Alkaloids - Wiley-VCH

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1 12299 - 07 - 325361 27.08.1999 08:09 Uhr Seite 327 7 Alkaloids NEIL C. BRUCE Cambridge, UK 1 Introduction 328 2 Tropane Alkaloids 332 2.1 Tropane Alkaloid Biosynthesis 332 2.2 Microbial Metabolism of Tropane Alkaloids 335 3 Benzylisoquinoline Alkaloids 338 3.1 Benzophenanthridine Alkaloids 338 3.2 Morphine Alkaloids 339 3.2.1 Morphine Alkaloid Biosynthesis 342 3.2.2 Microbial Metabolism of Morphine Alkaloids 344 3.2.3 Transformations of Morphine Alkaloids by Pseudomonas putida M10 346 3.2.4 Biological Production of Hydromorphone and Hydrocodone 350 3.2.5 Microbial Transformation of Heroin 351 4 Monoterpenoid Indole Alkaloids 352 5 Summary 356 6 References 357

2 12299 - 07 - 325361 27.08.1999 08:09 Uhr Seite 328 328 7 Alkaloids 1 Introduction ever, results from a recent examination of ma- terials from the tomb of the Royal Architect KHA seem to refute earlier observations (BIS- Plants have the ability to produce tens of SET et al., 1994). It was not until the 19th centu- thousands of highly complex secondary me- ry that the active compounds, alkaloids, were tabolites to assist their survival in the environ- isolated from the opium. Morphine was also ment, many of which protect the plant from the first alkaloid to be identified and crystal- predators. Man has exploited these com- lized by the chemist SERTRNER in 1805. This pounds of self-defence as sources of medicinal was a significant achievement as not only was agents, poisons, and potions since time imme- it the first time that a nitrogenous base had morial. Throughout the world different com- been isolated from a biological source, but it munities have discovered plants with phar- was also the first time that such a substance macological properties, and many useful drugs had been shown to be intrinsically basic. This have their origins in indigenous ethnopharma- finding formed the basis of one of the earliest cologies. Some notable examples include: the definitions of an alkaloid which was attributed roots of the mandrake plant, known for their to the pharmacist W. MEISSNER (HESSE, 1981; sedative properties from the time of HIPPO- PELLETIER, 1983). The two authors, HESSE CRATES (ca. 400 BC) and also used as a deadly (1981) and PELLETIER (1983), differ on the ex- poison during Elizabethan times (MANN, act date of the coining of the term (1818 and 1989); the leaves of the coca plant, which were 1819 being cited, respectively) and the deriva- chewed as an aid to stamina and as part of ce- tion, but the general meaning was taken to be remonies in South America over 5000 years an alkali like compound of plant origin, or as ago (VAN DYKE and BYCK, 1982); and plants BENTLEY (1954) interpreted, a vegetable al- whose hallucinogenic properties were used in kali. This was extended by WINTERSTEIN and the preparation of magic potions by the Az- TIET (1910) to include a four part definition tec Indians. The compounds responsible for stating a true alkaloid can be characterized these physiological effects in man were isolat- by: (1) the possession of a nitrogen atom as ed during the 19th and early 20th centuries and part of a heterocyclic system; (2) a complex were identified as scopolamine, cocaine, and molecular structure; (3) significant pharmaco- amides of lysergic acid, respectively. SOCRA- logical properties; (4) its origin from the plant TES death in 399 BC was the result of con- kingdom (cited in PELLETIER, 1983). sumption of hemlock (Conium maculatum) The majority of alkaloids fit this four part which contains the alkaloid coniine (1) (Fig. 1; definition; however, a number of exceptions HENDRICKSON et al., 1970), while CLEOPATRA exist. The compounds samandarine (2) (Fig. 1), used extracts from Egyptian henbane samandarone, and cycloneosamandarine, iso- (Hyoscyamus muticus) during the last century lated from the skin glands of the European fire BC to dilate her pupils and increase her beau- salamander (Salamandra maculosa Laurenti) ty. Likewise, medieval European women used all exhibit the usual properties of an alkaloid extracts of deadly nightshade (Atropa bella- substance, but do not fit the definition of a donna) in their beauty preparations, hence the true alkaloid owing to their animal origin. name bella donna, fair lady. There are numerous examples of alkaloids fur- Other