Journal of Chemical Ecology, Vol. 13, No. 2, 1987
SYNTHESIS OF ENANTIOMERICALLY ENRICHED 2-HEPTANOL A N D 3-OCTANOL BY MICROBIAL REDUCTASES OF Curvularia falcata AND Mucor SPECIES
J O H N M. B R A N D , 1 D I A N A L. C R U D E N , MARKOVETZ
and A L L E N
J.
Microbiology Department, University of lowa Iowa City, Iowa 52242 (Received January 8, 1986; accepted February 28, 1986) Abstract--Certain insects produce 2-heptanol or 3-octanol in various glandular secretions and recent studies have shown that the 3-octanol of two different genera of ants (Crematogaster and Myrmica) can be either the (S)-(+) or mainly the (R)-(-) enantiomer, respectively. Synthesis of each of these alcohols can be achieved in relatively high enantiomeric purity by certain microbial reductases. The corresponding ketone of each alcohol is reduced by Curvularia falcata, giving an alcohol which is about 90% the (S)-(+) enantiomer, and two Mucor species give as much as 80% the ( R ) - ( - ) enantiomer. The synthesis of certain chiral alcohols from their corresponding ketones by microbial reductases can offer a simple procedure for obtaining sufficient amounts of these substances for certain behavioral studies. Key Words--Chiral alcohols, 2-heptanol, 3-octanol, microbial reductases, Curvularia falcata, Mucor species.
INTRODUCTION T h e m a n d i b u l a r glands o f three species o f M y r m i c a ants contain 3-octanol as a c o m p o n e n t o f their a l a r m p h e r o m o n e blend, and this alcohol is > 90% the (R)( - ) e n a n t i o m e r (Attygalle et al., 1983). Bioassays o f the p h e r o m o n a l activity o f the separate e n a n t i o m e r s o f 3 - o c t a n o l for three species o f these ants indicated that they respond o n l y to the ( R ) - ( - ) e n a n t i o m e r ; the ( S ) - ( + ) e n a n t i o m e r is 1Present address: Biochemistry Department, University of Fort Hare, Alice 5700 Ciskei, South Africa. 357 0098-0331/87/0200-0357505.00/0
9 1987 Plenum Publishing Corporation
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BRAND ET AL.
inactive (Cammaerts et al., 1985). The separate enantiomers of 2-octanol, which are commercially available, gave little or no response with these Myrmica ants. In contrast, two species of Crematogaster ants produce exclusively (S)-(+)-3octanol in their mandibular glands (Brand, 1985), but no bioassays have been conducted with the separate enantiomers on these species. The results of Cammaerts et al. (1985) illustrate the need for behavioral studies on the separate enantiomers of such simple alcohols as 3-octanol in insect species that produce them. However, the separate enantiomers generally are not available to the biologist wishing to bioassay them, and a chemist might not be persuaded easily to synthesize the enantiomers of such mundane alcohols. For their bioassays, Cammaerts et al. (1985) isolated (S)-(+)-3-octanol by preparative gas chromatography from oil of Japanese peppermint (Mentha japonica), and (R)-(-)-3-octanol from heads of worker ants of Myrmica ruginodis. However, in many cases, the preparation of microliter quantities of a compound from the insects themselves may not be feasible. The two alcohols, 2-heptanol and 3-octanol, occur in various insect secretions (Blum, 1981). While the separate enantiomers of 2-heptanol are available from Norse Laboratories, Newbury Park, California 91320, the separate enantiomers of 3-octanol are not available commercially. The enantiomers of each of these simple alcohols could be produced by the action of an appropriate dehydrogenase on the corresponding ketone, a reaction obviously taking place in vivo. Reduction of ketones by microbial reductases, using whole cells, often with high yields of only one enantiomer, is quite common, and this procedure can be used to obtain appreciable amounts of product (MacLeod et al., 1964; Sariaslani and Rosazza, 1984, and references therein; Wong and Drneckhammer, 1985, and references