METHOD FOR THE REMOVAL OF THE FORMYL GROUP FROM AN ESTER OF AN N-FORMYL PEPTIDE OR N-FORMYL AMINOACID

Abstract: A method for the removal of the formyl group from an ester of an N-formyl peptide or N-formyl aminoacid, in which the ester is reacted with urea in the presence of a polar solvent and a strong acid.

Description: The present invention relates to a method for the removal of the formyl group from an ester of an N-formyl peptide or N-formyl amino acid, particularly an ester of the aforesaid type having a free carboxyl group in the chain. Methods of the aforesaid type for removing formyl groups lay particular importance on the production of the methyl ester of alpha-L-aspartyl-L-phenylalanine (aspartame) which is obtained by a condensation reaction between the anhydride of N-formyl-L-aspartic acid with the methyl ester of L-phenylalanine. From U.S. patent No. 4,071,511 a deformylation method is known in which the N-formyl ester is reacted with a strong acid in aqueous solution and in the presence of an organic solvent, particularly alcohols. This method suffers from the disadvantage of poor selectivity since secondary reactions may take place, particularly hydrolysis of the ester group. U.K. patent no. 2,098,220 describes a deformylation method which uses hydrazine and derivatives of hydrazine which apparently result in greater selectivity and a greater deformylation yield, but however requires the pH to be controlled precisely within the range 1 to 3.5 during the reaction. The object of the present invention is to provide a new deformylation method of the type specified above which is highly selective and gives a high reaction yield, and which can be carried out with the use of reagents which are widely available on the market and of low cost. For this purpose, the subject of the present invention is a method for the removal of the formyl group from an N-formyl peptide ester or an N-formyl amino acid ester by reacting said ester in the presence of a polar solvent with a strong acid, characterised in that said reaction is carried out in the presence of urea of a pH less than 1. The method of the invention does not require rigorous control of the pH during the reaction. The reaction is preferably carried out with the use of hydrochloric acid, preferably in a ratio of 3-4 moles per mole of formyl group. However other acids such as sulphuric acid, nitric acid and phosphoric acid or their mixtures could be used. The polar solvent used in the reaction is preferably selected from the group consisting of monohydroxy alcohols having from 1 to 4 carbon atoms, acetone, acetonitrile and dimethylformamide, possibly in the presence of water. It is preferable to carry out the reaction with a stoichiometric excess of urea, preferably with 3-5 moles per mole of formyl group. The reaction temperature is not critical since it influences solely the reaction rate. Typically the reaction is carried out at a temperature in the region of from 30°C up to the temperature of reflux of the reaction solution. The reaction yield is kept at a value above 60% and analysis of the reaction products has shown the absence of appreciable quantities of undesirable by-products. The method of the invention will now be described with reference to the following examples which relate to the deformylation of N-formyl aspartame. Example 1 16.5g of a mixture of alpha+beta formyl aspartame, containing 13.4g (41.6 mmoles) of alpha-formyl aspartame are dissolved in a mixture of: 65ml CH₃CN 20ml methyl alcohol 11ml 37% HCl 12.5ml glacial acetic acid 10ml dimethylformamide 13g urea (216 mmoles) The clear, colourless, homogeneous solution obtained is agitated at 50°C for 30 hours. A final check on the products is effected by high pressure liquid chromatography (HPLC) and shows the following composition: alpha-aspartame (ASP): 8.83g (30 moles); alpha-formyl aspartame (FASP): 2.03g Yield: 72.1% Example 2 13.4g of alpha-formyl aspartame (41.6 moles) are dissolved atambient temperature in a mixture of: 100ml acetone 11ml 37% HCl 12.5ml glacial acetic acid 13g urea (216 mmoles) The clear, colourless, homogeneous solution obtained is agitated at 50°C for 55 hours and then for a further 64 hours at ambient temperature. The final composition determined by HPLC is as follows: alpha-ASP: 9.58g (32.6 mmoles); alpha-FASP: 1.97g Yield: 78.3% Example 3 16.5g of a mixture of alpha-beta-formyl aspartame, containing 13.6g (42 mmoles) of alpha-formyl aspartame, are dissolved in a mixture of: 65ml CH₃CN 20ml methyl alcohol 11ml 37% HCl 12.5ml glacial acetic acid 13g urea The initial pH of the solution is 0.15. The clear, homogeneous, colourless solution obtained is heated to 50°C and agitated at this temperature for 30 hours. The final pH is 0.3. The final composition determined by HPLC is as follows: alpha-ASP: 8.43g (28.7 mmoles); alpha-FASP: 0.88g Yield: 67.8% Example 4 13.5g of alpha-formyl aspartame are dissolved in a mixture of: 65ml CH₃CN 20ml tert. butyl alcohol 11ml 37% HCl 12.5ml glacial acetic acid 13g of urea The clear, colourless solution obtained is heated at 65°C for 24 hours. The final composition by HPLC is as follows: alpha-ASP: 8.15g (27.7 mmoles); alpha-FASP: 0.82g Yield: 66.1% Example 5 16.5g of a mixture of alpha- and beta-formyl aspartame, containing 13.6g of alpha-formyl aspartame are dissolved in a mixture of: 65ml CH₃CN 20ml H₂O 11ml 37% HCl 12.5ml glacial acetic acid 13g urea The solution obtained is agitated at 50°C for 12 hours and then kept for 64 hours at ambient temperature. Final composition by HPLC: alpha-ASP: 8.48g (28.8 mmoles); alpha-FASP: 1.1g Yield 62.3% Example 6 13.6g of alpha-formyl aspartame (42.2 mmoles) are dissolved in a mixture of: 65ml CH₃CN 20ml H₂O 11ml 37% HCl 12.5ml of glacial acetic acid 13g urea The clear, colourless solution obtained is heated to 65°C and agitated for 8 hours. Final composition by HPLC: alpha-ASP: 8.37g (28.5 mmoles); alpha-FASP: 0.62g Yield: 61.5% It is evaporated under vacuum to oil, diluted to 200ml with H₂O. The final water rich composition by HPLC: alpha-ASP: 7.97g; alpha-FASP: 0.59g.

Claims: A method for the removal of the formyl group from N-formyl peptide esters or N-formyl amino acid esters, by reacting said ester in the presence of a polar solvent with a strong acid, characterised in that said reaction is carried out in the presence of urea at a pH less than 1. A method according to Claim 1, characterised in that the solvent is selected from monohydroxy alcohols having from 1 to 4 carbon atoms, acetone, acetonitrile and dimethylformamide. A method according to Claim 1, characterised in that the acid present in solution is selected from hydrochloric acid, nitric acid, sulphuric acid, phosphoric acid and their mixtures. A method according to any one of Claims 1 to 3, characterised in that the urea is present in a stoichiometric excess of 3-5 moles of urea per mole of N-formyl group. A method according to any one of Claims 1 to 4, characterised in that the reaction temperature is from 30°C up to the temperature of reflux of the reaction solution. A method according to any one of Claims 1 to 5, characterised in that the ester is the methyl ester of alpha-L-aspartyl-L-phenylalanine (aspartame).

Inventors: BOLIS, GOFFREDO ANTONIO, CANTARINI, GIUSEPPE, DA COL, MARCO
Applicants: LARK S.P.A.
Application year: 1986
Main classification: C07K1
IPC codes: C07K1, C07K5