The diastereo- and enantioselective microbial oxidation of a mixture of racemic cis/trans-chrysanthemols to the corresponding stereoisomeric chrysanthemic ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1988, p. 2268-2272 0099-2240/88/092268-05$02.00/0 Copyright © 1988, American Society for Microbiology
Vol. 54, No. 9
Microbiologically Catalyzed Enantio- and Diastereoselective Oxidation of Chrysanthemol Stereoisomers to Chrysanthemic Acids MAHMUT MISKI AND PATRICK J. DAVIS* Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas 78713 Received 17 March 1988/Accepted 21 June 1988
The diastereo- and enantioselective microbial oxidation of a mixture of racemic cis/trans-chrysanthemols to the corresponding stereoisomeric chrysanthemic acids by Aspergillus species is described. Of the three microorganisms which were found capable of oxidizing racemic cis/trans-chrysanthemols, A. ochraceus ATCC 18500 showed complete enantioselectivity for (+)-stereoisomers [(+)-trans-chrysanthemol and (+)-cis-chrysanthemol), whereas A. flavipes ATCC 1030 and ATCC 11013 showed complete enantioselectivity for the (+)-cis-chrysanthemol but a time-dependent enantioselectivity during oxidation of trans-chrysanthemol [oxidation of (+)-trans-chrysanthemol prior to (-)-trans-chrysanthemol]. The diastereoselectivity of all three microorganisms was time dependent, in that the trans-stereoisomers were oxidized prior to the cis-isomers.
analogs, precursors of deltamethrin (i.e., based upon the 6,6-dibromo analog of cis-chrysanthemic acid). Herein, we report an alternative biotechnological approach for preparation of the active chrysanthemic acid analogs, based upon the enantio- and diastereoselective oxidation of the precursor chrysanthemyl alcohols (chrysanthemols), using selected microbial cultures. The overall results obtained are presented in Fig. 1. Regio- and stereospecific whole-cell microbial or enzymatic oxidoreduction reactions have been extensively used for the synthesis of chiral products from both chiral and achiral substrates (16, 21). The aforementioned restrictions in biotechnological approaches to the preparation of active chrysanthemic acid isomers have led us to investigate the regio- and stereoselective microbial oxidation of stereoisomeric chrysanthemol mixtures to the desired active acids. Efficient chemical oxidation of chrysanthemols to chrysanthemic acids is difficult owing to the chemical instability of the cyclopropane ring system. Thus, the development of an appropriate mild, microbial, oxidative method for this conversion would be beneficial even in the absence of diastereoor enantioselectivity.
The natural pyrethrins, insecticidal esters from Chrysanthemum cinerariaefolium (Compositae) (22), are superior to many other insecticides in their quick-killing power, low mammalian toxicity, biodegradability, and relatively low resistance development in insects (3, 6, 18). On the other hand, these compounds have limited applications due to their photoinstability and high volatility. These disadvantages and the growing demand for safe but effective pesticides have spurred the exploration of synthetic methods for the preparation of more active and stable pyrethroid derivatives in large quantities (2, 23). The acid component of pyrethroid esters is indispensable for insecticide activity. Structure-activity relationship studies have revealed marked differences in the insecticidal activity of (+) versus (-) optical isomers, as well as transversus cis-diastereomers (8). Thus, recent synthetic efforts have focused on the asymmetric syntheses of chrysanthemic acid derivatives. Conventional asymmetric syntheses typically require complex synthetic strategies and/or expensive chiral synthons and/or catalysts. An alternative approach involves the resolution of synthetic chrysanthemic acid mixtures or their precursors, with recycling of the less active isomers. Chemical resolutions often require tedious procedures and suffer from the loss of a considerable amount of product. Microorganisms, via their enzyme