Synthesis and Metabolism of Drugs by Means of Enzyme-Catalysed Reactions

ADVANCED BIOTECHNOLOGY 536 CHI MIA 1999, 53, No. 11

Chimia 53 (1999) 536-540 © Neue Schweizerische Chemische Gesellschaft ISSN 0009-4293

Hans Georg W. Leuenberger*), Peter K. Matzinger, and Beat Wirz

Abstract. The usefulness of enzyme catalysed-reactions is exemplified by recent results from research at Roche. Sequences of enzyme reactions, well organised in metabolic pathways of selected microorganisms, lead to secondary metabolites with innovative chemical structures. An example is the pancreas-lipase inhibitor lipstatin produced by Streptomyces toxytricini. Hydrogenation of lipstatin yields tetrahydrolipstatin, the active substance of the anti-obesity drug Xenical™. The biosynthetic pathway has been elucidated and an improved fermentation process for the production of lipstatin has been developed. Intermediates of the primary metabolism can be valuable building blocks for the chemical synthesis of drugs. Examples are quinic acid and shikimic acid, which are both suitable starting materials for the synthesis of the neuraminidase inhibitor GS 4104. Metabolic engineering of Escherichia coli with the goal to overproduce these two substances is briefly described Microorganisms or enzymes derived thereof are used in drug synthesis to catalyse single, highly specific reaction steps (biotransformations). Three examples yielding chiral precursors of a protein-kinase inhibitor, a collagenase inhibitor, and an antifungal compound are discussed Recombinant Escherichia coli strains expressing human drug-metabolising enzymes are suited to mimic drug metabolism and to produce intermediates of human drug metabolism. The desired hydroxylated drug derivatives could be obtained after incubation of drug substances with strains coexpressing one specific human cytochrome P450 isozyme together with human reductase.

1. Introduction catalyse single, highly specific reac- the anti-obesity drugXenical™. Tetrahy- tion steps (biotransformations) diffi- drolipstatin can be obtained by chemical Microorganisms with their great variety cult to perform by chemical methods. synthesis [3] as well as by fermentative of highly specific enzymes have proven to - Recombinant microorganisms express- production of lips tat in followed by hydro- be useful catalysts in medicinal chemis- ing human drug-metabolising enzymes genation. However, original fermentation try: are used to mimic drug metabolism yields of lipstatin amounted only to a few - Sequences of enzyme reactions, well and to produce intermediates of human milligrams per litre. organised in metabolic pathways, lead liver drug metabolism. The biosynthesis of lipstatin was in- to a variety of secondary metabolites Recent applications from the pharmaceu- vestigated in Streptomyces toxytricini by with innovative chemical structures. tical research at Hoffmann-La Roche will trace studies with crude (U_13C)-lipid mix- Many such secondary metabolites ex- be discussed. tures obtained from algal biomass cul-

hibit pharmaceutical activities, e.g. as tured with 13C02. The data showed that antibiotics or enzyme inhibitors. the carbon backbone oflipstatin is built up - Intermediates of the primary metabo- 2. Lipstatin, the Precursor of by Claisen condensation of a C14- and an lism can be valuable building blocks XenicafTM Cg moiety before the L-leucine residue is for the chemical synthesis of drugs. added [4]. Their overproduction in suitable mi- Lipstatin is a potent, irreversible inhib- The fermentation process for lipstatin croorganisms can be initiated by ge- itor of pancreas lipase, the key enzyme in could be significantly improved by estab- netic engineering. dietary fat absorption. It has been detected lishing a fed-batch process with feeding of - Microorganisms or enzymes derived in a screening for lipase inhibition in broths linoleic acid (which is readily degraded by thereof are used in drug synthesis to from soil microorganisms. Lipstatin is pro- twofold {J-oxidation into the required Cl4 duced by Streptomyces toxytricini [1][2]. unit), caprylic acid (C8 precursor) and L- The molecule consists of a hydrocarbon leucine [5]. Feeding of these precursors to 'Correspondence: Dr. H.G.W. Leuenberger backbone bearing two double bonds, a {3- a typical run with 81 of starting volume in F. Hoffmann-La Roche Ltd. Department PRPB lactone structure essential for the biologi- a 141 reactor was initiated after a growth p.e.Box cal activity and a hydroxy group substitut- phase of 47 h. In order to avoid inhibitory CH-4070 Basel ed with N-formyl-L-leucine (Fig.). effects, fatty acids were added in a way Tel.: into +41 61 6884561 Fax: int +41 61 6881673 Hydrogenation of lipstatin yields tet- that the concentration of each remained E-Mail: [email protected] rahydrolipstatin, the active substance of below 70 mg/I. Lipstatin concentration in ADVANCED BIOTECHNOLOGY 537

