Chemoenzymatic synthesis of neotame

Details Chemoenzymatic synthesis of neotame Chemoenzymatic synthesis of neotame

2000-05-12

2003-09-30

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Resolution of optical isomers or purification of organic...

C435S183000, C435S135000, C435S134000, C560S040000

Reexamination Certificate

active

06627431

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the chemoenzymatic synthesis of neotame (N-[N-(3,3-dimethylbutyl)-L-&agr;-aspartyl]-L-phenylalanine 1-methyl ester) via regioselective hydrolysis of neotame esters. Neotame is particularly useful as a sweetening agent.
2. Related Background Art
N-[N-(3,3-dimethylbutyl)-L-&agr;-aspartyl]-L-phenylalanine 1-methyl ester is a derivative of aspartame that has a sweetening potency that is about 40 times that of aspartame (and about 8,000 times that of sucrose). N-[N-(3,3-dimethylbutyl)-L-&agr;-aspartyl]-L-phenylalanine 1-methyl ester may be prepared from aspartame as described in U.S. Pat. No. 5,480,668, U.S. Pat. No. 5,510,508, and U.S. Pat. No. 5,728,862, all of which are incorporated by reference herein.
These patents describe methods for preparing neotame by treating a mixture of aspartame and 3,3-dimethylbutyraldehyde with reducing agents. There is a need, however, to develop more cost-effective and efficient methods of preparing high purity neotame from readily obtainable materials.
Neotame may be used for sweetening a variety of products, including drinks, foods, confectionery, pastries, chewing gums, personal care, hygiene products and toiletries, as well as cosmetic, pharmaceutical and veterinary products. Its superior sweetening potency makes neotame an attractive alternative to aspartame because it permits the use of neotame in substantially smaller quantities than is required for aspartame to achieve an equivalent sweetening effect.
Enzymes are becoming increasingly important as catalysts for asymmetric synthesis. Of particular importance are the hydrolytic enzymes, namely hydrolases. There are many examples of the synthetic utility of hydrolases, and there are many commercially available sources from which to obtain such enzymes. Lipases and esterases are widely used for ester hydrolysis, transesterification, acetylation and diastereomeric separation. Lipase and esterase catalyzed regioselective transesterification and de-esterification have been shown to be excellent alternatives to traditional esterification and de-esterification. U.S. Pat. No. 5,928,909 disclosed regioselective and chemoselective hydrolysis of an &agr;-ester group of an amino acid diester using pig liver esterase. Porcine pancreatic lipases and lipases from
Candida rugosa
and Pseudomonas sp. were used for enantioseletive transesterification in organic synthesis. Liisa Kanerva, et al. (
Tetrahedron: Asymmetry
, 7(6): 1705-1716, 1996), synthesized highly enantiopure &bgr;-amino acid esters using catalysis lipases from
Candida antarctica
and
Pseudomonas cepacia
. Adamczky et al. (
Tetrahedron Lett
., 35(7): 1019-22, 1994.) used lipases from Pseudomonas species to mediate hydrolysis of rapamycin 42-hemisuccinate benzyl and methyl esters. Stein et al. (
J. Org. Chem
., 60: 8110, 1995.) demonstrated that the hydrolysis of aspartate dimethyl ester using pig liver esterase resulted in the hydrolysis of both ester groups, i.e. the &agr;-ester group and the &bgr;-ester group. The selectivity of the &agr;-ester hydrolysis to the &bgr;-ester hydrolysis was found to be 98:2 for the formation of the corresponding aspartate monoesters. In contrast, both the (R)-aspartate diethyl ester and (S)-aspartate diallyl ester are converted to their respective &bgr;-monoesters with pig liver esterase with complete regioselectivity hydrolysis of the a-ester group (U.S. Pat. No. 5,928,909). In addition, it was found that the preferential hydrolysis for the &agr;-ester position is found to be partially reversed when the aspartate is N-protected as its formamide. The selectivity for the N-protected aspartame was found to be 55:45 for the &agr;-ester hydrolysis to the &bgr;-ester hydrolysis. Guibe-Jampel et al. (
J. Chem. Soc., Chem. Commun
., 1080, 1987) used porcine pancreatic lipase (PPL) for regioselective hydrolysis of dialkyl amino acid esters. Hydrolysis of N-protected dialkyl aspartates using PPL resulted in the regioselective hydrolysis of the &bgr;-ester group and formation of the corresponding N-protected &agr;-ester aspartate. However, in the case of N-alkylated aspartate ester which conjugated with phenylalanine ester, regioselective hydrolysis with hydrolases was not reported.
SUMMARY OF THE INVENTION
This invention relates to the chemoenzymatic synthesis of neotame (N-[N-(3,3-dimethylbutyl)-L-&agr;-aspartyl]-L-phenylalanine 1-methyl ester) via regioselective hydrolysis of neotame esters.


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