system(s), are also capable of performing a wide variety of regio- and stereoselective transformations (5, 9, 12). It is thus possible to select specific microorganisms to catalyze desired stereoselective chemical transformations, including resolutions. Several biotechnological approaches have been applied to the synthesis of the active chrysanthemic acid isomers. The use of chiral precursors to chrysanthemic acid, which are prepared by enzymatic or whole-cell microbial transformations (7, 10, 19), still requires multistep synthetic procedures. Moreover, strict dependence on a single precursor restricts the synthetic approach, even if low overall yields are obtained. The biological resolution of the synthetic chrysanthemic acid mixtures using stereoselective esterase activity (20) appears to be limited to the resolution of only the trans-isomers and is not practical for the resolution of the commercially important synthetic cis-chrysanthemic acid
MATERIALS AND METHODS General. 1H-nuclear magnetic resonance spectra were recorded on a Varian EM 390 spectrometer. Infrared spectra were taken on a Perkin-Elmer model 1330 spectrophotometer. Mass spectra were obtained on a Finnigan-MAT model 4023 instrument with a direct inlet system at 70 eV. Optical rotation measurements at the Na-D line were carried out on a Perkin-Elmer model 241 MC polarimeter using a microcell. Analytical gas chromatography (GC) analyses were performed on a Shimadzu GC-4CM instrument using a silanized glass column (5 m by 3 mm internal diameter) packed with 10% QF-1 on Chromosorb GHP (60/80 mesh) as described previously (14). It should be noted that, although the chromatographic conditions described (14) were capable of resolving cis- versus trans-chrysanthemyl alcohols, aldehydes, and acids, the aldehydes (logical oxidation intermediates) were not observed in any of the transformations described herein. Chiral derivatizations and GC separation of chrysanthemic acid stereoisomers were based on the procedure of Murano (15). Preparation of the racemic cis/trans-chrysan-
* Corresponding author. 2268
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CH20H
>e
t-
c OH20H
>e
COOH
A. ochraceus A. flavipes 2
6
CH20H
COOH A. ochraceus A.
flavlpes
3
7
~~CH OH
\=
COOH A.
ochraceus
A.
flavipes
4
8
FIG. 1. Summary of the overall stereochemical specificity of chrysanthemol oxidation to chrysanthemic acids using A. ochraceus ATCC 18500 and A. flavipes ATCC 1030. Solid arrow implies complete conversion, while conversion.
a
dotted
arrow
implies minor (or trace)
themol mixture and standards was as described previously (14). (+/-)-cis-Chrysanthemic acid was purified from a technical-grade sample (95%) (McLaughlin Gormley King Co., Minneapolis, Minn.) by fractional crystallization from
ethyl acetate. Preparation of racemic trans-chrysanthemol from cis/transchrysanthemic acid. Racemic trans-chrysanthemol was prepared by modification of a method described previously (24). A total of 15 g of cis/trans-chrysanthemic acid mixture,
obtained from alkaline hydrolysis of commercially available cis/trans-ethylchrysanthemate (isomeric ratio, ca. 35:65), was dissolved in dry benzene (100 ml), 1.5 ml of BF3etherate complex was added, and the solution was stirred overnight under a nitrogen atmosphere. The reaction mixture was quenched by the addition of 20 ml of ice-water, and stirring was allowed to continue for 2 h. The benzene layer separated and dried over anhydrous MgSO4. Evaporation of the solvent under reduced pressure yielded a 13.8-g mixture of racemic trans-chrysanthemic acid and cis-dihydrochrysanthemolactone. This mixture was reacted with excess of diazoethane in ether to convert the free transchrysanthemic acid to its ethyl ester. After the esterification, the ether layer was removed under reduced pressure, and the oily residue was redissolved in hexane and extracted with a 5% NaOH solution. The hexane layer was dried over anhydrous MgSO4 and evaporated under reduced pressure to yield racemic trans-ethylchrysanthemate (10.5 g, 92.3%). This compound was then reduced by lithium aluminum hydride (LAH) in dry ether to yield racemic trans-chrysanthemol (8.1 g, 98.2%). Racemic cis-chrysanthemol was obtained from LAH reduction of pure racemic cis-chrysanthemic acid in lieu of preparation from racemic cis-dihydrochrysanthemolactone. Cultures. The following cultures were screened for their ability to oxidize chrysanthemols to chrysanthemic acids:
