N-3, 3-dimethylbutyl-L-aspartic acid and esters thereof, the process of preparing the same, and the process for preparing N-(N-(3,3-dimethylbutyl) -.alpha. L-aspartyl)-L- phenylalanine 1-methyl ester therefrom

This invention relates to the chemical synthesis of N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl ester using peptide coupling methods. The coupling reaction is conducted by condensation of an activated derivative of novel N-neohexyl-L-aspartic acid with L-phenylalanine or L-phenylalanine methyl ester or by enzymatic coupling of N-neohexyl-L-aspartic acid with L-phenylalanine or L-phenylalanine methyl ester. This invention also relates to novel N-(3,3-dimethylbutyl)-L-aspartic acid, derivatives thereof and the preparation thereof. The activated derivative of N-neohexyl-L-aspartic acid may be an anhydride, mixed anhydride, active ester or an intermediate activated derivative thereof.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to N-3,3-dimethylbutyl-L-aspartic acid and 
derivatives thereof. The invention also relates to a process for preparing 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester (neotame) via peptide coupling of a derivative of the novel 
N-(3,3-dimethylbutyl) L-aspartic acid with L-phenylalanine or an 
L-phenylalanine ester. 
2. Related Background Art 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl ester 
(neotame) is a highly intense non-nutritive sweetening agent useful to 
impart sweetness to a wide variety of food products. This sweetener, 
disclosed in U.S. Pat. No. 5,480,668, is approximately 8,000 times as 
sweet as sucrose, on a weight basis. Thus, very small quantities of this 
sweetening agent may be used to produce foods and food products that are 
equi-sweet tasting to presently available high caloric food products. 
U.S. Pat. No. 5,510,508 describes a method for preparing 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester of the formula 
##STR1## 
comprising treating an aqueous acetic acid/alcoholic solution of aspartame 
and 3,3-dimethylbutyraldehyde, at room temperature, with hydrogen at a 
pressure less than or equal to 1 bar (0.1 MPa) in the presence of a 
catalyst based on platinum or palladium. The product is purified by 
precipitation and filtration after the alcohol is removed from the 
solution under vacuum. 
U.S. Pat. No. 5,728,862 describes a method comprising treating a solution 
of aspartame and 3,3-dimethylbutyraldehyde in an organic solvent with the 
reducing agent, hydrogen in the presence of a catalyst. After removal of 
the catalyst, water is added to form an aqueous/organic solvent solution 
containing about 17% to about 30% of the organic solvent, by weight, from 
which the neotame is obtained by precipitation and filtration. 
It would be desirable, however, to develop more efficient and 
cost-effective methods of preparing high-purity neotame from readily 
available or readily obtainable materials. 
SUMMARY OF THE INVENTION 
This invention relates to novel N-(3,3-dimethylbutyl)-L-aspartic acid and 
derivatives thereof, e.g., esters, anhydrides and the like. These novel 
aspartic compounds may be prepared by reductive alkylation of 
3,3-dimethylbutyraldehyde with L-aspartic acid or derivatives thereof. 
N-(3,3-Dimethylbutyl)-L-aspartic acid may also be prepared by reductive 
alkylation of 3,3-dimethylbutyraldehyde with L-aspartic acid dialkyl 
ester, followed by hydrolysis of the dialkyl ester moieties. 
This invention also relates to processes for the preparation of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester using peptide coupling methodology. In these processes, the peptide 
amide bond of N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 
1-methyl ester may be formed via peptide coupling of an activated 
derivative of N-(3,3-dimethylbutyl)-L-aspartic acid with L-phenylalanine 
or an L-phenylalanine ester. High purity 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester may be obtained by direct crystallization of the condensed product 
or by additional functional group transformation of that product, followed 
by crystallization or by column chromatography. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention is directed to novel N-(3,3-dimethylbutyl)-L-aspartic acid 
and derivatives thereof represented by the formula 
##STR2## 
wherein R.sup.1 and R.sup.2 are independently hydroxy or lower alkoxy 
having 1 to 6 carbon atoms or together are oxygen, forming an anhydride; 
and Q is hydrogen or (CH.sub.3).sub.3 C(CH.sub.2).sub.2 --. Preferably Q 
is hydrogen. Preferably R.sup.1 and R.sup.2 are hydroxy or methoxy. 
This invention is also directed to useful methods for the preparation of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester in high yield and in high purity via peptide coupling of activated 
derivatives of the novel N-(3,3-dimethylbutyl)-L-aspartic acid with 
L-phenylalanine, or the methyl ester thereof. 
Scheme I illustrates a method of preparation of 
N-(3,3-dimethylbutyl)-L-aspartic acid via reductive alkylation of 
L-aspartic acid using 3,3-dimethylbutyraldehyde. 
