The biosynthesis of papaverine proceeds via (S)-reticuline

Article abstract of DOI:10.1016/j.phytochem.2010.04.022

Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precursor alkaloid for a multitude of nitrogen containing p

Full text of DOI:10.1016/j.phytochem.2010.04.022

Phytochemistry 71 (2010) 1305–1312
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Phytochemistry
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The biosynthesis of papaverine proceeds via (S)-reticuline q
Xu Han a,1, Marc Lamshöft b,1, Nadja Grobe a, Xuan Ren a, Anthony J. Fist c, Toni M. Kutchan a,
Michael Spiteller b,2, Meinhart H. Zenk a,2,
*
a Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA
b Institute of Environmental Research, University of Technology Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany
c Tasmanian Alkaloids, Westbury, Tasmania 7303, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on
theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precur-
sor alkaloid for a multitude of nitrogen containing plant metabolites. This tetrahydroxylated compound
was proposed to be fully O-methylated. The resulting tetrahydropapaverine should then aromatize to
papaverine. In view of experimental data, this pathway has to be revised. Precursor administration to
8-day-old seedlings of Papaver followed by direct examination of the metabolic fate of the stable-iso-
tope-labeled precursors in the total plant extract, without further purification of the metabolites, led
to elucidation of the papaverine pathway in vivo. The central and earliest benzylisoquinoline alkaloid
is not the tetraoxygenated norlaudanosoline, but instead the trihydroxylated norcoclaurine that is further
converted into (S)-reticuline, the established precursor for poppy alkaloids. The papaverine pathway is
opened by the methylation of (S)-reticuline to generate (S)-laudanine. A second methylation at the 30
position of laudanine leads to laudanosine, both known alkaloids from the opium poppy. Subsequent
N-demethylation of laudanosine yields the known precursor of papaverine: tetrahydropapaverine.
Inspection of the subsequent aromatization reaction established the presence of an intermediate, 1,2-
dihydropapaverine, which has been characterized. The final step to papaverine is dehydrogenation of
the 1,2-bond, yielding the target compound papaverine. We conclusively show herein that the previously
claimed norreticuline does not play a role in the biosynthesis of papaverine.
Received 1 February 2010
Received in revised form 14 April 2010
Available online 20 May 2010
Keywords:
Tetrayhydropapaverine
Opium poppy
Papaver somniferum
P. somniferum ssp. setigerum
Papaveraceae
Metabolism
Papaverine
(S)-Reticuline
(S)-Norreticuline
Biosynthesis
Laudanine
Laudanosine
1,2-Dihydropapaverine
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Papaverine (1) is an interesting target for biosynthetic studies.
This molecule is fully O-methylated on the hydroxyl groups in
Papaverine (1) is a benzylisoquinoline alkaloid (Fig. 1) that was
discovered by Merck (1848) as a minor (ca. 1%) component in the
latex of the opium poppy (Papaver somniferum L.). It occurs also in
other members of the genus Papaver. Due to selection for morphin-
ans in cultivars of P. somniferum, papaverine (1) is either a very
minor alkaloid or totally absent. The pharmacological effect of
papaverine (1) is on cerebral blood flow; it possesses spasmolytic
and vasodilatory activity. The isolated natural product drug was
eventually replaced by the synthetic drug for commercial use after
development of an efficient chemical synthesis (Taylor and Martin,
1974). Papaverine (1) was also used to correct male impotence, but
has now been substituted by the more effective Sildenafil.
positions 6, 7, 30, and 40; it lacks, however, an N-methyl group
and its B-ring is aromatic. Winterstein and Trier in 1910 postulated
that plant-derived tetrahydrobenzylisoquinoline alkaloids were
derived from the condensation of dopamine and 3,4-dihydroxy-
phenylacetaldehyde to form norlaudanosoline (2) (Fig. 2). Follow-
ing this early biogenetic prediction, Battersby and co-workers
conducted experiments that seemingly verified and extended the
Winterstein and Trier hypothesis (Battersby and Binks, 1960; Batt-
ersby, 1963; Battersby et al., 1964). Application of position-specific
labeled tyramine as precursor and tedious specific degradation of
the labeled papaverine (1), established that this alkaloid was in-
deed formed from two C6–C2 units derived from dopamine and
3,4-dihydroxyphenylacetaldehyde (Battersby, 1963). Furthermore,
it was postulated that papaverine (1) is formed: (1) from norlaud-
anosoline (2) (Battersby et al., 1965), (2) from (S)-norreticuline (4)
(Battersby, 1963) as well as several other alkaloids from the nor-
series such as norlaudanine and tetrahydropapaverine (possessing
no N-methyl group). The latter, however, was claimed to be not
significantly incorporated into papaverine (1) due to the lack of
q
This paper is dedicated to Prof. Rolf Huisgen on the occasion of his 90th
birthday.
