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DOI:10.15227/orgsyn.069.0019

Organic Syntheses, Coll. Vol. 8, p.332 (1993); Vol. 69, p.19 (1990).

ENANTIOSELECTIVE SAPONIFICATION WITH PIG LIVER
ESTERASE (PLE): (1S,2S,3R)-3-HYDROXY-2NITROCYCLOHEXYL ACETATE[1,3-Cyclohexanediol, 2-nitro-, 1-acetate, [1S-(1α, 2β, 3α]]Submitted by Martin Eberle, Martin Missbach, and Dieter Seebach1.
Checked by David L. Coffen

1. Procedure
A. (1R,2r,3S)-2-Nitrocyclohexane-1,3-diol. A 1-L, round-bottomed flask, equipped with a magneticstirrer, a thermometer, and an ice–ethanol bath is charged with 175 mL (0.455 mol) of an aqueous 25%
solution of glutaric dialdehyde, 38 mL (0.708 mol) of nitromethane, and 600 mL of methanol (Note 1).
At 0–5°C 12 mL of aqueous 2 M sodium hydroxide is added gradually. The cooling bath is removed
and the reaction mixture is stirred for 4 hr at room temperature. The resulting yellow solution isneutralized by adding 15 g of acidic cation-exchange resin and stirring for an additional 20 min (Note2). The resin is filtered off and washed with a small volume of methanol. The filtrate is evaporated to asemisolid residue using reduced pressure and a 35°C water bath. The residue is dissolved in 100 mL ofabsolute ethyl alcohol with heating and diluted by gradual addition of 250 mL of toluene. The resulting
two-phase mixture (Note 3) is again evaporated, with azeotropic removal of water. The resulting residueis again taken up in 100 mL of hot ethyl alcohol and diluted with 250 mL of toluene (Note 3). The
almost colorless crystals are filtered and dried at high vacuum to yield 44–52 g (60–70%) of nitrodiol,
mp 152–155°C (dec).
B. (1R,2r,3S)-3-Acetoxy-2-nitrocyclohexyl acetate. In a 1-L flask 52 g (0.323 mol) of the nitrodiol
are suspended in 150 mL of acetic anhydride. Without cooling, 3–6 drops of concentrated sulfuric acidare added (Note 4). After 1 hr 500 mL of ice/water is rapidly added and stirring is continued for 60 min.
The resulting colorless crystals are filtered, washed with water, and air-dried. The product thus
obtained, 75.6 g (95.6%) of colorless crystals, is pure by TLC (4 : 1 hexane/ethyl acetate) and NMR,and melts at 89–90°C (Note 5).

C. (1S,2S,3R)-3-Hydroxy-2-nitrocyclohexyl acetate. A 500-mL flask equipped with a magnetic
stirrer is charged with 10 g (41 mmol) of powdered nitrodiacetate and 300 mL of 0.2 M phosphatebuffer of pH 7.0 prepared by dissolving 11 g of potassium dihydrogenphosphate and 3.3 g of potassiumhydroxide in 300 mL of deionized (or distilled) water. To the stirred suspension is added 30 mg of
purified PLE [Esterase (EC 3.1.1.1), suspended in 2.8 mL of 3.2 M ammonium sulfate buffer] (Note 6)
and the mixture is stirred for 24 to 48 hr (Note 7). The continuously measured pH drops to about 5.6
during the reaction and then remains almost constant, while the mixture has turned to a practically clear,pale-yellow solution (Note 8). The solution is filtered through a paper filter and the filtrate is extracted 3times with 100 mL of ether. The organic phase is dried over anhydrous magnesium sulfate and filtered.
Removal of the solvent and of the acetic acid under reduced pressure leaves 7–8 g (85–95%) ofcolorless monoacetate as a crystalline solid (Note 9). Recrystallization by dissolving in 100 mL of ether
and adding 250 mL of pentane gives 5–6 g (60–70%) of pure (1S,2S,3R)-3-hydroxy-2-nitrocyclohexylacetate (Note 10), mp 90–91°C, [α]D + 9.5° (CHCl3, c 1.0) (Note 11). From the mother liquor, another1–2 g (12–24%) of monoacetate, mp 89–90°C, can be obtained (Note 12).