historical uses include extracts from nished by animal, including batrachotoxinin A the bark of Cinchona officinalis which have (3), a steroidal alkaloid from the Colombian been employed as antimalarials. Extracts de- arrow-poison frog (Phyllobates aurotaenia), rived from the opium poppy Papaver somni- bufotenine (4), a tryptamine-type alkaloid ferum comprise another group of important from the common European toad, (P)-deoxy- pharmacologically active compounds which nuphradine and (P)-castoramine (5) from the possess powerful analgesic properties. It has Canadian beaver (Castor fiber L.), and musco- been reported that extracts of the milky latex pyridine (6) from the scent gland of the musk material that exudes from the cut unripe seed deer (Moschus moschiferus). Alkaloids have capsule of the opium poppy were used by the also been identified from arthropod, bacterial, early Egyptians for medicinal purposes; how- and fungal origins. For example, the quinazole

3 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 329 1 Introduction 329 Fig. 1. See text. alkaloids, glomerine (7a) and homoglomerine tral and, therefore, does not conform to the (7b) discharged from the dorsal glands of the original definition of an alkaloid. PELLETIER European millipede (Glomeris marginata), the (1983), however, provides a reasonable sum- deep-blue colored alkaloid pyocyanine (8), mary of an alkaloids properties as being an isolated from the bacterium Pseudomonas ae- alicyclic compound containing nitrogen in a ruginosa, and agroclavine (9), produced by the negative oxidation state which is of limited dis- fungi Claviceps purpurea and Aspergillus fu- tribution among living organisms. Over migatus. Other examples of alkaloids exist 10 000 compounds fall within this definition which also do not adhere to the criteria stipu- (SOUTHON and BUCKINGHAM, 1989) and new lated in the four part definition of an alkaloid. alkaloids are continually being reported from For example, the alkaloids colchicine (10) (au- various sources. These represent approximate- tumn crocus, Colchicum autumnale L.) and ly 20% of all known natural products; howev- mescaline (11) (Lophophora williamsii) do er, only about 30 of these with biological activ- not possess nitrogen as part of a heterocyclic ity are commercialized (FARNSWORTH, 1990). system. Also colchicine (10) is essentially neu-

4 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 330 330 7 Alkaloids Unlike any other group of compounds the alkaloids exhibit a vast array of skeletal types and are classified accordingly. A typical exam- ple is the scheme used by HESSE (1981), who describes 11 classes of heterocyclic alkaloids, differentiating by the nature of the carbon skeleton, e.g., the pyrrolidine and isoquinoline alkaloids. The majority of alkaloids are amino acid-derived, although terpenes, steroids, pu- rines, and nicotinic acid can also act as building blocks of, e.g., aconitine, solanidine, caffeine, and nicotine, respectively. If the anabolic route of an alkaloid is known, this can be used to classify the compound (DALTON, 1979). The tropane, and pyrrolidine alkaloids, for in- stance, are all derived from ornithine, a deriva- tive of arginine, and thus grouped together under this scheme. Alkaloids have provided a wealth of phar- macologically active compounds; approxi- mately 25% of the drugs used today are of plant origin. These are administered either as pure compounds or as extracts and have often served as model structures for synthetic drugs, e.g., atropine (13) for tropicamide, quinine for chloroquinine, and cocaine (12) (Fig. 2) for procaine (KUTCHAN, l995). Screening of plant extracts for pharmacologically active com- pounds still continues and results in new drug discoveries; recent examples include the anti- cancer drugs taxol from the western yew, Taxus brevifolia, and camptothecin from Camptothe- ca acuminata. Alkaloids are generally regard- ed as speciality chemicals; approximately 300500 metric tons of quinine and quinidine are produced each year; ajmalicine (98) pro- Fig. 2. Examples of tropane alkaloids. duction amounts to about 3600 kg, while com- pounds like vincristine (94) and vinblastine (95) (Fig. 21) are produced in the kilogram range. The annual market value of the major and MILNE, 1981). However, the synthesis of alkaloids has been estimated to be in the range such