* Corresponding author. Tel.: +1 314 587 1474; fax: +1 314 587 1574.
E-mail address: mhzenk@danforthcenter.org (M.H. Zenk).
1
X. Han and M. Lamshöft contributed equally to this work.
M. Spiteller and M.H. Zenk share senior author status for this work.
2
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.04.022

1306
X. Han et al. / Phytochemistry 71 (2010) 1305–1312
an unmethylated phenolic hydroxyl group (Battersby et al., 1964).
Independently, the group of Brochmann-Hanssen (Brochmann-
Hanssen et al., 1971, 1975, 1980) extended these suggestions and
claimed that norreticuline (4), norlaudanine and norlaudanosine
were easily incorporated into papaverine (1), while reticuline (9)
was not (Uprety et al., 1975). This proposal received support by
the recent claim that a SAM-dependent O-methyltransferase exists
that accepts norreticuline (4) as the sole substrate (Pienkny et al.,
2009). Our recent findings, however, are at variance to the above
claims. Norlaudanosoline (2), norreticuline (4), norlaudanine or
norlaudanosine have never been found in higher plants as natural
products despite meticulous phytochemical work. Isotope dilution
analysis showed that norlaudanosoline (2) and norreticuline (4)
were not detected during isoquinoline alkaloid synthesis (Stadler
and Zenk, 1990). Furthermore, the trihydroxylated (S)-norcoclau-
rine (5) is the central precursor of benzylisoquinoline alkaloids
and not the long postulated (S)-norlaudanosoline (2) (Stadler
et al., 1987; Stadler and Zenk, 1990; Frenzel and Zenk, 1990a).
The sequence of enzymatically catalyzed methylation and oxida-
tion steps of (S)-norcoclaurine (5) excludes the existence of (S)-
norreticuline (4), the postulated precursor of papaverine (1)
(Fig. 2). The sequence is 6-O-methylation, N-methylation, hydrox-
ylation of 30 position and 40-O-methylation.
Fig. 1. Papaverine (1) and its three aromatic rings A, B and C.
Hence the above-described hypothesis of a pathway for the for-
mation of papaverine (Fig. 2A), does not have experimental sup-
port. The incorporation of potential synthetic precursors such as
norreticuline (4), and even their correct position-specific incorpo-
ration into papaverine (1), may be attributed to the promiscuous
enzyme activities such as are found in the plant kingdom. All of
these inconsistencies in the hitherto published literature and text-
books justified revisiting the biosynthesis of papaverine (1). We
approached a solution to this problem by using high resolution
mass spectrometry together with stable-isotope-labeled potential
precursors and 8-day-old seedlings of an opium poppy hybrid that
accumulate sufficient amounts of papaverine (1) for trace analysis
of intermediates as well as end products.
2. Results and discussion
To begin our investigation, we analyzed the pertinent alkaloids
in the methanolic extract of 8-day-old poppy hybrid seedlings via
high resolution LC–MS. The alkaloid pattern and the molecular
characteristics are shown in Table 1.
As can be noted from the retention times in Table 1, the individ-
ual alkaloids are clearly separated from each other by high resolu-
tion LC–MS, which is a prerequisite for using isotopically labeled
compounds. No prediction can be made at this stage regarding
the stereochemistry of these alkaloids. Thebaine (10) was used as
an indicator for the presence of the morphine pathway. In order
to establish the origin of the aromatic ring systems in the alkaloids
Table 1
High resolution LC–MS/MS data and quantified amounts of pertinent alkaloids from
poppy hybrid seedlings.
Compound
[M+H]+ [m/z]
[M+H]+ [m/z] Retention Amount
(experimental) (theoretical) time
(min)
(nmol/g
dry
weight)
Reticuline (9)
Laudanine (11)
Laudanosine (12)
330.17011
344.18542
358.20112
330.16998
344.18563
358.20128
344.18563
7.78
12.74
14.79
11.59
2.53
0.06
0.06
0.40
Fig. 2. Postulated and verified sequence of reactions for the formation of benzyl-
isoquinoline alkaloids. (A) Previously postulated sequence of reactions for the
biosynthesis of (S)-norreticuline (4), as precursor to papaverine (1); (B) general
pathway and sequence of reactions verified for the biosynthesis of benzylisoquin-
oline alkaloids (via (S)-reticuline (9)).
