EP2914734A1 - Procédé pour la formation enzymatique de liaisons amide - Google Patents

Procédé pour la formation enzymatique de liaisons amide

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Publication number
EP2914734A1
EP2914734A1 EP13773248.3A EP13773248A EP2914734A1 EP 2914734 A1 EP2914734 A1 EP 2914734A1 EP 13773248 A EP13773248 A EP 13773248A EP 2914734 A1 EP2914734 A1 EP 2914734A1
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EP
European Patent Office
Prior art keywords
mixture
alkyl
aqueous buffer
alkylaryl
protease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP13773248.3A
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German (de)
English (en)
Inventor
Steffen Osswald
Martin BINDL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evonik Operations GmbH
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Evonik Degussa GmbH
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Publication date
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Publication of EP2914734A1 publication Critical patent/EP2914734A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the present invention is concerned with the formation of amide bonds with the aid of proteases in general.
  • One aspect of the present invention further, relates to the protease mediated synthesis of organogellant compounds (OG) as depicted below
  • L is a linking moiety of molecular weight from 14 g/mol to 500 g/mol, one of X 1 , X 2 is nitrogen and the other is carbon, and wherein R 1 are sidechain substituents.
  • Organogellant compounds as depicted above are known in the art to serve as gellants to thicken liquid compositions. Such gellants have, for example, been described in WO 201 1 /1 12912 A1 and WO 201 1 /1 12887 A1 .
  • Organogellant compounds also termed organogellants herein, in general are used to provide structure and a pleasant texture to liquid consumer products such as, for example, liquid detergent compositions. Furthermore, organogellants can be used to stabilize other components within such compositions such as, for example, enzymes and bleaches. However, organogellants need to be selected carefully for their respective application in order to prevent incompatibilities between organogellant and other components as well as unwanted side effects such as clouding.
  • Organogellants of the present invention offer significant advantages over other gellants currently in use, such as being compatible with a broad range of consumer products as well as not affecting product clarity.
  • amino acid esters connected via an amide bond on their amino terminus to nicotinic acid (pyridine-3-carboxylic acid )or isonicotinic acid (pyridine-4-carboxylic acid ) result in better yields during protease mediated coupling to amino compounds than amino acid esters carrying other moieties on their amino terminus such as for example typical protecting groups Benzoyl- or Z- (Carboxybenzyl).
  • Protease mediated amide bond formation employing amino acid esters connected via an amide bond on their amino terminus to nicotinic acid or isonicotinic acid, accordingly, provides efficient access to organogellants and peptides.
  • R 1 and R 2 are independently selected from hydrogen atom, Ci-C 4 alkyl, Ci-C 4 hydroxyalkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20
  • L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl;
  • X and Y are independently selected from -OH, -NH 2 , -NHR 3 , -NR 3 R 4 ;
  • R 3 and R 4 are independently selected from C1-C6 alkyl and C 7 -C20 alkylaryl; n is selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20;
  • R A is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl.
  • An alkyl is a linear, branched, or cyclic hydrocarbon chain. It may also be a combination of linear, branched, and cyclic hydrocarbon chains.
  • a C n -C m alkyl is an alkyl having n to m carbon atoms.
  • An aryl is an aromatic hydrocarbon.
  • An aryl may be monocyclic or polycyclic. In the case of polycyclic aryls, the individual aromatic rings may be fused or may be connected by single carbon-carbon bonds. Examples of suitable aryls are phenyl, biphenyl, naphtyl, anthryl, or phenanthryl.
  • a C n -C m aryl is an aryl having n to m carbon atoms.
  • a heteroaryl is an aromatic hydrocarbon that contains 1 to 4 heteroatoms, preferably 1 to 2 heteroatoms. Heteroatoms are independently selected from nitrogen, oxygen, sulfur.
  • a heteroaryl may be monocyclic or polycyclic.
  • a heteroaryl may be attached to the main molecule through any of its carbon or nitrogen atoms.
  • a C n -C m heteroaryl is a heteroaryl having n to m carbon atoms and 1 to 4 heteroatoms.
  • An alkylaryl is an aryl that is substituted with one or more alkyls.
  • An alkylaryl may be attached to the remainder of the molecule through any of its alkyl or aryl carbon atoms.
  • a C n -C m alkylaryl contains n to m carbon atoms.
  • An a Iky I heteroaryl is a heteroaryl that is substituted with one or more alkyls.
  • the alkyl substituents may be attached to the heteroaryl through any of the carbon- or heteroatoms of the heteroaryl.
  • the alkylheteroaryl group may be attached to the remainder of the molecule through any of the alkyl carbon atoms and/or the heteroaryl carbon- or heteroatoms.
