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Applied and Environmental Microbiology, August 2001, p. 3716-3719, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3716-3719.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Transformation of 2,2'-Bimorphine to the Novel
Compounds 10-
-S-Monohydroxy-2,2'-Bimorphine and
10,10'-,'-S,S'-Dihydroxy-2,2'-Bimorphine by
Cylindrocarpon didymum
Peter J.
Stabler,
Peter J.
Holt, and
Neil C.
Bruce*
Institute of Biotechnology, University of
Cambridge, Cambridge CB2 1QT, United Kingdom
Received 16 January 2001/Accepted 17 May 2001
 |
ABSTRACT |
Whole-cell suspensions of Cylindrocarpon didymum were
observed to transform 2,2'-bimorphine to the compounds
10-
-S-monohydroxy-2,2'-bimorphine and
10,10'-,'-S,S'-dihydroxy-2,2'-bimorphine.
Mass spectrometry and 1H nuclear magnetic resonance
spectroscopy confirmed the identities of these new morphine alkaloids.
| |
TEXT |
The morphine alkaloids are the major
alkaloid components of the opium poppy, Papaver somniferum,
and these compounds still provide some of the most potent analgesic
compounds in clinical use. Many synthetic derivatives of morphine have
been produced, and subtle changes in functionality can significantly
alter the pharmacological activity of these compounds. Microbial
transformations of morphine alkaloids have been investigated as a means
of producing opiate drugs and pivotal intermediates that are difficult
to produce chemically. These transformations have mainly comprised
either N-dealkylation, reduction of the C-7-C-8 vinylic
group, oxidation or reduction of the C-6 oxygroup, or hydroxylation of
the C-14 position of the morphinan skeleton (10, 11, 14, 15, 21, 22). Several of the morphine alkaloid-transforming enzymes have been purified and characterized (2, 3, 7, 16).
Furthermore, the genes encoding a number of these enzymes have been
cloned and expressed in Escherichia coli, which has
facilitated the engineering of recombinant cells to produce
biologically semisynthetic opiate drugs (1, 8, 9, 20).
Oxyfunctionalization of morphine and related compounds is of interest
as a means of providing both new synthons for the production of
existing semisynthetic drugs and new intermediates that have potential
as platforms for combinatorial synthesis of new opiate drugs. We
recently demonstrated that the fungus Cylindrocarpon didymum
is capable of converting morphine to pseudomorphine (2,2'-bimorphine)
(19). In this paper we describe the biological conversion
of bimorphine to two novel compounds, 10--S-monohydroxy-2,2'-bimorphine and
10,10'-,'-S,S'-dihydroxy-2,2'-bimorphine.
Biotransformation of morphine and 2,2'-bimorphine.
Mycelia of
C. didymum, obtained from the Institute of Biotechnology's
culture collection, were grown in mineral medium (pH 7.0) containing
(per liter) 10.0 g of KH2PO4, 5.0 g
of (NH4)2SO4, and 0.5 g of
MgSO4 and supplemented with 10.0 g of yeast extract per liter and trace elements as described by Rosenberger and Elsdon (18) (YL medium). Cultures were incubated in Erienmeyer
flasks (2 liters for 400 ml of medium or 250 ml for 40 ml of medium) at
30°C for 48 h with rotary shaking at 180 rpm. Mycelia were harvested by filtration through sterile Whatman no. 1 filter paper in
vacuo, washed with 100 ml of sterile mineral medium, and used immediately for whole-cell incubation.
Initially, morphine was transformed with washed mycelia resuspended in
40 ml of mineral medium (typically 12.5 g [wet weight]/liter) supplemented with 10 mM morphine. To monitor the transformation, samples (0.2 ml) were abstracted at regular intervals and diluted five-fold in 50 mM phosphoric acid (pH 3.5) to dissolve any
precipitated metabolites. Mycelia were removed by centrifugation at
14,000 × g, and the samples were analyzed by
high-performance liquid chromatography (HPLC) as described previously
(19). The 2,2'-bimorphine that was produced was not
transformed further under these conditions (that is, in a neutral pH
medium that contained untransformed morphine).