compounds is often difficult to achieve on of several hundred million dollars (VER- a commercial scale due to the chemical com- POORTE et al., 1993). plexity of the starting material, cost, and envi- The important pharmacological activity of ronmental issues, in addition to the precursors many alkaloids has spurred chemists to make being in limited supply. Biotransformations many derivatives of these natural compounds. can offer a number of advantages over con- The chemical preparation of such semisynthet- ventional chemical processes.The specificity of ic alkaloids has resulted in the production of enzyme-catalyzed reactions, e.g., allows the drugs with improved properties, such as the stereospecific transformation of defined func- addition of a 14-hydroxy group to the mor- tional groups. However, biotransformations of phine alkaloid structure which has been found alkaloids, unlike their steroid counterparts, to dramatically increase potency (JOHNSON have yet to meet their potential on an industri-

5 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 331 1 Introduction 331 al scale. This is in part due to the lack of suit- factors. Furthermore, it is now theoretically able enzymes and partly because no alkaloid- possible to manipulate alkaloid biosynthetic based drug commands a significant share of pathways to improve yields and to extend the therapeutic market, unlike the steroids. pathways to synthesize new bioactive mole- The rate at which new drugs derived from cules. Metabolic engineering of plants offers natural products are entering the therapeutic the capability of altering the pattern of alka- market has declined significantly over recent loid accumulation in the plant; in addition, the periods in contrast to synthetic molecules, pos- ability to house and express recombinant sibly due to the difficulty of modifying these genes in plants from other organisms offers often complex chemicals for the development the potential of both extending pathways and of new drugs and the difficulty of producing allowing the biological synthesis of semi- these natural products in a pure form. The dif- synthetic derivatives. It is now possible to de- ficulties associated with the development of sign strategies to alter the metabolic flux in a new drugs are being addressed by the ever in- variety of organisms, such as the introduction creasing interplay of chemistry and biology. of extra copies of genes encoding enzymes Undoubtedly combinatorial approaches which form bottlenecks in pathways affords a (AMATO, 1992) and genetic engineering will way to attain increases in yields of plant secon- play an important role in the development of dary products, or more globally through the new drugs. expression of one or more regulatory genes The use of recombinant DNA technology is (for reviews see BAILEY, 1991; NESSLER, 1994; beginning to have substantial impact on bio- HUTCHINSON, 1994; KUTCHAN, 1995). Due to transformation processes, resulting in the de- their complex structures, alkaloids are still velopment of new approaches using biological most efficiently produced by the plant and the systems. Recent advances in the understanding future success of metabolic engineering of of the genetic and biochemical basis of alka- plant secondary products is dependent on hav- loid biosynthetic pathways are now beginning ing a good understanding of the biochemistry to make biotransformations of complex alka- and regulation of the pathways under consid- loid molecules more plausible. The expression eration. of plant enzymes, which are often present at Plant cell culture has been invaluable as a very low levels in the plant, in heterologous means of providing suitable biomass for the hosts such as bacteria allows detailed examina- elucidation of pathways for secondary metabo- tion of mechanisms of reaction which are often lites, particularly for alkaloid synthesis. Cell unknown in synthetic organic chemistry. It is culture has also been examined for biotrans- possible to add to the genetic repertoire of a formation purposes (reviewed by VERPOORTE plant by incorporating genes from other spe- et al., 1993) and extensively investigated as a cies allowing the possibility of producing means of producing plant secondary products unique compounds with potential biotechno- on a large scale. Unfortunately, the level and logical