IE52242B1 - Preparation of amino protected-l-aspartyl-l-phenylalanine alkyl ester - Google Patents

Abstract

A process for the production of amino-protected-L-aspartyl-L-phenylalanine 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 phi -CH2- 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. 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

The present invention relates to a microbial enzymatic coupling (MEC) process for production of aminoprotected-L-aspartyl-L-phenylalanine alkyl ester. More particularly, the invention relates to a 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 microorganism 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 and the acid chloride method. 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 on 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 cannot 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 peptide 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. 1949). The reactions of this method are outlined in Chart A which follows the Examples.

The 'problem which is common to reactions I to III 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., 185,629 (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 of polypeptides.

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 aaspartyl-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 4,119,493, 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-A-OH is reacted with an amino acid or peptide having a C-terminal protective group or salt thereof of the formula Η-Β-Υ 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 metalloproteinase. 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. This patent discloses novel addition compounds of dipeptides composed of N-substituted iiionoaminodicarboxyl ic 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 4,212,945 and 4,212,946 where the protease is recovered by isolating the protease from an addition compound after a peptide; synthesis of peptidebonded amino acid derivatives in the presence of the protease.

The present invention provides a process for the production of an amino-protected-L-aspartyl-L-phenylalanine alkyl ester which comprises reacting aminoprotected-L-aspartic acid and L-phenylalanine alkyl ester in the presence of a protease-producing microorganism or an enzyme preparation obtained from a microorganism having protease activity, under conditions which favour the microbial enzymatic coupling thereof, the product being removed continuously as it is formed.

More particularly, the present invention relates to such a process for the production of an aminoprotected-L-aspartyl-L-phenylalanine alkyl ester corres- ponding to the following general formula: H 0 1 ll Η H I I 15 1 M R. - N - CH - CH_- C - 1 1 2 1 1 N - C - 1 I COj .PA c°2r. wherein represents an amino-protecting group; Rj represents an alkyl group containing from 1 to 6 carbon atoms; PA represents phenylalanine; and 0 represents phenyl; wherein a carboxyl-protected-L-phenylalanine corresponding to the following general formula: - CHj - CH - COjRj wherein 0 and Rj are as defined NHj.HCl above; is reacted with an amino-protected L-aspartic acid corresponding to the following general formula: Η Ο I II R. - Ν - CH - CH,- C - OH 1 I 2 co2h wherein R1 is as defined above in the presence of a protease-producing microorganism or an enzyme preparation obtained from a microorganism having protease activity under conditions which favour the microbial enzymatic coupling thereof to form the amino-protected-L-aspartylL-phenylalanine alkyl ester which is continuously removed. (In this connection reference may be made to IQ accompanying Chart B.) The enzyme source is preferably a proteaseproducing microorganism.

The process should be carried out at a pH which maintains enzyme activity. Further reactants are j5 generally added as the product is removed.

The amino group of L-aspartic acid may be protected by commonly-used protecting groups which include the following: aliphatic oxycarbonyl groups, such carbobenzoxy, t-butyl-oxycarbonyl ((CHjC-O-CO) and t-amyloxycarbonyl ((CH^)2C(C2Hg)-O-C0-); nuclear substituted carbobenzoxy groups, such as £-methoxy-carbobenzoxy (£-CH3O-0-CH2-O-CO-), 3,5-dimethoxycarbobenzoxy (3,5-(CHjO)2-0-CH2-O-CO-) and 2,4,6-trimethoxycarbobenzoxy (2,4,6-(CH3O)3-0-CH2-O-CO-), benzoyl group (0-CO-); £-toluenesulfonyl group (£-CH3"0-SO2-)j the urethane type{ and aromatic sulfinyl groups, such as 0-nitrosulfinvl group. A preferred amino protecting group is carbobenzoxy. The enzyme used in the present invention are proteases and are preferably metalloproteases which have a metal ion in the active centre. Suitable metalloproteases are enzymes originating from microorganisms, such as Bacillus, Streptomyces, Pseudomonas, marine and various other bacteria and certain fungi, particularly Aspergillus Where applicable the protease producing microorganism 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).

Pronase (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 hyperproteaseproducing 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 with isolating and recovering the enzyme can be eliminated. Additionally, an enzyme preparation from a proteaseproducing 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 wich 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 he 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 arc available and therefore two isomers theoretically arc possible 2242 ίο (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-12315 is thawed and 2 ml used to inoculate 200 ml of Trypticase Soy Broth (BBL, Cockeysville, Md.) contained in a 1 1 baffled flask. The culture is incubated at 30 ± 1GC for 16 - 24 hrs. at 200 rpm on a rotary shaker with a 2 inch (approx 5 cms) circular orbit.

