Kwan et al.
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evaporated to dryness, and reconstituted in 100 μL of H2O.
Analysis by chiral HPLC [column, Chirobiotic TAG (4.6 ꢀ 250
mm), Supelco; solvent, MeOH-10 mM NH4OAc (40:60, pH
5.39); flow rate, 0.5 mL/min; detection by ESIMS in positive ion
Advanced Marfey’s Analysis. The N-benzoyl O-methyl esters
of (2R,3R)- and (2R,3S)-2-methyl-3-aminobutyric acid (Maba)
were treated with 6 N HCl at 110 °C for 22 h. The products of
each reaction were dried down and made up to 50 mM solutions
in water. Then, 10 μL of 1 M NaHCO3 and 50 μL of 1-fluoro-2,4-
dinitrophenyl-5-L-leucinamide (L-FDLA, 1% w/v in acetone)
were added to 25 μL of the Maba stock solutions. The mixtures
were heated to 35 °C for 1 h with frequent mixing, then neutra-
lized with 5 μL of 2 N HCl, concentrated to dryness, and then
reconstituted with 250 μL of MeCN-H2O (1:1). The FDLA
derivative of the ozonolysis product of 3 was prepared in a similar
way using ∼45 μg of starting material. Derivatives were analyzed
by reversed phase HPLC [column, Kinetex 2.6 μm C18 100A
(4.6 mm ꢀ 100mm), Phenomenex; flowrate, 0.5mL/min; ESIMS
detection in negative ion mode], using a MeOH-H2O (both with
0.1% HCOOH) linear gradient (40-100% MeOH over 50 min).
Under these conditions two peaks were detected in the L-FDLA
derivative of the ozonolysate of 3, corresponding to (2R,3R)- and
(2S,3R)-Maba-L-FDLA, respectively, in the ratio 1.51:1. This is
consistent with the extent of epimerization at the 2-position
observed after treatment of the standards with acid,12 allowing
the assignment of (2R,3R)-Maba. The retention times (tR, min) of
the authentic standards were as follows: (2R,3S)-Maba-L-FDLA
(25.6), (2R,3R)-Maba-D-FDLA43 (25.6), (2R,3R)-Maba-L-
FDLA (27.1), (2R,3S)-Maba-D-FDLA44 (27.5).
mode (MRM scan)], revealed the presence of D-Aba at tR
=
15.4, but both N-Me-L-Phe and N-Me-D-Phe were detected at
tR = 25.3 and 47.3 min, respectively, in the ratio 1.28:1. The
retention times (tR, min; MRM ion pair, parent f product) of
the authentic amino acids were as follows: L-Aba (9.1, 104 f 58),
D-Aba (15.4), N-Me-L-Phe (25.3, 180 f 134), N-Me-D-Phe
(47.3). Compound-dependent MS parameters were as follows:
Aba, DP 41, EP 2, CEP 8, CE 14, CXP 2.3; N-Me-Phe, DP 30,
EP 10, CEP 13, CE 20, CXP 3. Source-dependent MS param-
eters were as follows: CUR 50, CAD medium, IS 5500, TEM
750, GS1 70, GS2 70.
Samples of both 1 and 3 (50 μg each) were subjected to
ozonolysis at room temperature for 30 min followed by oxida-
tive workup and then subjected to chiral HPLC analysis
[column, Chirobiotic TAG (4.6 mm ꢀ 250 mm), Supelco;
solvent, MeOH-10 mM NH4OAc (30:70, pH 5.14); flow rate,
0.5 mL/min; detection by ESIMS in negative ion mode (MRM
scan)]. For the ozonolysis product of 3, both N-Me-L-Asp
and N-Me-D-Asp were detected at tR = 6.6 and 10.6 min, respec-
tively, in the ratio of 1.22:1. For the ozonolysis product of 1, the
ratio was 1:1.85. The retention times (tR, min; MRM ion pair,
parent f product) of the authentic amino acids were as follows:
N-Me-L-Asp (6.6, 146 f 102), N-Me-D-Asp (10.6). Compound-
dependent MS parameters were as follows: DP -33, EP -4.5,
CEP -14, CE -18, CXP -5. Source-dependent MS conditions
were as follows: CUR 50, CAD high, IS -4500, TEM 750, GS1
70, GS2 70. The ozonolysis product of 3 was also examined
under different chiral HPLC conditions [column, Chirex phase
3126 (D) (4.6 mm ꢀ 250 mm), Phenomenex; solvent, 2 mM
CuSO4-MeCN (97.5:2.5); flow rate, 0.8 mL/min; UV detection
at 254 nm], to confirm the presence of D-allo-Thr, N-Me-L-Val,
L-Pro and D-Aba at tR = 14.5, 16.3, 17.4, and 21.0, respectively.
