METHYLATION OF ORGANIC COMPOUNDS

Abstract: A method of methylating organic compounds which is potentially cheaper than conventional esterification of carboxylic acids or methylation of phenols which comprises contacting the substance that is to be methylated within methyl donor material in the presence of a fungus or another organism into which an appropriate fungal gene is introduced.

Description: METHYLATION OF ORGANIC COMPOUNDS This invention relates to methylation of organic compounds. At present the industrial esterification of carboxylic acids to form the methyl ester is normally effected by refluxing the carboxylic acid with methanol in the presence of mineral acid and separating the ester from the reaction mixture by fractional distillation. Methylation of phenols to form anisoles is normally performed industrially by refluxing dimethyl sulphate with the sodium salt of the phenol. The resulting anisole is separated from the reaction mixture by solvent extraction or distillation techniques. Both methylation of carboxylic acids and of phenols is therefore energy and capital intensive. The object of the present invention is to provide a cheaper method of methylation. According to the invention there is provided a method of methylation comprising contacting an organic substance that is to be methylated with a methyl donor material in the presence of fungus or another organism into which an appropriate fungal gene is introduced. The invention, therefore, is a biological system and it offers the possibility of preparation of methylated compounds at ambient temperatures using a gaseous halomethane. It is believed that this system is potentially cheaper to operate than the known techniques. In a preferred embodiment of the invention a fungal preparation or immobilised enzyme preparation is produced using an appropriate fungus preferably selected from fungi which can biosynthesise chloromethane such as the Hymenochaetaceae, Polyporaceae and Corticiacae. Alternatively the invention employs an immobilised preparation of a bacteria into which a cloned copy of the fungal gene, or genes, coding for the methylation system has been introduced using standard genetic manipulation techniques. An aqueous solution of the organic substance to be methylated, for example a carboxylic acid or a phenol containing dissolved gaseous chloromethane are together cycled through the preparation. Insoluble methyl ester of the carboxylic acid or O-methyl phenol as the case may be, is formed and can be separated and the unreacted mixture re-cycled through the preparation. Additional starting materials can be added as necessary to the recycle stream. The reaction is carried out at ambient temperature for example between 15 and 25°C and preferably at a pH between 2 and 8. The invention can be applied to a wide range of organic substances, particularly acids such as benzoic, furoic, phenylacetic, cinnamic, propionic, butyric, isobutyric, valeric, isovaleric, hexanoic and heptanoic acids and halo-, methyl and hydroxy-, methoxy- and cyano- substituted derivatives of acids. The invention can also be applied to the methylation of many phenols and thiophenols including both halo- and methyl substituted compounds such as 2-, 3- and 4- fluorophenol, 2-, 3- and 4- chlorophenol, 2, 6- dichlorophenol, 2-, 3- and 4- bromophenol, 2- iodophenol and 2-, 3- and 4- methylphenol. The methyl donor material may be a halo methane such as chloro-, bromo- or iodo-methane. As mentioned above a gaseous donor material is preferably used. The following Examples further illustrate the invention. EXAMPLE 1 The fungus Phellinus pomaceus (NCWRF.FPRL 33A obtained from the National Collection of Wood Rotting Fungi, Princes Risborough Laboratory, Building Research Establishment, A lesbury, Bucks., England, or CBS 137.42 obtained from the Centraalbureau Voor Schimmelcultures, Baarn, The Netherlands, was used as a basis of the system. A 2 mM solution of butyric acid containing 0.1 mM chloromethane buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium for a period of 12 hours at a temperature of 25°C. Methyl butyrate was formed at a rate of 245 nanomoles/g fungal mycelium/hour. EXAMPLE 2 A 0.5 mM solution of benzoic acid containing 1.23 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus pomaceus, NCWRF.FPRL 33A or CBS 137.42) for a- period of 12 hours at a temperature of 25°C. Methyl benzoate was formed at a rate of 290 nanomoles/g fungal myce1ium/hour. EXAMPLE 3 A 10 