Preparation of amino acids from unsaturated hydantoins

Amino acids can be easily prepared by reducing unsaturated hydantoins to the corresponding saturated hydantoins by hydrogenating the unsaturated hydantoin using either Raney Nickel catalyst in the presence of more than a stoichiometric amount of caustic or by using zinc and hydrochloric acid followed by hydrolyzing the resultant composition with at least 3 molar equivalents of an alkali metal hydroxide to produce a racemate of an alpha amino acid. The amino acid in suitable derivative form can then be resolved particularly using a two-phase solvent system. The residual isomer of the amino acid remaining after the resolution process can then be racemized using either pyridoxal-5-phosphate or an aliphatic acid in combination with an aldehyde or a ketone. By these procedures, it is possible to obtain high yields of amino acids.

FIELD OF THE INVENTION 
The present invention relates to a novel process for preparing amino acids 
from unsaturated hydantoins. More particularly, this invention relates to 
a novel process for the preparation of amino acids by hydrogenating 
unsaturated hydantoins using inexpensive catalysts followed by hydrolysis 
of that product to the corresponding amino acid. The racemic mixture of 
amino acids thus produced can be resolved using a stereospecific enzyme in 
a two phase water immiscible organic solvent/water system. The present 
invention also relates to a process for racemizing the residual amino acid 
isomer from the resolution process. The process does not require high 
pressure, can be carried out in aqueous solvents, and results in 
substantially complete conversion of the unsaturated hydantoin in short 
reaction times using low levels of catalyst to amino acids. 
BACKGROUND OF THE INVENTION 
It has long been common practice to use hydantoin and substituted 
hydantoins are precursors and intermediates in the synthesis of amino 
acids. The use of substituted hydantoins in the synthesis of amino acids 
such as alanine, methionine, tryptophan and lysine is well documented in 
the prior art. (Kirk Othmer, Encyclopedia of Chemical Technology, Volume 
12, pages 694-695). These unsaturated hydantoins can be formed by any 
number of reactions with one of the more commonly used being a 
condensation reaction between an aldehyde and a substituted or 
unsubstituted hydantoin. In this reaction, an ethylenic bond is formed 
between the non-carbonyl, or C-5, carbon of the hydantoin moiety and the 
carbonyl carbon of the original aldehyde. Further reduction, or 
hydrogenation, of this ethylenic linkage is a necessary step in the 
synthesis of some amino acids. This step must be done without 
hydrogenation of any of the aromatic or aliphatic substituents of the 
hydantoin moiety other than at this ethylenic linkage. Previously, this 
hydrogenation step has been done using hydrogen and a nickel catalyst 
under high pressure or by using hydrogen and a very expensive noble metal 
catalyst such as palladium or platinum under little or no pressure. 
The use of one or more of these techniques is reported in a number of U.S. 
patents. In U.S. Pat. No. 2,605,282, 5-vanillylidenehydantoin is reduced 
to the 5-vanillylhydantoin by dissolving the unsaturated hydantoin in an 
aqueous solution containing 4 to 10 percent by weight of sodium hydroxide 
(75 mole % of the unsaturated hydantoin) and shaking the mixture with 
hydrogen under pressure in the presence of a palladium containing 
hydrogenation catalyst. The reduction is carried out at a temperature of 
25.degree. to 40.degree. C. at a pressure of 60 pounds per square-inch 
gauge or higher for 1 to 4 hours. 
In U.S. Pat. No. 2,479,065, 5-benzalhydantoin is reduced to 
5-benzylhydantoin using a caustic activated nickel aluminum alloy 
catalyst, methanol as a solvent and pressures of from 750 to 760 
atmospheres. One disadvantage of the above method is the use of extremely 
high pressures to complete the hydrogenation in a short reaction time. The 
above mentioned patent does not specifically define the type of nickel 
aluminum alloy to be caustic activated or the degree of caustic 
activation. Although nickel aluminum alloys are commonly employed 
catalysts in hydrogenation procedures, a distinction must be drawn between 
a nickel alloy catalyst and a particular class of nickel type catalyst 
called Raney Nickel catalyst. The accepted method of making the latter 
catalyst involves reacting the nickel-aluminum alloy with caustic to 
remove the aluminum and then washing the precipitated nickel with water to 
remove essentially all the caustic to produce a spongy nickel catalyst. 
[Ind. and Eng. Chem. 33 1199 (1940)]: Hereinafter, the term Raney Nickel 
catalyst refers to the form of nickel catalyst produced by the above 
procedure. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, amino acids can be easily 
prepared by reducing unsaturated hydantoins to the corresponding saturated 
hydantoins by hydrogenating the unsaturated hydantoin using either Raney 
Nickel catalyst in the presence of more than a stoichiometric amount of 
caustic or by using Zinc and hydrochloric acid followed by hydrolyzing the 
resultant composition with at least 3 molar equivalents of an alkali metal 
hydroxide to produce a racemate of an alpha amino acid. The racemate of 
the amino acid can then be resolved using a stereospecific enzyme in a two 
phase water immiscible organic solvent/water system. The residual amino 
acid isomer from the resolution process can then be racemized using either 
pyridoxal-5-phosphate or an aliphatic acid in combination with an aldehyde 
or a ketone. Using this method, it is possible to obtain high yields of 
the desired amino acid in short reaction times using low levels of 
catalyst. 
DETAILED DESCRIPTION OF THE INVENTION 
The process of the invention is directed to the production of amino acids 
from unsaturated hydantoins of the general formula. 
##STR1## 
where A is X or Y, and X is an unbranched or branched alkyl or alkenyl 
group, a cycloalkyl group, a cycloalkenyl group, an alkylthio group, a 
haloalkyl group, a haloalkenyl group, a hydroxyalkyl group, an aralkyl 
group, a mono- or dialkylaminoalkyl group, an acylaminoalkyl group, or a 
mercaptoalkyl group. Preferably the alkyl groups contain 1 to about 20, 
especially 1 to about 10 carbon atoms, the alkenyl group 2 to about 10, 
especially 2 to about 5 carbon atoms, the cycloalkyl and cycloalkenyl 
groups from about 3 to about 15, preferably from about 3 to about 10 
carbon atoms. In a given case in the cycloalkyl or cycloalkenyl group, one 
or more --CH.sub.2 -- units can also be replaced by --O--, --S--, or 
--NH--, or --C.dbd. can be replaced by --N-- so that there is present the 
corresponding heterocyclic ring with 3 to about 15, preferably from about 
3 to about 10 ring atoms. The alkoxy, alkylthio, hydroxyalkyl, 
mercaptoalkyl, mono- or dialkylaminoalkyl and acylaminoalkyl groups 
contain preferably 1 to about 10, especially 1 to about 6 carbon atoms in 
the alkyl or acyl groups, and Y is 
##STR2## 
in which Y.sub.1, Y.sub.2, and Y.sub.3 are the same or different and can 
be X as defined above, hydrogen, halogen, e.g. of atomic weight 9 to 80, a 
hydroxy group, a nitro group, a cyano group, an amino group, an aralkyl 
group, or an alkaryl group. Preferably, the aralkyl and the alkaryl groups 
contain from about 7 to about 15 carbons in the alkylene or alkyl groups. 
In a given case, two of the groups Y.sub.1 to Y.sub.3 together can form an 
alkylene or alkenylene group with from about 3 to about 5 carbon atoms 
whereby in this case one or more --CH.sub.2 -- units can be replaced by 
--O--, --S--, or --NH-- or --CH.dbd. can be replaced by --N.dbd.. 
R.sub.1 and R.sub.2 are the same or different and are hydrogen, alkyl, 
aryl, or amino. 
The unsaturated hydantoin can be purchased commercially or can be 
synthesized, for example, through the condensation reaction of an 
aliphatic or aromatic aldehyde with a substituted or unsubstituted 
hydantoin. 
One such condensation reaction is disclosed in the copending application of 
S. Mirviss. Ser. No. 641,888, filed Aug. 17, 1984, entitled "New 
Inexpensive Catalyst for the Production of Unsaturated Hydantoins", the 
subject matter of which is incorporated herein by reference. In this 
application, the condensation reaction of an aldehyde and hydantoin is 
carried out in the presence of a basic salt of an inorganic acid. In this 
process, there are employed aliphatic aldehydes having the formula 
EQU X--CHO 
wherein X is as defined above. Representative aldehydes include 
butyraldehyde, isobutyraldehyde, valeraldehyde, isovaleraldehyde, 
caproaldehyde, enanthaldehyde, nonaldehyde, cyclobutylaldehyde, 
cyclopentylaldehyde, cyclohexylaldehyde, furfural, 2-thiophenealdehyde, 
2-pyrrolealdehyde, imidazolealdehyde, oxazolealdehyde, 3-indolealdehyde, 
pyridylaldehyde, pyrimidylaldehyde, malonic acid half aldehyde and 
monoaldehyde derivatives of decarboxylic acids. 
