Structural analyses of metabolites of phenolic 1- benzyltetrahydroisoquinolines in plant cell cultures by LC/NMR, LC/MS, and LC/CD

Article abstract of DOI:10.1021/np0402219

The metabolism of the phenolic 1-benzyltetrahydroisoquinoline alkaloids was studied in cell cultures of Macleaya and Corydalis species. The crude alkaloid fraction obtained from feeding experiments was investigated by application of the combined LC/NMR and LC/APCI-MS (/MS) techniques. Several metabolites were detected, and their structures (6 and 8-14) were identified. Bioconversion of the phenolic 1-benzyltetrahydroisoquinoline (2) into the pseudoprotoberberine (8) was demonstrated for the first time. LC/APCI-MS and LC/CD experiments carried out on a chiral column permitted the deduction of the major enantiomeric form of the chiral metabolites. Thus, the combination of NMR, MS, and CD data permitted the structural elucidation and stereochemical analysis of the metabolites in the extract matrix solution, without isolation and sample purification prior to the coupling experiments.

Full text of DOI:10.1021/np0402219

992
J. Nat. Prod. 2005, 68, 992-1000
Structural Analyses of Metabolites of Phenolic 1-Benzyltetrahydroisoquinolines
in Plant Cell Cultures by LC/NMR, LC/MS, and LC/CD
Kinuko Iwasa,*,† Wenhua Cui,† Makiko Sugiura,† Atsuko Takeuchi,† Masataka Moriyasu,† and
Kazuyoshi Takeda‡
Kobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada-ku, Kobe-shi 658-8558, Japan, and Ebara Research Co.
Ltd.4-2-1, Honfujisawa, Fujisawa-shi 251-8502, Japan
Received November 28, 2004
The metabolism of the phenolic 1-benzyltetrahydroisoquinoline alkaloids was studied in cell cultures of
Macleaya and Corydalis species. The crude alkaloid fraction obtained from feeding experiments was
investigated by application of the combined LC/NMR and LC/APCI-MS (/MS) techniques. Several
metabolites were detected, and their structures (6 and 8-14) were identified. Bioconversion of the phenolic
1-benzyltetrahydroisoquinoline (2) into the pseudoprotoberberine (8) was demonstrated for the first time.
LC/APCI-MS and LC/CD experiments carried out on a chiral column permitted the deduction of the major
enantiomeric form of the chiral metabolites. Thus, the combination of NMR, MS, and CD data permitted
the structural elucidation and stereochemical analysis of the metabolites in the extract matrix solution,
without isolation and sample purification prior to the coupling experiments.
The bioconversion of isoquinoline alkaloids from Papav-
eraceae and Fumariaceae plants and their cell cultures
have been investigated by us in combination with assays
for certain types of biological activity.1-5 These studies have
resulted in the discovery of numerous metabolites with
antimicrobial, antimalarial, anti-HIV, and anticancer
activities.2-5 Although protoberberine alkaloids differ in the
number and position of various oxygen functions on the
aromatic rings A and D, the two oxygenation patterns most
frequently encountered within this class of alkaloids are
at carbons 2,3,9,10 and 2,3,10,11. The former group occurs
most commonly, while the latter has been labeled
“pseudoprotoberberine” and is not as widespread.6 Some
2,3,10,11-oxygenated alkaloids display higher activity in
biological tests (e.g., antimalarial activity) than the corre-
sponding 2,3,9,10-substituted analogues.3 The biosynthetic
conversion of the 2,3,9,10-oxygenated protoberberines into
other alkaloidal types, such as the protopines, benzophenan-
thridines, rhoeadines, benzindanoazepines, and spiroben-
zylisoquinolines, has been demonstrated.1,7 However, de-
spite its occurrence in some plant species, including
Corydalis species,6 only our earlier study on the biosyn-
thesis of 2,3,10,11-oxygenated protoberberines has been
reported.8 The applications of LC/NMR to drug metabolism,
identification of natural products in crude plant extracts,
and the characterization of isomeric mixtures prepared by
chemical reactions have been summarized.9-11 The applica-
tion of LC/NMR to biosynthetic studies was previously
demonstrated.8 The metabolism of 2,3,10,11-oxygenated
protoberberines (pseudoprotoberberine) alkaloids was stud-
ied in cell cultures of Corydalis species.8 Without prior
isolation, the structures of the metabolites were determined
by LC/NMR and LC/MS analyses. Some new alkaloids were
identified, and preliminary evidence for the metabolic
pathways leading to the formation of these alkaloids was
obtained.
quinolines, which might serve as precursors to pseudopro-
toberberines. Metabolites were identified by the comple-
mentary techniques of LC/NMR, LC/MS, and LC/CD.
Results and Discussion
The LC/APCI-MS (Figure 1) of the products obtained by
the acid-catalyzed ether cleavage of racemic tetrahydro-
papaverine (1) measured with SIM (selected ion monitor-
ing) and TIM (total ion monitoring) in the positive ion mode
(LC/APCI-MS I) indicate the presence of several products
having one or more phenolic hydroxyl groups besides the
starting material. The protonated molecular ions [M + H]+
of each peak were found to be m/z 344 for peak a (1), m/z
330 for peaks b-e, m/z 316 for peaks f-j, m/z 302 for peaks
k-n, and m/z 288 for peak o from the total ion chromato-
gram (TIC) of the LC/APCI-MS analysis. Peak a (1)
displayed the product ions at m/z 192 and 151 (Scheme 1).