##STR3## 
The reductive alkylation reaction is typically conducted in water, a lower 
alkyl alcohol solvent, or mixtures thereof. Lower alkyl alcohol solvents, 
e.g., methanol, ethanol, propanol, isopropanol, butanol, and the like, are 
preferred. The reduction is typically conducted using palladium on carbon 
in a hydrogen atmosphere. Other hydrogenation catalysts which may be used 
include platinum on carbon, platinum black, palladium black, nickel on 
silica and alumina homogeneous hydrogenation catalysts, Raney nickel, 
ruthenium black, ruthenium on carbon, palladium oxide, palladium hydroxide 
on carbon, rhodium black, rhodium on carbon or alumina. Other useful 
reducing agents include sodium cyanoborohydride, lithium or sodium 
borohydride. Addition of a base, e.g., sodium bicarbonate, sodium 
carbonate, potassium bicarbonate, potassium carbonate, lithium carbonate 
or an organic base, is optionally used to solubilize the aspartic acid. If 
the reaction is conducted in the absence of a base, dialklyation occurs 
and N,N-di-(3,3-dimethylbutyl)-L-aspartic acid may be obtained. 
Alternatively, as illustrated in Scheme II, 
N-(3,3-dimethylbutyl)-L-aspartic acid may be obtained via a two step 
process of reductive alkylation of a di-protected L-aspartic acid with 
3,3-dimethylbutyraldehyde followed by removal of the carboxyl protecting 
groups. Each of the carboxyl groups of L-aspartic acid are protected with 
a suitable protecting group. Carboxyl-protecting groups that are suitable 
for use in the present invention are those that are both stable to the 
reductive alkylation reaction conditions and that can be removed from the 
final product without racemization or degradation of that product. 
Optionally the carboxyl groups are protected by conversion into ester 
moieties. Exemplary ester groups include C.sub.1 -C.sub.4 alkyl esters, 
substituted C.sub.1 -C.sub.4 alkyl esters, silyl esters and the like. 
Methods of protecting and de-protecting carboxyl-containing compounds are 
well known to those skilled in the art and are described in T. W. Greene, 
et al., "Protective Groups in Organic Synthesis" Second Edition, John 
Wiley & Sons, New York, N.Y. (1991). In this reaction sequence, the 
reductive alkylation may be conducted in same manner without the addition 
of a base, and using the same hydrogenation catalysts or reducing agents 
as described above. The reaction may also be conducted in organic solvents 
such as ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, 
tetrahydrofuran (THF), diethyl ether, tert-butylmethyl ether, toluene and 
the like. 
##STR4## 
As previously noted, yet another embodiment of this invention is directed 
to methods of preparing 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester via peptide coupling of an activated derivative of 
N-(3,3-dimethylbutyl)-L-aspartic acid with L-phenylalanine or an 
L-phenylalanine ester. According to the present invention, an activated 
derivative of N-(3,3-dimethylbutyl)-L-aspartic acid includes the anhydride 
or mixed anhydrides of the aspartic acid as well as intermediate activated 
derivatives, which may or may not be isolated, formed by treatment of the 
aspartic acid, or a protected aspartic acid, with a peptide coupling 
agent. Methods of peptide coupling are well known to those skilled in the 
art and are described in M. Bodansky, et al., "The Practice of Peptide 
Synthesis, Reactivity and Structure, Concepts in Organic Chemistry," 
Volume 21, Second, Revised Edition, Springer-Verlag, New York, N.Y. 
(1994). 
In a first embodiment of the peptide coupling method of this invention, the 
peptide amide bond of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester is formed via condensation of L-phenylalanine or L-phenylalanine 
methyl ester with the anhydride of N-(3,3-dimethylbutyl)-L-aspartic acid, 
hereinafter N-neohexyl-L-aspartic anhydride. Scheme III illustrates the 
preparation of N-neohexyl-L-aspartic anhydride and Scheme IV illustrates 
the preparation of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester via this first embodiment. In the structures in the following 
Schemes III to VII, the N-(3,3-dimethylbutyl), or neohexyl, moiety is 
abbreviated "Neo". 
##STR5## 
N-Neohexyl-L-aspartic anhydride may be prepared by dehydration of 
N-neohexyl-L-aspartic acid using a dehydrating agent. Dehydration 
reactions are known to those skilled in the art and may be accomplished 
using known reaction conditions. 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. 
Optionally, prior to formation of the anhydride, the amino group of 
N-neohexyl-L-aspartic acid may be protected by forming an 
N-protected-N-neohexyl-L-aspartic acid. Amino-protecting groups that are 
suitable for use in this invention must be stable to the dehydration 
reaction conditions and must be removable from the 
N-neohexyl-L-.alpha.-aspartyl-L-phenylalanine or its methyl ester without 
racemization. Suitable amino-protecting groups are generally carbamate or 
amide type protecting groups. Exemplary amino-protecting groups, include 
formyl, acetyl, benzyloxycarbonyl, tert-butoxycarbonyl and 
p-methoxybenzyloxycarbonyl. Methods for protecting and deprotecting 
nitrogen-containing compounds using these groups are well known to those 
skilled in the art and are described in T. W. Greene, et al., supra. 