Tetrahydropapaverine 344.18546
(13)
Papaverine (1)
Thebaine (10)
340.15417
312.15929
340.15433
312.15942
16.76
12.61
0.98
3.71

X. Han et al. / Phytochemistry 71 (2010) 1305–1312
1307
of interest, we applied the chemically more stable 6-O-methylated
derivative, [1-13C]-(S)-coclaurine (6) (Fig. 2B), to the poppy seed-
lings. As a result of the precursor administration experiment, we
found 15.08 nmol labeled and 41.86 nmol unlabeled thebaine
(10) resulting in a ratio of 0.265 for the quotient of labeled to unla-
beled alkaloid. Calculation in an analogous manner for papaverine
(1) resulted in a quotient of 0.017. Based on dry weight, this calcu-
lation corresponds to 0.04% papaverine (1) and 0.4% thebaine (10).
The conclusion from the result of this precursor administration
experiment is that the universal precursor (S)-coclaurine (6) is
incorporated into both papaverine (1) and thebaine (10), but it
cannot be excluded that coclaurine (6) was processed further with-
out an N-methyl group to furnish papaverine (1).
As quoted above (Stadler and Zenk, 1990), norreticuline (4) has
never been found in plants, therefore we applied [1-13C, N-13CH3]-
(S)-reticuline (9) to the poppy hybrid. The reason for the use of the
double labeled compound was that the [1-13C, N-13CH3]-(S)-reticu-
line (9) ought to be incorporated into papaverine (1) while the N-
methyl group should be lost during the transition to the target
compound. (S)-Reticuline (9) is one of the central precursors for
a multitude of benzylisoquinoline alkaloids retaining the N-methyl
group such as morphinans, aporphines, protoberberines and
benzo[c]phenanthridines (Kutchan, 1998). The substitution pattern
of the phenolic groups means that a biosynthetic relationship to
papaverine (1) could be envisaged, if it is assumed that N-demeth-
ylation is necessary in the course of the pathway to papaverine (1).
To test this possibility, we observed the flow of [1-13C, N-13CH3]-
(S)-reticuline (9) into the hypothetical biosynthetic pathway of
papaverine (1).
binant SAM-dependent enzyme is known that methylates (S)-
reticuline (9) at the 7 position to yield laudanine (11) (Ounaroon
et al., 2003), it was tempting to test whether this precursor was
incorporated into papaverine (1). (S)-Laudanine (11) was synthe-
sized by methylating (D3-methyliodide) (S)-reticuline N-oxide
with subsequent deprotection (H2SO3) (Phillipson et al., 1976; Lu
et al., 1987). This method yielded both [7-D3]-laudanine (11), as
well as [30,7-D6]-laudanosine (12), the former having +3 mass units
due to the deuterium label while the latter had +6 mass units. The
alkaloids were separated by TLC and their constitution verified by
high resolution LC–MS (Fig. 3A and B). [7-D3]-laudanine (11) and
[30,7-D6]-laudanosine (12) were administered individually to pop-
py seedlings, as described in Section 4. The result of this applica-
tion experiment showed that [7-D3]-laudanine (11) was
incorporated into papaverine (1) to 53.6% of the total papaverine
(1) present, while [30,7-D6]-laudanosine (12) was incorporated to
66.7% of the total target compound (Fig. 3C and D). This experi-
ment and the high incorporations show without any doubt that
the N-methylated alkaloids are excellent precursors and that in
the biochemical event, the N-methyl group is removed and the
resulting alkaloid is dehydrogenated to yield papaverine.
Having established the precursor function of laudanine (11) by
efficient incorporation into papaverine (1), we set out to explore
the missing biochemical steps between laudanine (11) and papav-
erine (1). Precursor administration experiments using [7-D3]-lau-
danine (11) showed in the full scan the following four labeled
intermediates calculated in percentage based on the total amount
(labeled and unlabeled) of each alkaloid present in the seedling:
laudanosine (12) (98.6%), tetrahydropapaverine (13) (missing the
N-methyl group) (96.4%), an unknown metabolite (99.2%) and
papaverine (1) as shown before (53.6%), the obvious end product.
Administering [7-14C]-labeled laudanine (11) to poppy seedlings,
subjecting the total extract (without fractionation) to 2D TLC fol-
lowed by exposure on a phosphorimager screen revealed only
the four radioactive products, laudanosine (12), tetrahydropapa-
verine (13), the unknown metabolite and papaverine (1), and no
additional metabolites (data not shown).
The occurrence of stable-isotope-labeled tetrahydropapaverine
(13) demonstrates, that in the poppy plant, the fully methylated
alkaloid laudanosine (12) is subjected to demethylation. This is
the missing step between the N-methylated tetrahydrobenzyliso-
quinoline alkaloid and the N-demethylated papaverine skeleton.