therein). We have synthesized each enantiomer of 2-heptanol and 3-octanol from the corresponding ketone in 80-90 % purity with microbial reductases. The chirality of the alcohol obtained was determined by gas chromatography of the (R)(+)-trans-chrysanthemoyl esters as described previously (Attygalle et al., 1983; Brand, 1985). Curvularia falcata gives approximately 90 % (S)-(+)-2-heptanol and (S)-(+)-3-octanol, and two Mucor species produce 70-80% (R)-(-)-2heptanol and (R)-(-)-3-octanol. These studies have been done using whole cells in their culture medium and the procedure can be scaled up to produce sufficient quantities of material for behavioral work. METHODS AND MATERIALS
All fungal strains were obtained from the personal collection of J.P.N. Rosazza, University of Iowa. The successful strains used were Curvularia falcata QM-72 D, Mucor recurvatus UI-36, and M. mucedo 20094 P. The organisms were grown and maintained on soybean meal-glucose medium consisting of (grams per liter distilled water): glucose 20 g, yeast extract 5 g, soybean
359
ENRICHED 2-HEPTANOL AND 3-OCTANOL
meal 5 g, NaC1 5 g, and K2HPO4 5 g. The pH of the medium was adjusted to 7.0 before autoclaving. Cultures were inoculated from an actively growing culture (5 % inoculum), and grown with vigorous shaking in 100 ml medium in 250 ml Erlenmeyer flasks or 1 liter medium in Fernbach flasks. After 1 to 3 days of growth, the cultures were placed in an anaerobic chamber (Coy Mfg. Co.; gas phase: 85 % N2, 10% H2, 5% CO2) and allowed to become anaerobic for several hours before addition of either 2-hePtanone or 3-octanone (200/~1 for 100 ml medium or 2 ml for 1 liter medium). Preliminary studies showed that yields of the alcohol were higher with cultures kept anaerobic rather than aerobic. The period of exposure to either ketone varied from 6 hr to 8 days. After exposure to either ketone, all the cultures were extracted twice with ether and the ether dried over anhydrous Na2SO4. C. falcata cultures turn black with time, and it is only when the organism is black that it is extracted easily with ether. Extraction of the earlier yellowish-brown stage results in an emulsion that is extremely difficult to break. Mucor cultures did not give any extraction problems. Gas chromatographic determination of the ketone-alcohol ratio was done at 70~ using a Supelcowax 10 fused silica capillary column (15 m). Derivatization of the alcohols was done on the ketone-alcohol mixture with (R)-(+)trans-chrysanthemic acid as described previously (Brand, 1985). Separation of the 2-heptanyl esters was achieved at 115~ and the 3-octanyl esters at 130~ on the Supelcowax 10 column. GC-MS of the standard 2-heptanyl and 3-octanyl esters and of the same esters from microbially produced alcohols was carried out on a 5 % phenyl methyl silicone fused silica capillary column (25 m) at 140~ interfaced to a Finnigan 1015 quadrupole mass spectrometer. RESULTS
Gas chromatography of the ether extracts from C. falcata and Mucor cultures indicated that the added ketone and its corresponding alcohol were the only major volatile compounds in the extract. The formation of the (R)-(+)trans-chrysanthemates was performed on the ketone-alcohol mixture without any further purification. Confirmation of the structures of the (R)-(+)-transchrysanthemoyl esters was obtained by comparison of the retention times with standards on the Supelcowax 10 column, and by GC-MS on the 5% phenyl methyl silicone column interfaced to a Finnigan 1015 quadrupole mass spectrometer. Equivalent mass spectra were obtained from the esters of standard 2heptanol and 3-octanol and the microbially produced alcohols. The characteristic base peak at m/e 123 was obtained in all cases with a molecular ion at m/e 266 for the 2-heptanyl ester and at m/e 280 for the 3-octanyl ester. 2-Heptanone. C. falcata gave 2-heptanol that was 87-90% the (S)-(+) enantiomer, M. recurvatus gave about 76% the (R)-(-) enantiomer, and M.
