Page 278 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

254 11 Nitrile Converting Enzymes Involved in Natural and Synthetic Cascade Reactions

converting all monocyano amide into the acid. The enantioselectivity of the overall
hydrolysis seems to be derived from the stereospecific action of NHase. Use of a
cosolvent such as acetone was mandatory to obtain a satisfactory enantioselectivity
(ee 32–95% depending on the nature of the 3-substituent).
2-Aminonitriles are very interesting substrates for biotransformation reactions
because they can be easily prepared by the Strecker reaction (or related syntheses)
from aldehydes, ammonia, and cyanide. The classical Strecker synthesis couples
the formation of the aminonitriles in aqueous media with a subsequent acid
hydrolysis to the corresponding acids. This is one of the most efficient and
straightforward methods for the synthesis of various amino acids, but suffers from
the disadvantage that the reaction products are racemic [32]. Therefore, it has been
repeatedly attempted to enzymatically convert (racemic) aminonitriles by using
(enantioselective) nitrilases or NHase/amidase systems (see also Chapter 14). There
are several reports which demonstrate that organisms expressing NHase/amidase
systems could convert 2-aminonitriles. Thus different Rhodococcus, Pantoea,and
Klebsiella strains were found which convert racemic phenylglycinenitrile into (S)-
phenylglycine with rather high enantioselectivities. In all analyzed cases, the
enantiorecognition was caused by a highly enantioselective amidase whereas the
enantioselectivity of the NHase was low. Thus, these processes can lead to highly
enantioenriched (R)-(d)-phenylglycineamide and (S)-(l)(+)-phenylglycine [33–37].
R. erythropolis A4, Rhodococcus sp. R312, and R. erythropolis NCIMB 11540 were
used in the hydrolysis of five- and six-membered alicyclic trans-aminonitriles
(Figure 11.6), which are the precursors of cyclic β-amino acids. The enzymes
discriminated between the trans- and cis-isomers, the transformation of the former
proceeding slowly or stopping at the amide stage. Moreover, hydrolysis of the latter
resulted in excellent enantiopurity of the trans-amino acids or amides [38].
Nicotinic acid has a wide range of therapeutic uses, and can be manufactured
from 3-cyanopyridine using a NHase/amidase cascade. The control of this cascade
reaction in M. imperiale by operational parameters was studied in continuous reac-
tors to optimize the production of nicotinic acid ([39, 40] for details, see Chapter
13). Its analog, 2-chloronicotinic acid, a building block of pesticides and pharma-
ceuticals, was also produced from the corresponding nitrile by NHase/amidase
using R. erythropolis ZJB-09149 [41].
A very recent addition to the use of the natural NHase/amidase system in
synthetic applications was reported by D’Antona et al. [42]. They demonstrated

Nitrile
NHR NHR NHR
hydratase Amidase
) )
(CH 2 ) n (CH 2 n (CH 2 n
CN CONH 2 COOH
R = Bz, n = 1 ee 94% (conversion 40%) R = Bz, n = 2 ee >95% (conversion 36%)
R = Ts, n = 1 ee >99% (conversion 14%) R = Ts, n = 2 ee >99% (conversion 13%)
Figure 11.6 Biotransformations of racemic β-aminonitriles by nitrile hydratase and amidase
in whole cells of Rhodococcus erythropolis A4 [38]. The enantiomeric excess is only specified
for the reactions which proceeded with significant enantioselectivities.