Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1992-09-17
2002-03-05
Leary, Louise N. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S018700, C536S018500, C536S053000, C536S055000, C514S023000, C514S008100, C424S085100, C424S093200, C435S137000
Reexamination Certificate
active
06353095
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to ketoaldonic acids having formed stereogenic centers of R configuration, particularly octulosonic and nonulosonic acids, and methods for synthesizing such sugars using sialic acid aldolase.
BACKGROUND OF THE INVENTION
A major synthetic value of enzyme catalysis is its usually predictable stereoselectivity. See, e.g., Whitesides et al.,
Angew. Chem. Int. Ed. Engl.,
24:617 (1985); Jones,
Tetrahedron,
42:3351 (1986); Yamada et al.,
Angew. Chem. Int. Ed. Engl.,
27:622 (1988); Wong, C-H.,
Science,
244:1145 (1989); Ohno et al.,
Org. React.,
37:1 (1989); Chen et al.,
Angew. Chem. Int. Ed. Engl.,
28:695 (1989).
A change of stereoselectivity, however, may occur, though very unusual, with different substrate structures, temperatures or solvents. See. e.g., Mohr et al.,
Helv. Chim. Acta,
66:2501 (1983); Sabbioni et al.,
J. Chem. Soc. Chem. Commun.,
236 (1984); Ohno et al.,
J. Am. Chem. Soc.,
103:2405 (1983); Wang et al.,
J. Org. Chem.,
53:3127 (1988); Lalonde et al.,
J. Am. Chem. Soc.,
103:2405 (1981); Wang et al.,
J. Org. Chem.,
53:2323 (1988); Pham et al.,
J. Am. Chem. Soc.,
111:1935 (1989); Keinan et al.,
J. Am. Chem. Soc.,
108:162 (1986); Sakurai et al.,
J. Am. Chem. Soc.,
110:7236 (1988); Fitzpatrick et al.,
J. Am. Chem. Soc.,
113:3166 (1991). These selectivity changes are often not very significant, with some exceptions where the enantioselectivity is inverted.
In the case of enzymatic aldol reactions, the diastereofacial selectivity for the aldehyde component is often consistent and completely controlled by the enzyme as documented by numerous reactions catalyzed by fructose-1,6-diphosphate (FDP) aldolase or N-acetylneuraminic acid (or sialic acid) aldolase (EC 4.1.3.3). In most cases, the “D” isomer of an &agr;-substituted aldehyde reacts faster than the “L” isomer, both with si-facial selectivity. The Cram-Felkin mode of attack on the “D” aldehyde is therefore proposed for the transition state of the FDP aldolase reaction and the anti-Cram-Felkin mode for the sialic acid aldolase reaction. See. e.g., Toone et al.,
Tetrahedron,
45:5365 (1989); Bednarski et al.,
J. Am. Chem. Soc.,
111:627 (1989); Straub et al.,
J. Org. Chem.,
55:3926 (1990); Durrwachter et al.,
J. Org. Chem.,
53:4175 (1988); von der Osten et al.,
J. Am. Chem. Soc.,
111:3924 (1989); Kajimoto et al.,
J. Am. Chem. Soc.,
113:6187 (1991); Auge et al.,
New J. Chem.,
12:733 (1988).
Because of the stereoselectivity of enzymes such as aldolases that participate in the metabolism of carbohydrates, it is extremely difficult to design and make new carbohydrates that can be used to study carbohydrate metabolism. There is a need for such synthetic compounds for use as experimental tools in elucidating the molecular character of the numerous and varied pathways involved in carbohydrate anabolism and catabolism.
Of particular relevance to the present invention is the sugar, N-acetylneuraminic acid (NeuAc) or sialic acid. NeuAc is an integral component of most cells and is believed to play a major role in imparting electrical charge characteristics to such cells. Further, NeuAc-like compounds such as the eight and nine-carbon sugar moieties KDO and KDN are major constituents of non-mammalian tissues.
N-Acetylneuraminic Acid (NeuAc) aldolase, also commonly referred to as sialic acid aldolase is a type I aldolase known to form an enamine intermediate with pyruvate, which reversibly reacts with the second substrate N-acetylmannosamine to give NeuAc. See, e.g., Deijl et al.,
Biochem. Biophys. Res. Commun.,
111:668 (1983); and Shukla et al.,
Anal. Biochem.,
158:158 (1986).
