GB1593963A - Optical resolution of n-acyl-threonines - Google Patents

Description

(54) OPTICAL RESOLUTION OF N-ACYL-THREONINES (71) We, THE PROCTER & GAMBLE COMPANY, a Corporation organised and existing under the laws of State of Ohio, United States of America, of 301 East Sixth Street, Cincinnati, Ohio 45202, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statements: This invention relates to a process for providing an optically pure N - acyl L - threonine and L-threonine.

Threonine is one of the essential amino acids for human and animal nutrition.

Threonine has the structure of a - amino - 5 - hydroxybutyric acid.

This compound contains two asymmetric carbon atoms; therefore there are four optical isomers, L-threonine, D-threonine, L-allothreonine and D-allothreonine.

L-threonine is a naturally occurring a-amino acid and is nutritionally available. N-acylated L-threonines are also nutritionally available. D-threonine and D,L-allothreonine are not naturally occurring compounds and are not nutritionally available to animals. Therefore, when synthesizing a - amino - A hydroxy - butyric acid for nutritional purposes, the optical isomers must be separated to produce the optically pure, nutritionally-available L4hreonine or Nacylated L-threonines.

When a - amino - P - hydroxybutyric acid is synthesized, all four optical isomers are formed. Methods of converting D,L-allothreonine to D,L-threonine are known. See for example, U.S. 2,986,578 (1971), U.S. 2,846,439 (1958) and U.S.

2,446,192 (1948).

The interconversion of amides of D-threonine to L-threonine is described by Elliott, J. Chem. Soc., 62 (1950).

The resolution of N-acyl-D,L-methionine esters by hydrolysis using proteolytic enzymes is described by Stauffer, U.S. 3,963,573 (1976). The N-acyl-Dmethionine ester remains unchanged but the N-acyl-L-methionine ester is hydrolyzed to N-acyl-L-methionine. The enzymes disclosed by Stauffer are the same as those used in the present process.

It is an object of this invention to prepare optically pure L-threonine and/or N-acyl-L-threonines which can be used as a dietary supplement.

It is another object of this invention to provide a method of synthesizing Lthreonine and/or N-acyl-L-threonines by a multi-step reaction sequence beginning with an acetoacetate ester.

This invention provides an especially effective process for obtaining an Nacyl-threonines and L-threonine. The process for producing an optically pure N acyl-L-threonine comprises: (I) subjecting an N-acyl-D,L-threonine ester to the action of a proteolytic enzyme selected from the group of serine proteinases; and (2) separating the resulting N-acyl-L-threonine from the unreacted N-acyl-Dthreonine ester, as illustrated in Flow Chart 1.

Flow Chart I Serine Protease (1) N-acyl-D, L-threonine > N-acyl-L-threonine + ester mixture N-acyl-D-threonine ester Extraction (2) N-acyl-L-threonine + > N-acyl-L-threonine N-acyl-D-threonine ester In another aspect of this invention, the N-acyl group can be removed to provide L-threonine.

The N-acyl-D,L-threonine esters used herein can conveniently be prepared by esterifying a mixture of N-acyl-D,L-threonine with a C1-C10 alcohol, using standard techniques, or can be prepared de novo from an alkyl acetoacetate by a five-step process, comprising: Step (1) reacting an acetoacetate ester with sodium nitrite in the presence of an acid; Step (2) hydrogenating the a-oximino acetoacetate ester prepared in step (1) in the presence of an acyl anhydride; Step (3) reacting the mixture of N - acyl - p - hydroxy - butyrate ester formed in step (2) with a dehydrating agent under anhydrous conditions; Step (4) isomerizing the cis - D,L - 2 - alkyl - 5 - methyl - A2 - oxazoline 4 - carbonate formed in step (3) to trans - D,L - 2 - alkyl - 5 methyl - A - oxazoline - 4 - carboxylate; and Step (5) hydrolyzing the product of step (4) with dilute acid as illustrated in the Flow Chart II.

Flow Chart II

0 0 0 0 OR 0 II NaNQ II II II dehydratin Cc-0tl-c-o Th;- > & t5C -C -0-0'? Q) I aent N-OR II I 0 OH OH OR 0 + o=u e'=o ditttt Q II I u=O r id Cfrf3 OR NHC0$ u--2 L1' O=) I b~E r =0 (n tY'reo OH 0 oR 0 I II I 6 1 0 0 ci I 0 I OR 0 OH3-CR-OR o C-OH wt & ine O O 6 0=t w =o X 5 r > g) R=alkyl having from 1 to 9 carbon atoms or aryl R'=alkyl having from 1 to 9 carbon atoms or aryl R"=alkyl having from 1 to 4 carbon atoms This invention relates to a process for producing an optically pure N-acyl-Lthreonine comprising: (1) subjecting a mixture ofN-acyl-L-threoninc ester and Nacyl-D-threonine ester to the action of a proteolytic enzyme selected from the group of serine proteases; and (2) separating the resulting N-acyl-l-threoninc, from the unreacted N-acyl-D-threonine ester.