Preferably, the nitrogen protecting group is either benzyloxycarbonyl, 
formyl, or acetyl and more preferably formyl. 
N-Benzyloxycarbonyl-N-neohexyl-L-aspartic acid may be formed by reaction 
of N-neohexyl-L-aspartic acid with benzyl chloroformate. 
N-Formyl-N-neohexyl-L-aspartic acid may be prepared by reaction of 
N-neohexyl-L-aspartic acid with a formylating agent such as formic 
anhydride or formic acid and acetic anhydride. 
N-Acetyl-N-neohexyl-L-aspartic acid may be prepared by reaction of 
N-neohexyl-L-aspartic acid with an acetylating agent such as acetic 
anhydride. Advantageously, in the present invention, use of formic 
anhydride or acetic anhydride, as the dehydrating agent, in the presence 
of an acid such as formic acid or phosphoric acid, provides for formation 
of N-neohexyl-L-aspartic anhydride from either N-neohexyl-L-aspartic acid 
or an N-protected-N-neohexyl-L-aspartic acid. 
##STR6## 
Formation of the peptide amide bond of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester may be accomplished by condensation of N-neohexyl-L-aspartic 
anhydride with L-phenylalanine, as illustrated in Scheme IV. Amide bond 
formation occurs by reacting equi-molar amounts of the aspartic anhydride 
and phenylalanine in an inert solvent. The reaction may be conducted at 
about 20-100.degree. C. Exemplary inert solvents, suitable for use in the 
amide-bond forming reaction, include toluene, methyl acetate, ethyl 
acetate, tetrahydrofuran, acetonitrile, dioxane and dimethylformamide 
(DMF) containing either organic acids, such as acetic acid, propionic 
acid, butyric acid, isobutyric acid, or no acids. 
The condensation reaction of N-neo-L-aspartic anhydride with 
L-phenylalanine produces a mixture of two amide products, 
N-neohexyl-.alpha.-L-aspartyl-L-phenylalanine and 
N-neohexyl-.beta.-L-aspartyl-L-phenylalanine. N-Neo-L-aspartic anhydride 
is an unsymmetrical anhydride; that is, the aspartic anhydride, and the 
precursor aspartic acid, contain two different carboxyl groups designated 
.alpha. and .beta., based on their relationship to the nitrogen moiety. 
The .alpha.- and .beta.-amide products are formed by reaction of 
phenylalanine with either the .alpha.-carboxyl or the .beta.-carboxyl 
group of the anhydride. 
Both .alpha.- and .beta.-aspartic acid amides are obtained from the 
condensation of N-neohexyl-L-aspartic anhydride L-phenylalanine. The ratio 
of isomers obtained in this condensation reaction is generally about 2:1 
to about 3:1. 
Esterification of the mixture of .alpha.- and .beta.-aspartic acid amides 
with methanol, in acid, forms a mixture of the .alpha. and .beta. product. 
The desired N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 
1-methyl ester may be isolated in pure form by crystallization or column 
chromatography of this mixture. Use of methanol and water, such as 
described, for example, in U.S. Pat. No. 5,728,862, the disclosure of 
which is incorporated by reference herein, provides excellent recovery of 
high purity alpha product. 
Alternatively, N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 
1-methyl ester may be formed directly using the peptide coupling method of 
the present invention via condensation of N-neohexyl-L-aspartic anhydride 
with L-phenylalanine methyl ester, as illustrated in Scheme V. 
##STR7## 
The peptide coupling reaction may be conducted under the same conditions 
useful for coupling L-phenylalanine, described above. This coupling 
reaction forms a mixture of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester and N-[N-(3,3-dimethylbutyl)-L-.beta.-aspartyl]-L-phenylalanine 
1-methyl ester, generally, in a ratio of about 2:1 to about 3:1. The 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester may be obtained in high purity by recrystallization of the neotame 
mixture using methanol and water or by column chromatography using silica 
gel or resins. 
Scheme VI illustrates yet another embodiment of the peptide coupling method 
of this invention for the preparation of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester. 
##STR8## 
In this embodiment, formation of the peptide amide bond of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester is accomplished by condensation of L-phenylalanine methyl ester with 
a mixed anhydride of N-neohexyl-L-aspartic acid. A mixed anhydride is an 
anhydride derived from two different carboxylic acid-containing compounds, 
or may be formed in situ from a carboxylic acid-containing compound and a 
phosphoric acid derivative, such as o-phenylene phosphochloridite. A 
special category of mixed anhydrides is the N-carboxy- or Leuch's 
anhydrides, prepared by treatment of an amino acid, e.g., the 
.beta.-protected aspartic acid, with phosgene, or by heating 
benzyloxycarbonyl amino acid chlorides. These mixed anhydrides may be 
obtained in high efficiency using methods well known in the art. 
