Lipase CAL-B does not catalyze a promiscuous decarboxylative aldol addition or Knoevenagel reaction

Article abstract of DOI:10.1039/c0gc00505c

Recently it was reported that lipase B from Candida antarctica (CAL-B) shows catalytic promiscuity in the decarboxylative aldol addition and Knoevenagel reaction between 4-nitrobenzaldehyde (4-NBA) and ethyl acetoacetate. We here provide experimental evidence that CAL-B simply catalyzed hydrolysis of the ester yielding the free acetoacetic acid, which under the reaction conditions readily reacts with 4-NBA to the described products.

Full text of DOI:10.1039/c0gc00505c

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Lipase CAL-B does not catalyze a promiscuous decarboxylative aldol
addition or Knoevenagel reaction
Andrew S. Evitt and Uwe T. Bornscheuer*
Received 1st September 2010, Accepted 6th January 2011
DOI: 10.1039/c0gc00505c
Recently it was reported that lipase B from Candida
antarctica (CAL-B) shows catalytic promiscuity in the
decarboxylative aldol addition and Knoevenagel reaction
between 4-nitrobenzaldehyde (4-NBA) and ethyl acetoac-
etate. We here provide experimental evidence that CAL-
B simply catalyzed hydrolysis of the ester yielding the
free acetoacetic acid, which under the reaction conditions
readily reacts with 4-NBA to the described products.
(Scheme 1, Fig. 1a and b) as reported by Feng et al. and the
reaction without enzyme by starting with the already hydrolysed
ester – the lithium carboxylate 4 (Scheme 1, Fig. 1c). Initially
we performed the reaction as described, using CAL-B in the
presence of HPLC grade MeCN with no additional water. No
aldol product 5 and a very low formation of the Knoevenagel
product 6 were found, as confirmed by GC–MS analysis (Fig.
1a). In contrast – and as expected by us as outlined above –
an addition of water (10-fold excess) facilitated hydrolysis by
CAL-B and readily led to the formation of both the aldol 5,
as observed by Feng et al., and the Knoevenagel product 6
(Fig. 1b).
Furthermore, we repeated the authors’ reaction conditions us-
ing tert-butyl acetoacetate 3, an ester substrate that is known to
not be hydrolysed by CAL-B. Production of aldol/Knoevenagel
products did not proceed any faster than background levels,
further indicating the lack of role of CAL-B in promiscuous
catalysis (Fig. 1d).
The results gained in this study show that, from several
perspectives, the original authors have over-interpreted their
results. Firstly, the CAL-B catalysed reactions with and without
additional water suggest the requirement for hydrolysis prior
to the aldol/Knoevenagel reaction. Fresh HPLC grade MeCN
has a low, but present, water content. Observation of the
Knoevenagel product suggests that some level of hydrolysis
was occurring in this low water solvent, and even though each
molecule of 5 produced also restores the water molecule used
in hydrolysis, the water levels were considered too low to allow
full conversion within the reaction time reported by Feng et al.
The dehydrating nature of the low-water solvent may explain
why only the Knoevenagel product was obtained. In a 10-fold
excess of water, the reaction proceeds as described in the original
publication.
Catalytic promiscuity refers to enzymes catalyzing unexpected
reactions. This includes an enzyme’s ability to work under
unusual conditions (such as in organic solvents), but the most
important are examples in which more than one type of chemical
reaction – the natural as well as one or more unexpected
reactions – are catalyzed. Numerous examples can be found in
recent reviews.1,2 In addition, Mannich condensations, Michael,
and Markovnikov additions have been reported. Recently, it
was reported in this journal that a lipase (lipase B from
Candida antarctica, CAL-B) can catalyze the decarboxylative
aldol addition and Knoevenagel reaction between substrates
such as 4-nitrobenzaldehyde 1 and ethyl acetoacetate 2 (Scheme
1).3 At first glance, it appears that the authors have controls (i.e.
no conversion in the absence of lipase or with inactivated CAL-
B or bovine serum albumin); however, compound 2 is an ester
and hence can be hydrolyzed to the free carboxylic acid 4.
In order to prove that CAL-B does not show any promiscuous
activity and simply performs the well known hydrolysis of
ethyl ester 2, we examined both the CAL-B-catalysed reaction
Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis,
Greifswald University, Felix-Hausdorff-Str. 4, D-17487, Greifswald,
