A palladium-catalyst stabilized in the chiral environment of a monoclonal antibody in water

Article abstract of DOI:10.1039/c9cc08756g

We report the first preparation of a monoclonal antibody (mAb) that can immobilize a palladium (Pd)-complex. The allylic amination reaction using a supramolecular catalyst consisting of the Pd-complex and mAb selectively gives the (R)-enantiomer product w

Full text of DOI:10.1039/c9cc08756g

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Palladium-Catalyst Stabilized in Chiral Environment of Monoclonal
Antibody in Water†
Yuichiro Kobayashi,a Keisuke Murata,a Akira Harada*b and Hiroyasu Yamaguchi*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report the first preparation of a monoclonal antibody (mAb)
that can immobilize a palladium (Pd)-complex. The allylic
amination reaction using a supramolecular catalyst consisting of
the Pd-complex and mAb selectively gives the (R)-enantiomer
product with an enantiomeric excess (ee) of 98 ± 2%. This is in sharp
contrast to the reaction catalyzed by a conventional Pd-catalyst (ee
< 2%).
Palladium (Pd)-complexes have attracted much attention
because they promote various cross-coupling reactions such as
Negishi coupling,1-3 Suzuki-Miyaura cross-coupling,4-6 and
Mizoroki-Heck reaction.7-10 Recent efforts have focused on
controlling the synthesis products and increasing the catalytic
activity due to the importance of chirality for living systems. For
structural control of a product, both the first coordination
sphere, which is formed by metal ions and ligands, and the
second coordination sphere must be appropriately constructed.
Synthesizing a ligand with the desired structure can realize the
desired first coordination sphere. However, the second
coordination sphere is composed of multiple non-covalent
interactions such as hydrogen bonding, hydrophobic effects,
and electro-static interactions. Hence, it is difficult to produce
the desired second coordination sphere with a low molecular
weight system.
Fig. 1. Chemical structures of 1—5.
One example is the utilization of avidin-biotin interactions
during complex formation of avidin with a biotinylated Pd-
complex.14, 15 Because the space originally possessed by the
biomacromolecules is used as the second coordination sphere
of the Pd-complex, it is not suitable for the Pd-complex. If
biomacromolecules can directly recognize Pd-complexes,
complexation of the Pd-complex and biomacromolecules can
construct an appropriate second coordination sphere, realizing
a novel platform for a hybrid catalyst of the Pd-complex and
biomacromolecules.
To this end, hybrid catalysts with various combinations of Pd-
complexes and biomolecules11 such as ferritin,12 lipase,13 and
15
biotin-streptavidin14,
have been developed to obtain
structure-controlled products. A Pd-complex anchored in a
biomolecule provides asymmetric environmental fields in the
second coordination sphere. In this case, the Pd-complex is
incorporated into biomacromolecules by a non-direct method.
a. Department of Macromolecular Science, Graduate School of Science, Osaka
University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan.
E-mail: hiroyasu@chem.sci.osaka-u.ac.jp
Our work has focused on monoclonal antibodies (mAbs)
because the binding site of mAbs can be tailored to the antigen
specifications.16-29 This intriguing feature allows asymmetric
environmental fields to be constructed as the second
b. The Institute of Scientific and Industrial Research, Osaka University, 8-1
Mihogaoka, Ibaraki 567-0047, Japan.
E-mail: harada@chem.sci.osaka-u.ac.jp
† Electronic Supplementary Information (ESI) available: Experimental details, 1H and
13C NMR and affinities between mAb and catalyst. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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immunized with 2 with the same ligand as 1. VieTwhAerticl2e Ownlianes
prepared from the chloro(1,5-cyclooctadDieOnIe: 1)0rh.1o03d9iu/Cm9C(IC) 0d8im75e6rG.
The 2 was introduced to the proteins such as KLH and BSA in 0.1
M PBS buffer (pH 7.0) to obtain the antigens for immunization
and assay, respectively (2-KLH and 2-BSA) (Scheme S3). The
modification
ratio
was
estimated
by
2,4,6-
trinitrobenzenesulfonic acid (TNBS) method,34-36 and over six
hundred or eight of 2 were introduced into one KLH or BSA,
respectively (Table S1). Balb/c mice were immunized with 2-
KLH in saline emulsified 1:1 in Freund’s complete adjuvant four
times at two-week interval. To confirm antibodies production,
we performed ELISA measurement of the blood from
immunized and non-immunized mice using 2-BSA coated plate.