Peaks b and c showed the product ions at m/z 192 and 137,
while peaks d and e displayed ions at m/z 178 and 151 in
the LC/MS-MS analysis. The peaks b-e were separated
by repeated preparative HPLC, and the structures of the
1
isomers were determined by H NMR and MS analyses.
1
The position of the hydroxyl group was determined by H
NMR and MS analyses. The position of the hydroxyl group
was confirmed by NOESY and MS/MS data. The four
phenolic 1-benzyltetrahydroisoquinolines, 2, 3, 4, and 5,
corresponding to peaks b-e, were used as precursors. The
structures of peaks f-o will be presented elsewhere.
Callus tissues of M. cordata, C. platycarpa Makino, and
C. ochotensis var. raddeana were incubated at 25 °C on an
agar medium containing the substrate (Table 1). Following
incubation, media and cells were separated and extracted
according to the procedure shown in Figure 2. The alkaloid
fractions, E-1, E-2, C-1, and C-2 (Figure 2), were subjected
to LC/NMR, LC/MS, and LC/CD.
The LC/NMR spectra were measured in the stopped-flow
mode. The LC/APCI-MS(/MS) spectra were measured with
Q1 or product ion scan. Molecular weight information was
obtained on the basis of a protonated molecular ion [M +
H]+ or a product ion recorded in the LC/APCI-MS (/MS).
The LC/CD was carried out on a chiral reversed-phase
column.
This paper describes the structural analysis of the
metabolites obtained from the cell cultures of Macleaya and
Corydalis species fed with phenolic 1-benzyltetrahydroiso-
* Corresponding author. Tel: 078-453-0031. Fax: 078-435-2080. E-
mail: k-iwasa@kobepharma-u.ac.jp.
† Kobe Pharmaceutical University.
‡ Ebara Research Co. Ltd.
10.1021/np0402219 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/02/2005

Phenolic 1-Benzyltetrahydroisoquinolines
Journal of Natural Products, 2005, Vol. 68, No. 7 993
Figure 1. LC/APCI-MS I data of the acid-catalyzed ether cleavage products of tetrahydropapaverine (1).
Scheme 1
Table 1. Administration of 1-Benzyltetrahydroisoquinolines (2,
3, 4, and 5) to Cultured Cells of Macleaya and Corydalis
Species
substrate medium wt. of dry incubation period
no. callusa
(mg)
(mL)
cells (g)
(weeks)
1
2
3
4
5
6
A
B
C
B
B
B
2
2
2
3
21
50
50
22
800
800
800
800
800
800
7.0
6.6
5.5
5.4
5.0
4.4
3
4
4
4
4
4
3 and 4 50
25
5
a A: M. cordata. B: C. platycarpa. C: C. ochotensis var.
raddeana.
Figure 2. Preparation of samples for LC/NMR, LC/MS, and LC/CD
measurements.
deana. Fraction E-2 (Figure 2) obtained from M. cordata
showed peaks a1-e1 in the LC 1 (Figure 3), which displayed
the protonated ions [M + H]+ at m/z 370, 354, 342, 342,
and 340, respectively, in the LC/APCI-MS. The LC/MS-
MS analysis of peak a1 showed [M + H]+ at m/z 370 and
the product ions at m/z 222, 205, and 204. The formation
of the product ions is in accord with that of authentic
protopine (7) (Scheme 1). The stopped-flow 1H NMR
spectrum (A, Figure 4) of peak a1 displayed four aromatic
The precursor 2 was administered to the cultured cells
of M. cordata, C. platycarpa, and C. ochotensis var. rad-

994 Journal of Natural Products, 2005, Vol. 68, No. 7
Iwasa et al.
Figure 3. LC data of the alkaloid fraction (E-2) obtained from the feeding of 2 to M. cordata (LC 1), C. platycarpa (LC 2), and C. ochotensis var.
raddeana (LC 3). Gradient: A 0.1 M NH4OAc (0.05% TFA)/B CH3CN (0.05% TFA) LC 1 (A/B: initial 80/20; 25 min 55/45); LCs 2 and 3 (A/B:
initial 80/20; 30 min 0/100).
protons at δ 6.85 (3H) and 7.05 (1H), a methylenedioxy
group at δ 5.97, two O-methyl groups at δ 3.79 (6H), and
an N-methyl group at δ 2.97. On the basis of this evidence,
peak a1 was identified as cryptopine 6.
groups at δ 3.78 (9H), and nine aliphatic protons in the
region between δ 4.3 and 2.9. The LC/MS-MS analysis of
peak d1 showed [M + H]+ at m/z 342 and its product ion at
m/z 192 (Scheme 1). These data identified peak d1 as
tetrahydropalmatrubine 9. Comparison of the stopped-
flow 1H NMR data with those of synthetic 9, meas-
ured under the same conditions, confirmed this identifica-
tion.
