Table Of ContentCONTRIBUTORS
Numbers in parenthesesi ndicate the pages on which the authors ’ contributions begin.
STEFAN BIENZ (83), Organ&h-chemischesI nstitut der Universitat Ziirich, 8057
Ztirich, Switzerland
NEIL C. BRUCE (l), Institute of Biotechnology,U niversity of Cambridge,T ennis
Court Road, CambridgeC B2 lQT, United Kingdom
RICHARD DETTERBECK (83), Organisch-chemischeIsn stitut der Universittit Ziirich,
8057 Ziirich, Switzerland
CORINNEEN SCH(8 3), Organisch-chemischeIsn stitut der Universitiit Ziirich, 8057
Ziirich, Switzerland
ARMM GUGGISBERG (83), Organisch-chemischeIsn stitut der Universitit Ziirich,
8057 Ziirich, Switzerland
URSULA H~~USERMANN( 83), Organisch-chemischeIsn stitut der Universit%Zt tirich,
8057 Ztirich, Switzerland
MANFREHDE SSE (83), Organisch-chemischeIsn stitut der UniversitIt Ztirich, 8057
Ziirich, Switzerland
DIANE L. LISTER (l), Institute of Biotechnology,U niversity of Cambridge,T ennis
Court Road, CambridgeC B2 lQT, United Kingdom
CHRISTIAN MEISTERHANS (83), Organisch-chemischesIn stitut der Universittit
Ziirich, 8057 Ziirich, Switzerland
DEBORAH A. RATHBONE (l), Institute of Biotechnology,U niversity of Cambridge,
Tennis Court Road, CambridgeC B2 IQT, United Kingdom
BARBARAW ENDT( 83), Organisch-chemischesIn stitut der UniversitHt Ziirich,
8057 Ziirich, Switzerland
CHRISTA WERNER (83), Organisch-chemischesIn stitut der Universittit Ziirich,
8057 Ztirich, Switzerland
vii
PREFACE
In this volume of TheA lkaloids: Chemistry and Biology the recent progresso n
two quite different aspectso f alkaloids is presentedi n two chapters.
The first chapter, by Rathbone, Lister and Bruce, is reproduced from
Volume 57 becauseo f some production issuesw hich resulted in the omission
of certain parts of the text. Sincere apologies are offered to the authors for
this very unfortunate situation. The chapter updates an earlier chapter in the
series regarding the very substantial progress that has been made on the
biotransformationso f alkaloids of various classes,a nd the enzyme systemst hat
are involved. Theses tudiesa re very important in consideringh ow alkaloids used
as medicinal and biological agents may be produced in the future, and how
derivativesw ill be made availablef or biological evaluation.
The secondc hapter,b y Hessea nd his co-workersa t the University of Zurich,
also representsa n update of a chapter published in Volume 45 in the series.I t
provides a wonderful comprehensiver eview of the known polyamine alkaloids
based on their biogenesis,f ollowed by overviews of their detailed structural
analysis,t heir synthesisa nd their biosynthesis,a nd biology.
Geoffrey A. Cordell
University of Illinois at Chicago
-CHAPTER1-
BIOTRANSFORMATION OF ALKALOIDS
DEBORAH A. RATHBONE, DIANE L. LISTER’ AND NEIL C. BRUCE
Institute of Biotechnology, University of Cambridge,
Tennis Court Road, Cambridge. CB2 1Q T
United Kingdom
I. Introduction
II. Survey of alkaloid transformations
A. Indole alkaloids
B . Isoquinoline alkaloids
C . Pyridine alkaloids
D. Pyrrolizidine alkaloids
E . Quinoline alkaloids
F . Steroidal alkaloids
G . Tropane alkaloids
H. Miscellaneous alkaloids
III. summary
References
I. Introduction
The alkaloids have long intrigued both chemists and biologists. These
compounds, created via complex biosynthetic pathways, continue to provide
structural puzzles and a diverse range of therapeutic compounds. Alkaloids have
provided mankind with a wealth of medicines, poisons and potions for thousands of
years. As the majority of drugs are still derived from natural compounds, there will
’ Presenta ddress:D epartmento f Biochemistry, University of Cambridge,T ennis Court Road,
Cambridge, CB2 lQW, United Kingdom.