The intermediate is (S)-tetrahydropapaverine (13), a compound
first postulated in papaverine (1) biosynthesis (Brochmann-Hans-
sen et al., 1971) after it had been found as a natural product iso-
lated from P. somniferum. This postulate was verified by
Brochmann-Hanssen et al. (1975) who synthesized (ꢀ)-tetrahy-
dropapaverine (13) that was found to be incorporated into papav-
erine (1) with an, at that time, extremely high incorporation rate in
the poppy plant. However, the opinion was that (S)-norreticuline
(4) and norlaudanosoline (2) were the precursors of norlaudanine,
norlaudanosine, with the biosynthetic grid ending with tetrahy-
dropapaverine (13) and papaverine (1) (Battersby, 1963;
Interestingly, the majority of labeled [1-13C, N-13CH3]-(S)-reti-
culine (9) was transformed into thebaine (10) (4.35 nmol/g dry
weight) while the incorporation into (S)-laudanine (11) and (S)-
laudanosine (12), with the two labeled positions intact, was much
less (0.1 nmol/g dry weight, Table 2). This difference was due to the
fact that the metabolic route to thebaine (10) is the major pathway
in the used poppy cross, while the one to papaverine (1) is only a
side pathway. The detection of labeled laudanine (11) and lauda-
nosine (12) was not sufficient to demonstrate that both alkaloids
were precursors of papaverine (1) rather than simply natural prod-
ucts occurring in the plant. However, the same reticuline (9)
administration also resulted in single labeled tetrahydropapaver-
ine (13) and papaverine (1) at the original 1-13C position, while
the N-methyl group was lost.
This demonstrates that (S)-reticuline (9) is the precursor to tet-
rahydropapaverine (13) and papaverine (1). The question remains,
however, which pathway (S)-reticuline (9) takes on its way to
these two N-demethylated compounds. As a control, we analyzed
the incorporation of the doubly-labeled reticuline (9) into thebaine
(10), which gave an incorporation quotient of 0.54. This demon-
strated the dominance of the morphine pathway over the papaver-
ine pathway (Table 2).
Since laudanine (11), the O-trimethylated, N-methylated ben-
zylisoquinoline alkaloid, occurs in the opium poppy, and a recom-
Table 2
High resolution LC–MS/MS data and quantified amounts of pertinent alkaloids from the [1-13C, N-13CH3]-(S)-reticuline (9) administration experiment. Note: Administration of
doubly-labeled [1-13C, N-13CH3]-(S)-reticuline (9) to poppy plants leads to the formation of doubly-labeled (1-13C, N-13CH3) laudanine and laudanosine, but singly-labeled (1-13C)
tetrahydropapaverine (1) and singly-labeled (1-13C) papaverine (1) while thebaine is doubly-labeled (1-13C, N-13CH3) demonstrating the branch point.
Compound
[M+H]+ [m/z] (experimental)
[M+H]+ [m/z] (theoretical)
Retention time (min)
Amount (nmol/g dry weight)
[1-13C, N-13CH3]-Reticuline (9)
[1-13C, N-13CH3]-Laudanine (11)
[1-13C, N-13CH3]-Laudanosine (12)
[1-13C]-Tetrahydropapaverine (13)
[1-13C]-Papaverine (1)
332.17652
346.19247
360.20786
345.18873
341.15751
314.16595
332.17669
346.19234
360.20799
345.18899
341.15769
314.16613
7.78
12.74
14.79
11.59
16.77
12.61
95.30
0.10
0.02
0.01
0.01
4.35
[1-13C, N-13CH3]-Thebaine (10)

1308
X. Han et al. / Phytochemistry 71 (2010) 1305–1312
347.20442
364.23946
2
C
H
H O N= 347.20447
3 4
20 23
2
C
H
H
O N= 364.23895
4
21 22
6
100
90
80
70
60
50
40
30
20
10
0
-0.14065 ppm
1.42607 ppm
100
A
B
90
80
70
60
50
40
30
20
10
0
348.20768
2
-0.40003 ppm
13
C
CH
H O N= 348.20782
23 3 4
19
365.24268
2
H O N= 365.24230
22 6 4
13
CH
C
20
1.04476 ppm
349.21121
2
13
C
C
H
H O N= 349.21117
23 3 4
18
2
0.10683 ppm
346.5
347.0
347.5
348.0
348.5
349.0 349.5
364.0
364.5
365.0
365.5
366.0
366.5
m/z
m/z
346.19185
340.15425
N= 340.15433
2
C
H
H O N= 346.19200
C
H
O
20 16 6 4
20 22
4
-0.42363 ppm
-0.25121 ppm
100
100
90
80
70
60
50
40
30
20
10
0
343.17299
c
D
2
C 20 H19 H3 O4 N= 343.17316
-0.51293 ppm
90
80
70
60
50
40
30
20
10
0
340.15423
N= 340.15433
C
H
O
22 4
20
-0.32178 ppm
338
339
340
341
342
343 344
345
346
338
340
342
344
346
348
350
m/z
m/z
Fig. 3. High resolution mass spectra. (A) isotopically synthesized [7-D3]-laudanine (11); (B) isotopically synthesized [30,7-D6]-laudanosine (12); (C) biosynthetically obtained
unlabeled and [7-D3]-papaverine (1); (D) biosynthetically obtained unlabeled and [30,7-D6]-papaverine (1).