Peak b1 in the LC 1 (Figure 3) showed [M + H]+ at m/z
354 and the product ions at m/z 206, 189, and 188 in the
1
LC/MS-MS. Its stopped-flow H NMR spectrum exhibited
two methylenedioxy groups instead of one and two O-
methyl groups as observed in peak a1. Peak b1 in LC 1 was
identified to be a common component of the callus, pro-
topine 7.
Peak e1 in the LC 1 displayed [M + H]+ at m/z 340 and
its product ion at m/z 324 in the LC/MS-MS. The stopped-
flow 1H NMR spectrum (F, Figure 4) of peak e1 showed
two aromatic proton doublets (J ) 6.0 Hz) at δ 8.17 and
7.60, an aromatic two-proton AB quartet (J ) 8.0 Hz) at δ
6.81 and 6.78, three aromatic proton singlets at δ 7.46, 7.28,
and 6.93, four O-methyl signals at δ 3.91 (3H), 3.85 (3H),
and 3.68 (6H), and a methylene singlet at δ 4.48. These
data indicated that the structure responsible for peak e1
is papaverine 10.
A major metabolite in fraction C-2 (Figure 2) obtained
from the feeding of 2 to cultured cells of M. cordata
displayed [M + H]+ at m/z 338 and a product ion at m/z
322 in the LC/MS-MS. The stopped-flow 1H NMR spectrum
(see A, Figure 5) of the metabolite showed six aromatic
proton singlets at δ 8.70 (H-8), 8.01 (H-13), 7.45 (H-4), 7.27
(H-9), 6.96 (H-1), and 6.90 (H-12), three O-methyl groups
at δ 3.88 (6H) and 3.83 (3H), and four methylene protons
at δ 3.74 (CH2-6) and 3.09 (CH2-5). These assignments were
The LC/MS-MS of peak c1 displayed [M + H]+ at m/z
342 and its product ion at m/z 192 (Scheme 1). The stopped-
flow 1H NMR spectrum (B, Figure 4) of peak c1 showed
four aromatic protons as singlets at δ 6.84 (H-1), 6.80 (H-
4), 6.75 (H-9), and 6.70 (H-12), methylene protons as an
AB quartet (J ) 15.0 Hz) at δ 4.22 and 4.26, and three
O-methyl groups at δ 3.76 (9H), as well as the other
aliphatic protons. These data indicate that the structure
represented by peak c1 is corytenchine 8. The assigments
were confirmed by NOESY and by comparison of the
stopped-flow 1H NMR data with those of authentic S-
corytenchine measured under the same conditions.
The stopped-flow 1H NMR spectrum (D, Figure 4) of peak
d1 in the LC 1 exhibited two aromatic proton singlets at δ
6.86 (H-4) and 6.82 (H-1), two aromatic proton doublets (J
) 8.5 Hz) at δ 6.93 (H-11) and 6.77 (H-12), three O-methyl

Phenolic 1-Benzyltetrahydroisoquinolines
Journal of Natural Products, 2005, Vol. 68, No. 7 995
Metabolites associated with peaks b3, c3, d3, and f3 rep-
resent cryptopine (6), protopine (7), corytenchine (8), and
tetrahydropalmatrubine (9), respectively, from their indi-
vidual LC/NMR and LC/MS-MS data. The LC/MS-MS of
peak a3 in the LC 3 (Figure 3) displayed [M + H]+ at m/z
1
322 and its product ion at m/z 307. The stopped-flow H
NMR spectrum (B, Figure 5) of the metabolite displayed
four aromatic proton singlets at δ 9.51 (H-8), 8.53 (H-13),
7.47 (H-4), and 6.98 (H-1), two aromatic proton doublets
as an AB quartet (J ) 8.5 Hz) at δ 7.78 (H-11) and 7.74
(H-12), a methylenedioxy resonance at δ 6.34, one O-methyl
signal at δ 3.86, and four methylene protons at δ 4.74 (CH2-
6) and 3.15 (CH2-5). From these data the structure of the
metabolite was deduced to be dehydrocheilanthifoline (13).
The LC/MS-MS of peak e3 in the LC 3 (Figure 3) showed
[M + H]+ at m/z 336 and its product ion at m/z 320. The
1
stopped-flow H NMR spectrum (C, Figure 5) of peak e3
was similar to that of peak a3, but exhibits one more
O-methyl group than peak a3. The structure related to peak
e3 was defined as epiberberine (14). According to the LC/
NMR (A, Figure 5) and LC/MS-MS data, a main metabolite
obtained from fraction C-2 (Figure 2) was identical with
dehydrocorytenchine (11), which was also found as the
metabolite in M. cordata.
The precursors, 3, a mixture of 3 and 4, and 5 were
administered to the cultured cells of C. platycarpa. No
tetrahydroprotoberberine- or pseudotetrahydroprotober-
berine-type alkaloids or their dehydro derivatives were
detected in these feeding experiments. However, the cor-
responding N-methyl derivatives of 3, 4, and 5 were found
by the LC/MS-MS. The N-methyl derivatives of 4 and 5
exhibited [M + H]+ at m/z 344 and product ions at m/z
192 and 151. The N-methyl derivative of 3 displayed [M +
H]+ at m/z 344 and product ions at m/z 206 and 137
(Scheme 1).