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BRAND ET AL.
mucedo gave 70-80% the ( R ) - ( - ) enantiomer. The recovery of the ketone plus alcohol as a percentage of the initial ketone added was as high as 70%. 3-Octanone. C. falcata gave 3-octanol that was 86-90% the (S)-(+) enantiomer, M. recurvatus gave about 76% the ( R ) - ( - ) enantiomer, and M. mucedo gave 75-80% the ( R ) - ( - ) enantiomer. The recovery of the ketone plus alcohol as a percentage of the initial ketone added was as high as 90 %. In one experiment with 3 liters of C. falcata treated with 3-octanone and kept anaerobic for eight days, the ratio of ketone to alcohol recovered was 20: 80, with the 3-octanol being 90 % the (S)-(+) enantiomer. In another experiment, 3 liters of M. mucedo, kept anaerobic for 6 days, gave 10 : 90 ketone to alcohol but the 3-octanol was only 61% the ( R ) - ( - ) enantiomer. In each of these latter two experiments 0.3-0.4 ml of the alcohol was obtained. DISCUSSION
Cammaerts et al. (1985) conducted satisfactory bioassays on Myrmica ants using 3-octanol that is approximately 90% the ( R ) - ( - ) enantiomer. This illustrates that certain behavioral experiments are possible with an alcohol that is highly enriched in only one enantiomer and that absolute purity of the enantiomer is not necessary for some studies. The (S)-(+)-2-heptanol and the (S)(+)-3-octanol of about 90% purity produced by C. falcata and the ( R ) - ( - ) enantiomer of 75-80% purity produced by M. mucedo might be adequate for similar behavioral studies on some insects that produce these alcohols. However, the lack of activity of one enantiomer, as is the case with Myrmica (Cammaerts et al., 1985), may be an important criterion in the interpretation of bioassay data on alcohols of the above purity. An extensive literature documents the inhibitory effects of small percentages of unnatural enantiomers on the response of certain insects, and a cautionary note is offered on this point when bioassaying enantiomers that are only highly enriched and are not absolutely pure. Neither the C. falcata nor the Mucor species reduced all the ketone to the alcohol. The best conversions obtained were a 20 : 80 ratio of 3-octanone to 3octanol with C. falcata after anaerobic exposure to the ketone for eight days, and a 10:90 ratio with M. mucedo after six days. Sih and Chen (1984) have pointed out that a major complication in using intact cells for the reduction of ketones is that the process may be only partially enantioselective. They conclude that this usually arises from the combined action of competing enzymes of opposite chirality in intact cells. A ratio of 9 : 1 of the S: R enantiomers was obtained with C. falcata whether the exposure time was 6 hr or eight days, and in contrast, the M. mucedo usually gave about 80% the ( R ) - ( - ) enantiomer with an 18-hr exposure, but only 61% after six days. We have not attempted to optimize conditions for these organisms and do not know whether competing enzymes are present. Certain additional microorganisms tested, e.g., C. pallescens ATCC 12018 and bakers' yeast, did not produce any alcohol under the
ENRICHED 2-HEPTANOL AND 3-OCTANOL
361
usual conditions employed. However, it seems certain that suitable organisms and conditions could be found that would give better than 90 % of either enantiomer in good yield. As only one enantiomer of the alcohol was a major product, we wondered whether these organisms would selectively oxidize the same enantiomer if given the racemic alcohol under aerobic conditions. Our attempts to do this failed completely, and the reduction of the ketone seems irreversible with these organisms. This irreversibility of reduction has been mentioned by MacLeod et al. (1964) and by Sih and Chen (1984) and is understandable under anaerobic fermentative conditions. We have no explanation as to why our organisms failed to reoxidize any alcohol under aerobic conditions. In previous experiments we have found a fungus that would oxidize racemic trans-verbenol to verbenone quantitatively under aerobic conditions (Brand et al., 1976). A different approach employing the enantioselective hydrolysis of certain racemic acyclic alcohol acetates by microbial esterases, to yield chiral alcohols such as (S)-(+)3-octanol, has been described by Oritani and Yamashita (1980). As the reduction of the ketone is incomplete, solvent extracts of the culture medium contain variable amounts of the added ketone. Separation of the alcohol from the ketone could be achieved by adsorption column chromatography and preparative gas chromatography. REFERENCES
