Biotransformation of phenolic 1-benzyl-N-methyltetrahydroisoquinolines in plant cell cultures followed by LC/NMR, LC/MS, and LC/CD

Article abstract of DOI:10.1021/np068060r

(±)-1-Benzyl-N-methyltetrahydroisoquinolines 7-10 and 11-14 with one and two hydroxy groups on the aromatic rings, respectively, were fed individually to cultured cells of Corydalis and Macleaya species, respectively. The structures of the metabolites were determined by using combinatorial techniques, including LC/NMR, LC/MS-MS, and LC/CD. The enantiomeric excesses of the metabolites were derived from LC/CD and LC/MS-MS analyses. In cell cultures of Corydalis and Macleaya species, laudanine (7), with a hydroxy group at C-3′, can form the berberine bridge at C-2′ and C-6′ to produce S- and R-enantiomers of 2,3,9,10- and 2,3,10,11-oxygenated protoberberines (20 and 21), respectively, whereas reticuline (11) and protosinomenine (12), incoporating a hydroxy group at C-3′, form the berberine bridge at C-2′ to furnish the S-enantiomer of 2,3,9,10-oxygenated protoberberines (23 and 21), respectively.

Full text of DOI:10.1021/np068060r

J. Nat. Prod. 2007, 70, 1771–1778
1771
Biotransformation of Phenolic 1-Benzyl-N-methyltetrahydroisoquinolines in Plant Cell Cultures
Followed by LC/NMR, LC/MS, and LC/CD
Wenhua Cui,† Kinuko Iwasa,*,† Makiko Sugiura,† Atsuko Takeuchi,† Chisato Tode,† Yumi Nishiyama,† Masataka Moriyasu,†
Harukuni Tokuda,‡ and Kazuyoshi Takeda§
Kobe Pharmaceutical UniVersity, 4-19-1 Motoyamakita, Higashinada-ku, Kobe-shi 658-8558, Japan, Department of Biochemistry and
Molecular Biology, Kyoto Prefectural UniVersity of Medicine, Kawaramachi-dori, Kamigyo-ku, Kyoto-shi 602-0841, Japan, and Yokohama
College of Pharmacy, 601 Matanocyo, Hodogayaku, Yokohama-shi 245-0066, Japan
ReceiVed October 27, 2006
(()-1-Benzyl-N-methyltetrahydroisoquinolines 7–10 and 11–14 with one and two hydroxy groups on the aromatic rings,
respectively, were fed individually to cultured cells of Corydalis and Macleaya species, respectively. The structures of
the metabolites were determined by using combinatorial techniques, including LC/NMR, LC/MS-MS, and LC/CD. The
enantiomeric excesses of the metabolites were derived from LC/CD and LC/MS-MS analyses. In cell cultures of Corydalis
and Macleaya species, laudanine (7), with a hydroxy group at C-3′, can form the berberine bridge at C-2′ and C-6′ to
produce S- and R-enantiomers of 2,3,9,10- and 2,3,10,11-oxygenated protoberberines (20 and 21), respectively, whereas
reticuline (11) and protosinomenine (12), incoporating a hydroxy group at C-3′, form the berberine bridge at C-2′ to
furnish the S-enantiomer of 2,3,9,10-oxygenated protoberberines (23 and 21), respectively.
Within the biogenetic focus for the isoquinoline alkaloids, it has
been well documented that tetrahydrobenzylisoquinolines will lead
to protoberberines, which may undergo further transformations to
furnish an array of additional isoquinoline alkaloids.1–9
Studies on the biosynthesis of 2,3,10,11-oxygenated protober-
berines have been reported,10,11 including Corydalis species12,13
and others.
Our present focus was on establishing some of the structural
parameters for the eight 1-benzyl-N-methyltetrahydroisoquinolines
7–10 and 11–14, which possess one and two hydroxy groups,
respectively. To this end, we concentrated on the analysis of the
metabolites from the cell cultures of Corydalis (Fumariaceae) and
Macleaya (Papaveraceae) species.
Our conclusions show that the norlaudanosine analogue 7,
Figure 1. LC data of the crystalline products obtained by refluxing
possessing a 3′-hydroxy group, can serve as a precursor of 2,3,9,10-
N-methyltetrahydropapaverine (6) with 47% HBr for 13 min. Pump:
and 2,3,10,11-oxygenated protoberberines, whereas the remaining
three isomers, 8–10, were not metabolized to protoberberines.
Among species 11–14, with two hydroxy groups, 11 and 12, with
a 3′-hydroxy function, can form the berberine bridge to produce
Hitachi L-6200; column: Cosmosil 5 C18ARII (4.6 i.d. × 150 mm);
gradient: 0.1 M NH4OAc (0.05% TFA)/CH3OH (0.05% TFA),
initial 80/20, 30 min 0/100; flow rate: 1 mL/min; UV detection:
280 nm (preparative HPLC: initial 80/20, 80 min 0/100).
