2520
K. Kai et al. / Phytochemistry 68 (2007) 2512–2522
C18 (75 · 2 mm, Imtakt, Kyoto, Japan); flow rate, 200 ll/
min; solvent, 0.05% (v/v) acetic acid (A) and MeOH (B),
10ꢀ90% (v/v) B/(A + B) for 15 min; temperature, 30 ꢁC.
The data obtained were processed with Analyst 1.3 soft-
ware (Applied Biosyntems).
2), 3.88 (1H, m, Glc, H-6), 3.72–3.81 (5H, m, Glc H-
3,4,6, CH2C@ONH), 3.65 (1H, dd, J = 9.4, 9.2 Hz, Glc,
H-5), 2.89 (2H, m, Asp, H-b). 13C NMR (100 MHz,
D2O): d177.3 (C@O), 177.0 (C@O), 176.6 (C@O), 139.4
(IAA, C-7a), 130.6 (IAA, C-3a), 127.5 (IAA, C-2), 125.7
(IAA, C-6), 123.5 (IAA, C-5), 121.8 (IAA, C-4), 113.2
(IAA, C-7), 112.4 (IAA, C-3), 87.1 (Glc, C-1), 81.0 (Glc,
C-4), 79.2 (Glc, C-3), 74.4 (IAA, C-2), 72.0 (IAA, C-5),
63.3 (IAA, C-6), 51.9 (Asp, C-a), 38.1 (Asp, C-b), 34.7
(CH2C@ONH). HRESI-MS: m/z 453.1510 [M+H]+ (calc.
for C20H25N2O10, 453.1509). Physicochemical data for
IAA-Glu-N-Glc (11). [a]D22: +4.2ꢁ (EtOH; c0.23). 1H
NMR (400 MHz, D2O): d7.62 (2H, d, J = 8.3 Hz, IAA,
H-4,7), 7.47 (1H, s, IAA, H-2), 7.34 (1H, dd, J = 8.2,
7.2 Hz, IAA, H-6), 7.24 (1H, dd, J = 7.7, 7.3 Hz, IAA,
H-5), 5.62 (1H, d, J = 9.1 Hz, Glc, H-1), 4.31 (1H, dd, J=
10.4, 4.9 Hz, Glu, H-a), 4.07 (1H, dd, J = 9.2, 9.1 Hz,
Glc, H-2), 3.88 (1H, m, Glc, H-6), 3.73–3.81 (5H, m, Glc,
H-3,4,6, CH2C@ONH), 3.66 (1H, m, Glc, H-5), 2.36 (2H,
t, J = 7.3 Hz, Glu, H-c), 2.17 (1H, m, Glu, H-b), 1.92
(1H, m, Glu, H-b). 13C NMR (100 MHz, D2O): d179.7
(Glu, COOH), 177.8 (C@O), 177.6 (C@O), 139.4 (IAA,
C-7a), 130.6 (IAA, C-3a), 127.5 (IAA, C-2), 125.7 (IAA,
C-6), 123.5 (IAA, C-5), 121.8 (IAA, C-4), 113.2 (IAA, C-
7), 112.5 (IAA, C-3), 87.1 (Glc, C-1), 81.0 (Glc, C-4),
79.2 (Glc, C-3), 74.4 (Glc, C-2), 72.0 (Glc, C-5), 63.3
(Glc, C-6), 54.8 (Glu, C-a), 34.7 (CH2C@ONH), 32.6
(Glu, C-c), 28.3 (Glu, C-b). HRESI-MS: m/z 467.1684
[M+H]+ (Calc. for C21H27N2O10, 467.1665).
5.3. Chemical synthesis
1H and 13C NMR spectra were obtained with a Bruker
Avance 400 or Bruker ARX 500 spectrometer with tetra-
methylsilane (CD3CN, dimethylsulfoxide) or sodium 3-
(trimethylsilyl)-1-propansulfonate (D2O) as an internal
1
standard. Assignments of H and 13C signals were carried
out using 1H–1H COSY, HMQC, and HMBC spectro-
scopic analysis. High-resolution mass spectra were
recorded with
a LCMS-IT-TOF (Shimadzu, Kyoto,
Japan). Optical rotations were measured with a JASCO
P-1010 polarimeter.
IAA-N-Glc (9). IAA (1) was methylated with 1.0 equiv-
alent of methyl iodide and K2CO3 in N,N-dimethylform-
amide. Treatment of IAA methyl ester (IAA-OMe, 12)
with 2.1 equivalents of triethylsilane under strong-acidic
conditions gave 2,3-dihydroIAA-OMe (13) in 89% yield.
