FR2499098A1 - PROCESS FOR THE PRODUCTION OF (L-ASPARTYL AMINO-PROTECTED) ALKYL ESTER-L-PHENYLALANINE - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR THE PRODUCTION OF AN ALKYL ESTER OF (L-ASPARTYL AMINO-PROTECTED) -L-PHENYLALANINE BY ENZYMATIC REACTION OF L-ASPARTIC AMINO-PROTECTED ACID WITH ESTER HYDROCHLORIDE. L-PHENYLALANINE ALKYL AND ACID HYDROLYSIS OF THE PHENYLALANINE SALT OF DIPEPTIDE. THE PROCESS ACCORDING TO THE INVENTION INCLUDES THE FOLLOWING STEPS: CARBOXY-PROTECTED L-PHENYLALANINE IS REACTED WITH AMINO-PROTECTED L-ASPARTIC ACID UNDER CONDITIONS WHICH PROMOTE MICROBIAL ENZYMATIC COUPLING IN THE PRESENCE OF A PHENYLALANINE WITH AN ENZYME KEEP THE ENZYMATIC ACTIVITY, THE PRODUCT IS CONTINUOUSLY SEPARATED AS IT IS FORMED; AND THE NEW REAGENTS ARE ADDED AS THE PRODUCT IS SEPARATED.

Description

The present invention relates to an enzymatic coupling method

  microbial 'microbial enzymatic coupling process' (MEC)

  for the production of (L-aspartyl amino-protected) alkyl esters

  L-phenylalanine. More particularly, the invention relates to a process for the production of (L-aspartylamino-protected) -L-phenylalanine esters in which the product is separated and the reactants are added continuously using a protease-producing microorganism. or a crude or purified enzyme preparation having a

  protease activity obtained from a microorganism.-

  Conventional methods for the production of peptides include the azide method, the mixed anhydride method, the carbodiimide method, the reactive ester method, the acid chloride procedure, and the like. However, there are various problems on an industrial scale in conventional processes, such as

  racemisation of the carboxylic component on the C-terminal amino residue.

  Other problems include side reactions, temperature control, solvent choice, other properties of amino-protecting and carboxy-protecting groups, effects of functional groups on amino acid side chains, weak yields and difficulties in separating the desired end product. The fragment condensation method can be advantageously applied to compounds containing glycine (the only amino acid that can not be racemized) on the carboxyl end group. However, for compounds containing any other amino acid on the carboxyl end group, racemization can not be avoided. Currently, the problem of racemization is serious in any peptide synthesis. When racemization occurs, the purity of the product is diminished and it

  It is necessary to separate the undesired isomer from the desired product.

  This separation is a big disadvantage for any

industrial operation.

  Of the conventional methods for forming peptide bonds, the azide method is the only method in which racemization is not a major problem, and it is for this reason that this method is desirable. However, since the azide process involves complicated procedures and because there is production of a urea derivative in a side reaction, thereby reducing product yield, the azide process is also poorly controlled. satisfactory. In addition to the various methods of organic chemistry for preparing peptides, a particular peptide synthesis using an enzyme, papain or chymotrypsin has been described (see, for example, JS Fruton "Advances in Protein Chemistry", Academic Press Inc., New York, NY 1949). The reactions of this

  The process is described in Scheme A below.

  The problem which is common to reactions I to III in Scheme A is that it is necessary to separate the phenylamino group from peptide (III) under stringent conditions because the phenylamino group which is attached to the C-terminal group of the amino component (II) can not be easily separated from the peptide and the cleavage of the peptide chain is therefore possible and disadvantageous. Because of this defect, this mode of peptide synthesis can not be used in practice for peptide synthesis. On the other hand, reaction (4) is accompanied by side reactions of transamination and transpeptidation and is therefore not suitable in practice (see, for example, RB Johnston et al., J. Biol Chem. page 629 (1950) and JS Fruton et al., J. Biol Chem., 204, pg 891 (1953) In reaction (4), the primary amino group of the amide attached to the terminal group of the amino compound promotes Therefore, these methods are of only 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 a protective group for the terminal carboxyl group of the amino component.These methods give no indication of the possibility of oligopeptide synthesis or

desired polypeptides.

