Use of Microorganisms and Enzymes in the Synthesis and Production

Chapter 15

Use of Microorganisms and Enzymes in the Synthesis and Production of Optically Active Agricultural Chemicals

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Gary J. Calton Rhone-Poulenc Research Center, 8510 Corridor Road, Savage, MD 20763 The production of both s p e c i a l t y and commodity chemicals by enzyme reaction has become a r e a l i t y due to recent advances i n immobilization. These immobilization techniques have provided an economical system f o r reuse of enzyme and thus provide a route to o p t i c a l isomers i n high enantiomeric yields. This provides specific stereoisomers for agricultural synthesis at reasonable cost. The advantages of stereoisomers include high a c t i v i t y l e v e l s as w e l l as reduced t o x i c i t y due to the absence of the incorrect stereoisomer. Methods of immobilization will be reviewed with emphasis on immobilization by polyazetidine. Enzymatic reaction via immobilization enzymes and immobilized whole c e l l s w i l l be reviewed with emphasis on the production of a g r i c u l t u r a l chemicals.

The production of specialty chemicals by enzyme reaction has a high potential for having an impact upon agricultural chemical use, especially i n the area of pesticides and herbicides. Since enzymes may be used to introduce chemical moieties i n high yield and high enantiomeric excess at low temperatures and pressures, the use of enzymes as agents for introducing chirality into chemicals and producing chiral synthons w i l l play an increasing role i n agrichemlcals. To date, mainly due to the expense of their production, chiral compounds have not been used extensively i n agricultural chemicals. In fact, only two compounds are utilized commercially, Fusilade 2000 and the pyrethroids. Although agricultural chemicals may s e l l for significantly more than commodity chemicals, there are, nevertheless, limits to the sales price one can obtain when applying an herbicide or pesticide to a crop as opposed to the value of a lifesaving chemical which might be used i n the production of 0097-6156/87/0334-0181 $06.00/0 © 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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B I O T E C H N O L O G Y IN A G R I C U L T U R A L C H E M I S T R Y

pharmaceuticals. The pharmaceutical Industry has, of course, used chiral compounds for decades. I believe that ve are moving into an era i n which agricultural chemicals w i l l be based on compounds which have only one stereoisomer present. Examples of currently marketed herbicides and fungicides which have a chiral carbon that might be responsible for increased activity are the herbicides Dual (metolachlor, Ciba-Geigy),J[, Fusilade ( Fluazifop-butyl, ICI ) ,2, and Suffix bw (1 flam-prop isopropyl, Shell),3, and the fungicide, Ridomil(metalaxyl, Ciba-Geigy),jf. A l l of these materials are sold as the racemlc mixture;however, i t i s highly likely that one stereoisomer may have most of the activity for each of these compounds. Moser, et a l . have shown that of the k stereoisomers of metolachlor, the two having an S configuration of the chiral carbon atom have a stronger herbicidal effect. Although metolachlor has only a weak fungicidal action, tests showed that the R configuration, i n comparison to the S configuration, was 3 times more effective as a fungicide. An effective method for preparation or for resolution and racemization of these compounds could be a potentially lucrative chemical goal. One can envision several points In the synthesis of metolachlor at which such a resolution might be carried out enzymatically. The benefits of such a synthesis are numerous. In addition to lowered toxicity due to the lowered rate of application, one might also obtain lowered toxicity due to removal of the less active (or inactive) stereoisomer. Such a reduction occurred with the removal of the S isomer of thalidomide leaving a l l of the desired activity i n the R isomer. Had this fact been known at the time of the introduction of thalidomide i n Britain, a great tragedy could have been avoided. The main problem i n the enzymatic synthesis of optically active agricultural compounds has been the lack of enzymes appropriate for these syntheses and/or resolutions and the lack of stability of such enzymes. The advent of immobilized systems capable of producing chemicals i n large quantity at considerably reduced prices provides an attractive route to chiral synthons for the agriculturally oriented organic chemist. Ideally, a chiral catalyst, whether biologically active or not, should have the following properties: excellent engineering characteristics, good stereochemical control, excellent flow properties, and long life-times, thus providing low cost. Biological systems for catalysis have suffered i n a l l these areas. Life-times for most systems have been notoriously short. Commercial systems for the production of high fructose corn syrup have had life-times on the order of 20-40 days. These immobilization systems have often been soft gels which have had problems i n flow rate and i n structural rigidity. The latter aspect complicates plant engineering due to the Inability to stack these beads i n large columns or to use high flow rates. The lack of stability results i n Increased labor costs due to catalyst changeovers In the columns. Plant productivity shows excessive swings i n output due to the rapid 1

