Communications
nitroalcohol would then be deprotonated at the hydroxy
[2] a) K. Gruber, C. Kratky, J. Polym. Sci. Part A 2004, 42, 479 – 486;
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function by the catalytic triad Ser80/His235/Asp207 in the
cleavage direction, and the positive charge contributed by
Lys236 would facilitate the deprotonation of the nitroalkane
[
3]
in the synthesis direction. Whether enzyme kinetics follow
an ordered “uni–bi” mechanism as observed for cyanohydrin
[
16]
substrates is under investigation. The substrate binding site
of HbHNL is spacious enough to accept the more voluminous
nitro compounds in a similar fashion to the binding of the
corresponding cyanohydrins (Figure 1). Steric interactions,
however, may still be responsible for the observed reduced
catalytic rate.
[
4] a) N. Ono, The Nitro Group in Organic Synthesis, Wiley-VCH,
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[
For more information about the mechanism of the
enzymatic Henry reaction, we carried out experiments using
deuterated nitroalkanes. Although definitive conclusions will
have to await results from proper kinetics experiments, the
[6] L. Henry, C. R. Hebd. Seances Acad. Sci. 1895, 120, 1265.
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5
reduced yield obtained for the addition of [1,1-D ]nitroethane
2
3
to benzaldehyde already indicates the existence of a kinetic
isotope effect and suggests the deprotonation of the nitro-
alkane to be the rate-limiting step. In contrast, in the
HbHNL-catalyzed cyanohydrin reaction, the CÀC bond
2
hensive Asymmetric Catalysis, Vol. III (Eds.: E. N. Jacobsen, A.
Pfalz, H. Yamamoto), Springer, Berlin, 1999, pp. 1075 – 1090;
e) T. Marcelli, R. N. S. van der Haas, J. H. van Maarseveen, H.
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[
17]
formation was found to be rate limiting. In enzyme kinetics
studies of nitroalkane oxidases, true kinetic isotope effects of
8
–9 were measured for the formation of a nitroethane
À1
[
8] General procedure: Wt-HbHNL (4000 Ummol aldehyde;
[
18]
anion.
This is in reasonable agreement with our rough
activity determined for the cleavage of mandelonitrile; the
enzyme was kindly provided by DSM) was stirred in phosphate
buffer (pH 7, 50 mm) and TBME (1:1) until an emulsion was
established. Freshly distilled aldehyde (1–10 mmol) was added
to the mixture. The mixture was stirred for 5 min before the
estimate for the equivalent effect—on the order of 10—for
the HbHNL-catalyzed Henry reaction. These findings are
also in line with the well-known nitroalkane anomaly, which
describes the fact that the deprotonation rate of nitroalkanes
À1
[
19]
nitroalkane (10 mmolmmol aldehyde) was added. The reac-
is lower than that expected based on their pKa value. Thus,
a combination of steric and electronic effects may explain the
reduced rate of the HbHNL-catalyzed Henry reaction
compared to the rate of cyanohydrin formation.
tion mixture was stirred for 48 h at room temperature. After
centrifugation and separation of the layers, the aqueous phase
was extracted with TBME. The combined organic phases were
dried over Na SO and concentrated in vacuo. The crude
2
4
In summary, Henry reactions catalyzed by the hydroxyni-
trile lyase from Hevea brasiliensis involving aldehydes and
either nitromethane or nitroethane yielded the corresponding
nitroalcohols in good yields and reasonable-to-high enantio-
meric excess. In the case of the reaction with nitroethane, two
stereocenters are generated simultaneously with good dia-
stereo- and enantioselectivity, granting access to substances of
the ephedrine family. We are currently testing other carbonyl
compounds and nitroalkanes as starting materials for this
reaction, and studies to improve the efficiency of the process
with respect to both yields and stereoselectivity are underway.
products were purified by column chromatography.
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15] See the Supporting Information.
16] M. Bauer, H. Griengl, W. Steiner, Biotechnol. Bioeng. 1999, 62,
[
[
[
[
Received: November 28, 2005
Published online: April 24, 2006
20 – 29.
[
17] W.-M. Ching, R. G. Kallen, J. Am. Chem. Soc. 1978, 100, 6119 –
Keywords: biocatalysis · Henry reaction · hydroxynitrile lyase ·
molecular modeling · nitroalcohols
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Angew. Chem. Int. Ed. 2006, 45, 3454 –3456