Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
CHEMOENZYMATIC SYNTHESIS OF NEOTAME
Document Type and Number:
WIPO Patent Application WO/2001/085977
Kind Code:
A2
Abstract:
A chemoenzymatic method is disclosed for preparing N-[N-(3,3-dimethylbutyl)-L-a-aspartyl]-L-phenylalanine 1-methyl ester via the enzymatic regioselective hydrolysis of neotame esters using lipases or esterases.

Inventors:
Prakash, Indra (4731 Sunflower Lane Hoffman Estates, IL, 60195, US)
Zhao, Robert Y. (Apartment #1B 661 Pickwick Road Mount Prospect, IL, 60056, US)
Application Number:
PCT/US2001/040676
Publication Date:
November 15, 2001
Filing Date:
May 07, 2001
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
THE NUTRASWEET COMPANY (Suite 3200 10 South Wacker Drive Chicago, IL, 60606, US)
International Classes:
C07K5/072; C12P21/02; (IPC1-7): C12P21/02; C07K5/06
Domestic Patent References:
1998-03-05
2000-07-06
2000-06-08
Foreign References:
US5728862A1998-03-17
US3933781A1976-01-20
US4293648A1981-10-06
Other References:
DATABASE WPI Section Ch, Week 198719 Derwent Publications Ltd., London, GB; Class B05, AN 1987-133263 XP002180937 -& JP 62 074297 A (ASAHI CHEM IND CO LTD), 6 April 1987 (1987-04-06)
STEIN K A ET AL: "Enzyme-catalyzed regioselective hydrolysis of aspartate diesters" JOURNAL OF ORGANIC CHEMISTRY, vol. 60, no. 24, 1 December 1995 (1995-12-01), pages 8110-8112, XP002049344 ISSN: 0022-3263
NOFRE C ET AL.: "Neotame: Discovery, properties, utility." FOOD CHEMISTRY, vol. 69, no. 3, May 2000 (2000-05), pages 245-257, XP000922718 ISSN: 0308-8146
PANDEY A ET AL.: "The realm of microbial lipases in biotechnology." BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, vol. 29, no. 2, April 1999 (1999-04), pages 119-131, XP002180936 ISSN: 0885-4513
JAEGER K-E ET AL.: "Microbial lipases form versatile tools for biotechnology" TRENDS IN BIOTECHNOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 16, no. 9, September 1998 (1998-09), pages 396-403, XP004173187 ISSN: 0167-7799
Attorney, Agent or Firm:
Mandra, Raymond R. (Fitzpatrick, Cella Harper & Scinto 30 Rockefeller Plaza New York, NY, 10112-3801, US)
Download PDF:
Claims:
We claim:
1. A process for producing neotame comprising hydrolysing a neotame ester.
2. A process as in claim 1 wherein the neotame ester is hydrolysed by a regioselective enzyme.
3. A process as in claim 2 wherein the enzyme is selected from the group comprising lipases and esterases.
4. A process as in claim 3 wherein the lipase or esterase is obtained from a source selected from the group comprising Aspergillus niger, Aspergillus oryzae, Candida antarctica, Candida cylindracea, Candida lipolytica, Candida utilis, hog pancreas, Mucor javanicus, Mucor miehei, Penicillium roqueforti, Pseudomonas cepacia, Pseudomonase fluorescens, Rhizomucor miehei, Rhizopus arrhizus, Rhizopus delemer, Rhizopus niveus, Thermus aquaticus, Thermus flavus, Thermus thermophilus, wheat germ, Chromobacterium viscosum, Pseudomonas sp., Pseudomonas sp. type B, Bacillus sp., Bacillus stearothermophilus, Bacillus thermoglucosidasius, Candida lipolytica, Electrophorus electricus, hog liver, horse liver, rabbit liver, Mucor miehei, Saccharomyces cerevisiae and Thermoanerobium brockii.
5. A process as in claim 1 wherein the neotame ester has the formula: wherein R is selected from alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.
6. A process as in claim 5 wherein the alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is selected from methyl, ethyl, propyl, sopropyl, butyl, isobutyl, cyclohexyl, benzyl or phenyl.
7. A process as in claim 5 wherein the neotame ester is N[N(3, 3dimethylbutyl)Lßmethyl estera aspartyl]Lphenylalanine 1methyl ester.
8. A process for selecting an enzyme useful for producing neotame via hydrolysis comprising mixing neotame methyl ester with an enzyme to be evaluated and measuring the results of the hydrolysis reaction.
9. A process as in claim 1 wherein the neotame ester is produced by a chemical or an enzymatic method.
10. A process as in claim 9 wherein the method is the synthesis of neotame esters via peptide coupling agents, the synthesis of neotame esters via chloride of NneohexylLaspartic aesters, the synthesis of neotame esters via NneohexylL aspartic ßesters anhydride, or the synthesis of neotame esters via NneohexylLaspartic zester carboxy anhydride.
Description:
TITLE CHEMOENZYMATIC SYNTHESIS OF NEOTAME BACKGROUND OF THE INVENTION Field of the Invention This invention relates to the chemoenzymatic synthesis of neotame (N- [N- (3, 3-dimethylbutyl)-L-a-aspartyl]-L- phenylalanine 1-methyl ester) via hydrolysis of neotame esters. Neotame is particularly useful as a sweetening agent Related Background Art N- [N- (3, 3-dimethylbutyl)-L-a-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). <BR> <BR> <BR> <P>N-EN-(3, 3-dimethylbutyl)-L-a-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 solution of aspartame and 3,3- dimethylbutylaldehyde 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. Patent No. 5,928,909 disclosed regioselective and chemoselective hydrolysis of an a- 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 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 Pseudomonase 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 a-ester group and the P-ester group. The selectivity of the a-ester hydrolysis to the zester 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 monoesters with pig liver esterase with complete regioselectivity hydrolysis of the a-ester group (US.

