GB2092161A

GB2092161A - Preparation of Amino Protected-L-aspartyl-L-phenylalanine Alkyl Ester

Info

Publication number: GB2092161A
Application number: GB8202814A
Authority: GB
Original assignee: GD Searle LLC
Current assignee: GD Searle LLC
Priority date: 1981-02-02
Filing date: 1982-02-01
Publication date: 1982-08-11
Also published as: JPS57146595A; IE820220L; FR2499098A1; DE3203292C2; IE52242B1; DE3203292A1; GB2092161B

Abstract

A process for the production of amino-protected-L-aspartyl-L-phenylalanine alkyl ester addition compound of the formula <IMAGE> in which R1 represents an amino protecting group and R2 is an alkyl group containing from 1 to 6 carbon atoms, which comprises reacting a carboxyl- protected-L-phenylalanine of the formula phi -CH2- <IMAGE> in which R2 has the above-stated meaning in the presence of a protease producing microorganism or an enzyme obtained from a microorganism having protease activity under conditions which favour microbial enzyme coupling in the presence of an enzyme at a pH which maintains enzyme activity, to form an amino-protected-L-aspartyl-L-phenylalanine alkyl ester addition compound of the formula <IMAGE> and continuously removing said alkyl ester addition product. Compounds such as L-aspartyl-L-phenylanine methyl ester may be prepared from the amino-protected alkyl esters by removal of the protecting group, by catalytic hydrogenation.

Description

SPECIFICATION Preparation of Amino Protected-L-aspartyl-L-phenylalanine Alkyl Ester The present invention relates to a microbial enzymatic coupling (MEC) process for production of amino protected-L-aspartyl-L-phenylalanine alkyl ester. More particularly, the invention relates to process for producing amino protected-L-aspartyl-L-phenylalanine alkyl ester in which the product is continuously removed and reactants added using a protease producing micro-organism or a crude or purified enzyme preparation having protease activity obtained from a microorganism.

Conventional processes for producing peptides include the azide method, the mixed acid anhydride method, the carbodiamide method, the active ester method, the acid chloride method and the like. However, various industrial problems are encountered by the conventional processes, such as that racemization of the carboxyl component at the C terminal amino residue occurs. Other problems include side reactions, temperature control, selection of solvent, other properties of the amino protective groups and the carboxyl productive groups, the effects of functional groups in the side chains of amino acids, low yields and difficulty in removing desired end products. The fragment condensation method can be advantageously applied to compounds containing glycine (the only amino acid which can not be racemized) at the carboxyl terminal group.However, for compounds containing any other amino acid at the carboxyl terminal group, racemization cannot be prevented. In actual fact, in any peptide synthesis the racemization problem is serious. When racemization occurs, the purity of the product is decreased and it is necessary to separate the unwanted isomer from the product. This separation is very detrimental for any industrial operation.

Among the conventional methods for forming peptide bonds, the azide method is the only method in which racemization is not much of a problem, and it is for this reason that it is a desirable method. However, since the azide method involves complicated operational procedures and because an urea derivative is produced in a side reaction thereby decreasing the yield of the product, the azide method is also unsatisfactory. In addition to the various organic chemical processes for preparing peptides, a particular peptise synthesis using the enzyme papain or chymotrypsin has been disclosed (see, for example, J.S. Fruton "Advances in Protein Chemistry", 5, Academic Press Inc., New York, N.Y.

1 949). The reactions of this method are outlined in Chart A which follows the Examples.

The problem which is common to reactions I to Ill in Chart A is that it is necessary to remove the phenylamino group from the peptide (III) by severe conditions because the phenylamino group which is bonded to the C-terminal group of the amine component (II) cannot be easily separated from the peptide and thus cleavage of the peptide chain is possible and disadvantageous. Because of this deficiency, this mode of peptide synthesis cannot be practically used for peptide synthesis. On the other hand, reaction 4 is accompanied by transamination and transpeptidation side reactions and thus is not practically suitable. (See, for example, R.B. Johnston et al: J. Biol. Chem., 185629 (1950) and J.S. Fruton et al; J. Biol.Chem., 204,891(1953).). In reaction 4 the primary amino group of the acid amide bonded to the terminal group of the amine component, promotes the papain catalyzed amidase reaction. Accordingly, these processes provide only a theoretical interest in showing that papain and chymotrypsin act as catalysts for the synthesis of peptide bonds in which the phenylamino or primary amino group is used as the protective group for the terminal carboxyl group of the amine component.