1

2


15. C A L T O N

Production of Optically Active Agricultural Chemicals

4


183

184


decline of enzyme activity. In addition, there i s an increased cost of enzyme or microbial cells used i n production of the catalyst due to low stability. Recently we have developed novel immobilization systems^ which avoid these problems of lifetime, bead rigidity, flow rate and associated costs problems and thus allow the production of chiral synthons at considerably lower prices. One example i s the production of L-aspartic acid via immobilized aspartase contained i n microbial cells. Thus, _E. c o l l 11303 has been immobilized i n a number of systems by various companies. (Table I) TABLE I

Commercially Developed Methods for the Production of Aspartic Acid

Immobilization Method

Activity Half Life μ moles/hr/gm Retained cells Activity (Days at Co. (wet wt) 37°) (*)

Polyacrylamide 18,850 Ε Carrageenan 56,000 Cross linked Κ Carrageenan (Hexamethylenediamine + Gluteraldehyde) 48,000 Polyurethane 68,000 Polyazetidine 68,000

49 56

48 100 100

120 70

Ref.

Tanabe 4 Tanabe 5

Tanabe 6 680 Grace 7 37 8 1000-1400 PEI

Commercial production of pharmaceutical grade aspartic acid at levels of 4000 MT/yr has reduced selling prices to considerably less than $3.00 per kg, making i t one of the cheapest amino acids available. The most economic method for production of aspartic acid i s polyazetidine immobilization of J5. coll 11303, based on the reaction given i n Scheme 1.7 Thus, when polyazetidine prepolymer i s mixed with _E. c o l l 11303 and applied to a suitable support matrix (e.g. IRA 938 macroporous resin) and the polymer mixture cured by drying overnight i n a gentle flow of 25Î humidified a i r , a catalyst bead of approximately 0.3 mm diameter, which i s quite strong and possesses excellent flow characteristics (up to 60 space velocities (S.Y.)/hr. at less than 80 psi) can be generated. More than 97% of the aspartase activity of the free c e l l can be immobilized and retained. The half-life of this catalyst i s estimated to be 1100-1400 days (Figure 1). A flow rate of 5.5 S.V./hr allows a 98.5ί conversion of fumaric acid to L-aspartic acid with a final concentration of 19·8£. Based on this data, one can quickly calculate that a 200 l i t e r column would produce i n excess of 4 million lbs. of aspartic acid per year. Storage of the catalyst i n 1.5 M ammonium


C ALTON


H . ^ C Ο

CO

NH +

Η

Ο

I

II

185

H N — C — C0~

4

2

I "OC — CH

0 —C '

II ο

Η

2

SCHEME 1

70

60

hη

°ηηπ° ι Λ

Ο

ο Ο"

50

40

30

20

10

-L 20

40

60

80

100

TIME

(DAYS)

120

130

FIGURE 1 The stability of the aspartase catalyst vas determined by continuously passing 1.5M fumaric acid, pH 8.5 (adjusted with E H 3 ) , 1 mM MgSOi^ at 37°C at 6 volumes/volume/hr.


