cis-dihydrodiol, arene oxide and phenol metabolites of dictamnine: Key intermediates in the biodegradation and biosynthesis of furoquinoline alkaloids

Article abstract of DOI:10.1039/b506944k

Biotransformation of the parent furoquinoline alkaloid dictamnine and its 4-chlorofuroquinoline precursor, using the B8/ 36 bacterial mutant strain of Sphingomonas yanoikuyae, yielded, via biphenyl dioxygenase-catalysed dihydroxylation, the first isolable alkaloid cis-dihydrodiol metabolites; these metabolites were used in the chemoenzymatic synthesis of postulated arene oxide and phenol intermediates, and a range of derived furoquinoline alkaloids. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b506944k

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cis-Dihydrodiol, arene oxide and phenol metabolites of dictamnine: key
intermediates in the biodegradation and biosynthesis of furoquinoline
alkaloids
Derek R. Boyd,*ab Narain D. Sharma,ab Colin R. O’Dowd,a Jonathan G. Carroll,a Pui L. Lokea and
Christopher C. R. Allenc
Received (in Cambridge, UK) 18th May 2005, Accepted 6th June 2005
First published as an Advance Article on the web 11th July 2005
DOI: 10.1039/b506944k
observed using
a mutant strain (B8/36) of Sphingomonas
Biotransformation of the parent furoquinoline alkaloid dic-
tamnine and its 4-chlorofuroquinoline precursor, using the B8/
36 bacterial mutant strain of Sphingomonas yanoikuyae, yielded,
via biphenyl dioxygenase-catalysed dihydroxylation, the first
isolable alkaloid cis-dihydrodiol metabolites; these metabolites
were used in the chemoenzymatic synthesis of postulated arene
oxide and phenol intermediates, and a range of derived
furoquinoline alkaloids.
yanoikuyae (a source of biphenyl dioxygenase, BPDO).
Dictamnine 1a is the parent compound of the furoquinoline
alkaloid series. It is widely distributed among plants of the
Rutaceae family. To date, more than eighty furoquinoline
alkaloids have been identified, the majority having hydrogen
atoms on the benzo ring of dictamnine 1a replaced by OH, OMe,
OEt, OCH2O or OCH2CHLCMe2 groups and their derivatives.
Earlier biosynthetic labelling studies in Skimmia japonica and
Choisya ternata plants had shown that dictamnine 1a was
biotransformed into other furoquinoline alkaloids, e.g. skimmia-
nine 1g, via the putative arene oxide 2 as the biosynthetic precursor
(Scheme 1).14 Although, in some cases, these plant biosynthetic
sequences remain to be elucidated, the phenols, robustine 1b and
haplopine 1f, and ethers, c-fagarine 1c, haplophydine 1d,
skimmianine 1g, 7-isopentenyl-c-fagarine 1h and isohaplopine
3,39-dimethylallyl ether 1i are also among the range of quinoline
alkaloids that could be derived from dictamnine 1a via arene oxide
intermediate 2. In this context, it is evident that the enzyme-
catalysed oxidation of dictamnine 1a to yield arene oxide 2, and its
isomerisation to the corresponding phenol metabolite 1b could
lead to alkaloids 1c and 1d. Further aromatic hydroxylation of
alkaloids 1b or 1c, or possibly epoxide hydrolase-catalysed
hydrolysis of arene oxide 2 to yield trans-dihydrodiol 3 followed
by dehydrogenation to yield catechol 1e, could be involved in the
biosynthesis of the 7,8-disubstituted furoquinoline alkaloids, 1f–1i.
