Biotransformations for Fine Chemical Production

100 YEARS OF PROGRESS WITH LONZA 287 CHIMIA 51 (19Q7) Nr. 6 (Junil Chimia 51 (1997) 287-289 number of weaknesses, especially when it © Neue Schweizerische Chemische Gese/lschaft comes to functional group transformation ISSN 0009-4293 in multifunctional molecules, functionalisation of nonactivated carbon atoms, and construction of complex, high-molecular- Biotransformations for Fine weight molecules with multiple stereo- genic centres. Fortunately, biotechnology Chemical Production can complement organic chemistry to over- come some of these weaknesses. Biotech- Hans-Peter Meyer*, Andreas Kiener, Rene Imwinkelried, and Nicholas Shaw nological manufacturing processes are especially useful for larger molecules with a high degree of functionalisation, and the Abstract. Biotechnology has become an indispensable tool for the production of fine development of powerful new biocata- chemicals. The choice of route, chemical or biotechnological, for the manufacture of a lysts is resulting in important progress in given fine chemical is crucial. In general terms, biotechnology is the method of choice the field of chemo-, regio-, and stereose- for large molecules with a high degree of functionalisation and multiple stereocentres. lective transformation. The production of Most of LONZA' s biotechnological bioprocesses for the production of fine chemicals enantiomerically pure compounds bear- are whole cell processes using microorganisms which form very specific enzymes. ing chiral hydroxy, carboxy, amino, and Process improvement at LONZA is discussed in this paper on three levels: upstream sulfoxide groups are of particular com- processing, biotransformation/biosynthesis, and downsteam processing. mercial importance (Table 1),and research for biocatalysts for this area will retain a high priority. However, the potential of Introduction tion of Celltech Biologics (now LONZA enzymes for biotransformations has not Biologics), extending LONZA's expertise yet been fully exploited. According to The pharmaceutical industry is changing to the production of monoclonal antibod- Faber [6], of the 2500 known enzymes, for a number of reasons, one of which is ies and therapeutic proteins. 250 are used commercially in more or less the emergence of new drugs produced by purified forms, and merely 25 account for biotechnological means. The growth rate over 80% of all commercial applications for these biopharmaceuticals, such as ther- Biotechnology and Chemistry are (ca. USD 500 Mio. in 1990) mainly for apeutic proteins, peptides, antisense drugs, Complementary Technologies food and starch processing and as deter- and monoclonal antibodies is higher than gent additives. Forbiotransformations, the forclassical, chem ically synthesised drugs. The choice of route for the manufac- hydrolytic enzymes such as lipases and However, this trend could be reversed and ture of a given fine chemical is crucial. esterases have been used, because they are chemically synthesised drugs will defi- Each target molecule must undergo care- available in commercial quantities, often nitely continue to play an important role. ful retro-synthetic analysis, and in many have broad substrate spectra, and are usu- Fifteen of the top-selling 25 drugs in cases, the result is that a combination of ally simple, robust, have no cofactor re- 1994 were single isomers, and the trend chemistry and biotechnology will result in quirements and are therefore easy to use towards chiral pharmaceuticals and agro- the most efficient process. Often, a final (Fig. 1). The future use of other enzyme chemicals is increasing. The total market decision can be made only on the basis of types, e.g. lyases and reductases, will be for chiral synthons is estimated to be USD comparative laboratory experiments. driven by commercial requirements and 250-300 Mio. and is expected to grow to Synthetic organic chemistry offers a will depend on advances in such ru'eas as USD 1BiI. by the year 2000. Biocatalysis number of advantages including consider- cofactor recycling and protein engineer- will play an increasingly important role in able experience at both laboratory and ing. this area, because enzymes are selective industrial scales, predictability and relia- Even though the selectivity of enzyme- and chiral, and a synthetic route that incor- bility, alternative reagents and reactions catalysed reactions often exceeds those of porates one or more biocatalytic steps is for given transformations, rapid construc- chemical reactions, it does not necessarily often more efficient than a totally chemi- tion of carbon skeletons, and insertion of mean that the biological approach is more cal route. basic functional groups. Progress can also efficient. Other criteria, such as the avail- LONZA has a long tradition of