mM solution of phenol containing 1.23 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus pomaceus, NCWRF. FPRL 33A or CBS 137.42) at 25°C for 12 h. Anisole was formed at a rate of 30 nanomoles/g fungal mycelium/hour. EXAMPLE 4 A 2 mM solution of 2-chlorophenol containing 1.23 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus pomaceus, NCWRF. FPRL 33A or CBS 137.42) at 25°C for 12 h. 2-chloroanisole was formed at a rate of 16 nanomoles/g fungal mycelium/hour. EXAMPLE 5 A ImM solution of thiophenol containing 1.23 mM chloromethane and buffered at pH 4.0 with 20 M citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus pomaceus, NCWRF. FPRL 33A or CBS 137.42) at 25°C for 12 h. Thioanisole was formed at a rate of 113 nanomoles/g fungal mycelium/hour. EXAMPLE 6 The fungus Phellinus ribis (NCWRF. FPRL 42 obtained from the National Collection of Wood Rotting Fungi, Princes Risborough Laboratory, Building Research Establishment, Aylesbury, Bucks or CBS 175.29 obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was used as a basis of the system. A 2 mM solution of butyric acid containing 0.2 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium for a period of 15 hours at a temperature of 25°C. Methyl butyrate was formed at a rate of 197 nanomoles/g fungal mycelium/hour. EXAMPLE 7 A 0.5 mM solution of benzoic acid containing 0.2 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus ribis NCWRF. FPRL 42 or CBS 175.29) for a period of 15 hours at a temperature of 25°C. Methyl benzoate was formed at a rate of 77 nanomoles/g fungal ycelium/hour. EXAMPLE 8 A 2 mM solution of phenol containing 0.2 mM chloromethane and buffered at ph 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus ribis NCWRF. FPRL 42 or CBS 175.29) at a temperature of 25°C for a period of 15 hours. Anisole was formed at a rate of 11 nanomoles/g fungal mycelium/hour. EXAMPLE 9 A 1 mM solution of thiophenol containing 0.2 M chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus ribis NCWRF. FPRL 42 or CBS 175.29) at a temperature of 25°C for a period of 15 hours. Thioanisole was formed at a rate of 185 nanomoles/g fungal mycelium/hour. EXAMPLE 10 The fungus Phellinus lundelli (CBS 540.72 obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was used as a basis of the system. A 2 mM solution of butyric acid containing 0.2 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium for a period of 15 hours at a temperature of 25°C. Methyl butyrate was formed at a rate of 97 nanomoles/g fungal mycelium/hour. EXAMPLE 11 A 1 mM solution of thiophenol containing 0.2 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium (Phellinus lundelli (CBS 540.72)) at a temperature of 25°C for a period of 15 hours. Thioanisole was formed at a rate of 45 nanomoles/g fungal mycelium/hour. EXAMPLE 12 The fungus Phellinus robiniae (NCWRF-FPRL 180 obtained from the National Collection of Wood Rotting Fungi, Princes Risborough Laboratory, Building Research Establishment, Aylesbury, Bucks or CBS 211.36 obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was used as a basis of the system. A 2 mM solution of phenol containing 0.2 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium for a period of 15 hours at a temperature of 25°C. Anisole was formed at a rate of 8 nanomoles/g fungal mycelium/hour. EXAMPLE 13 The fungus Phellinus hippophaecola (CBS 168.31 obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was used as a basis of the system. A 2 mM solution of butyric acid containing 0.2 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium for a period of 15 hours at a temperature of 25°C. Methyl butyrate was formed at a rate of 49 nanomoles/g fungal mycelium/hour. EXAMPLE 14 The fungus Phellinus tremulae (CBS 123.40 obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was used as the basis of the system. A 2 mM solution of butyric acid containing 0.2 mM chloromethane and buffered at pH 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium for a period of 15 hours at a temperature of 25°C. Methyl butyrate was formed at a rate of 26 nanomoles/g fungal mycelium/hour. EXAMPLE 15 The fungus Fomitopsis pinicola (CBS 313.82 obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was used as the basis of the system. A 1 mM solution of benzoic acid containing 1.23 mM chloromethane and buffered at ph 4.0 with 20 mM citrate buffer was incubated with 0.035 g of fungal mycelium for a period of 15 hours at a temperature of 25°C. Methyl benzoate was formed at a rate of 6 nanomoles/g fungal mycelium/hour. ;< H3 International Application No: PCI - 12.