  It is known that peptide derivatives have various physiological activities and that these peptide derivatives can be produced

  by various methods. Peptides having amino acid residues

  acids such as the lower alkyl esters of α-aspartyl-L-phenyl-

  alanine, which are useful as sweetening compounds, may be

  obtained by removal of the amino-protecting group from a precursor

  having a carbobenzoxy group as a terminal protecting group. Accordingly, U.S. Patent Nos. 4,116,768 and 4,119,493 disclose methods for the synthesis of oligopeptides or polypeptides by a simple method. The process is a batch process in which an amino acid or peptide having an N-terminal protecting group or a salt thereof is reacted.

X-A-OH

  with an amino acid or peptide having a C-terminal protecting group or a salt thereof of the formula H-B-Y o A and B are identical or different and each represents a

  amino acid residue or a peptide residue, X represents a group

  protecting the amino group and Y represents a protective group

  carboxy group. They are reacted in the presence of a metallo-

  proteinase in an aqueous solution with a pH that maintains the activity

  enzymatic activity of said metalloproteinase. Although this method is effective, the reaction is limited as the

  reaction continues and filtration of the final product is difficult.

  Another method is described in U.S. Patent No. 4,165,311 which discloses novel dipeptide adducts composed of N-substituted monoaminodicarboxylic acid ester residues with aminocarboxylic acid esters and methods for producing the addition compound using an enzymatic reaction

  and to decompose the adduct.

  Corresponding methods for the recovery of proteases are described in US Pat.

  Nos. 4,212,945 and 4,212,946, where the protease is recovered by iso-

  the protease of an addition compound after a peptide synthesis of amino acid derivatives attached to the peptide in the presence of

protease.

  The present invention relates in particular to a

  process for the production of alkyl esters of (L-aspartylamino

  protected) -L-phenylalanine according to scheme B below in which the Laspartic acid bears an amino protecting group RI, the enzyme source is a protease producing microorganism or an enzyme preparation obtained from a microorganism having a protease activity, R is a C6-C6 alkyl group, which comprises the following steps: Carboxy-protected L-phenylalanine is reacted with L-aspartic acid amino-protected under conditions which promote microbial enzymatic coupling in the presence of an enzyme at a pH which retains enzymatic activity, the product is continuously separated as it is formed;

  and new reagents are added as the product is separated.

  The amino group can be protected from L-aspartic acid

  by means of commonly used protective groups which

  take but without limitation: aliphatic oxycarbonyl groups,

  such as carbobenzoxy, t-butoxycarbonyl (CH3) 3C-O-CO- and t-amyloxy-

  carbonyl (CH3) 2C (C2H5) -O-CO; substituted carbobenzoxy groups

  at the core, such as p-methoxycarbobenzoxy p-CH 3 O -CH 2 -O-CO-

  dimethoxycarbobenzoxy 3,5- (CH 3 O) 2 -O-CH 2 -O-CO-, and 2,4,6-trimethoxy-

  Carbobenzoxy 2,4,6- (CH 3 O) 3 -O-CH 2 -O-CO-, benzoyl O-CO-, p-toluene

  sulfonyl p-CH3-0-SO2-; the urethane type; and aromatic sulfinyl groups such as nitro-O-sulfinyl groups. A group

  The preferred amino-protector is carbobenzoxy.

