Enzymatic reductions with dihydrogen via metal catalyzed...

Details Enzymatic reductions with dihydrogen via metal catalyzed... Enzymatic reductions with dihydrogen via metal catalyzed...

2000-03-10

2003-07-29

Gitomer, Ralph (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing hydrocarbon

C435S167000

Reexamination Certificate

active

06599723

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to methods of employing H
2
as a reducing agent for an unsaturated organic compound, to form a reduced organic product, in the presence of a catalyst comprising a metal salt or complex, a nicotinamide cofactor; and a nicotinamide cofactor dependent enzyme.
BACKGROUND OF THE INVENTION
The present invention relates to catalytic processes for the use of H
2
as a reducing agent for organic compounds, in the presence of catalysts containing enzymes. Many man-made catalysts are known for reduction and/or hydrogenation reactions, but there are many limitations in the ability of the known catalysts to selectively reduce or hydrogenate one unsaturated functional group in the presence of another functional group. Moreover, most prior art catalysts and processes cannot selectively produce optically active products, as is highly desirable in the production of compositions for human or animal consumption, such as food or pharmaceuticals.
In contrast, many enzymes are capable of highly selective reduction of their natural substrates. In some cases enzymes catalyze unique transformations that require multiple steps by traditional synthetic methods. Moreover, a wide range of unnatural substrates, including a wide variety of unsaturated organic compounds, can be enzymatically reduced, with high chemo-, regio- and/or enantioselectivity, under mild reaction conditions. It is therefore highly desirable to employ enzymatic processes for reductions of unsaturated compounds of commercial interest, and especially for the preparation of chiral molecules, as taught by Simon et al. (
Ang. Chem. Int. Ed. Engl.
1985, 24, 539-53). Unfortunately, most enzymes capable of catalyzing such reduction reactions require the presence of cofactors, which function as biological reducing agents. One broad class of enzymes capable of selective reduction and/or hydrogenation of unsaturated organic compounds are the nicotinamide dependent oxidoreductases, as discussed by Walsh (
Enzymatic Reaction Mechanisms
Freeman & Co., N.Y., 1979; pages 311-521).
Nicotinamide dependent oxidoreductases require the presence of nicotinamide cofactors. The structures of the naturally occurring nicotinamide cofactors, (NAD
+
, NADP
+
, NADH and NADPH) are shown below.
A reduced nicotinamide cofactor (NADH or NADPH) binds to the nicotinamide cofactor dependent enzyme, and transfers a “hydride” (two electrons and one hydrogen nucleus) to reduce a substrate that also binds to the enzyme. After the substrate is reduced, the enzyme releases the oxidized form of the nicotinamide cofactor (NAD
+
or NADP
+
). Biological systems typically recycle the oxidized nicotinamide cofactors, by employing an external reducing agent, in combination with other enyzmes, to regenerate the reduced form of the nicotinamide cofactors. In these nicotinamide cofactors the nicotinamide ring (shown schematically immediately below) is the reactive group. To regenerate the reduced cofactor, an external reducing agent must transfer the equivalent of a “hydride” to the oxidized (pyridinium) form of the cofactor, regioselectively to form the reduced (1,4-dihydropyridine) form of the cofactor.
Although nicotinamide cofactor dependent enzymes and nicotinamide cofactors are present in all living organisms at low concentrations, they tend to be chemically unstable under non-biological conditions, and are extremely expensive in purified form. Because of their high cost, most industrial processes that seek to employ a combination of enzymes and nicotinamide cofactors must supply a method to regenerate the nicotinamide cofactors.
A number of methods for cofactor regeneration are known, as discussed by Chenault and Whitesides (
Appl. Biochem. Biotechnol.
1987, 14, 147-97), and in
Enzymes in Organic Synthesis
