Biocatalytic cascade for the production of tryptamine derivatives

Abstract

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(s) according to formula (I), wherein the tryptamine derivative(s) according to formula (I) in the mixture contain at least 10 % carbon of renewable sources.

Description

[0001] Biocatalytic cascade for the production of tryptamine derivatives

[0002] 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.

[0003] Serotonin and melatonin are well-known and important neurotransmitters, wherein particularly melatonin is involved in regulating the sleep-wake-cycle. Melatonin is thus used in the treatment of sleep disorders such as insomnia and circadian rhythm sleep disorders like delayed sleep phase disorder, jet lag disorder, and shift work disorder, but also as a sleeping aid in daily life.

[0004] Sumatriptan is frequently used to treat migraine headaches and cluster headaches, since it was found to be effective in reducing their intensities.

[0005] Psilocybin is a psychedelic prodrug compound, which itself is biologically inactive but is quickly converted to psilocin in the body. Psilocin has mind-altering effects, which are similar to those of LSD, mescaline, and DMT. The effects of psilocin include euphoria, visual and mental hallucinations, changes in perception, distorted sense of time, and perceived spiritual experiences. Therefore, the prodrug psilocybin has been selected as an interesting compound for the treatment of depression, particularly treatment-resistant depression. Today, psilocybin has obtained medical approval in some countries, whereas in other countries, such as Austria or Germany, the approval of this compound is intensely discussed. However, medical approval of psilocybin appears rather likely and is expected within the upcoming years. Considering suggested doses of psilocybin, it is expected that there will be an annual requirement of this compound in the range of from 500 to 1 ,000 kg.

[0006] Since medical approval procedures for further applications of psilocybin and similar compounds have been or will be initiated soon, a much higher annual requirement of this and similar compounds is expected.

[0007] Thus, there is already a high demand for tryptamine derivatives, which will intensely increase within the next years. Consequently, there is a strong need for methods for producing high amount of such compounds.

[0008] However, the efficiency of current methods for producing tryptamine derivatives requires improvement. Furthermore, current methods typically require large amounts of solvents or other reagents, which need to be disposed separately and under strict regulations.

[0009] One way of producing psilocybin in a synthetic process is a 4-step synthesis starting from 4-acetylindol:

[0010] With this approach it is possible to obtain psilocybin with a high purity (99.7 to 99.9 %), whereas the yield of psilocybin is rather low (17 %).

[0011] However, in this approach, dangerous and toxic chemicals such as oxalyl chloride ((COCI)2), lithium aluminium hydride (LiAIH4), triethyl amine (EtsN) and phosphor oxychloride (POCh) are required. Their reaction products, such as aluminium and lithium salts, need to be disposed under strict regulations. Furthermore, an isolation and purification of the intermediate products becomes necessary, which requires problematic solvents such as diethyl ether, n-heptane, tetrahydrofuran, dichloromethane, methanol and acetone, as well as further adjuvants such as silica gel. Thus, the amount of disposed solvents is very high (77 L per mol of the product, including up to 30 L of dichloromethane). Furthermore, the time for obtaining 1.2 kg of the product was reported as 226 h, which does not yet include the time for transferring the material, pauses and cleaning the equipment.

[0012] Since the educt 4-acetylindol is expensive and since the yield of psilocybin in this process is rather low, the costs for the obtained product are very high. For producing psilocybin on industrial scale, this disadvantage would need to be overcome. Furthermore, by selecting 4-acetylindol as educt, the product does not contain carbon of renewable sources.

[0013] Psilocybin is a natural compound, which is also synthesized by fungi of the genus Psilo- cybe. Thus, other approaches for obtaining psilocybin include growing and genetically modifying such fungi. The enzymes of this natural synthesis of psilocybin in these fungi have been isolated and characterized. In a first step, L-tryptophan is decarboxylated by the enzyme PsiD to tryptamine. Subsequently, tryptamine is enzymatically hydroxylated by PsiH and the introduced hydroxyl group is then phosphorylated by the kinase 4-hydroxytrypta- mine kinase (PsiK) using ATP. Finally, the methyl transferase PsiM successively introduces two methyl groups at the primary nitrogen atom to form psilocybin. The last step depends on the co-factor S-adenosyl-L-methionine (SAM), which provides the methyl groups to be transferred.

[0014] This approach has been described by Fricke et al., “Enzymatic Synthesis of Psilocybin", Angew. Chem. Int. Ed., 2017, 56, 12352.

[0015] Similar approaches, which are based on the fungal enzymes are also described in e.g. WO 2022 / 226493 A1 , US 2021 / 147888 A1 , WO 2022 / 047579 A1 , and WO 2021 / 052989 A1 .

[0016] Further metabolic engineering approaches have been developed, which are based on the above synthesis, and which apply parts of or variants of the process. In any of these approaches, the final step of SAM-dependent methylation is applied. For example, WO 2019 / 173797 A1 describes such an approach, which however requires a methylation step via SAM. WO 2023 / 043794 A1 describes another approach, which also requires a step of methylation.

[0017] However, the amount of SAM in fungal cells (or other microbial cells) is strictly limited. Thus, applying the above approaches inevitably includes a restriction of the produced amount of product. Adding SAM to the reaction may be an option to overcome this limitation, however, SAM is very expensive and thus increases the costs for producing psilocybin. Biocatalytic systems for recycling SAM are known and could theoretically be applied to overcome this deficiency. However, such systems require complex enzyme networks and / or toxic co-substrates such as methyl iodide.

