Lipase catalysed Michael addition of secondary amines to acrylonitrile

Article abstract of DOI:10.1039/b402244k

A new enzymatic process is described. Different preparations of lipase B from Candida antarctica are able to catalyse Michael-type addition of secondary amines to acrylonitrile. This new reaction widens the applicability of these biocatalysts in organic synthesis.

Full text of DOI:10.1039/b402244k

Lipase catalysed Michael addition of secondary amines to acrylonitrile
Oliver Torre, Ignacio Alfonso and Vicente Gotor*
Departamento de Química Orgánica e Inorgánica, Universidad de Oviedo, C/Julián Clavería, 8, E-33071
Oviedo, Spain. E-mail: vgs@sauron.quimica.uniovi.es; Fax: +34 985103448; Tel: +34 985103448
Received (in Cambridge, UK) 13th February 2004, Accepted 11th June 2004
First published as an Advance Article on the web 28th June 2004
A new enzymatic process is described. Different preparations of
lipase B from Candida antarctica are able to catalyse Michael-
type addition of secondary amines to acrylonitrile. This new
reaction widens the applicability of these biocatalysts in organic
synthesis.
also takes place with cyclic and non-cyclic amines and the reaction
rates depend on the amine structure. In this preliminary commu-
nication, specific data for pyrrolidine 1, piperidine 2 and diethyl
amine 3 are reported although other amines also react in very
similar reaction conditions. The reactivity order goes from cyclic to
non-cyclic amines (1 > 2 > 3), in good agreement with their
nucleophilicity.
Taking into account these results, we performed some experi-
ments focused on demonstrating the specific catalytic effect of the
enzyme. When the enzyme (Novozyme 435) is pre-treated with
urea at 100 °C in order to completely denaturalise the protein, the
rate is practically equal to the background reaction (entry 6),
The use of enzymes for organic synthesis has become an interesting
area for organic and bioorganic chemists. Since many enzymes
have demonstrated their activity with nonnatural substrates in
organic media, they have become widely used to carry out synthetic
transformations.1 Among all of them, hydrolases and especially
lipases are the most used enzymes due to their high stability and
activity with a broad spectrum of substrates. Besides, many of them
are commercially available; they work under mild reaction
conditions and without cofactor.
Table 1 Initial rates (V ) of Michael addition between secondary amines
o
(0.05 M) and acrylonitrile (0.1 M)
During our work devoted to exploring new synthetic applications
of biocatalysts, we have found that some hydrolases are able to
catalyse different reactions from the natural process that they
usually promote. For instance, some lipases are able to catalyse
Amount
(mg)
V
o
Entry
Amine Catalyst
(mM·min21) Vrela
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
2
2
2
3
3
3
—
—
100
50
100
50
100
50
23
300
267
467
200
40
1
2
aminolysis and ammonolysis reactions. In a recent paper, an
Novozyme 435
Chirazyme L-2
Chirazyme L-2
lyophilised CALB
denaturedb
B.S.A.
13.3
11.9
20.7
8.9
1.8
1
engineered mutant of CAL B was developed to catalyse carbon–
3
carbon bond forming reactions. The mutants Ser105Ala and
Ser105Gly, which lack the nucleophilic serine residue in the
catalytic triad, showed a clear aldolase activity. Surprisingly, the
wild type enzyme is also able to promote that reaction, although to
a lower extent. With the help of theoretical calculations, the authors
propose a mechanism in which the oxyanion hole of the active site
stabilizes the negative charge of the transition states and the His-
Asp pair serves as a proton shuttle. Considering that mechanism,
we envisioned that this enzyme could also catalyse Michael
addition. In a preliminary paper, a report regarding this possibility
was published, but in that case a covalent acyl-enzyme intermediate
20
—
—
8
1
Novozyme 435
Chirazyme L-2
—
Novozyme 435
Chirazyme L-2
100
100
—
100
100
100
300
3
100
300
12.5
37.5
1
33.3
100
10
11
12
13
a
c
c
c
Initial rates relative to the reaction in the absence of catalyst. b Pre-treated
with urea at 100 °C for several hours. These reactions were run at 0.3 M
c
4
can be formed. Here we report the unprecedented lipase-catalysed
in both amine and nitrile.
Michael addition of a secondary amine to acrylonitrile. In our case,
although a covalent acyl-enzyme intermediate is prevented, the
catalytic effect of the lipase is clearly demonstrated.
The reaction of acrylonitrile with different secondary amines in
the presence of lipase B from Candida antarctica led to the
corresponding Michael adduct faster than in the absence of the
biocatalyst. We have followed the formation of the products by GC
in order to quantify their concentration and compare reaction
rates.† Results of initial rates with different amines and biocatalysts
are summarised in Table 1. In all cases CAL B is able to catalyse the
reaction, up to 100-fold faster than the process in absence of
biocatalyst. Among two different CAL B preparations, Chirazyme
L-2 is more efficient than Novozyme 435 for the process. The