In contrast to 1, the absorbance ascribable to the enzymatic
reaction product in ELISA for immunized mouse was higher than
that of non-immunized mouse (Fig. 2b). Moreover, in the case
Fig. 2. Results of ELISA for blood obtained from the mouse immunized with transition-
metal complex modified KLH (red) and non-immunized mouse (black) [(a) 1-KLH and (b)
2-KLH].
coordination sphere for transition metal complexes, enabling a of immunized mouse, the absorbance of 2-BSA was higher than
31
stereospecific reaction.30,
However, to the best of our that of BSA (Fig. S7). These results clearly show preparation of
knowledge, a hybrid catalyst consisting of a Pd-complex and antibodies for 2. The hybridomas secreting antibodies specific
mAb has yet to be reported because typical Pd-complexes are to 2 were cloned twice by the limiting dilution method and then
unstable in water and degrade during immunization. Currently, the obtained mAbs were purified from the ascites fluid by
there is only one report on complexation of a Pd-complex of a affinity chromatography using HiTrap IgM Purification HP
porphyrin.32 This may be due to the difficulty preparing mAbs column (GE healthcare). The class of mAbs was determined
capable of recognizing Pd-complexes.
In this study, we describe our efforts to create
with Iso Strip Mouse Monoclonal Antibody Isotyping Kit. As a
result, the obtained mAb was Immunoglobulin M (IgM), and the
a
supramolecular catalyst using mAb to immobilize an unstable light chain was kappa chain.
Pd-complex (1) (Fig. 1 and S1-S3 and Scheme S1) for the allylic
To quantitatively investigate the affinity of mAb to 1, we
amination reaction. Because 1 degrades during immunization, determined the dissociation constant (Kd) of the complex
mAb capable of recognizing 1 is prepared by the cross-reactivity between mAb and 1 or 2. The Kd of the complexes between
of mAb binding to the rhodium (Rh)-complex (2) (Fig. 1, S4, and mAb and 1 or 2 were found to be 2.0 or 7.6 × 10-5 M, respectively
S5 and Scheme S2) with the same ligand as 1. A supramolecular (Figs. S8a and S8b), showing that the affinities of mAb to 1 and
catalyst consisting of 1 and mAb catalyzes the allylic amination that of mAb to 2 were almost same. To understand this reason,
reaction, which selectively affords a (R)-enantiomer product.
the binding form of mAb to 2 was estimated using Kd of the
We chose a η3-allyl palladium (II) complex as a late transition complex between mAb and the ligand of 2 (3), benzoic acid (4),
metal complex because they promote various coupling or the Rh-complex (5) whose structure is different from 2 (Fig.
reactions such as allylic amination reaction.33 To obtain mAbs 1). The mAb showed no affinity to 4 and 5 (>1.5 × 10-2 and >1.0
capable of recognizing 1, we prepared antigens in which 1 was × 10-3 M, respectively), whereas the mAb showed affinity to 3
modified to keyhole limpet hemocyanin (KLH) (1-KLH, Scheme (3.4 × 10-4 M) (Figs. S8c and S9). This result indicates that the
S3 and Table S1) for immunization and immunized 1-KLH in mAb recognizes the molecular elements of 3 in complex 2. The
saline emulsified 1:1 in Freund’s complete adjuvant for Balb/c ligand structure of 2 and that of 1 was the same, thus the mAb
mice four times at two-week interval. To investigate antibody can form complexes with 1. We prepared for the first time the
production, 1 was introduced into bovine serum albumin (BSA) mAb for Pd-complex using the cross-reactivity of mAb.