[0018] A fermentative production of psilocybin from 4-hydroxyindole in E. coll via a biosynthetic pathway was published in 2019. Therein, only moderate product titers of 1 .16 g / L in a fed- batch process were achieved, corresponding to 36% molar yield. Fermentation in the eukaryotic host Aspergillus nidulans yielded even lower product titers of 110 mg / L. Recently, also the in vivo production of DMT and bufotenine from tryptophan or glucose with very low yields between 14 and 75 mg / L in E. coli was shown. However, these methods are rather complex and laborious and only low yields are obtained.

[0019] Moreover, high amounts of the fungi are necessary to obtain relevant amounts of psilocybin. For obtaining 1 kg psilocybin, approximately 50 to 200 kg dried fungal material are required. This causes high variation of the product quality, which complicates the use of the natural product as a medical drug.

[0020] 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.

[0021] The primary object of the present invention is solved by a method for producing a tryptamine derivative according to formula (I), formula (I), wherein R1is selected from the group consisting of hydrogen, deuterium, COOH, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, wherein R2is selected from the group consisting of hydrogen, -H2C-CH=CH2, and linear or branched Ci-Cs-alkyl groups, preferably -CH3, -CD3 (D: deuterium), -CH2CH3, -CH(CH3)2, -CH2CH2CH3, or -CH2CH2CH2CH3, wherein R3is selected from the group consisting of hydrogen, -(C=O)-CH3, -H2C-CH=-CH2, and linear or branched Ci-Cs-alkyl groups, preferably -CH3, -CD3 (D: deuterium), -CH2CH3, -CH(CH3)2, -CH2CH2CH3, or -CH2CH2CH2CH3, and / or wherein R2and R3together with the nitrogen atom, to which they are bound, form a 5-, 6- or 7-membered, mono- or polyunsaturated heterocyclic ring, wherein R4is selected from the group consisting of hydrogen, deuterium, -OH, -OCH3, - O(C2H5), -CN, -OPO3H2, O-(C=O)-CH3, halogens, preferably -F, -Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, wherein R5is selected from the group consisting of hydrogen, deuterium, -OH, -OCH3, - O(C2H5), -CN, -C(=O)NHCH3, -CH2SO2NHCH3, halogens, preferably -F, -Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, wherein R6is selected from the group consisting of hydrogen, deuterium, -OH, -OCH3, - O(C2H5), -CN, -C(=O)NHCH3, -CH2SO2NHCH3, halogens, preferably -F, -Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, and / or wherein R5and R6together with the carbon atoms, to which they are bound, form a 5-, 6- or 7-membered, mono- or polyunsaturated heterocyclic ring, such as R5and R6both being oxygen and being connected by -(CH2)n- to form a corresponding ring, with n=1 , 2, 3, or 4, wherein R7is selected from the group consisting of hydrogen, deuterium, -OH, -OCH3, - O(C2H5), -CN, -CH2SO2NHCH3, halogens, preferably -F, -Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, wherein R8is selected from the group consisting of hydrogen, deuterium, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, the method comprising the steps

[0022] I) providing one or more compound(s) according to formula (II), wherein X is selected from the group consisting of oxygen and N(R9)(R10), wherein R9and R10are independently selected from the group consisting of hydrogen, -H2C-CH=CH2, and linear or branched Ci-Cs-alkyl groups, preferably -CH3, -CD3(D: deuterium), -CH2CH3, -CH(CH3)2, -CH2CH2CH3, or - CH2CH2CH2CH3, and / or wherein R9and R10together with the nitrogen atom, to which they are bound, form a 5-, 6- or 7-membered, mono- or polyunsaturated heterocyclic ring, wherein R1, R4, R5, R6, R7and R8are selected as described above,

[0023] II) providing a protein with reductive aminase- or imine reductase activity, and a nicotinamide cofactor, preferably NADPH, NADP+, NADH, NAD+or a mixture thereof, wherein in case X in formula (II) is oxygen, an amine nucleophile is further provided,

[0024] 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),

[0025] 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,

[0026] V) optionally: removing insoluble reaction components from the reacted mixture obtained after step IV), to obtain a purified mixture,

[0027] 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.

[0028] It was surprisingly found that 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.

[0029] Moreover, it is of particular advantage that in the method according to the invention no problematic I toxic solvents or other components need to be applied. Thus, the method does not comprise oxalyl chloride ((COCI)2), lithium aluminium hydride (LiAIF ), triethyl amine (EtsN) phosphor oxychloride (POCH), methyl iodide, diethyl ether, n-heptane, tetrahydrofuran, dichloromethane, methanol, acetone, silica gel, aluminium salts or lithium salts.

[0030] Furthermore, the amount of disposed solvent is reduced to a minimum in the method according to the invention.

[0031] It is further advantageous of the method according to the invention that it is possible to start from less expensive educts compared to methods in the prior art.

[0032] An exemplary reaction as in step IV) (including the optional step VI) is shown below: formula (II) formula (I)

[0033] Preferably, the term “protein with reductive aminase- or imine reductase activity”, as used herein, may refer to an enzyme characterized as reductive aminase and / or imine reductase, but may also refer to an enzyme with a reductive aminase- and / or imine reductase activity even though the enzyme is not classified as a reductive aminase and / or imine reductase. Particularly preferably, the term refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 1 .4.1 or E.C. 1 .5.1 . Furthermore, the term “providing a protein with reductive aminase- or imine reductase activity” may include or refer to providing a nucleic acid encoding such a protein, preferably an enzyme as described above.