THE ALKALOIDS, Vol. 58 Copyright 0 2002 Elsevier Science (USA)
0099-9598102$ 35.00 1 All rights reserved
2 RATHBONE ET AL.
continue to be interest in the identification of new alkaloids, both as drugs in their
own right, or as pivotal intermediates for the synthesis of new drugs. Synthetic
organic chemistry- :ontinues to yield a wealth of new preparative methods for the
synthesis of novel, semisynthetic alkaloid derivatives, and includes the use of
biotransformations. The success of steroid biotransformations in the 1950s heralded
an investigation into the ability of microorganisms to transform alkaloids, and the
modification of bioactive alkaloids provides a continued rationale for screening for
new biological catalysts. A considerable amount of effort has, therefore, been
directed by researchers towards the identification of whole cells or enzymes for the
transformation of alkaloids. Biotransformations are an attractive alternative to
chemical catalysis, particularly when high regioselectivity or functionality is required.
Biotransformations of alkaloids were last reviewed in The Alkaloids by Rosazza
and Duffel in 1986 (I). This is an excellent review that covers bioconversions of
alkaloids and provides a comprehensive and useful summary of the various classes of
enzymes that display activity towards alkaloids. Since the review by Rosazza and
Duffel, the development of molecular techniques that has allowed the cloning and
expression of genes from prokaryotic and eukaryotic organisms in heterologous hosts
has begun to have an impact on alkaloid transformations and is permitting a diverse
range of enzymes to become available to the synthetic chemist. Nature has evolved
remarkable enzymes that display some exquisite chemistries, as can be seen with the
variety of pathways for alkaloid biosynthesis. Molecular techniques are enabling these
enzymes to be individually expressed in heterologous hosts, such as bacteria,
permitting detailed examination of catalytic mechanisms which may be unknown in
synthetic organic chemistry. It is now feasible to consider using the enzymes
mediating alkaloid biosynthetic pathways, often present at very low levels in their
native hosts, within plants as recombinant biocatalysts. Over the next decade it is
foreseeable that complete biosynthetic pathways might be expressed in different
hosts, allowing the possibility of combinatorial synthesis of alkaloids in situ, and the
engineering of new biosynthetic pathways. Transgenic technology now makes it
possible to extend or divert pathways in plants by incorporating genes from other
species, enabling the production of unique compounds with potential biotechnological
applications. Rapid progress has been made in the technology for stable integration
and expression of recombinant DNA in plant cells. At the moment, this type of
approach is very hit-and-miss, since little is currently known about the regulation of
alkaloid biosynthetic pathways, or the metabolic flux of these multi-step enzymatic
processes.
Recent developments in the forced evolution of enzymes ate beginning to have a
considerable impact on biocatalysis and are being used to overcome the limitations
that might thwart the use of certain enzymes for biotransformations. These now allow
existing biocatalysts to be mutated/engineeredt o improve their activity, selectivity and
BIOTRANSFORMATION OF ALKALOIDS 3
specificity towards substrates. It is now feasible to swap domains between enzymes
to create novel catalysts with altered substrate specificity or reaction characteristics.
Studies into the structure and function of alkaloid-transforming enzymes along with
mutational work are providing insights into the operation and organization of catalytic
biomolecules in pathways of secondary metabolism. The information that is being
obtained concerning the genetics and biochemistry of alkaloid biosynthetic pathways
will continue to furnish us with new enzymes and will permit the rational redesign of
alkaloid biosynthetic pathways.
This review aims to provide an updated survey on alkaloid transformations and
concerns microbial transformations and plant enzymes where the enzyme has been
purified or obtained in a recombinant form. Past work has focussed on the use of
liver microsome and microbial transformation as models of human metabolism, and
such work has been well reviewed in the past. The work described here focuses on
single enzymatic transformations, either individually or as part of a pathway, and
consideration has been to made to identify biotransformations where a recombinant
source is available, or where the enzyme has been purified and characterized.
Likewise, biotransformations by plant cell cultures have not been covered unless key
enzymatic steps have been identified.
II. Survey of Alkaloid Transformations
A.THEINDOLEAXN.OJDS
1. Ellipticine derivatives
Ellipticine derivatives have found use as potent antitumor agents and, as a
consequence, these alkaloids have been the focus of much previously reviewed
research concerning their synthesis or modification. Meunier and Meunier detail what
they believe to be the first reported peroxidase-catalyzed O-demethylation reaction.
The cytotoxic agents 9-methoxyellipticine (1) and N2-methyl-9-methoxyellipticinium
acetate( 2) were O-demethylated to the corresponding quinone-imine derivatives 9-
oxoellipticine (3) and p-methyl-9-oxoellipticinium (4), respectively (Figure l), by a
peroxidase system which consisted of horseradish peroxidase and hydrogen peroxide
(2). One hydrogen peroxide molecule is consumed during the reaction, with the
concomitant elimination of the methoxy group of each alkaloid substrate as methanol.