ATTYGALLE,A.B., MORGAN,E.D., EVERSHED,R.P., and ROWLAND, S.J. 1983. Comparison of three derivatives for the enantiomeric separation of chiral alcohols and the absolute configuration of Myrmica ant 3-octanol. J. Chromatogr. 260:411-417. BLUM, M.S. 1981. Chemical Defenses of Arthropods. Academic Press. New York, 562pp. BRAND, J.M., BRACKE, J.W., MARKOVErZ, A.J., BRITTON, L.N. and BARRAS, S.J. 1976. Bark beetle pheromones: production of verhenone by a mycangial fungus of Dendroctonusfrontalis. J. Chem. Ecol. 2:195-199. BRAND, J.M. 1985. Enantiomeric composition of an alarm pheromone component of the ants Crematogaster castanea and C. liengmei. J. Chem. Ecol. 1 l: 177-180. CAIVIMAERTS,M.C., ATTYGALLE, A.B., EVERSHED, R.P., and MORGAN, E.D. 1985. The pheromonal activity of chiral 3-octanol for Myrmica ants. Physiol. Entomol. 10:33-36. MACLEOD, R., PROSSER, H., FIKENTSCIqER, L., LANYI, J., and MOSHER, H.S. 1964. Asymmetric reductions. XII Stereoselective ketone reductions by fermenting yeast. Biochemistry 3:838846. ORITANI, T., and YAMASHITA,K. 1980. Enzymic resolution of (_)-acyclic alcohols via asymmetric hydrolysis of corresponding acetates by microorganisms. Agrie. Biol. Chem. 44:2407-2411. SARIASLANI,F.S., and ROSAZZA,J.P.N. 1984. Biocatalysis in natural products chemistry. Enzyme Microb. Technol. 6:242-253. SIH, C.J., and CHE~, C.-S. 1984. Microbial asymmetric catalysis--enantioselective reduction of ketones. Angew. Chem. Int. Ed. Engl. 23:570-578. WONG, C.-H., and DRUECKHAMMER, D.G. 1985. Enzymatic synthesis of chiral hydroxy compounds using immobilized glucose dehydrogenase from Bacillus cereus for NAD(P)H regeneration. Bio/Technology 3:649-651.
SYNTHESIS OF ENANTIOMERICALLY ENRICHED 2-HEPTANOL A N D 3-OCTANOL BY MICROBIAL REDUCTASES OF Curvularia falcata AND Mucor SPECIES
J O H N M. B R A N D , 1 D I A N A L. C R U D E N , MARKOVETZ
and A L L E N
J.
Microbiology Department, University of lowa Iowa City, Iowa 52242 (Received January 8, 1986; accepted February 28, 1986) Abstract--Certain insects produce 2-heptanol or 3-octanol in various glandular secretions and recent studies have shown that the 3-octanol of two different genera of ants (Crematogaster and Myrmica) can be either the (S)-(+) or mainly the (R)-(-) enantiomer, respectively. Synthesis of each of these alcohols can be achieved in relatively high enantiomeric purity by certain microbial reductases. The corresponding ketone of each alcohol is reduced by Curvularia falcata, giving an alcohol which is about 90% the (S)-(+) enantiomer, and two Mucor species give as much as 80% the ( R ) - ( - ) enantiomer. The synthesis of certain chiral alcohols from their corresponding ketones by microbial reductases can offer a simple procedure for obtaining sufficient amounts of these substances for certain behavioral studies. Key Words--Chiral alcohols, 2-heptanol, 3-octanol, microbial reductases, Curvularia falcata, Mucor species.
INTRODUCTION T h e m a n d i b u l a r glands o f three species o f M y r m i c a ants contain 3-octanol as a c o m p o n e n t o f their a l a r m p h e r o m o n e blend, and this alcohol is > 90% the (R)( - ) e n a n t i o m e r (Attygalle et al., 1983). Bioassays o f the p h e r o m o n a l activity o f the separate e n a n t i o m e r s o f 3 - o c t a n o l for three species o f these ants indicated that they respond o n l y to the ( R ) - ( - ) e n a n t i o m e r ; the ( S ) - ( + ) e n a n t i o m e r is 1Present address: Biochemistry Department, University of Fort Hare, Alice 5700 Ciskei, South Africa. 357 0098-0331/87/0200-0357505.00/0
9 1987 Plenum Publishing Corporation
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BRAND ET AL.
