An Easy Approach to the Synthesis of Optically Active vic-Diols: A New Single-Enzyme System

Full text of DOI:10.1021/jo9618381

1854
J . Org. Chem. 1997, 62, 1854-1856
Ta ble 1. BSDR-Ca ta lyzed Red u ction s of r-Dik eton es by
An Ea sy Ap p r oa ch to th e Syn th esis of
th e Sin gle-En zym e System
Op tica lly Active vic-Diols: A New
Sin gle-En zym e System
Olga Bortolini, Giancarlo Fantin, Marco Fogagnolo,
Pier Paolo Giovannini, Alessandra Guerrini, and
Alessandro Medici*
Dipartimento di Chimica, Universita’ di Ferrara,
Via Borsari 46, 44100 Ferrara, Italy
Received September 25, 1996
The synthesis of enantiomerically pure vicinal diols is
an area of growing interest, since these compounds are
highly valuable chiral synthons. New efficient processes,
allowing for their synthesis, have been recently developed
by different authors using transition metal catalysis;1
however, the major drawback of these procedures is
represented by the use of heavy metal catalysts, which
may be source of pollution. In this context, enzymes
present attractive alternatives for asymmetric synthesis,2
and numerous illustrations of their applicability to a
broad spectrum of synthetic problems, including the
obtainment of enantiopure diols,3 are well documented.
Nicotinamide cofactor-dependent oxidoreductase, for ex-
ample, is a class of enzymes involved in the oxidation of
alcohols and reduction of ketones.4 For the oxidation of
alcohols that require nicotinamide adenine nucleotide
(NAD) or its phosphate analogue (NADP), or the reduc-
tion of carbonyl compounds mediated by the reduced form
NADH (NADPH), regeneration of the cofactor is neces-
sary. Enzymatic regeneration of NADH (or NADPH) for
synthesis usually employs the glucose/glucose dehy-
drogenase5a or the glucose 6-phosphate/glucose 6-phos-
phate dehydrogenase5b systems. An improvement in the
regeneration cycle is represented by the use of single-
enzyme systems, where one enzyme can catalyze a
desired reaction while simultaneously regenerating the
cofactor. Single-enzyme systems have been established
for the alcohol dehydrogenases from horse liver,6 Ther-
moanaerobium brockii,7 Lactobacillus kefir,8 Pseudo-
monas species,9 and Geotrichum candidum.10
a
Determined by GC analysis on a chiral column, when >98%
the (R,R) isomer is not detected; see the Experimental Section.
Reaction time 72 h, 8 mL of enzyme solution.
b
thermophilus (BSDR).11 This enzyme, also in its crude
form, is an efficient catalyst for the stereospecific redox
reactions of bicyclic octen- and heptenols and ones.12 In
this paper we present the synthesis of several chiral 1,2-
diols, two of them new as (S,S) isomers, that were
obtained from R-diketones by reduction with NADH
catalyzed by BSDR. In addition, the synthetic potential
of the system is illustrated by the synthesis of the
pheromone of the grape borer, Xylotrechus pyrrhoderus.13
The cofactor NADH is conveniently and simultaneously
regenerated by this single-enzyme system in the presence
of a cosubstrates of high synthetic utility.
Pursuing our interest in the development of new
enzymes for organic synthesis, we recently isolated and
characterized a diacetyl reductase from Bacillus stearo-
* To whom correspondence should be addressed: Tel. 39-532-
291172, Fax 39-532-240709, E-mail mda@ifeuniv.unife.it.
(1) (a) Kolb, H. C.; Van Nieuwenhze, M. S.; Sharpless, K. B. Chem.
Rev. 1994, 94, 2483. (b) Katsuki, T. Coord. Chem. Rev. 1995, 140, 189.
(2) Enzyme Catalysis in Organic Synthesis; Drauz, K., Waldmann,
H., Eds.; VCH: Weinheim, 1995; Vols I and II.
(3) (a) Faber, K.; Mischitz, M.; Kroutil, W. Acta Chem. Scand. 1996,
50, 249. (b) Pedragosa-Moreau, S.; Archelas, A.; Furstoss, R. Tetrahe-
dron 1996, 52, 4593. (c) Pedragosa-Moreau, S.; Archelas, A.; Furstoss,
R. Tetrahedron Lett. 1996, 37, 3319. (d) Bellucci, G.; Chiappe, C.;
Cordoni, A. Tetrahedron: Asymmetry 1996, 7, 197.
(4) Fang, J .-M.; Lin, C.-H.; Bradshaw, C. W.; Wong, C.-H. J . Chem.
Soc., Perkin Trans. 1 1995, 967.