applications. Microorganisms have manner of production of alkaloids in plants been used for the large-scale production of does not necessarily correlate with production high-value chemicals for many years, and the in cell cultures. The use of plant cell cultures use of microbial processes to make analogs of for biotransformations of alkaloids will not be naturally occurring alkaloids is achievable. It is considered in detail this chapter. The purpose now possible to assemble hybrid transforma- of this review is to introduce some of the tion pathways in microbes using structural newer technologies which are beginning to genes cloned from different organisms which make an impact on the area of alkaloid trans- mediate enzymic processes which are not in- formations. It also aims to introduce some of digenous to the host organism. These patch- the more recent developments in the biochem- work pathways can have the advantage of re- istry and genetic understanding of biosynthet- moving unwanted side reactions, they allow ic and catabolic routes of some of the more the possibility of increasing the activity of a pharmacologically important alkaloids in dif- cell by altering regulatory processes, and avoid ferent organisms, without which the rational the need to supply expensive exogeneous co- design of any recombinant alkaloid biotrans-

6 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 332 332 7 Alkaloids formation processes would not be feasible. The recent study suggested that the alkaloid has in- alkaloids discussed include the tropane alka- secticidal properties at naturally occurring loids, the benzylisoquinoline alkaloids, the concentrations due to potentiation of insect benzophenanthridine alkaloids, the morphinan octopaminergic neurotransmission (NATHAN- alkaloids, and the monoterpenoid indole alka- SON et al., 1993). loids. 2.1 Tropane Alkaloid Biosynthesis The biosynthetic pathways for the tropane 2 Tropane Alkaloids alkaloids have been studied in considerable detail and are associated with nicotine biosyn- The tropane alkaloids occur in the Solana- thesis, since the N-methyl-D1-pyrrolinium cat- ceae family, but they are also found in the ion (15) is a precursor to both classes of alka- plant families Erythroxylaceae, Convolvula- loids. The formation of the tropane nucleus ceae, Proteaceae, and Rhizophoraceae. Their from ornithine and acetoacetate was first in- common structural element is the azabicyc- vestigated in plants using radioactive tracers as lo[3.2.1]octane system, and over 150 tropane long ago as 1954, but it was not until the early alkaloids have been isolated. The 3-hydroxy 1980s that a biosynthetic scheme was finally aromatie ester derivatives form the parent al- elucidated in Erythroxylon coca (Fig. 3; LEETE, kaloids, examples of which include cocaine 1983). The pathway for biosynthesis of hyos- (12) (Erythroxylon coca, coca plant), hyoscy- cyamine (13) and scopolaminc (14) is quite amine (13), (Hyoscyamus niger, henbane), complex, since not only is an acetone unit re- atropine (13) (Atropa belladonna, deadly quired for the formation of tropinol (19), but a nightshade), and scopolamine (14) (Scopola second converging pathway is necessary for carniolica) (Fig. 2). It appears that in most the conversion of phenylalanine (20) to tropic cases atropine (13) is formed by racemization acid (23) (Fig. 4). Recent work with root cul- of hyoscyamine (13) during extraction. tures of Datura stramonium, suggests that hy- Long before the elucidation of their struc- grine (17) is not an intermediate, but an off tures, the pharmacological properties of sever- shoot from the main pathway (ROBINS et al., al tropane alkaloids were exploited. Atropine 1997). The biosynthesis of cocaine (12) is simi- (13), which typifies the action of tropane alka- lar to that of hyoscyamine (13). The N-methyl- loids, causes antagonism to muscarine recep- ation and cyclization of ornithine-derived pu- tors (parasympathetic inhibition) (CORDELL, trescine gives the N-methyl-D1-pyrrolinium 1981). cation (15), which condenses with acetoacetyl- These receptors are responsible for slowing CoA (16). Methylation of the free carboxylate of the heart rate, vasodilation, dilation of the group followed by ring closure, reduction of pupil, and stimulation of secretions. The heart the ketone group, and benzoylation results in rate altering