inactive (Cammaerts et al., 1985). The separate enantiomers of 2-octanol, which are commercially available, gave little or no response with these Myrmica ants. In contrast, two species of Crematogaster ants produce exclusively (S)-(+)-3octanol in their mandibular glands (Brand, 1985), but no bioassays have been conducted with the separate enantiomers on these species. The results of Cammaerts et al. (1985) illustrate the need for behavioral studies on the separate enantiomers of such simple alcohols as 3-octanol in insect species that produce them. However, the separate enantiomers generally are not available to the biologist wishing to bioassay them, and a chemist might not be persuaded easily to synthesize the enantiomers of such mundane alcohols. For their bioassays, Cammaerts et al. (1985) isolated (S)-(+)-3-octanol by preparative gas chromatography from oil of Japanese peppermint (Mentha japonica), and (R)-(-)-3-octanol from heads of worker ants of Myrmica ruginodis. However, in many cases, the preparation of microliter quantities of a compound from the insects themselves may not be feasible. The two alcohols, 2-heptanol and 3-octanol, occur in various insect secretions (Blum, 1981). While the separate enantiomers of 2-heptanol are available from Norse Laboratories, Newbury Park, California 91320, the separate enantiomers of 3-octanol are not available commercially. The enantiomers of each of these simple alcohols could be produced by the action of an appropriate dehydrogenase on the corresponding ketone, a reaction obviously taking place in vivo. Reduction of ketones by microbial reductases, using whole cells, often with high yields of only one enantiomer, is quite common, and this procedure can be used to obtain appreciable amounts of product (MacLeod et al., 1964; Sariaslani and Rosazza, 1984, and references therein; Wong and Drneckhammer, 1985, and references therein). We have synthesized each enantiomer of 2-heptanol and 3-octanol from the corresponding ketone in 80-90 % purity with microbial reductases. The chirality of the alcohol obtained was determined by gas chromatography of the (R)(+)-trans-chrysanthemoyl esters as described previously (Attygalle et al., 1983; Brand, 1985). Curvularia falcata gives approximately 90 % (S)-(+)-2-heptanol and (S)-(+)-3-octanol, and two Mucor species produce 70-80% (R)-(-)-2heptanol and (R)-(-)-3-octanol. These studies have been done using whole cells in their culture medium and the procedure can be scaled up to produce sufficient quantities of material for behavioral work. METHODS AND MATERIALS
All fungal strains were obtained from the personal collection of J.P.N. Rosazza, University of Iowa. The successful strains used were Curvularia falcata QM-72 D, Mucor recurvatus UI-36, and M. mucedo 20094 P. The organisms were grown and maintained on soybean meal-glucose medium consisting of (grams per liter distilled water): glucose 20 g, yeast extract 5 g, soybean
359
ENRICHED 2-HEPTANOL AND 3-OCTANOL
meal 5 g, NaC1 5 g, and K2HPO4 5 g. The pH of the medium was adjusted to 7.0 before autoclaving. Cultures were inoculated from an actively growing culture (5 % inoculum), and grown with vigorous shaking in 100 ml medium in 250 ml Erlenmeyer flasks or 1 liter medium in Fernbach flasks. After 1 to 3 days of growth, the cultures were placed in an anaerobic chamber (Coy Mfg. Co.; gas phase: 85 % N2, 10% H2, 5% CO2) and allowed to become anaerobic for several hours before addition of either 2-hePtanone or 3-octanone (200/~1 for 100 ml medium or 2 ml for 1 liter medium). Preliminary studies showed that yields of the alcohol were higher with cultures kept anaerobic rather than aerobic. The period of exposure to either ketone varied from 6 hr to 8 days. After exposure to either ketone, all the cultures were extracted twice with ether and the ether dried over anhydrous Na2SO4. C. falcata cultures turn black with time, and it is only when the organism is black that it is extracted easily with ether. Extraction of the earlier yellowish-brown stage results