  The enzymes used according to the invention are proteases and preferably metalloproteases which have in their active center a metal ion. Suitable metalloproteases are enzymes derived from microorganisms such as those produced by a number of strains of Bacillus, Streptomyces, Pseudomonas and various other bacteria and fungi, particularly Aspergillus. When used, protease-producing microorganisms can be used directly. We

  commercially available several metalloproteases of micro-

  such as Thermolysin or Thermoase (Bacillus thermo

  proteolyticus, from the Daiwa Kasai Company), Microprotease (B.

  cereus, from the Worthington Company), Dispase (B. polymyxa, from the Sigma Company), Pronase (Streptomyces griseus, from the Calbiochem Company) and fungal protease (Aspergillus oryzae, from the Sigma Company). A preferred source of enzyme for the present invention is the B. cereus NRRL B-12315 hyperprotease producing strain available from A.R.S. Culture Collection Investigations Fermentation Laboratory, 1815N University Avenue, Peoria, Ill., 61604. When the organism is used directly, the costs of isolation and recovery of the enzyme can be eliminated. In addition, an enzyme preparation can be used

  derived from a protease producing organism. This can

  to be in a number of different types of preparations. Examples of these are cell-free culture broths prepared by separating cells from culture medium by centrifugal filtration or other techniques.

  enzymatic enzymes in which additional steps are

  such as flocculation (which can eliminate colloids) or bacteriological filtration and partially purified enzymatic preparations which may involve precipitation of the enzyme by organic solvents or salts. The quantity of

  protease used in the process of the invention is not essential.

  although a certain minimal level of enzymatic activity is desired to maintain high rates of product formation.

  The utility of the invention comes from various fields.

  First, in the earlier processes which were batch processes, the reaction mixture solidified during the course of the reaction.

  time, increasing the difficulty of product recovery and reducing

  the speed at which the reaction takes place. The invention utilizes a continuous process which avoids solidification and provides a crystalline product which surprisingly and unexpectedly appears to be more effective in product handling, reaction time, and use of the enzyme. Secondly, lower enzyme concentrations can be used, which adds to the simplicity of handling the reaction while reducing the costs of

  production. Third, the direct use of the micro-enzyme

  Well reduces the time, effort and cost compared to the isolated enzyme. Finally, the MEC process according to the invention

  the advantage of specificity. There are two carboxyl groups available

  nibles and therefore two theoretically possible isomers (a and P).

  The isomer a is easily formed, which reduces the need for a separation step. The by-product can be used in the manufacture of L-aspartyl-L-phenylalanine methyl ester, an artificial sweetener about 200 times sweeter than table sugar. This is achieved by eliminating the amino-protective group

  by a process known as catalytic hydrogenation.

  The methods and products for the preparation and determination of B. cereus enzymatic broths used in the examples are as follows: A. Inoculum preparation A frozen culture of B. cereus NRRL B-12315 is thawed and 2 ml is used to inoculate 200 ml of trypticase-soy broth (BBL Company, Cockeysville, Md.) contained in a 1-liter baffled flask. The culture is incubated at 30 ° C. for 16-24 hours at 200 rpm on a rotary shaker having a circular orbit.

5.08 cm.

  B. Preparation of Enzyme Broth A medium consisting of 10.0 g of soluble starch, 0 g of soy flour, 2 is adjusted to pH 7.3 with 45% w / v potassium hydroxide. 0 g of yeast extract, 7.5 g of dibasic potassium phosphate, 1.0 g of Tween 80, 1.0 g of calcium chloride, 0.001 g of magnesium sulphate monohydrate, 0.001 g of sulphate ferrous iron and 0.001 g of zinc sulfate in 1 liter of distilled water, before autoclaving at 1 bar for 1 min.

  500 ml baffled flasks with 5 ml of the inoculum culture.

  After incubation for 24 h, the cultures are centrifuged at 12000 g for 10 min in a refrigerated centrifuge. The supernatant liquid is decanted and stored at 4 ° C. until use. If desired, concentrate the enzyme exempt from cells using

  CX filters (Millipore Corporation Corp., Bedford, Mass.).

  C. Determination of enzyme broth activity The enzymatic activity of the broth of

  cell-free culture in a spectrophotometric determination

  continuously, as described by Feder [Biochem.

  Biophys. Res. Commun., 32, p. 326 (1968)], except that the concentration of the substrate, furylacryloyl-L-glycyl-L-leucine -4-amide (FAGLA), is lowered to 5.M. enzymatic activity is

  expressed in FAGIA units (A345 / min.ml).