Germany. E-mail: uwe.bornscheuer@uni-greifswald.de; Fax: +49 3834
86 80066; Tel: +49 3834 86 4367
Scheme 1 Reaction of 4-nitrobenzaldehyde 1 with ethyl acetoacetate 2 using CAL-B (as reported by Feng et al.)3 or with lithium acetoacetate 4 in
the absence of lipase to yield aldol product 5 or Knoevenagel product 6.
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Fig. 1 (a) Reaction of 1 and 2 in the presence of CAL-B and cyclen in anhydrous MeCN;3 (b) reaction of 1 and 2 in the presence of CAL-B, cyclen
and H2O in MeCN;3 (c) reaction of 1 and 4 in the presence of cyclen in anhydrous MeCN (without CAL-B); (d) reaction of 1 and 3 in the presence of
CAL-B, cyclen and H2O in MeCN.
Distinct evidence for the lack of promiscuous CAL-B activity
was obtained when performing the reaction with the free
Experimental
CAL-B was purchased from Sigma–Aldrich (catalogue number
carboxylic acid (lithium acetoacetate 4) in the absence of CAL-
L4777 identical to that used by Feng et al.). Lithium acetoacetate
B and in dry MeCN (Fig. 1c). The similar timescales and
was purchased from Sigma–Aldrich (A8509) and ethyl acetoac-
product profiles of the reactions with and without enzyme
etate from Fluka (00410). MeCN was purchased at HPLC grade
strongly indicates that CAL-B was not providing a parallel
from Fisher chemicals and water was distilled. The reaction
and promiscuous mechanistic pathway between the carboxylic
was conducted in accordance with the conditions reported by
acid and reaction products. The lack of enantioselectivity in the
Feng et al.3 Reaction in Fig. 1a: to a 1.5 ml Eppendorf tube
was added MeCN (0.5 ml), 4-nitrobenzaldehyde 1 (0.1 mmol),
ethyl acetoacetate 2 (or tert-butyl acetoacetate 3; 0.12 mmol),
formation of 5 may also be attributed to the fact that the enantio-
determining step does not occur within the enzyme active-
site.
cyclen (0.01 mmol), and CAL-B (10 mg) and the mixture was
The mechanism detailing why acetoacetate 4 will undergo
incubated at 30 ◦C and 800 rpm. After 20 h, the reaction mixture
aldol reaction, while ethyl acetoacetate 2 does not, has not been
was diluted by a factor of 3 in MeCN and analysed by GC–
investigated by us. Deprotonation of the acetoacetic acid occurs
MS. Reaction in Fig. 1b: identical to the Fig. 1a procedure
but distilled water (1.2 mmol) was added. Reaction in Fig.
1c: identical to the Fig. 1a procedure but without CAL-B
and in the presence of lithium acetoacetate 3 (0.12 mmol).
first at the carboxylic acid functionality, resulting in a negatively
charged carboxylate, and the pKa of the free acid alpha protons
is therefore higher than the pKa of the ester. The pKa cannot,
therefore, underpin the preferential reaction of the free acid
Samples were analysed by GC–MS using a Shimadzu GC-2010
over the ester. We would hypothesise that, in line with previous
series gas chromatograph coupled with a Shimadzu GCMS-
investigations of the aldol/Knoevenagel reaction mechanism,
QP2010S mass detector. The stationary phase was a 5% phenyl
polysilphenylene-siloxane (BPX5) column and the mobile phase
the reaction of the enol form of both 2 and 4 with the aldehyde
is reversible.4 It is, however, only the acid that can perform the
◦
used was hydrogen. The injector was set at 250 C. The initial
irreversible decarboxylation.
◦
oven temperature was 60 ◦ C and th◦ is was held constant for 15
What the authors have discovered, therefore, is not a
min, then ramped to 180 C at 10 C min-1 and held at 180 ◦C
for a further 5 min.
reaction in which CAL-B performs aldol catalysis, but a
lipase-catalyzed “deprotection” that yields a reactive interme-
diate which is otherwise more difficult to prepare and work
Notes and references
1 U. T. Bornscheuer and R. J. Kazlauskas, Angew. Chem., Int. Ed., 2004,
43, 6032–6040.
2 K. Hult and P. Berglund, Trends Biotechnol., 2007, 25, 231–238.
3 X. W. Feng, C. Li, N. Wang, W. W. Zhang, Z. Wang and X. Q. Yu,
Green Chem., 2009, 11, 1933–1936.
with.5
Conclusions
4 M. Tanaka, O. Oota, H. Hiramatsu and K. Fujiwara, Bull. Chem. Soc.
Although CAL-B shows catalytic promiscuity in several cases,1,2
Jpn., 1998, 61, 2473–2479.
5 T. Kourouli, P. Kefalas, N. Ragoussis and V. Ragoussis, J. Org. Chem.,
2002, 67, 4615–4618.
this is not true for the decarboxylative aldol addition and
subsequent Knoevenagel reaction as reported.3
1142 | Green Chem., 2011, 13, 1141–1142
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The Royal Society of Chemistry 2011
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