Journal of Natural Products
Article
4
×
10− M, MeOH) λmax (Δε) 218 (−39.5) nm; IR (neat) ν 3410,
Analysis of the Absolute Configuration of the Glucose in
Mohangic Acid E (5). Mohangic acid E (3 mg) was dissolved in 3 N
HCl (0.5 mL) and stirred at 80 °C for 2 h. After the solution cooled to
rt, the HCl was evaporated in vacuo to yield the hydrolysate. HMDS
and TMSCl (50 μL, v/v = 2:1) were added to the hydrolysate with
pyridine (0.5 mL). After being stirred at 60 °C for 30 min, the mixture
max
−1
1
13
2
964, 1602, 1546, 1395 cm ; H and C NMR data, Table 1;
HRFABMS m/z 496.2681 [M + Na] (calcd for C H NO Na,
+
2
7
39
6
4
96.2675).
Mohangic acid C (3): yellow gum; [α]25 −7.1 (c 0.5, MeOH); UV
D
(
1
MeOH) λ (log ε) 270 (4.63) nm; 325 (4.32) nm; ECD (c 4.0 ×
max
4
−
0
M, MeOH) λmax (Δε) 218 (−39.1), 269 (−7.5), 275 (−6.2), 282
was dried in vacuo and separated using H
2 2 2
O and CH Cl (1 mL, v/v =
−
1 1
(
−9.2) nm; IR (neat) ν 3402, 2967, 1675, 1593, 1536 cm ; H and
1:1). The CH Cl layer was injected into a GC equipped with an HP5
2
2
max
1
3
+
C NMR data, Table 1; HRFABMS m/z 524.2629 [M + Na] (calcd
column (0.32 mm × 30 m). The temperature of the injector and the
detector in the GC was maintained at 200 °C. During analysis, the
temperature of the GC column was controlled (60 °C for 3 min, 60−
200 °C at 4 °C/min, and 200 °C for 3 min). The glucose derivative
from the hydrolysate of 5 was detected at a retention time of 32.65
min. The authentic D-glucose and L-glucose samples were treated and
analyzed using the same procedures. The derivatives of D-glucose and
L-glucose were detected at 32.67 and 32.85 min, respectively. Co-
injection of the silylated derivative of the hydrolysate and authentic D-
glucose gave a single peak at 32.64 min, thereby determining the
absolute configuration of the glucose in 5 as the D-form.
for C H NO Na, 524.2624).
28
39
7
25
Mohangic acid D (4): yellow gum; [α] −11.7 (c 0.5, MeOH);
D
−4
UV (MeOH) λmax (log ε) 270 (4.46) nm; ECD (c 4.0 × 10 M,
MeOH) λmax (Δε) 217 (−39.9), 259 (−4.4), 268 (−5.5), 280 (3.7)
−1
1
13
nm; IR (neat) νmax 3452, 2958, 1737, 1598, 1366 cm ; H and
C
+
NMR data, Table 1; HRFABMS m/z 526.2784 [M + Na] (calcd for
C H NO Na, 526.2781).
28
41
7
25
Mohangic acid E (5): yellow gum; [α] −25.7 (c 0.5, MeOH);
D
UV (MeOH) λ (log ε) 270 (4.19) nm, 325 (3.91) nm; ECD (c 3.2
max
−
4
×
10 M, MeOH) λmax (Δε) 218 (−41.3), 292 (−3.5) nm; IR (neat)
−1 1 13
Synthesis of Bidentate Chiral NMR Solvents. N-Bromosucci-
nimide (2.2 mmol) was added to a mixture of 2-methyl-1,3-
propanediol (1.0 mmol) and triphenylphosphine (2.2 mmol) in
νmax 3368, 2968, 1737, 1599, 1373 cm ; H and C NMR data, Table
+
1
6
; HRFABMS m/z 644.3052 [M + Na] (calcd for C H NO Na,
44.3047).
Hydrogenation of Mohangic Acid D. Mohangic acid D (4) (4
mg) was lyophilized for 24 h and subsequently suspended, along with
a 10% Pd/C catalyst, in absolute EtOH (1.5 mL). The mixture
solution of 4 was stirred for 2 h under 1 atm of H at rt. The reaction
mixture in EtOH was filtered using a PTFE syringe filter and
evaporated in vacuo to yield the hydrogenation product (6) of 4. The
product was isolated in pure form by reversed-phase HPLC
Phenomenex Luna 5 μm C18 (2) 250 × 10.0 mm, flow rate 2 mL/
min, UV 254 nm detection) using a gradient solvent system (10% to
00% CH CN/H O over 40 min). The hydrogenated product (6) was
32 47 11
CH Cl (10.0 mL) at 0 °C. The reaction mixture was stirred at rt for
2
2
3
0 min. The solvent was removed, and the dibromopropane was
purified by column chromatography (silica gel, 40−63 μm, pentane).
With the acquired dibromopropane, bidentate chiral solvents (R,R)-
and (S,S)-bis-α-methylbenzylamine-p-Me were synthesized according
2
1
1,20
to previously reported procedures.
Quinone Reductase Assay. QR activities were determined
21
spectrophotometrically according to a modified microtiter method.