It has also been found that the N-acyl-D,L-threonine esters can bc prcpared by a synthetic route starting with acetoacetate esters.

As used herein, "optically pure N-acyl-L-threonine" means an N-acyl-Lthreonine substantially free of the D-isomer.

By "aryl" herein is meant a hydrocarbon group which comprises an aromatic substituent. This term encompasses such groups as phenyl, benzyl, a-phenylethyl, p-phenylethyl, naphthyl, and the like.

The term "serine protease" as used herein includes all recognized serine proteases, especially the preferred microbially-derived strains and mutants disclosed hereinabove.

By the term "comprising" herein is meant that various other compatible ingredients may be present in the compositions in such proportion as will not adversely affect the formation of the optically pure N-acyl-L-threonine or threonine. This term encompasses and includes the more restrictive terms "consisting of" and "consisting essentially of".

It has been found that certain readily available microbially derived serine proteases exhibit high esterase activity for N-acyl-L-threonine esters and very low esterase activity for N-acyl-D-threonine esters. Furthermore, it has been found that the high esterase activity exhibited for the L-isomer is not inhibited by the presence of the D-isomer. Subjecting a mixture of an N-acyl-D-threonine ester and an N-acyl-L-threonine ester to the action of a microbially derived serine protease provides a mixture of untreated N-acyl-D-threonine ester and "free", i.e. de-esterified, N-acyl-L-threonine. The N-acyl-L-threonine can be readily separated from the mixture by conventional means, for example, by adjusting the pH of an aqueous mixture and extracting with an organic solvent such as chloroform ethyl acetate, or butyl acetate.

A variety of specific N-acyl-D,L-threonine ester compounds can be employed in this invention. Preferably, the acyl group is derived from a fatty acid containing from I to 9 carbon atoms, or an aromatic acid. More particularly, the N-acyl group will preferably be formyl, acetyl, propionyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, benzoyl or phenylmethanoyl. The ester group can be derived from a variety of alcohols containing from 1 to 10 carbon atoms, and preferably from 1 to 7 carbon atoms. Especially suitable examples of the alcohol components of the ester groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert. butyl and benzyl,. The most preferred compounds are Nacetyl-threonine methyl, ethyl and benzyl esters. Racemic N-acetyl,D,L-methyl ester is the most preferred ester for use in the process of this invention.

The microbially derived serine proteases are preferred for use in the process of this invention. These proteases are relatively inexpensive and commercially available. An example of the preferred serine proteases for use in this invention are those derived from the bacterial organism Bacillus subtilis. These serine proteases are termed subtilisins.

A preferred subtilisin for the practice of the present invention is the Bacillus subtilis-derived Carlsberg strain. The Carlsberg strain most preferred for the practice of this invention is a known subtilisin strain. The amino acid sequence of this subtilisin is described in Smith, et al., "The complete amino acid sequence of two type subtilisin, BPN Carlsberg", J. of Biol. Chem., 24/, 5974 (196). This subtilisin strain is characterized by a tyrosin to tryptophan ratio of about 13:1.

An X-ray mutated Bacillus subtilis-derived subtilisin constitutes another preferred subtilisin for use in the present invention. This mutation can be effected in accordance with U.S. Patent No. 3,031,330 issued Apr. 24, 1962 to Minagawa et al. by irradiation of a Bacillus subtilis organism with X-rays. Subsequent treatment in a conventional manner can be employed to result in the preparation of an enzymatic composition. The patent describes a process whereby an enzymatic composition is produced by subjecting Bacillus subtilis to X-rays of an intensity corresponding substantially to 2450 roentgens for an interval of at least half hour, selecting from the colony thus subjected to X-rays a strain identified by cells having hairless, rough, jagged, spotted and dull white characteristics, separating said strain and placing the separated strain in a culture selected from the group, consisting of wheat bran and corn meal, maintaining the culture for a period of at least 40 hours while aerating the culture substantially continuously, and drying the culture.