  • a hydroxyalkyl is an alkyl carrying one or more hydroxyl groups.
  • hydroxyalkyl group contains n to m carbon atoms.
  • a thioether is a moiety wherein two alkyls are linked by a thioether bond.
  • a C n -C m thioether group contains n to m carbon atoms in total. The thioether group may be attached to the remainder of the molecule through any of its carbon atoms.
  • An alkylhydroxyaryl is an alkylaryl, carrying hydroxyl groups on any of the aryl carbon atoms.
  • the alkylhydroxyaryl group may be attached to the remainder of the molecule through any of its alkyl and/or aryl carbon atoms.
  • a C n -C m alkylhydroxyaryl contains n to m carbon atoms.
  • An alkyl-C(O)Y is an alkyl carrying a C(O)Y-group, wherein C(O) is a carbonyl function and Y is selected from -OH, -NH 2 , -NHR 3 , -NR 3 R 4 ; and wherein R 3 and R 4 are independently selected from C1-C6 alkyl and C7-C20 alkylaryl.
  • a C n -C m alkyl- C(O)Y contains n to m carbon atoms within the carbonyl-bound alkyl excluding the carbonyl carbon atom itself.
  • Suitable solvents S are mixtures of from 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5.
  • Suitable aprotic organic solvents comprise dichloromethane, methyl terf-butyl ether, tetrahydrofuran, acetonitrile, 1 ,4-Dioxane, ethylene glycol dimethyl ether, methyl isobutyl ketone, terf-Butanol, methyl ethyl ketone, acetone or mixtures thereof.
  • terf-Butanol is considered as an aprotic organic solvent.
  • a suitable buffer in the context of the present invention is phosphate buffer.
  • suitable buffering agents are well known in the art. Suitable buffering agents comprise TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid), HEPES (4-2-hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid).
  • the buffers of the present invention contain 1 mM of calcium chloride. Further, preferably, the buffers of the present invention contain 1 %(w/v) of urea.
  • Suitable temperatures ⁇ are temperatures selected in relation to ⁇ , the temperature optimum of the protease P employed.
  • ⁇ the temperature optimum of a protease P is the temperature where the protease is most efficient at cleaving amide bonds in aqueous buffer.
  • the temperature optimum ⁇ is known for a number of proteases and can thus be determined from the literature by a person of skill.
  • the temperature optimum ⁇ of a protease P can be determined by a person of skill in the art with experiments regarding the temperature dependence of the velocity of proteolysis reactions involving model substrates of the respective protease.
  • Suitable temperatures ⁇ for performing the processes of the present invention are selected in the range from ( ⁇ - 50°C) to ( ⁇ + 10°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the
  • the process of the present invention is performed with the aid of a protease P.
  • the protease P can be any protease that is capable of forming the corresponding amide bond under the reaction conditions of the process of the present invention.
  • a person of skill in the art can for example perform an experiment analogous to examples 2, 3.1 1 or 3.12 as presented herein.
  • serine proteases and cysteine proteases are well suited for performing the process of the present invention, while aspartic acid proteases and metalloproteases are less/not suitable for performing the process of the present invention.
  • the protease P is a serine protease or a cysteine protease.
  • the protease P is a subtil isin-like serine protease (subtilisin-like serine proteases are defined in Siezen RJ and Leunissen JAM (1997) Protein Science 6: 501-523).
  • the protease P is selected from Achromopeptidase from Achromobacter lyticus, Ficin from fig tree latex, Papain from papaya latex, Protease (Subtilisin Carlsberg) from Bacillus licheniformis, Alcalase CLEA (Subtilisin) from Bacillus licheniformis, Protease from Streptomyces griseus, Proteinase K from Tritirachium album, Trypsin from bovine pancreas, a- Chymotrypsin from bovine pancreas, Clostripain from Clostridium histolyticum, Protease P "Amano" 6SD from Aspergillus melleus.
  • the protease P is Subtilisin, i.e. EC 3.4.21 .62 according to IUBMB nomenclature.
  • Reaction products according to formula (I) can be isolated from the reaction mixture according to standard procedures well known to a person of skill.
  • An exemplary procedure would be performed as follows: An amount of water about equal to the volume of the reaction mixture is added to the reaction mixture and the pH is adjusted to 1 -2 by addition of cone. HCI. The bulk of organic solvents is distilled off. The aqueous phase is then separated and washed with isopropyl acetate. The organic phases are discarded. The aqueous phase is concentrated and an amount of isopropyl acetate about equal to the volume of the concentrated aqueous phase is added and the pH is adjusted to 10-1 1 by addition of NaOH solution. Subsequently phase separation is performed at 70 °C.
  • the organic phase is separated and washed with water at 70 °C.
  • the organic phases are discarded.
  • the combined aqueous phases are cooled to 0 °C and the precipitate is filtered off, washed with cold isopropyl acetate and dried at 60 °C in vacuo.
  • L is selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl.
