flow—88 mL◦min-1, split—60 : 1; temperature program: 80◦◦C
(for 4) or 100 C (for 1) for 30 min, 15 ◦C min-1, final T = 180 C,
held for 5 min; detector: 300 ◦C; 30 mL min-1 H2, 400 mL min-1
air; retention times for enantiomers of 4: S at 24 min, R at 25 min;
retention times for enantiomers of 1: R at 22 min, S at 23 min.
acetate phases were concentrated in vacuo to give 61.6 g (93%)
of 1. The chemical purity as determined by GC was 99.5%
and the chiral purity was >99.5% e.e. for the R-enantiomer
(no S-enantiomer was detected).
References
Preparative enzymatic reduction of ECAA (3) to (S)-ECHB (4)
1 M. Mu¨ler, Angew. Chem., Int. Ed., 2005, 44, 362.
2 D. E. Butler, T. V. Le, A. Millar and T. N. Nanninga, US5155251
(1992).
3 G. DeSantis, Z. Zhu, W. A. Greenberg, K. Wong, J. Chaplin, S. R.
Hanson, B. Farwell, L. W. Nicholson, C. L. Rand, D. P. Weiner, D. E.
Robertson and M. J. Burk, J. Am. Chem. Soc., 2002, 124, 9024; G.
DeSantis, K. Wong, B. Farwell, K. Chatman, Z. Zhu, G. Tomlinson,
H. Huang, X. Tan, L. Bibbs, P. Chen, K. Kretz and M. J. Burk, J. Am.
Chem. Soc., 2003, 125, 11476.
4 F. H. Hoff and T. Anthonsen, Tetrahedron: Asymmetry, 1999, 10,
1401.
5 D. R. Yazbek, C. A. Martinez, S. Hu and J. Tao, Tetrahedron:
Asymmetry, 2004, 15, 2757.
6 H. Matsuda, T. Shibata, H. Hashimoto and M. Kitai, U.S. Patent,
5, 908,953, 1999 to Mitsubishi Chemical Corporation.
7 M. M. Elenkov, B. Hauer and D. B. Janssen, Adv. Synth. Catal., 2006,
348, 579.
8 T. Nakamura, T. Nagasawa, F. Yu, I. Watanabe and H. Yamada,
Biochem. Biophys. Res. Commun., 1991, 180, 124.
A 3 L jacketed three-neck flask equipped with a pH electrode
connected to a pH stat (Schott system) was charged with 570 ml
of 100 mM triethanolamine. The pH was adjusted to 7 using
96% H2SO4 (~5 ml) and D-glucose (298 g; 1.64 M) was added.
The temperature was raised to 25 ◦C. KRED (854 mg) and
GDH (578 mg) were charged as lyophilized powders. Then, Na-
NADP (98 mg) was added followed by butyl acetate (370 ml).
The reaction was started by the addition of 3 (240 g; 1.46 moles)
from an addition funnel while maintaining the pH at 6.9 0.05
using the pH stat with 4 N NaOH feeding. The reaction was
◦
completed in ~8 h at 25 C at which point 357 ml 4 N NaOH
was consumed. A sample was analyzed by GC to check for
completion. The reaction mixture was heated to 50 ◦C for 30 min
and 5 g of Celite was added to facilitate filtration. The reaction
mixture was cooled to 25 ◦C and filtered through a Celite pad.
The two layers were separated and the aqueous solution was
extracted with 350 mL butyl acetate. The two organic extracts
were combined and the solvents were removed to obtain 232.61 g
(96%) of 4, as a light yellow-colored liquid. The enantiomeric
excess of 4 was >99.5% (the R-enantiomer was not detected).
9 T. Nakamura, T. Nagasawa, F. Yu, I. Watanabe and H. Yamada,
Tetrahedron, 1994, 50, 11821.
10 J. H. Lutje Spelberg, L. Tang, M. van Gelder, R. M. Kellogg and
D. B. Janssen, Tetrahedron: Asymmetry, 2002, 13, 1083.
11 J. H. Lutje Spelberg, J. E. T. van Hylckama Vlieg, L. Tang, D. B.
Janssen and R. M. Kellogg, Org. Lett., 2000, 3, 41.
12 G. Hasnaoui, J. H. Lutje Spelberg, E. de Vries, L. Tang, B. Hauer
and D. B. Janssen, Tetrahedron: Asymmetry, 2005, 16, 1685.
13 R. A. Sheldon, Chirotechnology; Industrial Synthesis of Optically
Active Compounds, Marcel Dekker, New York, 1993; C. H. Wong and
G. M. Whitesides, Enzymes in Synthetic Organic Chemistry, Elsevier,
Amsterdam, 1994.