186


aspartate at k°C or at room temperature resulted In no decline in catalyst activity after a period of k years. Thus, the enzymatic approach to L-aspartic acid can provide a chiral synthon at extremely low prices. It i s of interest to note that metolachlor and metalaxyl can be considered as derivatives of a substituted amino acid, alanine. The most active form of metolachlor would correspond to an L-alanine derivative. L-alanlne i s a constituent of a number of biologically active compounds, among which are the pharmaceutical products Captopril (Squibb) and Alapril (Merck), both of which are angiotensin I converting enzyme inhibitors. Thus, L-alanlne may be considered as a primary example of a chiral synthon of value i n the production of both pharmaceutical and agricultural chemicals. The organic chemist w i l l quickly realize that neither metolachlor nor metalaxyl i s synthesized from L-alanine but rather their syntheses are based on substituted anilines. At the same time, i t should be pointed out that L-alanine i s not used i n the synthesis of agricultural chemicals. I believe that the primary reason for this discrepancy i s that, at the time of this writing, multi-ton quantities of L-alanine are quoted at $32-$45/kg. Based on these raw material costs, the use of L-alanlne i n the manufacture of most agricultural chemicals cannot be justified, even when a 50JÉ reduction of raw material consumed i s expected. L-alanine can also be produced i n a manner s1 miliar to that for aspartic acid. The enzyme aspartate-^-decarboxylase, catalyzes the loss of CO2 from aspartic acid, thus producing alanine. (Scheme 2) The main problem encountered In this reaction i s the CO2 production, which i s voluminous. Column reactions require pressure vessels'* and gel type catalysts w i l l not withstand the abrasion which occurs i n a continuous stirred tank reactor or fluldized bed reactor?. Pseudomonas dacunhae or Alcallgenes faecalis have been immobilized by the polyazetidine method, thus providing a catalyst having high activity which i s quite rigid. This catalyst can withstand the conditions of slow stirring i n a continuous stirred tank reactor without degradation of the enzyme activity (Figure 2). The projected h a l f - l i f e , based on this data, i s i n excess of 6 months. Low cost alanine i s a reality i f large quantities (> 1000 MT) are required. In fact the price would be equivalent or below that of the chemically produced racemic mixture at that level. In the area of pesticide chemistry the pyrethroids are an outstanding example of the use of chiral molecules on a commercial scale. Roussel-uclaf has commercialized Decis, which i s the d-cis-isomer of the pyrethroid, deltamethrin, _5. This insecticide i s reported to have sales i n excess of 500 million Franc i n 1982. The total plant capacity i s 225 metric tons per year. Decis i s prepared by resolution of DL trans chrysanthemic acid. This resolution utilizes the base of chloramphenicol which forms a diastereomer that can easily be separated by f i l t r a t i o n . Thus, chrysanthemic acid has been a chiral synthon which has been sought by a number of 10


15. C A L T O N


H O

Ι

ΝΑ

II

I

H Ν — C — CO" 2

•

CH —CH—COOH + C0 3

I

"OC — C H

187

2

2

SCHEME 2

o.6 H

1

5

10

15

20

25

30

TIME (Days)

Figure 2 The stability of the immobilized organism containing aspartate- β -decarboxylase was assessed by continuous infusion of 1.5M aspartate (pH 8.5), 0.1 mM pyridoxal-5-phosphate, 0.5 mM sodium pyruvate at 37°C through a 750 ml volume continuous stirred reactor containing 75 mL of catalyst (0.2g Pseudomonas dacunhae/ml beads) with the rate adjusted to provide 99% conversion.