  • L is selected from C6-C12 linear alkyl, 1 ,4-dimethylcyclohexyl, xylene.
  • L is selected from C2-C20 alkyl.
  • R A is selected from C2-C20 alkyl, C7-C20 alkylaryl.
  • R A is selected from C2-C20 alkyl.
  • R 1 is selected from hydrogen atom, Ci-C 4 alkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl.
  • R 1 is selected from a hydrogen atom, an n-butyl group, a f-butyl group, a propyl group, a cyclopropyl group, an ethyl group, or one of the side chains of the amino acids alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, glutamine, asparagine.
  • R 1 is selected from one of the side chains of amino acids alanine, valine, leucine, isoleucine, or phenylalanine.
  • side chain refers to the substituent group attached to the a-carbon atom of an ⁇ -amino acid.
  • the side chains are methyl, isopropyl, isobutyl, sec-butyl, 2- thiomethyl-ethyl, benzyl, 4-hydroxybenzyl, 3-methylindol, hydroxymethyl, 1 - hydroxyethyl, carboxamidoethyl, carboxamidomethyl.
  • R 1 is selected from Ci-C 4 alkyl.
  • S13 mixture of 95% (v/v) to 99% (v/v) acetonitrile and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8
  • S14 mixture of 95% (v/v) to 99% (v/v) 1 ,4-Dioxane and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8
  • S15 mixture of 95% (v/v) to 99% (v/v) 99% (v/v) ethylene glycol dimethyl ether and 5% (v/v) to 1 % (v/v) aqueous buffer at a pH between pH 7 and pH
  • the temperature ⁇ is selected in the range from ( ⁇ - 30°C) to ( ⁇ + 5°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the
  • the temperature ⁇ selected.
  • the temperature ⁇ is selected in the range from ( ⁇ - 10°C) to ( ⁇ + 5°C) with the proviso that the reaction mixture used for performing the process should be in the liquid state at the temperature ⁇ selected.
  • the temperature ⁇ is selected as 37°C.
  • the protease P is a serine protease or a cysteine protease.
  • the protease P is a subtil isin-like serine protease (subtilisin-like serine proteases are defined in Siezen RJ and Leunissen JAM (1997) Protein Science 6: 501-523).
  • the protease P is selected from Achromopeptidase from Achromobacter lyticus, Ficin from fig tree latex, Papain from papaya latex, Protease (Subtilisin Carlsberg) from Bacillus licheniformis, Alcalase CLEA (Subtilisin) from Bacillus licheniformis, Protease from Streptomyces griseus, Proteinase K from Tritirachium album, Trypsin from bovine pancreas, a-Chymotrypsin from bovine pancreas, Clostripain from Clostridium histolyticum, Protease P "Amano" 6SD from Aspergillus melleus.
  • the protease P is Subtilisin, i.e. EC 3.4.21 .62 according to lUBMB nomenclature.
  • L selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 selected from hydrogen atom, Ci-C 4 alkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl,
  • one of X 1 , X 2 is nitrogen, the other is carbon,
  • the protease P is a serine protease or a cysteine protease.
  • L selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 selected from hydrogen atom, Ci-C 4 alkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl,
  • one of X 1 , X 2 is nitrogen, the other is carbon,
  • protease P is a subtil isin-like serine protease.
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 selected from hydrogen atom, Ci-C 4 alkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl,
  • X 1 is carbon and X 2 is nitrogen
  • the solvent S selected from S2 mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8
  • S3 mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S4 mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S5 mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • the protease P is a serine protease or a cysteine protease.
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 selected from hydrogen atom, Ci-C 4 alkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl,
  • X 1 is carbon and X 2 is nitrogen
  • the solvent S selected from S2 mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8
  • S3 mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S4 mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S5 mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • protease P is a subtil isin-like serine protease.
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 and R 2 independently selected from hydrogen atom, Ci-C 4 alkyl, Ci- C 4 hydroxyalkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C 4 -C2o alkyl heteroaryl,
  • one of X 1 , X 2 is nitrogen, the other is carbon,
  • X selected from -OH, -NH 2 , -NHR 3 , -NR 3 R 4 ;
  • R 3 and R 4 independently selected from C1-C6 alkyl and C 7 -C20 alkylaryl;
  • n selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;
  • the protease P is a serine protease or a cysteine protease.
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 and R 2 independently selected from hydrogen atom, Ci-C 4 alkyl, Ci- C 4 hydroxyalkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C 4 -C2o alkyl heteroaryl,
  • one of X 1 , X 2 is nitrogen, the other is carbon,
  • X selected from -OH, -NH 2 , -NHR 3 , -NR 3 R 4 ;
  • R 3 and R 4 independently selected from C1-C6 alkyl and C7-C20 alkylaryl; and with
  • n selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10;