in an emulsion that is extremely difficult to break. Mucor cultures did not give any extraction problems. Gas chromatographic determination of the ketone-alcohol ratio was done at 70~ using a Supelcowax 10 fused silica capillary column (15 m). Derivatization of the alcohols was done on the ketone-alcohol mixture with (R)-(+)trans-chrysanthemic acid as described previously (Brand, 1985). Separation of the 2-heptanyl esters was achieved at 115~ and the 3-octanyl esters at 130~ on the Supelcowax 10 column. GC-MS of the standard 2-heptanyl and 3-octanyl esters and of the same esters from microbially produced alcohols was carried out on a 5 % phenyl methyl silicone fused silica capillary column (25 m) at 140~ interfaced to a Finnigan 1015 quadrupole mass spectrometer. RESULTS
Gas chromatography of the ether extracts from C. falcata and Mucor cultures indicated that the added ketone and its corresponding alcohol were the only major volatile compounds in the extract. The formation of the (R)-(+)trans-chrysanthemates was performed on the ketone-alcohol mixture without any further purification. Confirmation of the structures of the (R)-(+)-transchrysanthemoyl esters was obtained by comparison of the retention times with standards on the Supelcowax 10 column, and by GC-MS on the 5% phenyl methyl silicone column interfaced to a Finnigan 1015 quadrupole mass spectrometer. Equivalent mass spectra were obtained from the esters of standard 2heptanol and 3-octanol and the microbially produced alcohols. The characteristic base peak at m/e 123 was obtained in all cases with a molecular ion at m/e 266 for the 2-heptanyl ester and at m/e 280 for the 3-octanyl ester. 2-Heptanone. C. falcata gave 2-heptanol that was 87-90% the (S)-(+) enantiomer, M. recurvatus gave about 76% the (R)-(-) enantiomer, and M.
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mucedo gave 70-80% the ( R ) - ( - ) enantiomer. The recovery of the ketone plus alcohol as a percentage of the initial ketone added was as high as 70%. 3-Octanone. C. falcata gave 3-octanol that was 86-90% the (S)-(+) enantiomer, M. recurvatus gave about 76% the ( R ) - ( - ) enantiomer, and M. mucedo gave 75-80% the ( R ) - ( - ) enantiomer. The recovery of the ketone plus alcohol as a percentage of the initial ketone added was as high as 90 %. In one experiment with 3 liters of C. falcata treated with 3-octanone and kept anaerobic for eight days, the ratio of ketone to alcohol recovered was 20: 80, with the 3-octanol being 90 % the (S)-(+) enantiomer. In another experiment, 3 liters of M. mucedo, kept anaerobic for 6 days, gave 10 : 90 ketone to alcohol but the 3-octanol was only 61% the ( R ) - ( - ) enantiomer. In each of these latter two experiments 0.3-0.4 ml of the alcohol was obtained. DISCUSSION
Cammaerts et al. (1985) conducted satisfactory bioassays on Myrmica ants using 3-octanol that is approximately 90% the ( R ) - ( - ) enantiomer. This illustrates that certain behavioral experiments are possible with an alcohol that is highly enriched in only one enantiomer and that absolute purity of the enantiomer is not necessary for some studies. The (S)-(+)-2-heptanol and the (S)(+)-3-octanol of about 90% purity produced by C. falcata and the ( R ) - ( - ) enantiomer of 75-80% purity produced by M. mucedo might be adequate for similar behavioral studies on some insects that produce these alcohols. However, the lack of activity of one enantiomer, as is the case with Myrmica (Cammaerts et al., 1985), may be an important criterion in the interpretation of bioassay data on alcohols of the above purity. An extensive literature documents the inhibitory effects of small percentages of unnatural enantiomers on the response of certain insects, and a cautionary note is offered on this point when bioassaying enantiomers that are only highly enriched and are not absolutely pure. Neither the C. falcata nor the Mucor species reduced all the ketone to the alcohol. The best conversions obtained were a 20 : 80 ratio of 3-octanone to 3octanol with C. falcata