initial rate is practically proportional to the enzyme amount (entries
3
and 4), suggesting the catalytic effect of the enzyme. The reaction
Fig. 1 Plot of the concentration of Michael adduct product of pyrrolidine
(0.05 M) and acrylonitrile (0.1 M) versus time in the presence of: (a) no
catalyst (open squares); (b) 100 mg of CAL B Novozyme 435 (filled
squares); (c) 100 mg of CAL B Chirazyme L2 (filled diamonds); (d) 50 mg
of lyophilised CAL B (filled triangles); (e) 100 mg of denatured CAL B
Novozyme 435 (crosses); (f) 50 mg of bovine serum albumin (open
triangles).
Scheme 1
1
724
C h e m . C o m m u n . , 2 0 0 4 , 1 7 2 4 – 1 7 2 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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entering Michael donor nucleophile to the C-a position of the
6
Michael acceptor. In the catalytic site of a serine hydrolase both
moieties are present very close in the space. The proposed
mechanism would start with the accommodation of the Michael
acceptor (acrylonitrile) in the active site. Interaction of the nitrile
with the oxyanion hole would increase its electrophilic ability.
Then, conjugated addition of the nucleophile would lead to a
zwiterionic intermediate, which can be stabilised by both the
oxyanion hole and the His-Asp pair. This His-Asp pair, as proposed
7
in similar catalytic cycles, catalyses proton transference from the
incoming nucleophile to the a-carbon. Finally, a new Michael
acceptor would shift the final product, leading to a new catalytic
cycle.
In conclusion, here we report an unprecedented lipase catalysed
Michael addition reaction. Initial rates with different preparations
of the biocatalyst and different secondary amines are measured.
The catalytic effect of the enzyme is demonstrated by the
combination of different experiments. The importance of this new
amino-lyase activity ranges from the fundamental study of
enzymatic function to the potential synthetic applicability of this
new biotransformation. Studies in both directions are being carried
out in our laboratory and will be published in due course.
Notes and references
†
Typical experimental procedure: In an Erlenmeyer under nitrogen
atmosphere, containing the corresponding enzyme, a solution of internal
standard (anisol) and acrylonitrile in toluene was incubated at 30 °C and 250
r.p.m. (orbitally shaken) for 5 minutes. Then, the secondary amine was
added in order to initiate the reaction. The final concentrations of reactants
were 0.05 M amine and 0.1 M acrylonitrile for reactions with amines 1 and
2
, or 0.3 M in both amine and nitrile for 3. Samples were withdrawn from
Scheme 2 Proposed mechanism of lipase catalysed Michael addition.
the reaction and directly analysed by GC (100% dimethyl polysiloxane
capillary column, FID detection; oven temperature: from 60 °C to 160 °C,
suggesting that the tertiary structure of the biocatalyst is necessary
to promote the process. Moreover, this experiment suggests that the
polymeric support has little effect on the catalysis. This has been
further demonstrated by performing the reaction with lyophilised
CAL B, which is also able to catalyse the Michael addition (entry
21
rate of heating 20 °C min. ). All the compounds were spectroscopically
1
13
characterised ( H, C NMR and MS) and analytically compared (GC) with
true samples prepared by conventional methods. The reactions were run at
least twice.
5). Although the process is less efficient, the enzyme itself can
1 For some recent revisions, see: F. Secundo and G. Carrea, Chem. Eur. J.,
2
003, 9, 3194; N. M. Shaw, K. T. Robins and A. Kiener, Adv. Synth.
increase 9-fold the initial reaction rate. Another concern would be
if the catalysis takes place in a specific way or maybe some amino
acids on the enzyme surface can promote the process. Related to
that, we confirmed that bovine serum albumin (B.S.A.) is unable to
catalyse the same reaction (entry 7 in table 1, Fig. 1). Considering
that this protein has similar amino acid distribution on its surface,
this result suggests the reaction must take place in a specific fashion
on the catalytic site.
We also observed that, at long reaction times, some side products
coming from the acrylonitrile were formed. This presumably
polymerisation process can be avoided by adding an inhibitor such
us dihyroquinone to the reaction mixture. Both, reaction rate and
final conversion can be improved this way.
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2
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4
5
6
T. Kitazume, T. Ikeya and K. Murata, J. Chem. Soc., Chem. Commun.,
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1
With all these results in hand, we propose a tentative mechanism
for this new process (Scheme 2). Very common catalysts for
5
Michael reaction usually are Lewis acids (similar to the oxyanion
7 T. Ishida and S. Kato, J. Am. Chem. Soc., 2003, 125, 12035 and
hole) or an acid–base group for proton transference from the
references cited therein.
C h e m . C o m m u n . , 2 0 0 4 , 1 7 2 4 – 1 7 2 5
1725