The mixed anhydride of N-neohexyl-L-aspartic acid is preferably prepared by 
reaction of the aspartic acid with an acylating reagent, such as an alkyl 
or aryl acid chloride, chloroformate, chlorocarbonate or imide. The 
reaction is typically conducted in the presence of a non-nucleophilic base 
at ambient or reduced temperature. Exemplary non-nucleophilic bases 
include, alkali carbonates, such as sodium carbonate, alkyl amines, such 
as tertiary amines, triethylamine or N-methyl morpholine, aromatic amines, 
such as pyridine, and the like. The reaction may be conducted in a 
solvent, such as toluene, DMF, acetonitrile, methyl acetate, dioxane and 
the like. Alternatively, amine bases, e.g., triethylamine or pyridine, may 
also be used as suitable reaction solvents. Exemplary acylating reagents 
include but are not limited to acid chlorides such as pivaloyl chloride, 
isovaleryl chloride, benzoyl chloride, p-nitro-benzoyl chloride, 
pentachlorobenzoyl chloride, pentafluorobenzoyl chloride and the like, 
chloroformates, such as tertiary butyl chloroformate, isopropyl 
chloroformate, isobutyl chloroformate, and the like, chlorocarbonates, 
such as isobutyl chlorocarbonate, ethyl chlorocarbonate and the like, or 
imides such as N-hydroxy succinimide, N-hydroxysuccinimide and the like. 
As indicated above, N-neohexyl-L-aspartic acid contains two different 
carboxyl groups, .alpha.- and .beta.-, and either of these carboxyl groups 
may participate in the reaction to form the mixed anhydride. Accordingly, 
one of the carboxyl groups, the .beta.-carboxyl, is optionally protected 
so that formation of the mixed anhydride, and eventual formation of the 
peptide amide bond, occurs selectively at the .alpha.-carboxyl group. The 
.beta.-carboxyl group may be protected with any suitable 
carboxyl-protecting group that is not reactive in either the anhydride 
forming reaction or the peptide coupling reaction and can be removed from 
the final product, 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester, without racemization or degradation of that product. Optionally, 
the .beta.-carboxyl group is protected by conversion into an ester moiety. 
Exemplary ester groups, include tert-butyl ester, silyl ester, benzyl 
esters. Methods of protecting and deprotecting carboxyl-containing 
compounds are well known to those skilled in the art and are described in 
T. W. Greene, et al., supra. 
The .beta.-carboxyl group of N-neohexyl-L-aspartic acid is protected at a 
step prior to the anhydride-forming reaction. Optionally, the 
.beta.-carboxyl group is protected at a step prior to formation of 
N-neohexyl-L-aspartic acid by reductive alkylation. .beta.-Carboxylic 
esters may also be formed by the reaction of N-neohexyl-L-aspartic 
anhydride with an alcohol. Scheme VI illustrates the preparation of a 
.beta.-protected N-neohexyl-L-aspartic acid, via reductive alkylation of a 
.beta.-protected L-aspartic acid with 3,3-dimethylbutyraldehyde. The 
reductive alkylation reaction may be performed using conditions and 
reagents, as described above. The .beta.-protected N-neohexyl-L-aspartic 
acid, as prepared according to the method of Scheme VI, may be treated 
with an acylating agent, according to the method of Scheme V, described 
above, to form a mixed anhydride (R is alkyl or aryl). Formation of the 
peptide amide bond may be accomplished by condensation of the mixed 
anhydride of the .beta.-protected N-neohexyl-L-aspartate with 
L-phenylalanine methyl ester, using coupling conditions described above. 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester may be prepared by deprotection, that is, removal of the 
.beta.-protecting group, from the resulting .beta.-protected 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester using reaction conditions known in the art. If desired, the 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester may be further purified by crystallization from methanol/water or by 
column chromatography. 
Scheme VII illustrates yet another embodiment of the peptide coupling 
method of this invention for the preparation of 
N-[N-(3,3-dimethylbutyl)-L-.beta.-aspartyl]-L-phenylalanine 1-methyl 
ester. 