(5) (a) Wang, C.-H.; Drueckhammer, D. G.; Sweers, H. M. J . Am.
Chem. Soc. 1985, 107, 4028. (b) Wong, C.-H.; Whitesides, G. M. J . Am.
Chem. Soc. 1981, 103, 4890.
(6) Haslegrave, J . A.; J ones, J . B. J . Am. Chem. Soc. 1982, 104, 4666.
(7) (a) Keinan, E.; Hafeli, E. K.; Seth, K. K.; Lamed, R. J . Am. Chem.
Soc. 1986, 108, 162. (b) Keinan, E.; Seth, K. K.; Lamed, R. J . Am.
Chem. Soc. 1986, 108, 3474
(8) Bradshaw, C. W.; Hummel, W.; Wong, C.-H. J . Org. Chem. 1992,
57, 1532.
(9) (a) Bradshaw, C. W.; Fu, H.; Shen, G.-J .; Wong, C.-H. J . Org.
Chem. 1992, 57, 1526. (b) Shen, G.-J .; Wang, Y.-F.; Bradshaw, C. W.;
Wong, C.-H. J . Chem. Soc., Chem. Commun. 1990, 677.
(10) Nakamura, K.; Kitano, K.; Matsuda, T.; Ohno, A. Tetrahedron
Lett. 1996, 37, 1629.
Resu lts a n d Discu ssion
Bacillus stearothermophilus diacetyl reductase (BSDR)
is specific for 2,3-butanedione (diacetyl) and 2-hydroxy-
3-butanone (acetoin) substrates11 and, as most alcohol
dehydrogenases, catalyzes the transfer of a hydride from
the reduced cofactor to the re-face of the carbonyl function
to give (S) alcohols, according to Prelog’s rule.14 Taking
into account this specificity, the reduction of different
R-diketones bearing aliphatic, cyclic, or aromatic sub-
stituents has been studied. Table 1 lists six R-diketones
that have been reduced to (S,S)-1,2-diols by this catalytic
system. The overall reaction view is shown in Scheme
(11) Giovannini, P. P.; Medici, A.; Bergamini, C. M.; Rippa, M.
Bioorg. Med. Chem. 1996, 4, 1197.
(12) Giovannini, P. P.; Hanau, S.; Rippa, M.; Bortolini, O.; Fogag-
nolo, M.; Medici, A. Tetrahedron 1996, 52, 1669.
(13) (a) Mori, K. Tetrahedron 1989, 45, 3233. (b) Mori, K.; Otsuka,
T. Tetrahedron 1985, 41, 553. (c) Saki, T.; Nakagawa, Y.; Takahashi,
J .; Iwabuchi, K.; Ishii, K. Chem. Lett. 1984, 263.
(14) Prelog, V. Pure Appl. Chem. 1964, 9, 119.
S0022-3263(96)01838-5 CCC: $14.00 © 1997 American Chemical Society

Notes
J . Org. Chem., Vol. 62, No. 6, 1997 1855
Ta ble 2. BSDR-Ca ta lyzed Red u ction s of r-Dik eton es by
Sch em e 1
th e Dou ble-En zym e System
1. As the diketone is stereospecifically reduced to (S,S)-
1,2-diol by the diacetyl reductase, NAD is formed, which
in turn is reduced to NADH by the same enzyme in the
presence of the cosubstrate endo-bicyclo[3.2.0]hept-2-en-
6-ol, (() 3. BSDR accepts a large variety of side chains
of different size, including cyclic compounds, and all
diketones are reduced to 1,2-diols in high enantiomeric
excess and good chemical yield. A relatively easy workup
affords the diols 2a -f, in the configuration reported in
Table 1. In addition, the kinetic resolution of the
cosubstrate (()-3 is obtained during NADH regeneration.
The resulting ketone endo-(-)-(1S,5R)-bicyclo[3.2.0]hept-
2-en-6-one (4), an important starting material in the
synthesis of prostaglandins,15 and the remaining alcohol
(-)-(1R,5S,6R)-3 may be almost quantitatively recovered
from the reaction mixture in an enantiomeric excess
higher than 98%, by virtue of the strict stoichiometry of
the reaction.16
From a mechanistic point of view the reaction takes
place through two consecutive enantioselective reduc-
tions, with formation of the monoreduced product, R-hy-
droxy ketone, that is further reduced to 1,2-diol. By a
careful control of the experimental conditions, in par-
ticular cosubstrate concentration and reaction time, the
diketones may be chemo- and enantioselectively reduced
to (S)-R-hydroxy ketones; however there is a clear dis-
tinction between symmetric (R ) R′) and asymmetric (R
* R′) starting substrates. Symmetric diketones, due to
the statistical factor, are reduced faster but in poor yield,
and the product of monoreduction is always contaminated
by large amounts of diol. On the other hand, R-hydroxy
ketones are obtained, from asymmetric diketones, in good
enantiomeric and chemical yields. Particularly interest-
ing is the ability to reduce, in first place, the carbonyl
group proximal to the smaller substituent (methyl) in 1c,
1d , and 1f. The middle substrate was reported to be
reduced to (R)-hydroxy ketone at the carbonyl group close
to the phenyl substituent in the presence of Pseudomonas
sp. alcohol dehydrogenase.4,9a
a
Determined by GC analysis on a chiral column, when >98%
b
the (R) isomer is not detected; see the Experimental Section. The
reduction affords also the diol 2f in 30% yield.
