Synthesis of optically active hydroxyesters using biocatalysts

Article abstract of DOI:10.1023/A:1012538628323

Optically active hydroxyesters were prepared using the fungus Aspergillus niger and the yeast Sacharomyces cerevisiae. Substrate is converted highly specifically into optically active isomers with 95% purity. The R-hydroxyester was isolated by asymmetric reduction. (R,R)-Tartaric acid of 60% optical purity was used as an inductor.

Full text of DOI:10.1023/A:1012538628323

Chemistry of Natural Compounds, Vol. 37, No. 3, 2001
SYNTHESIS OF OPTICALLY ACTIVE HYDROXYESTERS
USING BIOCATALYSTS
G. S. Shakirzyanova, V. S. Abdukakharov,
and A. A. Abduvakhabov
UDC 542.91+547.473+632.936.2
Optically active hydroxyesters were prepared using the fungus Aspergillus niger and the yeast Sacharomyces
cerevisiae. Substrate is converted highly specifically into optically active isomers with 95% purity. The R-
hydroxyester was isolated by asymmetric reduction. (R,R)-Tartaric acid of 60% optical purity was used as
an inductor.
Key words: aggregation pheromones, grain beetles, synthesis, biocatalysts, Aspergillus niger, Sacharomyces
cerevisiae, asymmetric reduction.
Grain beetles (Coleoptera, Cucujidae) are agricultural pests. The aggregation pheromones of these insects are a
mixture of macrolide components, cucujolides with chiral centers [1]. Research has shown [2] that one of the factors leading
to species-specificity of grain-beetle pheromones is the chirality.
Therefore, we propose various approaches to the synthesis of optically active synthons of both R- and S-configuration.
The chiral center was introduced into the synthon structures using microorganisms as biocatalysts.
Microorganisms represent a large group of living organisms with a wide spectrum of metabolic processes owing to the
variety of enzymes they contain. It is not always necessary to isolate the enzymes from the bulk microorganism because the
substrate may be transformed chemically into product by the intact microorganism cells that contain the enzyme complex.
Therefore, undesired enzymes can be excluded from the process by regulating the temperature and pH [3].
We studied the reduction of ketones into alcohol with previously grown fungus Aspergillus niger and yeast
Sacharomyces cerevisiae.
The fungus was maintained under optimal growth conditions. The biomass was separated from the culture liquid by
centrifugation and then used in the experiment.
Substrate for biotransformation by A. niger was synthesized by the following scheme:
a
d
b
c
Br(CH2)3O THP
THP O(CH2)3MgBr
CH C(CH ) O THP
3
2 6
CH C(CH ) OTHP
3
2 6
Br(CH ) OH
2
3
1
2
O
HO H
4
3
o
a) 2,3-dihydropyran (DHP), HCl, Et O; b) EtMgBr/THF; c) 5-chloro-2-pentanone; d) A. niger, 27 C, 10 d.
2
The bioconversion was performed in aqueous medium and in culture liquid. The degree of conversion was 60%; optical
purity of the R-isomer, 95%.
The S-enantiomer 8 was obtained by treating 7 with S. cerevisiae. The yeast growth conditions were identical to those
for growing the fungus.
A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax
99871) 162 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 237-239, May-June, 2001. Original article
(
submitted February 19, 2001.
©
0
009-3130/01/3703-0279$25.00 2001 Plenum Publishing Corporation
279