  The products and procedures for the analysis of aAcPM by thin layer chromatography are as follows: A. E. Merck silica gel plate with fluorescent activated indicator

  254 nm (Company EM Laboratories, Inc., Elmsford, NY).

  B. Solvent system Chloroform 60% by volume Methanol 30% by volume Water 4% by volume Formic acid 2% by volume C. Determination techniques After applying bouillon stains to the plate and having developed them in the solvent system above, the plate is air-dried for 30 minutes and examined as follows: 1. exposure to short-wave ultraviolet light, and 2. exposure to excess t-butyl hypochlorite then evaporation of the excess reagent, spraying with 0.5% w / v potassium iodide and 0.5% w / v starch in water and

visualization in white light.

  The products and techniques used in the high performance liquid chromatography (HPLC) analyzes of the MEC reagents and products are as follows: A. "Partisil PXS 10/25 ODS" column (250 mm x 4.6 mm) of diameter

  interior) provided by Whatman, Inc. (Clifton, N.J.).

  B. Mobile phase 33% tetrahydrofuran (distilled in the glass); 67%

  of distilled water containing 0.005M pentanesulfonic acid.

  C. Liquid Chromatograph Instrument from Waters Associates (Milford, Mass.) With Model M-600 A pump, Model 440 detector (UV, 254 nm at 0.1 AUFS) and a autoinjector

  WISP Model 710A operating at a flow rate of 2 ml / min.

  D. Procedure Properly weighed portions of solids and measured volumes of liquid materials are suitably diluted in the mobile phase and chromatographed. We identify the peaks and we

  quantified by reference with known standards.

  In the following examples, all the yields are given in 7% relative to the theoretical amount corresponding to the starting amount of Z-Asp in the reaction mixture. The known infrared and NMR spectra of the APM are as follows: IR spectrum (KBr pellet method):

-1 -

  3350 cm (N-H stretching vibration); 3070, 3036, 2960 cm (al-

  length of NH3, broad and superimposed with the vibrations of

  C-H), 1742 cm1 (C = O, ester, elongation), 1672 cm (C = O, amide); 1553 (NH3 +, symmetrical deformation, C-N-H amide); 1500 and

  1450 cm1 (elongation of the aromatic ring); 1381 cm '(C-O2, allon-

  symmetrical composition), 1235 cm (C-O, ester, asymmetric elongation, NH + rock), 1037 cm-1 (C-O, ester, symmetrical elongation); 923 cm -1 (C-C-N, amino acid, elongation); 751 cm (twist, 5H neighbors, -1

  mono-substituted phenyl) and 702 cm (bending of the nucleus, phenyl mono-

substituted). NMR spectrum, &

0

  II (1) 3.07 ppm (apparent triplet, 4H, -CH2-C- and OCH2-); (2) 3.70 ppm (singlet, 3H, O-CH 3); (3) 4.52 ppm (triplet, 1H, J = 5.92 Hz, CH); (4) 4.83 ppm (triplet, 1H, J = 6.1Hz, CH);

  (5) 7.25 ppm (singlet, 5H, aromatic H).

  The following examples illustrate the invention without

however, limit its scope.

EXAMPLE 1

  Carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Z-APM): (Scheme B, where R1 = carbobenzoxy and R = methyl) 28 g (284 millimoles) of carbobenzoxy acid are dissolved in distilled water L-aspartic acid (Z-Asp), 77 g (714 mmol) of L-phenylalanine methyl ester hydrochloride (PH, HCI) and

  1.68 g of calcium acetate, the pH is adjusted to 6.8 by 21 g of carbon.

  sodium nate and the volume is made up to 250 ml with water. The resulting solution is added to an equal volume of culture broth.

