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J. J. La Clair, A. D. Rodríguez / Bioorg. Med. Chem. 19 (2011) 6645–6653
and CH3CN (500 mL). The CH3CN layer was collected and washed
with a second aliquot of hexanes (500 mL) and then dried by rotary
evaporation to afford 1.25 g of crude extract E1 that was stored at
ꢂ78 °C until used.
at 0 °C. The mixture was warmed to rt and stirred for 16 h. The
reaction was quenched by the addition of MeOH (0.5 mL) and dried
under N2 flow. The residue was purified by HPTLC using
5 cm ꢁ 20 cm Silica Gel 60 plate (EM Science) and eluted with a
mixture of 1:1 hexanes/EtOAc to afford
a-methyl-2,3,4,6-tetra-O-
4.5. Reverse affinity isolation
benzoyl-D-galactoside, which was identical to an authentic stan-
dard.24 1H NMR (d6-acetone) dH 8.13–7.33 (20H, m, Ar), 6.12 (1H,
dd, J = 1.5, 3.5 Hz, H-4), 6.03 (1H, dd, J = 3.6, 10.4 Hz, H-3), 5.75
(1H, dd, J = 3.5, 10.4 Hz, H-2), 5.40 (1H, d, J = 3.6 Hz, H-1), 4.81
(1H, dd, J = 1.6, 5.6 Hz, H-5), 4.51 (1H, dd, J = 5.5, 11.1 Hz, H-6a),
4.58 (1H, dd, J = 7.0, 11.0 Hz, H-6b), 3.58 (3H, s, –OCH3).
A sample of extract E1 (854 mg) dissolved in DMSO (5 mL) was
diluted to 1 L with PBS pH 7.2. After standing for 2 h, the resulting
solution was filtered through a 0.22 lm vacuum filter unit (Milli-
pore) to remove the particulates. This solution was then added to
the reservoir and air was bled from the system through a valve
on the chamber. An additional 50 mL of PBS pH 7.2 was required
to fill the tubing and dead space within the system. Once filled
and bled of air this system was pumped between 1 cm ID polycar-
bonate fritted columns (Ridout Plastics) loaded with 25 mL of
either resin R1–R5 using a low pressure liquid diaphragm pump
(KNF Neuberger) as depicted in Figure 2 and a flow rate of
0.5 mL/min at 4 °C until complete passage of the media. At this
point, the system was opened, the media was removed, and the
columns were washed by pumping PBS pH 7.2 (1 L) and deionized
water (2 ꢁ 1 L) for 2 h each. The columns were then removed from
the system and the bound materials were eluted from each column
using warm 95% EtOH (200 mL). The EtOH fractions were dried by
rotary evaporation.
4.8.3. Sphinganine analysis
Fraction A obtained from the methanolysis of 1 mg of 4 was dis-
solved in anhydrous pyridine (2 mL) containing 4-dimethylamino-
pyridine (50 lg) and treated with benzoylchloride (100 lL) at 0 °C.
The mixture was warmed to rt and stirred for 16 h. The reaction
was quenched by the addition of MeOH (1 mL) and dried under
N2 flow. The residue was purified by HPTLC using 5 cm ꢁ 20 cm Sil-
ica Gel 60 plate (EM Sciences) and eluting with a mixture of 1:1
hexanes/EtOAc to afford 180 lg of the perbenzoylated sphinganine
fragment (see D, Fig. 8) which was characterized by high-resolu-
tion MS. HR-ESI MS: M+Na+ at m/z 778.3724 (calcd for
C
48H53NNaO7, 778.3720) along with a
minor M+Na+ at m/z
764.3559 (calcd for C47H51NNaO7, 764.3563). Without an authentic
standard, detailed NMR studies were not conducted, rather the
resulting materials were examined by oxidative cleavage, as de-
scribed in the following section.