This report is thus focused on the chemoenzymatic synthesis of the
postulated arene oxide intermediate 2 of dictamnine 1a and
furoquinoline alkaloids derived from cis-dihydrodiol metabolite
Bacteria and fungi play an important role in the biotransformation
and biodegradation of plant alkaloids in the soil.1–3 Alkyl
monohydroxylations, N-oxidations and dealkylations are among
the most common types of microbial alkaloid oxidations to have
been reported.1–3 However, microbial aromatic hydroxylations of
alkaloids are relatively rare, and the isolation of arene cis-
dihydrodiol metabolites of alkaloids is, to our knowledge,
unprecedented. In prokaryotes, more than three hundred cis-
dihydrodiols have been isolated from dioxygenase-catalysed
dihydroxylation of arene substrates. Both mutant and recombi-
nant bacterial strains containing dioxygenases, e.g. toluene
dioxygenase (TDO), but lacking in the corresponding diol
dehydrogenase enzymes, have been used to produce substituted
benzene cis-dihydrodiols which have been widely utilised in
synthesis.4–7
The major objective of this study was to find the first evidence of
cis-dihydrodiol intermediate formation in the bacterial biotrans-
formation of an alkaloid. If the cis-dihydrodiol metabolite was
present, a further target was to isolate and utilise it for the
chemoenzymatic/biomimetic synthesis of other related alkaloids.
Earlier reports from these laboratories had shown that cis-
dihydrodiol metabolites could be isolated via dioxygenase-
catalysed asymmetric dihydroxylation of quinolines, using mutant
strains of bacteria.8,9 Furthermore, a relatively stable 7,8-arene
oxide derivative of quinoline was chemically synthesised10 and
isolated as a eukaryotic metabolite from monooxygenase-catalysed
epoxidation using liver microsomes.11 Dioxygenase-catalysed
asymmetric dihydroxylations of larger polycyclic azaarene sub-
strates e.g. acridine12 and benzo[c]phenanthridine13 were also
aSchool of Chemistry, The Queen’s University of Belfast, Belfast, UK
BT9 5AG. E-mail: dr.boyd@qub.ac.uk; Fax: +44 (0)2890 382117;
Tel: +44 (0)2890 681198
bCenTACat, The Queen’s University of Belfast, Belfast, UK BT9 5AG
cQUESTOR, The Queen’s University of Belfast, Belfast, UK BT9 5AG
Scheme 1
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precursors 4, 5, 7 and 8 (Scheme 2), as several of these
furoquinoline alkaloids have recently been reported to possess
interesting biological activities.15
A tentative assignment of an (R) absolute configuration was made
in each case.
The biotransformation of dictamnine 1a using rat liver
microsomal enzymes has also been reported to give the acyclic
diol 6 as a bioproduct of unspecified stereochemistry (Scheme 3).18
It was proposed, in the eukaryotic study, that the isolated
2-quinolone bioproduct 6 had resulted from the initial mono-
oxygenase-catalysed epoxidation of dictamnine 1a to yield the
transient arene oxide 10. The epoxide hydrolase-catalysed hydro-
lysis of arene oxide 10 to yield the trans-diol 11trans, followed by
spontaneous reversible ring opening to aldehyde 12, and then
enzyme-catalysed reduction could account for the formation of
acyclic diol 6 (Scheme 3). Similarly the cis-dihydrodiol metabolite
11cis, resulting from BPDO-catalysed oxidation of the furan ring in
dictamnine 1a, would also undergo ring opening to aldehyde 12
prior to being reduced enzymatically to diol 6 (Scheme 3). A
similar metabolic sequence (furan A furan cis-diol = hydro-
xyaldehyde = furan trans-diol A acyclic diol) was observed
during the dioxygenase-catalysed cis-dihydroxylation of benzo-
furan using P. putida UV4 to yield an equilibrating mixture of cis/
trans diols and a derived acyclic diol [55% ee and of (R)
configuration].16
Earlier successful bacterial biotransformations of the tricyclic
azaarene acridine,12 to yield a cis-dihydrodiol metabolite using S.
yanoikuyae B8/36, and of benzofuran16 to furnish cis-dihydrodiols
in both the carbocyclic and heterocyclic rings using Pseudomonas
putida UV4 (a source of TDO), influenced our choice in favour of
dictamnine 1a and its precursor 4-chlorofuroquinoline 1j as
suitable substrates for the study with whole cells of S. yanoikuyae
B8/36 (Scheme 2). Neither of these substrates has a substituent in
its benzo ring.