Η

Η 5


188


investigators. Brian Jones and h i s group, working i n the area of enzymes i n organic synthesis, have used commonly a v a i l a b l e enzymes for preparation of enantiomerically pure chiral compounds. His group has used horse l i v e r alcohol dehydrogenase (HLADH) f o r the production of chrysanthemic a c i d p r e c u r s o r s . unfortunately, HLADH i s an NAD dependent alcohol dehydrogenase. Although i t catalyzes oxido reductions on a broad spectrum of substrates, the cost of NAD, at approximately $685/KG, i s p r o h i b i t i v e since NAD i s used i n stoichiometric q u a n t i t i e s . The reaction was reported on a 2 gram in which à U m e t h y l - 1 , 2 b i s ( h y d r o x y m e t h y l ) c y c l o p r o p a n e (15.4mmol) was reacted with HLADH (35 mg) i n the presence of 1 mmol NAD (720 milligram) and 20.3 mmol FMN. FMN i n commercial grade i s approximately one h a l f the cost of NAD. Thus, although t h i s route i s an a d d i t i o n a l s o l u t i o n to the problem that Roussel-Uclaf has i n manufacturing Decis, i t i s , unfortunately, s t i l l not economically possible due to the high cost of regeneration of b i o l o g i c a l energy. Another example of the use of enzymes i s the resolution of racemic mixtures which are of value as c h i r a l synthons. This i s i s i l l u s t r a t e d by the stereoselective hydrolysis of 2-acyloxy-3-chloropropyl-2-toluenesulfonate. The l i p a s e from Psuedomonas aeruginosa was found to have a high h y d r o l y t i c a c t i v i t y and a s t e r e o s e l e c t i v i t y greater than 99% on this compound. This hydrolysis provides a route to the highly sought enantiomers of chloromethyloxirane. The novel feature involved in this stereoselective hydrolysis was the use of the toluenesulfonate as i t had been previously been found that the hydrolysis of other esters gave only a 90% enantiomeric excess. This enantiomeric excess i s not s u f f i c i e n t f o r the preparation of most a g r i c u l t u r a l and pharmaceutical chemicals. However, the enzymatic hydrolysis of the toluenesulfonate provides a method f o r c e r t a i n insect hormones amd pharmaceuticals v i a o p t i c a l l y active chloromethyloxLranes. The syntheses of the compounds above are provided to i l l u s t r a t e to the organic chemist, that the production of c h i r a l compounds can be accomplished i n an economically f e a s i b l e manner and i n large quantity. Additional efforts i n the use of c h i r a l synthons f o r the production of a g r i c u l t u r a l chemicals i s proposed as a method f o r reducing the t o x i c i t y and the cost of production of a g r i c u l t u r a l chemicals. In a d d i t i o n , these c h i r a l synthons w i l l open up new areas of i n t e r e s t to the organic chemist providing basic b u i l d i n g blocks which may be tapped to interrupt b i o l o g i c a l mechanisms v i a b i o s y n t h e t i c a l l y important precursors. 11

References 1. Moser, H.; Rihs, G.; Sauter, Η. Ζ. Naturforsch. 1982, 87B, 451-462. 2. Blaschke, G.; K r a f t , H. P.; Fickentscher, K.; Kohler, F. Arzneim. Forsch. 1979,29, 1640. 3. Wood, L.L. ; Calton, G.J. U.S. PAT 4,431,813, Mar 13, 1984. 4. Tosa, T.; Sato, T.; Nishida, Y.; Chibata, I . Biochem. Biophys. Acta. 1977, 483, 193.


C ALTON 5. 6. 7. 8. 9. 10. 11. 12.


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Nishida, Y.; Sato, T.; Tosa, T.; Chibata, I . Enzyme Microb. Technol. 1979, 1, 95. Fusee, M.C.; Swann, W.E.; Calton, G.J. Appl. Environ. Micro. 1981, 42, 672. Wood, L.L.; Calton, G.J. Bio/Technology. 1984, 2, 1081. Furui, M.; and Yamashita, K. J. Ferment. Tech. 1983, 61, 587. Yamamoto, K.; Tosa, T.; Chibata, I . Biotech. Bioeng. 1980, 22, 2045. Tessier, M.J., Informations chimie. 1982, 232, 93 Jakovac, I . J . ; Goodbrand, H.B.;Lok, K.P.; Jones, J.B. J . Am. Chem. Soc. 1982, 104, 4659. Hamaguchi, S.; Ohashi, T.; Watanabe, K. A g r i c . B i o l . Chem. 1986, 50, 375.

R E C E I V E D November21,1986


Use of Microorganisms and Enzymes in the Synthesis and Production

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