[0034] Particularly preferably, 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.

[0035] Preferably, the term “PfIRED” refers to the protein described by the UniProt ID: A0A848LF04 (SEQ ID NO:1).

[0036] Preferably, the term “IRED023” refers to the protein described by the UniProt ID: A0A1 L9AVJ5 (SEQ ID NO:2).

[0037] Preferably, the term “reacting [...] for a time [...] and at a temperature in the range of from as used herein, is to be understood such that for the time of the reaction the temperature is within the described range, wherein the temperature does not need to be constant but may vary within the described range. Preferably, the temperature is constant for the time of the reaction.

[0038] Preferably, a step of reacting at a described temperature (range), as described herein, starts as soon as the described temperature (range) is achieved.

[0039] Preferably, the temperature (range) described for a reaction refers to the temperature of the reaction mixture.

[0040] Preferably, a reaction, as described herein, 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.

[0041] Additionally or alternatively, it is preferred that 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.

[0042] Preferably, a reaction, as described herein, 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.

[0043] What is said herein with regard to reaction conditions for a reaction, as described herein, may apply to any reaction described herein, independent of other reactions described herein.

[0044] Preferably, the term “insoluble reaction components” refers to reaction components, which are not soluble in the reacted mixture obtained after step IV). Preferably, this term refers to precipitated reaction components in the reacted mixture. Particularly preferably, the term refers to or includes cell debris and inorganic salts.

[0045] Preferably, 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. Preferably, 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.

[0046] Further preferably, removing solid reaction components as described herein is performed with a method including a centrifugation step, wherein the supernatant is collected.

[0047] Furthermore, the method may also comprise a step

[0048] V.2) removing further side-products from the reacted mixture obtained after step IV).

[0049] Preferably, the term “removing further side-products from the reacted mixture” refers to removing some or all further side-products present in the reacted mixture. Preferably, the step of removing these components is or comprises a method selected from the group consisting of ion exchange chromatography, reversed-phase chromatography, normal phase chromatography, osmosis, size-exclusion chromatography (e.g. gel filtration), partitioning into water-immiscible organic solvents, precipitation by adjusting the pH-value and combinations thereof, preferably wherein the precipitation further includes removing the or some of the precipitates by a method selected from the group consisting of centrifugation, crystallization, solidphase adsorption, filtration, reverse osmosis, and combinations thereof.

[0050] Preferably, 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. Particularly preferably, 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.

[0051] Furthermore, the term “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.

[0052] Particularly preferably, the or one 4-hydroxyindole kinase provided in step VI) is 4-hydrox- ytryptamine kinase, preferably wherein the, one, two, three or all 4-hydroxytryptamine kinases is / are selected from the group consisting of 4-hydroxytryptamine kinase from Psilo- cybe cyanescens (pcy_PsiK), 4-hydroxytryptamine kinase from Psilocybe cubensis (pcu_PsiK) or 4-hydroxytryptamine kinases 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 pcy_PsiK or pcu_PsiK.

[0053] Preferably, the term “pcy_PsiK” refers to the protein described by the UniProt ID: A0A286LEZ6 (SEQ ID NO:3).

[0054] Preferably, the term “pcu_PsiK” refers to the protein described by the UniProt ID: P0DPA8 (SEQ ID NO:4).

[0055] The term “mixing the protein and the ATP with the other components in step III), or with the reacted mixture obtained after step IV) or with the purified mixture obtained after step V)” is to be understood such that the described components may be admixed in step III), and / or during and / or after step IV), and / or during and / or after step V). In case the described components are admixed in step III), the enzymatic reaction with regard to the 4-hydroxyindole kinase activity is obtained in the reaction in step IV).

[0056] In case the described components are admixed (at least) during or after step IV), and / or during or after step V), an enzymatic reaction with regard to the 4-hydroxyindole kinase activity may still be provided. Thus, the term “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” refers to those cases, in which the described components are admixed as described above, but in a step additionally to or other than step III).

[0057] Preferably, the term “providing a protein with reductive aminase- or imine reductase activity, wherein [...] 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.

[0058] It is preferred in the method according to the invention that in the mixture obtained in step III), 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).

[0059] It is preferred in the method according to the invention that in 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).

[0060] It is preferred in the method according to the invention that in 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).

[0061] It is preferred in the method according to the invention that in 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.

[0062] It is preferred in the method according to the invention that in 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 the mixture obtained in step III).

[0063] It is preferred in the method according to the invention that in 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).

[0064] It is preferred in the method according to the invention that in 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).

[0065] 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)), 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)).

[0066] 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)), 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)), 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 reacted mixture obtained after step IV), the protein provided in step VI) and optionally the ATP provided in step VI)).

[0067] It is preferred in the method according to the invention that in the 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 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 purified mixture obtained after step V), the protein provided in step VI) and optionally the ATP provided in step VI)).

[0068] It is preferred in the method according to the invention that in the 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 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)).

[0069] It is preferred in the method according to the invention that in the 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)).

[0070] It is preferred in the method according to the invention that in the mixture obtained in step III), 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 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 reacted mixture obtained after step IV), the ATP provided in step VI) and optionally the protein provided in step VI)).

[0071] It is preferred in the method according to the invention that in the 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 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)).

[0072] It is preferred in the method according to the invention that in the mixture obtained in step III), 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).