Brochmann-Hanssen et al., 1971; Uprety et al., 1975) but, as men-
tioned before, Brochmann-Hanssen and associates never found
norreticuline (4), norlaudanine or norlaudanosine.
We investigated further the precursor role of tetrahydropapa-
verine (13) in the biosynthesis of papaverine (1). The administra-
tion of deuterated (R,S)-tetrahydropapaverine (13) was carried
out as usual. The plants were extracted with methanol and sub-
jected to high resolution mass spectrometry. Quantitative mass
spectrometric analysis revealed a quotient of 0.81 for the incorpo-
ration of deuterium-labeled (R,S)-tetrahydropapaverine (13) (iso-
topic distribution < 0.1% [2H0]/ < 0.1% [2H1]/ < 0.1% [2H2]/ < 0.1%
[2H3]/3% [2H4]/21.5% [2H5]/39% [2H6]/29.9% [2H7]/5.5% [2H8]/1%
[2H9]) into papaverine (1) and an isotopic distribution for deute-
rium-labeled papaverine (1) of 11.8% [2H0]/2.9% [2H1]/12% [2H2]/

X. Han et al. / Phytochemistry 71 (2010) 1305–1312
1309
the detected product ion m/z 190 (Fig. 4A) was in good agreement
with the mass fragmentation of isoquinoline alkaloids shown by
Wu and Huang (2006). Papaverine (1), containing a double bond
H3CO
N+ H
H
H3CO
H3CO
H3CO
H3CO
between C1 and N and between C3 and C4, gives the major R-CHþ
N+
2
H
(R = isoquinoline) fragment m/z 202 (Wickens et al., 2006). A com-
pound with a double bond located between C1 and N would also
give a R-CHþ2 fragment, m/z 204 (Fig. 4B), due to its mesomeric sta-
bility. Because the dihydro-intermediate does not form a R-CH2þ
fragment (Fig. 4A), it can be inferred that the additional double
bond is located between C3 and C4. In addition, the MS/MS exper-
iment of tetrahydropapaverine (13) showed a product ion with m/z
192, which supports the structure of a 1,2-dihydro derivative (14)
(Fig. 4C).
A
H3CO
H3CO
m/z 190
N+
H
H3CO
H3CO
H3CO
H3CO
N
CH+2
H3CO
H3CO
For decades, norlaudanosoline (2) was assumed to be the first
alkaloidal precursor for the major classes of plant-derived isoquin-
oline alkaloids (Winterstein and Trier, 1910; Robinson, 1917;
Spenser, 1969). This hypothetical intermediate was seemingly ver-
ified, when it was shown that the expected labeled precursors
were incorporated into papaverine (1), the target compound (Batt-
ersby and Harper, 1959, 1962; Battersby et al., 1964), despite some
discrepancies in the labeling patterns (for review see Spenser,
1969) produced in poppy and other isoquinoline alkaloid accumu-
lating plants. Extensive phytochemistry, precursor synthesis and
experimentation by Brochmann-Hanssen and associates (Broch-
mann-Hanssen and Furuya, 1964; Brochmann-Hanssen et al.,
1965, 1971, 1975) not only discovered the now recognized true
precursors of papaverine (1), the opium poppy-derived laudanine
(11) (laudanidine) and laudanosine (12) alkaloids, but found in la-
beled (S)-tetrahydropapaverine (13) an excellent precursor for
papaverine (1), while (R)-tetrahydropapaverine is largely inactive.