##STR9## 
In this embodiment, formation of the peptide amide bond of 
N-[N-(3,3-dimethylbutyl)-L-.beta.-aspartyl]-L-phenylalanine 1-methyl ester 
is accomplished by direct condensation of an L-phenylalanine ester with an 
N-neohexyl-L-aspartic acid using a peptide coupling agent. Useful peptide 
coupling agents function to form an intermediate activated derivative of a 
carboxylic acid moiety of the N-neohexyl-L-aspartic acid. The intermediate 
activated aspartic acid derivative, which may or may not be isolated, 
undergoes reaction with the nitrogen moiety of L-phenylalanine 1-methyl 
ester to form the peptide amide bond of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester. An example of an activated aspartic acid derivative that may be 
isolated is a .beta.-protected aspartyl acid chloride, which undergoes 
peptide coupling via simple amide formation by reaction of the acid 
chloride and phenylalanine or phenylalanine methyl ester. Examples of 
other isolable activated derivatives include hydrazides and active esters. 
Hydrazide derivatives of .beta.-protected aspartic acid may be formed 
using hydrazine hydrate or derivatized hydrazines according to 
conventional procedures. Coupling of .beta.-protected aspartyl hydrazide 
derivatives may be accomplished by conversion of the hydrazide to the 
azide, followed by treatment with phenylalanine or phenylalanine methyl 
ester. Active ester derivatives of .beta.-protected aspartic acid may be 
formed using conventional esterification techniques. Active ester 
derivatives useful in peptide coupling reactions include, but are not 
limited to, cyanomethyl esters, p-nitrophenyl esters, o-nitrophenyl 
esters, 2,4-dinitrophenyl esters, 2,4,5-trinitrophenyl esters, 
pentachlorophenyl esters, pentafluorophenyl esters, 
tert-butyloxycarbonylamino acid pentafluorophenyl esters, 
N-hydroxyphthalimide esters, N-hydroxysuccinimide esters, 
1-hydroxypyridine esters, 5-chloro-8-hydroxy-quinoline esters, and the 
like. Coupling of active ester derivatives with phenyl alanine or 
phenylalanine methyl ester may be accomplished by simple reaction of the 
two materials under conventional acidic, neutral or basic reaction 
conditions. Alternatively, the active ester coupling reaction may be 
catalyzed using conventional coupling reagents, such as imidazole, 
1-hydroxybenzotriazole, or 3-hydroxy-3,4-dihydro-quinazoline-4-one. 
Typically, the activated aspartic acid derivative is not isolated, but is 
formed under the reaction conditions as an intermediate product that 
participates in the formation of the peptide amide bond. The intermediate 
activated aspartic acid derivative is preferably prepared by reaction of a 
.beta.-protected N-neohexyl-L-aspartic acid with a peptide coupling agent. 
The .beta.-carboxyl group of the N-neohexyl L-aspartic acid may be 
protected with any suitable carboxyl-protecting group that is not reactive 
in the peptide coupling reaction and that can be removed from the final 
product, N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 
1-methyl ester, without racemization or degradation of that product. 
Suitable .beta.-protected aspartic acids are described above. Peptide 
coupling agents that are useful in the method of the present invention 
include, but are not limited to dicyclohexyl carbodiimide (DCC), 
DCC/1-hydroxybenzotriazole, DCC/N-hydroxysuccinimide, 
1-isobutoxycarbonyl-2-isobutoxy-1,2-dihydroquinone (IIDQ), 
carbonyldiimidizole, N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's 
Reagent K), benzotriazolyl-N-hydroxytris(dimethyamino)phosphonium 
hexafluorophosphate (BOP) and the like. The above-described peptide 
coupling agents and methods of using these agents, and others, may be 
found in any of several reviews on the subject, for example M. Bodansky, 
et al., supra. 
In yet another embodiment of this invention, enzymatic methods may be used 
to form the peptide amide bond of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester, as illustrated in Scheme VIII. 
##STR10## 
According to this embodiment, N-neohexyl-L-aspartic acid may be reacted 
with L-phenylalanine methyl ester, in the presence of the protease enzyme, 
papain, to provide .alpha.-neotame. The N-protected N-neohexyl-L-aspartic 
acid may also be used to form the peptide coupled product. Removal of the 
nitrogen protecting group provides the 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester. The solvent in which the reaction is conducted may be selected such 
that the desired product is insoluble, or only slightly soluble in the 
solvent, resulting in the a shift in the reaction equilibrium to favor 
formation of the peptide. Nitrogen protecting groups, useful in the 
enzymatic method of this invention may include any of the protecting 
groups discussed above. Preferably, acetoacetyl, acetyl, benzoyl, 
benzyloxycarbonyl, tert-butyloxycarbonyl, formyl, isovaleryl, 
(p-methoxybenzyl)oxycarbonyl, or phenylacetyl. Preferably, the nitrogen 
protecting group is benzyloxycarbonyl. Useful solvents in which the 
enzymatic coupling reaction may be conducted include those that are 
miscible or immiscible with water. Exemplary solvents include organic 
co-solvents such as glycerol and other glycols, acetonitrile, ethyl 
acetate, tert-amyl alcohol, and triethyl phosphate. Mixed solvent systems, 
such as ethyl acetate and tert-amyl alcohol may also be used. 