These processes give no indication of the possibility of synthesizing a desired oligopeptides or polypeptides.

Prior Art It has been known that peptide derivatives have various physiological activities, and these peptide derivatives can be produced by various methods. The peptides having acidic amino acid residue, such as a-aspartyl-l-phenylalanine lower alkyl ester, which are useful as sweeting compounds, can be obtained from a precursor having a carbobenzoxy group as an end terminal protective group by removing the amino protective group. Accordingly, in U.S. Patents 4,116,768 and 4,1 19,493, processes for synthesizing oligopeptides or polypeptides by a simple method are described.

The process is a batch method wherein an amino acid or peptide having an end terminal protective group or salt thereof of the formula X--AA-OH is reacted with an amino acid or peptide having a C-terminal protective group or salt thereof of the formula H-B-Y wherein A and B are the same or different and represent an amino acid residue or a peptide residue, X represents an amino acid protective group and Y represents a carboxyl protective group. These are reacted in the presence of a metalloproteinase in an aqueous solution having a pH which maintains the enzyme activity of the said metallo-proteinase. While an effective process, the reaction is limited as the reaction proceeds and filteration of the final product is difficult. Another process is described in U.S.

Patent 4,165,311. The patent discloses novel addition compounds of dipeptides composed of Nsubstituted monoaminodicarboxylic acid ester residues with amino carboxylic acid esters and processes for producing the addition compound utilizing an enzymatic reaction and for decomposing the addition product.

Relevant processes for the recovery of proteases are outlined in U.S. Patents 4,21 2,945 and 4,212,946 where the protease is recovered by isolating the protease from an addition compound after a peptide synthesis of peptide-bonded amino acid derivatives in the presence of the protease.

The present invention particularly provides a process for the production of amino protected-Laspartyl-L-phenylalanine alkyl ester according to Chart B which follows the Example, wherein the Laspartic acid has an amino protecting group R1, the enzyme source is a protease producing microorganism or an enzyme preparation obtained from a microorganism having protease activity, R2 is alkyl of 1 to 6 carbon atoms, inclusive, which comprises: reacting carboxyl protected-L-phenylalanine under conditions which favour the microbial enzymatic coupling in the presence of an enzyme at a pH which maintains enzyme activity, removing the product continuously as it is formed; and adding additional reactants as the product is removed.

The amino group of L-aspartic acid may be protected by commonly used protecting groups which include but are not limited to the following: aliphatic oxycarbonyl groups such as carbobenzoxy, tbutyl-oxycarbonyl ((CH3C-O-CO-) and t-amyloxycarbonyl ((CH3)2C(C2H5)-0-CO-); nuclear substituted carbobenzoxy groups such as p-methoxy-carbobenzoxy (p-CH30-C H 2-O-CO-), 3,5- dimethoxycarbobenzoxy (3,5-(CHO)2-CH2-O-CO-). and 2,4,6-trimethoxycarbobenzoxy (2A,6-(CH3O)3-CH2-O-CO-), benzoyl group (-CO-); p-toluenesulfonyl group (p CH3-SO2-); the urethane type, and aromatic sulfinyl groups are as QI--nitrosulfinyl group. A preferred amino protecting group is carbobenzoxy.