was
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Aspergillus alliaceus Ul 315 and NRRL 317; Aspergillus flavipes ATCC 1030, ATCC 11013, and ATCC 16795; Aspergillus flavus ATCC 9170 and ATCC 24714; Aspergillus foetidus NRRL 337; Aspergillus niger UI-X-172, NRRL 328, ATCC 10548, ATCC 10581, ATCC 11394, and ATCC 16888; Aspergillus ochraceus ATCC 18500 and ATCC 22947; Aspergillus parasiticus ATCC 15517; Fusarium solani ATCC 12823; Rhizopus arrhizus ATCC 11145 and ATCC 20097; Rhizopus stolonifer NRRL 1478; Saccharomyces cerevisiae wild type, NCYC 240, and NRRL Y2034; and Saccharomyces lipolytica CBS 599. Sources of the culture strains were the American Type Culture Collection (ATCC) (Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS) (Baarn, The Netherlands), National Collection of Yeast Cultures (NCYC) (Nutfield, Surrey, England), Northern Regional Research Laboratories (NRRL) (USDA Laboratories, Peoria, Ill.), and J. P. Rosazza, College of Pharmacy, University of Iowa (UI) (Iowa City). All cultures used in these studies were maintained on refrigerated (4°C) slants of Sabouraud maltose agar or American Type Culture Collection medium 5 sporulation agar and were transferred every 6 months to maintain viability. Medium composition. The fermentation medium used in these studies consisted of the following: glucose (20 g), dibasic potassium phosphate (5 g), sodium chloride (5 g), yeast extract (Difco) (5 g), Bacto-Soytone (Difco) (5 g), cellulose powder (particle size, less than 10 jxm) (Macherey, Nagel & Co., Duren, Federal Republic of Germany) (2 g), and distilled water (1 liter). The pH was adjusted to 6.0 with 6 N HCl, and the medium was sterilized in individual flasks at 121°C for 15 min. General fermentation procedures. First-stage incubations were conducted in 125-ml Bellco DeLong culture flasks containing 25 ml of sterilized medium. Cultures were initiated by suspending surface growth or spores of each organism on agar slants in a portion of the medium and transferring the suspension to the culture flask under aseptic conditions. The culture flasks were placed in a New Brunswick environmental shaker (250 rpm) and maintained at 27°C. After incubation for 72 h, a portion of the first-stage culture was used to inoculate a second-stage flask. In smallscale studies, 125-ml culture flasks were used, while in the preparative-scale studies, 1-liter Bellco DeLong culture flasks containing 200 ml of the sterile medium were used. After 36 h of incubation in second stage, the appropriate substrate (0.5 mg in 2 ,ul of dimethylformamide per 1 ml of culture) was added, and the incubation was allowed to continue. Samples (1 to 2 ml) were aseptically harvested at the specified time periods and analyzed by GC. Time course studies of the microbial transformation of the racemic cis/trans-chrysanthemol mixture to the corresponding acids. Six second-stage cultures of A. ochraceus ATCC 18500 and A. flavipes ATCC 1030 were generated in 1-liter Bellco DeLong flasks containing 200 ml of the sterilized medium. After incubation in second-stage culture for 36 h, 100 mg of the chrysanthemol mixture (ca. 35% cisI65% trans) in 0.4 ml of dimethylformamide was added to each flask. The first whole-flask samples were harvested 1 h after substrate addition, and the initiation of transformation of chrysanthemols was monitored by harvesting 2-ml culture samples from each remaining flask every 6 h, followed by GC analysis. After the first detection of chrysanthemic acid formation by this procedure, whole-flask samples were harvested at 10-h intervals for A. ochraceus ATCC 18500 and 16-h intervals for A. flavipes ATCC 1030. Volumes of 20 ml of each culture were transferred to a 125-ml Erlenmeyer flask and acidified
