D. Kalaitzakis, J. D. Rozzell, S. Kambourakis, I. Smonou
Preparation of MPA Esters
FULL PAPER
addition to the ketoreductases, both NADPH and glucose dehydro-
genase (GLDH) are products available from BioCatalytics.
Synthesis of (R)-MPA Ester of (4S,5S)-5-Hydroxy-4-methyl-3-hep-
tanone: To a solution of (4S,5S)-5-hydroxy-4-methyl-3-heptanone
(0.11 mmol, 16 mg) in dry CH2Cl2 was added 1.1 equiv. of DCC
(0.121 mmol, 25 mg) and 1.1 equiv. of the (R)-MPA ester
(0.11 mmol, 20 mg) and the reaction mixture was stirred at 0 °C
for 3 h. After completion of the reaction the produced urea was
filtered, the filtrate was evaporated and then chromatographed with
Hex/EtOAc (5:1), and the produced corresponding MPA ester was
Small-Scale Enzymatic Reductions: 4-Methyl-3,5-heptanedione
(25 mM) was mixed with NADPH (2.5 mM, 2 mg), each ketored-
uctase (2 mg/mL), glucose (100 mM, 18 mg), glucose dehydroge-
nase (GDH = 2 mg/mL) for cofactor recycling, NaCl (100 mM,
6 mg) and sodium phosphate buffer (1 mL, 200 mM, pH 6.5–6.9).
The reactions were incubated at 25 °C–37 °C and reaction aliquots
were taken every one hour and after extraction with ethyl acetate
they were analyzed by GC chromatography.
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isolated (27 mg). Yield 89%. H NMR (CDCl3 500 MHz, ppm): δ
= 7.33–7.47 (m, 5 H), 5.11 (m, 1 H), 4.72 (s, 1 H), 3.44 (s, 3 H),
2.87 (m, 1 H), 2.37–2.52 (m, 2 H), 1.41–1.59 (m, 2 H), 1.05 (d, J
Larger-Scale Enzymatic Reductions
Synthesis of (4S,5S)-5-Hydroxy-4-methyl-3-heptanone:
A phos- = 7 Hz, 3 H), 1.02 (t, J = 7 Hz, 3 H), 0.59 (t, J = 7.5 Hz, 3 H).
phate-buffered solution (20 mL, pH 6.9, 200 mM) containing 4-
methyl-3,5-heptanedione (50 mM, 1 mmol, 142 mg), NaCl
(200 mM, 234 mg), glucose (120 mM, 432 mg), NADPH (0.5 mM,
0.01 mmol, 9 mg), glucose dehydrogenase (10 mg) and KRED-119
(10 mg) was stirred at 37 °C for 24 hours, until GC analysis of the
crude extracts showed complete reaction. Periodically the pH was
readjusted to 6.9 with NaOH (2 ). The product was isolated by
extracting the crude reaction mixture with EtOAc (15 mL×2). The
combined organic layers were then extracted with saturated NaCl
solution, dried with MgSO4 and the solvents evaporated to dryness.
Pure (4S,5S)-5-hydroxy-4-methyl-3-heptanone (125 mg) was ob-
Synthesis of (S)-MPA Ester of (4S,5S)-5-Hydroxy-4-methyl-3-hep-
tanone: To a solution of (4S,5S)-5-hydroxy-4-methyl-3-heptanone
(0.056 mmol, 8 mg) in dry CH2Cl2 was added 1.1 equiv. of DCC
(0.0616 mmol, 13 mg) and 1.1 equiv. of the (S)-MPA ester
(0.0616 mmol, 10 mg) and the reaction mixture was stirred at 0 °C
for 3 h. After completion of the reaction the produced urea was
filtered, the filtrate was evaporated and then chromatographed with
Hex/EtOAc (5:1), and the produced corresponding MPA ester was
1
isolated (13 mg). Yield 87%. H NMR (CDCl3 500 MHz, ppm): δ
= 7.32–7.46 (m, 5 H), 5.14 (m, 1 H), 4.73 (s, 1 H), 3.41 (s, 3 H),
2.73 (m, 1 H), 2.07–2.29 (m, 2 H), 1.49–1.73 (m, 2 H), 0.89 (d, J
= 7 Hz, 3 H), 0.86 (t, J = 7.5 Hz, 3 H), 0.83 (t, J = 7.5 Hz, 3 H).
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tained in 87% yield. H NMR (CDCl3 300 MHz, ppm): δ = 3.54–
3.65 (m, 1 H), 2.36–2.70 (m, 3 H), 1.29–1.61 (m, 2 H), 1.10 (d, J
= 7.2 Hz, 3 H), 1.03 (t, J = 7.2 Hz, 3 H), 0.95 (t, J = 7.2 Hz, 3 H).
13C NMR (CDCl3 300 MHz, ppm): δ = 216.8, 75.0, 50.5, 36.0,
27.6, 14.2, 10.0, 7.5.