Consequently, although 1-benzyltetrahydroisoquinolines,
3, 4, and 5, were N-methylated, in a culture of C. platy-
carpa, they were not bioconverted to any tetrahydropro-
toberberine-type alkaloid.
Figure 4. Stopped-flow 1H NMR spectra of authentic corytenchine
(8), tetrahydropalmatrubine (9), and metabolites (peaks a and c-e in
LC 1 of Figure 3) in the alkaloid fraction (E-2) obtained from the
feeding of 2 to M. cordata. (A) 1H NMR spectrum of peak a (6). (B) 1H
NMR spectrum of peak c (8). (B(2)) Use of mobile phases without TFA.
(C) 1H NMR spectrum of authentic 8. (D) 1H NMR spectrum of peak d
(9). (D(2)) Use of mobile phases without TFA. (E) 1H NMR spectrum
of authentic 9. (F) 1H NMR spectrum of peak e (10). *These signals
belong to CH3OH.
Of the 3′-, 4′-, 7-, and 6-hydoxy isomers (2, 3, 4, and 5)
of tetraoxygenated 1-benzyltetrahydroisoquinolines having
one hydroxyl and three O-methyl groups on the aromatic
rings A and D, only the 3′-hydroxy isomer 2 was converted
into the 2,3,10,11- and 2,3,9,10-oxygenated tetrahydropro-
toberberines. This might be due to the substrate specificity
of the enzyme which converts the 1-benzyltetrahydroiso-
quinolines to the tetrahydroprotoberberines. Bioconversion
of phenolic 1-benzyltetrahydroisoquinoline (2) into pseudot-
etrahydroprotoberberine (8) was demonstrated for the first
time. The metabolic transformations in cultured cells of
Macleaya and Corydalis species demonstrated in the
present investigations are summarized in Scheme 2.
The applicability of on-line coupling of gradient-eluted,
chiral reversed-phase HPLC to the APCI-MS and CD
spectroscopy for the stereochemical analysis of the me-
tabolites contained in the crude extracts was also investi-
gated. A detector simultaneously measuring the UV ab-
sorbances and the CD effects at constant wavelength in
the same detection cell was used for the LC/CD analysis.12
The S absolute configuration at C-13a of tetrahydroproto-
berberine alkaloids with levo rotation has been determined
by X-ray structure analysis.13 Changes in the nature of the
substituents in the aromatic rings A and D of the tetrahy-
droprotoberberines have little or no effect on the specific
rotation at the sodium D line.14 It follows that in general
levorotatory tetrahydroprotoberberines have an S-config-
uration. Consequently those that are dextrorotatory cor-
respond to an R-configuration.
confirmed by NOESY data. The structure of the metabolite
was deduced as dehydrocorytenchine 11.
Fraction E-2 (Figure 2) obtained from feeding of 2 to C.
platycarpa afforded the peaks a2-h2 in the LC 2 (Figure
3), which displayed [M + H]+ at m/z 330, 344, 370, 354,
344, 342, 342, and 342, respectively, in the LC/APCI-MS.
From individual LC/NMR and LC/MS-MS data, metabo-
lites associated with peaks c2, d2, f2, and h2 were deduced
to be cryptopine (6), protopine (7), corytenchine (8), and
tetrahydropalmatrubine (9), respectively. Their data were
identical to those of the corresponding metabolites obtained
from feeding experiment of 2 to M. cordata. The LC/MS-
MS of peak a2 in the LC 2 (Figure 3) showed [M + H]+ at
m/z 330 and product ions at m/z 192 and 137, while that
of peak b2 displayed [M + H]+ at m/z 344 and product ions
at m/z 206 and 137 (Scheme 1). From these data, peaks a2
and b2 were assumed to be the precursor 2 and its N-methyl
derivative, laudanine (12), respectively. The structures
related to peaks e2 and g2 are unknown.
Fraction E-2 (Figure 2) obtained from a feeding experi-
ment of 2 to C. ochotensis var. raddeana showed peaks a3-
f3 in the LC 3 (Figure 3), which displayed [M + H]+ at m/z
322, 370, 354, 342, 336, and 342, respectively, in the LC
/APCI-MS.

996 Journal of Natural Products, 2005, Vol. 68, No. 7
Iwasa et al.
Figure 5. Stopped-flow 1H NMR spectra of metabolites in the alkaloid fractions (E-2 and C-2) obtained from the feeding of 2 to C. ochotensis var.
raddeana. (A) 1H NMR spectrum of major peak (11) of fraction C-2. (B) 1H NMR spectrum of peak a (13) in the LC 3 (Figure 3) of fraction E-2. (C)
1H NMR spectrum of peak e (14) in the LC 3 (Figure 3) of fraction E-2. *These signals belong to CH3OH.
Figure 6. Mass chromatogram of the selected ion m/z 342 in the LC/APCI-MS of RS-corytenchine (8) and RS-tetrahydropalmatrubine (9) and in
the alkaloid fraction (E-2) obtained from the feeding of 2 to M. cordata.