2. Notes
1. Commercial-grade chemicals were used without further purification. The glutaric dialdehyde solution
should be fresh.
2. The checkers used Amberlite IR-120 (plus) acid form, capacity 1.9 meq/mL supplied by the Aldrich
Chemical Company, Inc.
3. Two layers will form unless the water content of the crude product is sufficiently reduced during thepreceding evaporation; in that case, the evaporation is to be repeated.
4. The solution turns clear and the temperature rises to 60–70°C when the reaction has started. Moresulfuric acid should be added if a sustained exotherm does not ensue.
5. The submitters recommend recrystallization from alcohol/water (2 : 1). This is essential if NMR andTLC analyses of the crude nitrodiacetate indicate the presence of monoacetate.
6. The checker used the contents of one 30-mg vial of Sigma material, rinsed in with ca. 1 mL ofdeionized water. The submitters originally developed the process with PLE purified according to aprocedure they had published previously.2
7. The submitters used 45 mg of enzyme and observed the reaction to be complete in 11.5–12.5 hr.
8. In addition to the monoacetate, a small amount of diacetate and an even smaller amount of diol could
be detected by TLC in the crude product.
9. The crude product is enantiomerically pure according to 19F NMR of the corresponding Mosher ester(>97% ee). The checker observed lower [α]D25 values (+8.72° and +8.61°) but confirmed theenantiomeric purity by HPLC analysis of the corresponding Mosher esters. By HPLC comparison withthe diastereomeric mixture of Mosher esters prepared from a sample of racemic monoacetate (oilysubstance obtained by partial hydrolysis of diacetate), the (−)-enantiomer content appears to be less than1%.
10. The absolute configuration of the product has been proved by X-ray analysis of the corresponding
camphanic ester.2,3
11. The monoacetate shows the following 1H NMR spectrum (90 MHZ) δ: 1.3–1.9 (m, 4 H), 2.0 (s, 3H), 2.1–2.2 (m, 2 H), 2.7 (d, J = 5.5, OH), 4.1 (m, 1 H), 4.4 (apparent, t, J = 10.5, 1H), 5.1–5.3 (m, 1 H).
12. Recrystallization of this fraction gave another 0.75–1.5 g (10–18%) of pure product.

3. Discussion
The use of ester-cleaving enzymes will probably be one of the most useful biological–chemicalmethods in the synthetic laboratory. No example of this type of reaction has hitherto been published in
the Organic Syntheses series of procedures. So far, the only biological–chemical Organic Syntheses
procedures are two yeast reductions,4,5 one oxidation with horse liver alcohol dehydrogenase,6 and adisaccharide synthesis catalyzed by emulsin.7 The procedure described here is also applicable with
crude enzyme powder, but the workup is a bit more complicated, because a continuous extractor must
be used to overcome problems with emulsions. Both crude enzyme concentrate and purified PLE as a
mixture of isoenzymes8 are commercially available, but the crude concentrate can easily be preparedfrom fresh pig liver and is thus very cheap.2 By using self-made PLE-powder, the submitters haveproduced amounts of 20–25 g of pure monoacetate per run.2

Several review articles containing discussions of enantioselective syntheses with ester-cleavingenzymes have appeared.9,10,11,12,13 Of the many examples, the ones in which meso substrates are
employed are most attractive since the theoretical yield is 100%. In many applications of PLE theenantiomeric excess (% ee) of the product depends crucially on the source of the enzyme. This effecthas not been noticed in the enantioselective saponifications of nitrodiol diacetates, either because thereaction is insensitive to it, or because this complication is overcome by the great crystallization
tendency of the products. The only problem we observed when the reaction was carried out withcommercial crude PLE-powder (but not with the self made one) was the production of a certain amountof diol that could not be removed by simple recrystallization. In this case, filtration over a short silica
gel column with methylene chloride as eluent gives after recrystallization pure monoacetate.
Other examples of enantiomerically pure monoacetates of meso-nitrodiols, which are available
using the procedure described above, are collected in Table I. Entries 1 and 3 in Table I refer to runs
following the above procedure, for all other cases the self made crude PLE-powder was used. Cases inwhich no reaction (a) or unsatisfactory selectivities (b) were observed, are shown below.

TABLE IYIELDS, MELTING POINTS, AND SPECIFIC ROTATIONS OF NITRODIOLMONOACETATES PREPARED BY PROCEDURE C USING PLE-POWDER2 INSTEAD OF
PURIFIED ENZYMEa
Entry No.