Appropriate aromatic aldehydes having the formula Y--CHO include 
benzaldehyde, tolylaldehyde, 4-isopropylbenzaldehyde, 
4-hydroxybenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 
3-bromo-4-methoxybenzaldehyde, 3,4-methylenedioxybenzaldehyde, 
2-hydroxy-4-nitrobenzaldehyde, 4,5-dimethoxy-2-nitrobenzaldehyde, 
salicylaldehyde, vanillin, 4-phenylbenzaldehyde, 4-benzylbenzaldehyde, 
4-fluorobenzaldehyde, 4-dimethylaminobenzaldehyde, 4-acetoxybenzaldehyde, 
4-acetaminobenzaldehyde, 4-methylthiobenzaldehyde, and 
3,5-dichloro-4-hydroxybenzaldehyde. Additional aldehydes include 
p-tolylaldehyde, m-tolylaldehyde, 4-chlorobenzaldehyde, 
4-hexylbenzaldehyde, 2-allylbenzaldehyde, 4-allylbenzaldehyde, 
2-vinylbenzaldehyde, 3-vinylbenzaldehyde, 4-methallylbenzaldehyde, 
4-crotylbenzaldehyde, 2-nitrobenzaldehyde, 3-nitrobenzaldehyde, 
4-nitrobenzaldehyde, 2-aminobenzaldehyde, 4-aminobenzaldehyde, 
4-cyclopropylbenzaldehyde, 2-cyclopropylbenzaldehyde, 
4-cyclohexylbenzaldehyde, 2,6-dichlorobenzaldehyde, anisaldehyde, 
3-hydroxybenzaldehyde, 2-hydroxybenzaldehyde, 
2-hydroxy-4-methylbenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde, 
veratraldehyde, 2,4-dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, 
4-cyclohexenylbenzaldehyde, 4-cyclooctylbenzaldehyde, 
4-piperidinylbenzaldehyde, 4-pyridylbenzaldehyde, 4-furylbenzaldehyde, 
4-thienylbenzaldehyde, 4-phenylethylbenzaldehyde, 4-sec.butylbenzaldehyde, 
4-morpholinobenzaldehyde, 4-isopropoxybenzalidehyde, 
2-propoxybenzaldehyde, 3-ethoxybenzaldehyde, 4-hexoxybenzaldehyde, 
2-isopropylaminobenzaldehyde, 4-hexylaminobenzaldehyde, 
4-diethylaminobenzaldehyde, 4-dipropylaminobenzaldehyde, 
4-methylethylaminobenzaldehyde, 3,4-ethylenedioxybenzaldehyde, 
4-acetylthiobenzaldehyde, 4-propionoxybenzaldehyde, 
4-formyloxybenzaldehyde, 4-butyroxybenzaldehyde, 
3,4-tetramethylenebenzaldehyde, 3,4-trimethylenebenzaldehyde, 
3,4-dihydroxybenzaldehyde, alpha napthaldehyde, beta naphthaldehyde, and 
3-indenecarboxaldehyde. 
In addition, hydantoins substituted at the N-1 or N-3 position can also be 
used in the condensation reaction. Examples of such hydantoins include, 
3-methylhydantoin, 1,3-dimethylhydantoin, 1-phenylhydantoin, 
3-benzylhydantoin, 1,3-dibenzylhydantoin and the like. 
The inexpensive basic salts of an inorganic acid to be employed in the 
reaction include ammonium bicarbonate or ammonium carbonate with the 
bicarbonate being the preferred compound. The basic salt can be derived 
from any inorganic acid with a pK.sub.a of above 5. For example, basic 
salts derived from carbonic acid (pK.sub.a =10.3), the bicarbonate of 
carbonic acid (pK.sub.a =6.4), or the monoacid phosphate of phosphoric 
acid (pK.sub.a =12.4) can be used. 
The basic salt used is dissolved in an aqueous solvent. Other solvents 
include water/alcohol, or water/glycol(s). Preferably, the aldehyde is 
added to the solution of catalyst, solvent and hydantoin. 
Generally, the condensation takes place at a temperature between about 
0.degree. to about 120.degree. C., especially at a temperature of about 
10.degree. to about 105.degree. C. The pressure at which the reaction is 
carried out is atmospheric but superatmospheric pressure can also be used. 
The molar ratio of aldehyde to hydantoin can be 0.8 to 1.2. Generally, it 
is advantageous to employ per mole of hydantoin from about 0.85 to 1.15 
moles, especially from about 0.9 to about 1.1 moles of the aldehyde. 
Per mole of hydantoin, there is suitably employed an effective amount, 
ranging from at least 0.10 mole, preferably from about 0.20 to about 1.0 
moles, especially from about 0.20 to about 0.6 moles, of the basic salt of 
the inorganic acid. 
The reaction can be carried out on a small scale or a large scale and can 
be done batchwise or in a continuous fashion. If a continuous reaction is 
chosen, the reaction is monitored and reactants are added when depleted. 
The unsaturated hydantoin as produced in the process of copending 
application Ser. No. 641,888, available commercially or produced through 
other names, can be rapidly reduced with little or no pressure to the 
corresponding saturated hydantoin or ring open derivative thereof by 
carrying out the hydrogentation of the unsaturated hydantoin using a Raney 
Nickel catalyst in the presence of more than a stoichiometric amount of 
caustic as described in copending application of S. Mirviss, Ser. No. 
641,886, filed Aug. 17, 1984, entitled "Hydrogenation of Substituted, 
Unsaturated Hydantoins to Substituted, Saturated Hydantoins". 
The Raney Nickel catalyst employed is available commercially (Davison 
Division of W. R. Grace). Briefly, the preparation of this catalyst 
involves fusing about 50 parts nickel with about 50 parts aluminum as 
described in U.S. Pat. Nos. 1,628,190 and 1,915,473, pulverizing the alloy 
and dissolving out most of the aluminum with sodium hydroxide solution [J. 
Am. Chem. Soc. 54, 4116 (1932)]. The nickel is then washed to remove any 
residual sodium hydroxyide [Ind. and Eng. Chem. 33 1199 (1940)]. The exact 
mechanism through which Raney Nickel exerts its catalytic activity is not 
known. Various theories have been put forth including absorbed hydrogen or 
the formation of a nickel hydride. A complete discussion of this subject 
can be found in Freifelder, Practical Catalytic Hydrogenation, Wiley 
Interscience, 1971, pp. 6-7, the discussion therein being incorporated by 
reference. As is known to those skilled in the art, the Raney Nickel 
catalyst must be kept under water. 
The hydrogenation reaction of the present invention is carried out in the 
presence of an effective amount of Raney Nickel catalyst ranging from 
about 0.1 to about 50, preferably from about 0.3 to about 40 percent by 
weight of the unsaturated hydantoin. The unsaturated hydantoin is 
dissolved in an aqueous solvent such as water or an alcohol, with water 
the preferred medium, and an effective amount of solid or liquid caustic 
of any strength from 10-100% by weight, ranging from about 101 to about 
300, preferably from about 105 to about 250, with optimum results at about 
105 to about 200 mole percent, based on the amount of unsaturated 
hydantoin, is added to the reaction mixture. The strength of the caustic 
solution produced can range from 0.1 to 15 weight percent (0.1N-2.5N) 
based on the amount of water used. Preferably, the process would comprise 
using a reaction mixture containing from about 0.5 to about 10 weight 
percent sodium hydroxide. Other caustics, such as the hydroxy derivatives 
of lithium, potassium, etc. may also be used. 
Hydrogen is bubbled in with vigorous stirring. The reaction can be 
performed at atmospheric or elevated pressures. The higher the pressure, 
the faster the reaction rate but preferably, the reaction is run at a 
pressure of 0 to about 100 psig. 
The temperature at which the reaction is run can range from 0.degree. to 
about 100.degree. C., preferably from about 10.degree. to about 65.degree. 
C. with optimum results being seen at from about 25.degree. C. to about 
40.degree. C. Too high a temperature causes hydrolysis of the 
benzahydantoin to phenylpyruvate before substantial hydrogenation is 
effected. 
The vessel in which the reaction is carried out in the laboratory may be a 
round bottom flask, a pressure resistant glass bottle, a Parr pressure 
bottle, a resin flask (bottle), a Morton flask, etc. The reaction may be 
performed in a batch fashion or a continuous fashion. 
When the reactants are introduced, the reaction can be summarized as 
follows: 
##STR3## 
in which it is theorized that an equilibrium exists between compound (V), 
the saturated hydantoin, and compound (VI), the alkali metal, e.g. sodium, 
salt of the ring opened saturated hydantoin. Compound (VI) can also be 
called a sodium salt of an N-carbamyl, beta-substituted alanyl derivative. 
Either or both products are desirable since the process is designed to 
hydrogenate the ethylenic linkage between a methine carbon of the 
aliphatic, aromatic or heterocyclic substituent on the hydantoin moiety 
and the non-carbonyl carbon of the hydantoin moiety or its original 
derivative. Accordingly, the amount of either compound (V) and/or compound 
(VI) may be measured by conventional methods, including liquid 
chromatography, melting point, UV analysis, etc. Both compounds of formula 
(V) and formula (VI) can be hydrolyzed to the beta-substituted alanine. 
An unsaturated hydantoin can also be reduced to the corresponding saturated 
hydantoin by carrying out the reduction in the presence of zinc plus 
hydrochloric acid. Through this method, almost complete reduction of the 
unsaturated hydantoin to the saturated form can be accomplished in short 
reaction times without the necessity of using hydrogen gas or pressure. 
This is disclosed in the copending application of S. Mirviss, Ser. No. 
641,890, filed Aug. 17, 1984, entitled "Reduction of Unsaturated 
Hydantoins to Saturated Hydantoins". 
The unsaturated hydantoin is dissolved and/or suspended in an appropriate 
solvent or diluent, with methanol being an example, although other 
solvents such as water, water/alcohol or water/glycol(s) may be used. 