2,3,9,10-oxygenated protoberberines.
By enzyme-catalyzed reaction, the structural requirements for
the formation of the berberine bridge to produce 2,3,9,10- and
1,2,9,10-oxygenated protoberberines from the tetrahydrobenzyl-
isoquinoline alkaloids are as follows:14 the N-methyltetrahydroben-
zylisoquinoline nucleus must have the S-configuration at C-1, and
the aromatic carbon ortho to C-2′ must carry a hydroxy group.
product ions representing the isoquinoline (ion A) and benzyl (ion
B) moieties as shown in Scheme 1. The structures of the compounds
obtained from peaks a-g and i (Figure 1) were determined by H
NMR (Table 1) and MS analyses (Table 2 and Scheme 1) to be
1
7–13 and 14, respectively. The structures of the deuterated
1
derivatives [N-CD3]-7 to [N-CD3]-14 were determined from H
NMR and MS data (Table 2, Scheme 1) and were identical to those
of the unlabeled compounds. 1-Benzyl-N-methyltetrahydroisoquino-
lines 7–14 and [N-CD3]-7–14 were used in the feeding experiments.
Feeding Experiments. Callus tissues of Macleaya cordata,
Corydalis platycarpa Makino, and C. ochotensis var. raddeana were
incubated at 25 °C on an agar medium containing the substrate for
four weeks (Table 3). Following incubation, media and cells were
separated and extracted according to the procedure shown in Figure
2. Alkaloid fractions, E-1, E-2, C-1, and C-2 (Figure 2), were
subjected to LC/NMR, LC/MS, and LC/CD.
Results and Discussion
Syntheses. N-Methyltetrahydropapaverine (6) and [N-CD3]-6
were obtained by reduction of N-methylpapaverinium salts 5 and
[N-CD3]-5, which were prepared by the N-methylation of com-
mercially available papaverine with CH3I and CD3I, respectively.
The LC/APCI-MS (Figure 1) of the products obtained from the
acid-catalyzed ether cleavage of 6 was measured in the positive
ion mode and showed the presence of several products bearing one
or more phenolic hydroxy groups as well as the presence of starting
material. The 1-benzyl-N-methyltetrahydroisoquinolines show two
Precursors 7–10. The metabolism of 1-benzyl-N-methyltet-
rahydroisoquinolines 7–10, possessing one hydroxy and three
methoxy groups on the aromatic A and D rings, respectively, was
examined (Table 3) in cultured cells of C. platycarpa or C.
ochotensis var. raddeana and M. cordata.
* Corresponding author. E-mail: k-iwasa@kobepharma-u.ac.jp. Tel: 081-
78-441-7544. Fax: 081-78-441-7544.
† Kobe Pharmaceutical University.
‡ Kyoto Prefectural University of Medicine.
§ Yokohama College of Pharmacy.
10.1021/np068060r CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/08/2007

1772 Journal of Natural Products, 2007, Vol. 70, No. 11
Cui et al.
Scheme 1
Table 1. 1H NMR Dataa of 1-Benzyl-N-methyltetrahydroisoquinolines (7-14)
compound
proton
7
8
9
10
4.56h
11
4.53i
12
4.50h
13
4.51i
14
4.54h
NOESYcorrelation
1-H
4.52b
4.50e
3.67g
2-NMe, 8-H
2′-H, 6′-H
1-H
2-NMe
2.96
2.92
2.53
2.98
2.92
2.95
2.93
2.95
3-H, 4-H and 9-H 3.0-3.77
3.01-3.65
6.79
3.85
2.56-3.17
3.05-3.78
3.02-3.70
2.98-3.75
2.95-3.71
2.99-3.71
5-H
6.81
3.81
3.50
6.03
6.64c
6.62
6.82
6.79
6.66
6.65
6.67
6-OMe
5-H
8-H
1-H, 7-OMe
3′-OMe, 1-H
2′-H
5′-H
4′-OMe, 6′-H
5′-H, 1-H 9-H
6-OMe
7-OMe
8-H
3.81
3.50
3.85
3.57
5.99
6.58c
3.79
3.85
6.77d
6.65f
3.52
5.99
6.64c
3.53
6.00
6.64c
3.76
6.18
6.69c
3.75
3.82
6.91d
6.74f
6.03
6.24
6.16
6.68c
3.75
3.82
6.91d
6.75f
2′-H
6.66c
3.76
6.68c
3′-OMe
4′-OMe
5′-H
3.84
3.85
3.84
6.89d
6.60f
6.77d
6.63f
6.89d
6.90d
6.77d
6.63f
6′-H
6.62f
6.61f
a CD3OD; δ ppm, 500 MHz. b m. c d, J ) 2.0 Hz. d d, J ) 8.0 Hz. e t, J ) 6.0 Hz. f dd, J ) 8.0, 2.0 Hz. g dd, J ) 8.5, 5.0 Hz. h dd, J ) 9.0, 5.0
Hz. i t, J ) 6.5 Hz. j t, J ) 7.0 Hz.