Other examples of suitable serine proteinases for use herein include the following. Serine proteinases derived from Aspergillus oryzae. Methods for producing and separating these mold derived enzymes are known to those skilled in the art. See, for example, Subramamian et al., Biochemistry, Vol. 3, No. 12, pages 1861-74 (1964), and Misaki et al., Agr. Biol. Chem., Vol. 34, No. 9 pages 1383-92 (1970). Serine proteinase derived from Streptomyces griseus (ATCC 3463). Such serine proteinases are available commercially under the tradename "Pronase" from Kaken Chemical Co., Japan. Methods for producing and separating the proteinases are known. See, for example, Narahashi et al., The J.

Biochem., Vol. 62, No. 6, pages 633A1 (1967). Serine proteinase derived from Aspergillus sydowi. Methods for producing and separating this fungally derived serine proteinase are known. See, for example, Danno et al., Agr. Biol. Chem., Vol.

31, No. 10, pages 1151-58 (1967).

Other suitable examples of microbially derived serine proteinases are Aspergillus alkaline proteinase (E.C. 3.4.21.15), Alternaria endopeptiadase (E.C.

3.4.21.16), Arthrobacter serine proteinase (E.C. 3.4.21.17). These particular enzymes have been identified according to a systematic nomenclature involving an "E.C. number". See "Enzyme Nomenclature", Commission of Biochemical Nomenclature, Elsevier Publishing Company (1973), U.S. Library of Congress Card No. 83-78247.

The action of the proteolytic enzyme on the N-acyl-D,L-threonine ester is very suitably conducted in an aqueous medium at a pH of from about 5 to about 10, preferably from about 7 to about 8. A temperature of from about 10"C to about 60"C is acceptable. Preferably, the temperature is maintained in the range of from about 20 to about 4000.

Because of the high selective esterase activity of the particular proteolytic enzymes employed in this invention toward the N-acyl-L-threonine ester and Nacyl-D,L-threonine ester mixtures, only small amounts of the proteolytic enzyme are required in order to rapidly produce an N-acyl-L-threonine. For example, aqueous solutions containing from about 0.5% to about 5.0%, by weight, preferably from about 1.0% to about 2.0%, by weight, of enzymes are employed.

(Amounts of enzymes referred to herein refer to pure crystalline enzyme.) The amount of the N-acyl-D,L-threonine ester employed will generally be at least about 5% to about 15% by weight of the aqueous solution. Preferably larger amounts are employed, for example, amounts up to and exceeding the maximum solubility of the N-acyl-D,L-threonine ester in the aqueous medium. (Amounts exceeding maximum solubility can be employed since as the L-ester is consumed by the action of the enzyme, more will enter solution.) The rate of the action of the enzyme on the material will depend on the concentration of the enzyme and ester in solution. In this regard, N-acetyl-D,Lthreonine methyl or ethyl esters are quite suitable in that they exhibit good solubility in water.

The racemic mixture of the N-acyl-D,L-threonine esters used herein can be synthesized by the multistep reaction sequence in good yields, about 65% overall based on the acetoacetate ester as follows: In Step I of the process an acetoacetate ester of the formula CH3CO CH2CO2R wherein R is an alkyl or aryl group having from 1 to 9 carbon atoms, is reacted with sodium nitrite in the presence of an acid, preferably glacial acetic acid. The sodium nitrite in water is slowly added to the alkyl acetoacetate in solution at a temperature of from about -30"C to about 30"C. The mixture is stirred for several hours after addition at temperatures of from about 20"C to about 35"C. The a oximino acetoacetate ester which is formed in about 85% yield by the reaction is separated by standard procedures.

In the second step of the process the a-oximino acetoacetate ester prepared in Step I is hydrogenated in the presence of an acyl anhydride of the formula (R'CO)2O wherein R' is an alkyl group having from 1 to 9 carbon atoms or an aryl group.

Any standard method of hydrogenation can be used. In a preferred method, the a oximino acetoacetate ester formed in Step 1 is hydrogenated in a Parr Hydrogenator using platinum on powered charcoal in about 950% yield.

A mixture of the acyl anhydride and corresponding carboxylic acid can also be used. The preferred anhydride is acetic anhydride. A preferred composition is a mixture of acetic anhydride and glacial acetic acid.

In Step 3 of the reaction, the mixture of N-acyl-B-hydroxybutyrate esters formed by the hydrogenation of the a-oximino acetoacetate ester is reacted with a dehydrating agent, preferably thionyl chloride, under anhydrous conditions.

Other reagents useful for this reaction are sulfonic acid ion exchange resin, anhydrous sulfonic acid esters, sulfonamides, sulfonyl chloride and methanesulfonic acid.

This reaction is preferably carried out at temperatures of from about -100C to about 40"C. The cis - D,L - 2 - alkyl - 5 - methyl - A2 - oxazoline - 4 carboxylate and the trans isomer are formed in a ratio of I part cis to about 4 parts trans in about 85% yield. It is the trans isomer which, on acidic hydrolysis, yields the N-acyl-D,L-threonine ester.