After the unsaturated hydantoin is added, the zinc and hydrochloric acid 
can be introduced in a variety of fashions. The zinc can be added first to 
the mixture containing the unsaturated hydantoin, and then the 
hydrochloric acid can be added over a period of time to the reaction 
mixture. Alternatively, the hydrochloric acid can be first added to the 
mixture containing the unsaturated hydantoin and then the zinc powder can 
be added over a period of time. In either case, after all the reactants 
have been added, the reaction is allowed to proceed for a period of time 
ranging from about 30 minutes to about 120 minutes to allow for complete 
reaction of the zinc and hydrochloric acid to take place. After this 
period, the reaction mixture is observed and if there is a quantity of 
zinc which remains unreacted, additional quantities of hydrochloric acid 
may be added to dissolve, or react with, the unreacted zinc. 
Once all the added zinc is dissolved, the reaction is run for an additional 
period of time ranging from about 30 minutes to about 3 hours to allow for 
reaction completion. 
The amount of zinc which is added to the reaction can range from about 100 
to about 400 mole percent of zinc based on weight of the unsaturated 
hydantoin. The amount of hydrochloric acid added is based on the amount of 
zinc to be added. The amount of hydrochloric acid can range from about 200 
to about 400 mole percent based on the amount of zinc added. The 
hydrochloric acid used is preferably concentrated hydrochloric acid 
containing over 30 weight percent HCl and concentrations ranging from 5 
weight percent HCl to about 40 weight percent HCl can be used. 
The zinc used can be in the form of a powder, or can be in any other form 
including filings, shavings, etc. The reaction may be carried out in any 
suitable vessel including round bottom flasks, Morton flasks, etc. 
The temperature at which the reaction is run is preferably room 
temperature. During the course of the reaction, the temperature may rise 
but it is not necessary to do the reaction in an ice bath. 
When all the reactants are present, the reaction may be summarized as per 
formulae (IV), (V) and (VI). 
The saturated hydantoin or its ring opened derivatives including salt forms 
thereof are then hydrolyzed to the corresponding amino acids by any known 
procedure. The hydrolysis is preferably conducted using the process 
described in copending application of M. Empie, Ser. No. 642,293, filed 
Aug. 17, 1984, entitled "Process for the Synthesis of Amino Acids from 
Saturated Hydantoins", the subject matter of which is incorporated herein 
by reference. In accordance with this process, a saturated hydantoin or 
its ring opened derivatives including salt forms thereof can be 
effectively hydrolyzed by heating the same with an hydroxide of an alkali 
metal, preferably having an atomic number within the range of from 2 to 
20, e.g., lithium, sodium and potassium hydroxides. The preferred 
hydroxide is sodium hydroxide. The hydroxide is used in an amount 
sufficient to provide at least about 3 and preferably at least about 5 
molar equivalents based on the hydantoin or derivatives thereof. More 
preferably, the hydroxide is used in an amount ranging from about 3 to 
about 7 and most preferably from about 5 to about 7 molar equivalents. The 
amount of alkali used is based on a neutral (pH 6.5-7.5) solution of the 
hydantoin or its derivatives. If the original solution is highly basic, 
less hydroxide is needed to achieve the required level. If the original 
solution were highly acidic, more base would be required. The total amount 
of hydroxide used is determined as if the solution of hydantoin or 
derivatives is neutral. 
The hydroxide must be used in an amount sufficient to provide about 1 molar 
equivalent for the reaction and 1-2 molar equivalents to neutralize the 
products of reaction, e.g., amino acid and CO.sub.2. Preferably, and to 
obtain maximum hydrolysis in a minimum period of time, it is preferred 
that the molar equivalency of the hydroxide range from about 3 to about 6. 
The addition sequence of hydroxide to hydantoin and derivatives is not 
critical. Sufficient agitation is needed to ensure uniform heating. The 
reaction vessel is preferably operated at atmospheric pressure though 
slight pressure of up to 3 atmospheres can be used to elevate heating 
temperature. 
The hydrolysis reaction is conducted in an aqueous medium. The amount of 
water is sufficient to form an hydroxide solution having from about 12% to 
about 50% hydroxide solids. Water can be added to a mixture of a solution 
of hydroxide and the hydantoin or derivative to obtain the desired final 
percentage. 
The aqueous solution is heated to a temperature within the range of from 
about 70.degree. C. to about 110.degree. C. as determined at atmospheric 
pressure. Generally, the reaction is conducted at reflux temperature. If 
additional heating is required above reflux, slight pressure can be used 
to elevate the reflux temperature. The temperature preferably ranges from 
about 90.degree. C. to about 140.degree. C. and more preferably from about 
95.degree. C. to about 105.degree. C. 
The reaction is conducted for a period of time sufficient to effect 
hydrolysis of at least about 70%. Preferably, the reaction is conducted 
for from about 4 to about 25 hours and more preferably from about 6 to 
about 20 hours. Hydrolysis yields of about 80% and even above 90% are 
obtainable. 
At the conclusion of the reaction, the amino acid can be isolated from the 
reaction mixture and any salt formed during the reaction by appropriate 
means. In the case of phenylalanine, the amino acid can be precipitated 
from the reaction mixture by neutralizing the same. A majority of the salt 
will remain in the mother liquor. The precipitate can then be alcohol 
washed and toluene washed to remove a majority of the salt and any organic 
impurities. The phenylalanine can then be processed further by drying or 
further reaction. Other amino acids which cannot be precipitated by pH 
adjustment can be purified by other means known to a skilled artisan such 
as by ion exchange. The isolated product can be sold as such or as a 
purified amino acid, e.g., phenylalanine. 
The amino acid so prepared is a racemate which must be converted to a form 
which can be resolved if a specific isomer is desired. Resolvable forms of 
the amino acids such as an amide or ester are those which are hydrolyzable 
by an enzyme capable of selectively forming an amino acid isomer of one 
optical rotation. The amino acid carbonyl can be substituted with a moiety 
hydrolyzable by an isomer selective enzyme. The amino nitrogen can also be 
modified with an enzyme hydrolyzable moiety. Both of these methods are 
well known to the skilled artisan. Preferably, the alpha amino nitrogen is 
underivatized (no covalent bonded moieties other than hydrogen) and the 
amino acid carbonyl is substituted with an enzyme hydrolyzable moiety. The 
preferred such moiety is the ester. Methyl and ethyl esters are most 
desirable though esters up to about 8 carbons can be used. Amino acids can 
be esterified by known methods including refluxing a solution of amino 
acid in methanol saturated with hydrogen chloride gas. 
Carbonyl-substituted amino acids wherein the alpha amino nitrogen is 
underivatized can be represented by the formula: 
##STR4## 
where R.sub.1 can be straight or branched chain alkyl, alkylthio, alkoxy, 
benzyl and indolylalkyl and the hydroxy, halo, alkyl and nitro substituted 
derivatives thereof; R.sub.2 is selected from the group consisting of 
YR.sub.3, wherein Y is oxygen or sulfur, or NHR.sub.4 ; R.sub.3 can be 
straight or branched chain aliphatic radicals having from 1 to about 8 
carbon atoms, aryl of up to 3 fused rings and the hydroxy, halo, alkyl and 
nitro substituted derivatives thereof and R.sub.4 can be the same as 
defined in R.sub.3 and hydrogen. As used herein the term alkyl used alone 
or in derivative form such as alkoxy, alkylthio, indolylalkyl and the like 
are intended to include groups ranging from 1 to about 8 carbon atoms. As 
used herein, the term "carbonyl-substituted" is intended to mean that the 
carbonyl group attached to the alpha carbon atom is substituted as is 
shown in the formula in this paragraph and halo includes fluoro, chloro, 
bromo and iodo. 
Examples of the represented parent amino acids, i.e. as if R.sub.2 were 
hydroxyl, include valine, leucine, isoleucine, methionine, phenylalanine 
(preferred), tyrosine, tryptophan, 3,4-dihydroxyphenylalanine, 
2,4-dihydroxyphenylalanine 3,4-methylenedioxyphenylalanine, 
3,4-dimethoxyphenylalanine; 3(4)-methoxy-4(3)-hydroxyphenylalanine, 
3,4-isopropylidenedioxyphenylalanine, 
3,4-cyclohexylidenedioxyphenylalanine, 5-hydroxytryptophan, 
5-methyltryptophan and 3,4,5-trihydroxyphenylalanine. 
The moiety attached to the carbonyl group of the amino acid must be 
hydrolyzable by an enzyme to form the corresponding optical isomer of the 
free amino acid. The hydrolyzable group must not be of sufficient 
molecular weight or structure to cause the amino acid modified with the 
group to become completely insoluble in the water in which the hydrolysis 
must occur. Some of the groups which are included in the term 
"hydrolyzable" include the preferred ester, as well as amides and 
substituted amides (R.sub.2 can be --NH.sub.2, --NHR and NRR' wherein R' 
and R are the same or different and are the same as R.sub.1) and 
thioesters (Y=S). The ester groups can be straight or branched chain 
aliphatic of from C.sub.1 to C.sub.8, aromatic up to three fused rings and 
substituted derivatives thereof such as halo, hydroxy, alkyl, nitro and 
the like. The amides are preferably prepared from straight or branched 
chain aliphatic amines of from C.sub.1 to C.sub.8, aryl of up to 3 fused 
rings and the alkyl, halo, hydroxy and nitro substituted derivatives 
thereof. 