After the coupling reaction has been completed using any of the 
above-described methods, any protecting groups that may have be present on 
the aspartic acid moiety (e.g., nitrogen protecting groups or the 
.beta.-carboxyl protecting group) may be removed using typical 
de-protection procedures. The 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester may be further purified by crystallization from methanol/water or by 
column chromatography. 
The examples which follow are intended as an illustration of certain 
preferred embodiments of the invention, and no limitation of the invention 
is implied.

EXAMPLE 1 
N-Neohexyl-L-Aspartic acid 
Process A 
L-aspartic acid (4 g, 0.03 mol) was added to a Parr bottle containing a 
solution of sodium bicarbonate (5.04 g, 0.06 mol) in water (50 mL) to form 
a clear solution, with effervescence. A solution of 
3,3-dimethylbutyraldehyde (3 g, 0.03 mol) in methanol (50 mL) was then 
added to the Parr bottle, followed by Pd/C (4% palladium on carbon, 50% 
wet, 0.4 g). The mixture was hydrogenated at 50 psi/room temperature for 2 
days. If the reaction was not complete, fresh Pd/C (0.2 g) would be added 
and hydrogenation continued for an additional 8 hrs. The mixture was 
filtered through a Celite.RTM. bed, and the bed was washed with methanol 
(20 mL). The filtrate and washings were combined and concentrated under 
reduced pressure at 40-50.degree. C. The pH of the remaining aqueous 
solution was adjusted to 3 with concentrated hydrochloric acid. The 
precipitated solid was filtered and dried in a vacuum oven at 50.degree. 
C. for 4 hrs to give 4.75 g (73%) of crude N-neohexyl-L-aspartic acid. The 
crude product was slurried with aqueous ethanol (47.5, 80% ethanol, 20% 
water) for 15 minutes and filtered to give 3.3 g (51%) of &gt;98% pure 
N-neohexyl-L-aspartic acid. M.P. 184-186.degree. C.; [.alpha.].sub.D 
+12.35 (c=1, water); NMR (D.sub.2 O) .delta.3.37(t, 3H); 2.25-2.65 (m, 
4H); 1.27-1.47 (m, 2H); 0.89(s, 9H); Anal. Calc'd. for C.sub.10 H.sub.19 
NO.sub.4 ; C, 55.28; H, 8.81; N, 6.45; Found: C, 55.35; H, 8.86; N, 6.50. 
EXAMPLE 2 
N-Neohexyl-L-Aspartic acid Hydrochloride 
Process B 
Sodium hydrogen carbonate (1.68 g, 0.02 mol) was added to a Parr bottle 
containing a solution of dimethyl L-aspartate hydrochloride (3.94 g, 0.02 
mol) in water (10 mL), followed by addition of 3,3-dimethylbutyraldehyde 
(2.2 g, 0.02 mol) in methanol (100 mL) and Pd/C (4% palladium on carbon, 
50% wet), 10% by weight of substrate. The mixture was hydrogenated at 50 
psi at room temperature for 1.5 hrs then filtered through a Celite.RTM. 
bed and the bed washed with methanol (10 mL). The filtrate and washings 
were concentrated under reduced pressure. The residual aqueous solution 
was extracted with diethyl ether (2.times.100 mL). The organic layer was 
washed with aqueous saturated sodium hydrogen carbonate solution (50 mL), 
dried over anhydrous magnesium sulfate, filtered and concentrated to give 
N-neohexyl-L-aspartic acid dimethyl ester (4.65 g, 95%) as an oil. NMR 
(CDCl.sub.3) .delta.3.67 (s, 3H); 3.50 (s, 3H); 3.57(t, 3H); 2.37-2.70 (m, 
4H); 1.20-1.40 (m, 2H); 0.80 (s, 9H). 
A mixture of N-neohexyl-L-aspartic acid dimethyl ester (15 g, 0.061 mol) in 
1N hydrochloric acid (350 mL) was heated at reflux for 4 hours to form a 
clear solution. The mixture was freeze dried to give N-neohexyl-L-aspartic 
acid hydrochloride as a white solid (14 g, 91%), M.P. 133-138.degree. C., 
[.alpha.].sub.D +14.69 (c=1, water). Anal. Calc'd. for C.sub.10 H.sub.19 
NO.sub.4 --HCl; C, 47.52; H, 7.92; N, 5.54; Cl, 14.05. Found: C, 47.84; H, 
8.14; N, 5.31; Cl, 13.08. 
