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WO2025073360A1 - Cascade biocatalytique pour la production de dérivés de tryptamine - Google Patents

Cascade biocatalytique pour la production de dérivés de tryptamine Download PDF

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Publication number
WO2025073360A1
WO2025073360A1 PCT/EP2023/077442 EP2023077442W WO2025073360A1 WO 2025073360 A1 WO2025073360 A1 WO 2025073360A1 EP 2023077442 W EP2023077442 W EP 2023077442W WO 2025073360 A1 WO2025073360 A1 WO 2025073360A1
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formula
mixture
protein
compound
range
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Stefan Payer
Mattia LAZZAROTTO
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Enzyan Biocatalysis GmbH
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Enzyan Biocatalysis GmbH
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    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • C07D209/16—Tryptamines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)

Definitions

  • the present invention relates to a method for producing a tryptamine derivative according to formula (I) and to a mixture comprising one or more tryptamine derivative ⁇ ) according to formula (I), wherein the tryptamine derivative ⁇ ) according to formula (I) in the mixture contain at least 10 % carbon of renewable sources.
  • Tryptamine derivatives are organic compounds, which include several members such as serotonin, melatonin as well as sumatriptan, psilocybin and psilocin.
  • a primary object of the present invention was thus to provide methods for producing psilocybin or other tryptamine derivatives, which do not include (or not all of) the disadvantages in the prior art.
  • step III mixing the compound(s) provided in step I) with the protein, and the nicotinamide cofactor, and optionally the amine nucleophile, provided in step II),
  • step IV) reacting the mixture obtained in step III) for a time in the range of from 1 to 48 h, preferably of from 5 to 40 h, particularly preferably of from 10 to 36 h, further preferably of from 18 to 30 h, more preferably of from 20 to 25 h, and at a temperature in the range of from 20 to 50 °C, preferably of from 22.5 to 40 °C, particularly preferably of from 25 to 30 °C, to obtain a reacted mixture,
  • step V) optionally: removing insoluble reaction components from the reacted mixture obtained after step IV), to obtain a purified mixture
  • VI) optionally: providing a protein with 4-hydroxyindole kinase activity and ATP, and mixing the protein and the ATP with the other components in step III), and/or with the reacted mixture obtained after step IV) or with the purified mixture obtained after step V), wherein in case the protein and the ATP are mixed with reacted mixture obtained after step IV) or with the purified mixture obtained after step V), the obtained mixture is reacted at a temperature in the range of from 20 to 50 °C, preferably of from 22.5 to 35 °C, particularly preferably of from 25 to 30 °C, for a time in the range of from 0.5 to 10 h, preferably of from 1 to 5 h, particularly preferably of from 1.5 to 2.5 h.
  • the tryptamine derivative according to formula (I) can be produced with the method according to the invention. This was particularly surprising, since the method according to the invention applies a reaction with protein with reductive ami- nase- or imine reductase activity, which is not present in the natural, e.g. fungal, biosynthesis of the tryptamine derivatives.
  • the or one reductive aminase or imine reductase provided in step II) or vi) is selected from the group consisting of imine reductase from Pyxidicoccus fallax (PfIRED), IRED023 and a reductive aminase with at least 90 %, preferably at least 91 %, preferably at least 92 %, preferably at least 93 %, preferably at least 94 %, preferably at least 95 %, preferably at least 96 %, preferably at least 97 %, preferably at least 98 %, preferably at least 99 % sequence identity to PfIRED or IRED023.
  • PfIRED Pyxidicoccus fallax
  • PfIRED refers to the protein described by the UniProt ID: A0A848LF04 (SEQ ID NO:1).
  • IRED023 refers to the protein described by the UniProt ID: A0A1 L9AVJ5 (SEQ ID NO:2).
  • the temperature (range) described for a reaction refers to the temperature of the reaction mixture.
  • a reaction is performed at a pH in the range of from 6 to 8.5, preferably of from 6.5 to 8, particularly preferably of from 6.75 to 7.75.
  • a reaction as described herein, is performed in an aqueous buffer. It is preferred that the reacted mixture comprises one or more of the solvents selected from the group consisting of water, DMSO, isopropanol, 2-methyl-2-butanol, methanol, ethanol, acetonitrile, glycerol, acetone, tert-butanol, methyl-tert-butyl-ether (MTBE), and n-heptane.