The enzyme used in the present, invention are proteases and are preferably metailoproteases which have a metal ion in the active centre. Suitable metalloproteases are enzymes originating from microorganisms, such as those produced by a number of bacilli, streptomyces, pseudomonads, marine and various other bacteria and certain fungi, particularly aspergilli. Where applicable the protease producing microorganisms may be used directly. Several metalloproteases of microbial origin such as Thermolysin or Thermoase (Bacillus Thermoproteolyticus, Daiwa Kasai), Microprotease (B. cereus, Worthington), Dispase (B. polymyxa, Sigma), P ronase (Streptomyces griseus, Calbiochem), fungal crude protease (Aspergillus oryzae, Sigma) are available from commercial sources.A preferred source of enzyme of the present invention involves the use of the hyperprotease producing strain of B. cereus NRRL B-12315. Bacillus cereus NRRL B-12315 is available from the A.R.S. Culture Collection Investigations Fermentation Laboratory, 1815 N. University Avenue, Peoria. Illinois, 61604. When the organism is used directly, the expenses concerned which isolating and recovering the enzyme can be eliminated. Additionally, an enzyme preparation from a protease producing organism may be used. This may consist of a number of different types of preparations.Examples might include cell-free culture broths prepared by removing the cells from the culture medium using centrifuge filtration or other techniques, crude enzyme preparation where additional steps such as flocculation (which can remove colloids) or bacteriological filtration are used and partially-purified enzyme preparations which could include precipitation of the enzyme with organic solvents or salts. The amount of protease used in the process of the present invention is not critical although a certain minimal level of enzyme activity is desired to maintain high rates of product formation.

The utility of this invention arises from several areas. First, in previous processes which were batch methods, the reaction mixture solidified with time, increasing the difficulty in recovery of the product and inhibiting the rate at which the reaction proceeds. The present invention utilizes a continuous process which prevents the solidification and produces a crystalline product which appears suprisingly and unexpectedly more efficient in terms of material handling, reaction time and enzyme utilization. Second, lower concentrations of enzymes may be used which further add to the simplicity of handling the reaction as well as decreasing costs. Thirdly, use of the microbial enzyme directly substantially reduces time, effort, and cost compared with the isolated enzyme. Lastly, the MEC process of the instant invention has the advantage of specificity.Two carboxyl groups are available and therefore two isomers theoretically are possibie (alpha and beta). The alpha isomer is readily formed alleviating the necessity of a separation step. The product derived hereby may be used in the making of L-aspartyl-L-phenylalanine methyl ester, an artificial sweetener about 200 times as sweet as table sugar. This can be accomplished by removing the amino protecting group by a known method such as catalytic hydrogenation.

The methods and materials for preparing and assaying the B. cereus enzyme broths used in the examples are as follows: A. Inoculum Preparation A frozen stock culture of B. cereus NRRL B-1 231 5 is thawed and 2 ml used to inoculate 200 ml of Trypticase Soy Broth (BBL, Cockeysville, Md.) contained in a 11 baffled flask. The culture is incubated at 30+1 OC for 16-24 hrs. at 200 rpm on a rotary shaker with a 2 inch circular orbit.

B. Enzyme Broth Preparation A medium consisting of soluble starch, 10.0 g; soy flour, 1 0.0 g; yeast extract, 2.0 g; dibasic potassium phosphate, 7.5 g; Tween 80, 1.0 g; calcium chloride, 1.0 g; magnesium sulfate monohydrate, 0.001 g; ferrous sulfate, 0.001 g; and zinc sulfate, 0.001 g in 11 of distilled water is adjusted to pH 7.3 with 45% (w/v) potassium hydroxide before autoclaving at 1 5 Ibs for 1 5 min. 1 00 ml portions of this medium in 500 ml baffled flasks are inoculated with 5 ml of the inoculum culture.

After 24 hours incubation, the cultures are centrifuged at 12,000xg for 10 min in a refrigerated centrifuge. The supernatant is decanted and stored at 40C until needed. if desired, the cell-free enzyme is concentrated using CX filters (Millipore Corp., Bedford, Mass.) C. Enzyme Broth Assay The cell-free culture broth is assayed for enzyme activity in a continuous spectrophotometric assay essentially as described by Feder [Biochem. Biophys, Res. Commun., 32, 326 (1 968)] except that the concentration of the substrate, furylacryoyl-L-Glycyl-L-leucine amide (FAGLA), is decreased to 5x10-4 M. Enzyme activity is expressed as FAGLA units (A345/min/ml).