Condensation of 13 with excess D-glucose in MeOH, fol-
lowed by acetylation with Ac2O-pyridine (1:2, v/v) gave
2,3-dihydroIAA-OMe-N-AcGlc (14) in 58% yield. Oxida-
tion of 14 with 1.0 equivalent of 2,3-dichloro-5,6-dicy-
ano-1,4-benzoquinone produced IAA-OMe-N-AcGlc (15)
in 49% yield, which was deprotected in mild alkaline to give
22
IAA-N-Glc (9) as a colorless solid quantitatively. [a]D
:
IAA-OMe-N-Glc (17). The carboxy group of IAA-N-
Glc (9) was methylated with (trimethylsily)diazomethane
+3.9 (EtOH; c0.16). 1H NMR (400 MHz, D2O): d7.61
(2H, d, J = 8.1 Hz, IAA, H-4,7), 7.43 (1H, s, IAA, H-2),
7.34 (1H, dd, J = 7.9, 7.4 Hz, IAA, H-6), 7.24 (1H, d, J=
7.7, 7.5 Hz, IAA, H-5), 5.60 (1H, d, J = 9.1 Hz, Glc, H-
1), 4.05 (1H, t, J = 9.1 Hz, Glc, H-2), 3.87 (1H, m, Glc,
H-6), 3.86 (2H, s, CH2C@O), 3.69–3.77 (3H, m, Glc, H-
3,4,6), 3.60 (1H, m, Glc, H-5). 13C NMR (100 MHz,
D2O): d179.8 (C@O), 139.2 (IAA, C-7a), 130.7 (IAA, C-
3a), 127.2 (IAA, C-2), 125.6 (IAA, C-6), 123.4 (IAA, C-
5), 121.8 (IAA, C-4), 113.2 (IAA, C-7), 112.3 (IAA, C-3),
87.2 (Glc, C-1), 81.0 (Glc, C-3 or 4), 79.1 (Glc, C-3 or 4),
74.5 (Glc, C-2), 72.0 (Glc, C-5), 60.1 (Glc, C-6), 33.2
(CH2C@O). HRESI-MS: m/z 338.1239 [M+H]+ (calc. for
C16H20NO7, 338.1239).
1
to afford 17 in 73% yield. H NMR (500 MHz, CD3CN):
d7.53 (1H, d, J = 7.9 Hz), 7.51 (1H, d, J = 8.3 Hz), 7.33
(1H, s), 7.21 (1H, ddd, J = 8.2, 7.1, 0.8 Hz), 7.12 (1H,
ddd, J = 7.9, 7.3, 0.7 Hz), 5.43 (1H, d, J = 9.1 Hz), 3.83
(1H, t, J = 8.9 Hz), 3.77 (2H, s), 3.76 (1H, dd, J = 12.1,
2.5 Hz), 3.66 (3H, s), 3.61 (1H, dd, J = 12.0, 5.4 Hz Hz),
3.50–3.56 (2H, m), 3.45 (1H, dd, J = 9.3, 9.2 Hz). 13C
NMR (125 MHz, CD3CN): d173.6, 137.9, 129.4, 125.4,
123.2, 121.0, 120.0, 111.5, 110.0, 85.9, 79.9, 78.6, 73.2,
71.1, 62.6, 52.6, 31.5. HRESI-MS: m/z 352.3586 [M+H]+
(Calc. for C17H22NO7, 352.3591).
IAA-N-[6,6-2H2]Glc (9-d2), IAA-Asp-N-[6,6-2H2]Glc
(10-d2), and IAA-Glu-N-[6,6-2H2]Glc (11-d2). 9-d2 was
synthesized using the same procedure as IAA-N-Glc (9),
except for the use of [6,6-2H2]D-glucose. 10-d2 and 11-d2
were synthesized from 9-d2 as described above. Their struc-
tures were confirmed by NMR spectroscopy and mass
spectrometry.
IAA-Asp-N-Glc (10) and IAA-Glu-N-Glc (11). IAA-N-
Glc (9) was acetylated with excess acetic anhydride, and
subsequently condensed with L-amino acid methyl esters
to give N-glucosides of IAA-amide (yield: Asp, 38%; Glu,
54%). Treatment of respective N-glucosides of IAA-amide
with 1M KOH afforded IAA-Asp-N-Glc (10) and IAA-
Glu-N-Glc (11) as colorless solids quantitatively. Physico-
chemical data for IAA-Asp-N-Glc (10). [a]D22: +12.5ꢁ
5.4. Quantitative analysis
1
(EtOH; c0.22). H NMR (400 MHz, D2O): d7.61 (1H, d,
Plant tissues were homogenized in liquid N2, to which
acetoneꢀH2O (4:1 v/v) containing 2.5 mM diethyldithio-
carbamic acid and the internal standards were added.
Extraction was conducted at 4 ꢁC for 2 h twice, and the
combined extract was evaporated to remove the solvent.
J = 8.4 Hz, IAA, H-7), 7.59 (1H, d, J = 8.1 Hz, IAA, H-
4), 7.46 (1H, s, IAA, H-2), 7.34 (1H, m, IAA, H-6), 7.23
(1H, m, IAA, H-5), 5.62 (1H, d, J = 9.2 Hz, Glc, H-1),
4.78 (1H, m, Asp, H-a), 4.06 (1H, t, J = 9.2 Hz, Glc, H-