enzymes.4 It will be exciting to see whether catalytic activities of
BAL and BFD can be interconverted by mutagenesis studies.
Turkish Scientific and Technical Research Council
(TUBITAK). E. J. is recipient of a Konrad Adenauer Stiftung
fellowship. The authors thank Professor R. Vicuña for provid-
ing the BAL gene.
Experimental
Preparation of BAL
Notes and references
1 (a) W. Boland, C. Frößl and M. Lorenz, Synthesis, 1991, 1049;
(b) K. Faber, Pure Appl. Chem., 1997, 69, 1613; (c) F. Thiel, Chem.
Rev., 1995, 95, 2203.
Hexahistidine-tagged BAL was obtained from recombinant
E. coli SG13009 cells following a procedure described previ-
ously.8a One unit of activity is defined as the amount of enzyme
which catalyses the cleavage of 1 µmol benzoin (1.5 mM) into
benzaldehyde in potassium phosphate buffer (50 mM, pH 7.0,
containing MgSO4 (2.5 mM), ThDP (0.15 mM) and 15% PEG
400 (v/v)) in 1 min at 30 ЊC.
2 (a) R. B. Herbert, B. Wilkinson, G. J. Ellames and E. K. Kunec,
J. Chem. Soc., Chem. Commun., 1993, 205; (b) R. B. Herbert,
B. Wilkinson and G. J. Ellames, Can. J. Chem., 1994, 72, 114;
(c) S. M. Roberts and P. W. H. Wan, J. Mol. Catal. B: Enzym., 1998,
4, 111 and references cited therein; (d) A. L. Zeitlin, M. S. Raju and
D. I. Stirling, Eur. Pat. Appl. EP 507 153, 1992 (Chem. Abstr., 1993,
118, 21078b); (e) T. Morikawa, M. Ikemi and T. Myoshi, JP Patent
06 125 786, 1994 (Chem. Abstr., 1994, 121, 81137s); ( f ) J. Q. Liu,
M. Odani, T. Dairi, N. Itoh, S. Shimizu and H. Yamada, Appl.
Microbiol. Biotechnol., 1999, 51, 586; (g) cf. J. M. Turner, T. Bui,
R. A. Lerner, C. F. Barbas III and B. List, Chem. Eur. J., 2000, 6,
2772.
3 cf. (a) D. Voet and J. G. Voet, Biochemistry, 2nd edn., Wiley,
New York, 1995; (b) Enzyme Nomenclature, Recommendations of the
Nomenclature Committee of the International Union of Biochemistry,
Academic Press, San Diego, 1992, p. 447.
4 (a) G. A. Sprenger and M. Pohl, J. Mol. Catal. B: Enzym., 1999, 6,
145; (b) U. Schörken and G. A. Sprenger, Biochim. Biophys. Acta,
1998, 1385, 229; and references cited therein.
5 (a) G. M. Coppola and H. F. Schuster, α-Hydroxy Acids in
Enantioselective Synthesis, VCH, Weinheim, 1997; (b) J. Polonský,
Prog. Chem. Org. Nat. Prod., 1985, 47, 221; (c) S. Hanessian, Total
Synthesis of Natural Products: The Chiron Approach, Pergamon,
New York, 1983, ch. 2; (d) W. Adam, M. Lazarus, C. R. Saha-
Möller and P. Schreier, Acc. Chem. Res., 1999, 32, 837; (e) F. A.
Davies and B.-C. Chen, Chem. Rev., 1992, 92, 919; ( f ) T.
Hashiyama, K. Morikawa and K. B. Sharpless, J. Org. Chem., 1992,
57, 5067; (g) A. S. Demir, H. Hamamci, C. Tanyeli, I. M.
Akhmedov and F. Doganel, Tetrahedron: Asymmetry, 1998, 9, 1673.
6 (a) B. Gonzáles and R. Vicuña, J. Bacteriol., 1989, 2401; (b) P.
Hinrichsen, I. Gómez and R. Vicuña, Gene, 1994, 144, 137.
7 M. Pohl and E. Janzen, unpublished results; parts of the originally
published incorrect DNA sequence coding for BAL6 have been
corrected. The corrected DNA-sequence has been submitted,
GenBank accession no. AY007242.