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MISKI AND DAVIS
with 2 ml of 1 N HCl, and the internal standard (borneol) was added (10 mg in 50 ,ul of dimethyl formamide). Samples were homogenized and extracted with 5 ml of ether. Ether extracts were dried over anhydrous MgSO4 and filtered, and the solvent was carefully evaporated to dryness in the cold under reduced pressure. After derivatization with diazoethane, samples were analyzed by GC. The transformation time courses of chrysanthemol diastereomers (i.e., cis versus trans) by A. ochraceus ATCC 18500 and A. flavipes ATCC 1030 are shown below (see Fig. 4 and 5, respectively). Preparative-scale microbial oxidation of racemic cis- and trans-chrysanthemols with A. ochraceus ATCC 18500. Five 1-liter second-stage cultures of A. ochraceus ATCC 18500, each containing 200 ml of culture, were generated for each substrate as described above. The substrate (racemic cis- or racemic trans-chrysanthemol; 80 mg per flask) was added at 36 h. The microbial conversions were carried out as described above and were monitored by GC. At 72 h after substrate addition, the transformations were complete. Flask contents for each substrate were combined, acidified with 1 N HCl, and extracted with ether (three times with 300 ml each). The ether layer was dried over anhydrous MgSO4 and carefully evaporated under reduced pressure in the cold. The oily residue was redissolved in pentane (200 ml) and extracted with 5% aqueous NaOH solution in a separatory funnel to separate chrysanthemic acid products from untransformed chrysanthemols. The pentane layer was separated, dried over anhydrous MgSO4, and carefully evaporated under reduced pressure in the cold to yield the residual chrysanthemol as an oily mixture. The aqueous layer was acidified with 1 N HCl and extracted with ether. The ether layer was processed as described above to yield an oily extract of chrysanthemic acid. Final purification of both extracts was accomplished using a silica gel column (1.5 by 15 cm, 100 to 200 mesh), using 10% ethyl acetate in hexane as the mobile phase. The purified products and residuals were characterized by infrared, nuclear magnetic resonance, and mass spectrometry (identical to standards) and then subjected to optical rotation measurements: trans-(+)-chrysanthemic acid, yield 94 mg, [aID23 +11.8° (c 6.1 in ethanol) (lit. +14.52° [13]) (83.33% optical purity and 91.7% enantiomeric excess); trans-(-)chrysanthemol, yield 32 mg, [ax]D23 -39.20 (c 1.07 in cyclohexane) [lit. for (+) isomer; +49.7° (1)] (78.9% optical purity and 89.45% enantiomeric excess); cis-(+)-chrysanthemic acid, yield 89 mg, [cxD23 +31.2° (c 4.4 in ethanol) (lit. +39.69° [13]); cis-(-)-chrysanthemol, yield 72 mg, [aID23 -33.4° (c 4.8 in CH2Cl2). Time-dependent stereoselective biotransformation of racemic trans-chrysanthemol with A. flavipes ATCC 1030. Six 1-liter second-stage flasks were used to examine the timedependent enantioselective transformation of racemic transchrysanthemol by A. flavipes ATCC 1030. The experiment was conducted as described above except that after the first detection of the chrysanthemic acid formation by GC analysis, single flasks were harvested at appropriate intervals. Each flask was extracted, and the acid product and residual substrate were separated as described above. The oily acid mixtures were subjected to chiral derivatization with R-(-)2-octanol and subjected to GC analyses (see Fig. 3). RESULTS AND DISCUSSION Twenty-five microorganisms were screened for their ability to oxidize a 36:65 cis/trans-chrysanthemol mixture to the
i5
50
45
50
Retention Time (min) Retention Time (min) FIG. 2. GC of stereoisomeric chrysanthemic acids [as the (R)-2octanol esters] produced in initial biotransformations with (A) A. ochraceus ATCC 18500 and (B) A. flavipes ATCC 1030. Structure numbers are specified in Fig. 1, and stereochemical assignments of chrysanthemic acids are indicated.