The problems inherent in the resolution of racemates of 
carbonyl-substituted amino acids can be overcome by contacting a solvent 
sulution of the carbonyl-substituted amino acid racemate dissolved in a 
substantially water-immiscible organic material with water; and, while 
portions of the so formed organic phase and aqueous phase are in contact, 
selectively hydrolyzing in at least a portion of the aqueous phase one of 
the isomers of the racemate with an enzyme capable of selectively 
hydrolyzing that optical isomer to the corresponding amino acid otpical 
isomer. The desired amino acid can be recovered by known techniques such 
as by precipitation from the aqueous solution. 
By using the two phase solvent system, the requirement for continuous 
neutralization and the problems of enzyme activity inhibition such as 
caused by the buildup of the unresolved isomer and peptide formation can 
be avoided. This resolution system is disclosed in copending application 
of M. Empie Ser. No. 641,887, filed Aug. 17, 1984, entitled "Resolution of 
Racemates of Amino Acids", the context of which is incorporated herein by 
reference. 
Preferably, the amino acid is phenylalanine or ring substituted derivatives 
thereof, i.e. hydroxy, alkyl, halo and nitro groups. The remaining 
description of the invention will be discussed in connection with the 
preferred amino acid, phenylalanine and its esters, though the teachings 
apply equally to the other named amino acids and hydrolyzable derivatives. 
The racemate of carbonyl-substituted phenylalanine is dissolved in a 
substantially water-immiscible inert organic material which is a solvent 
for the racemate but is not a solvent for the corresponding amino acid. By 
immiscible it is intended to mean that the organic material is miscible in 
the water up to no more that 15% under the conditions of reaction. The 
organic material can be any water-immiscible or partially miscible organic 
solvent which is non-reactive with the carbonyl-substituted amino acid, 
the amino acid, the hydrolytic enzyme or has substantially no inhibitory 
effect on the enzyme activity. The organic solvents can be illustrated by 
toluene, methylene chloride, cyclohexanone, ethyl acetate, butyl acetate, 
butanol, and the like, with toluene being preferred. The 
carbonyl-substituted amino acid also can act as the solvent itself. The 
solvent to water volume ratio can range from about 1:10 to about 10:1 with 
a ratio of from about 2:1 to about 1:2 being preferred. The 
carbonyl-substituted amino acid, e.g. phenylalanine ester, content of the 
organic solvent can range from about 100% to about 5%, with from about 30% 
to about 10% on a weight per volume basis being preferred. 
Carbonyl-substituted amino acids can also be used in the form of an 
inorganic salt. The inorganic salts can be specifically prepared or be the 
result of other processing such as racemization. The racemate is extracted 
into the immiscible organic material by neutralizing the salt with a 
sufficient amount of base such as sodium hydroxide, sodium bicarbonate, 
ammonia or sodium carbonate. The aqueous phase remaining which contains an 
inorganic salt can be removed or used as the aqueous phase for the enzyme 
hydrolysis. It is essential to the operation of the process of the 
invention that a substantial portion of the non-hydrolyzable isomer remain 
essentially in the organic phase during hydrolysis. Thus the 
non-hydrolyzed isomer of the racemate, which acts as an inhibitor to the 
esterase (protease) enzyme can be kept isolated from the enzyme while the 
hydrolysis reaction is proceeding. By "neutralized" is meant a pH within 
the range of from about 5.0 to about 8.0. 
Following extraction, an enzyme which can selectively hydrolyze the 
carbonyl-substituted amino acid to the corresonding amino acid, e.g. an 
esterase (protease), is added to the aqueous phase. The enzyme can be 
selected to resolve i.e. hydrolyze, either the D or the L forms as desired 
but must be specific to one in order for the resolution to be effected. 
Proteases will hydrolyze the ester, amide or thioester substituted amino 
acids listed hereinbefore. The protease include chymotrypsin (in all 
forms), fungal protease, pancreatic extracts such as pancreatin, papain, 
subtilisin as well as commercially available enzymes such as Pronase.TM. 
brand and yeast protease. One of the preferred enzymes for converting the 
ester of L-phenylalanine into its corresponding amino acid is 
chymotrypsin. As is obvious to a skilled artisan, care should be taken to 
avoid excess loss of enzyme activity due to the use of reaction conditions 
which can adversely effect enzyme activity. 
The enzyme can be added free or immobilized on a matrix or contained in an 
insolubilized enzyme column. In the case of a system utilizing the organic 
material and the aqueous phase in a single reactor, the enzyme can be 
added to the aqueous phase and the hydrolysis reaction allowed to proceed 
in that fashion. The enzyme could also be added in an immobilized state by 
stirring or suspending the immobilized enzyme in the aqueous portion of 
the reactor. The aqueous phase can also be pumped through an immobilized 
enzyme either contained totally within the aqueous phase or preferaby 
external to the reactor. In one preferred form of the present invention, 
portions of the aqueous phase are pumped out of the reaction vessel, 
through a filter to remove any solids, into the enzyme column where the 
hydrolysis reaction occurs followed by pumping the effluent from the 
column back through the organic phase and the aqueous phase. Any insoluble 
amino acid formed by the hydrolysis reaction can then be separated from 
the aqueous phase, preferably at the filter. Part of the aqueous phase 
remains in contact with the solvent phase to replenish the unresolved 
carbonyl-substituted amino acid. 
In another embodiment, immobilized enzyme is suspended in the aqueous phase 
and the hydrolysis reaction is then conducted while the aqueous phase is 
in contact with the organic phase under the appropriate conditions for 
enzymatic hydrolysis. After the hydrolysis reaction has proceeded to the 
desired end point, the aqueous phase is separated from the organic phase, 
the immobilized enzyme is separated, and the desired amino acid isomer 
isolated from the aqueous phase by normal methods, such as by 
crystallization at temperatures appropriate for the amino acid isomer to 
be separated, such as from about 0.degree. C. to about 10.degree. C. and 
preferably from about 2.degree. C. to about 6.degree. C., for 
L-phenylalanine. To prevent the immobilized enzyme from becoming 
inactivated by blocking the pores on the support with precipitated amino 
acid, the concentration of amino acid is at or below the limits of 
solubility at the reaction conditions used. 
The immobilized enzyme can be carried on any one of a number of supports 
well known to the prior art such as polymers of acrylic acid, or styrene 
crosslinked with divinyl benzene, or other supports such as wood, 
charcoal, glass, aluminum, silica, cellulose, starch, polyethylene 
terephthalate, agarose or dextran. The enzyme to substrate ratio can be 
within the range of from about 1:10 to about 1:10000 with a ratio of from 
about 1:2000 being preferred. Preferably, the enzyme is used in an amount 
sufficient to hydrolyze one of the optical isomers of the 
carbonyl-substituted racemate (50% of the total) in the two phase system 
to the corresponding amino acid within 3 hours. 
The temperature and pH conditions within the aqueous phase or within the 
immobilized enzyme column are maintained under such conditions as to be 
favorable to the hydrolysis reaction with the enzyme being used. In 
connection with enzymes in general and chymotrypsin in particular, the 
temperature of reaction can range from about 0.degree. C. to about 
60.degree. C. with from about 18.degree. C. to about 50.degree. C. being 
preferred. The pH of the reaction system can range from about 5 to about 8 
with a preferred pH ranging from about 6.0 to about 7.5. Other conditions 
would be obvious to a skilled artisan depending on the enzyme utilized. 
As the resolution reaction proceeds, the protease hydrolyzes the 
carbonyl-substituted amino acid, e.g. the L-phenylalanine ester, in the 
aqueous phase to the L-amino acid, e.g. L-phenylalanine. An equilibrium 
between the organic phase, which contains a majority of the racemate and 
the aqueous phase, which contains a small part of the racemate, is thus 
upset by the reduction of the isomer being hydrolyzed. To reestablish the 
equilibrum, the amino acid ester being hydrolyzed is partitioned from the 
organic phase into the aqueous phase. In this manner, the isomeric amino 
acid ester which is to be hydrolyzed is continuously drawn from the 
organic phase into the aqueous phase until substantially all of that 
isomer is hydrolyzed. While both isomers go from organic to aqueous only 
one returns to the organic layer unchanged while the other isomer is 
hydrolyzed. Thus the unhydrolyzed isomeric amino acid ester remains 
essentially in the organic layer so that the concentration of the 
unhydrolyzed isomer in the aqueous layer is kept constantly low so 
concentrations of the inhibiting isomer in the aqueous phase do not build 
up. In the course of partitioning the one isomer from the other isomer 
from the organic phase to the aqueous phase, the free amino group is 
protonated. This provides a buffering action needed to neutralize the acid 
produced from the ester hydrolysis. The free amino group absorbs a 
hydrogen ion, keeping the pH of the aqueous phase essentially constant 
without having to add an external neutralizing agent though an external 
weak neutralizing agent could be added if desired. 
The pH of the aqueous phase controls the amount of carbonyl-substituted 
amino acid in the aqueous phase. The lower the pH, the more is contained 
in the aqueous portion. This allows for control of the enzymatic reaction 
rate. Because of the lower concentration of the L-amino acid derivative, 
i.e., carbonyl-substituted amino acid, in the aqueous phase, the formation 
of phenylalanine peptide is controlled. 