4 RATHESONEETAL.
1 9-methoxyellipticine R= -
2 N2-methyl-9-methoxyellipticinium R= CHs
CH3
3 9-oxoellipticine R= -
4 N2-methyl-9-oxoellipticinium R=CH,
FIGURE 1. Methoxyellipticine derivatives
Peroxidase-catalyzed N-demethylation reactions involve the formation of
formaldehyde rather than methanol, which suggests that 0-demethylation proceeds
via a different reaction mechanism to N-demethylation. This was further suggested
by incubations utilising ‘*O-enrichedw ater in which the “0 was incorporated into the
oxidized ellipticine derivatives and not into methanol, indicating that the oxygen-
carbon aromatic bond is cleaved during the reaction with the incorporation of an
oxygen from water.
2. Ergot alkaloids
The ergot alkaloids and their derivatives have attracted long-term interest due to
the broad spectrum of pharmacological activities that they exhibit, being used to treat
a range of complaints including uterine atonia, migraine, orthostatic circulatory
disturbances, senile cerebral insufficiency, hypertension, hypergahtctinemia,
acromegaly, and Parkinsonism (3). No other group of natural products exhibits such
a wide spectrum of biological action. The majority of naturally occurring ergot
alkaloids is produced by ascomycetes from the genus CZaviceps, with further
examples seen in other filamentous fungi and plant species. Semisynthetic ergot
alkaloids are manufactured via chemical and biological modification of the extracted
BIOTFtANSFORMATIONO F ALKALOIDS 5
naturally occurring compounds (4). Ergot alkaloids have provided a vital stimulus in
the development of new drugs by providing structural prototypes of molecules with
pronounced pharmacological activities (5).
The ergot alkaloid structure comprises a lysergic acid molecule and a cyclole-
structured dipeptide linked by acid amide-type bond, forming a tetracyclic ergoline
ring system. The review by Kobel and Sanglier gives a detailed list of ergot structures
(6). Only two naturally occurring ergot alkaloids, ergotamine (5) and ergometrine (6)
(Figure 2) are used directly in therapy; the remainder have undergone some chemical
modification, such as elimination of the 9,10-double bond by hydrogenation,
halogenation, alkylation etc. Although the total chemical synthesis of ergot alkaloids
is possible (7), the prohibitive nature of the costs involved on a large scale have
promoted the importance of being able to specifically modify natural ergot alkaloids to
achieve the desired functionality.
The main emphasis of ergot bioconversions is that of achieving specific
oxidations. This work has been motivated by the need to produce more effective
drugs and also by the problems of the metabolism of ergot alkaloids in mammals.
Such oxidation reactions tend to be restricted to the alkaloid-producing Cluviceps, and
these organisms may be used in several ways: “xenobiotic” biotransformations (i.e. :
supplying alkaloids which are not native to the organism, “pressured”
biotransformations (i.e.: supplying alkaloids which are normally present at low
amounts at vastly increased concentrations, forcing the production of new
compounds), or “aggressive bioconversions”, in which the regulation of alkaloid
biosynthesis becomes unbalanced as a result of the presence of high concentrations of
the supplemented alkaloid (8).
CONHCH(CH3)CH20H
5 Ergotamine 6 Ergometrine
FIGURE2 . Ergotamine and Ergometrine
6 RATHBONE ET AL.
Agroclavine (7) is produced by certain C. fusifonnis and C. pwpurea strains.
Until fairly recently, the main emphasis of agroclavine research was its oxidation to
elymoclavine (8), which is an important substrate for ergot-based drug production
(8-11). This conversion is very desirable and economically important since it cannot
be achieved by chemical reactions. Industrial bioconversions use the high production
strains C. fisiformis or certain C. paspali strains (8, 9, 12).
The successful use of immobilized and permeabilized C. fusijomis cells in
biotransformations has been demonstrated, allowing the reuse, regeneration and
protection of the biological catalyst during the bioconversion (9). Low level
agroclavine oxidation to elymoclavine has been seen in other systems, but only
Claviceps strains are able to carry out this reaction at a reasonable rate (3).
Other recently documented bioconversions of agroclavine include its S-
hydroxylation by non-Claviceps species, such as horseradish peroxidase, forming
setoclavine (9) and isosetoclavine (10) (13).