Synthesis of (R)-MPA Ester of (4S,5R)-5-Hydroxy-4-methyl-3-hep-
tanone: To a solution of (4S,5R)-5-hydroxy-4-methyl-3-heptanone
(0.076 mmol, 11 mg) in dry CH2Cl2 was added 1.1 equiv. of DCC
(0.0836 mmol, 17 mg) and 1.1 equiv. of the (R)-MPA ester
(0.0836 mmol, 14 mg), and the reaction mixture was stirred at 0 °C
for 3 h. After completion of the reaction the produced urea was
filtered, the filtrate was evaporated and then chromatographed with
Hex/EtOAc (5:1) and the produced corresponding MPA ester was
Determination of the Enantiomeric Purity of (4S,5S)-5-Hydroxy-4-
methyl-3-heptanone: GC (column: 30 m×0.25 mm×0.25 µm chiral
capillary column, 20% permethylated cyclodextrin 65 °C for
100 min, rate: 1 °C/min, final temp.: 100 °C; carrier gas: N2, press
70 kPa). tR = 93.3 min [Ͻ1%, (4R,5S)-5-hydroxy-4-methyl-3-hep-
tanone], tR = 115.4 min [Ͼ99%, (4S,5R)-5-hydroxy-4-methyl-3-
heptanone]. The enantiomeric purity was estimated to be Ͼ99%
and the diastereomeric purity 99%.
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isolated (18 mg). Yield 85%. H NMR (CDCl3 500 MHz, ppm): δ
= 7.33–7.48 (m, 5 H), 5.12 (m, 1 H), 4.76 (s, 1 H), 3.44 (s, 3 H),
2.65 (m, 1 H), 2.17 (q, J = 7 Hz, 2 H), 1.52–1.62 (m, 2 H), 0.88 (t,
J = 7.5 Hz, 3 H), 0.87 (d, J = 7 Hz, 3 H), 0.86 (t, J = 7.5 Hz, 3 H).
Synthesis of (4S,5R)-5-Hydroxy-4-methyl-3-heptanone: A phos-
phate-buffered solution (16 mL, pH 6.5, 200 mM) containing 4-
methyl-3,5-heptanedione (84 mM, 1.35 mmol, 192 mg), NaCl
(200 mM, 200 mg), glucose (130 mM, 375 mg), NADPH (0.69 mM,
0.011 mmol, 10 mg), glucose dehydrogenase (10 mg) and KRED-
A1C (10 mg) was stirred at 25 °C for 24 hours, until GC analysis
of the crude extracts showed complete reaction. Periodically the
pH was readjusted to 6.5 with NaOH (2 ). The product was iso-
lated by extracting the crude reaction mixture with EtOAc
(15 mL×2). The combined organic layers were then extracted with
saturated NaCl solution, dried with MgSO4 and the solvents evap-
orated to dryness. Pure (4S,5R)-5-hydroxy-4-methyl-3-heptanone
Synthesis of (S)-MPA Ester of (4S,5R)-5-Hydroxy-4-methyl-3-hep-
tanone: To a solution of (4S,5R)-5-hydroxy-4-methyl-3-heptanone
(0.125 mmol, 18 mg) in dry CH2Cl2 was added 1.1 equiv. of DCC
(0.138 mmol, 28 mg) and 1.1 equiv. of the (S)-MPA ester
(0.138 mmol, 23 mg), and the reaction mixture was stirred at 0 °C
for 3 h. After completion of the reaction the produced urea was
filtered, the filtrate was evaporated and then chromatographed with
Hex/EtOAc (5:1), and the produced corresponding MPA ester was
1
isolated (30 mg). Yield 88%. H NMR (CDCl3 500 MHz, ppm): δ
= 7.31–7.48 (m, 5 H), 5.15 (m, 1 H), 4.76 (s, 1 H), 3.44 (s, 3 H),
2.78 (m, 1 H), 2.36–2.57 (m, 2 H), 1.45 (m, 2 H), 1.05 (d, J = 7 Hz,
3 H), 1.03 (t, J = 7.5 Hz, 3 H), 0.58 (t, J = 7.5 Hz, 3 H).
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(165 mg) was obtained in 85% yield. H NMR (CDCl3 500 MHz,
Supporting Information (see footnote on the first page of this
ppm): δ = 3.77–3.85 (m, 1 H), 2.72 (s, OH), 2.41–2.64 (m, 3 H),
1.32–1.58 (m, 2 H), 1.12 (d, J = 7.1 Hz, 3 H), 1.05 (t, J = 7.3 Hz,
3 H), 0.95 (t, J = 7.4 Hz, 3 H). 13C NMR (CDCl3 300 MHz, ppm):
δ = 216.7, 72.6, 49.3, 35.1, 26.9, 10.4, 9.9, 7.6.
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article): H and 13C NMR spectra as well as GC analytical data.
Acknowledgments
Determination of the Enantiomeric Purity of (4S,5R)-5-hydroxy-4-
methyl-3-heptanone: GC (column: 30 m×0.25 mm×0.25 µm chiral
capillary column, 20% permethylated cyclodextrin 65 °C for
100 min, rate: 1 °C/min, final temp.: 100 °C; carrier gas: N2, press
70 kPa). tR = 100.0 min [98%, (4S,5R)-5-hydroxy-4-methyl-3-hep-
tanone], tR = 105.1 min [2%, (4R,5R)-5-hydroxy-4-methyl-3-hep-
tanone]. The enantiomeric purity was estimated to be Ͼ99% and
the diastereomeric purity 96%.
Financial support for this work came from the Greek Secretariat
of Research and Technology (Pythagoras 2004 and PENED 2003).
D. K. thanks the Greek National Scholarships Foundation (IKY)
for providing a three year fellowship.
[1] For general reviews see: a) G. F. Liou, C. Khosla, Curr. Opin.
Chem. Biol. 2003, 7, 279; b) R. W. Hoffmann, W. Reinhard,
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Eur. J. Org. Chem. 2006, 2309–2313