  • the solvents selected from the group consisting of water, DMSO, isopropanol, 2-methyl-2-butanol, methanol, ethanol, acetonitrile, glycerol, acetone, tert-butanol, methyl-tert-butyl-ether (MTBE), and n-heptane.
  • a reaction is stopped by addition of a base, particularly a strong base, or an acid, particularly a strong acid, or an excess of a water immiscible or miscible organic solvent or removal of the protein by filtration.
  • a base particularly a strong base
  • an acid particularly a strong acid
  • reaction conditions for a reaction may apply to any reaction described herein, independent of other reactions described herein.
  • the term “insoluble reaction components” refers to reaction components, which are not soluble in the reacted mixture obtained after step IV).
  • this term refers to precipitated reaction components in the reacted mixture.
  • the term refers to or includes cell debris and inorganic salts.
  • the term “removing insoluble reaction components from the reacted mixture” refers to removing some or all of the insoluble reaction components present in the reacted mixture.
  • the step of removing these components is or comprises a method selected from the group consisting of centrifugation, crystallization, solid-phase adsorption, filtration, reverse osmosis, and combinations thereof, particularly preferably centrifugation, further preferably centrifugation in the range of from 18.000 to 24.000 x g, preferably in the range of from 20.000 to 22.000 x g.
  • removing solid reaction components as described herein is performed with a method including a centrifugation step, wherein the supernatant is collected.
  • the term “protein with 4-hydroxyindole kinase activity”, as used herein, may refer to an enzyme characterized as 4-hydroxyindole kinase, but may also refer to an enzyme with a 4-hydroxyindole kinase activity even though the enzyme is not classified as a 4-hydroxyindole kinase.
  • the term refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 2.7.1 .222.
  • providing a protein with 4-hydroxyindole kinase activity may include or refer to providing a nucleic acid encoding such a protein, preferably an enzyme as described above.
  • the term “providing a protein with reductive aminase- or imine reductase activity, whereintinct an amine nucleophile is further provided” includes cases, in which the amine nucleophile is provided separately to the protein and/or together with another component of the reaction. The same applies accordingly to the nicotinamide cofactor and ATP.
  • the amount of the compound according to formula (II) provided in step I) is in a range of from 0.5 to 200 mM, preferably of from 1 to 150 mM, particularly preferably of from 5 to 100 mM, further preferably of from 10 to 50 mM, based on the total volume of the mixture obtained in step III).
  • the amount of the protein provided in step II) is in a range of from 0.1 to 50 mg/mL, preferably of from 1 to 30 mg/mL, particularly preferably of from 5 to 15 mg/mL, based on the total volume of the mixture obtained in step III).
  • the amount of the protein provided in step II) is at least 1 U/mL, preferably at least 1 .5 U/mL, particularly preferably at least 2 U/mL, based on the total volume of the mixture obtained in step III).
  • the amount of the protein provided in step II) is in a range of from 1 to 10 U/mL, preferably of from 1 .5 to 7.5 U/mL, particularly preferably of from 2 to 5 U/mL, based on the total volume of the mixture obtained in step III). It is preferred in the method according to the invention that in the mixture obtained in step III), the molar ratio of the compound of formula (II) to the amine nucleophile provided in step II) is in a range of from 1 :1 to 1 :5, preferably of from 1 :1 to 1 :4, particularly preferably of from 1 :1 to 1 :2.5.
  • the amount of the protein provided in step VI) is in a range of from 1 to 500 pL/mL, preferably of from 10 to 250 pL/mL, particularly preferably of from 50 to 150 pL/mL, based on the total volume of the mixture obtained in step III).
  • the amount of the protein provided in step VI) is at least 0.25 U/mL, preferably at least 0.5 U/mL, particularly preferably at least 0.75 U/mL, based on the total volume of the mixture obtained in step III).
  • the amount of the protein provided in step VI) is in a range of from 0.25 to 10 U/mL, preferably of from 0.5 to 7.5 U/mL, particularly preferably of from 0.75 to 5 U/mL, based on the total volume of the mixture obtained in step III).
  • the amount of the protein provided in step VI) is in a range of from 1 to 500 pL/mL, preferably of from 10 to 250 pL/mL, particularly preferably of from 50 to 150 pL/mL, based on the total volume of said mixture described in step VI) (i.e. the mixture of the reacted mixture obtained after step IV), the protein provided in step VI) and optionally the ATP provided in step VI)).