B. Enzyme Broth Preparation A medium consisting of soluble starch, 10.0 g; soy flour, 10.0 g; yeast extract, 2.0 g; dibasic potassium phosphate, 7.5 g; Tween (Registered Trade Mark) 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 1 1 of distilled water is adjusted to pH 7.3 with 45% (w/v) potassium hydroxide before autoclaving at 15 lbs (6.8 kg) for 15 min. τι 100 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 12,000 x g for 10 min in a refrigerated centrifuge. The supernatant is decanted and stored at 4°C 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 [Eiochem. Biophys, Res. Commun., 32, 326 (1966)) except that the concentration of the substrate, furylacryoyl-L-glycyl-L-leucine amide -4 (FAGLA), is decreased to 5 x 10 M. Enzyme activity is expressed as FAGLA units ( A^^g/min/ml).

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

Solvent system Chloroform 60% (v/v) Methanol 30% n Water 4% tl Formic Acid 2% » I 242 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 visulization 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 (Registered Trade Mark) PXS 10/25 ODS(250- mm x 4.6 i.d.) supplied by Whatman, Inc. (Clifton, N.J.) B. Mobile Phase vol % tetrahydrofuran (distilled in glass): vol % distilled water containing 0.005M pentane sulphonic acid.

C. Instrument Water Associates (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 710A 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 chomatographed. Peaks are identified and quantitiated by referenbe to known standards.

In the examples that follow, all yields are given as [e of theory based on the starting amount of Z-Asp in the reaction mixture. The known infrared and NMR spectra of α APM are as follows: Infrared spectrum: (KBr Disc Method) 3550 em-1(N-H stretching vibration); 5070, 5056 and 1 + 2960cm (Nil- stretching vibration, broad and overlapped with C-H stretching vibrations), 1762cm-1 (C=0 ester, + stretching vibration), 1672ctn- (C=0, amide); 1555(NHj 1 symmetrical deformation vibration, C-X-II, amide); 1500 and 1650cm-1 (aromatic ring stretching vibration); ?1 cm (C-0o symmetrical stretching vibration), 1255cm-1 (C-0 ester, asymmetrical stretching vibration; + 1 Nil- rock), 1057cm (C-0 ester, symmetrical stretching vibration); 925cm-1 (C-C-N, ainino acid, stretching vibration); 751cm-1(5 ’adjacent II wag vibration, monosubstituted phenyl), and 702cm-1 (ring bending vibration, mono-substituted phenyl) .

NMR spectrum: g (1) 3.07 ppm (apparent triplet, 4H,-CH2-C- (2) 3.70 ppm (singlet, 3H, O-CH3); (3) 4.52 ppm (triplet, 1H, 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-aspartic-Lphenylalanine alkyl esters is exemplified in the following representative examples.

Example 1 Carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Ζ- APM): (Chart B wherein R^ is carbobenzoxy and Rj is methyl). g (284 mmol) of carbobenzoxy-L-aspartic acid (z-Asp), 77 g (714 mmol) of L-phenylalanine methyl ester hydrochloride (PM.HC1), 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 40°C 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 62242 1'ι and washed with water. After drying and recrystallizing from a solvent mixture of methanol and water (1:2 by volume), a total of 64.52 g of a 1:1 addition compound of Z-APM and PM 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 IN 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 2-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 (3.5 kg/sq cm) pressure in the presence of 4.48 g of 5 wt % 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 50°C. The a-aspartyl-L-phenylalanine (aAPM) is crystallized by cooling the solution to 0°C 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 liquors for a 96.5% total yield of aAPM of which 62% was shown to be of high quality by TLC analysis.

I is The physical properties and results of elementary analysis of the α-APM product are as follows: Melting point: 247° to 284°C Elementary analysis (C^g 1/2 HjO) C Η N Calculated: (%) 55.44 6.31 9.24 Found: (%) 55.56 6.15 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 enzyrae/reactant mixture is divided into two equal portions. After 6 hrs a total of 34.60 g of the 1:1 addition compound of Z-APM and PM 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 IN hydrochloric acid and stirred vigorously for 1,5 hrs.to give 16.82 g of Z-APM (over yield of 55.2% of theory).

Tho resultant line precipitate is 100# pure based on HPLC assay.

Tbe second portion of cnzyme/rcactant mixture is treated identically except that product is not removed during tbe 6 -hr reaction period (batch reaction). A total of 17.54 g of tbe addition compound is obtained and suspended in $5 ml of water and 55 ml of IN hydrochloric acid. After stirring vigorously for 1.5 hrs, 11.65 g of Z-APM (100# pure by IIPLC) is obtained (overall yield of 5S.2#).

Example 5 The process of example 2 is repeated except that the reaction is run at room temperature. The continuous reaction gives a total of 50.57 g of the addition compound which is suspended in 150 ml of water and 60.4 ml of IN 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 45.5 ml of IN hydrochloric acid. After stirring vigorously for 1 hr, 14.5 g of Z-APM (9S.7# pure by HPLC) is obtained foi· an overall yield of 46,2# of theory.