- ANNEX H3 International Aooiication No: PCT/ MICROORGANISMS Optional Sheet In connection with the microorganism raferred to en paga- . of the daacπoβon l A. IDENTIFICATION OF DEPOSIT - Further deeeelta are Identlfted en an additional aheet fyi > Nam* ol deeβaitarγ InsOtuflon * CENTRAALBUREAU VOOR SCHIMMELCULTURES Aάάreaa ai deooβitaηr InsUtutlon (Including postu coαe and country) * BAARN, THE NETHERLANDS , Oats of deooelt * Accaeaien Number * CBS 175 . 29 >. ADDITIONAL INDICATIONS 7 (ieeve biaπs if not applicable). Thia information la continued an a separata atlacneα aneet [~ C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE » (It th* indications sra not for all designates SUtea) D. SEPARATE FURNISHING OF INDICATIONS • (laav* oiank il not applicable) Th* indications Ustaα below will be submitted to the International Buraau lalar • (Specify th* gineral nature of th* Indications e.g.. ** Accaaaiαn Number of Deposi ") DATE OF DEPOSIT [7] This afteet wιι received with tha international aoolicatlαπ when filed (to be checked by the receiving Office) D. LOvVπv CS--: ) ROOM 5/6 INTERNATIONAL UNiT FYT RQ 0β (Authoπzsd Officer) ' The date of receiøt (from the aoølicant) by me Iπiernauonaf fiureeu lβ (Authorized Officer) 13 - ANNEX M3 International Aoplicstlon No: PCT/ MICROORGANISMS Optional Sheet In connection with the microorganism referred to on psβe_ 19 . of the deecnption ' A. IDENTIFICATION OF DEPOSIT > Further depoaits sre Identified on en additional aneet (yi > Nam* of depositary institution « CENTRAALBUREAU VOOR SCHIMMELCULTURES Address of deoeeiUry institution (including postal coαe and country) < BAARN, THE NETHERLANDS Date of depoalt * Aceaeeton Number * CBS 540 . 7 2 8. ADDITIONAL INDICATIONS ' (leave blank if not aopiicabie). Thle information la continued on a aeparata atuched aneet | C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE » (If the indications sre not for ail designsteα Sutas) O. SEPARATE FURNISHING OF INDICATIONS • (leave blank il net applicable) The indicationa listed below will be suomitted to the International Bureau later • (Specify the general nature of th* Indleationa e.g., - Accaaaion Numoer of Deposit ") DATE OF DEPOSIT E. p7] This sh*«l waa received with tn* international application when filed (to be checked by tne receiving Ofllca) """ . i: ' J ~ ■ • • • . '• -. -. : i NTΞΠNATJ Q Γ;ΛL υr::τ. Exτ....fignR (Authontsd OIDcar) i Th* date of receiot (from the aoolicant) by the International Buraau ' (Authorized Officer) - I H-- AHNEX M3 International Application No: PCT/ MICROORGANISMS Optional Sheet In connection with the mtcroorganiim referred to on page. 17 . — of the description * A. IDENTIFICATION OF DEPOSIT * Further deposits are Identified on an additional aneet Ham* at depositary Institution « CENTRAALBUREAU VOOR SCHIMMELCULTURES Address of deoosiury Institution (Inducing postal code end country) ' BAARN, THE NETHERLANDS. Oaie of deposit * Accession Number * CBS 211.36 M. ADDITIONAL INDICATIONS 7 (leev* blenk if not applicable). This informstiαn is continued an a separate anacneα aneet | r C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MAOE ' (II the indicationa are not far all daaignated SUtea) O. SEPARATE FURNISHING OF INDICATIONS • (laave plank if not applicable) The inoieatlens Bated belβw will be submitted to the International Bureau later ■ (Specify the general nature of e Indleationa e.g.. - Accession Numoer ol Oepssit"} DATE OF DEPOSIT £. {7j This sheet was received wilh tne international application whan filad (to be checked by the receiving Office) P\ I r-' - , • — \,- / . - * — m \ r~- r— ~^ . - r- i r- INTERNATIQΓ: L ur::τ. ET . G306 (Authonzed Officer} The date of receipt (from the applicant) by the International Buraau '• (Authorized Officer) Form PCT>'ROft34 (January JβatJ - IS- U M3 International Aopiicatlon No: PCT/ MICROORGANISMS Optional Sheet In connection ith the microorganism referred to on page- _2 ;,„ i. ..of the deecnption ι A. IDENTIFICATION OF DEPOSIT ' Further depoaita are identified on en additional aheet f Nam* βf deoosiUry InsUtudoπ * CENTRAALBUREAU VOOR SCHIMMELCULTURES Addreee of depositary institution (Including postal cooe and country) * BAARN, THE NETHERLANDS Date of deposit * Acceeeion Numeer * CBS 168 . 