  • the amount of the protein provided in step VI) is at least 0.25 U/mL, preferably at least 0.5 U/mL, particularly preferably at least 0.75 U/mL, based on the total volume of said mixture described in step VI) (i.e. the mixture of the reacted mixture obtained after step IV), the protein provided in step VI) and optionally the ATP provided in step VI)). It is preferred in the method according to the invention that in the mixture described in step VI) (i.e. the mixture of the reacted mixture obtained after step IV), the protein provided in step VI) and optionally the ATP provided in step VI)). It is preferred in the method according to the invention that in the mixture described in step VI) (i.e.
  • the amount of the protein provided in step VI) is at least 0.25 U/mL, preferably at least 0.5 U/mL, particularly preferably at least 0.75 U/mL, based on the total volume of said mixture described in step VI) (i.e. the mixture of the purified mixture obtained after step V), the protein provided in step VI) and optionally the ATP provided in step VI)).
  • the amount of the protein provided in step VI) is in a range of from 0.25 to 10 U/mL, preferably of from 0.5 to 7.5 U/mL, particularly preferably of from 0.75 to 5 U/mL, based on the total volume of said mixture described in step VI) (i.e. the mixture of the purified mixture obtained after step V), the protein provided in step VI) and optionally the ATP provided in step VI)).
  • the amount of the ATP provided in step VI) is in a range of from 0.05 to 10 mM, preferably of from 0.1 to 5 mM, particularly preferably of from 0.5 to 1.5 mM, based on the total volume of the mixture obtained in step III). It is preferred in the method according to the invention that in the mixture described in step VI) (i.e.
  • the amount of the ATP provided in step VI) is in a range of from 0.05 to 10 mM, preferably of from 0.1 to 5 mM, particularly preferably of from 0.5 to 1 .5 mM, based on the total volume of said mixture described in step VI) (i.e. the mixture of the reacted mixture obtained after step IV), the ATP provided in step VI) and optionally the protein provided in step VI)).
  • the amount of the ATP provided in step VI) is in a range of from 0.05 to 10 mM, preferably of from 0.1 to 5 mM, particularly preferably of from 0.5 to 1 .5 mM, based on the total volume of said mixture described in step VI) (i.e. the mixture of the purified mixture obtained after step V), the ATP provided in step VI) and optionally the protein provided in step VI)).
  • the amount of the nicotinamide cofactor provided in step II) is in a range of from 0.01 to 10 mM, preferably of from 0.05 to 5 mM, particularly preferably of from 0.1 to 1 mM, based on the total volume of the mixture obtained in step 3).
  • the, one, two, three or more or all amine nucleophile(s) optionally provided in step vi) is I are selected from the group consisting of methylamine, dimethylamine, diethylamine, methylethylamine, cyclopropylamine, piperidine, and propargylamine.
  • step I) of the method according to the invention one or more compound(s) according to formula (II) are provided. It is preferred that the compound(s) are provided by another enzymatic reaction, which may be performed before or together with the reaction in step IV). It is thus preferred that providing one or more compound(s) according to formula (II) in step I) comprises the steps a) providing one or more compound(s) according to formula (III), wherein X is oxygen, and R 1 is COOH, and wherein R 4 , R 5 , R 6 , R 7 and R 8 are selected as described above, b) providing a protein with 2-oxoacid decarboxylase activity, c) mixing the compound(s) provided in step a) with the protein provided in step b), d) reacting the mixture obtained in step c) for a time in the range of from 0.5 to 48 h, preferably of from 5 to 40 h, particularly preferably of from 10 to 36 h, further preferably of from 18 to 30 h, more preferably
  • protein with 2-oxoacid decarboxylase activity may refer to an enzyme characterized as 2-oxoacid decarboxylase, but may also refer to an enzyme with a 2-oxoacid decarboxylase activity even though the enzyme is not classified as a 2-oxoacid decarboxylase.
  • the term refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 4.1.1.
  • providing a protein with 2-oxoacid decarboxylase activity may include or refer to providing a nucleic acid encoding such a protein, preferably an enzyme as described above.
  • step b) further includes providing a cofactor of the protein with 2-oxoacid decarboxylase activity, preferably thiamine diphosphate.