Substrate 7 for the biotransformation by yeast was synthesized by the following scheme:
O
a
O
c, d
e
g
b
O
O
HOOC(CH ) COOCH
3
C(CH2)2COOCH3
CH3C(CH2)2COOCH3
CH3C(CH2)2COOCH3
2
2
Cl
H OH
5
7
8
6
o
o
a) MeOH; b) PCl ; c) MeMgI/Et O; d) CdMe /C H ; e) S. cerevisiae, 35 C, 7 d; g) NaBH , R,R-tartaric acid, -5 C, 24 h.
5
2
2
6
6
4
-Carbomethoxypropanoic acid (5) was synthesized by reacting methanol and succinic anhydride. Treatment with
PCl converted the acid to the acyl chloride 6 in 70% yield. Ketoester 7 was prepared by reacting an organocadmium compound
5
with 6.
o
The methyl ester of ketovaleric acid was treated with yeast and periodically stirred at 35 C for 7 d. The degree of
conversion was 20%. The optical purity of the S-isomer was 95%.
We attempted to prepare optical isomers via a chiral synthesis. Reduction of the ketone in alcohol with a suspension
of NaBH and the chiral inductor (R,R)-tartaric acid by the literature method [4] gave the R-isomer of 8 in 60% optical purity.
4
Thus, the biotransformation of asymmetric synthons was studied for the synthesis of chiral macrolides by A. niger and
S. cerevisiae. The results have showed that substrate is converted highly specifically to product by the microorganisms and
forms synthons with 95% optically pure R- and S-configurations.
The highest degree of conversion of substrate into product was observed with A. niger micelles and reaction in culture
medium.
EXPERIMENTAL
GLC was performed in an LKhM-8MD instrument over a column (1 m × 3 mm) packed with 15% Carbowax 20M on
Chromaton N-AW-DMCS and a Chrom 5 instrument over a column packed with 5% XE-60 Chromaton N-AW-DMCS with
He carrier gas. PMR spectra were obtained on a Varian XL-200 NMR spectrometer at working frequency 200 MHz in CCl4
with HMDS internal standard. Optical rotation was measured on a POLAMAT polarimeter in CHCl . Column chromatography
3
used Chemapol L 100/250 silica gel with elution by ether—hexane (2:1). Analyses for all compounds agreed with those
calculated.
1
-(Tetrahydropyran-2-yl)oxy-3-bromopropane (1). 3-Bromopropan-1-ol (27.8 g, 0.2 mole) and HCl (5 drops, conc.)
o
were placed in an Erlenmeyer flask, cooled to 0 C, treated dropwise with 2,3-dihydropyran (18.4 g, 0.22 mole) over 30 min,
stirred for 16 h at room temperature, diluted with ether (50 mL), washed with NaOH (100 mL, 1 N) and NaCl solution
(
1
100 mL), and dried over MgSO . Solvent was removed at reduced pressure. The residue was vacuum distilled, bp
4
02 C/7 mm. Yield 36.1 g (81%).
o
1-(Tetrahydropyran-2-yl)oxy-7-oxooctane (3). Ethylmagnesium bromide was prepared from metallic Mg (0.92 g,
0
.04 mole) and ethylbromide (4.36 g, 0.04 mole). The solution was heated, treated dropwise with 1 (8.43 g, 0.04 mole), stirred
o
and boiled for 5 and 6 h at room temperature, cooled (0 C) and treated dropwise with 5-chloro-2-pentanone (4.82 g, 0.04 mole),
and stirred for 4 h. The mixture was decomposed with HCl (20 mL, 1 N). The organic layer was separated. The aqueous layer
was extracted with ether (3×25 mL). The ether extracts were dried over MgSO . Solvent was removed at reduced pressure.
4
The residue was vacuum distilled. The distilled fractions were purified by column chromatography, R = 0.37. Retention time
f
o
2
2
.8 min (GLC). bp 98 C/8 mm. Yield 4.91 g (57%). PMR ( , ppm, J/Hz): 4.06 (1H, m, OCH–O), 3.2-3.8 (4H, m, O–CH ),
2
.50 (2H, t, J = 6.4, CH –C O), 2.04 (3H, s, CH –C O), 0.8-2.1 (14H, m, CH ).
2
3
2
1
-(Tetrahdyropyran-2-yl)oxy-7-hydroxyoctane (4). Moist mycelium of A. niger (8.77 g) and 3 (600 mg) were stored
o
in culture medium (100 mL) at 27 C and periodically stirred over 10 d. The temperature was maintained using a thermostat.
The solution was filtered and extracted with ether. The extract was dried over MgSO . The solvent was removed. Yield
4
0
.31 g (60% conversion) of crude product. The reaction product was analyzed using GLC at 160 C, retention time 0.5 min.
2
3
Optical purity of the R-isomer, 95%, [ ]D +10.5 (c 0.31, CHCl3).
Carbomethoxypropionic acid (5). A mixture of succinic anhydride (200 g, 2 mole) and methanol (97 mL, 2.4 mole)
was boiled on a water bath for 20 min and stirred for 15 min. When the mixture became homogeneous it was placed on a steam
-
2
80