[0073] It is preferred that, when determining the molar ratio of components as described herein, in case a component refers to two or more compounds or substances, both or, respectively, all compounds or substances are considered for determining the molar ratio. For example, in case the molar ratio of the compound(s) according to formula (II) to the amine nucleophile is determined in a mixture and two compounds according to formula (II) are present in the respective mixture, the ratio of both compounds (summed) to the amine nucleophile needs to be determined.

[0074] Particularly preferably, 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.

[0075] In 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 R1is COOH, and wherein R4, R5, R6, R7and R8are 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 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 (II).

[0076] What was described above with regard to the method according to the invention applies accordingly to steps a) to d).

[0077] An exemplary reaction as in step d) is shown below: formula (III) formula (II)

[0078] Preferably, the term “protein with 2-oxoacid decarboxylase activity”, as used herein, 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. Particularly preferably, the term refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 4.1.1.

[0079] Furthermore, the term “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.

[0080] Preferably, step b) further includes providing a cofactor of the protein with 2-oxoacid decarboxylase activity, preferably thiamine diphosphate.

[0081] Particularly preferably, the or one protein with 2-oxoacid decarboxylase activity provided in step b) is a phenylpyruvate decarboxylase or an indolepyruvate decarboxylase, preferably wherein the, one, two, three or more or all protein(s) with 2-oxoacid decarboxylase activity provided in step b) or v) is / are selected from phenylpyruvate decarboxylases and / or indolepyruvate decarboxylases from Saccharomyces cerevisae, Azospirilium brasil- iense, Enterobacter cloacae or Klyuvera intermedia.

[0082] It is preferred in the method according to the invention that in the mixture obtained in step c), 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).

[0083] 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 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).

[0084] As described above, the 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).

[0085] In this case, 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). In this case, 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.

[0086] For example:

[0087] One or more compound(s) according to formula (III) is / are provided (step a) ). 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 compounds are mixed (step c) ) and reacted (step d) ). In the reaction, one or more compound(s) according to formula (II) are produced (i.e. provided; 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) ).

[0088] It is thus preferred that steps IV) and d) are performed together, as described above.

[0089] In step a) of the method according to the invention, one or more compound(s) according to formula (III) 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 d).

[0090] It is thus preferred that providing one or more compound(s) according to formula (III) in step a) comprises the steps

[0091] 1) providing one or more compound(s) according to formula (IV), formula (IV) wherein R1is COOH and R2and R3are hydrogen, and wherein R4, R5, R6, R7and R8are selected as described above,

[0092] 2) providing 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,

[0093] 3) mixing the compound(s) provided in step 1) with the protein provided in step 2),

[0094] 4) reacting the mixture obtained in step 3) 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 (III).

[0095] What was described above with regard to the method according to the invention applies accordingly to steps 1) to 4).

[0096] An exemplary reaction as in step 4) is shown below: formula (IV) formula (III)

[0097] Preferably, the term “protein catalysing the transformation of an alpha-amino acid into a 2- oxoacid”, as used herein, may refer to an enzyme characterized as amino acid deaminase, an amino acid dehydrogenase or an amino acid transaminase, but may also refer to an enzyme with such an activity even though the enzyme is not classified as an amino acid deaminase, an amino acid dehydrogenase or an amino acid transaminase. Particularly preferably, the term refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 1 .4.1 . or 1 .4.3. or 2.6.1 .

[0098] Furthermore, the term “providing a protein catalysing the transformation of an alpha-amino acid into a 2-oxoacid” may include or refer to providing a nucleic acid encoding such a protein, preferably an enzyme as described above.

[0099] Particularly preferably, the, one, two, three or all protein(s) catalysing the transformation of an alpha-amino acid into a 2-oxoacid provided in step 2) or iv) is / are selected from the group consisting of L-amino-acid deaminase, L-amino-acid dehydrogenase and L-amino- acid transaminase, preferably wherein the, one, two, three or all protein(s) catalysing the transformation of an alpha-amino acid into a 2-oxoacid provided in step 2) or iv) is / are selected from the group consisting of L-amino-acid deaminases from Proteus myxofaciens (PmaLAAD or PmaLAAD-W2) or from Proteus mirabilis (PmiLAAD), and L-amino-acid deaminases 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 PmaLAAD or PmaLAAD-W2 or PmiLAAD.

[0100] Preferably, the term “PmaLAAD” refers to the protein described by the UniProt ID: A0A158RFS7 (SEQ ID NO:5).

[0101] Preferably, the term “PmaLAAD-W2” refers to the protein described in Y. Wu, S. Zhang, W. Song, J. Liu, X. Chen, G. Hu, Y. Zhou, L. Liu, J. Wu, ChemCatChem 2021 , 13, 4557, https: / / doi.Org / 10.1002 / cctc.202101067.

[0102] Preferably, the term “PmiLAAD” refers to the protein described by the UniProt ID: Q51890 (SEQ ID NO:6).

[0103] It is preferred in the method according to the invention that in the mixture obtained in step 3), 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).

[0104] It is preferred in the method according to the invention that in the mixture obtained in step 3), the amount of the protein provided in step 2) is at least 250 U / mL, preferably at least 400 U / mL, particularly preferably at least 500 U / mL, based on the total volume of the mixture obtained in step 3).

[0105] It is preferred in the method according to the invention that in 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).

[0106] As described above, 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). Thus, preferably one or more compound(s) according to formula (IV) are provided (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) ), all components are mixed and reacted according to the conditions described in step d).

[0107] In the reaction, 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) ).

[0108] It is thus preferred that steps 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 d).