  • the amount of the compound according to formula (III) provided in step a) is in a range of from 0.5 to 200 mM, preferably of from 1 to 150 mM, particularly preferably of from 5 to 100 mM, further preferably of from 10 to 50 mM, based on the total volume of the mixture obtained in step a). It is preferred in the method according to the invention that in the mixture obtained in step c), the amount of the protein provided in step b) is at least 2 U/mL, preferably at least 5 U/mL, particularly preferably at least 7.5 U/mL, based on the total volume of the mixture obtained in step c).
  • the amount of the protein provided in step b) is in a range of from 2 to 50 U/mL, preferably of from 5 to 40 U/mL, particularly preferably of from 7.5 to 30 U/mL, based on the total volume of the mixture obtained in step c).
  • reaction providing one or more compound(s) according to formula (II), as described in steps a) to d), may be performed together with the reaction in step IV).
  • providing one or more compound(s) according to formula (II) in step I) includes steps a) to d).
  • a protein with reductive aminase- or imine reductase activity, and a nicotinamide cofactor, preferably NADPH, NADP + , NADH, NAD + or a mixture thereof, and optionally an amine nucleophile, are provided in step II).
  • the reaction temperature in step IV) corresponds to the reaction temperature in step d) and thus allows the reaction described in step d) to be obtained within step IV).
  • step I), III) and IV) providing one or more compound(s) according to formula (II); mixing the compound(s) with the provided protein); reacting the mixture) are performed together.
  • step I) and are mixed with the protein with reductive aminase- or imine reductase activity, as well as the the nicotinamide cofactor (step III) ) and optionally the amine nucleophile, which are already present.
  • the reaction then continues and converts the one or more compound(s) according to formula (II) to one or more compound(s) according to formula (I) (step IV) ).
  • steps IV) and d) are performed together, as described above.
  • step a) comprises the steps
  • the amount of the compound according to formula (IV) provided in step 1) is in a range of from from 0.5 to 200 mM, preferably of from 1 to 150 mM, particularly preferably of from 5 to 100 mM, further preferably of from 10 to 50 mM, based on the total volume of the mixture obtained in step 3).
  • the amount of the protein provided in step 2) is in a range of from 250 to 5,000 U/mL, preferably of from 400 to 3,000 U/mL, particularly preferably of from 500 to 2,000 U/mL, based on the total volume of the mixture obtained in step 3).
  • the reaction providing one or more compound(s) according to formula (III), as described in steps 1) to 4), may be performed together with the reaction in step d). Furthermore, these steps may also be performed together with the reaction in step IV).
  • a protein catalysing the transformation of an alpha-amino acid into a 2-oxoacid preferably an amino acid deaminase, an amino acid dehydrogenase or an amino acid transaminase is provided (step 2) ), a protein with 2-oxoacid decarboxylase activity is provided (step b) ), all components are mixed and reacted according to the conditions described in step d).
  • the one or more compound(s) according to formula (IV) is/are converted to one or more compound(s) according to formula (III).
  • the reaction then continues and converts the one or more compound(s) according to formula (III) to one or more compound(s) according to formula (II) (step d) ).
  • step 1) a protein catalysing the transformation of an alpha-amino acid into a 2-oxoacid, preferably an amino acid deaminase, an amino acid dehydrogenase or an amino acid transaminase is provided (step 2) ), a protein with 2-oxoacid decarboxylase activity is provided (step b) ), a protein with reductive aminase- or imine reductase activity, and a nicotinamide cofactor, preferably NADPH, NADP + , NADH, NAD + or a mixture thereof, and optionally an amine nucleophile, are provided (step II) ), all components are mixed and reacted according to the conditions described in step IV).
  • a protein catalysing the transformation of an alpha-amino acid into a 2-oxoacid preferably an amino acid deaminase, an amino acid dehydrogenase or an amino acid transaminase is provided (step 2)
  • the one or more compound(s) according to formula (IV) is/are converted to one or more compound(s) according to formula (III).
  • the reaction then continues and converts the one or more compound(s) according to formula (III) to one or more compound(s) according to formula (II) (step d) ).
  • the reaction then continues and converts the one or more compound(s) according to formula (II) to one or more compound(s) according to formula (I) (step IV) ).
  • steps IV), d) and 4) are performed together, as described above. In this case, it is preferred that the reaction is performed at the conditions as described for step IV).
  • step 1) of the method according to the invention one or more compound(s) according to formula (IV) are provided. It is preferred that the compound(s) are provided by another enzymatic reaction, which may be performed before or together with the reaction in step 4).