The materials and procedures used in the thin layer chromatographic analysis of aAPM are as follows: A. Plate E. Merch Silica Gel with Fluorescent indicator activated at 254 nm (EM Laboratories, Inc., Elmsford, NY).

B. Solvent System Chloroform 60% (v/v) Methanol 30% (v/v) Water 4% (v/v) Formic Acid 2% (v/v) C. Detection Procedures After spotting and development in the above solvent system, the plate is air dried for 30 min and examined as follows: 1. by exposure to shortwave ultraviolet light, and 2. by exposure to excess t-butyl hypochlorite followed by evaporation of excess reagent, spraying with 0.5% (w/v) potassium iodide and 0.5% (w/v) starch in water and visualization under white light.

The material and procedures used in the high performance liquid chromatographic (HPLC) analyses of the reactants and products of the MEC process are as follows: A. Column Partisil PXS 10/25 ODS (250-- mmx4.6 i.d.) supplied by Whatman, Inc. (Clifton, N.J.) B. Mobile Phase 33% tetrahydrofuran (distilled in glass): 67% distilled water containing 0.005M pentane sulfonic acid.

C. Instrument Water Associated (Milford, Mass.) Liquid Chromatograph equipped with a model M-600A pump, Model 440 detector (UV, 254 nm at 0.1 AUFS) and a WISP Model 71 0A Auto injector operated at a flow rate of 2 ml/min.

D. Procedure Weighed portions of solid materials and measured volumes of liquid materials are diluted appropriately in the mobile phase and chromatographed. Peaks are identified and quantitiated by reference to known standards.

In the examples that follow, all yields are given as % of theory based on the starting amount of Z Asp in the reaction mixture. The known infrared and NMR Spectra of a APM are as follows: Infrared Spectrum: (KBr Disc Method) 3350 cam~' (N-H stretching vibration); 3070, 3036 and 2960 cm-l (NH3 stretching vibration, broad and overlapped with C-H stretching vibrations),1742 cam~' (C=O ester, stretching vibration), 1 672 cam~' (C=O, amide); 1 553(NH+3, symmetrical deformation vibration, C-N-H, amide); 1 500 and 1450 cm-l (aromatic ring stretching vibration); 1381 cm-' (C-O2 symmetrical stretching vibration), 1 235 cm-' (C--O ester, asymmetrical stretching vibration; NH+ rock),1037 cm-' (C-O ester, symmetrical stretching vibration); 923 cm-' (C-C-N, amino acid, stretching vibration); 751 cm-' (5 adjacent H wag vibration, mono-substituted phenyl), and 702 cm-' (ring bending vibration, monosubstituted phenyl).

NMR spectrum: S (1) 3.07 ppm (apparent triplet, 4H,

and CH2-); (2) 3.70 ppm (singlet, 3H,O-CH3); (3) 4.52 ppm (triplet, 1 H, J=5.9Hz, CH); (4) 4.83 ppm (triplet, 1H,J=6.1Hz, CH); (5) 7.25 ppm (singlet, 5H, aromatic H).

The preparation of amino protected-L-asparatic-L-phenlalanine alkyl esters is exemplified in the following representative examples.

Description of the Preferred Embodiments Example 1 Carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Z-APM): (Chart B wherein R1 is carbobenzoxy and R2 is methyl).

38 g (284 mmol) of carbobenzoxy-L-aspartic acid (z-Asp), 77 g (714 mmol) of L-phenylalanine methyl ester hydrochloride (PM HCI), and 1.68 g of calcium acetate are dissolved in distilled water, the pH is adjusted to 6.8 with 21 g of sodium carbonate, and the volume made up to 250 ml with water. The resultant solution is added to an equal volume of cell-free B. cereus culture broth with a FAGLA activity of 4.0 units. This mixture is then placed in a suitable glass vessel and stirred at 400C for 6 hrs. The product is collected continuously by filtration as it forms and the filtrate is pumped back into the reaction vessel. The reaction is discontinued and the remaining solid residue is combined with the collected precipitate and washed with water.After drying and recrystallizing from a solvent mixture of methanol and water (1 :2), a total of 64.52 g of an addition compound of Z-APM and PM (1:1) is obtained for a 74.7% yield.