Table 2. MS Data of 1-Benzyl-N-methyltetrahydroisoquinolines (7–14) and [N-CD3]-l-Benzyltetrahydroisoquinolines ([N-CD3]-7-[N-CD3]-
14
HRSIMS
product ionsa m/z
compound
SIMS m/z
formula
calcd
found
A
B
7
344
347
344
347
344
347
344
347
330
333
330
333
330
333
330
333
C20H26NO4
C20H23D3NO4
C20H26NO4
C20H23D3NO4
C20H26NO4
C20H23D3NO4
C20H26NO4
C20H23D3NO4
C19H24NO4
C19H21D3NO4
C19H24NO4
C19H21D3NO4
C19H24NO4
C19H21D3NO4
C19H24NO4
344.1860
347.2049
344.1860
347.2049
344.1860
347.2049
344.1860
347.2049
330.1704
333.1892
330.1704
333.1892
330.1704
333.1892
330.1704
333.1892
344.1862
347.2052
344.1873
347.2066
344.1863
347.2059
344.1875
347.2056
330.1702
333.1702
330.1707
333.1904
330.1714
333.1910
330.1726
333.1908
206
209
192
195
192
195
206
209
192
195
192
195
178
181
192
195
137
137
151
151
151
151
137
137
137
137
137
137
151
151
137
137
[N-CD3]-7
8
[N-CD3]-8
9
[N-CD3]-9
10
[N-CD3]-10
11
[N-CD3]-11
12
[N-CD3]-12
13
[N-CD3]-13
14
[N-CD3]-14
C19H21D3NO4
a These product ions were derived by APCI-MS/MS spectra.
First, precursor 7 was administered to cultured cells of C.
platycarpa (Table 3, no. 1). Fraction E-2 (Figure 2) showed nine
peaks, a1-i1, in LC 1 (Figure 3). From the LC/NMR and LC/MS-
MS data, peaks a1 ([M + H]+: m/z 344, product ion: m/z 206), b1
([M]+: m/z 322, product ion: m/z 307), d1 ([M + H]+: m/z 354,
product ions: m/z 206, 189, and 188), and h1 ([M + H]+: m/z 326,
product ion: m/z 178) were attributed to the precursor 7, dehydro-
cheilanthifoline (15), protopine (16), and cheilanthifoline (17),
respectively. Dehydrocheilanthifoline (15), protopine (16), and
cheilanthifoline (17) were identified by comparison of their LC/
NMR data with those of authentic samples from C. platycarpa and
C. ochotensis var. raddeana, and 16 was also identified as a
component of M. cordata.
21. Compounds 18–21 have also been identified in the feeding
experiment of 1.11 Likewise, the stopped-flow H NMR spectrum
1
of peak i1 is due to tetrahydroepiberberine 22. Laudanine (7) and
epiberberine (19) were also found in fraction C-2 (Figure 3).
Furthermore, the metabolites obtained from the feeding of 7 to
C. ochotensis var. raddeana (Table 3, no. 2) were found to be
cryptopine (18), corytenchine (20), tetrahydropalmatrubine (21), and
tetrahydroepiberberine (22). Thus, compound 7 was bioconverted
into corytenchine (20) and tetrahydropalmatrubine (21), which was
further converted via tetrahydroepiberberine (22) to cryptopine (18)
(Scheme 2). Tetrahydroepiberberine (22) might simply arise from
the reduction of epiberberine (19). These conversions were con-
firmed by the feeding of [N-CD3]-7 to C. platycarpa (Table 3, no.
13). The deuterated metabolites cryptopine (m/z 372), corytenchine
(m/z 344), tetrahydropalmatrubine (m/z 344), and tetrahydroepiber-
berine (m/z 342), and unlabeled epiberberine (m/z 336), were
Similarly, peak c1 (Figure 3) corresponded to cryptopine (18),
peak e1 (tR ) 9.54 min) to epiberberine (19), peak f1 (tR ) 9.82
min) to corytenchine (20), and peak g1 to tetrahydropalmatrubine

Phenolic 1-Benzyl-N-methyltetrahydroisoquinolines
Journal of Natural Products, 2007, Vol. 70, No. 11 1773
Table 3. Administration of 1-Benzyl-N-methyltetrahydroisoquino-
lines to Cultured Cells of Macleaya and Corydalis Species
no. callusa
substrate (mg)
medium (mL) wt of dry cells (g)
1
2
3
4
5
6
7
8
A
B
A
B
A
B
A
B
A
A
A
A
A
C
A
B
C
A
B
A
B
C
A
A
A
A
7 (38)
7 (19)
8 (42)
8 (38)
9 (39)
9 (39)
10 (35)
10 (35)
11 (34)
12 (20)
13 (39)
14 (34)
[N-CD3]-7 (29)
[N-CD3]-7 (26)
[N-CD3]-8 (18)
[N-CD3]-8 (18)
[N-CD3]-8 (18)
[N-CD3]-9 (17)
[N-CD3]-9 (16)
[N-CD3]-10 (17)
[N-CD3]-10 (19)
[N-CD3]-10 (18)
[N-CD3]-11 (19)
[N-CD3]-12 (19)
[N-CD3]-13 (6)
[N-CD3]-14 (35)
800
400
800
800
800
800
800
800
800
800
400
800
800
800
400
400
400
400
400
400
400
400
800
800
400
800
4.0
2.1
5.2
4.0
4.8
6.0
6.3
4.3
4.7
7.2
3.3
4.8
5.4
5.9
3.5
2.6
2.3
1.9
7.3
2.8
2.1
2.4
3.7
5.0
2.2
3.7
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
a A: Corydalis platycarpa Makino. B: Corydalis ochotensis var.
