Antibody-catalyzed asymmetric intramolecular Michael addition of aldehydes and ketones to yield the disfavored cis-product

Article abstract of DOI:10.1021/ja0536825

The development of new catalytic asymmetric reactions continues to be a major goal in organic chemistry. Here we report a novel antibody-catalyzed intramolecular Michael addition of aldehydes and ketones to enones. The reaction is enantioselective and dia

Full text of DOI:10.1021/ja0536825

Published on Web 08/30/2005
Antibody-Catalyzed Asymmetric Intramolecular Michael Addition of
Aldehydes and Ketones to Yield the Disfavored Cis-Product
Roy Weinstain,† Richard A. Lerner,‡ Carlos F. Barbas, III,‡ and Doron Shabat*,†
School of Chemistry, Raymond and BeVerly Sackler Faculty of Exact Sciences, Tel-AViV UniVersity,
Tel AViV 69978 Israel, and The Skaggs Institute for Chemical Biology, Departments of Molecular Biology and
Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received June 5, 2005; E-mail: chdoron@post.tau.ac.il
Scheme 1. Mechanism of Trapping the ꢀ-Amino-lysine Residue in
the Antibody Binding (2) Site Using the â-Diketone Derivative
Hapten 1 (R ) CH2CH2CH2COOH)
The world’s first commercially available catalytic antibody, 38C2
(Ab38C2), is perhaps the most promiscuous antibody catalyst
generated to date. The antibody was found to efficiently catalyze
aldol and retro-aldol reactions of a remarkably broad range of
substrates1 with excellent enantioselectivity through an enamine
class I aldolase mechanism.2 The antibody was raised against the
â-diketone hapten 1, which served as a chemical trap to imprint a
unique lysine residue (2) with an ꢀ-amino group with a pKa of 5.8
(Scheme 1). The X-ray structure3 of the antibody binding site
suggests an interaction between a tyrosine residue and one of the
carbonyl groups of the hapten (Scheme 1). Antibody 38C2 is also
capable of catalyzing retro-Michael reactions of â-alkoxy ketones,4-8
a direct Michael addition of acetone to a maleimide derivative,9a
and mimics the classic organocatalytic Wieland-Miescher ketone
synthesis.9
Scheme 2. Antibody-Catalyzed Intramolecular Michael Addition of
Ketones and Aldehydes to Enones
The recent report by List10 of chiral imidazolidinone-catalyzed
intramolecular Michael reactions has promoted us to determine
whether antibody 38C2 could catalyze the intramolecular Michael
addition of aldehydes and ketones to enones. To our delight,
incubation of formyl-enone I (R′ ) H, R ) H) or methyl ketone-
enone (R′ ) H, R ) CH3) with antibody 38C2 in phosphate-
buffered saline (PBS), pH 7.4, indeed generated the expected
Michael product II (Scheme 2).
Scheme 3. General Synthesis of the Enone Substrates for the
Antibody-Catalyzed Intramolecular Michael Addition Reactions
The substrates for the antibody-catalyzed reactions were syn-
thesized through a Wittig coupling in a biphasic solvent system
using methylene chloride and 2 N sodium hydroxide as shown in
Scheme 3. The Wittig salt III was dissolved in the aqueous phase
and quickly formed the corresponding ylide. The keto-aldehyde (R
) CH3) or the dialdehyde (R ) H) were added in methylene
chloride. The reaction was mixed for several hours, and after
workup the enone product was purified by standard column
chromatography techniques. Racemic reference type II products
were prepared by incubation of the enones with piperidine in DMF.
Several aldehyde-enones and methyl ketone-enones were suitable
substrates for the antibody-binding site and afforded the intramo-
lecular Michael products with excellent diastereo- and enantiose-
lectivity (Table 1). The cis/trans ratio and the enantioselectivity
were determinated by standard RP-HPLC and chiral-phase HPLC
(AD-RH column), respectively.
Interestingly, the antibody reaction product was, in all examples,
predominantly the thermodynamically unfavored cis-diastereoiso-
mer. In contrast, the asymmetric intramolecular Michael reaction
catalyzed by imidazolidinones of formyl-enone 3 afforded almost
exclusively the trans-diastereoisomer ((S)-proline gave a 2:1 trans/
cis ratio with only 15% ee of trans-3a).10 As presented in Table 1,
antibody 38C2 also catalyzed the intramolecular Michael addition
of methyl ketones (6-9) with very high enantio- and diastereose-
lectivity. Incubation of MacMillan imidazolidinone with ketone-
enone 6 in THF for 3 days did not afford any Michael addition
product.
The kcat and Km were determined from Lineweaver-Burk plots
using Michaelis-Menten analysis (Table 2). Full graphical data
are available in the Supporting Information. While the ketones
reacted at a relatively moderate rate, aldehyde 3 yielded the
intramolecular Michael product 3a with a kcat of 8.73 min-1 (Table
2). To date, this is the highest measured rate for antibody catalysis
of a C-C bond-forming reaction. The antibody-catalyzed reaction
