G Model
CCLET 3018 1–5
W.-X. Song et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
3
118
119
CH3CN–H2O (4:1) was used for TLC identification of glucose
(Rf = 0.39).
482 [M+K]+. HRFABMS at m/z 444.1512 [M+H]+ (calcd for 174
C19H26NO11
,
444.1506) indicated the molecular formula 175
C19H25NO11, which was supported by the NMR data (Table 1). 176
The 1H NMR spectrum of 1 in D2O showed signals attributed to a 177
trisubstituted olefinic proton at dH 7.47 (brs, H-3), two acetal 178
protons at dH 5.58 (d, J = 1.8 Hz, H-1) and 5.13 (dd, J = 9.6 and 179
3.6 Hz, H-7), and a monosubstituted vinyl group at dH 5.50 (ddd, 180
J = 16.8, 10.2, and 10.2 Hz, H-8), 5.39 (dd, J = 16.8 and 1.2 Hz, H- 181
10b), and 5.31 (d, J = 10.2 and 1.2 Hz, H-10b). In addition, the 182
spectrum showed resonances due to two methines at dH 3.10 183
(J = 13.2, 6.0, and 3.6 Hz, H-5) and 2.85 (J = 10.2, 6.0, and 1.8 Hz, H- 184
120
121
2.5. Determination of the absolute configurations of amino acid units
in 1 and 2
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
Compounds
hydrolyzed with 6 N HCl (200
110 8C for 16 h. The solutions were evaporated in vacuo. To the
residues, FDAA [(1-fluoro-2,4-dinitrophenyl)-5- -alanine amide]
solution in acetone (1%, 300 L) and 6% aqueous triethylamine
(150 L, 1 M) were added. The mixture was stirred at 40 8C for 1 h,
then diluted with H2O (500 L), and filtered. The standard FDAA-
amino acids were prepared in the same way, using -serine,
serine, -threonine, and -threonine (1.1–1.3 mg), respectively. The
1
(1.0 mg) and
2
(0.7 mg) were separately
m
L) in a sealed glass bomb at
L
m
m
9) and a methylene at
and 2.31 (ddd, J = 12.0, 3.6, and 3.6 Hz, H-6
due to a -glucopyranosyl unit were observed at dH 4.86 (d, 187
d
H 1.43 (ddd, J = 13.2, 12.0, and 9.6 Hz, H-6
a
)
185
m
b
). Characteristic signals 186
L
D
-
b
L
D
J = 8.4 Hz, H-10), 3.31 (dd, J = 9.6 and 8.4 Hz, H-20), 3.52 (t, J = 9.6 Hz, 188
H-30), 3.42 (t, J = 9.6 Hz, H-40), 3.54 (m, H-50), 3.94 (dd, J = 12.6 and 189
1.8 Hz, H-60a), and 3.75 (dd, J = 12.6 and 6.0 Hz, H-60b). Also 190
present was an ABX coupling system assignable to a serine unit at 191
FDAA-amino acid derivatives from the hydrolysate were compared
with the standard FDAA-amino acids by HPLC analysis: Alltech
Alltima C18 column (250 ꢁ 4.6, 5
mm), flow rate 1 mL/min, UV
detection at 340 nm, mobile phase CH3CN–H2O (18:82) containing
1% AcOH. The retention times tR, are as follows: FDAA derivative of
the hydrolysate from 1, 24.2 min; FDAA derivative of the
d
H 4.58 (dd, J = 9.0 and 8.4 Hz, H-300a), 3.95 (dd, J = 9.0 and 8.4 Hz, 192
H-300b), and 4.71 (t, J = 8.4 Hz, H-200). Besides the resonances 193
corresponding to the above protonated carbons (Table 1), the 13C 194
NMR and DEPT spectra of 1 showed resonances for two carbonyl 195
carbons at dC 176.6 (C-100) and 167.2 (C-11), and a quaternary 196
hydrolysate from 2, 32.4 min; FDAA-L-serine, 24.1 min; FDAA-
D-
serine, 26.5 min; FDAA-
L
-threonine, 32.4 min; and FDAA- -threo-
D
nine, 43.6 min.
olefinic carbon at dC 109.6 (C-4). These spectroscopic data suggest 197
that 1 is an unusual secoiridoid glycoside [14–16] containing a 198
serine unit. The suggestion was confirmed by comprehensive 199
analysis of the 2D NMR data, which resulted in an unambiguous 200
140
2.6. Synthesis of 1 and 2
141
142
143
144
145
146
147
148
149
150
Secologanin (3) or secologanic acid (4) (30–50 mg) was refluxed
with two molar equivalents of -serine or -threonine in acetoni-
structure determination of 1.