8 (a) H. Iding, T. Dünnwald, L. Greiner, A. Liese, M. Müller,
P. Siegert, J. Grötzinger, A. S. Demir and M. Pohl, Chem. Eur. J.,
2000, 6, 1483; (b) R. Kluger, J. F. Lam, J. P. Pezacki and C.-M. Yang,
J. Am. Chem. Soc., 1995, 117, 11383; (c) R. Kluger, Pure Appl.
Chem., 1997, 69, 1957; (d) D. Kern, G. Kern, H. Neef, K. Tittmann,
M. Killenberg-Jabs, C. Wikner, G. Schneider and G. Hübner,
Science, 1997, 275, 67.
Representative example of the synthesis of benzoin derivatives:
(R)-benzoin
Benzaldehyde (318 mg, 3 mmol) was dissolved in a mixture of
DMSO (20 mL) and potassium phosphate buffer (80 mL, 50
mM, pH 7.0, containing MgSO4 (2.5 mM) and ThDP (0.15
mM)). After addition of BAL (20 U) the reaction was allowed
to stand at 25 ЊC for 48 h and a further 20 U of BAL was
added. After 62 h the conversion was determined as 97%
(GC-MS). The reaction mixture was extracted with dichloro-
methane (250 mL) and the organic layer washed with water
(25 mL) and brine (25 mL) and dried over Na2SO4. Evapor-
ation of the solvent and purification of the crude product by
crystallisation afforded 305 mg (96%) (R)-benzoin (mp 134 ЊC);
[α]D22 Ϫ115 (c 1.5, CH3COCH3), ee > 99%; lit.11 [α]D25 Ϫ118.3
(c 2.4, CH3COCH3) for ee 99% (from commercially available
compound); HPLC: (Chiralpak AD, isohexane–propan-2-ol;
90 : 10, 0.75 ml minϪ1, 20 ЊC, 21 bar) tR: 26.95 min.
Representative example of the synthesis of (R)-2-hydroxy-1-
phenylpropanone derivatives: (R)-2-hydroxy-1-phenylpropanone
[(R)-2-HPP]
Benzaldehyde (212 mg, 2 mmol) was dissolved in a mixture of
DMSO (20 mL) and potassium phosphate buffer (80 mL, 50
mM, pH 7.0, containing MgSO4 (2.5 mM) and ThDP (0.15
mM)). To this solution 88 mg (2 mmol) acetaldehyde was
added. After addition of BAL (20 U) the reaction was allowed
to stand at 25 ЊC. After 24 h 20 U of BAL and 176 mg
(4 mmol) of acetaldehyde were added. This was repeated
every 24 h. After 96 h the conversion was determined as 97%
(GC-MS). Work-up according to the former procedure
afforded 285 mg (95%) (R)-2-HPP, [α]D22 ϩ76 (c 2, CHCl3),
ee > 99%; lit.12a [α]D22 ϩ70 (c 1.7, CHCl3) for ee 89%; lit.12b [α]D20
ϩ58 (c 2, CHCl3) for ee 62%; lit.5g [α]D20 ϩ82 (c 2, CHCl3) for ee
96%; HPLC: (Chiralpak AD, isohexane–propan-2-ol; 90 : 10,
0.75 ml minϪ1, 20 ЊC, 21 bar) tR: 15.78 min.
9 (a) A. S. Demir, T. Dünnwald, H. Iding, M. Pohl and M. Müller,
Tetrahedron: Asymmmetry, 1999, 10, 4769; (b) T. Dünnwald, A. S.
Demir, P. Siegert, M. Pohl and M. Müller, Eur. J. Org. Chem., 2000,
2161.
10 D. Kolter, R. Feldmann, P. Dünkelmann and M. Müller,
unpublished work.
11 J. Kenyon and R. L. Patel, J. Chem. Soc., 1965, 435.
12 (a) T.-H. Duh, Y. F. Wang and M.-J. Wu, Tetrahedron: Asymmetry,
1993, 4, 1793; (b) F. A. Davis, A. C. Sheppard, B. C. Chen and
M. S. Haque, J. Am. Chem. Soc., 1990, 112, 6679.
Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft
within the scope of SFB 380 is gratefully acknowledged. We
thank the Alexander von Humboldt Foundation, DAAD,
J. Chem. Soc., Perkin Trans. 1, 2001, 633–635
635