N-acyl-D-threonine ester which is recovered from the enzymatic hydrolysis of the mixture of N-acyl-D,L-threonine esters can be recycled at this step of the process.

In step (4), the mixture of the cis and trans isomers are mixed with cold alcoholic sodium alkoxide, preferably sodium methoxide in methanol under an inert atmosphere, for about a half hour. Under these conditions, the cis isomer is isomerized to the trans isomer.

In step (5) of the reaction, the oxazoline isomerized in the proceeding step is hydrolyzed to the corresponding N-acyl-D,L-threonine ester by reaction with dilute acid. In a preferred embodiment, IN hydrochloric acid is used to hydrolyze the oxazoline to the corresponding, N-acyl-D,L-threonine ester.

The ester formed by this reaction sequence is then converted to an N-acyl-Lthreonine by the enzymatic hydrolysis process previously described.

While the foregoing procedure is an effective means for obtaining N-acyl D,L-threonine esters for use herein, the source of these esters is not critical to the practice of this invention. Any mixture of D,L-threonine can be acylated, esterified and used herein; the sequence of the acylation and esterification reactions is not critical.

In an optional mode, the optically pure, nutritionally-available N-acyl-Lthreonine prepared in the foregoing manner can be hydrolyzed with dilute acid or an acylase enzyme to yield o tically pure L-threonine.

The following examples illustrate the various aspect of the present invention and are not intended to be limiting thereof.

EXAMPLE I N-acetyl-D,L-threonine methyl ester is added to an aqueous solution buffered at pH 7.5 with phosphate buffer. Subtilisin Carlsberg is added, to 5 grams of the threonine ester about 0.35 grams subtilisin Carlsberg is used. After 2 1/2 hours at 250C, the reaction is about 78% complete.

The reaction mixture is extracted with 20 equal volumes of chloroform to remove the unreacted N-acetyl-D-threonine methyl ester and any unreacted Lmethyl ester. The solution is then acidified to a pH 1.5 and the N-acetyl-Lthreonine is removed by extraction with 9 equal volumes of ethyl acetate. The activity of the N-acetyl-threonine methyl ester using subtilisin Carlsberg was 48 ,um/min/mg of enzyme.

When the N-acetyl-D,L-threonine benzyl ester is used, the activity is 80 m/min/mg of enzyme, when the ethyl ester is used, the activity is 40 1im/min/mg of enzyme, and when the isopropyl ester is used, the activity is 12 m/min/mg.

When N-benzoyl-D,L-threonine methyl ester is used in the above reaction, the activity is 32 m/min/mg of enzyme.

When subtilisin BPN is used in place of the subtilisin Carlsberg, similar results are secured.

EXAMPLE 11 A solution of 1.1 moles (76 grams) of sodium nitrite in 170 ml. of water was added over a period of from 45 minutes to one hour to a cooled mixture of I mole (116 grams) of methyl acetoacetate and 2-1/2 moles (151 grams) of acetic acid which was stirred under an inert atmosphere. The temperature of the reaction mixture was not permitted to exceed 25"C. After the addition of the sodium nitrite, the reaction mixture was stirred at room temperature for about three hours.

After this time the mixture was diluted with about 200 ml. of water and repeatedly extracted with diethyl ether. The combined ether extracts were separated from the water solution and washed with sodium carbonate (1020% solution) to remove any acid from the ether. The extracts were then dried over magnesium sulfate and ether removed by evaporation. A yellow oil remained (121 grams) representing an 84% yield of methyl a-oximino acetoacetate. The product was characterized by nuclear magnetic resonance spectra in deuterated chloroform (single peaks at 2.4 and 3.85 ppm) and by infrared spectrum having resonance frequencies at 1745, 1690 and 1630 cm-'.

A mixture of the methyl a-oximino acetoacetate (3.2 grams) prepared above, acetic anhydride (5 ml), acetic acid (5 ml) and 5% Platinum on powdered charcoal (1.0 g) was placed in a hydrogenation jar. Hydrogenation was carried out at 50 psi on a Parr Hydrogenator for about 20 hours. The catalyst was removed by filtration and the solvent evaporated under vacuum to yield a yellow oil (3.63 grams) representing 94% yield. The product was characterized by nuclear magnetic resonance spectrum and infrared spectrum.

The methyl N - acetyl - a - amino - p - hydroxybutyrate (2 grams) prepared above was dissolved in 25 ml. of acetonitrile. Thionyl chloride (2.09 grams) was slowly added to the cooled ester with stirring under an argon atmosphere. The reaction mixture was then stirred at about 5"C for from six to eight hours and then for about 12 hours at room temperature.

