Polyclonal antibodies: A cheap and efficient tool for screening of enantioselective catalysts

Article abstract of DOI:10.1039/c2cc31312j

Enantioselective polyclonal antibodies have been produced and characterized to develop a high-throughput screening method for lipase activity fingerprinting, with a view to the enantioselective hydrolysis of azlactones.

Full text of DOI:10.1039/c2cc31312j

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Chemical Communications
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Volume 48 | Number 37 | 11 May 2012 | Pages 4377–4508
ISSN 1359-7345
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Frédéric Taran et al.
Polyclonal antibodies: a cheap and efficient tool for screening of enantioselective catalysts

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Polyclonal antibodies: a cheap and efficient tool for screening of
enantioselective catalystsw
a
a
a
b
b
´
Cristian Macovei, Paola Vicennati, Julia Quinton, Marie-Claire Nevers, Herve Volland,
b
a
´ ´ ´
Christophe Creminon and Frederic Taran*
Received 21st February 2012, Accepted 12th March 2012
DOI: 10.1039/c2cc31312j
Enantioselective polyclonal antibodies have been produced and
characterized to develop a high-throughput screening method for
lipase activity fingerprinting, with a view to the enantioselective
hydrolysis of azlactones.
immunoassays (ELISA).8 Although very powerful (throughput
41000 yields and ee determination per day), this method
requires preparation of mAbs which involves time-consuming
and expensive techniques. Furthermore, since mAbs generally
display very specific binding properties, few analytes can be
detected. As a consequence, ELISA using mAbs allows catalyst
screening of only a few, structurally close substrates and there-
fore cannot evaluate the substrate scope of catalysts.
Combinatorial and parallel methods have become an important
focus of research in catalysis. In particular, techniques allowing the
simultaneous screening of numerous catalyst candidates have
gained much attention over the past two decades as they accelerate
the identification and optimization of interesting catalysts.1 To
fully realize the potential of combinatorial approaches to catalyst
discovery, general and powerful high-throughput screening (HTS)
methods are essential.2
On the other hand, polyclonal antibodies (pAbs) raised
against small molecules (haptens) are easily obtained from
serum after immunization of animals using hapten–protein
conjugates. Although constituted of thousands of specific and
non-specific antibodies, examples in the literature prove that
pAbs displaying enantioselective binding properties can be
obtained.9 It should therefore be possible to develop immuno-
assays based on the use of enantioselective pAbs allowing the
high-throughput screening of enantioselective catalysts. In this
contribution we show that such a method can be elaborated at
minimum cost and in only a few weeks.
A promising but still under-exploited HTS method relies on the
use of antibody-based techniques (i.e. immunoassays) routinely
used in the field of diagnosis but rarely exploited to solve chemical
problems. Pioneering work in this field was done by D. S. Tawfik
and D. Hilvert’s groups who developed a so-called Cat-ELISA
technique to identify biocatalysts.3 Variants of this technique were
then developed to reveal protease or esterase activities.4 An
interesting property of antibodies is their capacity to selectively
bind chiral molecules. Stereoselective binding of antibodies was
first demonstrated in 1928 when Karl Landsteiner developed the
classic understanding of immunochemical molecular recognition.5
Since this pioneering work, the capacity of antibodies to discrimi-
nate enantiomers was surprisingly largely ignored by the chemical
community and antibody-based approaches for the detection of
chiral compounds were developed only recently. Nowadays, the
use of monoclonal antibodies (mAbs) as chiral selectors for the
development of immunosensors allowing the precise and sensitive
detection of enantiomeric impurities is largely demonstrated.6
During the course of our work dealing with the development
of antibody-based screening techniques for the monitoring of
chemical reactions,7 our laboratory showed that enantio-
selective catalyst libraries can be easily screened by competitive
The critical point for the development of such immuno-
assays is the preparation of pAbs with appropriate binding
specificity. These pAbs should exhibit high chiral recognition
while binding a panel of analytes. In other words, the binding
selectivity of pAbs needs to be focused on the chiral center,
while low binding specificity to other parts of the analyte
structure is required. It is well known that the site of hapten–
protein linkage orients antibody specificity.10 Based on these
data, we decided to produce stereoselective pAbs raised against
N-Bz amino acids by immunizing rabbits with BSA-coupled
optically pure N-Bz (R)- or (S)-lysine. The linking of the lysine
haptens to BSA was carried out through the amine function in
order to maximize exposure of the chiral center. After a few
weeks, rabbit sera were collected and assayed for their ability to
bind N-Bz (R)- and (S)-lysine. Standard ELISA experiments
highlighted the excellent enantioselectivity of these pAbs (Fig. 1).
Relative affinities (B/Bo 50% = Kd, app E 0.1 mM) of pAbs
were in favor of the injected enantiomer by almost three orders
of magnitude.
a CEA, iBiTecS, Service de Chimie Bioorganique et de Marquage,
Gif sur Yvette, F-91191, France. E-mail: frederic.taran@cea.fr;
Fax: +33 169087991; Tel: +33 169082685
Control experiments proved that this high degree of stereo-
selectivity allowed ee determination of (R)/(S) mixtures of
N-Bz-lysine with a precision of Æ2% (Fig. 2).
b CEA, iBiTecS, Service de Pharmacologie et d’Immunoanalyse,
Gif sur Yvette, F-91191, France
w Electronic supplementary information (ESI) available: Experimental
procedure for screening, complete screening results and analytical data
of products. See DOI: 10.1039/c2cc31312j
We then looked at the scope of the method by determining
the binding properties of pAbs using a panel of N-Bz amino
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4411–4413 4411