[0109] Further preferably one or more compound(s) according to formula (IV) are provided (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).

[0110] In the reaction, 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) ).

[0111] It is thus preferred that 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).

[0112] In 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).

[0113] It is thus preferred that providing one or more compound(s) according to formula (IV) in step 1) comprises the steps

[0114] A) providing one or more compound(s) according to formula (V), formula (V), wherein R4, R5, R6and R7are selected as described above,

[0115] B) providing 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, C) providing a protein with tryptophan synthase activity,

[0116] D) mixing the compound(s) provided in step A) with the compound(s) provided in step B) and with the protein provided in step C),

[0117] 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).

[0118] What was described above with regard to the method according to the invention applies accordingly to steps A) to E).

[0119] An exemplary reaction as in step E) is shown below: formula (V) formula (IV)

[0120] Preferably, the term “protein with tryptophan synthase activity”, as used herein, may refer to an enzyme characterized as protein with tryptophan synthase activity, but may also refer to an enzyme with tryptophan synthase activity even though the enzyme is not classified as a protein with tryptophan synthase. Particularly preferably, the term refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 4.2.1.

[0121] Furthermore, the term “providing a protein with tryptophan synthase activity” may include or refer to providing a nucleic acid encoding such a protein, preferably an enzyme as described above. Particularly preferably, the, or one or two or all protein(s) with tryptophan synthase activity provided in step C) or iii) is a tryptophan synthase beta-subunit, preferably from Thermotoga maritima (TmaTrpB_2F3) or a tryptophan synthase from Salmonella typhimurium (StTrpS) or a tryptophan synthase 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 TmaTrpB_2F3 or StTrpS.

[0122] Preferably, the term “TmaTrpB_2F3” refers to the protein described by the UniProt ID: P50909 (SEQ ID NO:7), or the P19G mutation thereof, or the I69V mutation thereof, or the K96L mutation thereof, or the P140L mutation thereof, or the N167D mutation thereof, or the L213P mutation thereof, or the T292S mutation thereof. Such mutations are for example described in J. Am. Chem. Soc. 2017, 139, 31 , 10769-10776, https: / / doi.org / 10.1021 / jacs.7b05007.

[0123] Preferably, the term “StTrpS” refers to the protein described by the UniProt ID: P0A2K1 (SEQ ID NO:8).

[0124] It is preferred in the method according to the invention that in the mixture obtained in step D), the amount of the compound according to formula (V) 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 D).

[0125] It is preferred in the method according to the invention that in the mixture obtained in step D), 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).

[0126] It is preferred in the method according to the invention that in 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).

[0127] It is preferred in the method according to the invention that in the mixture obtained in step D), the molar ratio of the compound(s) according to formula (V) to the compound provided in step B) is in a range of from 20:1 to 1 :20, preferably of from 10:1 to 1 :10, particularly preferably of from 5:1 to 1 :5, further preferably of from 2:1 to 1 :2.

[0128] As described above, the 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).

[0129] Further preferably 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) ), all components are mixed and reacted according to the conditions described in step 4).

[0130] In the reaction, 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) ).

[0131] It is thus preferred that 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).

[0132] Further preferably 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).

[0133] In the reaction, 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) ).

[0134] It is thus preferred that 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).

[0135] Further preferably 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+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).

[0136] In the reaction, 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) ).

[0137] It is thus preferred that 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).

[0138] As described above, the or some of the 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.

[0139] Furthermore, it is of particular advantage that with the method according to the invention, several or all steps may be performed at the same temperature. Thus, the time and the required energy for producing tryptamine derivatives is significantly reduced, since no extensive heating or cooling steps are required.

[0140] It is thus preferred that 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,

[0141] 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

[0142] 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.

[0143] In case one or more reactions are performed together, any indications for amounts or ratios on the basis of a mixture obtained before the respective reaction steps are then based on the mixture obtained before the reaction step, which is performed together, i.e. comprising all the components described for both reaction steps. For example, in case reaction step 4) is performed together with reacting step E) and amounts or ratios as described herein are based on the mixture obtained in step 3) or D), the amounts or ratios are then based on a mixture obtained in step D), comprising or consisting of the compounds provided in step 1), 2), A), B), and C). The same applies accordingly for mixtures after the respective reaction steps.

[0144] It is furthermore preferred in the method according to the invention that the term “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. Preferably, 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-butyldimethylsilyl ether (TBDMS-OR), t-butyldiphenylsilyl ether (TBDPS-OR), acetic acid ester, and pivalic acid ester.

[0145] Thus, 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.

[0146] It is furthermore preferred in the method according to the invention that the, one, two, three or more or all tryptamine derivative^) according to formula (I) is I are selected from the group consisting of

[0147]

[0148] It is advantageous in the method according to the invention that the nicotinamide cofactor can be recycled, for example during the reaction in step IV), and be used again. Thus, the amount of required nicotinamide cofactor can be reduced.

[0149] It is thus preferred that in step II) 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.

[0150] An exemplary reaction as in step IV), wherein the nicotinamide cofactor (for example NADPH) is recycled is shown below:

[0151] Particularly preferably, the enzyme catalysing the reaction from NADP+ to NADPH and / or the reaction from NAD+ to NADH is provided together with one or more co-substrate(s) for the reaction, preferably wherein the, one or more or all co-substrate(s) is / are selected from the group consisting of Na2HPOs, glucose, formate, isopropanol, and combinations thereof.

[0152] Additionally or alternatively, it is preferred that in step II) 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+.