properties of atropine (13) have the formation of cocaine (12). The benzoic ac- led to its use in the initial treatment of myocar- id is derived from phenylalanine (20). dial infraction. Tropane alkaloids have also The molecular biology of tropane alkaloid been used to treat peptic ulcers, prevent mo- synthesis is being studied extensively and tion sickness, and as components of pre-an- holds considerable potential for alkaloid bio- esthetic drugs. Cocaine (12) is perhaps the transformations. An elegant example is the best known of all the tropane alkaloids mainly construction of a transgenic species of Atropa because of its use as an illicit drug; it is a pow- belladonna that was able to accumulate the erful central nervous system stimulant and ad- important pharmaceutical scopolamine (14) renergic blocking agent, and its hydrochloride instead of hyoscyamine (13) (YUN et al., 1992). salt has been used as a local and surface anes- The final two steps in the pathway for the bio- thetic in face, eye, nose, and throat surgery synthesis of scopolamine (14) (Fig. 5) are cata- (GERALD, 1981). The function of cocaine (12) lyzed by 2-oxoglutarate-dependent hyoscy- in leaves of the coca plant was unknown until a amine 6b-hydroxylase (hyoscyamine[6b]-di-

7 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 333 2 Tropane Alkaloids 333 Fig. 4. Biosynthesis of tropic acid (ROBINS et al., 1994). showed that catalysis of these two steps car- ried out was by the same enzyme (HASHIMOTO et al., 1987). Analysis of key enzymes of meta- bolic pathways at the molecular genetic level assists clarification of complex biochemical mechanisms, and hydrolysis and epoxidation of the tropane ring was later confirmed un- equivocally by molecular cloning and expres- sion of the structural gene of the 6b-hydroxyl- Fig. 3. Biosynthesis of tropane alkaloids (ROBINS ase in a heterologous host (MATSUDA et al., et al., 1994). 1991; HASHIMOTO et al., 1993b); A. belladonna accumulates hyoscyamine (13) instead of sco- polamine (14) because it lacks the 6b-hydrox- ylase. The cDNA encoding the 6b-hydroxylase oxygenase; EC This enzyme first from H. niger was transferred into Agrobacte- hydroxylates hyoscyamine in the 6b-position rium tumefaciens and introduced into A. bella- of the tropane ring (24), which is followed by donna. The regenerated transgenic plants were epoxidation. The use of purified 6b-hydroxy- found to contain elevaled levels of scopol- lase from root cultures of Hyoscyamus niger amine (14) (YUN et al., 1992). The change in

8 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 334 334 7 Alkaloids Fig. 5. See text. alkaloid composition in the transgenic A. bel- eospecificities were found in cultured roots of ladonna was considerable, with scopolamine H. niger (HASHIMOTO et al., 1992). (14) being almost the only alkaloid present in These two distinct enzyme activities reduc- the aerial parts of the plant. It was thus pos- ed tropinone (18) to 3a-hydroxytropane (19) sible to isolate pure scopolamine (14) by re- (tropinol, tropine) and 3b-hydroxytropane crystallization of the total alkaloid fraction, in- (pseudotropinol, c-tropine, pseudotropine), stead of conventional differential extraction respectively. Marked differences were ob- and chromatography. Analysis of expression of served between the two reductases in their af- the 6b-hydroxylase gene by measurements of finities for tropinone (18), substrate specificity, levels of mRNA and Western blot analysis of and in the effects of amino acid modification protein extracts from various tissues showed reagents. The cDNA clones for the two tropi- that enzyme expression in scopolamine pro- none reductases have been expressed in Es- ducing species of Hyoscyamus was lacking in cherichia coli and sequenced (NAKAJIMA et al., the stem or leaves, being localized in the roots 1993). Preparation of various chimeric forms of these plants, and explains why it has not of these two enzymes led to the identification been possible to produce these alkaloids in sig- of the domain conferring the stereospecificity nificant quantities by cell culture (HASHIMOTO of the reaction (NAKAJIMA et al., 1994). et al., 1991; MATSUDA et al., 1991). HASHIMOTO These elegant experiments demonstrate a et al. (1993b) have now engineered transgenic key to future alkaloid biotransformation pro- A. belladonna hairy root