corresponding chrysanthemic acids. Three Aspergillus species, A. ochraceus ATCC 18500, A. flavipes ATCC 1030, and A. flavipes ATCC 11013, were found capable of catalyzing the desired transformation. Initial biotransformation studies with A. ochraceus ATCC 18500 indicated that ca. 50% each of both cis- and transchrysanthemol were oxidized to the corresponding acids. In contrast, both A. flavipes strains (ATCC 1030 and 11013) were found to oxidize trans-chrysanthemol completely, but cis-chrysanthemol to only ca. 50%. Such an observation frequently suggests the possibility of enantioselectivity in the bioconversions (11). This possibility was then examined by chiral derivatization of chrysanthemic acid products with R-(-)-2-octanol, followed by GC analyses of the resultant diastereomers according to the method of Murano (15) (Fig. 2). The results of these analyses clearly supported a high degree of enantioselectivity in the oxidation of (+)-cis- and (+)-trans-chrysanthemols using A. ochraceus ATCC 18500 and enantioselectivity with A. flavipes ATCC 1030 and 11013 in the oxidation of (+)-cis-chrysanthemol (Fig. 1). Further evidence for highly enantioselective biotransformations was obtained by the preparative-scale microbial oxidation of chrysanthemol isomers using A. ochraceus ATCC 18500. After termination of the microbial transformation, both unreacted alcoholic substrates and acid products were extracted, purified, characterized, and subjected to optical rotation measurements. The optical rotations of trans- and cis-chrysanthemic acid products were +11.80 (83.33% optical purity and 91.7% enantiomeric excess of compound 5; see Fig. 1) and +31.20 (85.04% optical purity and 92.52% enantiomeric excess of compound 7), respectively. An approximate 50% biotransformation of cis-chrysanthemol using A. flavipes ATCC 1030 and ATCC 11013, as well as enantiodiscriminating GC analyses of the chrysanthemic acid products, were consistent with a highly enantioselective biotransformation of the (+)-cis isomer of chrysanthemol (compound 3, Fig. 1). In contrast, the racemic trans-chrysanthemol (compounds 1 and 2) was completely metabolized to the corresponding acids (compounds 5 and 6), and GC analyses indicated modest (+)-trans-chrysanthemic acid (compound 5) enrichment with these fungi. It
VOL. 54, 1988
MICROBIAL OXIDATION OF CHRYSANTHEMOL STEREOISOMERS
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Retention Time (min)
FIG. 3. GC of the time-dependent enantioselective production of trans-chrysanthemic acids [as the (R)-2-octanol esters] with A. flavipes at approximately (A) 45 h, (B) 55 h, and (C) 65 h. Structure numbers are specified in Fig. 1, and stereochemical assignment of chrysanthemic acids is indicated.
was recognized that, although this enrichment was modest, it could result from a time-dependent, highly enantioselective oxidation of trans-chrysanthemol with A. flavipes; that is, initial metabolism of (+)-trans-chrysanthemol (compound 1) could be accompanied by partial loss of (-)-trans-chrysanthemol (compound 2) awaiting metabolism, due to volatility. Preparative-scale microbial transformation of racemic trans-chrysanthemol with A. flavipes ATCC 1030 and GC analyses of the R-(-)-2-octanyl derivatives of acid products during the course of biotransformation confirmed the presence of time-dependent enantioselectivity as shown in Fig. 3. The initial biotransformation preference observed with A. flavipes ATCC 1030 was for (+)-trans-chrysanthemol (compound 1). Only after nearly complete transformation of this
enantiomer did biotransformation of the (-) enantiomer (compound 2) initiate. The biotransformation diastereoselectivity of all three fungi was not absolute, but was also found to be time dependent. During preliminary studies, GC analyses indicated preferential trans-chrysanthemol oxidation prior to biotransformation of the cis isomer with all three fungi. Time course studies of the microbial transformation of racemic cis/trans-chrysanthemol mixture were then conducted with A. ochraceus ATCC 18500 (Fig. 4) and A. flavipes ATCC 1030 (Fig. 5), which confirmed the preliminary observations; that is, initial biotransformation diastereoselectivity observed with both fungi was for trans-chrysanthemols (i.e., compound with A. ochraceus and compounds 1/2 with A.
