Journal of Natural Products
ARTICLE
V2 to V8 hypervariable regions within the 16S rRNA gene. Prior to
phylogenetic predictions, a statistical selection of best-fit models of
nucleotide substitutions for the 16S rRNA data set was made using
Akaike and Bayesian information criteria (AIC and BIC) in jModelTest
0.1.1.31 Phylogenetic trees were calculated using the minimum-evolu-
tion algorithm in MEGA 5 as well as the Bayesian (MrBayes)32 and
phylogenetic maximum likelihood (PhyML v3.0)33 algorithms in Geneious
5.3.34 The minimum evolutionary distances were determined using a
maximum composite likelihood method performed with 1000 bootstrap
replicates and the close-neighbor-interchange algorithm. All ambiguous
positions were removed for each sequence pair (pairwise deletion
option). The PhyML analysis was performed with 500 bootstrap
replicates using the GTR+I+G model (selected by AIC and BIC in
jModelTest; proportion of invariable sites (pINV) = 0.465, shape
parameter (R) = 0.387, number of rate categories = 4). Bayesian analysis
was performed with the GTR+I+G substitution model (pINV = 0.452,
R = 0.372, number of rate categories = 4). The Markov chain length (one
cold and three heated) was set to 3 million with sampling performed
every 100 generations (25% burn-in). The analysis was completed once
convergence was achieved (∼2.7 million generations), which was deter-
mined by an average standard deviation in split frequencies of <0.01.
Extraction and Isolation of Compounds 1ꢀ3 and Dolas-
tatin 12. The field collection of Leptolyngbya (500 mL, collection code
EHu5-27-07-1) for chemical extraction was stored in 2-propanol at
ꢀ20 °C until extraction to yield 1.26 g of organic extract (CH2Cl2ꢀ
MeOH, 2:1). The organic extract was subjected to bioassay-guided
fractionation via NP VLC using a stepped gradient of hexanes to EtOAc
to MeOH. The fraction eluting with 25% MeOHꢀEtOAc was further
separated by RP-SPE using a stepped gradient of MeOHꢀH2O from
50% MeOHꢀH2O to 100% MeOH, followed by 100% CH2Cl2.
Isocratic RP-HPLC (column: Synergi Fusion-RP, 10 ꢁ 250 mm, 70%
MeCNꢀH2O, 3 mL/min) of the SPE fraction C eluting in 70%
MeOHꢀH2O yielded two impure HPLC peaks targeted for further
purification. RP-HPLC (Chirobiotic TAG, 4.6 ꢁ 250 mm, 98%
EtOHꢀH2O, 0.5 mL/min) of the less polar of these fractions yielded
Ibu-epidemethoxylyngbyastatin 3 (3, 2.9 mg) and a mixture of grass-
ypeptolides D (1, 1.5 mg) and E (2, 0.5 mg), which were separated on
the Chirobiotic TAG column using 75% MeOHꢀH2O (0.5 mL/min).
Dolastatin 12 was also isolated from the 25% MeOHꢀEtOAc NP VLC
fraction H. Repeated isocratic RP-HPLC (column: Synergi Fusion-RP,
10 ꢁ 250 mm, 70% MeCNꢀH2O, 3 mL/min) of the SPE fraction (H2)
eluting in the 70% MeOHꢀH2O fraction yielded dolastatin 12 as the
major component. Subsequent LC-MS profiling (Synergi Fusion-RP,
2 ꢁ 100 mm, 0.2 mL/min, linear gradient of 65% to 100% MeCN in 0.1%
(v/v) aqueous TFA) of extracts from monoclonal Leptolyngbya cultures (2 ꢁ
1.5 L) yielded m/z1138.6 (1and 2, [M+Na]+) and 1005 (3, [M+Na]+) at
the same retention times as 1, 2, and 3 purified from the original field
collection.
Dolastatin 12: white, amorphous solid; [R]21D ꢀ79.8 (c0.5, CHCl3);
data, see Supporting Information;13 HR-TOFMS m/z [M + H]+ 969.6035
(calcd for C50H81N8O11, 969.6019).
1
UV (MeOH) λmax (log ε) 212 (3.71), 256 (3.57); H and 13C NMR
Absolute Configuration of Grassypeptolides D (1) and E
(2). The amino acid standards relevant to compounds 1 and 2 were
obtained commercially or as gifts and prepared as 50 mM solutions
in H2O. Standards for (2R)- and (2S)-methylcysteine were kindly
provided by Dr. W. H. Gerwick, Scripps Institution of Oceanography,
University of California, San Diego. A portion of each standard (5.0 mg)
was dissolved in 720 μL of HCO2H at 0 °C. Next, 80 μL of H2O2 (30%)
was added dropwise with continuous stirring, and the reaction was
carried out at 0 °C for 2 h to yield either (2R)- or (2S)-methylcysteic acid
(MeCysA). The product mixture was dried under a steady stream of N2
gas and resuspended in H2O (50 mM). The N-benzoyl O-methyl ester
of (2R,3S)-2-methyl-3-aminobutyric acid was gratefully received from
Dr. Hendrik Luesch, Department of Medicinal Chemistry, University of
Florida. Approximately 0.4 mg was deprotected with 500 μL of 6 N HCl
at 110 °C for 24 h, evaporated to dryness, and resuspended in H2O
(50 mM). Each standard was derivatized for Marfey’s analysis by adding
10 μL of 1 M NaHCO3 and 50 μL of N-R-(5-fluoro-2,4-dinitrophenyl)-
L-leucinamide (L-FDLA or D-FDLA, 1% w/v in acetone) to 25 μL of
each standard solution. The mixture was heated at 40 °C for 1 h with
continuous stirring, cooled to room temperature, acidified with 5 μL
of 2 N HCl, evaporated to dryness, and resuspended in 250 μL of
MeCNꢀH2O (1:1).