Yield (%)

Mp (°C)[α]D (CHCl3, c 1)

150–70

58–60

−10.5

240–50Oil−10.7

360–70

90–91

+9.8

480–90

106–107−9.4

570–80

91–92

+29.1

660–70

130–131−1.3

720–30

111–112+14.7

860–70

130–131 (dec)+28.1

960–70

93–94

+6.0

Entries 1 and 3 refer to runs following the above procedure; for all other cases theself made crude PLE-powder was used. The configuration and the sense of chiralityof the products of entries 1, 3, and 4 were determined by x-ray crystal structure
analysis of the camphanic esters, those of the other are inferred by analogy and by
NMR comparison. The open-chain compounds (entries 1 and 2) were obtained using
TES buffer at pH 6.5.
aThe meso-nitrodiol starting materials for the preparation of the PLE diacetate substrates are readily
obtained from nitromethane or nitroethane and aldehydes or dialdehydes. They crystallize readily. The
above procedures for the preparation of nitrocyclohexanediol and its diacetate from glutaraldehyde andnitromethane are modifications of published methods.14,15
The chiral monoacetates now available are useful multiple coupling reagents16,17,18 for syntheses ofenantiomerically pure target molecules. They can be converted to nitroolefinic allylic esters, achiral orracemic analogs of which we have previously shown16,17,18 to combine sequentially with two (different)
nucleophiles (see 1 and 2 in Scheme 1).
In a first approach to an enantioselective version of this method, we employed19 chiral enamines
derived from proline,20 (see 1–3 in Scheme 1). In this stoichiometric, enantioselective reaction, thevaluable auxiliary used has to be recovered (i.e., recycled) in preparative-scale applications.21

We then used22 nitroallylic pivalates for the alkylation of hydroxy acid-derived enolates to prepare
enantiomerically pure compounds (EPC), (see 4 and 5 in Scheme 2), an example of the use of the poolof chiral building blocks for EPC syntheses.23,24,25 Finally, the procedure described here allows forsyntheses of EPC with a catalytic enantioselective step:26 dehydration of the monoacetate from the PLE
saponification leads to (S)-nitrocyclohexenyl acetate 6, and pivaloylation followed by acetate hydrolysis
and dehydration leads to the pivalate 8 of the enantiomeric alcohol. These compounds can be used forsubstitutions with a variety of nucleophiles.2,3,26 Thus, starting from the enantiomerically pure
Michael acceptors 6 and 8, 3-alkyl nitrocyclohexenes 7 9, respectively, of high enantiomeric excess areavailable (see Scheme 2, Nu = methyllithium, phenyllithium and the morpholinoenamine ofacetophenone):

References and Notes
1. Laboratorium für Organische Chemie der ETH, Universitätstr. 16, CH-8092, Zürich,
Switzerland.
2. Seebach, D.; Eberle, M. Chimia 1986, 40, 315.
3. Eberle, M.; Egli, M.; Seebach, D. Helv. Chim. Acta 1988, 71, 1.
4. Levene, P. A.; Walti, A. Org. Synth., Coll. Vol. II 1943, 545.
5. Seebach, D.; Sutter, M. A.; Weber, R. H.; Züger M. F. Org. Synth., Coll. Vol. VII 1990, 215.
6. Jones, J. B.; Jakovac, I. J. Org. Synth., Coll. Vol. VII 1990, 406.
7. Helferich, B.; Leete, J. F. Org. Synth., Coll. Vol. III 1955, 428.
8. Horgan, D. J.; Stoops, J. K.; Webb, E. C.; Zerner, B. Biochemistry 1969, 8, 2000.
9. Sih, C. J.; Chen. C.-S. Angew. Chem. 1984, 96, 556; Angew. Chem., Int. Ed. Engl. 1984, 23, 570.
10. Simon, H.; Bader, J.; Günther, H.; Neumann, S.; Thanos, J. Angew. Chem., Int. Ed. Engl. 1985,24, 539.
11. Whitesides, G. M.; Wong, C. H. Angew. Chem. 1985, 97, 617; Angew. Chem., Int. Ed. Engl.
1985, 24, 617.
12. Jones, J. B. In "F.E.C.S. Third International Conference on Chemistry and Biotechnology ofBiologically Active Natural Products," Sept. 16–21, 1985, Sofia, Bulgarian Academy ofSciences, Ed.; VCH Publ.: New York, 1987, Vol. 1; pp. 18–39; Breitgoff, D.; Essert, T.; Laumen,K.; Schneider, M. P., Vol. 2, pp. 127–147.
13. See also the three articles on this subject published simultaneously in Chimia: Adachi, K.;
Kobayashi, S.; Ohno, M. Chimia 1986, 40, 311; Seebach, D.; Eberle, M. Chimia 1986, 40, 315;
Whitesell, J. K.; Lawrence, R. M. Chimia 1986, 40, 318. For more recent reviews about enzyme
catalyzed reactions, see: Ohno, M.; Otsuka, M. Org. React. 1989, 37, 1; Crout, D. H. G.;
Christen, M. in "Modern Synthetic Methods 1989;" Scheffold, R., Ed.; Springer Verlag:
Heidelberg, 1989; Chen, C.-S.; Sih, C. J. Angew. Chem. 1989, 101, 711.
14. Lichtenthaler, F. W.; Nakagawa, T.; El-Scherbiney, A. Chem. Ber. 1968, 101, 1837 (diol);
Lichtenthaler, F. W. Chem. Ber. 1963, 96, 845 (diacetate).
15. Baer, H. H.; Shields, D. C.; Dawson, G. G. Can. J. Chem. 1973, 51, 2843.
16. Knochel, P.; Seebach, D. Nouv. J. Chim. 1981, 5, 75; Knochel, P.; Seebach, D. Tetrahedron Lett.
1981, 22, 3223; Knochel, P.; Seebach, D. Tetrahedron Lett. 1982, 23, 3897.
17. Seebach, D.; Knochel, P. Helv. Chim. Acta 1984, 67, 261.
18. Seebach, D.; Calderari, G.; Knochel, P. Tetrahedron 1985, 41, 4861.
19. Seebach, D.; Calderari, G.; Meyer, W. L.; Merritt, A.; Odermann, L. Chimia 1985, 39, 183.
20. Blarer, S. J.; Schweizer, W. B.; Seebach, D. Helv. Chim. Acta 1982, 65, 1637; Blarer, S. J.;