Preparative enzymatic cyanation of (S)-ECHB (4) to HN (1)
A 1 L jacketed three-neck round bottom flask equipped with
a rubber septum, a pH electrode connected to a pH stat,
and a mechanical stirrer was charged with 400 ml 500 mM
NaCN. The whole system was airtight since gaseous HCN is
generated. The pH of the solution was ~11.2. The pH electrode
14 U. T. Strauss, U. Felfer and K. Faber, Tetrahedron: Asymmetry, 1999,
10, 107.
15 A. Bommarius and B. Bommarius-Riebel, Fundamentals of Biocatal-
ysis, Wiley-VCH, Weinheim, 2005.
16 A. Liese, K. Seelbach, and C. Wandrey, Industrial Biotransforma-
tions, Wiley-VCH, Weinheim, 2000; G. J. Lye, P. A. Dalby and J. M.
Woodley, Org. Process Res. Dev., 2002, 6, 434.
◦
was calibrated at room temperature 20–23 C before use. The
vessel was sealed and the pH was adjusted to ~7.5 using conc.
H2SO4 (~7 mL). HHDH was charged as an aqueous solution
(1.05 g in 20 mL de-ionized water) and the reaction mixture was
heated to 40 ◦C. Then, the pH stat control was started and 70 g
(0.42 moles) of 4 was added with a syringe over ~10 min. The
pH stat maintained the pH at 7.3 0.05 with the addition of
a 25% NaCN solution containing 0.25% NaOH. After ~18 h,
73 mL of NaCN/NaOH solution was consumed. At that point,
the progress of the reaction was checked by GC and no ECHB
was detected. The pH of the reaction mixture was adjusted to ~3
with conc. H2SO4 (~2.5 mL). For safety reasons, the reaction was
cooled to 25 ◦C. The pH electrode and the rubber septum were
removed [caution: HCN gas present]. The flask was equipped
with a condenser and nitrogen dip tube extending into the liquid.
Vacuum (~100 mm Hg) was applied from a diaphragm pump
(a caustic trap containing ~4 M NaOH was used to collect the
HCN in the off gas). The reaction mixture was heated to 40◦C. A
nitrogen bleed was used to facilitate the removal of HCN. After
2.5 h, HCN removal was complete (<5 ppm in the off gas). The
mixture was cooled and treated with 3.5 g of Celite and 3.5 mL
bleach (containing 6.1% sodium hypochlorite). This mixture
was filtered through a Celite pad. The filtrate was extracted
four times with 280 mL of ethyl acetate. The combined ethyl
17 K. A. Powell, S. W. Ramer, S.B. del Cardayre, W. P. C. Stemmer, M. B.
Tobin, P. F. Longchamp and G. W. Huisman, Angew. Chem., Int. Ed.,
2001, 40, 3948; M. T. Reetz and K.-E. Jaeger, Chem.–Eur. J., 2000,
6, 407; S. B. Rubin-Pitel and H. Zhao, Combinatorial Chemistry &
High Throughput Screening, 2006, 9, 247.
18 R. J. Fox, S. C. Davis, E. C. Mundorff, L. M. Newman, V. Gavrilovic,
S. K. Ma, L. M. Chung, C. Ching, S. Tam, S. Muley, J. Grate, J.
Gruber, J. C. Whitman, R. A. Sheldon and G. W. Huisman, Nat.
Biotechnol., 2007, 25, 338.
19 US 7125693 and US 7132267 to Codexis.
20 W. P. Stemmer, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 10747–51;
W. P. Stemmer, Nature, 1994, 370, 389–91; J. E. Ness, M. Welch, L.
Giver, M. Bueno, J. R. Cherry, T. V. Borchert, W. P. Stemmer and J.
Minshull, Nat. Biotechnol., 1999, 17, 893–6.
21 J. F. Chaparro-Riggers, K. M. Polizzi and A. S. Bommarius,
Biotechnol. J., 2007, 2, 180; R. J. Fox and G. H. Huisman, Trends
Biotechnol., 2008, 26, 132.
22 P. Anastas and J. Warner, Eds., Green Chemistry: Theory and Practice,
Oxford University Press, New York, 1998.
23 R. A. Sheldon, Chem. Ind. (London, UK), 1992, 903; R. A. Sheldon,
Chem. Commun., 2008, 3352; R. A. Sheldon, Green Chem., 2007, 9,
1273.
24 B. M. Trost, Science, 1991, 254, 1471; B. M. Trost, Angew. Chem.,
Int. Ed. Engl., 1995, 34, 259.
25 P. A. Wender, M. P. Croatt and B. Witulski, Tetrahedron, 2006, 62,
7505; P. A. Wender, V. A. Verma, T. J. Paxton and T. H. Pillow, Acc.
Chem. Res., 2008, 41, 40.
26 US 6,140,527 to Kaneka.
86 | Green Chem., 2010, 12, 81–86
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