BIOTECHNOLOGY FOR INDUSTRIAL PRODUCTION OF FINE CHEMICALS 431

C'HIMIA 50(1996) Nr. 9 (SCpIC11l~CI') nates of the native wild-type apoenzyme cal applications from the development of menea, S.D. Varfolomeyev, l.R. Wild, Bi- (fromH. Holden, U. Wisconsin) and mod- transgenic soil fungi and plants to whole osensors and Bioelectronics] 996, in press. eling of important features at the active cell hydrolysis in slurry-bed bioreacter [4] K. Lai, N.J. Stolowich, Arch. Biochem. Biophys. 1995,318,59. site suggest critical attributes that can be systems to air stream purging and devel- [5] F. Yang, J.R. Wild, A.L. Russell, Biotecll- further modified to continue the rational opment of neurotoxin-specific bioreac- nol. Prog. 1995, 11, 71. design of this enzyme for bioremediation tors (see references) is quite pronounced. [6] F.c.G. Hoskin, J.E. Walker, W-D. Dett- of pesticide contaminants and CW A stock- bam,J.R. Wild, Biochem. Pharnwcol.1995, piles. OPH is one of the few enzymes 49, 711. which has been shown to be capable of [7] lE. Kolakowski, J.J. DeFrank, K. Lai, l.R. [I) B. Xu, l.R. Wild, C.M. Kenerley, J. Fer- Wild, 1996, submitted for publication. hydrolyzing the P-S bond of various OP mentation and Bioengineering 1996, 81, [8] KJ. Dave, L. Phillips, VA Luckow, J.R. pesticides; however, it possesses a wide 473. Wild, Biotech. and Appl. Biochem. ]994, range of catalytic rates (0.0067-167 S-I). [2] J.K. Grimsley, V.K. Ratogi, J.R. Wild, 19,271. Nonetheless, it has been possible to en- 'Biological Systems for the Detoxification [9] K. Lai, K.I. Dave, J.R. Wild, J. Bioi. Chem. hance the unique P-S bond hydrolysis of of Organophosphorus Neurotoxins: Poten- 1994,269, 16579. this enzyme by selecting specific changes tial for Environmental Remediation Appli- [10] L. Pei, G. Omburo, W.O. McGuinn,. I. cations', in 'Bioremediation: Principles and in the amino acids bordering the active Petrikovics, K.J. Dave, F.M. Raushel, J.R. Practice', Eds. S.K. Sikdar and R.L. Irvine, Wild, J.R. DeLoach, l.L. Way, Toxicol. site. Thus, it appears that the capacity for Technomic Publishing Co. Inc., 1996, in Appl. Pharmacal. ]994, 124,296. further improvement is remarkable, and press. [II] K.I. Dave, C.E. Miller, J.R. Wild, Clzem.- the opportunity for a variety ofbiotechni- [3] E.!. Rainina, A.L. Simonian, E.N. EIre- Bioi. Interact. 1993, 87, 55.

Chimia 50 (1996) 431-432 The Chemferm Process © Neue Schweizerisclze Cizemische Gesellschaft ISSN 0009-4293 In the Chemferm process (Fig.) only six steps are needed, whereby biocatalysis is involved in three of them [4]; a major Biocatalysis and Process improvement through the eyes of the or- ganicchemist [5]. However, when itcomes Integration in the Synthesis of to the design of the production plant for the final coupling step, the engineers are Semi-synthetic Antibiotics faced with an equilibrium process requir- ing recycle of starting materials and han- dling of many solids: Aile Bruggink* - crystallization and isolation of the de- sired cefalexin, - crystallization and recovery of excess Introduction - the low entry barrier, i.e. low invest- 7-ADCA (= 7-aminodeacetoxycepha- ments, for new technologies in this losporanic acid), The fine chemical industry is one of small scale industry, facilitated by the - crystallization and isolation of phe- the industry segments where the impact of fact that fine chemical companies be- nylglycine from undesired hydrolysis biocatalysis is felt most profoundly. Pos- come increasingly part oflarger indus- of both end product and side-chain sible explanations are [1][2]: trial conglomerates. precursor. - the need to replace traditional, stoichio- So far, in our developments at Chem- metric processes in order to improve ferm, the environment has been the main the product to waste ratio, Synthesis of Semi-synthetic Antibiotics winner. Only aqueous waste streams con- the failure to translate chemocatalytic taining some simple inorganic salts are processes from petrochemicals to fine The industrial production of semi-syn- produced, whereas the traditional process chemicals, thetic antibiotics, with a history of some releases methylene chloride and other sol- - the ready acceptance of enzymes by 30 years, is an outstanding example of the vents and needs stoichiometric amounts the organic chemist as part of his tool- development of biocatalysis. Cefalexin, of silylating agents, Dane-salt-protected box, whereas organic chemistry is still with an annual consumption of almost side chains and acylating promotors (such the dominating discipline in fine chem- 2000 t, the largest cephalosporin on the as pivaloyl chloride) which all end up as ical industry [3], world market, serves as a useful illustra- waste. tion. The original synthesis (see Fig.) start- ing from benzaldehyde and fermentation of penicillin G was a ten-step process The NOVO Process *Correspondence: Prof. Dr. A. Brugginka)b) employing stoichiometric chemistry only ") Chernferm. Industrial Pharmaceuticals and causing a waste stream of 30-40 kg An alternative process was developed P.O. Box 3933 per kg of end product. Often 4-6 different by NOVO, acquired and further improved NL-4800 OX Breda companies were involved to serve the chain by Chemferm. Using /3-naphthol as com- b) University of Nijmegen Department of Organic Chemistry from basic raw materials to bulk drug; plexing agent, which surprisingly is com- Toernooiveld nowadays one or two companies cover the patible with the enzymatic condensation NL-6525 ED Nijmegen full production column. conditions, an almost quantitative yield of BIOTECHNOLOGY FOR INDUSTRIAL PRODUCTION OF FINE CHEMICALS 432