The control of the equilibrium rate on partition from the organic phase 
into the aqueous phase as well as the pH control is also maintained in 
like manner when the hydrolysis is conducted external to the vessel 
containing the aqueous phase and the organic phase. 
The process can be conducted statically or under agitation. Agitation can 
be strong enough to form finely dispersed droplets of one phase in the 
other. The two phase system should not be agitated sufficiently to form a 
stable emulsion as the two phases must be separated to isolate the 
product. The concentration of the desired amino acid can be above or below 
the limits of solubility under the reaction conditions. If the amino acid 
does precipitate, it can be easily filtered off. Agitation formed by 
pumping fractions in and out of the reactor is acceptable. 
The process of the invention can be operated as a batch process or 
continuously, as desired. In a continuous process, precipitated L-amino 
acid can be continuously removed such as by filtration of the aqueous 
phase. Portions of the organic phase can be removed, racemized and 
returned to provide more L-isomer or, if the racemate is present as a 
water-soluble salt such as the hydrochloride, an organic solution can be 
prepared by dissolving the racemate in water, contacting with organic 
solvent, neutralizing the hydrochloride salt and separating off the 
aqueous phase. If a racemizing agent is used that can retard the 
effectiveness of the enzyme, good manufacturing practices may dictate 
removal of the agent before resolution. 
The non-hydrolyzed isomer of the carbonyl-substituted amino acid can also 
be recovered from the organic solvent by any known separation technique, 
such as distillation, precipitation, or extraction as a hydrochloride 
salt. These HCl salts can be isolated and sold as such or the hydrolyzable 
group hydrolyzed. After hydrolysis has proceeded to the desired point, the 
aqueous solution can be concentrated and neutralized whereby the D-isomer 
amino acid precipitates. 
After substantially all of the L-isomer is separated from the organic 
phase, the concentrated D-isomer can be racemized by any known method such 
as high temperature. The enzymatic hydrolysis of the racemate can then be 
continued in the normal fashion. 
In order to make the two phase solvent system resolution process 
economical, it is necessary that the non-hydrolyzed isomer, which remains 
substantially in the water-immiscible organic solvent after resolution, be 
racemized to allow further production of the amino acid of the other 
isomer. Any process for racemizing one isomer of a carbonyl-substituted 
amino acid in a water-immiscible solvent system must be under such 
conditions that the substituent group is not destroyed. Destruction of the 
substituent group would prevent further resolution in the protease enzyme 
hydrolysis procedure. 
This beneficial result can be obtained by heating the solution of the 
carbonyl-substituted amino acid with an effective amount of an aliphatic 
acid in combination with an effective amount of an aldehyde or ketone at a 
temperature sufficient to effect racemization and preferably at reflux 
temperature for a period of time sufficient that at least 10% of the 
largest optical isomer is racemized. This is more fully disclosed in 
copending application of M. Empie, Ser. No. 642,212, filed Aug. 17, 1984, 
entitled "Method for Racemizing Derivatives of Alpha Amino Acids in 
Organic Media". 
The optical isomer of the carbonyl-substituted alpha amino acid that is 
racemized can be either the L form, the D form or mixtures thereof. Any 
mixture less than the theoretical ratio of 50:50 can be racemized. Any 
degree of racemization, even partial racemization can be obtained in 
accordance with the present invention. By partial racemization is meant 
that at least 10% of the largest quantity of optical isomer has been 
converted to the corresponding D or L isomer. Preferably, at least 50% of 
the largest amount of optical isomer is racemized. By racemized is meant 
the conversion of the optically active form to an optically inactive form, 
consisting of equal mixtures of D and L forms. 
The optically active isomers of hydrolyzable amino acid derivatives with 
underivatized alpha amino nitrogen that can be racemized can be 
represented by the formula: 
##STR5## 
wherein R.sub.1 and R.sub.2 are as defined hereinbefore. 
Examples of the represented parent amino acids, i.e. as if R.sub.2 were 
hydroxyl, include valine, leucine, isoleucine, serine, threonine, 
methionine, phenylalanine, tyrosine, tryptophan, 
3,4-dihydroxyphenylalanine, 2,4-dihydroxyphenylalanine, 
3,4-methylenedioxyphenylalanine, 3,4-dimethoxyphenylalanine, 
3,4-isopropylidenedioxyphenylalanine, 
3,4-cyclohexylidenedioxyphenylalanine, 5-hydroxytryptophan, 
5-methyltryptophan and 3,4,5-trihydroxyphenylalanine. 
The amino acids used in the invention are generally in the form of 
hydrolyzable derivatives. The moiety attached to the carbonyl group of the 
amino acid is preferably hydrolyzable such as by an enzyme in order to 
form the desired isomer of the free amino acid. 
The D- and L-isomeric derivatives must be soluble in water and organic 
solvent, and one of the isomers must be hydrolyzable by an isomer 
selective enzyme at the carbonyl group to produce the corresponding amino 
acid. Preferably, the hydrolyzable group is the ester and preferably the 
ester is methyl or ethyl. 
Preferably the amino acid is phenylalanine or ring-substituted derivatives 
thereof including hydroxy, halo, alkyl and nitro groups. The preferred 
hydrolyzable group is the ester. Preferably the ester is the methyl or 
ethyl ester. The remaining description of the invention will be discussed 
in connection with the preferred amino acid, phenylalanine, and its esters 
though the teachings will apply equally to other named amino acids and 
hydrolyzable derivatives. 
The racemization reaction is conducted in a substantially anhydrous organic 
medium of a substantially water-immiscible organic material as defined 
above which is a solvent for the racemate but is not a solvent for the 
corresponding amino acid under the conditions of the reaction. By 
"substantially anhydrous" it is intended to mean that the organic solvent 
medium contains less than 5% moisture on a weight basis. The organic 
material can be any immiscible or partially miscible organic solvent which 
is not reactive with the ester as can be illustrated by toluene, methylene 
chloride, cyclohexanone, ethyl acetate, butyl acetate, butanol and the 
like, and mixtures thereof with toluene being preferred. The 
carbonyl-substituted amino acid can also act as the solvent itself. The 
carbonyl-substituted amino acid, e.g. a phenylalanine ester, content of 
the organic solvent can range from about 100% to about 2% with from about 
30% to about 2% on a weight per volume basis being preferred. 
The solution to be racemized can be prepared separately or it can occur as 
the result of a resolution process. An organic solvent system can be used 
to dissolve the carbonyl-substituted amino acid or the 
carbonyl-substituted amino acid as its hydrochloride can be extracted into 
the organic solvent, e.g. toluene, by neutralization. The solution to be 
racemized can also be the result of a resolution reaction utilizing an 
oranic solvent phase containing unhydrolyzed isomer. 
In a preferred embodiment of the present invention, the solution to be 
racemized is the result of an enzyme hydrolysis process for resolving 
amino acids, e.g. D,L-phenylalanine, by the use of an organic 
solvent-aqueous system wherein a majority of the carbonyl-substituted 
amino acid concentrates in the solvent phase. 
As the racemization reaction is more conducive at elevated temperatures, it 
is preferred that the apparatus be adapted to allow for sufficient heating 
of the organic solvent system. Glass apparatus can also be adapted to 
accommodate pressures up to 5 psig (0.3 atmospheres) as a means of 
increasing the temperature of reaction above that of reflux. Still higher 
temperatures can be attained in metal equipment resistant to still higher 
pressures. 
The aliphatic acids usable in the invention can be depicted by the formula: 
EQU R.sub.5 COOH 
wherein R.sub.5 is hydrogen or an aliphatic carbon chain of from C.sub.1 to 
about C.sub.6 and preferably from C.sub.1 to about C.sub.3. Suitable 
examples of aliphatic carboxylic acids usable in the invention include 
formic, acetic, propionic, butyric and valeric acids. 
Suitable aldehydes and ketones for use in the invention can be depicted by 
the formula: 
EQU R.sub.6 C(O)R.sub.7 
wherein R.sub.6 and R.sub.7 can be hydrogen; from about C.sub.1 to about 
C.sub.8 alkyl; from C.sub.1 to about C.sub.8 alkenyl; phenyl, substituted 
phenyl having a substituent selected from the group of hydroxy, nitro, 
halo (F, Cl, Br and I), sulfoxy, amino and alkoxy (from C.sub.1 to about 
C.sub.5), phenylvinyl, hydroxynaphthyl and nitrogen-containing 
heteromonocyclic. R.sub.6 is preferably from C.sub.1 to about C.sub.8 
alkyl, and phenyl. Representative examples of aldehydes and ketones 
included within this group can be illustrated by: acetaldehyde, 
propionaldehyde, n-butyraldehyde, isobutyraldehyde, n-valeraldehyde, 
caproaldehyde, n-heptylaldehyde, acrylaldehyde (i.e. acrolein), 
methacrylaldehyde (i.e. methacrolein), salicylaldehyde, benzaldehyde, 
polyvinyl methyl ketone, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, 
o-nitrobenzaldehyde, m-nitrobenzaldehyde, nitrosalicylaldehyde, 
anisaldehyde, vanillin, alpha-naphthylaldehyde, dihydroxybenzaldehyde, 
p-methyl, m-hydroxybenzaldehyde, 2-thienylcarboxaldehyde, 
2-pyridinylcarboxaldehyde, nicotinic aldehyde and the like, 
phenylacrolein, furfural and nitrofurfural, cyclohexanone, 
methylethyl-ketone. Cyclohexanone as sole solvent can form non-reactive 
adducts but can be used in limited amounts with acetic acid as catalyst. 