  • step 1) comprises the steps
  • step E) reacting the mixture obtained in step D) for a time in the range of from 0.5 to 48 h, preferably of from 5 to 40 h, particularly preferably of from 10 to 36 h, further preferably of from 18 to 30 h, more preferably of from 20 to 25 h, and at a temperature in the range of from 20 to 50 °C, preferably of from 22.5 to 40 °C, particularly preferably of from 25 to 30 °C, to obtain a reacted mixture comprising one or more compound(s) according to formula (IV).
  • the amount of the protein provided in step C) is at least 50 mU/mL, preferably at least 75 mU/mL, particularly preferably at least 100 mU/mL, based on the total volume of the mixture obtained in step D).
  • the amount of the protein provided in step C) is in a range of from 50 to 1 ,000 mU/mL, preferably of from 75 to 750 mU/mL, particularly preferably of from 100 to 500 mU/mL, based on the total volume of the mixture obtained in step D).
  • reaction providing one or more compound(s) according to formula (IV), as described in steps A) to E), may be performed together with the reaction in step 4). Furthermore, these steps may also be performed together with the reaction in step d). Furthermore, these steps may also be performed together with the reaction in step IV).
  • the one or more compound(s) according to formula (V) is/are converted to one or more compound(s) according to formula (IV).
  • the reaction then continues and converts the one or more compound(s) according to formula (IV) to one or more compound(s) according to formula (III) (step 4) ).
  • steps 4) and E) are performed together, as described above. In this case, it is preferred that the reaction is performed at the conditions as described for step 4).
  • step A) one or more compound(s) according to formula (V) are provided (step A) ), one or more compound(s) selected from L-serine, or L-threonine, a mixture of pyruvate and ammonium salts or a mixture of 2-oxobutyrate and ammonium salts, are provided (step B) ), a protein with tryptophan synthase activity is provided (step C) ), a protein catalysing the transformation of an alpha-amino acid into a 2-oxoacid, preferably an amino acid deaminase, an amino acid dehydrogenase or an amino acid transaminase is provided (step 2) ), a protein with 2-oxoacid decarboxylase activity is provided (step b) ), all components are mixed and reacted according to the conditions described in step d).
  • the one or more compound(s) according to formula (V) is/are converted to one or more compound(s) according to formula (IV).
  • the reaction then continues and converts the one or more compound(s) according to formula (IV) to one or more compound(s) according to formula (III) (step 4) ).
  • the reaction then continues and converts the one or more compound(s) according to formula (III) to one or more compound(s) according to formula (II) (step d) ).
  • steps d), 4) and E) are performed together, as described above. In this case, it is preferred that the reaction is performed at the conditions as described for step d).
  • step A) one or more compound(s) according to formula (V) are provided (step A) ), one or more compound(s) selected from L-serine, or L-threonine, a mixture of pyruvate and ammonium salts or a mixture of 2-oxobutyrate and ammonium salts, are provided (step B) ), a protein with tryptophan synthase activity is provided (step C) ), a protein catalysing the transformation of an alpha-amino acid into a 2-oxoacid, preferably an amino acid deaminase, an amino acid dehydrogenase or an amino acid transaminase is provided (step 2) ), a protein with 2-oxoacid decarboxylase activity is provided (step b) ), a protein with reductive aminase- or imine reductase activity, and a nicotinamide cofactor, preferably NADPH, NADP + , NADH, NAD +
  • the one or more compound(s) according to formula (V) is/are converted to one or more compound(s) according to formula (IV).
  • the reaction then continues and converts the one or more compound(s) according to formula (IV) to one or more compound(s) according to formula (III) (step 4) ).
  • the reaction then continues and converts the one or more compound(s) according to formula (III) to one or more compound(s) according to formula (II) (step d) ).
  • the reaction then continues and converts the one or more compound ⁇ ) according to formula (II) to one or more compound(s) according to formula (I) (step IV) ).
  • steps IV), d), 4) and E) are performed together, as described above. In this case, it is preferred that the reaction is performed at the conditions as described for step IV).
  • reaction steps may be performed together. It is thus of particular advantage in the method according to the present invention that it is not required to isolate (instable) intermediates for a further reaction. Thus, the method according to the invention significantly facilitates the production of tryptamine derivatives.
