738
T. Purkarthofer et al. / Tetrahedron 60 (2004) 735–739
required amount of HCN was freshly formed by dropping a
saturated NaCN solution into aqueous sulfuric acid (60%) at
3.2.5. Reaction in organic solvents. 0.5 g of Celitew
were swollen in 4 mL of 50 mM phosphate buffer (pH
7.0) for 2 h. After filtration, 4.2 mL of a solution of
recombinant PaHNL (175 U/mL) and 2 mL of phos-
phate buffer (pH 7.0) were added and the mixture was
stirred for 10 min at room temperature. After freezing in
liquid nitrogen the preparation was lyophilized. To a
suspension of the dried enzyme formulation and 1
8
0 8C and trapping HCN at 212 8C in a cooling trap. For
continuous warning, an electrochemical sensor for HCN
detection was used. Waste solutions containing cyanides
were treated with aqueous sodium hypochlorite (10%).
Subsequently the pH was adjusted to 7.0 with aqueous
sulfuric acid.
(
toluene or CH Cl ) were added either 60 or 120 mL of
250 mL, 2.46 mmol) in 6 mL of dry solvent (either
3.2.1. Racemic 2-acetoxy-2-phenyl acetonitrile (acetyl-
ated racemic 3). To a stirred solution of 3 (2.00 g,
2
2
phosphate buffer [pH 7.0, 1 or 2% (v/v)] and 2 (610 mL,
6.17 mmol) and the mixture was stirred at room
temperature. The course of the reaction was followed
by GC after acetylation. In the case of HCN-triggering,
HCN (95 mL, 2.46 mmol) was added prior to the
addition of 2.
1
1
5.02 mmol) and pyridine (2.90 mL, 36.04 mmol) in
5 mL dry CH Cl , acetyl chloride (1.30 mL, 18.30 mmol)
2
2
was added dropwise at 0 8C under an argon atmosphere.
Stirring was continued for 1 h. Subsequently, the solution
was poured onto ice water. The organic phase was extracted
with NaHSO 5% and sat. NaHCO , dried (Na SO ) and
4
3
2
4
concentrated under reduced pressure. Purification using
column chromatography (cyclohexane/EtOAc¼50:1–20:1)
1
yielded acetylated 3 (1.81 g, 69%) as a colourless oil. H
NMR (CDCl ): d (ppm)¼2.17 (s, 3H), 6.42 (s, 1H), 7.46–
References and Notes
3
1
3
7
.51 (m, 5H); C NMR (CDCl ): d (ppm)¼20.71, 63.09,
1. (a) Gotor, V. Org. Proc. Res. Dev. 2002, 6, 420–426. (b)
Gotor, V. J. Biotechnol. 2002, 96, 35–42. (c) Effenberger, F.;
F o¨ rster, S.; Wajant, H. Curr. Opin. Biotechnol. 2000, 11,
3
116.37, 128.12, 129.50, 130.66, 131.99, 169.18. The NMR
data were consistent with those previously reported.
1
2
5
32–539. (d) Johnson, D. V.; Zabelinskaja-Mackova, A. A.;
3
.2.2. Racemic 2-ethoxycarbonyloxy-2-phenyl aceto-
nitrile (racemic 4). To a stirred solution of 1 (1.90 mL,
8.69 mmol) in 10 mL dry THF under an argon atmosphere
were added 2 (3.70 mL, 37.45 mmol) and DABCO (210 mg,
Griengl, H. Curr. Opin. Chem. Biol. 2000, 4, 103–109.
2. (a) Fechter, M. H.; Griengl, H. In Enzyme Catalysis in Organic
Synthesis; Drauz, K., Waldmann, H., Eds.; Wiley-VCH:
Weinheim, 2002; pp 974–989. (b) Griengl, H.; Schwab, H.;
Fechter, M. TIBTECH 2000, 18, 252–256. (c) Griengl, H.;
Hickel, A.; Johnson, D. V.; Kratky, C.; Schmidt, M.; Schwab,
H. Chem. Commun. 1997, 1933–1940. (d) Effenberger, F.
Angew. Chem. 1994, 106, 1609–1619.