CHI MIA 1999, 53. No. 11 such a fed-batch experiment was 150 mg/ Iafter an incubation period of 138 hours [5]. Further strain improvement and process optimisation is in progress. ••.. NHCHO

3. Quinic Acid and Shikimic Acid as Starting Materials for the Synthesis of the Neuraminidase Inhibitor GS 4104

Neuraminidase is an enzyme essential for the propagation of the influenza virus. The sialic-acid analogue substance GS 4104 (Ro 64-0796; Tamiflu™) is a potent inhibitor of neuraminidase and therefore a ••--NHCHO promising drug substance for the preven- tion and treatment of influenza [6]. It is o under advanced development jointly by 9 Hoffmann-La Roche Ltd. and Gilead Sci- ences Inc. .J~ Starting materials for the synthesis of GS 4104 [6-8] are either quinic acid (QA) Figure. Structures of the pancreas-lipase inhibitors lipstatin (a fermentation product from Strep- or shikimic acid (SA) (Scheme 1) which tomyces toxytricim) and tetrahydrolipstatin (XenicaI™) are both available by extraction from plant sources. Scheme 1. (-)-Quinic Acid (QA) or (-)-Shikimic Acid (SA) as Precursors of the Neuraminidase QA and SA are both intermediates in Inhibitor GS-4104 (for details, see [6-8]) the common biosynthetic pathway lead- ing to aromatic amino acids (Scheme 2). Therefore, it was obvious to consider mi- ,')(,ooH crobial fermentation as a production meth- QA od. H(t'~OH Recombinant Escherichia coli strains • which overproduce QA or SA have been 5H eOOEI engineered by streamlining carbon flow into the respective metabolic pathway, by repeated cloning of the genes encoding for -- ~","H2H3P04 rate-limiting key enzymes as well as by eOOH knocking out the genes encoding the two ) ~HAC isozymes of shikimate kinase [9]. The latter step prevents further processing of GS-41 04-02 the metabolic intermediate SA (or its pre- SA Ro 64-0796/002 cursor QA) to shikimk acid-3-phosphate H~~ and further towards aromatic amino acids OH (Scheme 2). The overproduced intermediates, QA or SA, are excreted and the strains become auxotrophic for aromatic of a precursor of protein-kinase inhibitor brevis, yielding the isolated product in amino acids. Resulting strains produced in Ro 43-2759. The synthetic pathway (de- 82% from regioisomerically pure substrate a fed-batch process 60 gIl ofQA within 60 signed by Ulrich Zutter, Roche, Basel la. h (side product: 3-dehydro-QA) or 27.2 gI [10]) is based on asymmetric microbial For economic reasons, the attempt was I of SA in 42 h (side products: QA and 3- reduction of the fl-keto ester la to the made to use directly the unpurified keto dehydro-SA) [9]. Further strain and proc- respective cis-(3R,4S)-configured hydroxy ester substrate containing la and about ess development is in progress. ester 2 (Scheme 3). The theoretical yield is 25% of the unwanted regioisomer lb. If 100% because of in situ racemisation of the microorganism is able to reduce selec- substrate la. tively the desired regioisomer la, the re- 4. Biotransformations A microbial screening with various maining regioisomer lb can easily be re- yeasts and fungi was carried out. Most of moved by decarboxylation under alkaline 4.1. Microbial Hydrogenation of the strains tested afforded preferentially conditions and subsequent extraction of Racemic p-Keto Ester 1a in the the cis-configured product. The best five the reaction product. Kloeckera brevis Synthesis of the Protein-Kinase strains generated both centres of asymme- conveniently did exhibit this additional Inhibitor Ro 43-2759 try with excellent specificity. The enanti- regioselectivity: only the desired isomer A microbial hydrogenation with a use- omeric excess as well as the cis/trans- la was reduced and afforded the cis-hy- ful combination of regio- and enantiose- purity was 97% or better. The best result droxy ester in excellent steric purity. In a lectivity was employed for the synthesis was obtained with the yeast Kloeckera typical fermentation run, 29 g of 1 (3.5 g/ ADVANCED BIOTECHNOLOGY 538