NeuAc aldolase is known to accept many aldoses as acceptor substrates. In all previously known aldol condensation reactions with such acceptor substrates, the eneamine intermediate approaches the si face of the incoming aldehyde substrate to form a new stereogenic center of S configuration. Anti-Cram-Felkin attack is generally observed for good chiral aldehyde substrates and Cram-Felkin attack is observed for weak substrates. In both cases, a si-facial selectivity was observed. See. e.g., Auge et al.,
New J. Chem.,
12:733 (1988); and Auge et al.,
Tetrahedron,
46:201 (1990).
Based on such current knowledge concerning aldolase stereoselectivity, therefore, NeuAc aldolase is considered to be useful only for the production of D-sugars having S configuration. As is disclosed hereinafter, NeuAc aldolase has now unexpectedly been found to be capable of the production of certain ketoaldonic acids having a formed stereogenic center of R configuration.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention contemplates ketoaldonic acids, and particularly octulosonic or nonulosonic acids, having a formed stereogenic center of R configuration. A contemplated ketoaldonic acid is a sialic acid aldolase-catalyzed condensate of pyruvate and an acceptor substrate aldose for that enzyme. The ketoaldonic acid contains a stereogenic center of the R configuration other than present in the acceptor substrate aldose. Exemplary acceptor substrate aldoses include D-gulose and a five or six carbon L-configured acceptor substrate aldose other than L-arabinose, which form an octulosonic or nonulosonic acid. In another aspect, the present invention contemplates a compound having the Formulae I-VIII, below, in which compounds of Formulae V-VIII, are particularly preferred.
Although L-arabinose forms an octulosonic acid with a new S rather than R stereogenic center in the above sialic acid aldolase-catalyzed condensation with pyruvate, the product of that reaction, 3-deoxy-L-manno-octulosonic acid (L-KDO), a compound of Formula IX, below is new and unexpectedly produced.
One aspect contemplates a process for preparing a ketoaldonic acid having a new stereogenic center of the R configuration, relative to the aldose starting material. This process comprises the steps of:
(a) admixing in an aqueous solvent (i) pyruvate (typically in excess), (ii) a catalytic amount of sialic acid aldolase and (iii) an acceptor substrate aldose for that enzyme, such as D-gulose or a five or six carbon L-configured acceptor substrate aldose other than L-arabinose, to form a reaction mixture; and
(b) maintaining the reaction mixture for a time period and under biological reaction conditions sufficient for condensation of the pyruvate with the acceptor substrate aldose and the formation of a ketoaldonic acid product.
That product is preferably recovered. Use of D-gulose or a five or six carbon L-configured acceptor substrate aldose forms an octulosonic or nonulosonic acid.
In another process aspect, the present invention contemplates a process for synthesizing a compound of Formulae I-VIII comprising the steps of:
(a) admixing pyruvate (typically in excess), in the presence of a catalytic amount of sialic acid (NeuAc) aldolase, with an acceptor substrate L-rhamnose, L-mannose, L-talose, D-gulose, 2-deoxy-L-glucose, 2-deoxy-L-rhamnose, N-acetyl-L-mannosamine or 2-azido-2-deoxy-L-mannose, respectively, (the latter four aldoses being preferred) to form a reaction mixture; and
(b) maintaining the reaction mixture for a time period and under biological reaction conditions sufficient for condensation of the pyruvate with the acceptor substrate and formation of a compound of Formulae I-VIII, above.
In a preferred embodiment, the synthetic method further comprises recovering the synthesized compound of Formulae I-VIII.
In another embodiment, the invention contemplates an enhanced process for synthesizing any ketoaldonic acid such as an octulosonic or nonulosonic acid like sialic acid. In accordance with this process, an excess of pyruvate, e.g. about 2 to about 10 fold excess, and an acceptor substrate aldose for sialic acid aldolase (EC 4.1.3.3) and a catalytic amount of that aldolase are admixed in an aqueous solvent to form a reaction mixture. That reaction mixture is maintained for a time period and under biological reaction conditions sufficient for the condensation of the pyruvate with the
Lin Chun-Hung
Wong Chi-Huey
Leary Louise N.
The Scripps Research Institute
Welsh & Katz Ltd.
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