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Table 1 Enantioselective binding properties of pAbs
Anti (R) pAbs, Kd,appa/mM Anti (S) pAbs, Kd,appa/mM
R
0.25
0.11
96.61
40.80
58.02
25.60
0.16
0.04
0.06
0.43
0.06
167.45
150.23
10.05
103.91
150.62
175.68
0.30
1.42
0.09
Fig. 1 Competitive ELISA of (R)- and (S)-N-Bz-lysine with pAbs
(see ESIw for details).
0.03
2.74
13.35
0.19
0.67
0.53
125.36
141.25
117.75
148.69
0.16
0.81
0.21
0.23
33.62
39.30
0.10
0.19
111.82
185.24
0.08
0.14
4.47
108.62
43.52
0.14
0.07
92.42
Fig. 2 ee measurements of (R) and (S)-N-Bz-lysine mixtures by
a
ELISA using stereoselective pAbs.
Kd,app determined by competitive ELISA (B/Bo = 50%; see ESI).
acids. As depicted in Table 1, the pAbs did not bind only lysine
derivatives, but exhibited good affinities and high enantio-
meric specificities for a series of aliphatic or aromatic N-Bz
amino acids.
As the side chains of N-Bz amino acids are structurally
different from the lysine hapten, lower binding affinities were
observed as expected. However, in all cases the binding
properties of the pAbs toward all tested N-Bz amino acids
were good enough to allow the development of a screening
procedure for the fast identification of enantioselective
catalysts.
To this end, we were interested in the enantioselective ring
opening of azlactones by water using lipases. This particular
biocatalyzed dynamic kinetic resolution reaction leading to
N-Bz amino acids has been previously reported under buffered
conditions.11 Because spontaneous hydrolysis of azlactones
might cause low ee values,12 we were interested in exploiting
lipase activity in organic solvents. 11 azlactones were thus
reacted with 9 lipases in 5 anhydrous solvents in the presence
of 3 equivalents of H2O. A series of experiments were also
carried out in the presence of TEA to facilitate racemization.
To look at possible spontaneous hydrolysis, control experiments
performed without lipase were also carried out.
A total of 660 experiments were thus conducted in parallel
and screened in one day with two jointly run ELISAs
(one using anti-(R) pAbs and the other using anti-(S) pAbs).
Fig. 3 Activity fingerprinting of 9 lipases for the water-promoted
ring opening of azlactones.
c
4412 Chem. Commun., 2012, 48, 4411–4413
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on the lipase activities and a dramatic effect of TEA which
decreased both yields and ee (see ESIw for complete results).
The best results, obtained in CH3CN, are summarized in Fig. 3.
To confirm the validity of the method, 40 samples were
randomly chosen across the range of ee and yield values and
reanalyzed by chiral HPLC. The ELISA and HPLC measure-
ments varied on average by B10% and afforded excellent linear
correlations (Fig. 4).
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Candida antarctica B was the lipase that displayed broader
substrate tolerance and generated the best yields and ee results.
The activity of this enzyme was reproduced on a mmol scale
reaction using three different azlactones. As expected, the desired
N-Bz-amino acids were isolated in high yields and ees (Scheme 1).
In summary, we report a screening method that has at least two
major advantages: it is cheap and fast to develop (stereoselective
pAbs can be obtained in only 2 weeks, see ESIw) and it allows
exploration of the scope of enantioselective catalysts using a panel
of substrates. Since antibodies can be raised against virtually any
kind of compound, we think that the technique can be extended to
a panel of interesting chemical reactions.
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This work was supported by the European Community
(IBAAC Project).
Notes and references
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c
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Chem. Commun., 2012, 48, 4411–4413 4413