CHIMIA 1999, 53, No. 11

Scheme 2. Metabolic Pathway Towards Aromatic Amino Acids. DAHP: 3-Deoxy-o-arabino- I) were fed to a pre-grown stirred culture of heptulosonic acid-7-phosphate; DHQ: 3-Dehydro-quinic acid; DHS: 3-Dehydro-shikimic acid; Kloeckera brevis supplemented with 5% S3P: Shikimic acid-3-phosphate glucose. After] 2 h of incubation at 27 ec, the culture broth was extracted with ethyl OOH0,\ XOOH H acetate. The yield of isolated product 2 /HXO was 80% (98% ee and 99% cis-purity). Thus, Kloeckera brevis exhibited excel- . H ~H HO·l.AH lent regio- and stereoselectivity in this H203P;+6H 6H 6H hydrogenation reaction and afforded the DAHP DHO OA desired product 2 in high chemical yield t and steric purity. t 4.2. Enzymatic Resolution of t Racemic Ester 3 Yielding a Chiral glucose Precursor of the Collagenase XH Inhibitor Ro 32-3555 Collagenase inhibitor Ro 32-3555 had to be prepared in larger amounts as a ~H candidate for clinical trials. Among sever- 6H al synthetic approaches, the most advanced DHS at that time proceeded via the benzyI- protected (R)-cyclopentyllactate (R)-3. En- zymatic kinetic resolution of racemic es- ]' ter 3 with preferential hydrolysis of the (S)-ester was investigated as an approach to produce (R)-3 (Scheme 4). In a preliminary enzyme screening with XH cheap bulk enzymes, only subtilisin aromatic Carlsberg showed reasonable enantiose- amino acids --- lectivity (91 % ee). The reproducibility of H,o,Pif~H the reaction was, however, unsatisfactory 6H and did not improve upon altering several S3P chemical and physical parameters. Only the introduction of tert-butyl methyl ether as a biphasic co-solvent led to improved Scheme 3. Microbial Hydrogenation of {3-Keto Ester 1a by Kloeckera brevis. The chiral product reproducibility as well as enantioselectiv- 2 serves as a building block for the synthesis of the protein-kinase inhibitor Ro 43-2759. ity (-96% ee at53% conv.). Most interest- ingly, at higher substrate concentrations (20%), the enantioselectivity further in- creased, and the co-sol vent could be omit- ted. The substrate was emulsified in aque- Kloeckera brevis ous buffer by vigorous stirring and the liquid commercial enzyme Protease L660 was applied in 10% with respect to the substrate. The cheap bulk enzyme was 18 2 discarded after use. Product (R)-3 was obtained in 44% yield (>98% ee at 53% conv.) after extraction and subsequent dis- EtOO tillation to remove formed benzylic alco- hol. The reaction was - with some modi- K fications - successfully carried out also on the multi-kg scale, and a total of more than ~JBOC 100 kg of (R)-3 have been produced. HO~ 1b 4.3. Enzymatic Resolution of Racemic Ester 5 Yielding a Chiral ~ HN, Precursor of the Antifungal HHC1 Compound Ro 09-3355 (R)-Configured 2,4-difluorophenyl a- hydroxyethyl ketone 6 is a key intermedi- d-O ate in a synthetic pathway (designed by HO Ao 43-2759 Milan Soukup, Roche, Basel) of the sys- temic antifungal Ro 09-3355. Two ap- ADVANCED BIOTECHNOLOGY 539 CHI MIA 1999, 53, No, 11 proaches were examined for its prepara- Scheme 4. Enzymatic Resolution of Racemic 3 with Protease L660 Yields Chiral (R)-3, a Building tion (Scheme 5): enantioselective acyla- Block of the Collagenase Inhibitor Ro 32-3565 tion of racemic 2,4-difluorophenyl a-hydroxyethyl ketone and enantioselective hydrolysis of its acylate. The first approach N s;:: led to the (R)-acetate in excellent enantio- Protease L660 R~O + meric excess, however, since subsequent HO I HOJyO hydrolysis led to isomerisation, we 0"-0 OH switched to the alternative hydrolysis approach affording 6 directly (Scheme 5). (R)-3 4 As hydrolysis experiments inmonopha- sic organic systems (as well as alcoholysis experiments in various organic solvents) gave only poor results, biphasic hydrolysis was chosen. Butyrate 5 was selected as the substrate due to its superior discrimi- nation (98% ee for 6) as compared to the corresponding acetate (70% ee). In addi- tion, the butyrate strongly facilitated work- upby allowing product separation by means of distillation. Hydrolysis with Candida antarctica lipase solution (Chirazyme L2, ]5% with respect to the substrate) in a biphasic substrate/buffer system proceed- Ro 32-3555 ed smoothly. At pH 8.5, a substrate concentration around 20% could be employed. The procedure was successfully carried Scheme 5. Enzymatic Resolution of Racemate 5 by Chirazyme L2 Yielding Optically Active 6, a out on the multi-kg scale: 5 was converted Chiral Precursor of the Antifungal Compound Ro 09-3355 to give 6 in 42-44% yield after extraction and distillation. The procedure yielded 46 kg of enantiomerically highly pure product 6. Chirazyme L2 +