manu- be expected in asymmetric synthesis, es- ability of starting materials and product facturing fine chemicals for the life sci- pecially in the field of transition-metal recovery, can be critical. For a company to ence industries by chemical synthesis. catalysis, which has not been fully exploit- be successful in the fine chemicals busi- Since starting a small biotechnology re- ed. However, organic synthesis also has a ness, expertise in both organic synthesis search group 12 years ago, our company has successfully developed a number of Table I. The Occurrence of Functional Groups at Chiral Centres in Pharmaceuticals [5 J processes incorporating one or more biocatalytic steps for the manufacture of fine lin tioonl group O~ unen c (a. ~ of hiral (cntn:~) chemicals [1-4]. This innovative approach Hydr H has been furthered by the recent acquisi- - 40 arb xy --COOl I 2 mine - H~ 16 *Corre.\pondence: Dr. H.-P. Meyer ........ Department of Biotechnology ulfo. ide ./ =0 3 LONZA AG CH-3930 Visp .her 19 100 YEARS OF PROGRESS WITH LONZA 288 CHIMIA 5/ (1997) Nr. 6 (Juni) and biotechnology is essential. LONZA is for biosynthesis and biotransformation are Numberof publications proud to be in this position. produced by fermentation. Fermentation 800 Care of the environment is also impor- and biotransformation can take place at tant, and in some cases bioprocesses pro- the same time (precursor fermentation), as 600 duce less waste than chemical routes. A e.g. with 5-methylpyrazine-2-carboxylic comparison of the waste stream from the acid (MPCA), or separately, as with 6- LONZA L-carnitine bioprocess [7] with hydroxynicotinic acid (Table 2). 400 that from the LONZA chemical synthesis The only industrial scale biotransfor- [8] is shown in Fig. 2. mation that LONZA carries out with a 200 commercially available enzyme is the production of the chiral intermediate (R)- a Whole Cell Processes glycidyl butyrate by resolution of the ra- 55 60 65 70 75 80 85 90 95 00 cemic compound with porcine pancreas Year Biosynthesis is the production of a lipase (Table 2). The reason that commer- substance de novo by living cells, and the cial enzymes are not used more often is production of a secondary metabolite that they are not always suitable for a Fig. I. Number of publications per year in the specific biotransformation. Consequent- area ofbiotransformations. Thegraphshowsthat would be a typical example. Biotransfor- the number of publications has reached a steady mation is the conversion of one substance ly, microbial strains with the desired activ- state. Many papers on hydrolyticenzymes, espe- into another, catalysed either by whole ity must be found, either by screening or cially proteases, esterases, and lipases were pub- cells or by isolated enzymes. The starting selection. Most of the bioprocesses devel- lished. material can be synthetic or natural. Cells oped by LONZA for secondary metabo- lites, vitamins, steroids, and intermediates therefore rely on whole cells and make use Table 2. Commercial Biotransformations Developed at LONZA Using Various Reaction Types and Biocatalysts of their high selectivity and productivity. E.g., the regioselective functionalisation of nicotinic acid by purely chemical meth- BiOlr:.mI"onnation nz)me class BlOcatulyst ods is difficult to control, and therefore the hydroxylation of nicotinic acid to 6-hy- Whole Cell droxynicotinic acid by Achromobacter I HO 0 xyloxidans opens up a novel route for the dehydrogenase Agrobacterlum -N I~ -~-N~ production of substituted nicotinic-acid "" 0 hydratase HK1349 "" 0 derivatives. The biotransformation of 3- Butyrobetalne L-Carnitine cyanopyridine illustrates the use of an 0 0 immobilised biocatalyst by LONZA for dOH dOH 'hydroxylase' Archromobacler the continuous multistage production of xyloxldans 3000 t of nicotinamide per year. N HO N NlcoUnicacid 6-Hydroxynlcolinlcaeld 0 Strain Selection --~ monooxygenase Pseudomonas )::(OH 2 dehydrogenases pulido A strain suitable for a specific biotrans- )::( formation may be found by screening, 2,5-Dlmethylpyrazine 5--Methylpyrazine- selection, and classical mutagenesis, and (DMPY) 2-carboxylicacid then genetic engineering techniques can (MPCA) A)nltrile- A)Rhodococcus be used to overexpress a single enzyme or B hydratase equi a whole pathway [9]. The strategy de- >- XCNA NH~ B)amdase B) Escherichia pends on the complexity and economics of ~N~ coIl DHS/pCAR6 the process, the time available, and the 0 (R,S)-Drv1CP-Nllrlle (S) OMCP-CarboxalTid market volume of the final product. A first generation process will often be put into Immobllised Biocatalyst place, followed by a second generation 0 process based on an improved biocatalyst. CN Rhodococcus E.g., (+)-(S)-2,2-dimethylcyclopropane- (fN~ nllrile~ rllodochrous carboxamide

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