1
1
1
.87 mmol) at 0 8C. The ice bath was removed and after
.5 h the reaction was diluted with 30 mL of ether. The single
phase was washed with sat. NaHCO3 (2£20 mL). The
aqueous phase was re-extracted with ether (2£20 mL). The
combined organic phases were dried (Na SO ) and con-
2
4
3. For reviews see: (a) Gregory, R. J. H. Chem. Rev. 1999, 99,
3649–3682. (b) North, M. Tetrahedron: Asymmetry 2003, 14,
147–176.
centrated under reduced pressure. Purification using column
chromatography (cyclohexane/EtOAc¼15:1) yielded 4
1
(
3.22 g, 84%) as a colourless oil. H NMR (CDCl ): d
3
4. Hanefeld, U.; Straathof, A. J. J.; Heijnen, J. Mol. Catal. B
Enzym. 2001, 11, 213–218.
(
ppm)¼1.34 (t, J¼7 Hz, 3H), 4.29 (q, J¼7 Hz, 2H), 6.27 (s,
1
3
1
1
1
6
H), 7.44–7.57 (m, 5H); C NMR (CDCl ): d (ppm)¼
5. (a) Inagaki, M.; Hiratake, J.; Nishioka, T.; Oda, J. Am. Chem.
Soc. 1991, 113, 9360–9361. (b) Inagaki, M.; Hiratake, J.;
Nishioka, T.; Oda, J. Org. Chem. 1992, 57, 5643–5649. (c)
Inagaki, M.; Hatanaka, A.; Mimura, M.; Hiratake, J.;
Nishioka, T.; Oda, J. Bull. Chem. Soc. Jpn 1992, 65, 111–120.
6. (a) Kanerva, L. T.; Rahiala, K.; Sundholm, O. Biocatalysis
1994, 10, 169–180. (b) Paizs, C.; Tosa, M.; Majdik, C.;
T a¨ htinen, P.; Irimie, F. D.; Kanerva, L. T. Tetrahedron:
Asymmetry 2003, 14, 619–627. see also (c) Li, Y.-X.;
Straathof, A. J. J.; Hanefeld, U. Tetrahedron: Asymmetry
2002, 13, 739–743.
3
4.34, 65.86, 66.60, 116.02, 128.12, 129.51, 130.87, 131.49,
53.67. Anal. calcd for C H NO : C, 64.38; H, 5.40; N,
.83; Found: C, 64.60; H, 5.41; N, 6.74.
1
1
11
3
3.2.3. NMR measurements. For stability studies 40 mL of 2
were dissolved in 0.5 mL of D O containing 1% (v/v) of
DMSO-d . Spectra were recorded every 15 min over a
6
period of 16.5 h at 22 8C. For the biocatalytic reaction, to
0.6 mL of a solution of recombinant PaHNL (80 U/mL after
dilution with D O and pH adjustment to 3.3 with an aqueous
2
2
citric acid solution) containing 1% (v/v) of DMSO-d were
6
added 30 mL of 1 (0.30 mmol) and 90 mL of 2 (0.91 mmol).
Spectra were recorded every 15 min over a period of 5 h at
22 8C. The residual water signal was suppressed by the
transmitter presaturation method.
7. For reviews see: (a) El Gihani, M. T.; Williams, J. M. J.
Curr. Opin. Chem. Biol. 1999, 3, 11–15. (b) Huerta, F. F.;
Minidis, A. B. E.; B a¨ ckvall, J.-E. Chem. Soc. Rev. 2001,
30, 321–331.
8. (a) Tian, S.-K.; Deng, L. J. Am. Chem. Soc. 2001, 123,
6
195–6196. (b) Tian, J.; Yamagiwa, N.; Matsunaga, S.;
3.2.4. Reaction in water. 2.5 mL of a solution of
recombinant PaHNL (300 U/mL) were adjusted to pH 3.3
with an aqueous solution of citric acid and diluted with
Shibasaki, M. Angew. Chem. 2002, 114, 3788–3790. (c)
Casas, J.; Baeza, A.; Sansano, J. M.; N a´ jera, C.; Sa a´ , J. M.
Tetrahedron: Asymmetry 2003, 14, 197–200.
2
3
.5 mL of 50 mM potassium phosphate/citrate buffer (pH
.3). 1 (106 mg, 1 mmol) and 2 (297 mL, 3 mmol) were
9. Schwab, H.; Glieder, A.; Kratky, C.; Dreveny, I.; Poechlauer,
P.; Skranc, W.; Mayrhofer, H.; Wirth, I.; Neuhofer, R.; Bona,
R. EP 1223220 (DSM Fine Chemicals, Austria).
10. Weber, H.; Brecker, L. Curr. Opin. Biotechnol. 2000, 11,
572–578.
added and the mixture was stirred at room temperature. The
course of the reaction was monitored by GC after
acetylation.