Patent No. 5,928,909). In addition, it was found that the preferential hydrolysis for the a-ester position is found to be partially reversed when the aspartate is N-protected as its formamide. The selectivity for the N-protected aspartate was found to be 55: 45 for the a- ester hydrolysis to the zester 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 ester group and formation of the corresponding N-protected a-ester aspartate. However, in the case of N-alkylated aspartate ester which conjugated with phenylalanine ester, selective 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-a-aspartyl]-L- phenylalanine 1-methyl ester) via hydrolysis of neotame esters.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the time dependant production of neotame via hydrolysis of 150 mM of neotame methyl ester by the lipase from Pseudomonase species type B in a buffer of 15% CH3CN and 250 mM Tris-Cl, pH 7.5 at room temperature.

Fig. 2 shows the time dependant production of neotame via hydrolysis of 150 mM of neotame methyl ester by the esterase from porcine liver in a buffer of 15% CH3CN and 250 mM Tris-Cl, pH 7.5 at room temperature.

Fig. 3 shows the time dependant production of neotame via hydrolysis of 150 mM of neotame methyl ester by the

lipase from Pseudomonase species type B in a buffer of 15% DMSO and 250 mM Tris-Cl, pH 7.5 at room temperature.- Fig. 4 shows the time dependant production of neotame via hydrolysis of 150 mM of neotame methyl ester by the esterase from porcine liver in a buffer of 15% DMSO and 250 mM Tris-Cl, pH 7.5 at room temperature.

DETAILED DESCRIPTION OF THE INVENTION This invention is directed to an improved method of preparing an N-alkylated aspartame derivative, namely <BR> <BR> <BR> <BR> N-[N-(3t3-dimethylbutyl)-L-a-aspartyl]-L-phenylalanine 1-methyl ester (neotame). This N-alkylated aspartame derivative is a highly potent sweetening agent.

This invention includes an alternative cost-effective strategy for preparing neotame via the enzymatic regioselective hydrolysis of neotame esters as shown in the following general reaction: COOR COOH O O N N !'OCH N ! OCH H o-O \ H H Ph Ph Neotame ester Neotame The neotame ester of the present invention is a compound of the formula:

wherein R is alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. Preferably, R is methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, cyclohexyl, benzyl or phenyl.

The neotame esters useful in the present invention can be synthesized according to the following general reaction scheme: COOR COOR H N ° (CH3) 3CCHCHO r OH OCH3 : HZNOH Hzpd/C NHJ Ph 0 O Coupling R =alkyl or aryl COOR COOH _NH Enzymatic XNH OCH3 Hydrolysìs XNH oOCH3 0 Ph 0 Ph Neotame ester Neotame The starting materials of the above reaction, L- aspartic acid a-esters, are commercial available and can also be cost-effectively produced from regioselective hydrolysis of L-asparticacid di-esters with pig liver esterase according to the method of U.

S. Patent 5,928,909. U. S. Patent application No.

09/219,898, which has been allowed, discloses methods of synthesizing the t-butyl ester of neotame and N-

neohexyl-L-aspartic acid B-t-butyl ester and is hereby incorporated by reference in its entirety herein.

N- (3, 3-Dimethylbutyl)-L-aspartic acid a-esters (N- neohexyl-L-aspartic acid -esters) may then be prepared by reductive alkylation of 3,3- dimethylbutylaldehyde with L-aspartic acid (3-esters.

The chemical coupling of N- (3, 3-dimethylbutyl)-L- aspartic acid -esters with L-phenylalanine methyl ester to form neotame esters can be accomplished through any of the following four strategies: 1. Synthesis of neotame esters via peptide coupling agents There are many state of the art peptide coupling agents, including, without limitation, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), carbonyldiimidazole (CDI), 1- isobutyloxylcarbonyl-2-isobutyloxy-1, 2-dihydroquinoline (IIDQ), benzotriazole-l-yl-oxyl-tri (dimethylamino)- phosphonium hexafluorophosphate (BOP), benzotriazole-1- yl-oxyl-tri-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium. hexafluorophosphate (PyBrOP), 2- (lH-benzotriazole-l- yl)-1, 1, 3,3-tetramethyluronium hexafluorophosphate (HBTU), 2- (lH-9-azabenzotriazole-1-yl)-1, 1,3,3- tetramethyluronium hexafluorophosphate (HATU), 2- (5- norbornene-2,3-dicarboximido)-1,1,3,3- tetramethyluronium tetrafluoroborate (TNTU), 0- (N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). However, these coupling

reagents are extremely expensive for industrial scale production of peptide based sweeters.