The Z-APM.PM salt is ground to a fine powder in a mortar, suspended in 350 ml water and 127 ml of 1 N hydrochloric acid and shaken vigorously for 2 hrs at room temperature. The resulting suspension is filtered and the precipitate is washed with several portions of water. After drying there is obtained 44.8 g of Z-APM (overall yield of 73.6%) and 22.63 g of PM (29% recovery).

The entire 44.8 g portion of Z-APM is added to 746 ml of water and 2,240 ml of methanol and the resultant mixture is hydrogenated at 50 psi pressure in the presence of 4.48 g of 5% palladium on charcoal catalyst. After 2 hrs, the catalyst is removed by filtration and filtrate is concentrated to a volume of 500 ml under reduced pressure at a temperature not exceeding 500 C. The a-aspartyl-Lphenylalanine (APM) is crystallized by cooling the solution to OOC for 2 hrs. Filtration afforded 25.14 g of product, shown to be aAPM by TLC analysis. A second crop, 3.60 g, is obtained by further concentration of the iiquors for a 96.5% total yield of aAPM of which 82% was shown to be of high quality by TLC analysis.

The physical properties and results of elementary analysis of the a-APM product are as follows: Melting point: 2470 to 2840C Elementary analysis (C4H18N205TH2O) C H N Calculated: (%) 55.44 6.31 9.24 Found: (%) 55.56 6.1 5 9.24 Infrared and NMR spectra of the product shown the same characteristics as described above.

Example 2 The process of example 1 is repeated with a B cereus cell-free culture broth containing 3 un/ml of FAGLA activity except that the resultant enzyme/reactant mixture is divided into two equal portions.

After 6 hrs, a total of 34.600 g of the addition compound of Z-APM and PM (1 :1) is obtained from the reaction in which the product is removed as it is formed (continuous reaction). The Z-APM PM salt is suspended in 125 ml water and 51 ml of 1 N hydrochloric acid and stirred vigorously for 1.5 hrs. to give 1 6.82 g of Z-APM (over yield of 55.2% of theory). The resultant fine precipitate is 100% pure based on HPLC assay.

The second portion of enzyme/reactant mixture is treated identically except that product is not removed during the 6 hr reaction period (batch reaction). A total of 1 7.34 g of the addition compound is obtained and suspended in 85 ml of water and 35 ml of 1 N hydrochloric acid. After stirring vigorously for 1.5 hrs, 11.65 g of Z-APM (100% pure by HPLC) is obtained (overall yield of 38.2%).

Example 3 The process of example 2 is repeated except that the reaction is run at room temperature. The continuous reaction gives a total of 30.57 g of the addition compound which is suspended in 150 ml of water and 60.4 ml of 1 N hydrochloric acid and stirred vigorously for 1 hr. There is obtained 20.08 g of Z-APM (90.6% pure by HPLC) for an overall yield of 59.7% of theory.

The corresponding batch reaction gives 21.94 g of the addition compound which is suspended in 110 ml of water and 43.3 ml of 1 N hydrochloric acid. After stirring vigorously for 1 hr, 14.3 g of Z-APM (98.7% pure by HPLC) is obtained for an overall yield of 46.2% of theory.

Example 4 The process of example 1 is repeated with a B. cereus cell-free culture broth containing 4.7 units of FAGLA activity except that the reaction is run at room temperature and a second 500 ml of enzyme/reactant mixture is also prepared. The additional mixture is added to the reactant vessel containing the original 500 ml of enzyme/reactant mixture in 1 5 ml portions at about 10 min intervals starting 3 hrs after the reaction is begun. After 11 and one-half hours, the residue remaining in the reaction mixture is combined with the collected precipitate, washed with water and dried. A total of 115.4 g of the addition compound is obtained which consists of 75.6% (87.3 g) of Z-APM and 30.9% (35.7 g) of PM as determined by HPLC for an overall yield of 71.56% of Z-APM.