[0153] It is preferred in the method according to the invention that in the mixture obtained in step III), 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).

[0154] It is preferred in the method according to the invention that in the mixture obtained in step III), the amount of the enzyme catalysing the reaction from NADP+ to NADPH and / or the reaction from NAD+ to NADH is in a range of from 500 to 5,000 U / mL, preferably of from 750 to 3,000 U / mL, particularly preferably of from 1 ,000 to 2,500 U / mL, based on the total volume of the mixture obtained in step III).

[0155] It is preferred in the method according to the invention that the mixture obtained in step III) further comprises the cofactor(s) for the reaction from NADP+ to NADPH and / or the reaction from NAD+ to NADH in an amount of from 0.1 to 75 mM, preferably of from 1 to 50 mM, particularly preferably of from 10 to 25 mM, based on the total volume of the mixture obtained in step 3).

[0156] It is further advantageous in the method according to the invention that the ATP in optional step VI) can be recycled, for example during and / or after the reaction in step IV), and be used again. Thus, the amount of required ATP can be reduced.

[0157] It is thus preferred that in step VI) 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.

[0158] An exemplary reaction as in optional step VI) and / or IV), wherein the ATP is recycled is shown below: formula (I)

[0159] It is preferred in the method according to the invention that in the mixture obtained in step III), the amount of the kinase catalysing the reaction from ADP to ATP is at least 50 U / mL, preferably at least 75 U / mL, particularly preferably at least 100 U / mL, based on the total volume of the mixture obtained in step III).

[0160] It is preferred in the method according to the invention that in the mixture obtained in step III), the amount of the kinase catalysing the reaction from ADP to ATP is in a range of from 50 to 500 U / mL, preferably of from 75 to 400 U / mL, particularly preferably of from 100 to 300 U / mL, based on the total volume of the mixture obtained in step III).

[0161] It is preferred in the method according to the invention that the mixture obtained in step III) further comprises the phosphate donor in an amount of from 1 to 300 mM, preferably of from 10 to 200 mM, particularly preferably of from 25 to 100 mM, based on the total volume of the mixture obtained in step 3).

[0162] The present invention further relates to a mixture comprising one or more tryptamine derivative^) according to formula (I), preferably obtainable or obtained by a method according to the invention, wherein the mixture, preferably the tryptamine derivative(s) according to formula (I) in the mixture contain at least 5 %, preferably at least 10 %, particularly preferably at least 12.5 %, further preferably at least 15 % carbon of renewable sources, based on the total carbon content of the mixture or, respectively, tryptamine derivative^) according to formula (I) in the mixture.

[0163] Advantageously in the method according to the invention, educts can be used, which originate from renewable sources. Thus, also the produced tryptamine derivative^) and further (side-)products of the method according to the invention comprised) carbon from renewable sources.

[0164] Preferably, whether carbon originates from renewable or fossil sources is determined by a method comprising or consisting of radiocarbon dating.

[0165] Preferably, for determining whether the carbon of the tryptamine derivative(s) according to formula (I) in the mixture originates from renewable or fossil sources, the tryptamine derivative^) according to formula (I) are isolated from the mixture and analysed with a method for determining whether carbon originates from renewable or fossil sources as described above.

[0166] Preferably, the mixture according to the invention comprises the tryptamine derivative^) according to formula (I) in an amount of from 10 to 99 wt.-%, preferably of from 20 to 95 wt.-%, further preferably of from 30 to 90 wt.-%, more preferably of from 50 to 80 wt.-%, even further preferably of from 60 to 75 wt.-%; based on the total weight of the mixture.

[0167] Preferably, the mixture according to the invention further comprises one or more components selected from the group consisting of compounds according to formula (II), compounds according to formula (III) compounds according to formula (IV) compounds according to formula (V), proteins with reductive aminase- or imine reductase activity, proteins with 4-hydroxyindole kinase activity, proteins with 2-oxoacid decarboxylase activity, proteins catalysing the transformation of an alpha-amino acid into a 2-oxoacid, proteins with tryptophan synthase activity, amine nucleophiles, nicotinamide cofactors, ATP, ADP, L- serine, L-threonine, pyruvate salts, and ammonium salts.

[0168] Preferably, the mixture according to the invention comprises less than 1 wt.-%, preferably less than 0.5 wt.-%, further preferably less than 0.1 , even further preferably less than 0.01 wt.-% of 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 and lithium salts, based on the total weight of the mixture. Particularly preferably, 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. What was said above with regard to the respective components, particularly preferred features, applies accordingly.

[0169] Preferably, whenever the present disclosure refers to sequence identities of nucleic acid or amino acid sequences in terms of percentages, such references are to values as can be calculated using EMBOSS Water Pairwise Sequence Alignments (nucleotides) (http: / / www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html) for nucleic acid sequences and EMBOSS Water Pairwise Sequence Alignments (proteins) (http: / / www.ebi.ac.uk / Tools / psa / emboss_water / ) for amino acid sequences, respectively. Local sequence alignment tools provided by the European Molecular Biology Laboratory (EMBL) European Bioninformatics Institute (EBI) use a modified Smith-Waterman algorithm (see http: / / www.ebi.ac.uk / Tools / psa / and Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195- 197). Furthermore, here, when performing the respective pairwise alignment of two sequences using the modified Smith-Waterman algorithm, reference is made to the default parameters currently given by EMBL-EBI. These are (i) for amino acid sequences: Matrix = BLOSUM62, Gap open penalty = 10 and Gap extend penalty = 0.5 and (ii) for nucleic acid sequences: Matrix = DNAfull, Gap open penalty = 10 and Gap extend penalty = 0.5.