Dictamnine 1a, synthesised from 4-chlorofuroquinoline 1j by
the literature method,17 was oxidised using S. yanoikuyae B8/36 to
yield a mixture of bioproducts (4–6). These metabolites were
separated and purified by PLC (7% MeOH in CHCl3) to furnish
cis-dihydrodiols 4 (Rf 0.37, 20–29% yield), 5 (Rf 0.32, 0–3% yield)
and acyclic diol 6 (Rf 0.26, 0–1% yield). Under similar conditions,
the chlorofuroquinoline 1j yielded the corresponding cis-dihydro-
diols 7 (Rf 0.6, 10% yield), 8 (Rf 0.5, 30% yield) and 2-quinolone 9
(Rf 0.4, 2% yield). Treatment of cis-dihydrodiols 4 ([a]D +94,
MeOH), 5 ([a]D 2110, MeOH), 7 ([a]D +138, CHCl3), and 8
([a]D +214, MeOH), with (R) and (S)-2-(1-methoxyethyl)-
phenylboronic acid (MEPBA) yielded the corresponding
diastereoisomeric boronates. 1H-NMR analysis of the MEPBA
diastereoisomers confirmed that all these cis-dihydrodiols were
enantiopure (.98% ee). Using both the MEPBA method and
circular dichroism spectral comparison, it was concluded that the
absolute configurations of the cis-dihydrodiol metabolites 4 and 7
(7S,8R), 5 and 8 (5R,6S) were similar to those found for cis-
dihydrodiol metabolites of quinoline and acridine substrates.8,12
Catalytic hydrogenation of 5,6-cis-dihydrodiol 5 (H2, 5% Pd/C)
gave 5,6-dihydroxy-5,6,7,8-tetrahydrodictamnine ([a]D +15,
MeOH, 95% yield). The latter compound was also formed by (i)
catalytic hydrogenation (H2, 5% Pd/C) of chloro-cis-dihydrodiol 8
to give the corresponding tetrahydrodiol ([a]D 255, MeOH, 95%
yield), (ii) protection as an acetonide ([a]D +129, CHCl3, 98%
yield), (iii) substitution of the acetonide chlorine atom by a
methoxy group ([a]D +151, CHCl3, 85% yield) and (iv) deprotec-
tion to give 5,6-dihydroxy-5,6,7,8-tetrahydrodictamnine ([a]D +16,
MeOH, 75% yield), thus confirming the identical (5R,6S) absolute
configurations of both cis-dihydrodiols 5 and 8 by stereochemical
correlation.
The proposed intermediacy of arene oxide metabolites, during
both plant biosynthesis (e.g. compound 2, Scheme 1) and
eukaryotic metabolism (e.g. compound 10, Scheme 3) of
dictamnine 1a, prompted the synthesis of arene oxide 2 from the
corresponding major cis-dihydrodiol 4 (Scheme 4). The synthetic
sequence involved (i) catalytic hydrogenation of 7,8-cis-dihydrodiol
4 to give tetrahydrodiol 13, ([a]D 225, CHCl3), (ii) bromoacetyla-
tion to produce the trans-bromoacetate 14 ([a]D 238, CHCl3), (iii)
benzylic bromination to provide the unstable dibromoacetate 15
and (iv) dehydrobromination/cyclisation to yield the required
enantiopure 7,8-arene oxide 2 ([a]D +93, CHCl3).
The small quantities of acyclic diols 6 and 9 obtained from
furoquinolines 1a and 1j, were reacted with either (R) and
(S)-MEPBA or (R)-(2)-a-methoxy-a-(trifluoromethyl)phenylace-
tyl chloride (MTPA chloride) to form the corresponding
Scheme 3
1
diastereoisomeric esters. H-NMR analysis of the MEPBA and
diMTPA esters showed the enantiopurity values to be variable
(75% ee, [a]D +9, MeOH for 6 and 18% ee, [a]D 25, MeOH for 9).
Scheme 4 Reagents: i H2, Pd/C (96%); ii AcOCMe2COBr, MeCN
(90%); iii NBS, CCl4 (68%); iv NaOMe (90%); v TFA (95–98%); vi CH2N2
(90%).