raddeana. C: Macleaya cordata.
Just as with benzylisoquinoline 7, precursors 8–10 were also
administered to cultured cells of C. platycarpa and C. ochotensis
var. raddeana (Table 3, no. 3–8). The major metabolite obtained
from the feeding of 9 and 10 was identified as N-methyltetrahy-
dropapaverine (6) by analysis of LC/NMR and LC/MS-MS data.
A trace amount of 6 was also found as a metabolite of 8. The
bioconversion of 8–10 into 6 was confirmed by the feeding of
[N-CD3]-8, -9, and -10 (Table 3, no. 15–22) (Scheme 2). Signifi-
cantly, compounds 8–10 were not converted into tetrahydroproto-
berberine- or pseudotetrahydroprotoberberine-type compounds.
Scheme 2 summarizes the metabolic transformations of 1-benzyl-
N-methyltetrahydroisoquinolines 7–10 in cultured cells of Macleaya
and Corydalis species. Among the four isomers (3′-, 4′-, 7-, and
6-hydroxy, respectively), only the 3′-hydroxy isomer 7 was
converted into 2,3,10,11- and 2,3,9,10-oxygenated tetrahydropro-
toberberines (20 and 21). This observation suggests that the presence
of the hydroxy group at C-3′ is essential for the biotransformation
of 1-benzyl-N-methyltetrahydroisoquinolines into 2,3,10,11- and
2,3,9,10-oxygenated tetrahydroprotoberberines. Thus, the enzyme
responsible for the conversion likely shows substrate specificity.
This behavior has also been found in the bioconversion of phenolic
1-benzyltetrahydroisoquinoline 1 into 2,3,10,11- and 2,3,9,10-
oxygenated tetrahydroprotoberberines.11 The structural requirement
for the formation of the berberine bridge to produce 2,3,9,10- and
1,2,9,10-oxygenated protoberberines has been studied using enzyme-
Figure 2. Preparation of samples for LC/NMR, LC/MS, and LC/
CD measurements.
identified from the LC/MS-MS of fraction E-2. Deuterated tetrahy-
droepiberberine likely underwent dehydrogenation with the loss of
deuterium to give unlabeled epiberberine. A trace amount of
deuterated N-methyltetrahydropapaverine ([N-CD3]-6) was also
confirmed by LC/MS-MS ([M + H]+ at m/z 361, product ions at
m/z 209 and 151) (Scheme 1).
Finally, in cultured cells of M. cordata (Table 3, no 14),
deuterated 7 was metabolized to deuterated cryptopine (m/z 372),
corytenchine (m/z 344), tetrahydropalmatrubine (m/z 344), N-
methyltetrahydropapaverine (m/z 361), and tetrahydroepiberberine
(m/z 342).

1774 Journal of Natural Products, 2007, Vol. 70, No. 11
Cui et al.
Figure 3. LC data of the alkaloid fractions (E-2 and C-2) obtained from the feeding of 7 to C. platycarpa. Pump: Shimadzu LC-10ADvp;
column: Cosmosil 5 C18ARII (4.6 i.d. × 150 mm); gradient: A 0.1 M NH4OAc (0.05% TFA)/B CH3CN (0.05% TFA) A/B: initial 80/20,
25 min 55/45, 30 min 0/100; flow rate: 1 mL/min; UV detection: 280 nm.
Scheme 2
catalyzed reactions.14 Metabolite 21 was further biotransformed via
tetrahydroepiberberine (22) into cryptopine (18). All four 1-ben-
zyltetrahydroisoquinolines 7–10 were O-methylated to give N-
methyltetrahydropapaverine (6). However, with 9 and 10, N-me-
thyltetrahydropapaverine (6) was the major metabolite, suggesting
that the hydroxy groups at C-6 and C-4′ in 9 and 10, respectively,
might be O-methylated more readily than those at C-7 and C-3′ in
8 and 7, respectively.

