Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-12-10
2002-08-27
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S147000, C435S188500, C435S345000, C435S346000
Reexamination Certificate
active
06440710
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to a catalytic antibody and, more particularly, to a catalytic antibody effective in deuteration or tritiation of carbonyl compounds.
Deuterium- and tritium-labeled organic compounds have become increasingly important for the role they play in structure determination, mechanistic studies, elucidation of biosynthetic pathways and in biochemical studies. One of the most commonly used deuteration/tritiation methods is the acid or base-catalyzed exchange of protons &agr; to a carbonyl function with D
2
O. Unfortunately, the fairly strenuous aqueous conditions required to complete this exchange are incompatible with substrates that contain acid/base-sensitive functional groups (
5
,
6
). These difficulties have inspired much activity and innovation at the level of process engineering (
7
). Deuteration or tritiation of aldehydes is a particularly difficult task because most aldehydes are incompatible with both basic and acidic conditions required for the exchange reaction (
8
). The use of amine catalysts that form intermediate enamines could represent a solution to this problem. This approach, however, is applicable only in rare cases where the enamine is sufficiently stable in water (
9
).
There is thus a widely recognized need for, and it would be highly advantageous to have, a catalytic antibody effective in deuteration or tritiation of carbonyl compounds.
Enzymes are fascinating tools in synthetic organic chemistry mainly due to their high levels of catalytic efficiency under mild reaction conditions (
1
). While antibodies have been shown to catalyze a remarkable range of chemical reactions with impressive rates (
2
), the question remains whether these biocatalysts, which are evolved over a few weeks time, would ever exhibit the same efficiency of highly evolved natural enzymes (
3
).
Here it is shown that there is no fundamental limit to the efficiency of antibody catalysis in terms of rate enhancement and turnover numbers.
Specifically it is reported herein that antibody 38C2 (
4
) catalyzes the deuterium exchange reaction with a variety of ketones and aldehydes. The catalytic rates (k
cat
) and rate-enhancement values (k
cat
/k
un
) represent the highest values ever observed with catalytic antibodies. Such values are reminiscent of the efficiency of many natural enzymes.
SUMMARY OF THE INVENTION
While reducing the present invention to practice it was found that antibody 38C2 catalyzes a deuterium exchange reaction with a variety of ketones and aldehydes. All reactions were carried out in D
2
O under neutral conditions (pD 7.4). The exchange reaction was followed either by mass spectrometry (GCMS) or by
1
H-NMR. In all cases the deuterium exchange reaction followed Michaelis-Menten kinetics and was effectively inhibited by acetylacetone. In addition to the regio- chemo- and enantioselectivity of these reactions, the catalytic rates (k
cat
) and rate-enhancement values (k
cat
/k
un
) represent the highest values ever observed with catalytic antibodies. The observed regioselectivity is consistent with the formation of a protonated Schiff base intermediate at the lysine residue in the antibody active site followed by a reversible rearrangement to the corresponding enamine.
Thus, according to the present invention there is provided a method of deuteration or tritiation of a carbonyl compound comprising the step of contacting the carbonyl compound with a catalytic antibody being capable of catalyzing an aldol addition reaction in a presence of an aqueous solution enriched with an isotopically enriched water molecule to thereby exchange at least one hydrogen atom in the carbonyl compound with a deuterium or tritium atom.
According to further features in preferred embodiments of the invention described below, the isotopically enriched water molecule is selected from the group consisting of deuterium hydrogen oxide, dideuterium oxide, tritium hydrogen oxide, ditritium oxide and deuterium tritium oxide.
According to still further features in the described preferred embodiments the catalytic antibody being capable of catalyzing the aldol addition reaction between an aliphatic ketone donor and an aldehyde acceptor.
According to still further features in the described preferred embodiments the catalytic antibody is characterized by having a lysine with an &egr;-amino group.
According to still further features in the described preferred embodiments the catalytic antibody is subject to inhibition with a molecule containing a &bgr;-dicarbonyl group by formation of a complex between the &bgr;-dicarbonyl group and the &egr;-amino group of the lysine of the catalytic antibody.
According to still further features in the described preferred embodiments the complex is selected from the group consisting of a stable covalent vinylogous amide, a conjugated enamine and a Schiff base.
According to still further features in the described preferred embodiments the catalytic antibody controls the diastereofacial selectivity of the aldol addition reaction in both Cram-Felkin and anti-Cram-Felkin directions.
According to still further features in the described preferred embodiments the aliphatic ketone donor is selected from a group consisting of acetone, fluoroacetone, chloroacetone, 2-butanone, 3-pentanone, 2-pentanone, and dihydroxyacetone.
According to still further features in the described preferred embodiments the aldehyde acceptor is selected from a group represented by the following formulae:
AcNH—(C
6
H
6
)—CH
2
—CH(↑CH
3
)—C(═O)—H,
AcNH—(C
6
H
6
)—CH
2
—CH(↓CH
3
)—C(═O)—H
and
CH
3
—CO—NH—(C
6
H
6
)—CH
2
—CH
2
—C(═O)—H
According to still further features in the described preferred embodiments the catalytic antibody is secreted by hybridoma 38C2, having ATCC accession number HB 12005.
According to still further features in the described preferred embodiments the catalytic antibody is secreted by hybridoma 33F12, having ATCC accession number HB 12004.
According to still further features in the described preferred embodiments the catalytic antibody is elicited against a &bgr;-dicarbonyl group containing hapten.
According to still further features in the described preferred embodiments the carbonyl compound is selected from the group consisting of ketones and aldehydes.
According to still further features in the described preferred embodiments the carbonyl compound is selected from the group consisting of acetone, butanone, 2-heptanone, valeraldehyde, heptanal, decanal, ethyl 5-oxohexanoate, cyclopentanone, cyclohexanone and cycloheptanone.
Further according to the present invention there is provided a method of dedeuteration or detritiation of a carbonyl compound comprising the step of contacting the carbonyl compound with a catalytic antibody being capable of catalyzing an aldol addition reaction in a presence of an aqueous solution to thereby exchange at least one deuterium or tritium atom in the carbonyl compound with a hydrogen atom.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a method for deuteration and tritiation of carbonyl compounds which is readily effected under room temperature and mild pH conditions.
REFERENCES:
patent: 5733757 (1998-03-01), Barbas et al.
Efficient Aldolase Catalytic Antibodies That Use the Enamine Mechanism of Natural Enzymes; By Jurgen Wagner, Richard A. Lerner, Carlos F. Barbas; Science * vol. 270 * Dec. 15, 1995.
Keinan Ehud
Shulman Avidor
Friedman Mark M.
Naff David M.
The Scripps Research Institute
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