was up to 5 orders of magnitude faster than the background reaction
in buffer alone. This relatively high ratio will be adequate for
preparative-scale antibody reactions to generate enantiomerically
pure products.12 In general, the aldehyde-enones were less stable
(or more reactive) under these buffer conditions than the methyl
ketone-enones, and their reactions had higher kcat values. Ketone-
enone 6 exhibited the highest rate enhancement with a kcat/kuncat
ratio of 350,000, very high cis/trans product selectivity (90/1), and
an excellent enantiomeric excess value of 97%.
Scheme 4 illustrates our proposed mechanism for the antibody
catalysis of the intramolecular Michael addition. The ꢀ-amino group
of the lysine residue (IV) presumably reacts with the formyl or the
methyl ketone to form a nucleophilic enamine (V), that then reacts
with the enone to generate the Michael addition product (VI). The
imine in VI is hydrolyzed to release a carbonyl moiety (VII) and
to regenerate the free ꢀ-amino-lysine residue. The X-ray structure
† Tel-Aviv University.
‡ The Scripps Research Institute.
9
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J. AM. CHEM. SOC. 2005, 127, 13104-13105
10.1021/ja0536825 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Enantioselective Intramolecular Michael Addition
Catalyzed by Antibody 38C2
Scheme 4. Suggested Mechanism for Antibody 38C2-Catalyzed
Intramolecular Michael Addition of Aldehydes and Ketones to
Enones
with high ee values and cis/trans ratios. To the best of our
knowledge, no other Michael-addition methods offer such selectivity
for the thermodynamically unfavored cis-diastereoisomers.
While organocatalytic Michael addition products are known,
antibody 38C2 is the only catalyst known to act on both aldehyde
and ketone substrates to produce enantiomeric pure cis-cyclopentane
products. This study highlights the dynamic interplay between
bioorganic chemistry and organocatalysis.
Acknowledgment. Financial support has been provided by Tel-
Aviv University and the Israel Science Foundation.
a The cis/trans ratio of aldehydes 3a-5a decreased over time since the
antibody also catalyzes epimerization at the R carbon.11 This phenomenon
is not observed in ketones 6a-9a.
Supporting Information Available: Full experimental details,
characterization data of all new compounds and antibody assay
conditions, full graphical data used to generate Table 2. This material
is available free of charge via the Internet at http://pubs.acs.org.
Table 2. Kinetic Parameters for Intramolecular Michael Addition
Catalyzed by Antibody 38C2a
1
substrate
K
m (mM)
k
cat (min-
)
kcat/kuncat
3
4
5
6
7
8
9
47
235
36
336
709
656
44
8.73
0.18
2.92
0.14
0.09
0.09
0.025
244,428
3,333
References
51,228
350,000
30,479
134,285
22,910
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(2) Wagner, J.; Lerner, R. A.; Barbas, C. F., III. Science 1995, 270 (5243),
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a Antibody reactions were performed in PBS (50 mM buffer, 100 mM
sodium chloride), pH 7.4 with 5% acetonitrile at room temperature.
of antibody 33F12, a very similar antibody to 38C2, shows a
tyrosine residue near the active lysine.3 In our proposed mechanism,
the tyrosine hydroxyl residue acts as a Brønsted acid catalyst that
activates the enone toward the Michael addition of the nucleophilic
enamine. The antibody binding site tolerates a variety of ketone-
and aldehyde-enone substrates with different para substituents on
the aromatic ring. However, the substrates with the fastest reaction
rates among the aldehydes and the ketones were the nonsubstituted
enones 3 and 6, respectively. We have used only aryl-enones as
substrates since they are easily monitored using HPLC with a UV
detector.
(6) Shabat, D.; Rader, C.; List, B.; Lerner, R. A.; Barbas, C. F., III. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 6925-6930.
(7) Shamis, M.; Lode, H. N.; Shabat, D. J. Am. Chem. Soc. 2004, 126, 1726-
1731.
(8) Tanaka, F.; Kerwin, L.; Kubitz, D.; Lerner, R. A.; Barbas, C. F., III.
Bioorg. Med. Chem. Lett. 2001, 11, 2983-2986.
(9) (a) Tanaka, F.; Thayumanavan, R.; Barbas, C. F., III. J. Am. Chem. Soc.
2003, 125, 8523-8528. (b) Zhong, G.; Hoffmann, T.; Lerner, R. A.;
Danishefsky, S.; Barbas, C. F., III. J. Am. Chem. Soc. 1997, 119, 8131-
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(11) Shulman, A.; Sitry, D.; Shulman, H.; Keinan, E. Chem. Eur. J. 2002, 8,
229-239.
In summary, we have described novel antibody-catalyzed in-
tramolecular Michael addition reactions of aldehydes and ketones
to enones. The reactions were enantioselective and diastereoselective
(12) Turner, J. M.; Bui, T.; Lerner, R. A.; Barbas, C. F., III; List, B. Chem.
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