Phenolic 1-Benzyl-N-methyltetrahydroisoquinolines
Journal of Natural Products, 2007, Vol. 70, No. 11 1775
Peak e2 in the LC 2 (Figure 5) showed [M + H]+ at m/z 330
and a product ion at m/z 178 in the LC/MS-MS, thus corresponding
to [8-D2]-scoulerine ([8-D2]-23). This structure was confirmed by
comparison of the stopped-flow 1H NMR data with those of
authentic (S)-scoulerine measured under the same conditions. Chiral
phase LC/CD (A and B in Figure 6) was also performed on fraction
E-2 obtained from the feeding of [N-CD3]-reticuline ([N-CD3]-11),
with authentic S-scoulerine used as a reference. The CD of the
[8-D2]-scoulerine metabolite at 20 min in the LC/CD (Figure 6,
A) showed a negative Cotton effect at 236 nm, which matched
that of S-scoulerine (Figure 6, B).
Precursor 12 (protosinomenine) and the deuterated precursor
[N-CD3]-12, which have the two hydroxy groups at C-6 and C-3′,
was also administered to cultured cells of C. platycarpa (Table 3,
nos. 10 and 24). Fraction E-2 obtained from C. platycarpa showed
peaks a3-h3 in the LC 3 (Figure 7). From comparison of individual
LC/MS-MS data with those of the corresponding alkaloids obtained
from the feeding of [N-CD3]-7, compounds associated with peaks
a3, b3, d3, and g3 were determined to be [N-CD3]-protosinomenine
([N-CD3]-12), [N-CD3]-laudanine ([N-CD3]-7), [8-D2]-cryptopine
([8-D2]-18), and [8-D2]-tetrahydropalmatrubine ([8-D2]-21), respec-
tively. [N-CD3]-Laudanine ([N-CD3]-7), [8-D2]-cryptopine ([8-D2]-
18), and [8-D2]-tetrahydropalmatrubine ([8-D2]-21) are metabolites
of [N-CD3]-protosinomenine ([N-CD3]-12). The isomer of scoulerine
(23) predicted to be formed by ring closure of 12 was not detected.
This may be due to readily occurring O-methylation at C-6 to
furnish 7. The peaks at 15.3 and 18.2 min in the MS of the selected
ion at m/z 342 have been assigned as R- and S-enantiomers of (()-
tetrahydropalmatrubine (21), respectively.11 The MS of the selected
ion at m/z 344 ([8-D2]-21) of 18.2 min was identical with that of
(S)-tetrahydropalmatrubine (Figure 8), thus indicating that the
[8-D2]-tetrahydropalmatrubine metabolite was the S-enantiomer.
From the LC/MS-MS analysis, the compound associated with peak
f3 was found to be epiberberine (19), which may be a metabolite
of [N-CD3]-12. The compounds associated with peaks c3, e3, and
h3 were deduced as dehydrocheilanthifoline (15), protopine (16),
and cheilanthifoline (17), respectively, which are components of
the callus.
In a similar fashion, 6,7-dihydroxylated 13 and [N-CD3]-13 were
administered to cultured cells of C. platycarpa (Table 3, nos. 11 and
25). Codamine (8) and [N-CD3]-8 were identified (Table 2). In the
same way, isoorientaline (14) and [N-CD3]-14, with the two hydroxy
groups at C-6 and C-4′, were administered to C. platycarpa (Table 3,
nos. 12 and 26). Pseudocodamine (10) and N-methyltetrahydropapav-
erine (6) were characterized as the metabolites of 14; [N-CD3]-10 and
[N-CD3]-6, as the metabolites of [N-CD3]-14.
Scheme 3 summarizes the biotransformations demonstrated by the
results of feeding experiments of 1-benzyl-N-methyltetrahydroiso-
quinolines 11–14 to cultured cells of Macleaya and Corydalis species.
Among the four 1-benzyl-N-methyltetrahydroisoquinolines with
two hydroxy and two methoxy groups on the aromatic A and D
rings (11–14), respectively, compounds 11 and 12 were converted
into the S-enantiomer of 2,3,9,10-oxygenated tetrahydroprotober-
berines 23 and 21, respectively, while 13 and 14 were not
biotransformed to tetrahydroprotoberberines. These observations
with dihydroxy-substituted compounds confirm that the presence
of a hydroxy group at C-3′ is essential for the biotransformation of
1-benzyl-N-methyltetrahydroisoquinolines into 2,3,9,10-oxygenated
tetrahydroprotoberberines. The product formed by ring closure at
C-6′ was not detected. This is identical with the result reported by
Kutchan et al.14
Figure 4. Mass chromatogram of the selected ions m/z 344 in the
LC/APCI-MS of the alkaloid fraction (E-2) obtained from the
feeding of [N-CD3]-7 to C. platycarpa. Pump: Shimadzu LC-
10ADvp; column: Chiralcel OJ-RH (4.6 i.d. × 150 mm); gradient:
A 0.1 M NH4OAc (0.05% TFA)/B CH3CN (0.05% TFA) A/B:
initial 80/20, 10 min 60/40, 20 min 60/40, 30 min 0/100; flow rate:
0.5 mL/min; UV detection: 280 nm.