Agroclavine was also reported to be oxidized by a haloperoxidase from
Streptomyces aureofaciens in the presence of sodium acetate and bromide ions,
forming 2-oxo-3-acetoxyagroclavine (11) (24). Mass spectral analysis of the
transformation product excluded bromination of the molecule, and ‘H-NMR detected
the addition of acetate at the C-3 position. Both stereoselective oxidation and acetate
incorporation were thought to be catalyzed by the haloperoxidase. Under conditions
optimum for the maximum production of 2-0x0-3-acetoxyagroclavine, minor
products also accumulated which were determined to be setoclavine and
isosetoclavine, respectively. Rropionate was tested as an alternative carboxylate to
acetate in incubations and resulted in a major product identified as 2-0x0-3-
propionoxyagroclavine (12), indicating that propionate had been incorporated into the
agroclavine molecule. In addition, a higher oxidation product was identified with
incubations containing propionate, (4aS),(lObS)-7-amino-3,4,4a,5,6,1Ob-hexa-
hydro-2,4-dimethyl-6-oxobenzo-V]quinoline (13) (Figure 3). This compound had
previously been isolated as the degradation product formed in the later stages of ergot
alkaloid biosynthesis in Claviceps species, probably due to peroxidase activity (15).
The C. purpurea agroclavine 17-hydroxylase activity is able to oxidize a number
of analogues to their corresponding 8a-hydroxy derivatives; for example,
noragroclavine (14) and lysergine (16) am oxidized to norsetoclavine (15) and
setoclavine respectively (Scheme 1) (13). The stereospecificity of this reaction
contrasts with that of the mixture of 8a- and 8l3-isomers that arise when horseradish
peroxidase is used. Kren reviews in more detail the analogues tested (3).
Elymoclavine research has focused on a number of areas of bioconversion,
including the introduction of hydroxyl groups by peroxidase action (for example at C-
8 or C-lo), isomerization to lysergol (17), reduction to agroclavine (all reviewed by
Kren (3)) and glycosylation, although perhaps the most extensively studied area
BIOTRANSFORMATIONO F ALKALOIDS 7
& $ gC H3
H 0
7 Agroclavine R=CH, 11 2-Oxo-3-acetoxyagroclavine R=COCH,
8 Elymoclavine R=CH,OH 12 2-Oxo-3-propionoxyagroclavine R=COCH,CH,
$$ fj$CH3
9 Setoclavine R,=OH R&H, 13 (4a5’),(lObS)-7-Amino-3,4,4a,5,6,10b-hexa-
10 Isosetoclavine RI=CH, R,=OH hydro-2,4-dimethyl-6oxobenzo-V]quinoline
FIGURE3 . Agroclavine and its oxidationlhydroxylation products
has been that of oxidation of the molecule to lysergic acid (18) or paspalic acid (19)
(Figure 4). As for agroclavine oxidation, this has been demonstrated most practically
only by selected members of the Cluviceps genus, namely C. purpurea and C. paspali
strains (IO, 16, 17), with the bioconversion being achieved successfully on an
industrial scale (16). The enzymes responsible are cytochromes P-450 located in the
microsomal fraction (10). Derivatives of elymoclavine are also converted by C.
paspali to their corresponding lysergic acid derivatives (17).
The enzymatic glycosylation of ergot alkaloids has been the focus of much
research by Kren and coworkers over recent years. This work aimed to produce
glycosylated derivatives of ergot alkaloids which were proposed to possibly have
different pharmacological activities than their non-glycosylated counterparts.
The fructosylation of supplemented ergot alkaloids both in free and alginate-
immobilised cultures of C. purpurea in sucrose-containing media was reported by
Kren et aZ., who observed a mixture of fructosides, namely mono, di-, and probably
tri- and tetra-fructosides (18). The glycosylating reaction was highly pH-dependent
(optimum pH 6.5 for elymoclavine) and required a sucrose concentration of 75 g/l.
RATHBONEETAL.
14 Noragroclavine 15 Norsetoclavine
9 Setoclavine
16 Lysergine
SCHEME1 . The hydroxylation of noragroclavine and lysergine
H
17 Lysergol 18 Lysergic acid 19 Paspalic acid
FIGURE4 . Reaction products of elymcclavine conversions
Such glycosylating strains produce elymoclavine in its glycosidic form; thus, the
feedback inhibition of alkaloid biosynthesis by elymoclavine is strongly reduced,
helping to further improve total elymoclavine yields. However, this may cause
complications in the isolation of the product, as it is not possible to use HCl to
hydrolyse fructosides since it is too aggressive and causes losses of elymoclavine. A
more simple method is to use a biological hydrolytic process; one example might be
to add a suspension of Saccharomyces cerevisiae to the medium at the end of the
incubation, which causes complete hydrolysis in 1 h at 37’C (18).