after anaerobic exposure to the ketone for eight days, and a 10:90 ratio with M. mucedo after six days. Sih and Chen (1984) have pointed out that a major complication in using intact cells for the reduction of ketones is that the process may be only partially enantioselective. They conclude that this usually arises from the combined action of competing enzymes of opposite chirality in intact cells. A ratio of 9 : 1 of the S: R enantiomers was obtained with C. falcata whether the exposure time was 6 hr or eight days, and in contrast, the M. mucedo usually gave about 80% the ( R ) - ( - ) enantiomer with an 18-hr exposure, but only 61% after six days. We have not attempted to optimize conditions for these organisms and do not know whether competing enzymes are present. Certain additional microorganisms tested, e.g., C. pallescens ATCC 12018 and bakers' yeast, did not produce any alcohol under the
ENRICHED 2-HEPTANOL AND 3-OCTANOL
361
usual conditions employed. However, it seems certain that suitable organisms and conditions could be found that would give better than 90 % of either enantiomer in good yield. As only one enantiomer of the alcohol was a major product, we wondered whether these organisms would selectively oxidize the same enantiomer if given the racemic alcohol under aerobic conditions. Our attempts to do this failed completely, and the reduction of the ketone seems irreversible with these organisms. This irreversibility of reduction has been mentioned by MacLeod et al. (1964) and by Sih and Chen (1984) and is understandable under anaerobic fermentative conditions. We have no explanation as to why our organisms failed to reoxidize any alcohol under aerobic conditions. In previous experiments we have found a fungus that would oxidize racemic trans-verbenol to verbenone quantitatively under aerobic conditions (Brand et al., 1976). A different approach employing the enantioselective hydrolysis of certain racemic acyclic alcohol acetates by microbial esterases, to yield chiral alcohols such as (S)-(+)3-octanol, has been described by Oritani and Yamashita (1980). As the reduction of the ketone is incomplete, solvent extracts of the culture medium contain variable amounts of the added ketone. Separation of the alcohol from the ketone could be achieved by adsorption column chromatography and preparative gas chromatography. REFERENCES
ATTYGALLE,A.B., MORGAN,E.D., EVERSHED,R.P., and ROWLAND, S.J. 1983. Comparison of three derivatives for the enantiomeric separation of chiral alcohols and the absolute configuration of Myrmica ant 3-octanol. J. Chromatogr. 260:411-417. BLUM, M.S. 1981. Chemical Defenses of Arthropods. Academic Press. New York, 562pp. BRAND, J.M., BRACKE, J.W., MARKOVErZ, A.J., BRITTON, L.N. and BARRAS, S.J. 1976. Bark beetle pheromones: production of verhenone by a mycangial fungus of Dendroctonusfrontalis. J. Chem. Ecol. 2:195-199. BRAND, J.M. 1985. Enantiomeric composition of an alarm pheromone component of the ants Crematogaster castanea and C. liengmei. J. Chem. Ecol. 1 l: 177-180. CAIVIMAERTS,M.C., ATTYGALLE, A.B., EVERSHED, R.P., and MORGAN, E.D. 1985. The pheromonal activity of chiral 3-octanol for Myrmica ants. Physiol. Entomol. 10:33-36. MACLEOD, R., PROSSER, H., FIKENTSCIqER, L., LANYI, J., and MOSHER, H.S. 1964. Asymmetric reductions. XII Stereoselective ketone reductions by fermenting yeast. Biochemistry 3:838846. ORITANI, T., and YAMASHITA,K. 1980. Enzymic resolution of (_)-acyclic alcohols via asymmetric hydrolysis of corresponding acetates by microorganisms. Agrie. Biol. Chem. 44:2407-2411. SARIASLANI,F.S., and ROSAZZA,J.P.N. 1984. Biocatalysis in natural products chemistry. Enzyme Microb. Technol. 6:242-253. SIH, C.J., and CHE~, C.-S. 1984. Microbial asymmetric catalysis--enantioselective reduction of ketones. Angew. Chem. Int. Ed. Engl. 23:570-578. WONG, C.-H., and DRUECKHAMMER, D.G. 1985. Enzymatic synthesis of chiral hydroxy compounds using immobilized glucose dehydrogenase from Bacillus cereus for NAD(P)H regeneration. Bio/Technology 3:649-651.