Table 4. Enantiomeric Excess (%) of [8-D2]-Corytenchine
([8-D2]-20) and [8-D2]-Tetrahydropalmatrubine ([8-D2]-21) Obtained
from the LC/MS-MS Data
selected ions [M + H]+
alkaloid m/z 344 ([8-D2]-20) m/z 344 ([8-D2]-21)
precursor callusa fractionb
R
S
7
A
A
B
B
E-1
E-2
E-1
E-2
52
30
nd
46
80
84
nd
80
a A: Corydalis platycarpa Makino. B: Macleaya cordata. b See
Figure 2. nd: not determined.
Figure 4 shows the MS chromatogram of selected ions (m/z 344)
in the LC/APCI-MS of the alkaloid fraction E-2 obtained from the
feeding of [N-CD3]-7 to C. platycarpa. The peaks at 11.1 and 11.6
min were assigned to enantiomers of [8-D2]-20, with the R- and
S-configuration, respectively, at C-13a on the basis of reported
data.11 Likewise, the peaks at 14.6 and 17.2 min were assigned as
enantiomers of [8-D2]-21, with R- and S-configuration, respectively,
at C-13a.11 The enantiomeric excesses of [8-D2]-20 and -21 were
determined from the ratio of the peak areas of the MS chromato-
grams of the selected ion (m/z 344). The results summarized in
Table 4 indicate that metabolites 20 and 21 contain different
amounts of the S- and R-isomers. Metabolite 21 showed a high
predominance of the S-isomer, while metabolite 20 showed a lower
predominance of the R-isomer. Consequently, it is suggested that
a stereoselective bioconversion occurs in the metabolic processes
in cultured cells of Macleaya and Corydalis species.
In the next phase of our study, the metabolism of 1-benzyl-N-
methyltetrahydroisoquinolines 11–14 and their [N-CD3]-derivatives
([N-CD3]-11-[N-CD3]-14), which have two hydroxy and two
methoxy groups on the aromatic A and D rings, respectively, were
examined in C. platycarpa.
First, 7,3′-dihydroxylated 11 (reticuline) was administered to
cultured cells of C. platycarpa (Table 3, no. 9). Trace amounts of
laudanine (7) and scoulerine (23) were identified. In order to verify
this result, [N-CD3]-11 was administered to C. platycarpa (Table
3, no. 23), and the resulting fraction E-2 showed peaks a2-e2 in
LC 2 (Figure 5). The compounds associated with peaks a2, b2, and
c2 were deduced as [N-CD3]-reticuline ([N-CD3]-11), [N-CD3]-
laudanine ([N-CD3]-7), and protopine (16), respectively, and peak
d2 corresponded to dehydrocheilanthifoline (15).
The following sequences of O-methylation were thus identified in
the present study. Reticuline (11) was biotransformed to (S)-scoulerine
(23), since the hydroxy group at C-7 was not O-methylated readily.
However, protosinomenine (12) was readily O-methylated at C-6 to
provide laudanine (7), which in turn was bioconverted into (S)-
tetrahydropalmatrubine (21). Eventually, (S)-tetrahydropalmatrubine

1776 Journal of Natural Products, 2007, Vol. 70, No. 11
Cui et al.
Figure 5. LC data of the alkaloid fraction (E-2) obtained from the feeding of [N-CD3]-reticuline ([N-CD3]-11) to C. platycarpa. Pump:
Varian Prostar model 230; column: Chiralcel OJ-RH (4.6 i.d. × 150 mm); gradient: A 0.1 M NH4OAc/D2O (0.05% TFA)/B CH3CN
(0.05% TFA) A/B: initial 80/20, 10 min 60/40, 20 min 60/40; flow rate: 0.5 mL/min; UV detection: 280 nm.
Figure 6. UV and CD chromatograms from the LC/CD of (S)-scoulerine (S-23) and the alkaloid fraction (E-2) obtained from the feeding
of [N-CD3]-reticuline ([N-CD3]-11) to C. platycarpa. A: UV and CD chromatograms of the alkaloid fraction (E-2). B: UV and CD
chromatograms of authentic S-23.
(21) was converted to cryptopine (18). Compound 13 was O-
methylated at C-6 to give codamine (8), indicating that the hydroxy
group at C-6 is more readily O-methylated than that at C-7. Finally,
isoorientaline (14) underwent O-methylation at C-6 to produce
pseudocodamine (10), which was in turn O-methylated at C-4′ to yield
N-methyltetrahydropapaverine (6).