cultures that express cesses by the manipulation of biosynthetic the H. niger gene encoding hyoscyamine 6b- routes in plants with the use of recombinant hydroxylase which exhibited up to 5 times DNA technology not just for alkaloids but also higher activity. These transgenic roots may other secondary products. It is becoming pos- prove to be useful for enhancing scopolamine sible to design strategies to advantageously productivity in vitro. Recombinant strains of manipulate the metabolic flux in organisms, or Escherichia coli expressing the gene encoding to decrease or increase the production of phy- hyoscyamine hydroxylase were also capable of tochemicals; however, with regard to biotrans- transforming hyoscyamine (13) to scopol- formations, it is the possibility of manipulating amine (14) (HASHIMOTO et al., 1993a; LAY et pathways by altering enzyme function by di- al., 1994). rected mutagenesis and extending/altering ex- Tropinone reductase acts at a branch point isting pathways by heterologous gene expres- of biosynthetic pathways leading to a variety sion to alter the spectrum of plant alkaloids of tropane alkaloids. It is an NADPH-de- which is particularly challenging and exciting. pendent enzyme which reduces the 3-keto Although the genetic tools for manipulating group of tropinone (18) (ROBINS et al., 1994). biosynthetic pathways in plants lag behind Two tropinone reductases with different ster- those for prokaryotic organisms, the success of

9 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 335 2 Tropane Alkaloids 335 scopolamine production in transgenic plants ring cleavage and the formation of tropinic ac- will, hopefully, encourage interest and further id (25), though no enzymes or cofactors were development. identified. Isolation of a picrate derivative of methylamine from whole cell incubations with tropinone, indicated that nitrogen debridging was taking place. More recent investigations 2.2 Microbial Metabolism of into the microbial metabolism of atropine Tropane Alkaloids showed that a strain of Pseudomonas sp. (termed AT3) isolated from the rhizosphere of Microorganisms possess an incredible varie- atropine plants was able to utilize tropinol (19) ty of metabolic pathways which enables them as a sole carbon and nitrogen source (LONG et to degrade a plethora of natural and man- al., 1993). Growth studies revealed a diauxic made organic compounds. The elucidation of growth pattern. When this organism was sup- alkaloid dissimulating pathways has consider- plied with atropine (13) and an exogenous ni- able potential for the identification of bio- trogen source, tropic acid (23) was utilized transformation routes for new and existing during the first phase of growth and the heter- therapeutic compounds (BRUCE et al., 1995). ocyclic moiety, tropinol (19), was utilized in the The most extensively studied tropane alka- second. The enzymes responsible for tropinol loid, in terms of microbial metabolism is atro- (19) degradation appeared to be strongly re- pine (13). Several bacterial species have been pressed during the first phase of growth. shown to possess an esterase that catalyzes the Under nitrogen limitation, however, the nitro- esterolytic hydrolysis of the atropine molecule, gen must be stripped from the tropane ring be- to form tropinol (19) and tropic acid (23) (Fig. fore growth can occur and under these condi- 6). tions tropinol (19) was utilized in the first The interest in atropine esterase lies in its growth phase. Pseudomonas sp. AT3 initiated similarity to mammalian serine proteases and the degradation of tropinol (19) by attacking its use as a possible model of mammalian chol- the nitrogen atom, yielding a dinitrophenyl hy- inergic receptors. RRSCH et al. (1971) re- drazine positive intermediate, identified as 6- ported the isolation of a number of Pseudo- hydroxycyclohepta-1,4-dione (28), which was monas strains capable of utilizing atropine as a oxidized by an NADc-dependent dehydrog- sole source of carbon and nitrogen. The atro- enase activity to cyclohepta-1,3,5-trione (29). pine esterase from one of these strains, Pseu- The subsequent cleavage of this compound re- domonas putida PMBL-1, has been purified sulted in the formation of 4,6-dioxoheptanoic and extensively characterized (VAN DER acid (succinylacetone) (30) which was, in turn, DRIFT, 1983; VAN DER DRIFT et al., 