  • the reacting step 4) is performed together with reacting step E), preferably wherein the reaction is performed for a time in the range of from 0.5 to 48 h, preferably of from 5 to 40 h, particularly preferably of from 10 to 36 h, further preferably of from 18 to 30 h, more preferably of from 20 to 25 h, and at a temperature in the range of from 20 to 50 °C, preferably of from 22.5 to 40 °C, particularly preferably of from 25 to 30 °C, d) is performed together with reacting step 4) and optionally reacting step E), preferably wherein the reaction is performed for a time in the range of from 0.5 to 48 h, preferably of from 5 to 40 h, particularly preferably of from 10 to 36 h, further preferably of from 18 to 30 h, more preferably of from 20 to 25 h, and at a temperature in the range of from 20 to 50 °C, preferably of from 22.5 to 40 °C, particularly preferably of from 25 to 30 °C
  • reaction IV) is performed together with reacting step d) and optionally reacting step 4) and optionally reacting step E), or preferably wherein the reaction is performed for a time in the range of from 0.5 to 48 h, preferably of from 5 to 40 h, particularly preferably of from 10 to 36 h, further preferably of from 18 to 30 h, more preferably of from 20 to 25 h, and at a temperature in the range of from 20 to 50 °C, preferably of from 22.5 to 40 °C, particularly preferably of from 25 to 30 °C, or
  • VI) is performed together with reacting step IV) and optionally reacting step d) and optionally reacting step 4) and optionally reacting step E), preferably wherein the reaction is performed for a time in the range of from 0.5 to 48 h, preferably of from 5 to 40 h, particularly preferably of from 10 to 36 h, further preferably of from 18 to 30 h, more preferably of from 20 to 25 h, and at a temperature in the range of from 20 to 50 °C, preferably of from 22.5 to 40 °C, particularly preferably of from 25 to 30 °C.
  • tryptamine derivative(s) according to formula (I) refers to a compound as defined herein according to formula (I), wherein one or more chemical group(s) is/are protected by a protecting group.
  • the, one, two, three or more or all protecting group(s) is/are selected from the group consisting of 9-fluorenylmethyl carbamate (Fmoc-NRR'), t-butyl carbamate (Boc- NRR'), benzyl carbamate (Z-NRR', Cbz-NRR'), acetamide, trifluoroacetamide, phthalimide, benzylamine (Bn-NRR'), triphenylmethylamine (Tr-NRR'), benzylideneamine, p-toluenesul- fonamide (Ts-NRR'), N,N-dimethylhydrazone, 2-alkyl-1 ,3-oxazoline, methoxymethyl ether (MOM-OR), tetrahydropyranyl ether (THP-OR), t-butyl ether, allyl ether, benzyl ether (Bn- OR), t-butyld
  • the method according to the invention preferably further includes one or more step(s) of protecting one or more chemical group(s) of a tryptamine derivative(s) according to formula (I) with a protecting group, preferably as described above.
  • the nicotinamide cofactor can be recycled, for example during the reaction in step IV), and be used again.
  • the amount of required nicotinamide cofactor can be reduced.
  • the nicotinamide cofactor is NADPH, NADP + , NADH, NAD + or a mixture thereof, and is provided together with an enzyme catalysing the reaction from NADP+ to NADPH and/or the reaction from NAD+ to NADH, preferably wherein the enzyme is selected from the group consisting of phosphite dehydrogenase (PDH) (preferably of Pseudomonas stutzeri) glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, transhydrogenase, and mixtures thereof.
  • PDH phosphite dehydrogenase
  • step IV An exemplary reaction as in step IV), wherein the nicotinamide cofactor (for example NADPH) is recycled is shown below:
  • the nicotinamide cofactor is NADPH, NADP + , NADH, NAD + or a mixture thereof, and that the method according to the invention includes an electrochemical regeneration of NADH and/or NADPH from NADP+ and/or NAD+.
  • the amount of the enzyme catalysing the reaction from NADP+ to NADPH and/or the reaction from NAD+ to NADH is at least 500 U/mL, preferably at least 750 U/mL, particularly preferably at least 1 ,000 U/mL, based on the total volume of the mixture obtained in step III).
  • the ATP in optional step VI) can be recycled, for example during and/or after the reaction in step IV), and be used again.
  • the amount of required ATP can be reduced.
  • ATP is provided together with a kinase catalysing the reaction from ADP to ATP, and a phosphate donor, preferably wherein the, one, two, three or more or all kinase(s) is/are selected from the group consisting of polyphosphate kinases, adenylate kinases, acetate kinases, creatine kinases, carbamate kinases, pyruvate kinases, acid phosphatases and alkaline phosphatases, and/or preferably wherein the, one, two, three or more or all phosphate donor(s) is/are selected from the group consisting of polyphosphate, Graham’s salt, creatine phosphate, acetyl phosphate, phosphoenolpyruvate, carbamoyl phosphate, methoxycarbonyl phosphate, and methoxycarbonyl phosphate.