The aliphatic acid is preferably used in an amount ranging from about 5 to 
about 150 mole percent of the amino acid ester. The aldehydes are 
preferably used in an amount ranging from about 0.6 to about 21 mole 
percent based on the amino acid ester. The aliphatic acid is used in a 
ratio to the aldehyde in an amount ranging from about 250:1 to about 1:1. 
The organic solvent amino acid derivative solution has less than 20 weight 
percent aliphatic acid and less than 10 weight percent aldehyde or ketone 
and preferably from about 0.3 to about 5 weight percent aliphatic acid and 
from about 0.3 to about 1 weight percent aldehyde or ketone. The 
derivative such as the ester in the organic solvent concentration can be 
from about 1% to about 30% and preferably from about 3% to about 15%. 
The racemization reaction is preferably carried out by blending an 
optically active alpha amino acid derivative such as the ester with an 
inert solvent, the aliphatic acid and the aldehyde. Agitation is 
preferably applied to insure even blending and uniform heat transfer. 
It is desirable to tie up the free amino group to prevent peptidization 
during racemization. A convenient method for accomplishing this result is 
bubbling hydrogen halide gas such as hydrogen chloride gas through the 
liquid before racemization. Other convenient methods include treating the 
amino acid with mineral acids such as H.sub.2 SO.sub.4. 
The temperature conditions within the organic system are such as to be 
conducive to the racemization reaction. The temperature of reaction can 
range from about 50.degree. to about 120.degree. C. and preferably from 
about 90.degree. C. to about 115.degree. C. and most preferably at reflux. 
The reaction is preferably conducted at atmospheric pressure though 
pressures of up to 15 atmospheres can be used. The pH of the reaction 
system can range from about 1 to about 8, preferably from 1 to about 5. 
The racemization reaction is conducted for a period of time sufficient to 
racemize at least 10% of the optical ester of the larger quantity and 
preferably for a period of time sufficient to racemize at least 50%. The 
time for racemization generally ranges from about 1 to about 5 hours, 
though less or more time may be used depending on conditions. 
The process of the invention can be operated as a batch process or as a 
continuous process, especially in combination with an optical isomer 
resolution process as part of the overall process. Portions of the organic 
phase can be withdrawn from the system either totally or in part depending 
on whether a batch process or a continuous process is being undertaken. It 
is not necessary that the resolution be undertaken on a pure optical 
isomer since blends of the D and L-isomers wherein one is substantially 
larger than the other can be racemized by the present invention to the 
limit of 50:50 D to L isomer. 
It is desirable that the organic solvent, e.g. toluene, be essentially 
anhydrous to avoid hydrolyzing the carbonyl-substituted amino acid ester. 
This can be accomplished by drying the toluene solution using any known 
method such as by drying over a drying agent such as magnesium sulfate 
(anhydrous). 
The racemate can be recovered in a form for further resolution. Also, the 
D,L-racemate can be directly recovered by conventional means such as 
distillation and crystallation. The racemate can be recovered as the water 
soluble mineral acid salt or the salt can be neutralized to obtain the 
solvent-soluble compound. 
The optical isomer of the carbonyl-substituted alpha amino acid in a 
substantially anhydrous organic solvent can also be racemized without 
destruction of the substituent group by heating the solution with an 
effective amount of pyridoxal phosphate a temperature sufficient to effect 
racemization and preferably at reflux temperature for a period of time 
sufficient that at least 10% of the largest optical isomer is racemized. 
This is disclosed in copending application of M. Empie, Ser. No. 641,889, 
filed Aug. 17, 1984, entitled "Method for Racemizing Derivatives of Alpha 
Amino Acids in Organic Media". Preferably, the pyridoxal-5-phosphate is 
immobilized such as on an ion exchange resin. By this method, a solution 
of optical isomer in organic solvent substantially free of water can be 
racemized without destruction of the carbonyl substituent group 
functionality. 
The racemization reaction is conducted in a substantially anhydrous organic 
medium of a substantially water-immiscible organic material as defined 
hereinbefore. 
The solution to be racemized can be prepared separately as defined 
hereinbefore or it can occur as the result of a resolution process. An 
organic solvent system can be used to dissolve the amino acid or the 
carbonyl-substituted amino acid as its hydrochloride can be extracted into 
the organic solvent, e.g. toluene, by neutralization. The solution to be 
racemized can also be the result of a resolution reaction utilizing an 
organic solvent phase containing unhydrolyzed isomer. 
In a preferred embodiment of the present invention, the solution to be 
racemized is the result of an enzyme hydrolysis process for resolving 
amino acids, e.g. D,L-phenylalanine, by the use of an organic-aqueous 
solvent, or two phase system wherein a majority of the 
carbonyl-substituted amino acid concentrates in the solvent phase while 
the hydrolysis occurs in the aqueous phase. 
As the racemization reaction is more conducive at elevated temperatures, it 
is preferred that the apparatus be adapted to allow for sufficient heating 
of the organic solvent system. Apparatus can also be adapted to 
accommodate pressure up to 5 atmospheres as a means of increasing the 
temperature of reaction above that of reflux. 
To effect racemization of the optical isomer the pyridoxal-5-phosphate can 
be added directly to the organic phase though this is less preferred since 
the catalyst is expensive and is desirably used in a recoverable form. 
Preferably, the pyridoxal-5-phosphate is immobilized on a matrix such as 
an anion exchange resin which will bind the negatively charged phosphate. 
Illustrative of an ion exchange resin is the type sold under the trademark 
WHATMAN DE52, a brand of cellulosic anion exchange resin. Other resins 
which could be used are well within the knowledge of a skilled artisan. 
The immobilized catalyst is prepared using standard techniques which 
include washing the ion exchange resin with dilute acid followed by 
soaking the ion exchange resin in a neutral pH (6.5-7.5) solution of the 
pyridoxal-5-phosphate to attach the same thereto. 
The immobilized catalyst can be placed directly into the vessel containing 
the organic solvent and the carbonyl-substituted amino acid or the organic 
solvent system can be pumped through a vessel containing the immobilized 
catalyst. As the racemization reaction is a high temperature reaction, it 
is preferred that the apparatus be adapted to allow heating at the same 
time that the catalyst is in contact with the organic solvent system. 
Agitation may be required to allow even blending and uniform heat 
transfer. 
The amount of pyridoxal-5-phosphate used can range from about 0.1 to about 
200 moles percent ester to pyridoxal-5-phosphate based on the ester to be 
racemized with a preferred molar percent of from about 1 to about 100. 
The temperature conditions within the organic system are such as to be 
conducive to the racemization reaction. The temperature of reaction can 
range from about 50.degree. C. to about 120.degree. C. and preferably from 
about 90.degree. C. to about 115.degree. C. and most preferably at reflux. 
The reaction is preferably conducted at atmospheric pressure though 
pressures of up to 15 atmospheres can be used. The racemization reaction 
is conducted for a period of time sufficient to racemize at least 10% of 
the optical ester of the larger quantity and preferably for a period of 
time sufficient to racemize 50%. The time for racemization generally 
ranges from about 1 to about 5 hours, depending on conditions though less 
or more time may be used. 
The process of the invention can be operated as a batch process or as a 
continuous process, preferably in combination with an optical isomer 
resolution process. Portions of the organic phase can be withdrawn from 
the system either totally or in part depending on whether a batch process 
or a continuous process is being undertaken. It is not necessary that the 
racemization be undertaken on a pure optical isomer since blends of the D 
and L-isomers wherein one is present in substantially larger 
concentrations than the other can be racemized by the present invention to 
the limit of 50:50 D to L isomer. 
It is desirable that the organic solvent, e.g. toluene, be anhydrous to 
avoid hydrolyzing the carbonyl-substituted amino acid. This can be 
accomplished by drying the toluene blend using any known method such as by 
drying over a drying agent such as magnesium sulfate (anhydrous). 
The racemate can be recovered in a form for recycling for further 
resolution. Also, the D,L-racemate can be directly recovered by 
conventional means such as distillation or crystallization. The racemate 
can be recovered as the water soluble mineral acid salt or the salt is 
neutralized to obtain the solvent soluble compound. 
The L or single isomer amino acid product of the process of the invention 
finds many known uses, particularly L-phenylalanine which is a precursor 
for the preparation of aspartame, an artifical sweetner.

The invention will be illustrated in the examples which follow: 
EXAMPLE 1 
This example illustrates the preparation of 5-benzalhydantoin from 
hydantoin and benzaldehyde. 
A mixture of 25 g. hydantoin (0.25 mole), 29.3 g. benzaldehyde (0.275 mole) 
and 125 ml water as solvent were placed in a round bottom flask fitted 
with stirrer, condenser, thermometer, and heating mantle. Then, 9.9 g. of 
ammonium bicarbonate (0.125 mole-50 mole % based on hydantoin) was added 
with stirrring over a period of 10 minutes. A considerable amount of white 
solid formed. The mixture was stirred at reflux for 4 hours. Solids 
crystallized out on cooling to room temperture. The solid was suction 
filtered, water washed and then ethanol washed. 45 g. of a white solid 
determined by UV analysis to be 5-benzalhydantoin was obtained. The 
theoretical yield based on starting material was 96%. The 
5-benzalhydantoin had a melting point of 215.degree.-221.degree. C. which 
corresponds to the melting point of 218.degree.-220.degree. C. reported in 
BIOCHEM J. 29, 542 (1935). 