The LC/APCI-MS of synthetic corytenchine (8) and
tetrahydropalmatrubine (9) were measured by chiral chro-
matography. The protonated molecular ions [M + H]+ with
m/z 342 for the regioisomers 8 and 9 were selected and
reconstructed (Figure 6). The MS chromatograms at 11.3
and 11.8 min were attributed to the enantiomers with the
R- and S-configurations at C-13a of 8, respectively, which
were derived by comparison with the MS chromatogram
of enantiomerically pure natural reference 8 with levo
rotation (S-configuration).
The MS chromatograms at 15.3 and 18.2 min were
assigned as enantiomers with the R- and S-configurations
at C-13a of 9, respectively. The configuration at C-13a of 9
was determined by the optical rotation of each enantiomer
separated by preparative HPLC on a chiral column. From
the MS chromatograms (Figure 6) of the selected ions (m/z

Phenolic 1-Benzyltetrahydroisoquinolines
Journal of Natural Products, 2005, Vol. 68, No. 7 997
Scheme 2
342) in the LC/APCI-MS of the alkaloid fraction, fraction
E-2 (Figure 2) obtained from feeding 2 to M. cordata
contained racemic 8 and 9. The stereochemical composition
of 8 and 9 was determined by the ratio of the peak area of
the MS chromatograms.
Of the four isomers of the tetraoxygenated 1-benzyltet-
rahydroisoquinolines having one hydroxyl group, only the
3′-hydroxy isomer was transformed to 2,3,10,11- and 2,3,9,-
10-oxygenated protoberberine alkaloids in cultured cells of
Macleaya and Corydalis species. Bioconversion of 1-ben-
zyltetrahydroisoquinoline into 2,3,10,11-oxygenated pro-
toberberine in the culture was demonstrated for the first
time in the present work. This conversion might also occur
in intact plants, because the pseudo-type alkaloid coryten-
chine (8) has been isolated from C. ochotensis Turcz.15 Some
N-methylating conversions were also clarified. The stere-
ochemical composition of the chiral metabolites was de-
termined, and it was found that one enantiomeric form
dominates, suggesting stereospecific bioconversion. Thus,
it is possible to characterize metabolites in crude extacts
obtained from biosynthetic experiments without isolation
procedures by a combination of the LC/NMR, LC/MS-MS,
and LC/CD techniques.
In the LC/CD (A and B in Figure 7) of racemic 8 and 9
and their S- or R-enantiomers carried out on a chiral phase,
the molecules of retention times at 13.0 and 20.0 min in 8
and 9, respectively, with levo rotation show a negative
Cotton effect at 236 nm, while those at 12.5 and 16.8 min
in 8 and 9 (dextro rotation), respectively, display a positive
Cotton effect. The LC/CD data (C in Figure 7) of the
alkaloid fraction, E-2 (Figure 2), obtained from the feeding
of 2 to M. cordata indicates that the metabolites 8 and 9
are stereoisomeric mixtures. The enantiomeric composition
of 8 and 9 was derived from the ratio of the peak areas
from the CD spectrum. The results summarized in Table
3 indicate that the S- and R-isomers are major in the
metabolites 8 and 9, respectively. This is identical with the
results obtained from the LC/APCI-MS analysis (Table 2).
The enantiomer with S- and R-configurations in coryten-
chine (8) and tetrahydropalmatrubine (9), respectively, was
found to be the major enantiomer from the LC/APCI-MS
and LC/CD analyses. Consequently, it was suggested that
a stereospecific bioconversion might occur in the metabolic
processes in the cultured cells of Macleaya and Corydalis
species. It was demonstrated that the combined use of
HPLC coupled to MS and CD is able to characterize chiral
metabolites without an isolation step.
Experimental Section
General Experimental Procedures. Conventional 1H
NMR and NOESY spectra were obtained on a Varian VXR-
500S spectrometer (1H: 499.99 MHz) in CD3OD or DMSO-d6.
Mass spectra were determined on a Hitachi M 80 instrument
at 75 eV.
Materials. In 1974, 1989, and 1981, respectively, the calli
of M. cordata, C. platycarpa Makino, and C. ochotensis var.
raddeana were derived from the stems of wild plants grown
in Kobe (Japan) on Murashige and Skoog’s medium containing

998 Journal of Natural Products, 2005, Vol. 68, No. 7
Iwasa et al.
Figure 7. UV and CD chromatograms from the LC/CD of corytenchine (8), tetrahydropalmatrubine (9), and their R- or S-enantiomers, and the
alkaloid fraction (E-2) obtained from feeding of 2 to M. cordata. (A) UV and CD chromatograms of RS-8 and RS-9. (B) UV and CD chromatograms
of the R- or S-enantiomer of 8 and 9. (C) UV and CD chromatograms of the alkaloid fraction (E-2).