bath for 20-25 min. The excess of methanol was removed at reduced pressure (water aspirator). The residue was poured into
o
a marble dish for crystallization. The reaction product was dried in a desiccator to constant mass for 5 d, mp 56-64 C,
1
66.14 g (70%).
Carbomethoxypropionylchloride (6). Acid 5 (49 g, 0.37 mole) was treated with portions of ground PCl (80.2 g,
-
5
0
4
.39 mole) with stirring and cooling. The reaction was very violent and exothermic. The reaction mixture was stirred for
o
h. POCl was removed at reduced pressure (water aspirator). The residue was distilled, bp 76-77 C/7 mm, yield 23.84 g
3
(42.6%).
Methyl Ester of Ketovaleric Acid (7). CH MgI was prepared from CH I (19.88 g, 0.14 mole) and metallic Mg
3
3
o
(
0
3.22 g, 0.14 mole) in absolute ether (100 mL), cooled (0 C), treated with portions of previously calcined CdI (27.54 g,
.0752 mole), stirred at room temperature for 1.5 h, cooled (0 C), and treated dropwise with 6 (20.83 g, 0.14 mole). The
2
o
reaction mixture was stored at this temperature for 12 h. The excess of organocadmium reagent was decomposed with H SO
2
4
(
9 mL, 10%). The resulting two layers were separated. The aqueous layer was extracted with benzene (3×50 mL). The
combined extracts were dried over MgSO . The solvent was removed. The residue was vacuum distilled. The product was
4
further purified by column chromatography with elution by ether—hexane (5:1), R = 0.4, bp 70-72 C/4 mm, yield 5.08 g
f
(
28.3%). PMR ( , ppm, J/Hz): 3.63 (3H, s, COOCH ), 2.64 (2H, d, J = 4.1, CH ), 2.47 (2H, d, J = 4.1, CH ), 2.13 (3H, s,
3 2 2
O C–CH3).
Methyl Ester of 4-Hydroxyvaleric Acid (8). a. A suspension of NaBH (1.26 g, 0.02 mole) and R,R-tartaric acid
4
o
(
4.5 g, 0.03 mole) in THF (100 mL) was boiled for 4 h, cooled to 0 to -5 C, treated dropwise over 5 min with 7 (2 g, 0.02 mole)
in THF (10 mL), held at this temperature for 29 h using a thermostat, cooled, and decomposed with ethylacetate (50 mL) and
HCl (25 mL, 1 M). The organic layer was separated, washed with NaHCO , and dried over K CO . The solvent was removed
3
2
3
o
under reduced pressure. The residue was vacuum distilled. The product was purified by column chromatography, bp 60-62 C,
yield 0.6 g. PMR ( , ppm, J/Hz): 3.71 (1H, m, CH), 3.61 (3H, s, COOCH ), 2.35 (2H, t, J = 6.7, CH CO), 1.67 (2H, m, CH ),
1
3
2
2
2
3
o
.15 (3H, d, J = 6.6, CH ). The degree of conversion was 30%; optical purity of the R-isomer, 60%; [ ]
+45.5 (c 1.22,
3
D
CHCl3).
b. Compound 7 (540 mg) was treated with S. cerevisiae biomass (80 g) and glucose (0.04 g). The mixture was stored
o
and periodically stirred at 35 C for 7 d. The temperature was maintained using a thermostat. The reaction mixture was filtered.
The filtrate was treated with (NH ) SO and extracted with ether. The extract was dried over MgSO . The solvent was
4
2
4
4
2
3
removed. The degree of conversion was 20%; optical purity of the S-isomer, 95%; [ ]D -44.8 (c 0.06, CHCl3).
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1.
2.
3.
4.
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