  B. cell-free cereus with FAGLA activity of 4.0 units.

  This mixture is then placed in a suitable glass container and stirred at 40 ° C. for 6 hours. The product is continuously collected by filtration as it is formed and the filtrate is pumped back into the reaction vessel. The reaction is stopped and the remaining solid residue is combined with the collected precipitate and washed with water. After drying and recrystallization from a methanol-water solvent mixture (1: 2), 64.52 g are obtained (yield 74.7%).

  addition of Z-APM and PM (1: 1).

  The Z-APM, PM salt is ground into a fine powder in a mortar, suspended in 350 ml of water and 127 ml of acid.

  1N hydrochloric acid and vigorously stirred for 2 hours at room temperature.

  ambient temperature. The resulting suspension is filtered and the precipitate is washed with several portions of water. After drying, we get

  44.8 g of Z-APM (overall yield 73.6%) and 22.63 g of PM (recovered

ration 29%).

  44.8 g of Z-APM are added to 746 ml of water and 2240 ml of methanol and the resulting mixture is hydrogenated.

  3.5 bars in the presence of 4.48 g of 5% palladium

  lyst. After 2 hours, the cacalyser is filtered off and the filtrate is concentrated to a volume of 500 ml under reduced pressure at a temperature not exceeding 50 ° C. The α-aspartyl-L-phenylalanine (aAPM) is crystallized by cooling. the solution at 0 ° C. for 2 hours 25.14 g of product are obtained by filtration, the thin layer chromatography shows that it is cAPM, a second jet of 3.60 g is obtained while still concentrating the liquors. ,

  for a total yield of 96.5% oAPM including chromatic analysis

  thin film shows that 82% consist of high quality product. The physical properties and results of the cAPM elemental analysis are as follows

F. 247-2480C

  Elemental analysis (C 14 H 18 N 2 O 5/2 H 2 O) C (%) H ("/) N (1) Calcd. 55.44 6.31 9.24 Found 55.56 6.15 9.24 IR and NMR spectra of the product show the

  same characteristics as above.

EXAMPLE 2

  The procedure of Example 1 is repeated with a cell-free B. cereus culture broth containing 3 FAGLA / ml units, except that the resulting enzyme / reagent mixture is divided into two equal portions. After 6 h, a total of 34.60 g of the Z-APM adduct and of PM (1: 1) are obtained in the reaction or

  product is separated as it forms (continuous reaction).

  The salt Z-APM, PM is suspended in 125 ml of water and 51 ml of 1N hydrochloric acid and stirred vigorously for 1 h 30 min.

  to obtain 16.82 g of Z-APM (overall yield 55.2% of theory).

  HPLC determination shows that the resulting fine precipitate has a

100% purity.

  The second portion of the enzyme / reagent mixture is treated identically except that the product is not separated during the reaction time of 6 h (batch reaction). A total of 17.34 g of the addition compound is obtained and suspended in 85 ml of water and 35 ml of 1N hydrochloric acid. After having

  stirred vigorously for 1 h 30 min, 11.65 g (yield

  38.2%) of Z-APM (100% purity from CLUP analysis).

1l

EXAMPLE 3

  Example 2 is repeated except that the reaction is carried out at room temperature. The continuous reaction gave a total of 30.57 g of the addition compound which was suspended in 150 ml of water and 60.4 ml of 1N hydrochloric acid and stirred vigorously for 1 hour. 20.08 g (overall yield is obtained

  59.7% of theory) of Z-APM (purity 90.67% based on HPLC analysis).

  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 1N hydrochloric acid. After stirring vigorously for 1 hour, 14.3 g (overall yield 46.2% of theory) are obtained.

  Z-APM (purity 98.7% according to HPLC analysis).

EXAMPLE 4

  The procedure of Example 1 is repeated with a cell-free B. cereus culture broth containing 4.7 FAGLA units / ml, except that the reaction is carried out at room temperature and

  also prepares a second batch of 500 ml of enzyme / reagent mixture.