EXAMPLE 3 
N,N-Di-neohexyl L-Aspartic Acid 
To a slurry of L-aspartic acid (4 g, 0.03 mol) in 1:1 methanol/water (80 
mL) was added neat 3,3-dimethylbutyraldehyde (3 g, 0.03 mol), followed by 
Pd/C (4%, 50% wet, 0.5 g). The mixture was hydrogenated using H.sub.2 (50 
psi) at room temperature for 2 days. The resulting reaction mixture was 
filtered through a Celite.RTM. bed, and the bed washed with methanol (50 
mL) The filtrate and washings were combined and the methanol removed using 
a rotary evaporator under reduced pressure at 40-50.degree. C. to provide 
the N,N-di-neohexyl L-aspartic acid as a solid precipitate in water. The 
solid was filtered, washed with water (2 mL) and dried in a vacuum oven at 
50.degree. C. for 4 hrs to provide 3.2 g of N,N-di-neohexyl L-aspartic 
acid (36%), M.P. 197-198.degree. C., [.alpha.].sub.D -3 (c=1, methanol). 
Anal. Calc'd. for C.sub.16 H.sub.31 NO.sub.4 ; C, 63.78; H, 10.29; N, 
4.65. Found: C, 63.43; H, 10.29; N, 4.70 
EXAMPLE 4 
N-Neohexyl-L-Aspartic Anhydride 
Process A 
Phosphorous trichloride (3.7 ml, 43 mmol) was added, dropwise, to a 
10.degree. C. slurry of N-neohexyl-L-aspartic acid (11.6 g, 53.4 mmol) in 
glacial acetic acid (50 ml). After addition was complete (0.5 hr), the 
resulting clear solution was stirred at room temperature overnight. The 
resulting solid precipitate was collected by vacuum filtration, washed 
with acetic acid and methylene chloride, then dried at 35-40.degree. C. 
for 3 hours to give 3.62 g (69.8%) of pure N-neohexyl-L-aspartic 
anhydride. 
EXAMPLE 5 
N-Neohexyl-L-Aspartic Anhydride 
Process B 
N-Neohexyl-L-aspartic acid hydrochloride is dissolved in an aqueous 
solution of sodium bicarbonate with stirring. The solution is cooled, and 
treated with benzyloxycarbonyl chloride and aqueous sodium hydroxide, 
added in portions, alternatingly, with vigorous stirring. The resulting 
reaction mixture is stirred at room temperature for about 3 hours. The 
solution is extracted twice with diethyl ether and acidified to the Congo 
blue endpoint with concentrated hydrochloric acid. The 
N-benzyloxycarbonyl-N-neo-hexyl-L-aspartic acid separates as a solid or an 
oil that slowly solidifies. The acidified reaction mixture is cooled at 
10-15.degree. C. for several hours and the resulting solid is collected by 
filtration, is washed with water and is dried under vacuum. The 
N-benzyloxycarbonyl-N-neo-hexyl-L-aspartic acid may be used as recovered 
or may be recrystallized from a suitable solvent. 
A solution of N-benzyloxycarbonyl-N-neohexyl-L-aspartic acid, in acetic 
anhydride with a catalytic amount of phosphoric acid, is heated at reflux 
for 0.5 to 5 hours, is cooled to room temperature, and is concentrated 
under reduced pressure, with heating. The residual material is dissolved 
in water, then neutralized with aqueous sodium hydroxide. The resulting 
precipitate is collected by vacuum filtration, and is recrystallized to 
provide substantially pure N-neohexyl-L-aspartic anhydride. 
EXAMPLE 6 
N-Neohexyl-.alpha.-Aspartame 
Process A 
N-Neohexyl-L-aspartic anhydride and L-phenylalanine are added to dry 
dioxane and the solution is stirred at 20-80.degree. C. for 1-8 hours. The 
resulting reaction mixture is diluted with an organic solvent that is not 
miscible with water, washed successively with aqueous KHCO.sub.3, then 
aqueous HCl, dried over anhydrous Na.sub.2 SO.sub.4 and evaporated to 
dryness under reduced pressure, providing a mixture of amides: 
N-neohexyl-.alpha.-L-aspartyl-L-phenylalanine and 
N-neohexyl-.beta.-L-aspartyl-L-phenylalanine. The amide mixture is 
dissolved in a methanol/hydrochloric acid solution and the resulting 
reaction mixture is stirred at 20-80.degree. C. for 1-8 hours to form a 
mixture of N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 
1-methyl ester and 
N-[N-(3,3-dimethylbutyl)-L-.beta.-aspartyl]-L-phenylalanine 1-methyl 
ester. Recrystallization of the (.alpha.- and .beta.-aspartame mixture 
from methanol/water or column chromatography on silica gel using ethyl 
acetate:methanol:hexane (60:30:10) as an eluent provides pure 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester. 