Subsequently, the effect of pH on the transformation of 2,2'-bimorphine
was tested. Whole-cell preparations (400 ml) of mycelia
with morphine
were used to generate 2,2'-bimorphine. These preparations
were
harvested after 48 h, and the 2,2'-bimorphine precipitates
were
collected along with the mycelia. Samples of the harvested
mycelia and
2,2'-bimorphine (2.5 mM) were then incubated in 40
ml of mineral medium
adjusted to pH 4.0, 6.0, or 8.0. Samples
were removed and assayed for
2,2'-bimorphine by HPLC. After 150
h, the 2,2'-bimorphine had
disappeared from the pH 4.0 preparation,
and the amount of
2,2'-bimorphine was reduced by one-half in the
pH 6.0 and 8.0
preparations.
The effect of pH on the transformation of morphine by mycelia was also
tested. The morphine transformation at pH 4.0 proved
to be similar to
that observed previously at pH 7.0, with similar
morphine depletion
rates and initial 2,2'-bimorphine accumulation
rates. However, whereas
at pH 7.0 accumulation of 2,2'-bimorphine
continued with no sign of
further transformation, after incubation
for 30 h at pH 4.0, the
2,2'-bimorphine concentration was observed
to decrease (Fig.
1). The use of an acidic medium was
clearly
necessary for further transformation of 2,2'-bimorphine.
Consequently,
all subsequent whole-cell incubations were carried out at
pH 4.0
in mineral medium buffered with 20 mM 2,2-dimethyl succinic acid
(S medium). When cyclohexamide (0.5 mg/ml) was added to whole-cell
preparations containing mycelia that had not been challenged previously
with morphine, no transformation of morphine or 2,2'-bimorphine
was
observed. Including cyclohexamide in whole-cell preparations
with
mycelia that had been previously challenged with morphine
did not
prevent transformation of either compound. This indicated
that the
activities responsible for transforming both morphine
and
2,2'-bimorphine were induced by morphine. HPLC, thin-layer
chromatography (TLC), and spectrophotometric analyses indicated
that
two significant metabolites were produced during whole-cell
incubation
with 2,2'-bimorphine at pH 4.0. One of these metabolites
(DP584)
appeared in culture media prior to the other (DP600),
and both were
eventually depleted during extended incubation.
The maximum levels of
DP584 observed were significantly higher
than the levels of DP600. The
levels of DP600 observed proved
to be insufficient for effective
quantitation, whereas the DP584
levels could be monitored by HPLC (Fig.
2). Although the conversion
of morphine
to 2,2'-bimorphine is stoichiometric (
19), accumulation
of
DP584 and accumulation of DP600 appeared to be transient. This
indicates that these oxidation products are themselves transformed
prior to depletion of 2,2'-bimorphine and that although no other
metabolites of 2,2'-bimorphine were detected, other transformation
activities may occur.
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FIG. 1.
Transformation of morphine and 2,2'-bimorphine by
C. didymum at pH 7.0 and 4.0. Whole-cell incubation with
C. didymum mycelia in mineral media was carried out with
morphine (10 mM) at pH 7.0 (a) and pH 4.0 (b). Samples were assayed in
triplicate for morphine concentration ( ) and 2,2'-bimorphine
concentration ( ) by HPLC. Error bars indicate ±1 standard
deviation.
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FIG. 2.
Transformation 2,2'-bimorphine and transient
accumulation of DP584. C. didymum mycelia were challenged
with morphine in mineral medium buffered at pH 4.0 with 2,2-dimethyl
succinic acid (20 mM) for 24 h. The mycelia were washed and used
for whole-cell incubation in mineral media with 2,2'-bimorphine (2.5 mM). Samples were assayed in triplicate for 2,2'-bimorphine
concentration () and DP584 concentration () by HPLC. Error bars
indicate ±1 standard deviation.
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|
Identification of 10--S-monohydroxy-2,2'-bimorphine
and
10,10'-,'-S,S'-dihydroxy-2,2'-bimorphine.
