We reduced freshly-prepared geranial 1 on a 15 mmole scale
using an ammonium sulfate-treated crude extract containing
GST-OYE 2.6 in a total volume of 100 mL.15 Conversion
reached 95% after 5.75 h when the reaction was terminated
and the product isolated by solvent extraction followed by
column chromatography. (R)-Citronellal 2 was isolated in
67% yield with 98% ee. No citronellol was observed. To test
the generality of the process design, we applied analogous
conditions to neral 4. A crude extract was prepared from cells
overexpressing E. coli GST-NemA. Because this preparation
showed very high specific activity toward neral, competing
carbonyl reduction was not significant and ammonium sulfate
fraction was omitted. Using the methodology described above,
(S)-citronellal 2 was obtained in 69% yield with 99% ee after a
4 h reaction (>98% conversion).16
D. Leys and N. S. Scrutton, ChemBioChem, 2010, 11, 197–207;
(r) C. Stueckler, T. C. Reiter, N. Baudendistel and K. Faber,
Tetrahedron, 2010, 66, 663–667.
2 (a) M. A. Swiderska and J. D. Stewart, Org. Lett., 2006, 8,
6131–6133; (b) A. Fryszkowska, H. Toogood, M. Sakuma,
J. M. Gardiner, G. M. Stephens and N. S. Scrutton, Adv. Synth.
Catal., 2009, 351, 2976–2990; (c) D. J. Bougioukou and
J. D. Stewart, in Enzyme Catalysis in Organic Synthesis,
ed. K. Drauz, H. Groger and O. May, Wiley-VCH, Weinheim,
3rd edn, 2010.
3 H. Kumobayashi, N. Sayo, S. Akutagawa, T. Sakaguchi and
H. Tsuruta, Nippon Kagaku Kaishi, 1997, 835–846.
4 (a) A. Muller, B. Hauer and B. Rosche, J. Mol. Catal. B: Enzym.,
2006, 38, 126–130; (b) M. Hall, B. Hauer, R. Stuermer, W. Kroutil
and K. Faber, Tetrahedron: Asymmetry, 2006, 17, 3058–3062.
5 (a) A. Muller, B. Hauer and B. Rosche, Biotechnol. Bioeng., 2007,
98, 22–29; (b) B. Yin, X. P. Yang, G. D. Wei, Y. S. Ma and
D. Z. Wei, Mol. Biotechnol., 2008, 38, 241–245.
6 D. J. Bougioukou, PhD thesis, University of Florida, 2006.
7 (R)-selective enzymes (accession number in parentheses):
Saccharomyces pastorianus OYE1 (Q02899), 61% ee; Saccharomyces
cerevisiae OYE2 (Q03558), 87% ee; S. cerevisiae OYE3 (P41816),
21% ee; Schizosaccharomyces pombe OYEA (Q09670), 45% ee;
Kluyveri marxianus OYE (Q6I7B7), 44% ee; Pseudomonas putida
NemA (Q88I29), 17%; P. stipitis OYE 2.6 (XP_001384055.1), 90%.
8 (S)-selective enzymes (accession number in parentheses):
Escherichia coli NemA (P77258), 79%; Pseudomonas putida OYE
(Q88K07), 55%; Synechococcus elongatus OYE (Q31R14), 60% ee;
Arabidopsis thaliana OPR1 (Q8LAH7), 99%; A. thaliana OPR3
(Q9FUP0), 99%; Solanum lycopersicon OPR (Q9XG54), 78%;
Rat Ltb4 dehydrogenase (P97584), 19%.
The conditions developed here open the possibility of
employing alkene reductases in preparative-scale reactions.
We focused mainly on volumetric productivity since scaling
up reaction volumes provides a straightforward means to
converting larger substrate quantities. The lack of carbonyl
reduction is an important advantage of our methodology. No
special equipment is required and the reactions are carried out
in standard organic glassware. The methodology is currently
being used successfully in our laboratory for additional
substrates and we anticipate that it will be broadly applicable,
making the ever-increasing number of available alkene
reductases useful in synthetic planning and execution.
9 W. A. M. Wolken, R. ten Have and M. J. van der Werf, J. Agric.
Food Chem., 2000, 48, 5401–5405.
10 R. A. Sheldon, Biochem. Soc. Trans., 2007, 35, 1583–1587.
11 R. A. Scism and B. O. Bachmann, ChemBioChem, 2009, 11, 67–70.
12 J. F. Riordan and B. L. Vallee, Methods Enzymol., 1972, 25,
494–499.