hydrogenase.5b This second set of results is collected in
Table 2. The chemoselective reduction of the carbonyl
group closer to the smaller substituent and the formation
of R-hydroxy ketones with (S) stereochemistry are pecu-
liar to the double-enzyme catalytic cycle, as well.
The synthetic potential of the single- and double-
enzyme systems has been illustrated during the synthesis
of the male sex pheromone of the grape borer Xylotrechus
pyrrhoderus13 identified as a two-component mixture of
2f and 5f.13c The synthetic route to 2f and 5f employed
the commercially available, low-cost 1-octyn-3-ol that is
transformed to 3-hydroxy-2-octanone and oxidized to the
R-diketone 1f. The latter product is chemo- and enan-
tioselectively reduced to 2f, Table 1, or 5f, Table 2, by
the appropriate single- or double-enzyme system, in high
chemical and enantiomeric yield.
In conclusion, two concise and technically simple
procedures for the synthesis of chiral 1,2-diols and
R-hydroxy ketones have been uncovered. Work is in
progress to further exploit their synthetic utility.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Bacillus stearothermophilus is
available from American Type Culture Collection (ATCC 2027)
and was grown as previously described.12 For synthetic reac-
tions, the wet cells (20 g) obtained from four portions of 250 mL
cultures are separated upon centrifugation and washed with 200
mL of 0.15 M NaCl. The cells are suspended in 100 mL of TEA-
HCl buffer, treated with 40 mg of lysozyme for 60 min at 22 °C,
and recentrifuged. The supernatant (enzyme solution) was used
without further treatment. Typically 1 mL of enzyme solution
contains approximately 0.2 unit of enzyme (1 unit ) 1 µmol of
endo-bicyclo[3.2.0]hept-2-en-6-ol oxidized per minute).12 The
TEA-HCl buffer is composed of 50 mM triethanolamine, contain-
ing 0.1 mM EDTA, 1 mM â-mercaptoethanol, and HCl to adjust
the pH at 7.5. NADH and glucose 6-phosphate were purchased
from Sigma. Leuconostoc mesenteroides glucose 6-phosphate
dehydrogenase is from Boehringer Mannheim. endo-Bicyclo-
[3.2.0]hept-2-en-6-ol, (()-3, was obtained from the commercially
available bicyclo[3.2.0]hept-2-en-6-one (Merck) by reduction with
NaBH4 and separation of the endo:exo isomers (9:1 ratio) by
chromatography on SiO2.17 Alternatively, the bicyclo[3.2.0]hept-
2-en-6-one may be synthesized by cycloaddition of cyclopenta-
diene with dichloroketene, followed by reduction, in 80% yield.18
Contrary to the facile workup found during 1,2-diol
isolation, the synthetic-scale preparation of R-hydroxy
ketones by this single-enzyme system showed several
unexpected difficulties, related to product separation. To
overcome the problem, the single-enzyme system has
been replaced with a double-enzyme system consisting
of the substrate, NADH-dependent BSDR and the clas-
sical glucose 6-phosphate/glucose 6-phosphate de-
(15) (a) Newton, R. F.; Roberts, S. M. Tetrahedron 1980, 36, 2163.
(b) Hart, T. W.; Comte, M.-T. Tetrahedron Lett. 1985, 26, 2713. (c)
Riefling, B. F. Tetrahedron Lett. 1985, 26, 2063.
(16) The use of the less valuable but more common 2-hexanol, 2,3-
butanediol, or propan-2-ol as cosubstrate for NADH regeneration gave
unsatisfactory results.