However, (R,S)-reticuline (9) was very poorly incorporated into
papaverine (1), but most importantly the highest incorporation
rates into papaverine (1) were achieved with norreticuline (4),
which is missing the N-methyl group. These results led to the erro-
neous assumption that, as predicted by Winterstein and Trier
(1910) and then experimentally shown by Battersby (1963), nor-
laudanosoline (2) and norreticuline (4) were precursors for papav-
erine (1). It was not yet known at that time that plant
methyltransferases, and especially those from poppy, are promis-
cuous (Brochmann-Hanssen et al., 1980; Wat et al., 1986; Frenzel
and Zenk, 1990b). Thus exogenously provided norlaudanosoline
(2) and norreticuline (4) could easily be methylated at the nitrogen
and phenolic groups by these methyltransferases. This promiscuity
was demonstrated by us when poppy seedlings were administered
[1-13C]-(S)-norreticuline (4), which resulted in formation of singly-
labeled (S)-reticuline (9) and singly-labeled thebaine (10), from
which we found a ratio of 0.01 for the quotient of labeled to unla-
beled compound. Supporting this is the fact that the unnatural
alkaloid nororientaline is an excellent precursor to papaverine
(1), since presence of the 7-O-methyl group prevents funneling
into the thebaine (10) pathway (Brochmann-Hanssen et al., 1975).
With the knowledge of the biosynthesis of isoquinoline alka-
loids, the use of alkaloid producing seedlings, methanolic total
plant extracts and state-of-the-art mass spectrometry, we were
able to decipher the biosynthesis of papaverine (1), one of the old-
est targets of phytochemistry. The biosynthetic pathway as it
stands now is shown in Fig. 5. None of the predicted previous inter-
mediates with the exception of (S)-tetrahydropapaverine (13), is
present in the new scheme depicted here.
B
m/z 204
N+ H
H
H3CO
H3CO
H3CO
N+
H3CO
H
C
H3CO
m/z 192
Fig. 4. Two possible structures for the biosynthetic intermediate between tetrahy-
dropapaverine (13) and papaverine (1) and mass fragmentation of tetrahydropa-
paverine (13). (A) 1,2-Dihydropapaverine (14), the naturally occurring compound,
and its product ion m/z 190; (B) 3,4-Hydropapaverine and its predicted product ion
m/z 204; (C) Tetrahydropapaverine (13) and its product ion m/z 192.
32.2% [2H3]/30% [2H4]/11% [2H5]/0.1% [2H6]. This high incorporation
rate is explained by the immediate precursorship of tetrahydropa-
paverine (13) to papaverine (1). The loss of three deuterium atoms
during the aromatization process is reflected by the isotopic distri-
bution pattern of labeled tetrahydropapaverine (13) and labeled
papaverine (1) with the major peaks changing from D6 and D7 to
D3 and D4, respectively. The precursor administration experiment
with deuterated (R,S)-tetrahydropapaverine (13) established, in
addition to labeled papaverine (1), the presence of an intermediate
m/z 342.16992 (C20H24O4N) with an isotopic distribution of <0.1%
[2H0]/ < 0.1% [2H1]/3% [2H2]/1% [2H3]/18% [2H4]/32% [2H5]/24%
[2H6]/21% [2H7]/1% [2H8]. The intermediate was identified as la-
beled 1,2-dihydropapaverine (14) showing an incorporation quo-
tient of 0.28 and, for m/z 347.20137 carrying five deuterium
atoms, a fragment ion of m/z 195.11800 (C11H72H5O2N). The forma-
tion of labeled 1,2-dihydropapaverine (14) from labeled tetrahy-
dropapaverine (13) goes along with the loss of two deuterium
atoms. Indeed, the isotopic distribution patterns of both alkaloids
established that a decrease in the number of D6- and D7-labeled
isotopes by 15% and 8%, respectively, leads to an equivalent in-
crease in the number of 1,2-dihydropapaverine (14) isotopes carry-
ing four and five deuterium atoms. This experiment thus clearly
demonstrated tetrahydropapaverine (13) as a substrate for the aro-
matization process in poppy plants.
This result was confirmed by a detailed analysis of the [D6]-lau-
danosine (12) administration, which gave an additional peak be-
tween tetrahydropapaverine (13) and papaverine (1) in the mass
chromatograms at 13.66 min with quasimolecular ions at m/z
342.16992 (C20H24O4N) and 348.20760 (C20H182H6O4N) showing
that an intermediate with two hydrogens less than tetrahydropa-
paverine (13) was formed. The MS/MS spectrum of the new com-
pound showed fragments at m/z 190.08629 (C11H12O2N) and
193.10512 (C11H92H3O2N), respectively. As discussed earlier (Batt-
ersby et al., 1977), the dehydrogenation of tetrahydropapaverine
(13) to papaverine (1) may involve either a double bound forma-
tion between C1 and N or between C3 and C4. The formation of
3. Conclusion
By administering-heavy-isotope labeled potential precursors to
seedlings of a poppy cross that produces both thebaine (10) and
papaverine (1), and analyzing extracts by HPLC-high resolution

1310
X. Han et al. / Phytochemistry 71 (2010) 1305–1312
Fig. 5. New biosynthetic pathway to papaverine (1) in poppy plants. Shown in bold is the common pathway leading to both papaverine (1) and thebaine (10).