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TIME (HOURS) FIG. 4. Time-dependent diastereoselective oxidation of a 35:65 mixture of cis/trans-chrysanthemols to the corresponding chrysanthemic acids with A. ochraceus ATCC 18500.
0
20
40
60
80
100
120
TIME (HOURS) FIG. 5. Time-dependent diastereoselective oxidation of a 35:65
mixture of cis/trans-chrysanthemols to the corresponding chrysanthemic acids with A. flavipes ATCC 1030.
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flavipes). Biotransformation of cis-chrysanthemols was delayed in both cases until nearly total conversion of the trans-isomer(s) had taken place. These and the previous results are summarized in Fig. 1. The enantio- and diastereoselective bio-oxidative capacities of A. ochraceus ATCC 18500 and A. flavipes ATCC 1030 were also examined with alcohol precursors of the important synthetic halogenated chrysanthemic acid analogs permethrinic acid and K-othrinic acid. Similar results were obtained (data not shown). Although the synthesis of (2S, 3R)-cis-chrysanthemic acid from chiral precursors, which were prepared either by the horse liver alcohol dehydrogenase-mediated oxidation of cyclopropane meso-diols (10) or by microbial reduction of a symmetric 1,4-cyclohexadione derivative (4, 7), has been described, preparation of (2R, 3R)-trans-chrysanthemic acid with microbial (or enzymatic) oxidoreductase systems has not been reported. In addition, the stereoselective hydrolysis of racemic trans-chrysanthemyl acetate using microbial esterase activity has been reported (17); however, low optical enrichment was attained. The microbial oxidative resolution of racemic cis/trans-chrysanthemol mixtures using A. ochraceus ATCC 18500, A. flavipes ATCC 1030, and A. flavipes ATCC 11013 should allow for the simultaneous separation and generation of all of the desired geometric and enantiomeric stereoisomers of chrysanthemic acid derivatives in a single system. ACKNOWLEDGMENT supported by McLaughlin Gormley King ComMinneapolis, Minn.
This work pany,
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was
LITERATURE CITED 1. Alexander, K., and W. W. Epstein. 1975. Studies on biogenesis of non-head-to-tail monoterpenes. The isolation of (1R, 3R)chrysanthemol from Artemisia ludoviciana. J. Org. Chem. 40: 2576. 2. Arnt, D., M. Jautelat, and R. Lantzsch. 1981. Syntheses of pyrethroid acids. Angew. Chem. Int. Ed. Engl. 20:703-722. 3. Barthel, W. F. 1973. Toxicity of pyrethrum and its constituents to mammals, p. 123-140. In J. E. Casida (ed.), Pyrethrum, the natural insecticides. Academic Press, Inc., New York. 4. Buisson, D., R. Azerad, G. Revial, and J. d'Angelo. 1984. Stereoselective synthesis of (1R, 3S)-cis-chrysanthemic acid through microbiological reduction of 2,2,5,5-tetramethyl-1,4cyclohexadione. Tetrahedron Lett. 25:6005-6006. 5. Butt, S., and S. M. Roberts. 1986. Recent advances in the use of enzyme-catalyzed reactions in organic research: the synthesis of biologically active natural products and analogues. Nat. Prod. Rep. 3:489-503. 6. Camougis, G. 1973. Mode of action of pyrethrum on arthropod nerves, p. 211-221. In J. E. Casida (ed.), Pyrethrum, the natural insecticides. Academic Press, Inc., New York. 7. D'Angelo, J., G. Revial, R. Azerad, and D. Buisson. 1986. A short, efficient, highly selective synthesis of (1R, 3S)-cis-chry-
8.