21.
22.
23.
24.
25.
26.

Seebach, D. Chem. Ber. 1983, 116, 2250 and 3086.
Seebach, D.; Imwinkelried, R.; Weber, T. Mod. Synth. Methods 1986, 4, 125–259.
Calderari, G.; Seebach, D. Helv. Chim. Acta 1985, 68, 1592, and unpublished results.
Seebach, D.; Kalinowski, H. O. Nachr. Chem. Techn. 1976, 24, 415.
Seebach, D.; Hungerbühler, E. Mod. Synth. Methods 1980, 91; Conf. Pap. Int. Semin., 2nd 1980,91–171, Scheffold, R., Ed.; Otto Salle Verlag: Frankfurt/Main, Fed. Rep. Ger.
Seebach, D.; Roggo, S.; Zimmermann, J. in "Stereochemistry of Organic and BioorganicTransformations," Bartmann, W.; Shapless, K. B., Eds.; VCH: Weinheim, 1986, New York,1987; pp. 85–126.
Ph. D. Thesis of Martin Eberle, ETH Dissertation No. 8398, Zürich 1987, and part of the
projected Dissertation of Martin Missbach.

Appendix
Chemical Abstracts Nomenclature (Collective Index Number);(Registry Number)
methyllithium, phenyllithium and the morpholinoenamine of acetophenone
1,3-Cyclohexanediol, 2-nitro-, 1-acetate, [1S-(1α, 2β, 3α]ethyl alcohol (64-17-5)
sulfuric acid (7664-93-9)
acetic acid (64-19-7)ethyl acetate (141-78-6)
methanol (67-56-1)
ether (60-29-7)
acetic anhydride (108-24-7)
sodium hydroxide (1310-73-2)
potassium hydroxide (1310-58-3)
toluene (108-88-3)
ammonium sulfate (7783-20-2)
Pentane (109-66-0)
Nitromethane (75-52-5)
methylene chloride (75-09-2)
magnesium sulfate (7487-88-9)

potassium dihydrogenphosphate (7778-77-0)
proline (147-85-3)
hexane (110-54-3)
glutaraldehyde,
glutaric dialdehyde (111-30-8)
nitroethane (79-24-3)
meso-nitrodiol
nitrocyclohexanediol
pivalate(1S,2S,3R)-3-Hydroxy-2-nitrocyclohexyl acetate(S)-nitrocyclohexenyl acetate(1R,2r,3S)-2-Nitrocyclohexane-1,3-diol (38150-01-5)(1R,2r,3S)-3-Acetoxy-2-nitrocyclohexyl acetate
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