CHIMIA 50 (1996) Nr. 9 (Septemher)

NEW ROUTE TO CEFALEXIN WITH FOUR FEWER STEPS AND MORE ENZYMATIC REACTIONS

FORMER ROUTE NEW ROUTE

[BENZALDEHYDE ~ PENICI~LUM---· ~ BENZALDEHYDE IPENI~.ILLlU~_- _ J STRECKER SYNTHESIS FERMENTATION FERMENTATION * CHEMICAL * BIOCA TAL YTIC STRECKER SYNTHESIS * BIOCATALYTIC * STiOCHIOMETRIC * CHEMICAL 1 1 STOICHIOMETRIC * ,- Dl.-PHENYLGL YCINE J PENICILLIN G~ I l_fENICI!-L1~ G -~j RING ENLARGEMENT RING ENLARGEMENT * CHEMICAL DL-PHENYLGL YCINAMIoE-i * CHEMICAL 1 * STOICHIOMETRIC OR ESTER I I* STOICHIOMETRIC r CEPHALOSPClRlN-- - -] KINETIC RESOLUTION [-C;I;PHALOSf>QFUN DEACYLATION * ENZYMATIC OR 1 DEACYLATION * CHEMICAL * ASYMMETRIC * ENZYMATIC 1 I * STOICHIOMETRIC TRANSFORMATION DANE SAL T ADCA . . ~~ I -~J D-(-)-PHENYLGL YCINAMIDE [_ADCA----- ACTIVATION J PROTECTION * CHEMICAL * CHEMICAL J * STOICHIOMETRIC * STOICHIOMETRIC ORES~/ L~~~~~YDRID-Cl [pROTECTED -ADCA--l

COUPLING I * ENZYMATIC ~~~Al I~--C-E-F-A~L-EX-I-N=--=--_~j I DEPROTECTION I * CHEMICAL r--CEFALEXIN~~ J ADCA = 7-AMINODEACETOXYCEPHALOSPORANIC ACID

by eliminating organic waste. The overall production process, however, is more com- plicated, because of several recycle streams and a large number of solids to be handled. A better understanding of enzyme ac- tion on a molecular level and a rational design of improved biocatalysts (includ- ing immobilization) are needed to reach more ideal processes where recycles are eliminated. Also, more adequate down- stream processing including, e.g. selec- tive absorption of end products through modern molecular recognition techniques, are required to achieve feasable processes at plant scale.

the 2: I cefalexin-l3-naphthol complex is optimization of all process parameters is obtained [6]. required and extra investments in equip- [I] a.M. Birch, 1.M. Brass, A. Kiener, K. Rob- Again, from a synthetic point of view, ment for handling solids can not be avoid- ins, D. Schmidhalter, N.M. Shaw, T. Zim- an excellent process, but once more the ed. mermann, Chim. Oggi 1995, Sept., 9. chemical engineers have to solve a few [2] S.c. Stinson, Chem. Eng. News 1995, OCI., tough separation and recycle problems: 44. - separation of phenylglycine from the Conclusions [3] A. Bruggink, CPhI Conference Proceedings 1993, p. 38. cefalexin-,B-naphthol complex, [4] A. Bruggink, CPhI Conference Proceedings Integration of biocatalysis in the syn- - isolation of cefalexin, free of I3-naph- 1994, p. 76. thol, from the complex, thesis of s.s. antibiotics, i.e. cefalexin, [5] Patents pending to Chel11ferl11,Gist-hrocades, - isolation and recycling of ,B-naphthol. greatly reduces the number of steps and is DSM Research. All this can be done, but a very careful of considerable benefit to the environment [6] US Pat. 5.470.717.