(17) Newton, R. F., Paton, J .; Reynolds, D. P.; Young, S.; Roberts,
S. M. J . Chem. Soc., Chem. Commun. 1979, 908.
(18) (a) Depres, J .-P.; Greene, A. E.; Crabbe, P. Tetrahedron 1981,
37, 621. (b) Ghosez, L.; Montaigne, R.; Roussel, A.; Vanlierde, H.;
Mollet, P. Ibid. 1971, 27, 615. (c) Corey, E. J .; Arnold, Z.; Hutton, J .
Tetrahedron Lett. 1970, 307.

1856 J . Org. Chem., Vol. 62, No. 6, 1997
Notes
Except for 2,3-octanedione, all other diketones and 2a ,e are
commercially available products purchased from Aldrich and
used upon distillation. Enantiomeric separations and excesses
were determined by GC on a Megadex 5 column containing
dimethyl-n-pentyl-â-cyclodextrin in OV 1701 from Mega s.n.c.
The absolute configurations are determined by comparison of
the sign of the specific rotations with the reported values, when
available. In the other cases, the absolute configurations are
assigned on the basis of the GC retention time in a homologous
series, upon analysis of the racemates. The entry ee > 98% of
Tables 1 and 2 means that the (R,R)-diols or the (R)-R-hydroxy
ketones are not detected in our experimental conditions. The
1H and 13C NMR spectra were obtained on a 300 MHz instru-
ment.
Gen er a l P r oced u r e for 1,2-Diols (Sin gle-En zym e Sys-
tem ). A portion of the enzyme solution (4 mL) was added to a
flask containing 1 mmol of ketone substrate, 20 mg of NADH,
and 4 mmol of cosubstrate in 120 mL of TEA-HCl buffer. Note
that, due to kinetic resolution, half of the cosubstrate is
elaborated, and consequently 4 equiv is required to complete the
reduction. After the proper time (48 h) the reaction mixture was
saturated with NaCl and extracted with ethyl acetate (3 × 20
mL). The combined and dried organic layers were evaporated
and the products separated by chromatography on SiO2, using
ethyl ether-cyclohexane (1:1) as eluent.
Gen er a l P r oced u r e for r-Hyd r oxy Keton es (Dou ble-
En zym e System ). A portion of the enzyme solution (2 mL) was
added to a flask containing 1 mmol of ketone substrate, 20 mg
of NADH, 1 mmol of glucose 6-phosphate, and 10 µL of glucose
6-phosphate dehydrogenase in 100 mL of TEA-HCl buffer. After
the proper time (24 h) the reaction mixture was saturated with
NaCl and extracted with ethyl acetate (3 × 20 mL). The
combined and dried organic layers were evaporated and the
products separated by chromatography on SiO2, using ethyl
ether-cyclohexane (1:1) as eluent.
Syn th esis of 2,3-Octa n ed ion e (1f). 1-Octyn-3-ol (32 mmol)
was added dropwise to a well-stirred boiling solution composed
of HgO (32 mmol), H2SO4 (1.7 mL), and H2O (50 mL), with
continuous steam-distillation.19 The distillate was saturated
with NaCl, and the upper layer was separated and combined
with the ethereal extracts of the aqueous layer. The combined
and dried organic layers were evaporated, affording the 3-hy-
droxy-2-octanone as yellow oil, in 90% yield: 1H NMR (CDCl3)
4.20 (t, J ) 6 Hz, 1H), 2.20 (s, 3H), 1.80 (s, 1H, exchanges with
D2O), 1.60-1.40 (m, 2H), 1.40-1.20 (m, 6H), 0.90 (t, J ) 7 Hz,
3H); 13C NMR (CDCl3) 210.0, 76.8, 33.4, 31.6, 25.1, 24.3, 22.4,
13.9. The crude 3-hydroxy-2-octanone was oxidized to the
R-diketone 1f by the J ones reagent20 and purified by column
chromatography on SiO2, using ethyl acetate as eluent, in 85%
yield: 1H NMR (CDCl3) 2.70 (t, J ) 7 Hz, 2H), 2.15 (s, 3H), 1.65-
1.55 (m, 2H), 1.40-1.20 (m, 6H), 0.90 (t, J ) 6 Hz, 3H); 13C NMR
(CDCl3) 199.5, 197.6, 35.6, 31.2, 23.7, 22.7, 2.3, 13.8. Anal.
Calcd C, 67.57; H, 9.93, O, 22.49. Found: C, 68.30; H, 9.85.
(S,S)-2,3-Bu ta n ed iol (2a ). The synthesis of this product
followed the general procedure described above. After the proper
time the reaction mixture was evaporated under vacuum, the
residue was dissolved in ethyl acetate methanol (10%) and dried
over Na2SO4, and the solvent was removed under reduced
pressure. The low chemical yield (30%) is due to the high
solubility of 2a in water. Racemization or formation of complex
stereoisomeric mixtures is not observed:21 [R]20 D) +13.0 (neat);
1H and 13C NMR are consistent with commercial sample.