  The additional mixture is added to the reaction vessel containing

  500 ml of the starting enzyme / reagent mixture in 15 ml portions at intervals of about 10 min from 3 hours after the start of the reaction. After 11 h 30 min, the residue remaining in the reaction mixture is combined with the precipitate collected, washed with water and dried. A total of 115.4 g of the addition compound consisting of 87.3 g (75.67% Z-APM) and 35.7 g (30.9X of PM) was obtained by HPLC analysis for yield

overall 71.56% of Z-APM.

EXAMPLE 5

  The procedure of Example 4 is repeated with another cell-free B. cereus culture broth containing 4.7 FAGLA units / ml, except that an additional 750 ml batch of enzyme / reagent mixture is prepared. The resulting mixture is added in 25-50 ml portions from 4 hours after the start of the reaction. The pH is periodically monitored during the reaction and additional 5-10 ml portions of the reaction mixture of the uncontrolled pH reagent mixture are added as needed to maintain the pH below 7 (total ml is added). mixture of uncontrolled pH reagents). After 17 h, the reaction is terminated by combining the remaining residue in the reaction mixture with the collected precipitate and then washing and drying. 203.05 g of the addition compound consisting of 66.2% (134.42 g) of Z-APM and 28.4% (57.66 g) of PM are obtained.

  by HPLC analysis, for an overall yield of Z-APM of 73.4%.

  It is to be understood that the invention is not limited to the preferred embodiments described above by way of illustration and that those skilled in the art may make modifications without

depart from the scope of the invention.

SCHEME A

  + H-Leu-NHO II Papalne Bz-Leu-Leu-NHO III + Papatnhe Bz-Leu-Gly-NH0 III Chymotrypsin H-Gly-NH0 II H-Gly-NH0 II Bz-Tyr-Gly-NH0 III Z-Phe -Gly-OH I + PapaTin Z-Phe-Gly-Tyr-NH2 III (1) Bz-Leu-OH I (2) (3) Bz-Leu-OH I BzTyr-OH I (4) H-Tyr-NH2 II

SCHEME B

H O

! he

R -N-CH-CH-CH-OH

1, 2

  Co2H (amino-protected L-aspartic acid) +

0-CH 2 -CH-CO 2R 2

NH21HC1

  (L-phenylalanine alkyl ester hydrochloride,

PA, HC1)

H O H H

  Enzyme Enzyme, R -N-HCH-CH-CN-CH-O CO2, PA Co2R2 (L-Aspartyl-amino protected phenylalanine alkyl ester adduct, 1APA, PA )

(2) R1APA, PA

R1APA + PA

(1)

Claims (5)

R E V E N D I C A T IONS   1. Process for the production of (L-aspartyl aminoprotected) -L-phenylalanine alkyl ester according to the following scheme (1) H O ! I! R 1 -N-CH-C --OH + O-CH 2 -CH-CO2R 2 C02H NH2) HC1   L-aspartic acid (amino-protected ester hydrochloride) of L-phenylalanine alkyl, PA, HC1) H OHH1 enzyme H. H H R1-N-CH-CH 2-C-N-C-CH 2-0 R1, 2 C, C2 C02PA C02R2   (L-aspartyl amino-protected phenylalanine alkyl ester addition compound, R1APA, PA) e + (2) 2) R1APA, PA,> RAPA + PA   wherein the L-aspartic 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, R is a C1-C4 alkyl group 2 1 6   embedded image, said method being characterized in that it comprises the following steps: carboxy-protected L-phenylalanine is reacted   amino-protected L-aspartic acid under conditions which favor   If microbial enzymatic coupling is present in the presence of an enzyme at a pH which retains enzymatic activity, the product is continuously separated as it is formed; and we add the new reagents   as the product is separated.   2. Method according to claim 1, characterized in that   that the enzyme source is a protease-producing microorganism.   3. Method according to claim 2, characterized in that the protease-producing microorganism is a Bacillus, Streptomyces or Aspergillus.   4. The method of claim 3, characterized in that   what the protease-producing microorganism is Bacillus cereus NRRL B-12315.   5. Process according to claim 1, characterized in that the enzyme is a crude or partially purified enzyme preparation obtained from a microorganism having an activity of protease.