mass spectrometry, it was possible to resolve the controversial bio-
synthesis of papaverine (1) (Fig. 5). This methodology may be ap-
plied to other biosynthetic problems in the future to resolve
similar types of questions in plant and animal systems. It is advis-
able to use a quotient of the incorporation of diverse unlabeled and
position-labeled precursors. This procedure allows the study of a
biosynthetic pathway using only small amounts of seedling tissue
or plant cell suspension culture and omits tedious purification
steps of the target compounds.
MeOH (100 ll) and directly injected (10 ll) without fractionation
onto the LC–MS instrument.
4.2. Mass spectrometry
High resolution mass spectra were obtained using an LTQ-Orbi-
trap Spectrometer (Thermo Fisher, USA). The spectrometer was
operated in the positive mode (1 spectrum sꢀ1; mass range: 50–
1000) with a nominal mass resolving power of 60,000 at m/z 400
at a scan rate of 1 Hz using automatic gain control to provide
high-accuracy mass measurements (62 ppm deviation). The inter-
nal calibration standard bis-(2-ethylhexyl)-phthalate (m/z
391.28428) was used for the determination of elemental composi-
tion. The spectrometer was equipped with a Surveyor HPLC system
(Thermo Scientific, USA) consisting of LC-Pump, UV detector
4. Experimental
4.1. Papaver
Hybrid seeds were produced by pollinating emasculated buds of
P. somniferum with pollen of P. somniferum ssp. setigerum. Seeds of
the F1 P. somniferum ꢁ P. somniferum ssp. setigerum cross were sur-
face sterilized with hypochlorite. Twenty-five seeds were grown on
filter paper in Petri dishes in a minimum quantity of Linsmaier
Skoog mineral solution to allow uninhibited growth. The seedlings
were grown in a 12-h day/night cycle under incandescent lamps at
20 °C. After a growth period of 4 days, the developing seedlings
(k = 254 nm) and autosampler (injection volume 10
of samples was achieved by using a Machery–Nagel Nucleodor
Gravity column (1.8
m, 50 ꢁ 3 mm) combined with an associated
ll). Separation
l
guard column (4 ꢁ 3 mm). The mobile phase total flow was set to
0.4 ml/min with binary gradient elution, using solvents A (0.1%
HCO2H, 10 mM (NH4)OAc) and B (0.1% HCO2H in CH3CN) (all v/
v). The gradient started with 5% B for 4 min and was increased to
30% B over 20 min. Elution was continued for 10 min at 100% B fol-
lowed by a 7-min equilibration with the starting condition. MS/MS
spectra were obtained from the corresponding parent ions
([M+H]+) with a collision energy of 35 V. The quantitation of the
individual labeled compound was achieved by integration of the
peak area in the extracted ion chromatogram with a mass range
(25) were administered 350 ll of a sterile 0.5 mM solution of po-
tential precursor which was taken up by the roots. The seedlings
were supplied fresh precursor solution every 24 h for the following
3 days. On day 8, the seedlings were harvested and extracted with
boiling MeOH–H2O (20 ml, 1:4) (extracted dry weight 10.1 mg).
The soluble extract was evaporated to dryness, taken up with

X. Han et al. / Phytochemistry 71 (2010) 1305–1312
1311
of 2 ppm. All calibration graphs were linear (R2 > 0.99) from
4.3.2. Deuterated tetrahydropapaverine (13)
20 ng mlꢀ1 up to a concentration of 20,000 ng mlꢀ1
.
Deuterated tetrahydropapaverine (13) was synthesized from
papaverine (1) according to Taylor (1952) with slight modifica-
tions. To introduce a deuterium label at positions C1, C3 and C4,
papaverine (1) was refluxed in deuterium chloride and zinc dust.
After 30 h of reflux, the reaction mixture was made alkaline and
extracted with Et2O (2 ꢁ 20 ml). The presence of tetrahydropapa-
verine (13) in the combined Et2O phase was confirmed by TLC in
the solvent system xylene:butanone:MeOH:Et2NH (20:20:3:1).