(
Hepa-1c1c7 cells were plated at a density of 20 000 cells/mL and
cultured for 24 h in a humidified incubator containing 5% CO at 37
1
2
3
2
°
C. The cells were then exposed to mohangic acids (1−5) and
obtained at a retention time of 27 min (3 mg, yield: 75%) under
HPLC conditions. The molecular formula of product 6 was confirmed
incubated for an additional 24 h. The media was subsequently
decanted, and the cells in each well were lysed by incubation at 37 °C
for 10 min with 250 μL of a solution containing 10 mM Tris-HCl pH
+
as C H NO on the basis of ESIMS data ([M + H] m/z at 510).
2
8
47
7
1
Hydrogenation product 6: H NMR (600 MHz, CD OD) δ 7.49
d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 4.58 (m, 1H), 3.95 (m,
3
H
8
6
.0, 140 mM NaCl, 15 mM MgCl , and 0.5% NP-40 (IGEPAL CA-
30) (Sigma-Aldrich). The plates were then agitated on an orbital
(
2
1
1
1
4
0
3
H), 3.75 (m, 1H), 3.04 (dd, J = 7.5 Hz, 4.5 Hz, 1H), 2.42 (dd, J =
5.5 Hz, 5.5 Hz, 1H), 2.34 (dd, J = 15.5 Hz, 5.5 Hz, 1H), 2.10 (s, 3H),
.75−1.72 (m, 2H), 1.66−1.62 (m, 3H), 1.56 (m, 1H), 1.47−1.42 (m,
H), 1.38−1.30 (m, 5H), 1.24−1.20 (m, 2H), 1.12−1.05 (m, 2H),
.88 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.5 Hz,
H).
shaker for an additional 10 min at 25 °C, after which 1 mL of the
complete reaction mixture containing 12.5 mM Tris-HCl pH 7.4, 0.67
mg/mL bovine serum albumin, 0.01% Tween-20, 50 μM flavin
adenine dinucleotide, 1 mM glucose-6-phosphate, 2 U/mL glucose-6-
phosphate dehydrogenase, 30 μM NADP, 50 μg/mL 3-(4,5-
dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and
Preparation of Acetonide Derivative of 6 (7). The hydro-
5
0 μM menadione was added. A blue color developed, and the
genation product of 4 (6) (3 mg) was prepared in a 40 mL vial and
dried under high vacuum for 24 h. After adding pyridinium p-
toluenesulfonate (1 mg), the mixture was dissolved in anhydrous
MeOH (200 μL) and anhydrous CH Cl (2 mL) at rt. Then, 2,2-
dimethoxypropane (4 mL) was added to the reaction vial. The
solution was stirred at rt under argon for 48 h. To quench the reaction,
a saturated NaHCO3 aqueous solution was added. The reaction
mixture was fractionated by flash column reversed-phase chromatog-
reaction was stopped after 10 min. The rate of the NADPH-
dependent, menadiol-mediated reduction of MTT was measured at
6
10 nm. Protein was detected by crystal violet staining of an identical
2
2
set of test plates. The specific activity (SA) of quinone reductase
(nmol/min/mg of protein) was calculated from the equation
SA = [MTT /CV ] × 3345
abs
abs
raphy using a gradient solvent system of MeOH/H O (20%, 40%,
2
where MTTabs is the change in the absorbance of MTT per min, CVabs
is the absorbance of crystal violet, and 3345 is the ratio of the
proportionality constant determined for crystal violet and the
extinction coefficient of MTT and has units of nmol/mg.
6
0%, 80%, and 100%). The acetonide derivative (7) eluted in the 80%
and 100% MeOH/H O fractions. The final product (7) was purified
2
by reversed-phase HPLC (Phenomenex Luna 5 μm C18 (2) 250 ×
10.0 mm, flow rate 2 mL/min, UV 254 nm detection) with a gradient
solvent system (10% to 100% MeOH/H O) for 40 min. The
2
acetonide product (7) eluted at a retention time of 35 min (2.5 mg,
yield: 83%) under the HPLC conditions. The molecular formula of 7
ASSOCIATED CONTENT
■
*
S
Supporting Information
+
was confirmed as C H NO by ESIMS analysis ([M + H] m/z at
31
51
7
5
50).
1
Acetonide product (7): H NMR (600 MHz, CD OD) δ 7.52 (d, J
3
H
=
8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 3.95 (dd, J = 10.0 Hz, 6.0 Hz,
1
2
2
H), 3.92 (m, 1H), 3.74 (m, 1H), 3.06 (dd, J = 7.0 Hz, 4.0 Hz, 1H),
.38 (dd, J = 15.0 Hz, 5.0 Hz, 1H), 2.30 (dd, J = 15.0 Hz, 8.5 Hz, 1H),
.12 (s, 3H), 1.98 (m, 1H), 1.87 (m, 1H), 1.77 (m, 1H), 1.69−1.67
1
D and 2D NMR spectra for 1−5, homodecoupling
1 1
NMR experiment data of 1, H NMR spectrum of 6, H
and HSQC NMR spectra of 7, C NMR spectra for 1 in
a 5:2 mixture of (R,R)-, (S,S)-BMBA and pyridine-d5,
13
(
m, 2H), 1.57−1.55 (m, 2H), 1.47−1.42 (m, 4H), 1.39 (s, 6H), 1.38−
.30 (m, 5H), 1.22 (m, 1H), 1.12−1.06 (m, 2H), 0.89 (d, J = 6.5 Hz,
H), 0.88 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H).
1
3
and detailed quinone reductase assay (PDF)
G
J. Nat. Prod. XXXX, XXX, XXX−XXX