  • protease P is a subtil isin-like serine protease.
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 and R 2 independently selected from hydrogen atom, Ci-C 4 alkyl, Ci- C 4 hydroxyalkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C 4 -C2o alkylheteroaryl,
  • X 1 is carbon and X 2 is nitrogen
  • n 0, 1 , 2, 3,
  • the solvent S selected from S2 mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8
  • S3 mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S4 mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S5 mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • the protease P is a serine protease or a cysteine protease.
  • R A selected from C2-C20 alkyl, C6-C20 aryl, C7-C20 alkylaryl, and with
  • R 1 and R 2 independently selected from hydrogen atom, Ci-C 4 alkyl, Ci- C 4 hydroxyalkyl, Ci-C 4 thioether, C6-C20 aryl, C7-C20 alkylaryl, C7-C20 alkylhydroxyaryl, C 4 -C2o alkyl heteroaryl,
  • X 1 is carbon and X 2 is nitrogen
  • n 0, 1 , 2, 3,
  • the solvent S selected from S2 mixture of 90% (v/v) to 99.9% (v/v) aprotic organic solvent and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 7 and pH 8
  • S3 mixture of 90% (v/v) to 99.9% (v/v) acetonitrile and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S4 mixture of 90% (v/v) to 99.9% (v/v) 1 ,4- Dioxane and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • S5 mixture of 90% (v/v) to 99.9% (v/v) ethylene glycol dimethyl ether and 10% (v/v) to 0.1 % (v/v) aqueous buffer at a pH between pH 6.5 and pH 8.5
  • protease P is a subtilisin-like serine protease.
  • R 2 is selected from a hydrogen atom or one of the side chains of amino acids alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, glutamine, asparagine.
  • R 2 is selected from one of the side chains of amino acids alanine, valine, leucine, isoleucine, or phenylalanine.
  • the expression “side chain” refers to the substituent group attached to the a-carbon atom of an ⁇ -amino acid.
  • the side chains are methyl, isopropyl, isobutyl, sec-butyl, 2-thiomethyl-ethyl, benzyl, 4-hydroxybenzyl, 3-methylindol, hydroxymethyl, 1 -hydroxyethyl,
  • ester substrates The amount of ester substrates, mono- and di-amide products and hydrolysis byproducts were measured by HPLC using a C18 column and acetonitrile /phosphate buffer pH2.3 as solvent.
  • Isonicotinoyl-L-valine methylester to Isonicotinoyl-L-valine and of Z-L-valine methylester to Z-L-valine was analysed after 22 hours by HPLC.
  • Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500 ⁇ of Isonicotinoyl-L-valine methylester and 950-990 ⁇ acetonitrile, depending on the volume of buffer were prepared.
  • 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10-50 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and l OOOrpm.
  • Substrate mixtures containing 225 ⁇ of 1 ,12-Diaminododecane, 500 ⁇ of Isonicotinoyl-L-valine methylester, the desired amount of additive and 950 ⁇ (with DMSO) or 990 ⁇ (with Urea) acetonitrile were prepared.
  • 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and l OOOrpm.
  • a substrate mixture containing 225 mole of 1 ,12-Diaminododecane, 500 ⁇ of Isonicotinoyl-L-valine methylester and 990 ⁇ acetonitrile was prepared.
  • 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at different temperatures and 1000rpm.
  • Substrate mixtures containing the desired amounts of 1 ,12-Diaminododecane and Isonicotinoyl-L-valine methylester and 950 ⁇ acetonitrile were prepared.
  • licheniformis preparation dissolved in 50 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added to the substrate mixture and the vials were shaken for 24h at 37°C and 1000rpm.
  • a substrate mixture containing 225 mole of 1 ,12-Diaminododecane, 500 ⁇ of Isonicotinoyl-L-valine methylester and 990 ⁇ acetonitrile was prepared.
  • the desired amount of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and 10 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added. The substrate mixture was added and the vials were shaken for 24h at 37°C and l OOOrpm.
  • a substrate mixture containing 225 mole of 1 ,12-Diaminododecane, 500 ⁇ of Isonicotinoyl-L-valine methylester and 950 ⁇ acetonitrile was prepared.
  • the desired amount of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and 50 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride) was added. The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.
  • Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500 ⁇ of the desired Isonicotinoyl-L-valine ester and 990 ⁇ acetonitrile were prepared.
  • 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.
  • Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500 ⁇ of the desired N-protected/acylated L-valine methylester and 990 ⁇ acetonitrile were prepared.
  • 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.
  • Substrate mixtures containing 225 mole of 1 ,12-Diaminododecane, 500 ⁇ of the desired N-protected/acylated L-valine phenylester and 990 ⁇ acetonitrile were prepared.
  • 5.0mg of Subtilisin Carlsberg from Bacillus licheniformis preparation was weighed in 1 .5ml vials and dissolved in 10 ⁇ phosphate buffer pH 7.5 (containing 250mM potassium phosphate and 1 mM calcium chloride). The substrate mixture was added and the vials were shaken for 24h at 37°C and 1000rpm.