The following examples illustrate the preparation of 5-benzylhydantoin from 
benzalhydantoin using a Raney Nickel catalyst and sodium hydroxide. 
EXAMPLE 2 
A 250 ml round bottom flask, fitted with a stirrer, dip tube, thermometer, 
and condenser was charged with 5 g. of benzalhydantoin, 75 ml methanol and 
75 ml water, 1.2 g. of NaOH (100 mole % based on benzalhydantoin) and 1 g. 
of No. 2800 Raney Nickel (Davison Div.--W. R. Grace). Hydrogen was bubbled 
in with vigorous stirring. The temperature was held at approximately 
40.degree. C. After 7 hours of stirring, the hydrogenation as measured by 
UV analysis was 42% complete; after 14 hours, 64% complete and at 23 
hours, 95% complete. Liquid chromatography showed the reaction to be 
96-98% complete. The product consisted of 21% 5-benzylhydantoin, 71% 
N-carbamylphenylalanine, 2% phenylalanine and 4% unreacted 
benzalhydantoin. All were present as sodium salts. 
EXAMPLE 3 
Example 2 was repeated using 5 grams of 5-benzalhydantoin, 150 milliliters 
of distilled water as diluent, 1.4 grams of NaOH (113 mole % based on 
benzalhydantoin) and 1.0 gram of No. 2800 Raney Nickel. After 8 hours of 
hydrogen addition with stirring, there was observed over 95% complete 
hydrogenation by UV analysis and 100% completion as measured by liquid 
chromatography analysis. The product consisted mainly of the sodium salt 
of N-carbamylphenylalanine with a small amount of 5-benzylhydantoin 
present. 
EXAMPLE 4 
A 500 milliliter round bottom flask fitted as above was charged with 300 
milliliters of deoxygenated distilled water, 30 grams of benzalhydantoin, 
9.5 grams of NaOH (150 mole % based on benzalhydantoin) and then 1.5 grams 
of No. 2800 Raney Nickel (5 wt. % based on benzalhydantoin) were added. 
After 5 hours of stirring and H.sub.2 flow, the reduction was 53% 
complete; at 12.75 hours, 92% complete by UV analysis; at 19 hours, the 
reaction was 100% complete by liquid chromatography and UV analysis. The 
product was essentially all N-carbamylphenylalanine with a trace of 
5-benzylhydantoin. 
EXAMPLE 5 
Similar to Example 3 above except 1.8 grams (172 mole %) of NaOH was used. 
The reaction was done in 7 hours (over 98% complete based on UV analysis). 
Liquid chromatographic analysis showed the product to consist of 47.8% 
5-benzylhydantoin, 50.2% N-carbamylphenylalanine, 0.3% phenylalanine and 
1.7% 5-benzalhydantoin. The catalyst was then filtered off. The filtrate 
was neutralized with hydrochloric acid to pH 7-8. Upon evaporation to 
dryness, the filtrate gave 8 g. of white solid. The amount of NaCl present 
was determined by titration and the yield of 
benzylhydantoin/N-carbamylphenylalanine was essentially quantitative. 
After thorough washing with water to remove salt, the remaining solid had 
a melting point of 188.degree. C.-190.degree. C. 
EXAMPLE 6 
A 500 milliliter Parr pressure bottle was charged with 30 grams of 
benzalhydantoin, 300 milliliters of distilled water, 12.7 grams of NaOH 
and 1.5 grams No. 2800 Raney Nickel. The bottle was pressurized with 
hydrogen to 50 psig and shaken on a Parr apparatus. The pressure was 
maintained at 30-50 psig with an average of 40 psig. After 8 hours of 
hydrogenation at 25.degree. C. the reaction was 92% complete based on UV 
analysis and 100% complete after 10 hours. Liquid chromatographic analysis 
showed 0.8% phenylalanine, 92.0% N-carbamylphenylalanine, 2.3% 
5-benzylhydantoin, 1.3% phenylpyruvic acid and 1.6% benzalhydantoin, all 
present as the sodium salts. 
EXAMPLE 7 
Same as Example 6 except a 500 ml round bottom flask was used and the 
H.sub.2 was added at atmospheric pressure and 50.degree.-55.degree. C. 
After 7 hours, the reaction was 76% complete; at 14.5 hours, over 95% 
complete based on UV analysis. 
EXAMPLE 8 
Similar to Example 7 except for the higher temperature of 
50.degree.-80.degree. C. The reaction was 100% complete in 15-16 hours 
based on UV analysis. The product contained 9% phenylpyruvic acid and 4% 
phenylacetic acid as the sodium salts. 
These Examples illustrate the reduction of 5-benzalhydantoin to 
benzylhydantoin or its ring opened derivative, N-carbamylphenylalanine 
using zinc and hydrochloric acid. 
EXAMPLE 9 
This Example shows the process in which hydrochloric acid is added first 
and then the zinc is added. A round bottom flask fitted with a stirrer, 
thermometer and condenser was charged with 10 grams of 5-benzalhydantoin 
(0.053 mole), 100 milliliters of methanol and then 21.0 grams of 
concentrated (37 weight percent) hydrochloric acid (0.213 mole of hydrogen 
chloride, 400 mole percent based on 5-benzalhydantoin) was added. To this 
was added very slowly with stirring 6.93 grams of zinc powder (0.106 mole, 
200 mole percent based on 5-benzalhydantoin) over a period of 45 minutes. 
After 30 minutes of additional stirring, some of the zinc powder remained 
unreacted and so 5 grams more of concentrated hydrochloric acid was added. 
After about 60 minutes more of stirring, no unreacted zinc powder 
remained. The reaction was stirred an additional 2 hours. By UV analysis, 
the reaction mixture showed 98% reduction of the carbon-carbon double bond 
of the 5-benzalhydantoin. By workup of the reaction mixture 
N-carbamylphenylalanine was isolated. The melting point was determined to 
be 186.degree.-188.degree. C. corresponding to the literature melting 
point of 190.degree. C., reported in Am. Chem. J. 45, 368 (1911) (compound 
referred to as 4-benzylhydantoic acid). 
EXAMPLE 10 
This Example shows the effect of adding the hydrochloric acid to zinc. 
Similar to Example 9 above, the flask was charged with 10.0 grams of 
5-benzalhydantoin and 100 milliliters of methanol. Seven grams of zinc 
powder (0.106 mole) was then added. Twenty-one grams of concentrated (37 
weight percent) hydrochloric acid (0.213 mole of hydrogen chloride, 400 
mole percent based on 5-benzalhydantoin) was added with stirring over a 30 
minute period. The temperature rose to 52.degree. C. All of the insoluble 
benzalhydantoin was dissolved after about 75% of the hydrochloric acid was 
added. The reaction mixture was stirred for 1 hour at 50.degree. to 
55.degree. C. but some zinc was still unreacted. Therefore, 5 grams more 
of hydrochloric acid was added and after 30 minutes more of stirring, 
essentially all of the zinc had reacted. UV analysis showed that over 95% 
of the 5-benzalhydantoin had reacted yielding the saturated hydantoin 
and/or ring opened saturated N-carbamylphenylalanine. 
These Examples illustrate the hydrolysis of benzylhydantoin to 
phenylalanine using sodium hydroxide. 
EXAMPLE 11 
Three grams (0.016 mole) benzylhydantoin was mixed with 6 grams of 30% 
sodium hydroxide (2.8 molar equivalents) and 1.5 milliliters water in a 
round bottom flask equipped with a magnetic stirrer and reflux condenser. 
After the mixture was heated at reflux for 6 hours, 81% of the 
benzylhydantoin had been converted to phenylalanine as determined by high 
pressure liquid chromatography. After 14 hours heating, 84% hydrolysis had 
been achieved. 
EXAMPLE 12 
This Example shows that the rate of hydrolysis is increased when greater 
than 5 molar equivalents of an alkali metal hydroxide are used. 
The procedure of Example 11 was repeated using 12 milliliters of 30% 
sodium hydroxide (5.6 molar equivalents) and 3 milliliters water. After 6 
hours at reflux, the extent of hydrolysis was 73% and 100% after 14 hours. 
Using 4 molar equivalents, the hydrolysis reaction proceeded to completion 
whereas in Example 11 using 2.8 molar equivalents hydrolysis appeared to 
have ceased at 84% yield after the same time. 
EXAMPLE 13 
The procedure of Example 11 was repeated using 12 grams of 20% sodium 
hydroxide (3.75 molar equivalents) and no extra water. Hydrolysis yield of 
96% was achieved after 7 hours of heating at reflux. 
EXAMPLE 14 
The procedure of Example 11 was repeated using 5 grams of 50% sodium 
hydroxide (3.9 molar equivalents) and 4.5 grams water. After 11 hours, the 
extent of hydrolysis was 92%. 
These Examples illustrate the hydrolysis of benzylhydantoin to 
phenylalanine using acid conditions. 
EXAMPLE 15 
0.1 grams (0.00052 mole ) of benzylhydantoin was admixed with 0.16 grams 
H.sub.2 SO.sub.4 (98% concentrated) and 0.24 gram water. This was placed 
in a glass tube sealed at the bottom. The top was sealed by melting the 
glass using a torch. The sealed tube was placed in a lead pipe for 
explosion protection and then in a silicone oil bath and heated to 
150.degree. C. The tube was maintained in the bath for 5.5 hours. After 
removing and cooling, the tube was broken open. Analysis of the contents 
by high pressure liquid chromatography showed 92% hydrolysis (two peaks of 
starting materials and final product) as compared to standards. 