to obtain antigens for assays (1-BSA), and then enzyme-linked
A reaction in a nanoconfined geometry often gives products
immunosorbent assay (ELISA) measurements of the blood from whose symmetries differ from those obtained in a solution
immunized and non-immunized mice using 1-BSA coated plate reaction. We carried out an allylic amination reaction using 1 in
were performed. However, there were no difference of the the absence and presence of the mAb (Table 1). Firstly, reaction
absorbance ascribable to the enzymatic reaction product in concentrations of 1 and mAb were optimized. 3-Buten-2-yl
ELISA for the immunized and non-immunized mice, showing acetate (6) (10 mM) and benzyl amine (7) (20 mM) were reacted
that no antibodies for 1 has been produced (Fig. 2a). This is with 1 (1 μM) and the mAb (0.1 μM, binding site: 1 μM) in a
most probably due to the low stability of Pd-complex in water. mixed solution of 0.1 M phosphate borate buffer (PBB; pH 9.0)
In fact, the proton nuclear magnetic resonance (1H NMR) and DMSO (9:1) at r.t. for 24 h (Figure S10 and Schemes S4).
spectrum of
1 was changed by immersed in N,N- Under this conditions, the binding site of mAb and 1 are
dimethylformamide-d7/ deuterium oxide for 7 days, showing equivalent. The yield and ee of the product (8) were
decomposition of 1. (Fig. S6). The 1 was decomposed during determined by comparing the area ratio of 8 and allylbenzene
immunization, which could not allow for creation of antibodies. as an internal standard in HPLC analysis (Fig. S11) and calculated
To overcome this problem, we employed cross-reactivity of from the peak area ratio of (R)- and (S)-enantiomers of 8 of HPLC
antibody. Because Rh-complexes are stable in water, we spectra (Fig. S12). The yield and ee of 8 were decreased with
2 | J. Name., 2012, 00, 1-3
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Table 1. Results of the 1-catalyzed allylic amination reaction.
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Yield of 8
ee
(%)b
< 2
98 ± 2
< 2
Entry Catalyst
Conc. of Pd (μM)/mAb (μM)
(%)a
38
6
8.
1
2
3
1
1/0
1/0.1c
1/0.1
1+mAb
1+BSA
9.
11
a
Calculated from the peak area ratio of HPLC spectra using allylbenzene as an
10.
11.
internal standard. b Calculated from the peak area ratio of (R)- and (S)-enantiomers
of 8 of HPLC spectra. C Since the mAb is IgM with 10 binding sites, concentrations
of 1 and the binding site of mAb are equivalent.
increasing of the concentration of 1 and mAb. These results
suggest that the aggregation of IgM causes 1 to be adsorbed at
locations other than where the product structure is controlled.
The optimum reaction concentration of 1 and mAb was found
to be 1 and 0.1 μM, respectively. The yield of 8 in the presence
of the mAb (6%) was lower than that in the absence of the mAbs
(38%) (Table 1, Entries 1 and 2). Owing to the complex
formation between 1 and mAb, it is difficult for the substrates
such as 6 and 7 to access 1. The decline in the yield of 8 was
also observed in the presence of BSA (11%) (Table 1, Entry 3),
suggesting that the presence of protein reduces the catalytic
activity of 1. In the case of the absence of mAb, 1 gave racemic
12.
13.
14.
15.
16.
8 (ee: <2%) (Table 1, Entry 1 and Fig. S12a). In contrast, 17.
interestingly, the reaction catalysed by 1 incorporated into the
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binding site of mAb was found to proceed with excellent (R)-
enantioselectivity with a 98 ± 2% ee (Table 1, Entry 2 and and
Fig. S12b). These result indicate that no catalytic reaction
occurred outside the binding sites. Since the catalytic activity of
free 1 is reduced by the presence of protein, the catalytic
activity of free 1 would be negligible in the mAb system. This
asymmetric allylic amination was not observed in the presence
of BSA (ee < 2%) (Table 1, Entry 3 and and Fig. S12c). These
results indicate that the binding site of mAb functions as a
reaction field for the asymmetric reaction.
In summary, we have demonstrated that Pd-complex
catalysed allyl amination in the presence of mAb is a useful
synthetic strategy to control the resultant product asymmetry.
The substrate is converted to a racemic product by Pd-catalyst
without mAb. In contrast, the (R)-enantiomer (98 ± 2% ee) is
obtained in the presence of the complex between mAb and Pd-
catalyst.
20.
21.
22.
23.
24.
25.
This work was supported by JSPS KAKENHI Grant Numbers
JP15H05807 in Precisely Designed Catalysts with Customized
Scaffolding.
26.
27.
28.
There are no conflicts to declare.
Notes and references
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