Table 2. Composition (%) of the Enantiomers of Corytenchine
(8) and Tetrahydropalmatrubine (9) Obtained from the LC/
APCI-MS Data
Table 3. Composition (%) of the Enantiomers of Corytenchine
(8) and Tetrahydropalmatrubine (9) Obtained from the LC/CD
Data
selected ions [M + H]+
8
9
alkaloid
fractionb
precursor
callusa
R
S
R
S
m/z 342 (8)
m/z 342 (9)
alkaloid
fractionb
precursor
callusa
R
S
R
S
2
A
A
B
B
C
E-1
E-2
E-1
E-2
E-1
31
35
24
69
65
76
98
64
nt
72
28
2
A
A
B
B
C
C
E-1
E-2
E-1
E-2
E-1
E-2
25
36
38
12
46
33
75
64
62
88
54
67
63
71
56
60
61
48
37
29
44
40
39
52
nt
nt
2c
36
71
29
a A: M. cordata. B: C. platycarpa. C: C. ochotensis var.
raddeana. b Each fraction is shown in Figure 2. c Shoulder nt:
not determined.
a A: M. cordata. B: C. platycarpa. C: C. ochotensis var.
raddeana. b Each fraction is shown in Figure 2.
tetrahydropapaverine (1) was purchased from Sigma Chemical
Co.
2,4-dichlorophenoxyacetic acid (1 mg/L), kinetin (0.1 mg/L),
yeast extract (0.1%), and agar (1%). The callus tissues were
subcultured every 3 or 4 weeks on the same fresh medium at
25 °C in the dark.
S-Corytenchine (8) [[R]D -304.8° (c 0.31, CHCl3)] is a natural
product obtained from Monanthotaxis fornicata. Racemic
HPLC Parameters for LC/NMR, LC/MS, and LC/CD.
Chromatographic preparations were performed using a Cos-
mosil 5 C18-AR (4.6 i.d. × 150 mm) reversed-phase column.
As a mobile phase (A) 0.1 M NH4OAc (0.05% TFA, D2O for
LC/NMR) and (B) CH3CN (0.05% TFA) were used by linear or
nonlinear gradient elution. The chiral analytical separation

Phenolic 1-Benzyltetrahydroisoquinolines
Journal of Natural Products, 2005, Vol. 68, No. 7 999
was carried out on a chiral OJ-RH column (2.0 i.d. × 150 mm,
Daicel Chemical Ltd.) at room temperature for the LC/MS and
40 °C for the LC/CD.
rated in vacuo. The residue (330 mg) was dissolved in DMSO
(0.5 mL) and was separated by preparative HPLC (a Hitachi
L-6250 intelligent pump and a Hitachi L-4000 UV detector),
which was performed on a Cosmosil 5 C18-AR (20 i.d. × 250
mm) reversed-phase column. As a mobile phase (A) 0.1 M NH4-
OAc (0.05% TFA) and (B) CH3OH (0.05% TFA) were used by
a linear gradient: initial 20% of B and 80 min 100% of B. The
flow rate was 6 mL/min (detection: 280 nm). The eluent
obtained from the peaks b-e in the LC (Figure 1) was
evaporated, and the residue was further purified by HPLC
[H2O (0.05% TFA)-CH3OH (0.05%TFA)] to give the trifluo-
roacetates of 2 (11 mg), a mixture of 3 and 4 (26 mg), and 5
(17 mg). The mixture of 3 and 4 was separated by repeated
preparative HPLC to afford a small amount of 3 and a mixture
of 3 and 4. 2: 1H NMR (CD3OD, 500 MHz) δ 6.93 (1H, d, J )
8.3 Hz, H-5′), 6.80 (1H, s, H-5), 6.78 (1H, d, J ) 2.0 Hz, H-2′),
6.73 (1H, dd, J ) 8.3, 2.0 Hz, H-6′), 6.54 (1H, s, H-8), 4.65
(1H, t, J ) 7.0 Hz, H-1), 3.86 (3H, s, 4′-OCH3), 3.82 (3H, s,
6-OCH3), 3.67 (3H, s, 7-OCH3), 3.53 (1H, m, H-3), 3.33 (2H,
m, H-3 and H-9), 3.1-2.99 (3H, m, H-4 and H-9). 3: 1H NMR
(CD3OD, 500 MHz) δ 6.84 (1H, d, J ) 1.5 Hz, H-2′), 6.81 (1H,
d, J ) 8.3 Hz, H-5′), 6.80 (1H, s, H-5), 6.75 (1H, dd, J ) 8.3,
1.5 Hz, H-6′), 6.56 (1H, s, H-8), 4.68 (1H, t, J ) 7.5 Hz, H-1),
3.82 (6H, s, 3′-and 6-OCH3), 3.68 (3H, s, 7-OCH3), 3.52 (1H,
m, H-3), 3.36 (1H, dd, J ) 14.0, 7.0 Hz, H-9), 3.34 (1H, m,
H-3), 3.07 (1H, dd, J ) 14.0, 8.0 Hz, H-9), 3.07-2.99 (2H, m,
H-4). 5: 1H NMR (DMSO-d6, 500 MHz) δ 6.89 (1H, d, J ) 2.0
Hz, H-2′), 6.78 (1H, s, H-5), 6.75 (1H, d, J ) 8.0 Hz, H-5′),
6.70 (1H, dd, J ) 8.0, 2.0 Hz, H-6′), 6.62 (1H, s, H-8), 4.57
(1H, t, J ) 7.0 Hz, H-1), 3.75 (3H, s, 3′- OCH3), 3.74 (3H, s,
4′-OCH3), 3.62 (3H, s, 7-OCH3), 3.36 (1H, m, H-3), 3.26 (1H,
dd, J ) 14.0, 6.5 Hz, H-9), 3.19 (1H, m, H-3), 2.95 (1H, dd, J
) 14.0, 8.0 Hz, H-9), 2.96-2.84 (2H, m, H-4).