2. Synthesis of neotame esters via chloride of N-neohexyl-L-aspartic p-esters The peptide amide bond of N- [N- (3, 3-dimethylbutyl)-L-a- aspartyl P-ester]-L-phenylalaine 1-methyl ester can be formed via condensation of L-phenylalanine methyl ester with the acid chloride of N- (3, 3-dimethylbutyl)-L-a- aspartic (3-ester. The following reaction illustrates the preparation of N- (3, 3-dimethylbutyl)-L-a-aspartic acid chloride a-ester : COOR COOR OH HCI/PC cl NHOH or HCUSOCI 0 cri 0 O ZOOS NHNHOC R= Me, Et, i-Pr, t-Bu, Bz, lf Et3N/0-4*C 0 Ph Pli, All, cyclohexyl, etc.

To avoid the possible intra molecular condensation of the second amine and the acid chloride, the N-neohexyl aspartic acid a-ester should be acidified with hydrochloride gas in inert solvents for a short period.

3. Synthesis of neotame esters via N-neohexyl-L- aspartic a-ester anhydride Neotame esters may be prepared through a coupling of N-neohexyl-L-aspartic (3-ester anhydride and L- pheneylalanine methyl ester as follows: COOR COOR OH Dehydrating agents O ° 2 H2NoCH3 COOR Ph » XNHg OCH3 R= Me, Et, i-Pr. t-Bu, B7- Base 0 Ph Ph, All, cyclohexyl, etc

The N-neohexyl-L-aspartic a-ester anhydride may be prepared by dehydration of two molecular N-neohexyl-L- aspartic acid (3-esters using dehydrating agents.

Exemplary dehydrating agents include phosphorus pentoxide, phosphorous trichloride, phosphoric acid, acid anhydrides, such as acetic anhydride and formic anhydride, carbodiimides, such as dicyclohexyl carbodiimide (DCC) and the like.

4. Synthesis of neotame esters via N-neohexyl-L- aspartic a-ester carboxy anhydride Synthesis of neotame esters may also be accomplished through a coupling of N-neohexyl-L-aspartic D-ester carboxy anhydride and L-phenylalanine methyl ester as follows : COOR COOR OH COC1/EtOAc N O NH O O o O H2N 0 COOR o- NHNHOC R= Me, Et, i-Pr, t-BuB ; Ph Ph, All, cyclohexyl, etc The N-carboxy anhydride can be prepared by treatment of the (3-ester with phosgene.

The neotame esters of the present invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, t-butyl, isobutyl, cyclohexyl, benzyl and phenyl esters. The tert-butyl or benzyl esters achieve lower coupling yields than the other esters due to the instability of the esters in the strong acid conditions.

The present invention also includes the development of an effective method for the regioselective hydrolysis of peptide esters without effecting the peptide bond cleavage.

The present invention includes processes for the preparation of neotame through enzymatic regioselective hydrolysis of neotame ester using lipases or esterases from, for example, Aspergillus niger, Aspergillus oryzae, Candida antarctica, Candida cylindracea, Candida lipolytica, Candida utilis, hog pancreas, Mucor javanicus, Mucor miehei, Penicillium roqueforti, Pseudomonas cepacia, Pseudomonase fluorescens, Rhizomucor miehei, Rhizopus arrhizus, Rhizopus delemer, Rhizopus niveus, Thermus aquaticus, Thermus flavus, Thermus thermophilus, wheat germ, Chromobacterium viscosum, Pseudomonas sp., Pseudomonas sp. type B, Bacillus sp., Bacillus stearothermophilus, Bacillus thermoglucosidasius, Candida lipolytica, Electrophorus electricus, porcine (hog) liver, horse liver, rabbit liver, Mucor miehei, Saccharomyces cerevisiae, Thermoanerobium brockii.

In a particularly preferred embodiment, the neotame ester is N-[N-(3, 3-dimethylbutyl)-L-ß-methyl ester-a- aspartyl]-L-phenylalanine 1-methyl ester of-the formula: In particularly preferred embodiments of this invention the hydrolases are lipases from Pseudomonas sp. type B or esterases from porcine liver.

The preparation of neotame esters, (N- [N- (3, 3- dimethylbutyl)-L--ester-a-aspartyl]-L-phenylalanine 1- methyl ester, can be achieved via coupling of a derivate of the N- (3, 3-dimethylbutyl)-L--ester-a- aspartic acid with L-phenylalaine methyl ester. The preferred methods of coupling are through N-(3,3- dimethylbutyl)-L-ß-ester-a-aspartyl choloride, or N-neohexyl-L-p-ester aspartyl anhydride, or N-neohexyl- L-p-ester carboxy aspartyl anhydride. To avoid the possible intra molecular condensation of the second amine and the acid chloride, the N-neohexyl aspartic acid zester is acidified with hydrochloride gas in inert solvents for a short period.

Lipases and esterases useful in the present invention were selected by screening using hydrolysis reactions

which were conducted by mixing neotame esters (50 mM- 300 mM) and a protein (0.005-10,000 U/ml) in 5%-50% either acetonitrile or DMSO or isopropanol or DMF or THF, buffered by 0.1 M-0.4 M either Tris-Cl, or NaH2PO4, or MES or Bis Tris propane, with a pH 5-8 at temperature 22°C to 40°C for approximately 2 to 48 hours. The results of the hydrolysis were evaluated by C18 column HPLC. A summary of the regioselective hydrolysis reactions with different lipases and esterases are provided in Table 1, Table 2 and Table 3.