Whole-cell incubation mixtures after 100 h were acidified to pH
3.5 with 1.0 M HCl in order to dissolve any insoluble metabolites. Fungal mycelia were then removed by filtration through Whatman no. 1 filter paper in vacuo. The pH values of the supernatants were raised to
neutral with 1.0 M NaOH, which resulted in reprecipitation of insoluble
metabolites. These metabolites were collected by centrifugation at
6,000 × g for 10 min at 4°C, dissolved in ammonia (specific gravity, 0.88; typically at a concentration of 50 mg/ml), and
purified by preparative TLC. For preparative TLC we used silica gel
glass-backed plates (20 by 20 cm; silica gel thickness, 250 µm)
treated to fluoresce (K6F Silica Gel 60 A; Whatman Laboratory Division,
Clifton, N.J.) and a chloroform-methanol-ammonia (specific gravity,
0.88) (6:3:1, by volume) solvent system. The relevant bands were
visualized by fluorescence quenching at 254 nm and removed from the
plates. DP584 and DP600 were eluted from the silica with ammonia
(specific gravity, 0.88; 3 to 4 ml), and the silica was removed by
centrifugation. Samples were freeze-dried after the volume of solvent
was reduced to 1 ml under a stream of nitrogen.
Positive fast atom bombardment mass spectra of the samples were
obtained with a model MF890 mass spectrometer. The spectra
of DP584 and
DP600 showed molecular ions of
m/z 585.26 and
m/z 601.25, respectively. This indicated that DP584 and DP600 were
oxidation products of 2,2'-bimorphine, with DP584 containing one
additional oxygen atom compared to 2,2'-bimorphine and DP600 containing
two additional oxygen
atoms.
1H nuclear magnetic resonance spectra of the metabolites
DP564 and DP600 were obtained by using a Brucker AM-400 spectrometer,
D-6 dimethyl sulfoxide solvent, and tetramethylsilane as an internal
standard. The following signals were obtained for DP584: H-6.64
(H, d,
1-H); 6.32 (H, d, 1'-H); 5.56 (H, m, J 10.0, 7'-H); 5.46
(H, m, J 10.0, 7-H); 5.41 (H, m, J 10.0, 8-H); 5.24 (H, m, J 10.0,
8'-H); 4.71 (H,
broad 6-OH); 4.68 (H, dd, J 4.8, 5'-H); 4.60 (2H,
10
qe-H & 5-H); 4.07 (H, dd, J 5.25, 2.74, 6-H); 4.05 (H, dd, J
5.25, 2.74, 6'-H); 3.24 (H, dd, J 5.94, 3.2, 9'-H); 3.11 (H, d,
J 2.74, 9-H); 2.90 (H, d, J 18.27, 10
qe'-H); 2.52 (H, m, J 2.51
14'-H); 2.48 (2H, m, H-16
eq and H-16
eq' overlapping); 2.44 (3H,
s, N-Me'); 2.42 (1H, m, 14-H); 2.35 (H, m, H-16
ax');
2.31 (3H,
s, N-Me); 2.22 (2H, m, 10
qa' and 16
ax
overlapping); 1.97 (H, m,
H-15
eq'); 1.92 (H, m,
H-15
eq); 1.66 (H, m, H-15
ax'); 1.64 (H,
m,
H-15
ax). The following signals were obtained for DP600:
H-6.73
(2H, s, 1-H & 1'-H); 5.49 (2H, m, J 10.0, 7-H & 7'-H); 5.46 (2H,
m, J 10.0, 8-H & 8'-H); 4.71 (2H, dd, J 5.96, 1.38, 5-H & 5'-H);
4.65 (2H, s, 10
qe-H & 10
qe'-H); 4.10 (2H, m, 6-H & 6'-H); 3.18
(2H, d, 2.98, 9-H & 9'-H); 2.49 (10 H, N-Me & N-Me', H-14 & H-14',
H-16
eq, and H-16
eq' overlapping); 2.27 (2H, ddd, J 24.27, 12.14,
3.44, H-16
ax & H-16
ax'); 1.99 (2H, ddd, J 24.95, 12.59, 5.27,
H-15
eq & H-15
eq'); 1.72 (2H, m, J 14.19, 1.37).