Notes and references
1 (a) M. A. Swiderska and J. D. Stewart, J. Mol. Catal. B: Enzym.,
2006, 42, 52–54; (b) J. F. Chaparro-Riggers, T. A. Rogers,
E. Vazquez-Figueroa, K. M. Polizzi and A. S. Bommarius,
Adv. Synth. Catal., 2007, 349, 1521–1531; (c) M. Hall,
C. Stueckler, W. Kroutil, P. Macheroux and K. Faber, Angew.
Chem., Int. Ed., 2007, 46, 3934–3937; (d) C. Stueckler, M. Hall,
H. Ehammer, E. Pointner, W. Kroutil, P. Macheroux and
K. Faber, Org. Lett., 2007, 9, 5409–5411; (e) R. Stuermer,
B. Hauer, M. Hall and K. Faber, Curr. Opin. Chem. Biol., 2007,
11, 203–213; (f) M. Hall, C. Stueckler, H. Ehammer, E. Pointner,
G. Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, W. Kroutil,
P. Macheroux and K. Faber, Adv. Synth. Catal., 2008, 350,
411–418; (g) M. Hall, C. Stueckler, B. Hauer, R. Stuermer,
T. Friedrich, M. Breuer, W. Kroutil and K. Faber, Eur. J. Org.
Chem., 2008, 1511–1516; (h) B. Kosjek, F. J. Fleitz, P. G. Dormer,
J. T. Kuethe and P. N. Devine, Tetrahedron: Asymmetry, 2008, 19,
1403–1406; (i) P. Schweiger, H. Gross, S. Wesener and
U. Deppenmeier, Appl. Microbiol. Biotechnol., 2008, 80,
13 D. J. Bougioukou, J. D. Stewart, unpublished results.
14 E. coli JW0317, CGSC 8516.
15 An 85 mL sample of 100 mM KPi, pH 7.5 containing glucose
(44.4 mmoles, 8.0 g) was degassed for 1 h, then transferred to a
500 mL round bottom flask under Ar. GDH 102 (100 U, 1 mg),
NADPH (12 mmoles, 10 mg) and ammonium sulfate-purified
GST-OYE 2.6 (100 U, 13 mL) were added. After stirring at 300 rpm
for 15 min at room temp., geranial (5.2 mmoles, 0.79 g) dissolved in
0.83 mL of EtOH was added. The pH was maintained at 7.5 using
a pH stat (1 M KOH titrant) and additional portions of geranial
were added after 1.5 and 3.0 h. After 5.75 h, GC/MS analysis
showed 95% conversion. The mixture was acidified to pH 4 with
1 M HCl and stirred overnight with CH2Cl2 (100 mL). The aqueous
portion was extracted with additional CH2Cl2 (2 ꢀ 50 mL), then the
organic layers were combined, filtered over Celite, washed with
brine (3 ꢀ 50 mL), dried over Na2SO4 and passed over a small bed
of silica gel. After concentration under vacuum, the crude
product (2.12 g) was adsorbed onto silica gel deactivated with
10% H2O (5 g) and then added to a silica column (60 g)
equilibrated with hexanes. The desired product was eluted with
1 : 9 Et2O/hexanes to yield 1.59 g of a pale yellow liquid
(99% purity by GC) whose spectral data matched those of
995–1006;
(j)
B.
V.
Adalbjornsson,
H.
Toogood,
A. Fryszkowska, C. R. Pudney, T. A. Jowitt, D. Leys and
N. S. Scrutton, ChemBioChem, 2010, 11, 197–207;
(k) D. J. Bougioukou, S. Kille, A. Taglieber and M. T. Reetz,
Adv. Synth. Catal., 2009, 351, 3287–3305; (l) E. Burda,
M. Kraußer, G. Fischer, W. Hummel, F. Muller-Uri, W. Kreis
and H. Groger, Adv. Synth. Catal., 2009, 351, 2787–2790;
(m) T. Hirata, A. Matsushima, Y. Sato, T. Iwasaki, H. Nomura,
T. Watanabe, S. Toyoda and S. Izumi, J. Mol. Catal. B: Enzym.,
2009, 59, 158–162; (n) N. J. Mueller, C. Stueckler, B. Hauer,
N. Baudendistel, H. Housden, N. C. Bruce and K. Faber,
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J. M. Gardiner and N. S. Scrutton, ChemCatChem, 2010, 2,
892–914; (p) Y. Yanto, M. Hall and A. S. Bommarius, Org.
Biomol. Chem., 2010, 8, 1826–1832; (q) B. V. Adalbjornsson,
H. S. Toogood, A. Fryszkowska, C. R. Pudney, T. A. Jowitt,
24
(R)-citronellal. [a]D = +18.22 (c = 7.30, CHCl3).
16 GDH 102 (100 U, 1 mg), NADPH (12 mmoles, 10 mg) and a crude
extract containing E. coli GST-NemA (480 U, 11 mL) were stirred
for 15 min in 85 mL of degassed KPi, pH 7.5, containing glucose
(44.4 mmoles, 8.0 g). Neral was added in three equal portions (total
15.5 mmoles, 2.38 g) as an EtOH solution (total 2.5 mL) at 0,
1.5 and 2.5 h. The reaction was maintained at pH 7.5 and GC/MS
indicated complete consumption of neral after 3.5 h. Product
isolation as described above yielded 1.62 g of a pale yellow oil
(98% purity by GC) whose spectral data matched those of
(S)-citronellal. [a]2D4 = ꢁ13.71 (c = 5.55, CHCl3).
c
8560 Chem. Commun., 2010, 46, 8558–8560
This journal is The Royal Society of Chemistry 2010