Approximately 0.1 mg of 1 and 0.2 mg of 2 were dissolved separately
in 3 mL of CH2Cl2 (ꢀ78 °C). Ozone was then bubbled through each
solution for 15 min. The solution was dried under a stream of N2 gas,
followed by an oxidative workup of the residue (0.6 mL of H2O2ꢀ
HCOOH, 1:2, at 70 °C for 20 min). The oxidation product was con-
centrated under vacuum and hydrolyzed with 1 mL of 6 N HCl at 110 °C
for 18 h. The hydrolyzed products were resuspended in 25 μL of H2O
and derivatized for Marfey’s analysis in a similar manner to the deriv-
atized chromatographic standards. The Marfey’s products of 1 and 2
were resuspended in 50 μL of MeCNꢀH2O (1:1) and analyzed by RP-
HPLC (Gemini C18 110 A, 4.6 ꢁ 150 mm, 5 μm, 1.0 mL/min, UV
detection at 340 nm) using a linear gradient of 30% to 70% MeCN in
0.1% (v/v) aqueous TFA over 50 min. The retention time (tR, min) of
the residues in the hydrolysate of 1 matched standards for D-Aba (21.9;
L-Aba, 17.1), L-Cya (7.1; D-Cya, 6.5), (2S)-MeCysA [6.2; (2R)-MeCysA,
7.8], N-Me-D-Leu (27.8; N-Me-L-Leu, 24.5), (2R,3R)-Maba L-FDLA
[18.8; (2R,3R)-Maba D-FDLA, 25.6; (2R,3S)-Maba L-FDLA, 18.6;
(2R,3S)-Maba D-FDLA, 20.8], L-Pro (14.1; D-Pro, 17.0), N-Me-L-Phe
(22.7; N-Me-D-Phe, 24.6), N-Me-L-Val (21.2; N-Me-D-Val, 25.6), D-allo-
Thr (12.6; L-Thr, 10.2; L-allo-Thr, 11.1; D-Thr, 14.4). The retention
times for the residues in the hydrolysate of 2 were consistent with the
results for 1, with the exception of N-Me-L-Leu (24.5) and L-Thr (10.2)
standards matching the corresponding residues in the hydrolysate of 2.
The configuration of the Pla residue in the hydrolysates of both 1 and 2
was determined by chiral LC-MS. The retention time [Chirobiotic TAG,
4.6 ꢁ 250 mm; MeOHꢀ10 mM NH4OAc (3:2, pH 5.50); flow rate,
0.4 mL/min; detection by ESIMS in negative ion mode] of the natural
product hydrolysate matched that for L-Pla (7.4 min; D-Pla, 8.5).
Absolute Configuration of Ibu-epidemethoxylyngbyastatin
(3). Approximately 0.4 mg of 3 in 0.2 mL of anhydrous MeOH was
added to a solution of NaBH4 (2 mg) in anhydrous MeOH at 0 °C. After
stirring for 30 min, the solution was acidified with 1 N HCl until pH 6.
The solution was then partitioned between EtOAc and H2O, and the
organic layer concentrated to dryness for analysis by RP-HPLC (Synergi
Fusion-RP, 10 ꢁ 250 mm, 62% MeCNꢀH2O, 3.5 mL/min). A single
product consistent with the dihydro form of 3 (tR 18.9 min) was present,
while unreduced compound 3 (tR 21.3 min) was not detected. This
reduction product and an additional ∼0.4 mg of 3 were separately
Grassypeptolide D (1): colorless, amorphous solid; [R]21D +25.9
(c 0.15, CH2Cl2); UV (MeOH) λmax (log ε) 212 (3.82), 260 (3.56);
1H and 13C NMR data, see Table 1, Tables S1 and S4; HR-FTMS
m/z [M + Na]+ 1138.5515 (calcd for C57H81N9O10S2Na, 1138.5445),
m/z [M + H]+ 1116.5458 (calcd for C57H82N9O10S2, 1116.5620).
Grassypeptolide E (2): colorless, amorphous solid; [R]21D +13.2
(c 0.15, CH2Cl2); UV (MeOH) λmax (log ε) 212 (3.73), 256 (3.48); 1H
and 13C NMR data, see Table 1, Table S2; HR-TOFMS m/z [M + Na]+
1138.5413 (calcd for C57H81N9O10S2Na, 1138.5445), m/z [M + H]+
1116.5603 (calcd for C57H82N9O10S2, 1116.5620).
Ibu-epidemethoxylyngbyastatin 3 (3): white, amorphous
solid; [R]21 ꢀ48.6 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 212
D
1
(3.98), 256 (3.81); H and 13C NMR data, see Table S3; HR-FTMS
m/z [M + Na]+ 1005.5983 (calcd for C51H82N8O11Na, 1005.6001),
m/z [M + H]+ 983.6162 (calcd for C51H83N8O11, 983.6175).
1683
dx.doi.org/10.1021/np200270d |J. Nat. Prod. 2011, 74, 1677–1685
Thornburg, Christopher C.