Scheme 2
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Thermal- or acid-catalysed isomerisation of arene oxide 2, and
dehydration (TFA) of cis-dihydrodiol 4 yielded the phenolic
alkaloid 8-hydroxydictamnine (robustine, 1b) exclusively, which
was in turn methylated to give c-fagarine 1c. The alkaloids 1f–1i
could also be formed from dictamnine 1a via further P-450-
catalysed monohydroxylation of robustine 1b or c-fagarine 1c.
While no direct evidence was obtained for the formation of trans-
dihydrodiol 3 from acid treatment of arene oxide 2, the possibility
of its involvement during the biosynthetic pathway in plants, where
both P-450 monooxygenase and epoxide hydrolase are likely to be
present, cannot be excluded. It is noteworthy that during
comprehensive studies of the analogous 7,8-oxide of quinoline
under carefully controlled conditions, while 8-hydroxyquinoline
was the sole product under acid conditions (pH , 7.0), the
7,8-trans-dihydrodiol derivative of quinoline was the only product
under basic conditions (pH . 12).19 Acid-catalysed dehydration
of 5,6-cis-dihydrodiol 5 also yielded the phenol derivative
6-hydroxydictamnine 16, a possible eukaryotic metabolite of
dictamnine 1a.18 Although 6-hydroxydictamnine 16 has not yet
been isolated as a plant alkaloid, it is assumed to be an
intermediate during the biosynthesis of pteleine 17 which was
obtained after methylation of phenol 16 (Scheme 4).
Scheme 5 Reagents: i E. coli nar B; ii CH2N2; iii NaOMe; iv BBr3; v
ClCH2CHLCMe2, K2CO3.
We thank the HEA North–South Programme for Collaborative
Research (NDS), the European Social Fund (JGC) and Oxford
Glycosciences (PL) for financial support. We also acknowledge the
support provided by Overseas Research and Dunville Studentships
(PL), and Larmor and Musgrave Studentships (JGC) from The
Queen’s University of Belfast.
Notes and references
A small sample of cis-dihydrodiol 4, was used as a substrate
with whole cells of the recombinant bacterial strain Escherichia coli
nar B (a source of naphthalene cis-diol dehydrogenase, NDD),20
and gave catechol 1e but only in very poor yield (,5%).
Surprisingly, the 7,8-cis-dihydrodiol of chlorofuroquinoline, 7,
proved to be a much better substrate for E. coli nar B; it formed
catechol 18 in y40% yield and this provided an indirect route to
skimmianine 1g (Scheme 5). Thus, catechol 18 was methylated
(CH2N2) to give dimethoxy derivative 19 (95% yield) which
allowed substitution of the chlorine atom by the methoxy group
(NaOMe) and yielded skimmianine 1g (20% yield). A further
supply of catechol 1e was then synthesised from skimmianine 1g
by selective demethylation using BBr3 (85% yield). Partial
methylation of catechol 1e by reacting with CH2N2 (60 s) occurred
mainly at the OH group on C-8 to give haplopine 1f (55% yield);
further methylation (CH2N2) yielded skimmianine 1g (95% yield
from catechol 1e). Prenylation of haplopine 1f with 1-chloro-3-
methyl-but-2-ene in the presence of K2CO3 gave the alkaloid
7-isopentenyl-c-fagarine 1h (85% yield). The reverse sequence
involving initial prenylation of catechol 1e to yield phenol 1k
followed by methylation (CH2N2) gave the isomeric alkaloid
isohaplopine 3,39-dimethylallyl ether 1i (63% yield from catechol
1e).
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In conclusion, the isolation of cis-dihydrodiol metabolites of an
alkaloid, dictamnine 1a, using dioxygenase enzymes, has been
accomplished. Based on the above observations, the formation of
arene cis-diol metabolites may be of considerable significance in
the general context of bacterial biodegradation of plant alkaloids
in the environment. cis-Dihydrodiol 4 proved to be a remarkably
stable precursor of the corresponding arene oxide 2. Both arene
oxide 2 and cis-dihydrodiols 4, 5, and 7 yielded phenolic derivatives
from which a range of furoquinoline alkaloids were synthesised via
biomimetic routes.
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Chem. Commun., 2005, 3989–3991 | 3991