EXAMPLES 16, 17 AND 18 
Example 15 was repeated using the materials and conditions listed below. 
Yields of 84% to 97% were obtained. 
TABLE I 
______________________________________ 
Benzyl- Time Yield 
Ex. Hydantoin H.sub.2 SO.sub.4 
H.sub.2 O 
Temp. Hours 
% 
______________________________________ 
16 0.1 g .08 g 32 g 60.degree. C.24 
84 
(.00052 M) 
(.00082 M) 
(.0178 M) 
17 0.2 g 0.16 g* 0.24 g 150.degree. C.5.5 
97 
18 0.1 g 0.16 g* 0.24 g 110.degree. C.4 
93 
______________________________________ 
*0.4 gram 40% H.sub.2 SO.sub.4 = 0.16 gram H.sub.2 SO.sub.4 and 0.24 gram 
H.sub.2 O 
The following Examples illustrate the resolution of an ester of 
D,L-phenylalanine to L-phenylalanine. D,L-phenylalanine esters can be 
prepared by (a) forming a 20% solution of phenylalanine in methanol and 
saturating the solution with HCl gas (whereupon the phenylalanine 
dissolves--a noticeable temperature rise to 50.degree.-70.degree. C. 
occurs); (b) cease HCl addition and (c) reflux the solution for 4-8 hours. 
The methanol is stripped thereby removing the excess HCl and a dry solid 
phenylalanine methyl ester hydrochloride is obtained. The hydrochloride 
can be dissolved in water preparatory to resolution. 
EXAMPLE 19 
A mixture of 1.15 grams of D,L-phenylalanine methyl ester in 2.5 
milliliters of water was added to 5.0 milliliters of ethyl acetate. The pH 
was adjusted to 6.5 with 2.5 milliliters of sodium hydroxide. Two 
milligrams of chymotrypsin was added and the reaction was stirred gently 
at room temperature. A white precipitate appeared after 1.5 hours. The 
precipitate was isolated after 2 hours yielding 0.22 grams with an optical 
rotation equivalent to 98% pure L-phenylalanine. 
EXAMPLE 20 
A 0.2 molar solution phenylalanine methyl ester in 10 milliliters of water 
was prepared. Toluene was added and the pH of the system adjusted to 7.0 
with sodium hydroxide. Chymotrypsin in the amount of 1 milligram was 
added. The concentration of phenylalanine in the aqueous phase was 
monitored. The initial concentration was 0.05 molar and the final 
concentration at the end of the hydrolysis was 0.15 molar, consistent with 
only one isomer being extracted. 
EXAMPLE 21 
A solution of 2.29 grams of D,L-phenylalanine methyl ester hydrochloride in 
10 milliliters of water was prepared. Ten milliliters of toluene was added 
and the pH of the aqueous phase of the biphase mixture was adjusted to 6.8 
with sodium hydroxide. The mixture was allowed to separate and the aqueous 
phase (or lower phase) was connected to a pump via tubing. The pump was 
then connected to a filter. A portion of the aqueous phase was pumped 
through the filter into a column containing chymotrypsin enzyme 
immobilized in Sepharose.TM. 4B (Pharmacia). The effluent from the column 
was then pumped back into the biphase reaction mixture. The initial pH of 
the enzyme column effluent was 6.2. After 5 hours the pH increased to 6.8. 
The pH in the biphase reaction mixture was constant at 6.8. 
L-phenylalanine precipitated in the aqueous phase and was recovered by the 
filter with an overall yield of 85% based on the theoretical amount of 
recoverable phenylalanine after correcting for water saturation. 
EXAMPLE 22 
To 50 milliliters of a 3% weight per volume presaturated solution of 
L-phenylalanine was added 10.0 grams D,L-phenylalanine ethyl ester 
hydrochloride and 50 milliliters of toluene. The biphase mixture was 
neutralized to a pH of 6.8 with 50% sodium hydroxide and 25 milligrams 
chymotrypsin enzyme was added. The reaction proceeded for 3.5 hours 
whereupon the precipitate which formed during the reaction was filtered 
out and washed with small amounts of water. A total of 1.9 grams of 
L-phenylalanine was obtained with 98% purity by optical rotation. 
The toluene from the reaction was separated from the aqueous layer and the 
D-phenylalanine ester contained therein was racemized. The toluene and 
racemized ester were returned to the aqueous phase where a second 
hydrolysis reaction was undertaken to further resolve the racemized 
D,L-phenylalanine ester. A second precipitate was obtained in an amount of 
1.5 grams which was 96% L-phenylalanine. 
EXAMPLE 23 
D,L-phenylalanine methyl ester hydrochloride was extracted into toluene by 
neutralization with sodium hydroxide to give a 9.2% solids solution. An 
aliquot of 300 milliliters was placed in contact with 210 milliliters of 
water. The pH of the mixture was adjusted to 6.8. The temperature was 
raised to 40.degree. C., chymotrypsin immobilized as in Example 21 was 
added and the reaction mixture was stirred for 6 hours until the reaction 
was complete. The enzyme was filtered out and the aqueous portion 
separated. The aqueous portion was cooled overnight at 4.degree. C. 4.6 
grams of precipitated L-phenylalanine of 92% purity by optical rotation 
was obtained. This represents 84% yield after correcting for water 
saturation. 
The following Examples illustrate the racemization of an optical isomer of 
phenylalanine ester using glacial acetic acid and benzaldehyde. 
EXAMPLE 24 
To a solution of 2 grams L-phenylalanine ethyl ester dissolved in 30 
milliliters of toluene was added 1 milliliter glacial acetic acid and 0.1 
milliliters benzaldehyde. The solution was refluxed for 4 hours. The ester 
was completely recovered and optical rotation measurements showed complete 
racemization. 
EXAMPLE 25 
A mixture of 1 gram of D-phenylalanine methyl ester in 10 milliliters of 
toluene resulting from an enzymatic resolution of a racemic mixture of the 
esters was treated by bubbling dry HCl gas through the solution until an 
aliquot of the toluene yielded a pH below 4 when added to water. 0.2 
milliliters of glacial acetic acid and 0.1 milliliter of benzaldehyde was 
then added to the solvent mixture and the mixture was refluxed for 3 
hours. The solvent was then stripped and the 1.12 grams of recovered solid 
was dissolved in 10 milliliters of water. The pH of the latter solution 
was adjusted to 6.2 and placed on a pH stat (Brinkman). Chymotrypsin 
enzyme was then added. The acid released was titrated to yield 93% of a 
theoretical racemic mixture. 
EXAMPLE 26 
D,L-phenylalanine ethyl ester in an amount of 76.5 grams was enzymatically 
resolved to L-phenylalanine using chymotrypsin in a two-phase 
toluene-water system. At the end of the resolution, substantially all of 
the D-phenylalanine ethyl ester was contained in the toluene (300 
milliliters). The toluene was separated and dried over MgSO.sub.4. 
The dry toluene was acidified by bubbling dry HCl through it until a pH of 
an aliquot mixed with water was about 2.3. One milliliter of glacial 
acetic acid and 0.1 milliliter benzaldehyde was added and the mixture 
refluxed for 3 hours. The toluene-ester mixture was cooled, neutralized 
and added back to the aqueous portion of the enzyme reaction system. A 
second resolution was allowed to proceed resulting in the recovery of 
additional L-phenylalanine. 
The following Examples illustrate the racemization of an optical isomer of 
phenylalanine ester using pyridoxal-5-phosphate. 
EXAMPLE 27 
Pyridoxal-5-phospate ion exchange resin was prepared by adding 500 
milligrams of the compound to 3 grams of an ion exchange resin (WHATMAN 
brand DE52). After standing, the resin was filtered and washed two times 
with water followed by dehydrating with ethyl alcohol. L-phenylalanine 
ethyl ester hydrochloride in an amount of 3 grams was dissolved in water 
and extracted into toluene by neutralization. The toluene was removed and 
dried over MgSO.sub.4 (anhydrous). The pyridoxal-5-phosphate ion exchange 
resin was added to the dehydrated toluene containing the L-phenylalanine 
ethyl ester. The mixture was refluxed at a temperature of about 
110.degree. C. for about 1.5 hours. Forty-nine and one-half percent of the 
original L-phenylalanine ester was converted to the D isomer as determined 
by optical rotation measurements. 
EXAMPLE 28 
10.0 grams D,L-phenylalanine ethyl ester hydrochloride was dissolved in a 
two-phase enzyme resolution system consisting of equal 50 milliliter 
volumes of toluene and water saturated with L-phenylalanine. The water 
phase also contained chymotrypsin enzyme. The resolution reaction was 
allowed to proceed to completion whereupon the toluene containing the 
D-phenylalanine ethyl ester was removed, dried with MgSO.sub.4 and added 
to 3 grams of pyridoxal resin prepared as in Example 27. The 
toluene-ester-resin solution was refluxed for 6 hours. Optical rotation 
measurements indicate 85% conversion to a racemic mixture. The resin was 
filtered from the toluene and the toluene returned to the enzyme 
resolution system. A second enzymatic resolution took place, resulting in 
a second crop of L-phenylalanine. 
Additional features of the preferred and most preferred embodiments of the 
present invention are found in the claims.