201
L
L
The proton and protonated carbon signals in the NMR spectra of 202
trile or pyridine for 30–50 h, respectively. The reaction mixtures
were evaporated to give corresponding residues which were
separately isolated by preparative thin layer chromatography
(PTLC) over silica gel, eluting with the mobile phase CHCl3–MeOH–
HOAc (3:1:0.3), to afford 1 and 2 (64–93% yields) from the
1 were unequivocally assigned by interpreting the 1H–1H COSY and 203
HMQC spectroscopic data. The 1H–1H COSY spectrum of
1
204
displayed homonuclear vicinal coupling correlations: H-1/H-9/ 205
H-5/H2-6/H-7 and H-9/H-8/H2-10, in addition to W-type correla- 206
tions H-1/H-3/H-5 (Fig. 2, thick lines). These, combined with two- 207
and three-bond correlations in the HMBC spectrum (Fig. 2, red 208
arrows): H-1/C-3, C-5, C-8, and C-9; H-3/C-1, C-4, C-5, and C-11; H- 209
5/C-1, C-3, C-4, C-6, C-7, C-8, C-9, and C-11; H2-6/C-4, C-5, C-7, and 210
C-9; H-7/C-5; H-8/C-1, C-5, C-9, and C-10; H-9/C-1, C-4, C-5, C-6, C- 211
8, and C-10; and H2-10/C-8 and C-9; as well as the chemical shifts 212
of these proton and carbon resonances, revealed unambiguously 213
the presence of a secoiridoid parent nucleus with 7-acetalic and 214
8(10)-olefinic functionalities in 1. The COSY correlations H-10/H-20/ 215
H-30/H-40/H-50/H2-60 and the HMBC correlations H-1/C-10 and H- 216
reactions of 3 or 4 with
1H NMR, ESIMS, and [
L
-serine and
L
-threonine, respectively. The
20
a
]
data of 1 and 2 were consistent with
D
those of the natural products.
151
2.7. Determination of -glucosidase resistance of 1 and 2
b
152
153
154
155
156
157
158
159
160
161
162
163
164
Standard solutions (10 mmol/L) of 1, 2, and sweroside, and a
solution (0.66 mg/mL) of -glucosidase (from almonds) were
prepared with a KH2PO4/K2HPO4 buffer (pH 6.8) [19]. A mixture of
the standard solutions of sweroside (100 L) and enzyme
(100 L), as well as a mixture of the standard solutions of
sweroside (100 L), enzyme (100 L), and 1 (100 L) or 2 (100 L)
b
m
10/C-1 verified the presence of a
b-glucopyranosyl moiety at C-1 of 217
m
the nucleus. Additionally, the COSY correlations H-200/H2-300 and 218
the HMBC correlations from H-200 to C-100, C-300, C-7, and C-11 and 219
from H2-300 to C-100 and C-7, together with their chemical shifts, 220
demonstrated the presence of the serine unit, of which C-200 and C- 221
300 were connected through nitrogen and oxygen atoms to C-7 of 222
the nucleus. The HMBC correlation from H-7 to C-11, combined 223
with the molecular composition, indicated that the carbonyl 224
carbon (C-11) was linked through the nitrogen atom to C-7 to form 225
a lactam in 1. Accordingly, the planar structure of compound 1 was 226
m
m
m
m
were incubated at 37 8C. After incubation for 10, 30, and/or 60 min,
the mixtures were analyzed by RP-HPLC using an Altech Brava C18
column (250 mm ꢁ 4.6 mm i.d., 5
H2O (15:85) containing 0.1% HOAc. The chromatograms indicated
that 1 and 2 were not hydrolyzed by -glucosidase, whereas
mm) and mobile phase CH3CN–
b
sweroside was hydrolyzed. In addition, the hydrolysis of sweroside
with the enzyme was not disturbed by the presence of 1 and 2.
elucidated as shown in Fig. 2.
In the NOE difference spectrum, irradiation of H-5 enhanced the 228
H-6 , H-7, and H-9 resonances, while the H-5, H-6
, and H-300b 229
resonances were enhanced upon irradiation of H-7 (Fig. 3, red 230
dashed lines). The enhancements revealed that H-5, H-6b, H-7, H- 231
9, and H-300b were cofacial on one side of the ring system. The 232
227
165
2.8. Assays for pharmacological activities of 1 and 2
b
b
166
167
Details may be found in Refs. [14–16] and the references cited
therein.
168
3. Results and discussion
enhancements of the H-1, H-8 and H2-10 resonances upon 233
irradiation of H-6
on the other side of the ring system. This suggested that the 235
secoiridoid nucleus in 1, with a -oriented H-7, had the same 236
configuration as that of the co-occurring secologanic acid, for 237
which the absolute configuration was determined by a single- 238
crystal X-ray crystallographic analysis using anomalous scattering 239
a indicated that these protons were cofacial 234
20
169
170
171
172
173
Compound 1 was obtained as a white amorphous solid, [a]
D
ꢂ183.4 (c 0.40, H2O). Its IR spectrum showed absorption bands for
b
hydroxy (3326 cmꢂ1
)
and carbonyl [1720 (sh), 1657, and
1584 cmꢂ1] functionalities. The positive FABMS of 1 exhibited
pseudo molecular ion peaks at m/z 444 [M+H]+, 466 [M+Na]+, and
Please cite this article in press as: W.-X. Song, et al., Two new
b
-hydroxy amino acid-coupled secoiridoids from the flower buds of
Lonicera japonica: Isolation, structure elucidation, semisynthesis, and biological activities, Chin. Chem. Lett. (2014), http://dx.doi.org/