It can, therefore, be concluded that in cell cultures of Corydalis
and Macleaya species, laudanine (7), with a hydroxy group at C-3′,
can form the berberine bridge at both positions C-2′ and C-6′ to
produce S- and R-enantiomers of 2,3,9,10- and 2,3,10,11-oxygenated
protoberberines (20 and 21), respectively, whereas reticuline (11)
and protosinomenine (12), incorporating a hydroxy group at C-3′,
form the berberine bridge at C-2′ to furnish the S-enantiomers of
2,3,9,10-oxygenated protoberberines (23 and 21), respectively.
There are differences in the position of ring closure between
laudanine (7) and reticuline (11) or protosinomenine (12) and in
the configuration at C-13a of the protoberberine metabolites
obtained from 7 and 11 or 12. These may be due to difference in
the berberine bridge-forming enzyme. Formation of the protober-
berines from reticuline (11) and protosinomenine (12) parallel the
results demonstrated by Kutchan et al.14
Experimental Section
Materials. In 1974, 1989, and 1981, the calli of M. cordata, C.
platycarpa Makino, and C. ochotensis var. raddeana, respectively, were
derived from the stems of wild plants grown in Kobe (Japan) on

Phenolic 1-Benzyl-N-methyltetrahydroisoquinolines
Journal of Natural Products, 2007, Vol. 70, No. 11 1777
Figure 7. LC data of the alkaloid fraction (E-2) obtained from the feeding of [N-CD3]-protosinomenine ([N-CD3]-12) to C. platycarpa.
Pump: Varian Prostar model 230; column: Cosmosil 5 C18ARII (4.6 i.d. × 150 mm); gradient: A 0.1 M NH4OAc/D2O (0.05% TFA)/B
CH3CN (0.05% TFA) A/B: initial 80/20, 25 min 55/45, 26 min 0/100; flow rate: 1 mL/min; UV detection: 280 nm.
The callus tissues were subcultured every three or four weeks on fresh
medium at 25 °C in the dark.
The natural product (S)-scoulerine (23) {[R]D -270 (c 0.12,
CH3OH)} was obtained from C. platycarpa Makino. Papaverine
hydrochloride and CD3I were purchased from Sigma Chemical Co.
HPLC Parameters for LC/NMR, LC/MS, and LC/CD. Chro-
matographic preparations were performed using a Cosmosil 5 C18-AR
(4.6 i.d. × 150 mm) reversed-phase column. The mobile phases [(A)
0.1 M NH4OAc (0.05% TFA, D2O for LC/NMR) and (B) CH3CN
(0.05% TFA) or CH3OH (0.05% TFA)] were used for linear or
nonlinear gradient elution.
The flow rate was 1 mL/min (detection: 280 nm). The chiral
analytical separation was carried out on a chiral OJ-RH column (4.6
i.d. × 150 mm, Daicel Chemical Ltd.) at room temperature for LC/
MS and 40 °C for LC/CD. The flow rate was 0.5 mL/min (detection:
LC/MS 280 nm, LC/CD 236 nm).
LC/APCI-MS Method. The LC/APCI-MS (/MS) spectra were
measured with Q3 and product ion scans. Molecular weight information
was obtained through the protonated molecular ions [M + H]+, and
product ions were recorded by LC/MS-MS. LC/APCI-MS for acid-
cleavage products of N-methyltetrahydropapaverine (6) 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 conditions:
nebulizer and vaporizer temperatures were 320 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
Figure 8. Mass chromatograms of the selected ions. A: alkaloid
added by a linear gradient (initial 20% B, 30 min 100% B). The flow
rate was 1 mL/min (detection: 280 nm).
LC/APCI-MS (/MS) was measured on an Applied Biosystems API
fraction (E-2) obtained from the feeding of [N-CD3]-12 to C.
platycarpa. B: Authentic (()-tetrahydropalmatrubine (21). Pump:
Shimadzu LC-10ADvp; column: Chiralcel OJ-RH (4.6 i.d. × 150
3000 triple quadrapole mass spectrometer (MS/MS) with a heated
nebulizer interface as described in the previous paper.11
mm); gradient: A 0.1 M NH4OAc (0.05% TFA)/B CH3CN (0.05%
TFA) A/B: initial 80/20, 10 min 60/40, 20 min 60/40, 30 min 0/100;
flow rate: 0.5 mL/min; UV detection: 280 nm.
LC/NMR Method. The LC/NMR spectra were measured in the
stopped-flow mode. 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. 1D 1H NMR spectra were obtained in
stopped-flow mode as described in the previous paper.11
Murashige and Skoog’s medium containing 2,4-dichlorophenoxyacetic
acid (1 mg/L), kinetin (0.1 mg/L), yeast extract (0.1%), and agar (1%).

1778 Journal of Natural Products, 2007, Vol. 70, No. 11
Cui et al.