K. Drauz, H. Waldeman, Eds.; VCH: Weinheim, 1995; pages. 596-665. The most widely used methods for cofactor regeneration employ a chemical reducing agent and second enzyme to regenerate the nicotinamide cofactors. For example, using glucose as a reducing agent, glucose oxidase has been shown to successfully regenerate NADP
+
/NADPH through up to 4×10
4
turnovers (see Wong, and Whitesides
J. Org. Chem.
1982, 47, 2816-18; Wong et al.,
J. Am. Chem. Soc.
1985, 107, 4028-31; Obon et al.,
Biotech. Bioeng.
1998, 57, 510-17). Hummel et al., (
Appl. Microbiol. Biotechnol.
1987, 26, 409-416) have shown that a combination of formate dehydrogenase and formate salts regenerates NADH from NAD
+
with turnover numbers for the reduced cofactor as high as 6×10
5
. In these methods, a second enzyme couples the regenerated NADH to substrate reduction. In cases where activity of two separate enzyme systems can be accomplished in vitro without undue complexity or expense, reduction of substrates with a chemical reducing agent and two enzymes can be a viable cofactor regeneration method.
Prior art attempts to electrochemically regenerate the nicotinamide cofactors avoid the need for a second enzyme, but direct electrochemical methods have typically not achieved adequate cofactor regeneration, primarily due to formation of inactive nicotinamide-dimers. The addition of certain types of electron transfer catalysts or “mediators” to electrochemical methods can greatly improve electrochemical regeneration, as disclosed by Steckhan (
Topics in Current Chemistry,
1994, 170, 83-111). The most successful mediators are the rhodium complexes disclosed by Steckhan et al (
Ang. Chem,.
1982, 94, 786; U.S. Pat. No. 4,526,661 and
Organometallics
1991 10, 1568-77). Although these electrochemically-based systems have been successfully coupled to enzymatic reduction reactions, thus far cofactor turnover numbers remain too low to be commercially viable.
Photochemically assisted methods for chemical reduction of NAD(P)
+
to NAD(P)H in the presence of similar rhodium electron transfer catalysts, and successful coupling to enzymes has been reported (Willner, et al., in
J. Am. Chem. Soc.,
1984, 106, 5352-53, and
J. Chem. Soc., Perkin Trans.,
2 1990, 559-64; Franke and Steckhan in
Angew. Chem. Intl. Ed. Engl.,
1988, 27, 265; and Aono and Okura in
Inorg. Chim. Acta,
1988, 152, 55-59). Nevertheless, an economically competitive and long-lived photo-chemical cofactor regeneration system which achieves cofactor regeneration at rates and efficiencies competitive with enzymatic methods has remained an elusive goal.
Cofactor regeneration with non-biological chemical reducing agents is a simple approach, but most chemical reducing agents are not desirably selective for production of 1,4-dihydro isomers of the cofactor nicotinamide ring, as discussed by Ohnishi and Tanimoto (
Tetrahedron Lett.
1977, 1909-12). Dithionite salts are preferred reducing agents in this regard, providing up to about 10
2
turnovers of the nicotinamide cofactor, as described by Jones, et al. (
J. Chem. Soc., Chem. Commun.,
1972, 856-57). Nevertheless, dithionite salts are incompatible with many enzymes and react directly with many substrates, are expensive, and generate undesirable sulfur-containing wastes. Steckhan reported the use of formate salts to directly reduce PEG-NAD
+
in a membrane reactor, in the presence of homogeneous rhodium catalysts having covalently bound polyethyleneglycol tails (
Angew. Chem.,
1990, 102, 445-7). Keinan, et al. (
J. Am. Chem. Soc.
1986, 108, 162-9) reported the use of hydride donor alcohols (such as isopropanol) and an alcohol dehydrogenase from
T. brockii,
in a “coupled substrate” method to reduce certain organic substrates. In the “coupled substrate” method one enzyme catalyzes both (a) reduction of NADP
+
to NADPH by the hydride donor alcohol, and (b) reduction of ketone substrates such as 2-heptanone by NADPH.
Dihydrogen (H
2
), is a highly desirable chemical reducing agent. H
2
is a strong reducing agent, and can be inexpensively produced and stored in high purity on a large scale. H
2
is typically innocuous towards enzymes and cofactors, and because it is completel

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