[0170] Further aspects and advantages of the invention result from the subsequent description of preferred examples.

[0171] Examples

[0172] Example 1 : Production of dimethyltryptamine from indole, varying several reaction conditions

[0173] Example 1.1

[0174] A reaction mixture was provided comprising

[0175] 10 mM indole (i.e. compound according to formula (V)),

[0176] 12 mM L-serine,

[0177] 2.5 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) (200 mU / mL),

[0178] 5 mg / mL of a lyophilized E. coli whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (750 mU / mL),

[0179] 5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phenylpyruvate decarboxylase from Saccharomyces cerevisae (ScPPDC), (25 U / mL)

[0180] 10 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed imine reductase from Pyxidicoccus fallax (PfIRED) (2.362 U / mL),

[0181] 20 mM dimethylamine,

[0182] 0.5 mM NADP+,

[0183] 5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phosphite dehydrogenase from Pseudomonas stutzeri (PDH) (2060 U / mL),

[0184] 20 mM Na2HPO3, and furthermore, 50 mM KPi-buffer pH 7.5, DMSO (5% v / v), 0.75 mM MgCI2, and 0.5 mM Thiamine diphosphate.

[0185] 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.

[0186] It was thus found that dimethyltryptamine was obtained starting from indole.

[0187] Example 1 .2

[0188] The approach of Example 1.1 was performed, however in this approach the reaction mix- ture instead contained

[0189] 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 30 °C. 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.

[0190] It was found that a higher concentration of the tryptophan synthase and slightly increasing the temperature provided a higher yield of dimethyltryptamine. Example 1 .3

[0191] 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.

[0192] It was found that a higher concentration of the tryptophan synthase but slightly lowering the pH provided a slightly lower yield of dimethyltryptamine.

[0193] Example 1 .4

[0194] The approach of Example 1.1 was performed, however in this approach the reaction mixture instead contained

[0195] 2.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed tryptophan synthase from Salmonella typhimurium (StTrpS) (190 mU / mL),

[0196] 2.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phenylpyruvate decarboxylases from Saccharomyces cerevisae (ScPPDC) (12.5 U / 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.

[0197] It was found that StTrpS was less active than TmaTrpB_2F3. Furthermore, these conditions provided a lower yield of dimethyltryptamine.

[0198] Example 1 .5

[0199] The approach of Example 1.1 was performed, however in this approach the reaction mixture instead contained

[0200] 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)

[0201] 10 mg / mL of a lyophilized E. co / / whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (1.5

[0202] U / mL), isopropanol (5% v / v) instead of DMSO, and the mixture was reacted at 30 °C. 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.

[0203] It was found that the solvent DMSO can be changed with isopropanol without providing strong changes in the yield of dimethyltryptamine. Example 1 .6

[0204] The approach of Example 1.1 was performed, however in this approach the reaction mixture instead contained

[0205] 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)

[0206] 10 mg / mL of a lyophilized E. co / / whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (1.5 U / mL),

[0207] 0.5 mM NAD+instead of NADP+, and the mixture was reacted at 30 °C. 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.

[0208] It was found that the preferred nicotinamide cofactor for this IRED is NADPH / NADP+.

[0209] Example 2: Production of psilocin from 4-hydroxyindole, varying the L-amino-acid deaminase

[0210] Example 2.1

[0211] A reaction mixture was provided comprising

[0212] 10 mM 4-hydroxyindole (i.e. compound according to formula (V)),

[0213] 12 mM L-serine,

[0214] 2.5 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) (200 mU / mL), 5 mg / mL of a lyophilized E. coli whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (750 mll / mL),

[0215] 5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phenylpyruvate decarboxylase from Saccharomyces cerevisae (ScPPDC) (25 U / mL),

[0216] 10 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed imine reductase from Pyxidicoccus fallax (PfIRED) (2.362 U / mL),

[0217] 20 mM dimethylamine,

[0218] 0.5 mM NADP+,

[0219] 5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phosphite dehydrogenase from Pseudomonas stutzeri (PDH) (2060 U / mL),

[0220] 20 mM Na2HPO3, and furthermore, 50 mM KPi-buffer pH 7.5, DMSO (5% v / v), 0.75 mM MgCI2, and 0.5 mM Thiamine diphosphate.

[0221] 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.

[0222] It was thus found that psilocin was obtained starting from 4-hydroxyindole.

[0223] Example 2.2

[0224] The approach of Example 2.1 was performed, however in this approach the reaction mixture instead contained 10 mg / mL of a lyophilized E. co / / whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (1.5 U / mL), and the mixture was reacted at pH 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.

[0225] It was found that higher amounts of the PmaLAAD significantly increased the yield of psilocin. Example 2.3

[0226] The approach of Example 2.1 was performed, however in this approach the reaction mixture instead contained

[0227] 10 mg / mL of a lyophilized E. co / / whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (1.5 U / mL), and the mixture was reacted at 30 °C. 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. It was found that higher amounts of the PmaLAAD significantly increased the yield of psilocin.

[0228] Example 2.4

[0229] The approach of Example 2.1 was performed, however in this approach the reaction mixture instead contained

[0230] 10 mg / mL of a lyophilized E. coli whole-cell preparation containing a heterologously expressed engineered variant of L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD-W2), and the mixture was reacted at 30 °C. 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.