In addition, both L- and D-Cya were detected at tR = 19.8 and
23.7, respectively, consistent with previous results for 1 (ratio
∼2:1).12 The retention times (tR, min) of the authentic standards
were as follows: L-Thr(10.3), D-Thr (11.6), L-allo-Thr (13.9), D-allo-
Thr (14.5), L-Aba (15.0), D-Aba (21.0), N-Me-L-Val (16.3), N-Me-
D-Val (22.2), L-Pro (17.4), D-Pro (33.6), L-Cya (19.8), D-Cya (23.7).
Thiazoline Cleavage and Oxidation. Both 1 and 3 were treated
with RuCl3/NaIO4 using the procedure of Jayaramann et al.,42
with minor differences. Portions of both compounds (100 μg of
1 and 200 μg of 3) were dissolved in CH3CN/CCl4/H2O (2:2:3,
150 μL total). To this was added powdered NaIO4 (3 equiv)
followed by RuCl3 (0.7 mg), and the reactions were stirred at rt
for 1 h. The reaction mixtures were then diluted with water and
extracted with CH2Cl2. The combined CH2Cl2 fractions were
dried under N2 and then treated with 6 N HCl at 110 °C for
16 h and evaporated to dryness. The resulting hydrolysates were
subjected to chiral HPLC analysis [column, Chirobiotic TAG
(4.6 mm ꢀ 250 mm), Supelco; solvent, MeOH-10 mM NH4OAc
(40:60, pH 5.39); flow rate, 0.5 mL/min; detection by ESIMS in
positive ion mode (MRM scan)]. For 1, both N-Me-L-Phe and N-
Me-D-Phe were detected at tR = 25.3 and 47.3 min, respectively,
in the ratio 1:4.88. For 3, the ratio was reversed, with N-Me-L-Phe
and N-Me-D-Phe detected in the ratio 3.35:1. The retention times
(tR, min;MRMionpair, parentfproduct) of the authentic amino
acids were as follows: N-Me-L-Phe (25.3, 180 f 134), N-Me-D-Phe
(47.3). Compound-dependent MS parameters were as follows: DP
30, EP 10, CEP 13, CE 20, CXP 3. Source-dependent MS param-
eters were as follows: CUR 50, CAD medium, IS 5500, TEM 750,
GS1 70, GS2 70.
Attempted Interconversion of Grassypeptolides A (1) and C (3).
Compounds 1 and 3 were dissolved in CDCl3 (150 μL) and
transferred to a 2.5 mm NMR tube. Pyridine-d5 (0.5 μL) was
added, and the tube was heated at 60 °C for 72 h. Conversion
was assessed by 1H NMR.
Molecular Modeling of Grassypeptolide C (3). Because of the
general similarity of proton and carbon chemical shifts of 1 and
3, the same distance and angle constraints were used as in the
previously reported modeling of 1,12 except that the amide bond
between N-Me-Phe and Pro was set to cis due to a ROESY
correlation between H-28 and H-38 in 3. Also, four additional
distance constraints were added on the basis of ROESY correla-
tions unique to 3. The modeling procedure used was identical to
that previously used for 1,12 with 10 random structures of 3
being subjected to (1) minimization, (2) distance geometry, (3)
minimization, and (4) molecular dynamics in Sybyl 7.3. Con-
straints were applied at every step, and the distance geometry
procedure consisted of bounds generation, bounds smoothen-
ing, and embedding of coordinates, followed by an optimization
procedure where the structures were minimized and subjected to
simulated annealing (SA, 2000 to 200 K over 100,000 fs, step
time 0.3 fs) and then another round of minimization. Dynamics
were run at 500 K for 1 ns, with a step size of 1 fs. Following
simulation, structures were overlaid in PyMol by pair-fitting
backbone carbons only.
Cell Viability Assays. HT29 and HeLa cells were cultured
in Dulbecco’s modified Eagle medium (DMEM; Invitrogen)
containing 10% fetal bovine serum (Hyclone) in a humidified
atmosphere containing 5% CO2 at 37 °C. Cells were seeded into
96-well plates at a density of 10 000 cells/well (HT29) or 3 000
cells/well (HeLa) in 100 μL of medium. After 24 h, compounds
1-3 were added to wells at varying concentrations (as 1 μL stock
solutions in DMSO). Taxol was used as a positive control for
cytotoxicity, and DMSO alone was used as a negative control.
After 48 h of treatment the plates were developed with MTT dye
according to the manufacturer’s protocol (Promega). Taxol
exhibited IC50s of 2.2 and 1.7 nM for HT29 and HeLa cells,
respectively.
Cell Cycle Analysis. HT29 cells were seeded in 6 cm dishes
(650 000 cells/dish in 3 mL of DMEM). After 24 h of incubation,
(42) Jayaraman, M.; Srirajan, V.; Deshmukh, A. R. A. S.; Bhawal, B. M.
Tetrahedron 1996, 52, 3741–3756.
(43) Corresponding in tR to its enantiomer (2S,3S)-Maba-L-FDLA.
(44) Corresponding in tR to its enantiomer (2S,3R)-Maba-L-FDLA.
8022 J. Org. Chem. Vol. 75, No. 23, 2010