Example 5 The process of example 4 is repeated with another B. cereus cell-free culture broth containing 4.7 units of FAGLA activity except that an additional 750 ml of enzyme/reactant mixture is prepared. The resultant mixture is added in 25-50 ml increments starting 4 hrs after the reaction is begun. The pH is monitored periodically during the reaction and additional 5-1 0 ml portions reaction mixture of nonpH adjusted reactant mixture are added as required to maintain the pH below 7, (total of 125 ml of non-pH adjusted reactant mixture are added). After 17 hrs, the reaction is terminated by combining the remaining residue in the reaction mixture with collected precipitate which is then washed and dried.A total of 203.05 g of the addition compound is obtained which consists of 66.2% (134.42 g) of Z-APM and 28.4% (57.66 g) of PM as determined by HPLC for an overall Z-APM yield of 73.4%.

Chart A

Papain (1) Bz-Leu-OH + H-Leu-NH 2ăin Bz-Leu-Leu-NH(d II Ill Papain (2) Bz-Leu-OH + H-Gly-NH(ii > Bz-Leu-Gly-NH 11 Ill Chymotrypsin (3) Bz-Tyr-OH + H-Gly-NH > Bz-Tyr-Gly-NH 11 Ill Papain (4) Z-Phe-Gly-OH + H-Tyr-NH2 ' Z-phe-Gly-Tyr-NH2 11 III Chart B

(amino protected-L- (L-phenylalanine aspartic acid,) alkyl ester hydro-chloride, PA.HCl)

(amino protected-L-aspartyl L-phenylalanine alkyl ester addition compound, RrAPA PA)

Claims

1. A process for the production of amino protected-L-aspartyl-L-phenylalanine alkyl ester according to Chart B herein, in which the L-aspartic acid has an amino protecting group, R, the enzyme source is a protease producing microorganism or an enzyme preparation obtained from a microorganism having protease activity, R2 is alkyl of 1 to 6 carbon atoms, inclusive, which comprises: reacting carboxyl protected-L-phenylalanine under conditions which favour the microbial enzymatic coupling in the presence of an enzyme at a pH which maintains enzyme activity, removing the product continuously as it is formed, and adding additional reactants as the product is removed.

2. A process for the production of amino-protected-L-aspartyl-phenyalanine alkyl ester addition compound of the formula

in which R1 represents an amino protecting group and R2 is an alkyl group containing from 1 to 6 carbon atoms, which comprises reacting a carboxyl-protected-L-phenylalanine of the formula

in which R2 has the above-stated meaning in the presence of a protease producing microorganism or an enzyme obtained from a microorganism having protease activity under conditions which favour microbial enzyme coupling in the presence of an enzyme at a pH which maintains enzyme activity to form an amino-protected-L-aspartyl-L-phenylalanine alkyl ester addition compound of the formula

and continuously removing said alkyl ester addition product.

3. A process as claimed in claim 1 or claim 2 in which the enzyme source is a protease producing microorganism.

4. A process as claimed in claim 2 in which the protease producing microorganism is Bacillus, Streptomyces or Aspergillus.

5. A process as claimed in claim 4 in which the protease producing microorganism is Bacillus cereus NRRL B-12315.

6. A process as claimed in claim 1 or claim 2 in which the enzyme is a crude or partially purified enzyme preparation obtained from a microorganism having protease activity.

7. A process for the production of an amino-protected-L-aspartyl-L-phenylalanine alkyl ester substantially as herein described with reference to any one of the Examples.

8. An amino-protected-L-aspartyl-L-phenylalanine alkyl ester when prepared by a process as claimed in any of claims 1 to 7.

9. Compounds prepared by the removal of the amino-protecting group from compounds as claimed in claim 8.

1 0. L-aspartyl-L-phenylalanine methyl ester prepared by the removal of an amino protecting a group from amino protected precursor as claimed in claim 8.