Table 1. HPLC Analysis of the production of neotame via hydrolysis of neotame methyl ester by lipases and esterases at pH 7.5,250 mM TrisCl with 150 mM neotame methyl ester, 20 mM CaCl2, 15% CH3CN, room temperature, 14h No. Sources of Lipases or Esterases Specific activity in the Yields mixture 1 Lipase from Pseudomonas fluorescens 6180 U/ml 2.15% 2 Lipase from Penicillium roaueforti 0.755 U/ml 1. 76% 3 Lipase from Mucorjavanicus 0. 0245 U/ml 1. 75% 4* Lipase from Candida lipolytica 0. 005 U/mg 0. 10% 5* Lipase from Aspergillus oryzae 290 U/ml 0. 51% 6 Lipase from Pseudomonas species, type B 690 U/ml 92. 45% 7 Lipase from Pseudomonas species 10, 160 U/ml 2. 53% 8 Lipase from Chromobacterium viscosum 14,520 U/ml 2.03% 9* Lipase from wheat erm 0. 40 U/ml 1. 34% 10* Lipase from Rhizomucor miehei 2.55 U/ml 2.27% 11 Lipase from Pseudomonas cepacia 200 U/ml 1. 76% 12* Lipase from Mucor miehei 6. 5 U/m ! 0.45% 13 Lipase from Candida cylindracea 12. 0 U/ml 1. 73% 14* Lipase from Candida antartica 14. 5 U/ml 0.13% 15* Lipase from Aspergillus niger 7.0 U/ml 0.90% 16 Lipase from Pseudomonas fluorescens 212.5 U/ml 1.72% 17 Lipase from Rhizopus arrhizus 0. 010 U/ml 1.71 % 18 Lipase from Rhizopus niveus 0. 013 U/ml 1.73% 19* Lipase from hog pancreas 84. 0 U/ml 0. 15%

No. Sources of Lipases or Esterases Specific activity in the Yields mixture 20* Esterase from porcine liver (crude) 100 U/ml 28. 46% 21 Esterase from porcine liver esterase (pure) 350 U/ml 58. 71% 22 Esterase from rabbit liver 40.0 U/ml 13.50% 23 Esterase from Thermoanaerobium brockii 0.020 U/mtt. 28% 24 control 0 0.05% *Peptide bonds were hydrolyzed as well.

Table 2. HPLC analysis of the production of neotame via hydrolysis of neotame methyl ester by lipases and esterases at pH 7.5,250 mM TrisCl with 150 mM neotame methyl ester, 20 mM CaCl2, 15% isopropanol, room temperature, 14h No. Sources of Lipases or Esterases Specific activity in the Yields mixture Lipase from Pseudomonasfluorescens 6180 U/ml 2.49% 2 Lipase from Penicillium roqueforti 0. 755 U/ml 1. 91% 3 Lipase from Muscor javanicus 0. 0245 U/ml 1. 96% 4* Lipase from Candida lipolytica 0. 005 U/mg 0. 15% 5* Lipase from Aspergillus oryzae 290 U/ml 1. 95% 6 Lipase from Pseudomonas species, type B 690 U/ml 78. 60% 7 Lipase from Pseudomonas species4, 060 U/ml 2. 47% 8 Lipase from Chromobacterium viscosum 14,520 U/ml 2. 13% 9* Lipase from wheat germ 0. 40 U/ml 1. 47% 10* Lipase from Rhizomucor miehei 2. 55 U/ml 2.33% 11 Lipase from Pseudomonas cepacia 200 U/ml 1. 99% 12* Lipase from Mucor miehei 6.5 U/ml 0. 50% 13 Lipase from Candida cylindracea 12. 0 U/ml 1. 91% 14* Lipase from Candida antartica 14. 5 U/ml 0. 11% 15* Lipase from Aspergillus niger 7. 0 U/ml 1. 34% 16 Lipase from Pseudomonasfluorescens 212. 5 U/ml 1. 98% 17 Lipase from Rhizopus arrhizus 0. 010 U/ml 1. 86% 18 Lipase from Rhizopus niveus 0. 013 U/ml 1. 83% 19* Lipase from hog pancreas 84. 0 U/ml 0. 75% 20* Esterase from porcine liver (crude) 100 U/ml 20.65% 21 Esterase from porcine liver (pure) 350 U/ml 65.28% 22 Esterase from rabbit liver 40.0 U/ml 13.72% 23 Esterase from Thermoanaerobium brockii 0. 020 U/ml 1.19% 24 control 0 0.10% *Peptide bonds were hydrolyzed as well.