5. Recombinant Escherichia coli strains Expressing Human Cytochrome P450 Isozymes as 5 6 (R)-5 Biocatalysts for the Preparation of Drug Metabolites

Drug metabolism in the liver is mainly dependent on cytochrome P450 monoox- ygenases. Thus, human liver microsome preparations are widely used to establish metabolic routes of drug candidates. Larger amounts of drug metabolites are often required in order to assess their chemical structure and to evaluate activity/toxicity patterns or possible interfer- Ro 09-3355 eN ences with other drugs. In many cases, chemical syntheses of such drug metabolites are difficult and time consuming, and, from the University of Dundee, Scotland. tions have been evaluated for their biocat- therefore, a biotransformation approach is Fermentations with these strains have been alytic potential and successfully used for highly desirable. However, the availabili- carried out on the 10-1 and 100-1 scale. the production of required human drug ty of human liver preparations with cyto- They yield between 3 and 18 gil of wet metabolites on the 10-100-mg scale. If chrome P4S0 activities is very limited. cells. The amount ofexpressed cytochrome necessary, a scaling up is possible. This dilemma has been overcome by using and reductase varies greatly within the In several examples with model com- recombinant microorganisms expressing diverse constructs: 30-1000 nmol for cy- pounds (midazolam, tacrine, diclofenac, human cytochrome P450 isozymes tochromes and 2-750 nmol for the reduct- dextromethorphane) and with drug candi- [11][12]. ase. dates actually under development at Ro- A series of recombinant E. coli strains Intact biomass (growing or resting cells che, the desired drug derivatives (e.g., - each of them co-expressing one specific in the broth or isolated wet cells), sphero- hydroxylation products) could readily be human cytochrome P450 isozyme as well plasts after lysozyme treatment, as well as obtained in reasonable yield after biotrans- as human reductase - has been obtained partly or totally disrupted cell prepara- formation with wet cells from recombinant ADVANCED BIOTECHNOLOGY 540