Preparations of Racemic Corytenchine (8), Tetrahy-
dropalmatrubine (9), and Their Enantiomers. To a solu-
tion of 2 (50 mg) dissolved in EtOH (6 mL) was added 37%
formaldehyde (200 mL). The solution was allowed to stand for
one week at room temperature to give a mixture of 8 and 9
(59 mg). The mixture of 8 and 9 (50 mg) was separated by
preparative HPLC on a Cosmosil 5 C18-AR (20 i.d. × 250 mm)
reversed-phase column (a Hitachi L-6250 intelligent pump and
a Hitachi L-4000 UV detector). The mobile phases (A) 0.1 M
NH4OAc (0.05% TFA) and (B) CH3OH (0.05% TFA) were used
for a linear gradient: initial 20% of B and 40 min 100% of B.
The flow rate was 6 mL/min (detection: 280 nm). Each eluent
obtained from the peaks (tR ) 14.46 and 15.95 min) corre-
sponding to 8 and 9 was evaporated, and the residue was
further purified by HPLC [H2O (0.05% TFA)-CH3OH (0.05%
TFA)] to give 8 (13.9 mg) and 9 (34.3 mg) as trifluoroacetates.
8: 1H NMR (CD3OD, 500 MHz) δ 6.94 (1H, s, H-1), 6.82 (1H,
s, H-4), 6.77 (1H, s, H-9), 6.75 (1H, s, H-12), 4.55 (1H, brd, J
) 11 Hz, H-13a), 4.42 (1H, d, J ) 15.0 Hz, H-8), 4.38 (1H,
brd, J ) 15.0 Hz, H-8), 3.86 (3H, s, 3-OCH3), 3.85 (3H, s, 10-
OCH3), 3.83 (3H, s, 2-OCH3), 3.72-3.66 (2H, m, H-6 and H-13),
3.4-3.19 (2H, m, H-5 and H-6), 3.04-2.93 (2H, m, H-5 and
H-13); SIMS m/z 342 [M + H]+ (100), 192 (53); HRSIMS m/z
342.1719 (calcd for C20H24O4N, 342.1704). 9: 1H NMR (CD3-
OD, 500 MHz) δ 6.95 (1H, s, H-1), 6.94 (1H, d, J ) 8.3 Hz,
H-11), 6.81 (1H, s, H-4), 6.78 (1H, d, J ) 8.3 Hz, H-12), 4.62
(1H, d, J ) 16.0 Hz, H-8), 4.47 (1H, brd, J ) 10 Hz, H-13a),
4.16 (1H, brd, J ) 16.0 Hz, H-8), 3.86 (3H, s, 10-OCH3), 3.856
(3H, s, 3-OCH3), 3.84 (3H, s, 2-OCH3), 3.75-3.69 (2H, m, H-6
and H-13), 3.33-3.19 (2H, m, H-5 and H-6), 3.03-2.96 (2H,
m, H-5 and H-13); SIMS m/z 342 [M + H]+ (100), 192 (10);
HRSIMS m/z 342.1715 (calcd for C20H24O4N, 342.1704). Chiral
separation of tetrahydropalmatrubine (9) was performed using
a chiral OJ-RH reversed-phase column (2.0 i.d. × 150 mm) (a
Jasco 880-PU intelligent pump and a Jasco 875-UV detector).
As a mobile phase (A) 0.1 M NH4OAc (0.05% TFA) and (B)
CH3CN (0.05% TFA) were used for nonlinear gradient: initial
20% of B, 10 min 40% of B, and 20 min 40% of B. The flow
rate was 0.5 mL/min (detection: 280 nm). Each eluent
obtained from the peaks corresponding to R-9 (tR ) 16.6 min)
and S-9 (tR ) 19.2 min) was evaporated, and the residue was
LC/APCI-MS Method. LC/APCI-MS (I) for acid-cleavage
products of tetrahydropapaverine (1) was carried out using a
Hitachi M-1000H connected to a Hitachi L-6200 intelligent
pump and a Hitachi L-4000 UV detector. APCI-MS (I) condi-
tions: nebulizer and vaporizer temperatures were 330 and 399
°C, respectively. The drift voltage was 40 V. The quasi-
molecular ions were monitored in the SIM method. LC was
performed on a Cosmosil 5 C18-AR (4.6 i.d. × 150 mm)
reversed-phase column. The mobile phase was 0.1 M NH4OAc
(0.05% TFA, A), to which CH3OH (0.05% TFA, B) was added
by a linear gradient: initial 20% of B and 80 min 100% of B.
The flow rate was 1 mL/min (detection: 280 nm). LC/APCI-
MS(/MS) was measured on an Applied Biosystems API 3000
Triple Quadrapole mass spectrometer (MS/MS) with a heated
nebulizer interface.