Table 3. HPLC analysis of the production of neotame via hydrolysis of neotame methyl ester by lipases and esterases at pH 7.5,250 mM TrisCl with 150 mM neotame methyl ester, 20 mM Caca2, 15% DMSO, 37°C, 8h No. Sources of Lipases or Esterases Specific activity in the Yields mixture I Lipase from Pseudomonas fluorescens 6180 U/ml 2. 40% 2 Lipase from Penicillium roaueforti 0. 755 U/ml 1. 05% 3 Lipase from Mucor javanicus 0. 0245 U/ml 0. 62% 4* Lipase from Candida lipolytica 0. 005 U/mg 0. 63% 5* Lipase from Aspergillus oryzae 290 U/ml 0.085% 6 Lipase from Pseudomonas species, type B 690 U/ml 73.60% 7 Lipase from Pseudomonas species4, 060 U/ml 1. 94% 8 Lipase from Chromobacterium viscosum 14,520 U/ml 2. 22% 9* Lipase from wheat germ 0. 40 U/ml 0. 43% 10* Lipase from Rhizomucor miehei 2. 55 U/ml 3. 61 % lipase from Pseudomonas cepacia 200 U/ml 1. 28% 12* Lipase from Mucor miehei 6. 5 U/inl 0. 45% 13 Lipase from Candida cylindracea 12. 0 U/ml 1.04% 14* Lipase from Candida antartica 14.5 U/ml 0.88% 15* Lipase from Aspergillus niger 7.0 U/ml 1.29% 16 2l2. 5 U/ml 1.04% 17 Lipase from Rhizopus arrhizus 0. 010 U/ml 1. 18% 18 Lipase from Rhizopus niveus 0. 013 U/ml 0. 59% 19* Lipase from hog pancreas 84. 0 U/ml 0. 52% 20* Esterase from porcine liver (crude) 100 U/ml 35. 91% 21 Esterase from porcine liver esterase (pure) 350 U/ml 64. 95% 22 Esterase from rabbit liver 40. 0 U/ml 10. 25% 23 Esterase from Thermoanaerobium brockii 0. 020 U/ml 1. 14% 24 control 0 0. 57% *Peptide bonds were hydrolyzed as well.

Low yields often resulted from the fact that the commercial hydrolases used were not pure and some preparations showed protease activities such as the cleavage of the dipeptide bond to form neohexyl aspartic acid and L-phenylalanine methyl ester. These proteolytic properties contributed to the poor

selective hydrolysis evident with some enzymes as demonstrated in Tables 1 to 3. A lipase from Pseudomonase species, type B (No. 6 in Tables 1-3) and an esterase from Porcine liver (No. 21 in Tables 1-3) provide high hydrolysis yields (approximately 65%-95%) within 15 hours as shown in Figures 1 to 4. Other enzymes and purified forms of the enzymes listed in Tables 1-3 may provide substantially improved yields and are included in this invention.

Several factors effecting catalytic performance of these lipases and esterases were investigated. Organic solvents, pH value, concentration of the buffer, and temperature have significant effects on enzyme activity but have little effect on enzyme selectivity.

Preferably, 5%-50% organic solvents, including acetonitrile, dimethyl sulfoxide (DMSO), tetrahydofuran, acetone, dimethyl foramide (DMF), dioxane, isopropanol, ethanol, methanol, or the like, should be present in the reaction mixture to help solubilize the neotame ester and accelerate the hydrolysis. However, over 50% of the organic co- solvents, in particular, over 50% of methanol, ethanol and isopropanol in the hydrolysis mixture can reduce the hydrolysis rate due to the denaturation of the enzymes.

All methods described herein may be performed in the presence of a suitable buffer. The buffer may be selected from the group comprising 10 mM-500 mM of

phosphate, acetate, Tris, MES, MES and Bis Tris Propane at pH 4-10, preferably at pH 5.5-8.0.

The enzymatic hydrolysis reaction can be conducted at 20-60°C. The hydrolysis rate is increased with temperature increase. But selectivity is slightly decreased with temperature increase. In a preferred embodiment of the present invention the temperature is 20°C to 40°C.

Empirical observation has shown that metal ion cofactors (such as Ca2+, Mg2+, Zn2+) have little effect on the hydrolases selectivity and do not significantly increase the hydrolase activities.

A further advantage of the present invention is that the lipases and esterases may be immobilized on clay, silica gel, Celite, DEAE-Sephadex, or CM-Sephadex, or other similar supports in order to facilitate the recycling of the enzymes and therefore accelerate the production of neotame. The enzymatic hydrolysis can be accomplished in a batch system as well as in a fluidized bed column. Hydrolases may be recombinant enzymes which are cloned and expressed in E. Coli, yeast or other suitable hosts known in the art.

The examples below are provided in order to impart a better understanding of the present invention, however, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention and no limitation of the invention is implied.

EXAMPLE 1 General screening of the lipases and the esterases for production of neotame via hydrolysis of neotame esters In a typical experiment, a lipase or an esterase from, for example, Aspergillus niger, Aspergillus oryzae, Candida antarctica, Candida cylindracea, Candida lipolytica, Candida utilis, hog pancreas, Mucor javanicus, Mucor miehei, Penicillium roqueforti,.

Pseudomonas cepacia, Pseudomonase fluorescens, Rhizomucor miehei, Rhizopus arrhizus, Rhizopus delemer, Rhizopus niveus, Thermus aquaticus, Thermus flavus, Thermus thermophilus, wheat germ, Chromobacterium viscosum, Pseudomonas sp., Pseudomonas sp. type B, etc.