Et2O was removed in vacuo, with the residue reconstituted in
MeOH–CH3CN (2:1). Deuterated tetrahydropapaverine (13) was
further purified by HPLC (Merck Hitachi) using a Hibar Pre-
4.3. Chemical synthesis
4.3.1. [7-D3]-laudanine (11) and [30,7-D6]-laudanosine (12)
m-Chloroperbenzoic acid (100 mg) dissolved in CH2Cl2 (5 ml)
was added dropwise to a stirred solution (0°) of (S)-reticuline (9)
(100 mg) in CH2Cl2 (15 ml) to yield (S)-reticuline N-oxide (66%)
after 2.5 h (Phillipson et al., 1976; Lu et al., 1987). The (S)-reticuline
N-oxide was purified via thin layer chromatography (TLC) on Poly-
gram silica G/UV254 plates (Macherey–Nagel, layer: 0.2 mm silica
gel with fluorescent indicator UV254) using the solvent system,
acetone:H2O:NH4OH (80:15:5). The purified N-oxide was sus-
pended in a mixture of EtOH (3 ml) and THF (9 ml) and reacted
with D3-methyliodide (10 equiv) and KOH (7.5 equiv) overnight
at room temperature to methylate the N-oxide. After methylation,
the reaction mixture was dried and dissolved in EtOH–H2O (8 ml,
1:1). To remove the N-oxide protecting group, NaHSO3 (400 mg)
was added and stirred for 5 h. The reaction mixture was washed
with CH2Cl2 and purified via the TLC system toluene:EtOAc:Et2NH
(7:2:1) to obtain [7-D3]-laudanine (11) in 10% yield (Rf: 0.17) and
[30,7-D6]-laudanosine (12) in 30% yield (Rf: 0.58). Several other
compounds carrying deuterium label were noted on the chromato-
gram and clearly separated from each other, but their structures
were not determined. Rechromatography was done on [7-D3]-lau-
danine (11) using the TLC system xylene:2-butanone:2-propa-
nol:Et2NH (20:20:3:1). The isotopic distribution of d3- and
d6-labeled alkaloids exhibited an isotopic purity >99.5%. [7-D3]-
Laudanine (11) and [30,7-D6]-laudanosine (12) were identified by
comparison with unlabeled authentic standards, exhibiting the
same chromatographic and spectroscopic data (Fig. 3A and B), ex-
cept for their mass spectra, which show the calculated shift of their
quasimolecular ion [M+H]+ either +3 or +6, respectively (Fig. 6).
Packed column RT250-25 (Merck, 7 lm, LiChrosorb RP-18). The
mobile phase total flow was set to 8 ml/min with binary gradient
elution using 0.1% CF3CO2H (solvent A) and CH3CN (solvent B).
The gradient started with 20% B and was increased to 50% B over
60 min. Elution was continued for 20 min at 100% B followed by
a 20 min equilibration under standard conditions. The fraction
containing deuterated tetrahydropapaverine (13) was collected
and lyophilized. Deuterated tetrahydropapaverine (13) was ob-
tained in 56% yield and completely free of papaverine (1) as con-
firmed by TLC, HPLC and high resolution LC–MS. The isotopic
distribution of deuterium-labeled tetrahydropapaverine (13)
was <0.1% [2H0]/ < 0.1% [2H1]/ < 0.1% [2H2]/ < 0.1% [2H3]/3% [2H4]/
21.5% [2H5]/39% [2H6]/29.9% [2H7]/5.5% [2H8]/1% [2H9]. This ob-
served isotopic distribution was largely in agreement with the
expected isotopic distribution.
All other labeled or unlabeled alkaloids were from our labora-
tory collection.
The following equation was used for the calculation of the quo-
tient between labeled and unlabeled alkaloid:
nmol of labeled alkaloid
¼ Q
nmol of labeled and unlabeled alkaloid
Fig. 6. High resolution MS/MS fragmentation spectra of synthesized [7-D3]-laudanine (11) (A) and synthesized [30,7-D6]-laudanosine (12) (B).

1312
X. Han et al. / Phytochemistry 71 (2010) 1305–1312
Kutchan, T.M., 1998. Molecular genetics of plant alkaloid biosynthesis. In: Cordell,
Acknowledgements
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chloroperbenzoic acid. J. Chin. Chem. Soc. 34, 33–42.
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Chem. 66, 125–128.
This research was sponsored by NIH Grant #5R21DA024418-02
and the Mallinckrodt Foundation, St. Louis. X. Han thanks the REU
program at the Donald Danforth Plant Science Center for the edu-
cational experience and the Science and Research Office at Macal-
ester College for financial support.
Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F., Kutchan, T.M., 2003. (R,S)-
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7-O-methyltransferase
and
(R,S)-norcoclaurine
6-O-
methyltransferase of Papaver somniferum – cDNA cloning and characterization
of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 36,
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