EXAMPLE 7 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl ester 
Process B 
A mixture of N-neohexyl-L-aspartic anhydride hydrochloride (2 g, 8.5 mmol) 
and L-phenylalanine methyl ester in dry toluene (13 ml) was heated at 
55-60.degree. C. for about 3 hours, cooled to room temperature, and 
allowed to stand overnight. The resulting reaction mixture was 
concentrated to dryness under reduced pressure at 40-45.degree. C. The 
residual solid material was dissolved in ethyl acetate (20 mL), washed 
successively with water (10 mL), aqueous KHCO.sub.3, and aqueous HCl, 
dried over anhydrous Na.sub.2 SO.sub.4 and evaporated to dryness under 
reduced pressure to provide a mixture of 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester and N-[N-(3,3-dimethylbutyl)-L-.beta.-aspartyl]-L-phenylalanine 
1-methyl ester (3 g). Recrystallization of the .alpha.- and 
.beta.-aspartame mixture from methanol/water or column chromatography on 
silica gel using ethyl acetate:methanol:hexane (60:30:10) as an eluent 
provides pure N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 
1-methyl ester. 
EXAMPLE 8 
N-Neohexyl-.alpha.-Aspartame 
Process C 
To a methanol solution of L-aspartic acid .beta.-t-butyl ester (1 mmol) in 
a Parr bottle, was added a solution of 3,3-dimethylbutyraldehyde (1 mmol) 
in methanol (30 mL), followed by addition of Pd/C (0.1 mmol). The mixture 
was hydrogenated at 50 psi at room temperature for 1 hr. If the reaction 
is not complete, fresh Pd/C may be added and hydrogenation continued. The 
mixture was filtered through a Celite.RTM. bed, and the bed washed with 
methanol. The filtrate and washings were combined, and the methanol 
removed under reduced pressure. The pH of the remaining aqueous solution 
was adjusted to 3 with concentrated hydrochloric acid. The precipitated 
solid was filtered and dried in a vacuum oven to give crude 
N-neohexyl-L-aspartic acid .beta.-t-butyl ester hydrochloride. The crude 
product was slurried with aqueous ethanol and filtered to give pure 
N-neohexyl-L-aspartic acid .beta.-t-butyl ester hydrochloride in 90% 
yield. 
Benzotriazolyl-N-hydroxytris(dimethyamino)phosphonium hexafluorophosphate 
(BOP, 3.7 mmol) and triethyl amine (7.4 mmol) were added sequentially to a 
stirred solution of N-neohexyl-L-aspartic acid .beta.-t-butyl ester (3.7 
mmol) and phenylalanine methyl ester hydrochloride (3.7 mmol) in 
DMF/acetonitrile (15/20 mL) and the resulting reaction mixture was allowed 
to stir at room temperature overnight. Addition of brine (60 mL) resulted 
in instantaneous formation of a white precipitate. The resulting 
suspension was stirred for 2 hours and transferred to a separatory funnel. 
The aqueous phase was extracted ethyl acetate (1.times.200 mL, 
2.times.50-80 mL). The combined ethyl acetate extracts were combined and 
washed successively with 1N HCl (2.times.100 mL), water (1.times.100 mL), 
saturated NaHCO.sub.3 (2.times.100 mL), and brine (1.times.100 mL), dried 
over MgSO.sub.4, filtered and evaporated to a light yellowish oil in 
quantitative yield. This oil was dissolved in ethyl acetate (30 mL) and 
treated with hydrochloric acid (4.3 M, 20 mL), with stirring, for 6 hrs. 
at room temperature. The pH of the mixture adjusted to 5.2 and transferred 
to a separatory funnel. The organic layer was separated. The aqueous layer 
was extracted with ethyl acetate (3.times.30 mL) and the combined ethyl 
acetate extracts were dried over anhydrous MgSO.sub.4, filtered and 
evaporated to dryness. Crystallization of the crude product from 25% 
methanol- 75% water (30 mL) gave 2.5 g of pure 
N-[N-(3,3-dimethylbutyl)-L-.alpha.-aspartyl]-L-phenylalanine 1-methyl 
ester. 
EXAMPLE 9 
Enzymatic Synthesis of N-3,3-dimethylbutyl-L-aspartyl-L-phenylalanine 
methyl ester (Neotame) 
Papain (180 mg) was added to a mixture of Mc Ilvaine buffer (pH 5.5) and 
ethanol (60%buffer/40%ethanol, total volume: 3 mL) containing 
mercaptoethanol (100 mmol), neohexyl-L-aspartic acid (200 mmol) and 
L-phenylalanine methyl ester (200 mmol). The mixture was agitated for 20 
hours at room temperature. The reaction products may be separated by 
chromatography using ethyl acetate:methanol:hexane (60:30:10) as an eluent 
to provide pure .alpha.-neotame. 
Other variations or modifications, which will be obvious to those skilled 
in the art, are within the scope and teachings of this invention. This 
invention is not to be limited except as set forth in the following 
claims.