Bacillus sp., Bacillus stearothermophilus, Bacillus thermoglucosidasius, Candida lipolytica, Electrophorus electricus, hog liver, horse liver, rabbit liver, Mucor miehei, Saccharomyces cerevisiae, Thermoanerobium brockii, or the like. (Fluka Chemical Company, Milwaukee, WI.; and Sigma, St. Louis, MO., USA) was added to a suspension of neotame esters (0.05 M-0.3 M, its synthesis is shown in Example 2) in a mixture of 5%-25% of acetonitrile or 5%-25% of DMSO or 5%-15% isopropanol with a buffer of 0.05 M-0.4 M phosphate, or TrisCl, or MES, or Bis Tris Propane, or Acetate at pH 5.5-8.0 buffer at room temperature to 40°C in centrifuge tubes. After shaking for 4 hours to 48 hours, two times amount methanol or DMSO was added to the reaction mixture. The denatured protein was spun down and the supernatant was subjected to C-18 column HPLC for hydrolysis analysis. Exemplary results are presented in Table 1, Table 2 and Table 3. The neotame product can be isolated either by crystallization from

methanol/water, ethanol/hexane or by silica gel chromatography (HOAc/CH30H/CHCl3 0.1: 1: 7).

EXAMPLE 2 General preparation of neotame via the hydrolysis of neotame methyl ester with a lipase (1) Synthesis of N- (3, 3-Dimethylbutyl)-L-aspartic acid ß-methyl ester.

In a Parr bottle containing a solution of L-aspartic acid ß-methyl ester (10 g, 54.4 mmol), triethyl amine (8 ml), 3, 3-dimethylbutyraldehyde (5.5 g, 55.0 mmol) and methanol (80 ml) was added 0.75 g of Pd/C (4% palladium on carbon, 50% wet). After stirring for 30 minutes, the mixture was hydrogenated at 50 psi/room temperature for 8 hours. The mixture was filtered through a Celite bed, and the bed was washed with methanol (50 ml) and water (50 ml). The filtrate and washings were combined and concentrated. The pH of the aqueous solution (50 ml) was adjusted to pH 3 with concentrated hydrochloric acid, followed by extraction with chloroform (20 ml). The remaining aqueous solution was concentrated and the crude product was crystallized from water/acetone to afford 8.6 g (72%) of the N- (3,3-Dimethylbutyl)-L-aspartic acid p-methyl ester. 1H NMR (D2O), 3.80 (t, 1H, J = 5.6 + 5.8 Hz), 3.59 (s, 3H), 2.97 (dd, 2H, J = 7.8,14.0 Hz), 2.89 (d, 2H, J =5.6 Hz), 1.47 (m, 2H), 0.77 (s, 9H) ;"C NMR 172.62,172.46,58.19,53.07,44.65,39.14,34.20, 29.26,28.53.

(2) Synthesis of neotame methyl ester via N-neohexyl- L-aspartic acid chloride:

A rapid stream of hydrogen chloride (dried through a concentrated sulfuric acid) was bubbled through (ca. 5 bubbles/sec) a stirred suspension of N-neohexyl-L- aspartic acid -methyl ester (5.0 g, 19.6 mmol) in 80 ml of anhydrous chloroform, at 0-4°C, for 15 minutes.

Finely powdered phosphorus pentachloride (4.08 g, 19.57 mmol) was then added and the reaction was kept at 0- 10°C for 4-5 hours. The solvent was evaporated and the mixture was dried on an oil pump for 45 minutes.

The acid chloride obtained was re-dissolved in anhydrous dichloromethane or chloroform and used directly for next step without further purification.

To a solution of L-phenylalaine methyl ester (4.5 g, 20.86 mmole) in anhydrous triethylamine (10 ml) and anhydrous dichloromethane (30 ml) at 0-4°C was added dropwise the crude N-neohexyl-L-aspartic acid chloride (generated from 4.0 g of N-neohexyl-L-aspartic acid D-methyl ester) in 10 ml anhydrous dichloromethane.

The reaction was stirred at 0-10°C for 6 hours and the solvent was evaporated. The remaining oil was re- dissolved in chloroform (100 ml) and washed with 100 ml of 0.2 M NaH2PO4, pH 3.0 and 100 ml of H2O. The organic phase was dried with anhydrous MgS04, filtered, evaporated and passed through a short silica gel column using 45% ethyl acetate in hexane as an elvant. The neotame ß-methyl ester was eluted out and evaporated to an oil (6.01 g, 79% from neohexyl aspartic acid D-methyl ester). Rf = 0.46 (1: 1 EtOAc/Hexane), lH NMR (CDC13) 7.80 (d, 1H, J =8.1 Hz), 7.28 (m, 3H), 7.13 (m, 2H), 4.83 (dd, 1H, J = 6.1,14.6 Hz), 3.70 (s, 3H), 3.67 (s, 3H), 3.40 (dd, 1H, J = 4.0,8.1 Hz), 3.11 (m,

2H), 2.70 (dd, 1H, J = 4.0,16.0 Hz), 2.51 (m, 3H), 1.30 (m, 2H), 0.85 (s, 9H) ; 13C NMR (CDC13) 172.11, 171.72,135.94,129.24,128.53,127.08,59.14,52.69, 52.23,51.89,44.45,44.00,37.98,35.93,29.75,29.51.