After this time the reaction mixture was slowly added to 75 ml. of a 10% sodium carbonate solution; the pH of the reaction mixture is maintained above 7.

The organic layer is separated and the aqueous phase extracted with chloroform.

The combined organic extracts were dried over magnesium sulfate and the solvent removed by rotary evaporation to yield 1.53 grams, 86%, of methyl D,L-2,5 dimethyl-A -oxazoline-4-carboxylate. The product was identified by infrared and NMR spectra.

The methyl D,L - 2,5 - dimethyl - A2 - oxazoline - 4- carboxylate is dissolved in dry methanol. Cold methanolic sodium methoxide is added to the methanol solution of the cis and trans isomer mixture at 50C under an inert atmosphere with stirring. The reaction mixture continues to stir after the addition for about 30 minutes. A phosphate buffer of pH 7.3 is then added to the reaction mixture to stop the reaction.

Extraction of the aqueous mixture with chloroform followed by evaporation yields a tan oil which was characterized by both nuclear magnetic resonance and infrared spectra as methyl trans - D,L - 2,5 - dimethyl - A2 - oxazoline - 4 carboxylate.

The methyl trans - D,L - 2,5 - dimethyl - A2 - oxazoline - 4 - carboxylate formed in the previous step can be hydrolyzed in an aqueous solution with one normal hydrochloric acid to yield the N-acetyl-D,L-threonine methyl ester.

The oxazoline does not have to be isolated. A mineral acid solution, for example, phosphoric acid or hydrochloric acid, can be added to the methanol solution to open the oxazoline to the N-acetyl ester within about 1/2 hour. The acid is then neutralized and the methanol distilled off.

The N-acetyl-D,L-threonine methyl ester is hydrolyzed in the manner of Exampler I.

EXAMPLE III N-acetyl-L-threonine prepared in the manner of Example I is hydrolyzed using one normal hydrochloric acid for 2 hours at 1000C. Optically pure Lthreonine is produced.

Claims (10)

WHAT WE CLAIM IS:

1. A process for producing optically pure N-acyl-L-threonine comprising: (1) subjecting a mixture of an N-acyl-L-threonine ester and an N-acyl-Dthreonine ester to the action of a proteolytic enzyme selected from the group of serine proteases; and (2) separating the resulting N-acyl-L-threonine from the unreacted N-acyl-Dthreonine ester.

2. A process according to Claim 1 wherein the acyl group comprises a C1-C9 alkyl group or an aryl group.

3. A process according to Claim 1 or Claim 2 wherein the alcohol portion of the ester group contains from I to 10 carbon atoms.

4. A process according to Claim 2 and Claim 3 wherein thc acyl group is acetyl or benzoyl and the ester is the methyl, ethyl, or benzyl ester.

5. A process according to any one of Claims 1--4, wherein the serine protease is selected from the group consisting of subtilisin Carlsberg and suhtilisin BPN.

6. A process according to Claim I for preparing pure N-acelyl-L-threonine comprising: (I) subjecting a mixture of an N-acetyl-L-threonine methyl ester and an Nacetyl-D-threonine methyl ester to the action of a proteolytic enzyme selected from the group of microbially derived serine proteases, at a pH range of from 5 to 10; and (2) separating the resulting N-acetyl-L-threon ine.

7. A process for preparing optically pure L-threonine comprising carrying out a process according to any one of Claims 1-5 and, as an additional step, hydrolysing the N-acyl-L-threonine to L-threonine. -

8. A process according to any one of Claims I 5 wherein the mixture of Nacyl-L-threonine ester and N-acyl-D-threonine ester is prepared by Step (I) reacting an acetoacetate ester with sodium nitrite in the presence of an acid; Step (2) hydrogenating the a-oximino acetoacetate ester prepared in step (1) in the presence of an acyl anhydride; Step (3) reacting the mixture of N - acyl - p - hydroxy - butyrate esters formed in step (2) with a dehydrating agent under anhydroux conditions; Step (4) isomerizing the cis - D,L - 2 - alkyl - 5 - methyl - A2 - oxazoline 4 - carboxylate formed in step (3) to trans - D,L - 2 - alkyl - 5 methyl A2 - oxazoline - 4 - carboxylate; and Step (5) hydrolyzing the product of step (4) with dilute acid.

9. A process according to Claim 8 wherein the unreacted N-acyl-D-threonine ester is recycled with the N-acyl-p-hydroxybutyrate esters in step (3).

10. A process for preparing optically pure L-threonine when carried out substantially as described in any one of the Examples.