (14.8), D-allo-Thr
(16.9), D-Thr (19.1), D-Val (32.5), D-Phe (35.5), N-Me-D-Tyr (42.6),
D-Ala (22.3), D-Htyr (48.4), and D-Glu (17.6).
Note Added after ASAP Publication: HPLC analysis was done
with 0.05% aqueous TFA, not 0.5%. This appears correctly in the
version posted on October 9, 2007.
Supporting Information Available: Figure S1 depicting key
ROESY correlations in compounds 1 and 2 in comparison with
lyngbyastatin 4. This material is available free of charge via the Internet
Portions of the hydrolyzates derived from compounds 1 and 2 were
also subjected to chiral HPLC analysis (Phenomenex Chirex phase 3126
N,S-dioctyl-(D)-penicillamine, 4.60 × 250 mm, 5 µm; solvents, 2 mM
CuSO4–MeCN (85:15); flow rate 1.0 mL/min; detection at 254 nm),
Taori, Kanchan