4.6. LC/MS screening
The fractions eluted from resins R1–R5 were evaluated by LC/
MS analysis on an Agilent 1100 LC equipped with
a
4.8.4. Microscale oxidation of sphinganine fraction D
50 mm ꢁ 4.6 mm Xterra reversed-phase column with matching
guard cartridge (Waters). Exemplary LC assay conditions: mobile
phase A: 0.1% HCO2H in H2O, B: 0.1% HCO2H in CH3CN, gradi-
ent = 5–95% B in 30 min with post-column injection of C: 20 mM
The sample of fraction D (180
l
g) was dissolved in t-BuOH
(300
(50
l
L) and treated by the sequential addition of 40 mM K2CO3
l
L), 25 mM KMnO4 (200 lL) and 100 mM NaIO4 (200 lL). The
mixture was warmed to 37 °C in a sealed vial. After 18 h, the mix-
ture was acidified with 5 M H2SO4 and decolorized by the addition
of a saturated solution of Na2SO3. The mixture was diluted with
1 mL of H2O and extracted with Et2O (3 ꢁ 5 mL). The combined ex-
tracts were dried with Na2SO4. The resulting solution was treated
NH4OAc in CH3CN/H2O (3:1), 0.2 mL/min; 2.5
lL injection volume
(ꢀ10
l
M sample); diode array detection was monitored at 254 nm.
Data from this analysis were processed in Microsoft Excel and ren-
dered in line format as depicted in Figure 4.
with 2 M TMSCHN2 in Et2O (500
lL) for 1 h at rt at which point
4.7. Purification
it was concentrated to ꢀ200 L by rotary evaporation. The result-
l
ing solution was evaluated by GC/MS. The results were as follows:
methyl-heptanoate with tR = 2.5 min with M+ at m/z 144, methyl-
6-methylheptanoate, tR = 3.6 min with M+ at m/z 158, methyl-octa-
noate, tR = 4.4 min with M+ at m/z 158. Relative abundances were
determined by peak integration and were compared to a quantita-
tive 1:1:1 mixture of methyl-heptanoate, methyl-6-methylhept-
anoate, and methyl-octanoate. These results are summarized in
Figure 8.
Compounds were purified by HPTLC on 5 cm ꢁ 20 cm Silica Gel
RP-18 plate (EM Science) using a mixture of 3:1 CH3CN/H2O for
elution. Samples of compounds 1–5 were eluted from the plates
with CH3CN and dried via rotary evaporation. The purity of each
material was determined to be P95% by HPLC analysis.
4.8. Degradation analyses
Degradation studies were conducted using procedures devel-
oped by Fattorusso and Mangoni.13 The following sections provide
a description of the experimental procedures and observations.
4.8.5. Microscale oxidation of fraction B
Fraction B obtained from the methanolysis of 400
dissolved in t-BuOH (150 L) and treated by the sequential addi-
tion of 40 mM K2CO3 (25 L), 25 mM KMnO4 (100 L) and
100 mM NaIO4 (100 L). The mixture was warmed to 37 °C in a
lg of 4 was
l
l
l
4.8.1. Methanolysis of 4
l
Compound 4 (100
l
g) dissolved in 500
l
L of 1 M HCl in 95%
sealed vial. After 18 h, the mixture was acidified with 5 M H2SO4
and decolorized by the addition of a saturated solution of Na2SO3.
The mixture was diluted with 1 mL of H2O and extracted with Et2O
(3 ꢁ 3 mL). The combined extracts were dried with Na2SO4. The
resulting solution was treated with 2 M TMSCHN2 in Et2O
MeOH was incubated for 12 h at 80 °C in a sealed tube. The reac-
tion mixture was dried under N2 flow and the resulting material
was partitioned between CHCl3 (2 mL) and 20% MeOH (2 mL). Both
layers were dried by N2 flow to deliver fraction A (from the 20%
MeOH layer) and fraction B (from the CHCl3 layer). This process
was also scaled to 1 mg of 4.
(250
ꢀ200
l
l
L) for 1 h at rt at which point it was concentrated to
L by rotary evaporation. The resulting solution was evalu-
ated by GC/MS. The results were as follows: methyl-heptanoate
with tR = 2.5 min and M+ at m/z 144, methyl-6-methylheptanoate,
tR = 3.6 min, M+ at m/z 158, methyl-octanoate, tR = 4.4 min with
M+ at m/z 158, methyl-decanoate, tR = 6.8 min with M+ at m/z
186, methyl-undecanoate, tR = 7.3 min with M+ at m/z 200,
4.8.2. Carbohydrate analysis
Fraction A obtained from the methanolysis of 100
dissolved in anhydrous pyridine (1 mL) containing 4-dimethyl-
amino-pyridine (10 g) and treated with benzoylchloride (50 L)
lg of 4 was
l
l