(3) Preparation of neotame via the hydrolysis of neotame methyl ester with a lipase.

In a typical experiment, a lipase from Pseudomonas sp. type B (139 Units/mg, 50 mg, Fluka Chemical Company, Milwaukee, WI., USA) was added to a suspension of neotame methyl ester (5.01 g, 12.75 mmol) in a mixture of 10 ml of acetonitrile and 65 ml of 0.05 M sodium phosphate, pH 7.0 buffer at room temperature. The pH was kept at 7.0 by addition of 0.1 M NaOH with an automatic pH titrator (Brinkman, Westbury, NY). After hydrolysis was complete, confirmed by HPLC (about 8- 12 hours with yield of about 80%-95%), the mixture was carefully acidified with 0.1 M HC1 to pH 3.0. The product was extracted with ethyl acetate (5 x 50 ml).

The organic phase was dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The subject compound was crystallized from ethanol/hexane at 4°C to give 2.90 g (61%) of neotame. The remaining mother liquid was concentrated and subjected to silica gel chromatographic purification (1: 7 CH30H/CHC13) to obtain 0.86 g (18%) of additional neotame.

EXAMPLE 3 Preparation of neotame via the hydrolysis of neotame methyl ester with an esterase.

In a typical experiment, an esterase from porcine liver (5,340 Units, Sigma, St. Louis, MO., USA) was added to a suspension of neotame methyl ester (2.19 g, 5.58 mmol) in a mixture of 6 ml of acetonitrile and 30 ml of 0.05 M sodium phosphate, pH 7.0 buffer at room temperature. The pH was kept at 7.0 by addition of 0.1 M NaOH with an automatic pH titrator (Brinkman, Westbury, NY). After hydrolysis was complete, as confirmed by HPLC (about 10 hours with-yields around 75%-90%), the mixture was carefully acidified with 0.1 M HC1 to pH 3.0. The product was extracted with ethyl acetate (5 x 50 ml). The organic phase was dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The crude compound was subjected to silica gel chromatographic purification (1: 7 CH30H/CHCl3) and crystallized from ethanol/hexane at 4°C to give 1.28 g (60%) neotame.

EXAMPLE 4 Preparation of neotame via the hydrolysis of neotame methyl ester with a lipase.

In a typical experiment, a lipase from Pseudomonas sp. type B (139 Units/mg, 25 mg, Fluka Chemical Company, Milwaukee, WI., USA) was added to a suspension of neotame methyl ester (2.00 g, 5. 48 mmol) in a mixture of 3 ml of DMSO and 10 ml of 20 mM CaCl2 at room temperature. The pH value was kept at 7.0 by addition of 0.1 M NaOH with an automatic pH titrator (Brinkman,

Westbury, NY). After hydrolysis was complete, confirmed by HPLC (about 10 hours with yields around 75%-90%), the mixture was carefully acidified with 0.1 M HCl to pH 3.0. The product was extracted with ethyl acetate (5 x 50 ml). The organic phase was dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The crude compound was subjected to silica gel chromatographic purification (1: 7 CH30H/CHCl3) and crystallized from ethanol/hexane at 4°C to give 1.22 g (58%) neotame.

EXAMPLE 5 Preparation of neotame via the enzymatic hydrolysis of neotame t-butyl ester.

(1) Synthesis of N- (3, 3-Dimethylbutyl)-L-aspartic acid (3-t-butyl ester.

In a Parr bottle containing a solution of L-aspartic acid (3-t-butyl ester (10.0 g, 52.85 mmol), triethyl amine (8 ml), 3,3-dimethylbutyraldehyde (5.31 g, 53.0 mmol) and methanol (80 ml) were added to 0.75 g of Pd/C (d4% palladium on carbon, 50% wet). After stirring for 30 minutes, the mixture was hydrogenated at 50 psi/room temperature for 8 hours. The mixture was filtered through a Celite bed, and the bed was washed with methanol (50 ml) and water (50 ml). The filtrate and washings were combined and concentrated. The aqueous solution was extracted with chloroform (20 ml) followed by adjustment to pH 3 with 0.1 M hydrochloric acid.

The solid precipitate was filtered and crystallized from methanol/water to afford 10.1 g (70%) of the N- (3, 3-Dimethylbutyl)-L-aspartic acid ß-t-butyl ester.

1H NMR (CDC13), 3.57 (dd, 1H, J = 3.8,9.3 Hz), 2.89-

2.75 (m, 3H), 2.60 (dd, 1H, J = 9.3,17.0 Hz), 1.58 (m, 2H), 1.47 (m, 9H), 0.90 (s, 9H) ; 13C NMR 172.33,169.48, 83.65,57.57,44.12,41.68,35.43,29.81,-29.29,27.91.