Scheme 3
LC/CD Method. The LC/CD analysis was carried out on a chiral
reversed-phase column at 236 nm. Chromatographic separations were
performed using a Jasco PU-2080Plus intelligent pump with a column
oven (Jasco 860-CO), a Jasco Browin NT, HSS-2000 data processor,
and a Jasco CD-2095Plus CD chiral detector (Hg-Xe lamp), simulta-
neously monitoring the CD and UV signals at one specific wavelength
(range 220–420 nm). A nonlinear gradient (initial 20% B, 10 min 40%
B, 20 min 40% B, 30 min 100% B) was programmed. The flow rate
was 0.5 mL/min (detection: 236 nm).
11 (19 mg), [N-CD3]-12 (19 mg), [N-CD3]-13 (6 mg), and [N-CD3]-14
(35 mg). 1H NMR data were identical with those of the unlabeled
derivatives, except for the N-methyl group. For MS data, see Table 2.
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 four weeks (Table
3). Cells and medium were separated and extracted with CH3OH at 60
°C. Extracts were worked up as described in Figure 2.
Preparation of 1-Benzyl-N-methyltetrahydroisoquinolines 7–14.
N-Methylpapaverinium salt (5) and N-methyltetrahydropapaverine
References and Notes
(6) were prepared according to the procedure described before.15
A
solution of racemic N-methyltetrahydropapaverine (6) (500 mg) in 47%
HBr (1 mL) was refluxed for 13 min. Hydrobromic acid was evaporated
in Vacuo. The residue was dissolved in DMSO (0.5 mL) and was
separated by preparative HPLC (Hitachi L-6250 intelligent pump and
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 NH4OAc (0.05% TFA) and (B) CH3OH (0.05% TFA) were
used in a linear gradient (initial 20% B, 80 min 100% B). The flow
rate was 6 mL/min (detection: 280 nm). The eluent obtained from the
peaks a-l 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 trifluoroacetates of peaks a (7, 42 mg), b (8, 24 mg), c (9,
23 mg), d (10, 66 mg), e (11, 35 mg), f (12, 20 mg), g (13, 10 mg), h
(1) Battersby, A. R.; Binks, R.; Foulkes, D. M.; Francis, R. J.; McCaldin,
D. J.; Ramuz, H. Proc. Chem. Soc. 1963, 203.
(2) Barton, D. H. R. Pure Appl. Chem. 1964, 9, 35–47.
(3) Battersby, A. R. Pure Appl. Chem. 1967, 14, 117–136.
(4) Leete, E.; Murrill, S. J. B. Phytochemistry 1967, 6, 231–235.
(5) Müller, M. J.; Zenk, M. H. Planta Med. 1992, 58, 524–527.
(6) Zenk, M. H. Pure Appl. Chem. 1994, 66, 2023–2028.
(7) De-Eknamkul, W.; Tanahashi, T.; Zenk, M. H. Phytochemistry 1992,
31, 2713–2717.
(8) Kammerer, L.; De-Eknamkul, W.; Zenk, M. H. Phytochemistry 1994,
36, 1409–1416.
(9) Iwasa, K. In The Alkaloids; Cordell G. A., Ed.; Academic Press: New
York, 1995; Vol 46, pp 273–346.
(10) Iwasa, K.; Kuribayashi, A.; Sugiura, M.; Moriyasu, M.; Lee, D.-U.;
Wiegrebe, W. Phytochemistry 2003, 64, 1229–1238.
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1
(71 mg), i (14, 34 mg), j (35 mg), k (20 mg), and l (10 mg). For H
NMR and MS data, see Tables 1 and 2.
Preparation of [N-CD3]-1-Benzyltetrahydroisoquinolines [N-CD3]-
7-[N-CD3]-14. [N-CD3]-5 was prepared by treatment of papaverine
with CD3I according to the method used for the preparation of 5.15
[N-CD3]-6 was prepared from [N-CD3]-5 according to the method used
for the preparation of 6.15 [N-CD3]-6 (1 g) in 47% HBr (1 mL) was
refluxed for 9.5 min. Hydrobromic acid was evaporated in Vacuo. The
residue was dissolved in DMSO (0.5 mL) and was separated by
preparative HPLC to give the trifluoroacetates of [N-CD3]-7 (80 mg),
[N-CD3]-8 (48 mg), [N-CD3]-9 (38 mg), [N-CD3]-10 (86 mg), [N-CD3]-
(12) Preininger, V. In The Alkaloids; Brossi, A., Ed.; Academic Press: New
York, 1986; Vol. 29, pp 1–98.
(13) Lu, S.-T.; Su, T.-L.; Kametani, T.; Ujiie, A.; Ihara, M.; Fukumoto,
K. J. Chem. Soc., Perkin Trans. 1 1976, 63–68.
(14) Kutchan, T. M.; Dittrich, H. J. Biol. Chem. 1995, 270, 24475–24481.
(15) Cui, W. H.; Iwasa, K.; Tokuda, H.; Kashihara, A.; Mitani, Y.;
Hasegawa, T.; Nishiyama, Y.; Moriyasu, M.; Nishino, H.; Hanaoka,
M.; Mukai, C.; Takeda, K. Phytochemistry 2006, 67, 70–79.
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