Example 4 .

The process of example 1 is repeated with a B. ccrons cell-free culture broth containing 4.7 units of FAGLA activity except that the reaction is run at room 53242 is 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 enzvwe/reactant mixture in 15 ml portions at about 10 min intervals starting 5 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% (87.5g) of Z-APM and 50.9% (55.7 g) of PM as determined by 11PL-C for an overall yield of 71.56% of Z-APM.

Example 5 The process of example 4 is repeated with another B. ccrcus cell-free culture broth containing 4.7 units of FAGLA activity except that an additional 750 ml of enzymc/rcactant 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 - 10 ml portions reaction mixture of non-pll adjusted reactant mixture are added as required to maintain the pH below 7, (total of 125 uil of. non-pll adjusted reactant mixture arc 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 205.05 g of the addition compound is 22 42 obtained wliicli consists of 66.2% (154.42 g) of Z-APM and 2S.4% (57.66 g) of PM as determined by.HPLC for an overall Z-APM yield of 75.4%. 53242 (1) Bz-Leu-OH I Chart Λ l-Leu-ΝΗφ II Papain (2) Bz-Leu-OH I Bz-Leu-Leu-NH φ III + 1 l-Gly-ΝΗφ II Papain (3) Bz-Tyr-OH I Bz-Leu-Gly-NHφ III + H l-Gyl-ΝΗφ II Chvwotrynsi n (4) Bz-Tyr-Gly-NH φ III Z-P)ie-Gly-0H + h I -Tyr-Nll2 II Papn i n Z-I’he-Gly-Tyr-».’ll2 III (1) Chart B I II R,-N-CH-CH.-C-OH + 1 I 2 co2h $-CH2-CH-CO2R2 NH2·HCl (amino protected-L-aspartic acid,) (L-phenylalanine alkyl ester hydro-chloride, PA·HCl) Η Ο 11 11 I tl I I enzyme \ R, -N-CH-Ch\-C-N-C-CH„-4i ) 1 j 2 j 2 CO2*PA c°2R2 (amino protected-L-aspartyl-L-phenylalanine alkyl ester addition compound, RjAPA’PA)) (2) R^APA'PA 11+ Rj^APA+PA 2242

Claims (12)

1. A process for the production of an amino-protectedL-aspartyl-L-phenylalanine alkyl ester which comprises reacting amino-protected-L-aspartic acid and L-phenylalanine alkyl ester in the presence of a protease-producing microorganism or an enzyme preparation obtained from a microorganism having protease activity, under conditions which favour the microbial enzymatic coupling thereof, the product being removed continuously as it is formed.

2. A process as claimed in claim 1 for the production of an amino-protected-L-aspartyl-L-phenylalanine alkyl ester corresponding to the following general formula; H II I CH - CH,- C - N I 2 C0 2 .PA H wherein Rj represents an amino-protecting group; R 2 represents an alkyl group containing from 1 to 6 carbon atoms; PA represents phenylalanine; and 0 represents phenyl; wherein a carboxyl-protected-L-phenylalanine corresponding to the following general formula: 0 - CH 2 - CH - CO 2 R 2 wherein 0 and R 2 are as NH 2 .HC1 defined above; is reacted with an amino-protected L-aspartic acid corresponding to the following general formula; I U R. - N - CH - CH, - C - OH 1 I 2 co 2 h wherein R^ is as defined above in the presence of a protease-producing microorganism or an enzyme preparatibn obtained from a microorganism having protease activity under conditions which favour the microbial enzymatic coupling thereof to form the amino-protected-L-aspartylL-phenylalanine alkyl ester which is continuously removed.

3. A process as claimed in claim 1 or claim 2 wherein the alkyl ester is a methyl ester.

4. A process as claimed in any of claims 1 to 3 wherein the enzyme source is a protease-producing microorganism.

5. A process as claimed in claim 4 wherein the protease-producing microorganism is Bacillus, Streptomyces or Aspergillus.

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

7. A process as claimed in any of claims 1 to 3 wherein a crude or partially purified enzyme prepar52242 ation obtained from a microorganism having protease activity is used. B. A process as claimed in any of claims 1 to 7 5 for the production of an amino-protected-L-aspartyl-Lphenylalanine alkyl ester substantially as herein described with particular reference to the Examples,

8. 9. An amino-protected-L-aspartyl-L-phenylalanine

9. 10 alkyl ester when produced by a process as claimed in any of claims 1 to 8.

10. An L-aspartyl-L-phenylalanine alkyl ester prepared by the removal of the amino-protecting group 15 from a compound as claimed in claim 9.

11. An L-aspartyl-L-phenylalanine alkyl ester ester as claimed in claim 10 which is a methyl ester. 20

12. A process for the removal of the amino-protecting group from a compound as claimed in claim 9 substantially as herein described with particular reference to the