  • a kinase catalysing the reaction from ADP to ATP
  • the mixture does not comprise one, two, three or more or all compounds selected from oxalyl chloride ((COCI)2), lithium aluminium hydride (LiAIFU), triethyl amine (EtsN) phosphor oxychloride (POCH), methyl iodide, diethyl ether, n-heptane, tetrahydrofuran, dichloromethane, methanol, acetone, silica gel, aluminium salts or lithium salts.
  • oxalyl chloride (COCI)2)
  • LiAIFU lithium aluminium hydride
  • EtsN triethyl amine
  • POCH triethyl amine
  • methyl iodide diethyl ether
  • n-heptane n-heptane
  • tetrahydrofuran dichloromethane
  • dichloromethane methanol
  • acetone silica gel
  • aluminium salts or lithium salts
  • Example 1 Production of dimethyltryptamine from indole, varying several reaction conditions
  • a reaction mixture was provided comprising
  • Example 1.1 The approach of Example 1.1 was performed, however in this approach the reaction mixture instead contained 5 mg/mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed engineered tryptophan synthase beta-subunit from Thermotoga maritima (TmaTrpB_2F3) (400 mU/mL) and the mixture was reacted at a pH of 6.8. The obtained, reacted mixture was then analysed by HPLC-MS. The amount of the compounds of formulae (V), (IV), (III), (II), and (I) were compared to each other.
  • TmaTrpB_2F3 Thermotoga maritima
  • Example 1.1 The approach of Example 1.1 was performed, however in this approach the reaction mixture instead contained
  • Example 1.1 The approach of Example 1.1 was performed, however in this approach the reaction mixture instead contained
  • the mixture was reacted for 20 h at 25 °C and pH 7.5 and was shaken at 800 rpm. The obtained, reacted mixture was then analysed by HPLC-MS. The amount of the compounds of formulae (V), (IV), (III), (II), and (I) were compared to each other.
  • Example 2.1 The approach of Example 2.1 was performed, however in this approach the reaction mixture instead contained
  • Example 2.1 The approach of Example 2.1 was performed, however in this approach the reaction mixture instead contained
  • Example 2.1 The approach of Example 2.1 was performed, however in this approach the reaction mixture instead contained
  • Example 3 Varying amine nucleophiles and reductive aminases / imine reductases
  • a reaction mixture was provided comprising
  • the mixture was reacted for 20 h at 30 °C and pH 7.5 and was shaken at 800 rpm.
  • a panel of six reductive aminases and imine reductases was screened against seven primary and secondary amines to see if they can be used as nucleophiles in the reductive amination reaction with indole-3-acetaldehyde. Because this compound is unstable and spontaneously oxidizes to indole 3-acetic acid, the screening was carried out in the cascade assembly starting from indole. The screening was carried out in a 96-deep well plate, with duplicates for each amine I enzyme pair. For each enzyme, an amine blank was included. The outcome was assessed by flow-injection mass-spectrometry (FIA-MS), and positive hits were validated by LC-MS measurements.
  • FIA-MS flow-injection mass-spectrometry
  • reaction mixture comprising - M -
  • the mixture was reacted for 23 h at 30 °C and pH 7.5 and was shaken at 290 rpm.
  • a panel of six compounds according to formula (V) was screened against seven primary and secondary amines. For each of the combinations, PfIRED was used, except for the amine nucleophile methylethylamine, for which IRED023 was used.
  • the mixture was reacted for 22.5 h at 30 °C and pH 7.5 and was shaken at 800 rpm.
  • the mixture was reacted for 2 h at 30 °C.

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Abstract

La présente invention concerne un procédé de production d'un dérivé de tryptamine selon la formule (I) et un mélange comprenant un ou plusieurs dérivés de tryptamine selon la formule (I), le ou les dérivés de tryptamine selon la formule (I) dans le mélange contenant au moins 10 % de carbone de sources renouvelables.
PCT/EP2023/077442 2023-10-04 2023-10-04 Cascade biocatalytique pour la production de dérivés de tryptamine Pending WO2025073360A1 (fr)

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