9.
10.
11.
12.
13. 14.
15. 16.
17. 18.
19.
20.
21. 22. 23. 24.
santhemic acid through the microbiological reduction of 2,2,5,5tetramethyl-1,4-cyclohexadione. J. Org. Chem. 51:40-45. Elliott, M. 1977. Synthetic pyrethroids. ACS (Am. Chem. Soc.) Symp. Ser. 42:1-29. Fischli, A. 1980. Chiral building blocks in enantiomer synthesis using enzymatic transformations, p. 269-350. In R. Scheffold (ed.), Modern synthetic methods. Otto Salle Verlag, Frankfurt am Main, Federal Republic of Germany. Jakovac, I. J., H. B. Goodbrand, K. P. Lok, and J. B. Jones. 1982. Enzymes in organic synthesis. 24. Preparation of enantiomerically pure chiral lactones via stereospecific horse liver alcohol dehydrogenase catalyzed oxidations of monocyclic meso diols. J. Am. Chem. Soc. 104:4659-4665. Jones, J. B. 1986. Enzymes in organic synthesis. Tetrahedron 42:3351-3403. Kieslich, K. 1976. Microbial transformation of non-steroid cyclic compounds. Georg Thieme Publishers, Stuttgart, Federal Republic of Germany. Matsui, M., and F. Horiuchi. 1971. Resolution of the chrysanthemic acids with L-lysine. Agric. Biol. Chem. 35:1984-1985. Miski, M., and P. J. Davis. 1988. Gas chromatographic determination of racemic cis- and trans-chrysanthemols and their potential aldehyde and carboxylic acid microbial metabolites. J. Chromatogr. 437:436-441. Murano, A. 1972. Determination of the optical isomers of insecticidal pyrethroids by gas-liquid chromatography. Agric. Biol. Chem. 36:2203-2211. Ng, G. S. Y., L.-C. Yuan, I. J. Jakovac, and J. B. Jones. 1984. Enzymes in organic synthesis. 29. Preparation of enantiomerically pure cis-2,3- and 2,4-dimethyl lactones via horse liver alcohol dehydrogenase-catalyzed oxidations. Tetrahedron 40: 1235-1243. Oritani, T., and K. Yamashita. 1975. Microbial resolution of some racemic monocyclic alcohols. Agric. Biol. Chem. 39:8996. Pillmore, R. E. 1973. Toxicity of pyrethrum to fish and wildlife, p. 143-164. In J. E. Casida (ed.), Pyrethrum, the natural insecticides. Academic Press, Inc., New York. Sabbioni, G., and J. B. Jones. 1987. Enzymes in organic synthesis. 39. Preparation of chiral cyclic acid-esters and bicyclic lactones via stereoselective pig liver esterase catalyzed hydrolyses of cyclic meso diesters. J. Org. Chem. 52:4565-4570. Schneider, M., N. Engel, and H. Boensmann. 1984. Enzymatic syntheses of chiral building blocks from racemates: preparation of (1R, 3R)-chrysanthemic, -permethrinic and -caronic acids from racemic, diastereomeric mixtures. Angew. Chem. Int. Ed. Engl. 23:64-66. Simon, H., J. Bader, H. Gunther, S. Neumann, and J. Thanos. 1985. Chiral compounds synthesized by biocatalytic reductions. Angew. Chem. Int. Ed. Engl. 24:539-553. Staudinger, H., and L. Ruzicka. 1924. Insektentotende Stoffe. I. Uber Isolierung und Konstitution des wirksamen Teiles des dalmatinischen Insektenpulver. Helv. Chim. Acta 7:177-200. Storck, W. J. 1987. Demand for home and garden pesticides spurs new products. Chem. Eng. News. 65(14):11-17. Ueda, K., and M. Matsui. 1971. Studies on chrysanthemic acid. XXI. Photochemical isomerization of chrysanthemic acid and its derivatives. Tetrahedron 27:2771-2774.