(2) Synthesis of neotame t-butyl ester via a peptide coupling agent A solution of N- (3, 3-Dimethylbutyl)-L-aspartic acid t-butyl ester (4.0 g, 14.65 mmol), L-phenylalanine methyl ester (3.2 g, 14.83 mmol) and triethylamine (2.8 ml, 20 mmol) in dichloromethane (100 ml) was stirred while Benzotriazole-1-yl-oxyl-tri (dimethylamino)- phosphonium hexafluorophosphate (BOP, 6.48 g, 14.65 mmol) was added. Stirring was continued at room temperature for 2 hours. A saturated sodium chloride solution (200 ml) was added and the product extracted with ethyl acetate (3 x 50 ml). The combined extracts were washed with 0.1 M NaH2PO4 pH 3.0,10% NaHCO3 solution and water. The dried (MgS04) solution was evaporated in vacuo and the crude product was subjected to silica gel chromatographic purification to obtained 5. 1 g (81%) of neotame t-butyl ester. Rf = 0. 65 (1: 1 EtOAc/Hexane), 1H NMR (CDC13) 7.78 (d, 1H, J =8.3 Hz), 7.20 (m, 3H), 7.06 (d, 2H, J = 7. 6 Hz), 4.77 (m, 1H,), 3.63 (s, 3H), 3.31 (dd, 1H, J = 4. 0,8.1 Hz), 3.04 (m, 2H), 2.60 (dd, 1H, J = 4.0,16.1 Hz), 2.46 (m, 2H), 2.30 (dd, 1H, J =9. 0, 16.0 Hz), 1.37 (s, 3H),), 1.25 (t, 2H, J = 8.3 + 8.1 Hz), 0.79 (s, 3H) ; 13C NMR (CDC13) 171.68,170.83,135.94,129.22,128.50,127.03,81.27, 59.49,52.67,52.18,44.55,43.97,37.92,37.72,29.72, 29.47,28.00.

(3) Synthesis of neotame t-butyl ester via N-neohexyl- L-aspartic acid chloride A rapid stream of hydrogen chloride (dried using concentrated sulfuric acid) was bubbled through (ca. 5 bubbles/sec) a stirred suspension of N-neohexyl-L- aspartic acid a-t-butyl ester (2.5 g, 9.15 mmol) in 60 ml of anhydrous chloroform at 0-4°C for 5 minutes.

Finely powdered phosphorus pentachloride (1.91 g, 9.15 mmole) was then added and the reaction was kept at 0- 10°C for 4 hours. The solvent was evaporated and the mixture was dried with oil pump for 1 hours. The acid chloride obtained was re-dissolved in anhydrous dichloromethane and used directly for next step without further purification.

To a solution of L-phenylalaine methyl ester (2.0 g, 9.27 mmol) in anhydrous triethylamine (10 ml) and anhydrous dichloromethane (20 ml) at 0-4°C was added dropwise the crude N-neohexyl-L-aspartic acid chloride (generated from 4.0 g of N-neohexyl-L-aspartic acid -t-butyl ester) in 10 ml anhydrous dichloromethane.

The reaction was kept at 0-10°C for 6 hours and the solvent was evaporated. The left oil was re-dissolved in chloroform (100 ml), washed with 100 ml of 0.2 M NaH2PO4, pH 3.0 and 100 ml of H2O. The organic phase was dried with anhydrous MgSO4, filtered, evaporated and run through a short silica column with 35% ethyl acetate in hexane. The neotame ß-t-butyl ester was eluted out and evaporated to oil (1.25 g, 31% from neohexyl aspartic acid P-t-butyl ester).

(4) Preparation of neotame via the hydrolysis of neotame t-butyl ester with a lipase.

In a typical experiment, a lipase from Pseudomonas sp. type B (139 Units/mg, 35 mg, Fluka Chemical Company, Milwaukee, WI., USA) was added to a suspension of neotame t-butyl ester (2.07 g, 4.76mmol) in a mixture of 5 ml of acetonitrile and 25 ml of 0.02 M sodium phosphate, 0.01 M Cal2, pH 7.0 buffer at room temperature. The pH was kept at 7.0 by addition of 0.1 M NaOH with an automatic pH titrator (Brinkman, Westbury, NY). After hydrolysis was complete, confirmed by HPLC (about 8 hours), the mixture was carefully acidified with 0.1 M HC1 to pH 3. 0. The product was extracted with ethyl acetate (4 x 40 ml).

The organic phase was dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The crude compound was subjected to chromatographic purification (1: 7 CH30H/CHC13) and crystallized with ethanol/hexane to give 0. 60 g (33%) of neotame.

EXAMPLE 6 Preparation of neotame via the hydrolysis of neotame t-butyl ester with an esterase.

In a typical experiment, an esterase from porcine liver (5340 Units, Sigma, St. Louis, MO., USA) was added to a suspension of neotame t-butyl ester (2.01 g, 4.63 mmol) in a mixture of 5 ml of acetonitrile and 25 ml of 0.05 M sodium phosphate, pH 7.0 buffer at room temperature.

The pH was kept at 7.0 by addition of 0.1 M NaOH with an automatic pH titrator (Brinkman, Westbury, NY After

hydrolysis was complete, confirmed by HPLC (about 10 hours with yield of 45%), the mixture was carefully acidified with 0.1 M HC1 to pH 3.0. The product was extracted with ethyl acetate (5 x 50 ml). The organic phase was dried over anhydrous magnesium sulfate and evaporated under reduced pressure. The crude compound was subjected to chromatographic purification (1: 7 CH30H/CHCl3) and crystallized from ethanol/hexane at 4°C to give 0.51 g (29%) of neotame.