For comparison,
the signals for 2,2'-bimorphine are H 6.31 (2H, s, 1-H
& 1'-H);
5.58 (2H, dd, J 9.6 and 2.5, 7-H & 7'-H); 5.26 (2H, d, J 9.6,
8-H & 8'-H); 4.70 (2H, d, J 5.7, 5-H & 5'-H); 4.10 (2H, dd, J
5.7 and
2.5, 6-H & 6'-H); 3.29 (2H, dd, J 6.2 and 2.6, 9-H & 9'-H);
2.91 (2H,
d, J 18.6, 10-H & 10'-H); 2.57 (2H, d, J 2.6, 14-H &
14'-H); 2.50 (2H,
dd, J 12.5 and 3.5, 16-H & 16'-H); 2.32 (6H,
s, N-Me & N-Me'); 2.28 (2H, d, J 12.5, 16-H & 16'-H); 2.23 (2H,
dd, J 18.6 and 6.2, 10-H & 10'-H); 1.99 (2H, dd, J 11.4 and 3.5,
15-H & 15'-H); 1.68 (2H, d, J
11.4, 15-H & 15'-H) (
19). Structures
and numbers are shown
in Fig.
3. The data for DP600, comprising
a single set of signals that integrate doubly, clearly indicate
that
the compound is a dimer of equivalent monomers. The data
for DP584 show
that there are two sets of signals with values
integrating singly,
which are equivalent to a dimer comprising
one monomer identical to the
monomers forming 2,2'-bimorphine
and one monomer identical to the
monomers forming DP600. The C-10(10')
quasi-equatorial signal obtained
for the nonsubstituted monomers
of 2,2'-bimorphine, occurring at 2.90 ppm, was absent from DP600
and occurred singly for DP584. The spectrum
for DP600 has the
appearance of a double proton signal at 4.65 ppm, and
the spectrum
for DP584 has the appearance of a single proton signal at
4.68
ppm; both spectra are consistent with hydroxymethine protons.
The
combination of these spectra and the mass spectrometric data
identified
DP584 and DP600 as 10-
-
S-monohydroxy-2,2'-bimorphine
and
10,10'-
,
'-
S,
S'-dihydroxy-2,2'-bimorphine,
respectively.
Hydroxy functionalization of codeine, morphine, and
bimorphine.
Although C-14 hydroxylation has been described
relatively frequently, reports of hydroxylation at the C-10 position of
the morphinan structure, under any circumstances, are scarce.
Consequently, the effects on the properties of morphine that such a
change has have not been determined. 10-Hydroxymorphine is known to be
a product of morphine oxidation and has been identified in mixtures of
morphine oxidation products (17) and as an active impurity in morphine (6). An adduct of morphine,
10--S-glutathionyl morphine, has been reported in rat
hepatic microsomal studies. It was suggested that the reaction of
morphine with cytochrome P-450 generated a reactive intermediate,
10--S-hydroxymorphine, which rapidly and spontaneously
reacted with the thiol groups of proteins and amino acids. The
postulated 10--S-hydroxymorphine intermediate was not
isolated and characterized (4, 5, 12). In vivo studies
with human patients located a morphine metabolite thought to be
10--S-hydroxymorphine but did not locate any thiolated products (13). The possibility that 2,2'-bimorphine,
DP584, and DP600 could form thiol adducts with glutathione in acidic media was tested by using conditions described previously (4, 5,
12), but no such adducts were detected. To the best of our
knowledge, hydroxylation of 2,2'-bimorphine has not been described previously.
The potential applications of the novel compounds described here,
whether as pharmaceuticals themselves or as intermediates
in the
synthesis of other novel alkaloids, remain to be
investigated.
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ACKNOWLEDGMENTS |
This work was supported by the Biotechnology and Biological Science
Research Council.
We acknowledge the comments and advice of Adam Walker with respect to
nuclear magnetic resonance spectrometry.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biotechnology, University of Cambridge, Tennis Court Road, Cambridge
CB2 1QT, United Kingdom. Phone: 44 (0) 1223 334168. Fax: 44 (0) 1223 334162. E-mail: n.bruce{at}biotech.cam.ac.uk.
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Applied and Environmental Microbiology, August 2001, p. 3716-3719, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3716-3719.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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