1985a, b, the substrate for a second hydrolase yielding 1987). This esterase showed activity with both succinate (31) and acetone(32) (BARTHOLO- enantiomers of hyoscyamine (13), but not with MEW et al., 1993, 1996). cocaine (12) (RRSCH et al., 1971), despite the BARTHOLOMEW et al. (1995) identified an close similarity of structure of those com- NADPc-dependent tropinol dehydrogenase pounds. NIEMER et al. (1959) and NIEMER and in cell-free extracts of Pseudomonas sp. AT3 BUCHERER (1961) reported a breakdown that was induced by growth on atropine (13), route of atropine (13) by Corynebacterium bel- tropinol (19) or tropinone (18). The product of ladonnae, which involves the formation of the reaction was tropinone (18) and the reac- tropinol (19) and tropic acid (23) by esterase tion was shown to be freely reversible. The de- action, followed by dehydrogenation, ring hydrogenase showed activity only with tropi- opening, and deamination of the tropane ring nol (19) and nortropinol (27); no activity was (Fig. 6). The first step in their proposed route detected with a number of closely related com- of tropine catabolism involves a tropine dehy- pounds including atropine (13), scopine, pseu- drogenase. Activity, however, was only demon- dotropinol, ecgonine (34) (Fig. 7) and 6-hy- strated in the reverse direction. The step pos- droxycyclohepta-1,4-dione (28), which sug- tulated by NIEMER and BUCHERER (1961) gests thal this inducible enzyme is involved in which follows tropinone formation involves the metabolism of tropinol (19) in Pseudomo-

10 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 336 336 7 Alkaloids Fig. 6. Proposed pathway for the degradation of atropine (13) by Pseudomonas sp. AT3 (BARTHOLOMEW et al., 1996). ----P Pathway proposed by NIEMER and BUCHERER (1959) for C. belladonna.

11 12299 - 07 - 325361 27.08.1999 08:10 Uhr Seite 337 2 Tropane Alkaloids 337 nas sp. AT3. However, the occurrence of 6-hy- droxycyclohepta-1,4-dione (28) during the me- tabolism of tropinol seemed to dispute this (BARTHOLOMEW et al., 1993). An elegant set of experiments with tropinol (19) and pseudo- tropinol labeled with deuterium in the C-3 po- sition provided an answer (BARTHOLOMEW et al., 1995).The labeled alcohol group at C-3 was shown to remain intact past the point of re- moval which indicated that tropinone (18) is not an intermediate in the pathway of tropinol (19) metabolism. What then is the role of trop- inol dehydrogenase in the metabolism of trop- inol? Tropinone (18) serves as a growth sub- strate for Pseudomonas sp. AT3 and it is likely to be encountered in nature, along with atro- pine (13) and tropinol (19), as it is an interme- diate in the biosynthesis of the tropane alka- loids in plants (LANDGREBE and LEETE, 1990). A mutant strain of Pseudomonas sp. AT3 blocked in 6-hydroxycyclohepta-1,4-dione de- hydrogenase activity was grown on tropinone; this resulted in the accumulation of 6-hydroxy- cyclohepta-1,4-dione (28), an indication that tropinone (18) is metabolized via the same route as tropinol (19) and that its keto group is reduced in the process. Thus, the tropinol de- hydrogenase may function primarily as a re- ductase in order to channel tropinone (18) and nortropinone into the pathway of tropinol (19) metabolism in Pseudomonas sp. AT3. The elucidation of the pathway for microbi- al metabolism of the related alkaloid cocaine (12) has proven to be slightly more elusive. A strain of Pseudomonas maltophilia (termed MB11L) was isolated from samples taken in and around a pharmaceutical company that processes cocaine. P. maltophilia MB11L was capable of utilizing cocaine (12) as its sole source of nitrogen and carbon for growth. The bacterium possessed an inducible cocaine esterase which converted cocaine (12) to ecgo- nine methyl ester (33) (Fig. 7), and benzoic ac- id. Both degradation products supported growth of P. maltophilia MB11L, although on- ly cocaine induced high activities of the co- caine esterase (BRITT et al., 1992). Benzoic ac- id was further metabolized via catechol and the 3-oxoadipate pathway; however, the path- way for the metabolism of ecgonine methyl es- Fig. 7. Proposed pathway for the metabolism of co- ter (33) was not further elucidated. caine (12) by P. fluorescens MBER and C. acidovor- ans MBLF (LISTER et al., 1995).

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