31 B. ADDITIONAL INDICATIONS : (leeve blank If not appiicaple). This information la continued on a sepsrste atuched eneet | ! C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE > (If the indications ara not tor ail deaignated SUles) D. SEPARATE FURNISHING OF INDICATIONS • (leave Olenk if not applicable) The indications liated below will be auomrtted to the International Bureau later • (Specify the general nature of th* Indication* e.g., ** Accaawoπ Numoer of Depoait "] DATE OF DEPOSIT E. ra Thia eheat was r*c*ιved with the international application when filed do be checked by in* receiving Office) D f fV ' " V t * - ' - m r. \ r > /-ι « /! - r n (Authorized Officar) i The date of receipt (from the applicant) by ihe inte netionei βureeu '• (Authorized Officer) ANNEX H3 International Appiication No: PCT/ MICROORGANISMS Optional Sheet in connection with tha microorganism referred to on page- 14 . of the deecπodαn ι A. IDENTIFICATION OF DEPOSIT ' Further deposits ara identified on an additions! aheet fyj * Nam* ol depositary institution * CENTRAALBUREAU VOOR SCHIMMELCULTURES Address of depositary institution (Inducing postal code and country} * BAARN , THE NETHERLANDS . Date of deposit * Accession Number * CBS 123 . 40 B. ADDITIONAL INDICATIONS * (Isav* blank if not applicable). Thla Information Is continued on a aeparata atucneα aneet f C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE * (If the inolcstlons sre not for ail daaignateα SUtea) D. SEPARATE FURNISHING OF INDICATIONS • (leave blank if not applicable) Th* indications liated beiew will be submitted to th* International Bureau later * (Specify the general nature of the Indleationa e.g., M cceaaιon Number ol Deposit"} DATE OF DEPOSIT ϊ. r~) This sheet waa received with the international application whan filed (to be checked by the receiving Office) D. Li; . • . t s .- . ^ ) -..- sT.: I u/o (Authonzed Officer) I ■ The date αf receipt (from the aαolicant) by tne International Buraau t° (Authonzed Officer) π- ANNEX H3 International Application No: PCT/ MICROORGANISMS Octloπsl Sheet In conneeflon with the microorganism raferred to on r*T 10 ""» 2 αf the deacπoDon > A. IDENTIFICATION OF OEPOSIT » Further deposits are identified on an additional aneet f ϊ » Nam* of dβooaitary Institution * CENTRAALBUREAU VOOR SCHIMMELCULTURES Addreea of deoosittry Institution (Including posul code end country) * BAARN , THE NETHERLANDS . Date of daoαeit • Aceeeeion Number * I CBS 313 . 82 B. ADDITIONAL INDICATIONS 7 (leave blank it not appiicaple). Thle Informetlon le continued on a aeparata arucned aneet | i C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE = (If the mαlcailons sre not far all αeaignsteo SUtee) O. SEPARATE FURNISHING OF INDICATIONS • (leave blank if not applicable) The indications Msted below will be suomitted to the International Bureau later • (Spacity the general nature of th* Inolcstlons e.g.. ■ * Aceeeeion Number of Deposit **) DATE OF DEPOSIT E. fπ Thie eneei waa received with the imernetional application when filed (to be checked by the receiving Office) D | ,-< ■ " - -. / / I • • <- ■ '> \ n "i >- . . r- r, (Authonzed Officer) i The date df receiot (from the aoolieant) by the Internactonel Bυreeu ιo (Authonzed Officer)

Claims: - IS - CLAIMS 1. A method of methylation comprising contacting an organic substance that is to be methylated with a methyl donor material in the presence of fungus or another organism into which an appropriate fungal gene is introduced. 2. A method as claimed in Claim 1, wherein the fungus is selected from the Hymenochaetaceae, Polyporaceae and Corticiaceae. 3. A method as claimed in Claim 2, wherein the fungus is selected from Phellinus pomaceus, (CBS 137.42), Phellinus ribis (CBS 175.29), Phellinus lundelli (CBS 540.72), Phellinus robiniae (CBS 211.36), Phellinus hippophaecola (CBS 168.31), Phellinus tremulae (CBS 123.40) and Fomitopsis pinicola (CBS 313.82). 4. A method as claimed in any preceding claim, wherein the reaction is carried out at a temperature of from 15° to 25°C. 5. A method as claimed in any preceding claim, wherein the pH of the reaction mixture is from 2 to 8. 6. A method as claimed in any preceding claim, wherein the methyl donor material is a halomethane. - - 7. A method as claimed in any preceding claim, wherein the methyl donor material is gaseous. 8. A methylated substance produced by the method as claimed in any preceding claim.

Inventors: HARPER DAVID BENJAMIN, HARPER, DAVID, BENJAMIN
Applicants: UNIV BELFAST, THE QUEEN'S UNIVERSITY OF BELFAST
Application year: 1989
Main classification: C12P1
IPC codes: C12P1, C12P7, C12P11