Data were collected and processed using Sciex Analyst 1.3
software. Conditions: ion source, APCI; scan type, Q1 scan or
product ion scan; source temperature, 400 °C; ion source
voltage, 5000 V; nebulizer and curtain gases (nitrogen) 10 and
10, respectively; Q1 scan and product ion scan using IDA
method modes were used in MS acquisition method; mass
range, 130-430; collision energy, 35-45 V. The mass spec-
trometer was connected to a Shimadzu LC-10ADVP intelligent
pump and a Shimadzu SPD-10AVP UV detector. A nonlinear
gradientsinitial 20% of B, 25 min 45% of B, 26 min 100% of
Bswas programmed. The flow rates were 1 and 0.5 mL/min
for achiral (Cosmosil 5 C18-AR) and chiral (Chiralcell OJ-RH)
chromatography, respectively (detection: 280 nm).
LC/NMR Method. LC-NMR data were acquired using a
Varian UNITY-INOVA-500 spectrometer (1H: 499.83 MHz)
equipped with a 60 µL triple-resonance microflow NMR probe.
H-1 1D NMR spectra were obtained in stopped-flow mode.
Varian WET solvent suppression16 and related sequences were
used to suppress the peaks of CH3CN, its C-13 satellites, and
the residual HOD in D2O. The WET technique used a series
of variable tip-angle solvent-selective radio frequency (rf)
pulses, where each selective rf pulse is followed by a dephasing
field gradient pulse. FIDs were collected with 32K data points,
a spectral width of 9000 Hz, a 3 ms 90° pulse, a 1.82 s
acquisition time, and a 0.08 s pulse delay. Typically, 8-392
scans were accumulated (1-13 min). Prior to Fourier trans-
formation, an exponential appotization function was applied
to the FID corresponding to a line broadening of 1 Hz. The
NOESY spectra were obtained using a WET-NOESY pulse
sequence, in which the WET element was incorporated into
the 700 ms mixing time. A total of 128 hypercomplex t1
increments with 16-96 (depending on the sample concentra-
tion) transients and 2K data points were acquired with a
spectral width in both dimensions of 3500 Hz with an acquisi-
tion time of 0.15 s, with total acquisition time of 2-14 h. The
data were Gaussian weighted in f2 and f1 and zero filled in f1
to 2K × 2K. The HPLC system consisted of a Varian Pro Star
Model-230, solvent delivery system, and Varian Pro Star
Model-310 variable-wavelength UV-vis detector. The outlet
of the UV detector was connected via a sampling unit (Rheo-
dyne) to the LC-NMR probe. A nonlinear gradientsinitial 20%
of B, 5 min 30% of B, 15 min 30% of B, and 25 min 100% of
Bswas programmed. The flow rate was 1 mL/min (detection:
280 nm).
LC/CD Method. Chromatographic separation was per-
formed using a Jasco PU-2080Plus intelligent pump with a
column oven (Jasco 860-CO) and a Jasco Browin NT, HSS-
2000 data processor, and a Jasco CD-2095Plus CD chiral
detector (Hg-Xe lamp), simultaneously monitoring the CD and
UV signals at one specific wavelength (range 220-420 nm).
A nonlinear gradientsinitial 20% of B, 10 min 40% of B, 20
min 40% of B, 30 min 100% of Bswas programmed. The flow
rate was 0.5 mL/min (detection: 236 nm).
Preparation of Phenolic 1-Benzyltetrahydroisoquino-
lines 2, 3, 4, and 5. A solution of racemic tetrahydropapav-
erine hydrochloride (1) (Sigma) (500 mg) in concentrated HCl
(5 mL) was refluxed for 5.5 h. Hydrochloric acid was evapo-

1000 Journal of Natural Products, 2005, Vol. 68, No. 7
Iwasa et al.
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further purified by HPLC [H2O (0.05% TFA)-CH3OH
(0.05%TFA)] to give R-9 [14.2 mg, [R]D +136.4° (c 0.286, CH3-
OH)] and S-9 [13.5 mg, [R]D -146.9° (c 0.715, CH3OH)] as
trifluoroacetates.
Feeding Experiments. Substrates were dissolved in H2O
(2-4 mL) and introduced through a sterile bacterial filter into
100 mL conical flasks containing 40 mL of autoclaved MS
medium, identical with that employed in the subculture. Calli
(ca. 4-5 g) were transferred to each conical flask and incubated
at 25 °C in the dark for an appropriate time (Table 1). Cells
and medium were separated and extracted with CH3OH at
60 °C. Extracts were worked up as described in Figure 2.
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References and Notes
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York, 1995; Vol. 46, pp 273-346.
(2) Iwasa, K.; Nanba, H.; Lee, D.-U.; Kang, S.-I. Planta Med. 1997, 64,
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(3) Iwasa, K.; Nishiyama, Y.; Ichimaru, M.; Moriyasu, M.; Kim, H.-S.;
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1999, 34, 1077-1083.
(4) Iwasa, K.; Moriyasu, M.; Yamori, T.; Turuo. T.; Lee, D.-U.; Wiegrebe,
W. J. Nat. Prod. 2001, 64, 896-898.
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