(S,S)-3,4-Hexa n ed iol (2b): [R]20 ) (-) 12.4 (c ) 2.0,
D
chloroform); 1H NMR (CDCl3) 3.35 (m, 2H), 2.40 (br s, 2H,
exchange with D2O), 1.65-1.40 (m, 4H), 1.00 (t, J ) 7.5 Hz, 6H);
13C NMR (CDCl3) 75.5, 26.4, 9.9; IR (neat) 3400.
(S,S)-2,3-Hexa n ed iol (2c): [R]20 ) (-)16.1 (c ) 1.2, chloro-
D
1
form); H NMR (CDCl3) 3.60 (m, 1H), 3.35 (m, 1H), 2.40 (br s,
1H, exchanges with D2O), 2.30 (br s, 1H, exchanges with D2O),
1.40-1.60 (m, 4H), 1.20 (d, J ) 7.0 Hz, 3H), 0.90 (t, J ) 7.5, Hz,
3H); 13C NMR (CDCl3) 75.0, 69.9, 34.5, 18.2, 17.8, 13.1; IR
(CHCl3) 3400.
(S,S)-1,2-Dih yd r oxy-1-p h en ylp r op a n e (2d ): [R]20D ) +55.9
(c ) 1.9, chloroform);22 1H NMR (CDCl3) 7.30-7.38 (m, 5H), 4.36
(d, J ) 7.3 Hz, 1H), 3.84 (dq, J ) 6.4 Hz, 1H), 2.78 (m, 1H,
exchanges with D2O), 2.62 (m, 1H, exchanges with D2O), 1.05
(d, J ) 6.4 Hz, 3H); IR (neat) 3580.
(S,S)-1,2-Cycloh exa n ed iol (2e): [R]20 ) +39.0 (c ) 1.6,
D
H2O), 1H and 13C NMR consistent with commercial sample.
(S,S)- 2,3-Oct a n ed iol (2f). [R]20 ) -17.9 (c ) 1.14,
D
cholroform);13b 1H (CDCl3) 3.60 (m, 1H), 3.30 (m, 1H), 2.70 (br
s, 2H, exchange with D2O), 1.10-1.70 (m, 8H), 1.35 (d, J ) 6.0
Hz, 3H), 0.90 (t, J ) 7.0 Hz, 3H); 13C NMR (CDCl3) 76.3, 70.9,
33.3, 32.0, 25.3, 22.6, 19.5, 14.1.
(S)-2-Hyd r oxy-3-h exa n on e (5a ): [R]20 ) +57 (c ) 2.5,
D
chloroform); 1H NMR (CDCl3) 4.10 (q, J ) 7.5 Hz, 1H), 3.10 (br
s, 1H), 2.35-2.55 (m, 2H), 1.60-1.79 (m, 2H), 1.40 (d, J ) 7.0
Hz, 3H), 0.90 (t, J ) 7.5 Hz, 3H); 13C NMR (CDCl3) 212.0, 72.5,
39.4, 19.8, 17.0, 13.7; IR (CHCl3) 3450, 1700.
(S)- 2-Hyd r oxy-3-p h en yl-3-p r op a n on e (5d ): [R]20D ) -64.4
(c ) 7.8, chloroform);22 1H NMR (CDCl3) 7.26-7.90 (m, 5H), 5.10
(q, J ) 7.0 Hz, 1H), 3.60 (s, 1H, exchanges with D2O), 1.40 (d, J
) 7.0, 3H); 13C NMR (CDCl3) 202.3, 133.9, 128.6-128.8, 69.2,
22.2; IR (neat) 3450, 1670.
(S)-2-Hyd r oxy-3-octa n on e (5f): [R]20 ) +65.8 (c ) 1.8,
D
chloroform);13b 1H NMR (CDCl3) 4.20-4.30 (m, 1H), 3.70 (s, 1H,
exchanges with D2O), 2.30-2.60 (m, 2H), 1.20-1.85 (m, 6H), 1.35
(d, J ) 7.0 Hz, 3H), 0.90 (t, J ) 6.0 Hz, 3H); 13C NMR (CDCl3)
212.8, 72.6, 37.5, 31.4, 23.3, 22.4, 19.8, 13.9.
Ack n ow led gm en t. We are grateful to Professor
Mario Rippa, this University, for the helpful and
stimulating discussion.
J O9618381
(19) Ansell, M. F.; Hickinbottom, W. J .; Hyatt, A. A. J . Chem. Soc.
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