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  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
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Abstract

La présente invention concerne des procédés pour la formation enzymatique de liaisons amide. Plus spécifiquement, l'invention concerne la formation de liaisons amide entre des esters d'acides aminés acylés sur l'extrémité amino par de l'acide nicotinique ou de l'acide isonicotinique et des composés amino à l'aide de protéases. L'invention concerne des procédés pour la formation d'amides à l'aide d'esters d'acides aminés, liés à des groupes nicotinoyle ou isonicotinoyle via une liaison amide sur l'extrémité amino, qui sont plus efficaces que les procédés de formation d'amides à l'aide d'esters d'acides aminés portant des groupes de protection standard tels que des groupes benzoyle ou Z sur leur extrémité amino. Dans un aspect, la présente invention concerne en outre la synthèse médiée par des protéases de composés organogélifiants (OG) tels que représentés ci-dessous, dans lesquels L représente un fragment de liaison de poids moléculaire de 14 g/mole à 500 g/mole, un parmi X1, X2 représente azote et l'autre représente oxygène et dans lesquels R1 sont des substituants de chaîne latérale.
EP13773248.3A 2012-11-01 2013-10-04 Procédé pour la formation enzymatique de liaisons amide Withdrawn EP2914734A1 (fr)

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PCT/EP2013/070714 WO2014067746A1 (fr) 2012-11-01 2013-10-04 Procédé pour la formation enzymatique de liaisons amide

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AU2017273857B2 (en) 2016-06-01 2021-08-19 Athira Pharma, Inc. Compounds
CN106366033B (zh) * 2016-09-07 2019-05-07 南京理工大学 一种双子型结构超分子凝胶因子及其制备方法
US11427814B2 (en) 2019-03-26 2022-08-30 Encodia, Inc. Modified cleavases, uses thereof and related kits
EP4127157A4 (fr) * 2020-03-24 2024-04-24 Encodia Inc Clivases dipeptidiques modifiées, utilisations correspondantes et kits correspondants

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US6677427B1 (en) * 2000-06-13 2004-01-13 Hercules Incorporated Enzyme-catalyzed polyamides and compositions and processes of preparing and using the same
DE102004058073A1 (de) * 2004-12-01 2006-06-08 Basf Ag Verfahren zur Herstellung einer wässrigen Polyamid-Dispersion
CA2792767C (fr) 2010-03-12 2014-07-08 The Procter & Gamble Company Gelifiant d'amidon a ph reglable utilisable dans des compositions de produit de consommation
WO2011112887A1 (fr) 2010-03-12 2011-09-15 The Procter & Gamble Company Gélifiant di-amido utilisé dans des compositions de produit de consommation

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WO2014067746A1 (fr) 2014-05-08

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