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Escherichia coli expressing the appropri- [4] W. Eisenreich, E. Kupfer, W. Weber, A. ez,1. Bruhin, H. Estermann, C.Gandert, V. ate cytochrome P450 isozyme. The results Bacher, J. Biol, Chern. 1997,272, 867. Gockel, S. Gotzo, U. Hoffmann, G. Huber, show the usefulness of this technology for [5] A. Bacher, P. Stohler, W. Weber, Europ. G. Janatsch, S. Lauper, O. Rockel-Stabler, Patentanrneldung 1997, 803 567. R. Tussardi, A.G. Zwahlen, Org. Process the production of human drug metabo- [6] C.U. Kim, W. Lew, M.A. Williams, H. Liu, Research & Development 1999, 3, 266. lites. L. Zhang, S. Swaminathan, N. Bischof- [9] K.M. Drahts, D.R. Knop,1.W. Frost,J. Am. Received: September II, 1999 berger, M.S. Chen, D.B. Mendel, c.y. Tai, Chem.Soc. 1999,121,1603. W.G. Laver, R.C. Stevens, J. Am. Chern. [] 0] U. Zutter, P. Matzinger, M. Scalone, Euop. [1] E.K. Weibel, P. Hadvary, E. Hochuli, E. Soc. 1997,119,681. Pat. Appl. 1996, No. 96 105998.7. Kupfer, H. Lengsfeld, 1. Antibiotics 1987, [7] J.c. Rohloff, K.M. Kent,M.J. Postich,M.W. [11] G. Urban, R. Gasser, M. Pritchard, M. 40, 1081. Becker, H.H. Chapman, D.E. Kelly, W. Bandera, T. Friedberg, Exp. Toxic. Pat/JOI. [2] E. Hochuli, E. Kupfer, R. Maurer, W. Lew, M.S. Louie, L.R. McGee, E.J. Prisbe, 1996,48, Suppl. II, 409. Meister, Y. Mercadal, K. Schmidt, J. Anti- L.M. Schultze, R.H. Yu, L. Zhang, J. Org. [12] J.A.R. Blake,M. Pritchard,S.Ding,G.C.M. biotics 1987, 40, 1086. Chern. 1998,63,4545. Smith, B. Burchell, C.R. Wolf, T. Fried- [3] P. Barbier, F. Schneider, Helv. Chirn. Acta [8] M. Federspiel, R. Fischer, M. Hennig, H.J. berg, FEBS Lett. 1999, 397, 210. 1987, 70, 196. Mair, T. Oberhauser, G. Rimmler, T. Albi-

Sperm Cells as DNA Vectors for the Preparation of Transgenic Animals

Ji'ff Janak

Abstract. Sperm-mediated DNA transfer strategy which has been used for the preparation of genetically modified, transgenic organisms is discussed, and sperm-mediated transgenesis of Xenopus laevis frogs with retroviral Rous sarcoma virus DNA is summarized.

1. Introduction were much less frequently reported in the Furthermore, it is difficult to ensure inte- past, and mechanisms of the development gration of the DNA into the host DNA in The process of transformation, both natu- of natura] eukaryotic competence have all cells of the transgenic organism oronly ral and artificial, continues to play an been only rarely studied and are poorly in cells of some 'target' tissues. Finally, important role in molecular biology. It is understood until now. Nevertheless, trans- the controlled expression of exogenous generally accepted that natural genetic fection of eukaryotic cells via an artificial- DNA in the whole organism or only in competence, the ability of cells to bind to ly induced competence (e.g., by chemical target tissues is the last essential step. and to take up exogenous DNA, is wide- or electrical treatment) is a widely used DNA Microinjection into fertilized egg spread among bacteria and might be an experimental procedure in laboratories of cells has been so far the most popular and important mechanism for the horizontal molecular and cellular biology. widely used method for the production of transfer of genes. In recent years, an excit- Transgenic animal and plant technolo- transgenic animals. However, it requires ing progress has been made in characteriz- gies represent some of the most powerful costly and sophisticated equipment and ing the pathways and signals that regulate. tools in functional studies of various genes considerable skills in micromanipulation, the development of bacterial competence and serve for the preparation of organisms while the efficiency of the method is rather as an active process [I]. Observations that with new properties. Furthermore, because low. It does not exceed more than 4-5% in DNA, RNA, and oligonucleotides may of many potential benefits, the research mice, the most successfully used organ- also naturally associate with the surface of into gene transfer has become a very rap- isms, and it is much lower with other eukaryotic cells and become internalized idly developing field with a special focus species such as livestock or marine ani- on 1) human gene therapy protocols and 2) ma]s. Moreover, microinjection is always production of biologically important mac- a non-physiological process, which may COffespondence: Dr. J.Jonak Institute of Molecular Genetics, Academy of Sciences romolecules by transgenic animals. There damage the cell. On the other hand, if the of the Czech Republic are still many problems in preparing a DNA uptake by spermatozoa developed Department of Protein Biosynthesis Flemingovo nam. 2 'perfect' trans geni c organ ism, and today' s into a well-controllable and defined proc- 16637 Praha 6 transgenic technology is far from being ess, these cells might become the most Czech Republic satisfactory. One of the crucial steps is to potent and, at the same time, the most Tel.: +420 2 20183273 Fax: +420224310955 find a simple way to efficiently introduce natura] too] forthe production oftransgen- E-Mail: [email protected] exogenous DNA into one-cell embryos. ic animals. Compared to microinjection, a