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X.-J. LU ET AL.
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Methyl (2-ethoxy-2-oxoacetyl) -L-leucinate (C2): light yellow oil; H-NMR (400 MHz,
DMSO-d6): δ 9.24 (1H, d, J = 8.0 Hz, 2-NH), 4.35 (2H, ddd, J = 10.7, 8.0, 4.0 Hz, H-2),
4.25 (2H, q, J = 7.1 Hz, 2′-OCH2CH3), 3.64 (3H, s, 1-OCH3), 1.76 (1H, td, J = 11.0, 4.0 Hz,
H-3a), 1.61–1.52 (2H, m, H-3b, H-4), 1.28 (3H, t, J = 7.1 Hz, 2′-OCH2CH3), 0.89 (3H, d,
J = 6.4 Hz, H-5 or 4-CH3), 0.85 (3H, d, J = 6.2 Hz, 4-CH3 or H-5).
To a solution of intermediate C in five volumes of ethanol cooled at 0 °C, 2–10 equivalents
of aq. ammonia (14.8 N) were added. Reaction mixture was slowly warmed up to room
temperature overlight. Concentration under reduced pressure gave target product as white
solid in good yields. Furthermore, the residue was purified by column chromatography on
silica gel (3:1 PE/EA) and HPLC (35% Methol-H2O) to give desired product as white powder.
Oxalamido-L-phenylalanine methyl ester (D1): white powder; CD (c 2.00 mM, MeOH)
Δε196 nm − 2.45, Δε219 nm + 7.43, Δε258 nm + 0.394; e 1H-NMR spectral data were same as
compound 1: δ 8.87 (1H, d, J = 8.5 Hz, 2-NH), 8.03 (1H, s, 2′-NH2), 7.80 (1H, s, 2′-NH2),
7.29–7.18 (5H, H-5–H-9), 4.56 (1H, dt, J = 8.5, 5.4 Hz, H-2). 3.65 (3H, s, 1-OCH3), 3.15
(1H, dd, J = 13.7, 5.4 Hz, H-3a), 3.11 (1H, dd, J = 13.7, 5.4 Hz, H-3b).
Oxalamido-L-leucine methyl ester (D2): white powder; CD (c 2.30 mM, MeOH) Δε196 nm
−
0.95, Δε209 nm + 1.31, Δε234 nm − 1.35; e 1H-NMR spectral data were same as natural isolates:
δ 8.91 (1H, d, J = 8.4 Hz, 2-NH), 8.10 (1H, s, 2′-NH2), 7.85 (1H, s, 2′-NH2), 4.33 (1H, ddd,
J = 8.4, 3.9, 3.0 Hz, H-2), 3.63 (3H, s, 1-OCH3), 1.84–1.78 (1H, m, H-3a), 1.59–1.51 (2H, m,
H-3a, H-4), 0.88 (3H, d, J = 6.2 Hz, H-5 or 4-CH3), 0.84 (3H, d, J = 6.0 Hz, 4-CH3 or H-5).
3.5. 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH·) scavenging assay
e DPPH· scavenging activity was assessed according to the described method with minor
modifications [25], using lower DPPH concentration (0.2 mM) and different sample to
DPPH ratio (1:1). To a 100 μl aliquot of the sample with different concentrations, 100 μl
of DPPH solution (0.2 mM) was added in a 96-well microplate. e mixture was shaken
vigorously and incubated in darkness for 30 min. e absorbance of the reaction solution at
517 nm was recorded using a varioskan flash multimode reader. Ascorbic acid was used as a
positive control. e percentage of scavenging DPPH versus concentration was plotted using
the following equation: DPPH scavenging activity (%) = [1 – (S – SB)/(C – CB)] × 100%,
where S, SB, C, and CB are the absorption of the sample, the blank sample, the control and
the blank control, respectively. All experiments were tested in triplicate.
3.6. 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) free radical (ABTS+)
scavenging assay
e determination of ABTS+ scavenging was carried out on the basis of the previous method
with minor modifications [25]. ABTS radical cations (ABTS+) were obtained by reacting
7 mM stock solution of ABTS+ with 2.45 mM potassium persulfate and allowing the mix-
ture to stand in the dark at room temperature for 14 h before use. e ABTS+ solution was
diluted with ethanol to the absorbance of 0.70 0.05 at 734 nm. To a 100 μl aliquot of the
sample with different concentrations, 150 μl ABTS+ solution in a 96-well microplate was
added. Afer reacting at room temperature for 30 min, the absorbance was recorded using
varioskan flash multimode reader at 734 nm. e percentage of ABTS+ scavenging activity