[0231] It was found that using the engineered variant PmaLAAD-W2 significantly increased the yield of psilocin.

[0232] Example 2.5

[0233] The approach of Example 2.1 was performed, however in this approach the reaction mixture instead contained

[0234] 10 mg / mL of a lyophilized E. co / / whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (1.5 U), 2.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed tryptophan synthase from Salmonella typhimurium (StTrpS) (153 mU), and the mixture was reacted at 30 °C. 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.

[0235] It was found that StTrpS was slightly less functional than TmaTrpB_2F3. Nevertheless the yield of psilocin was slightly increased.

[0236] Example 3: Varying amine nucleophiles and reductive aminases / imine reductases

[0237] A reaction mixture was provided comprising

[0238] 10 mM indole (i.e. compound according to formula (V)),

[0239] 12 mM L-serine,

[0240] 2.5 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) (200 mU / mL),

[0241] 10 mg / mL of a lyophilized E. co / / whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (1.5

[0242] U / mL),

[0243] 2.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phenylpyruvate decarboxylase from Saccharomyces cerevisae (ScPPDC) (12.5 U / mL),

[0244] 10 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed reductive aminase I imine reductase,

[0245] 20 mM amine nucleophile,

[0246] 0.5 mM NADP+,

[0247] 4 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phosphite dehydrogenase from Pseudomonas stutzeri (PDH) (1648 U / mL),

[0248] 20 mM Na2HPO3, and furthermore, 50 mM KPi-buffer pH 7.5, DMSO (5% v / v), 0.75 mM MgCh, and 0.5 mM Thiamine diphosphate.

[0249] The mixture was reacted for 20 h at 30 °C and pH 7.5 and was shaken at 800 rpm.

[0250] 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.

[0251] The following reductive aminases and imine reductases were tested:

[0252] IRED023, PfIRED, AspRedAm, IRED361 , NfRedAm, BaRedAm.

[0253] The following amine nucleophiles were tested:

[0254] A methylamine,

[0255] B dimethylamine,

[0256] C diethylamine,

[0257] D methylethylamine,

[0258] E cyclopropylamine,

[0259] F piperidine, and

[0260] G propargylamine.

[0261] It was found that PfIRED and IRED023, particularly PfIRED, provided the best results for most of the tested amine nucleophiles.

[0262] Furthermore, cyclopropylamine (E) and propargylamine (G) provided the best results.

[0263] Example 4: Varying amine nucleophiles and compounds of formula (V)

[0264] A reaction mixture was provided comprising - M -

[0265] 10 mM compound according to formula (V),

[0266] 12 mM L-serine,

[0267] 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),

[0268] 10 mg / mL of a lyophilized E. co / / whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (1.5 U / mL),

[0269] 2.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phenylpyruvate decarboxylase from Saccharomyces cerevisae (ScPPDC) (12.5 U / mL),

[0270] 10 mg / mL imine reductase from Pyxidicoccus fallax (PfIRED) (2.362 U / mL) or IRED023 (2.16 U / mL),

[0271] 20 mM amine nucleophile,

[0272] 0.5 mM NADP+,

[0273] 4 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phosphite dehydrogenase from Pseudomonas stutzeri (PDH) (1648 U / mL),

[0274] 20 mM Na2HPO3, and furthermore, 50 mM KPi-buffer pH 7.5, DMSO (5% v / v), 0.75 mM MgCI2, and 0.5 mM Thiamine diphosphate.

[0275] The mixture was reacted for 23 h at 30 °C and pH 7.5 and was shaken at 290 rpm.

[0276] 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.

[0277] 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, which yielded defined product mass peaks in 18 out of 19 cases.

[0278] The following compounds according to formula (V) were tested:

[0279] The following amine nucleophiles were tested:

[0280] A methylamine,

[0281] B dimethylamine,

[0282] C diethylamine,

[0283] D methylethylamine,

[0284] E cyclopropylamine,

[0285] F piperidine, and

[0286] G propargylamine.

[0287] It was found that indole (1 a) provided the best results.

[0288] Furthermore, cyclopropylamine (E) and propargylamine (G) provided the best results.

[0289] Example 5: Production of psilocybin from 4-hydroxyindole

[0290] Example 5.1 : Two-step approach

[0291] For converting 4-hydroxyindole (1e in Example 4) to psilocin, a reaction mixture was provided comprising

[0292] 10 mM 4-hydroxyindole (i.e. compound according to formula (V)),

[0293] 14 mM L-serine,

[0294] 2.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed tryptophan synthase from Salmonella typhimurium (StTrpS) (153 mll / mL),

[0295] 5 mg / mL of a lyophilized E. coli whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (750 mU / mL),

[0296] 2.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phenylpyruvate decarboxylase from Saccharomyces cerevisae (ScPPDC) (12.5 mU / mL), 10 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed imine reductase from Pyxidicoccus fallax (PfIRED) (2.362 U / mL), 20 mM dimethylamine,

[0297] 0.5 mM NADP+,

[0298] 5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phosphite dehydrogenase from Pseudomonas stutzeri (PDH) (2060 U / mL),

[0299] 20 mM Na2HPO3, and furthermore, 50 mM KPi-buffer pH 7.5, DMSO (5% v / v), 0.75 mM MgCI2, and 0.5 mM Thiamine diphosphate.

[0300] The mixture was reacted for 22.5 h at 30 °C and pH 7.5 and was shaken at 800 rpm.

[0301] The formation of psilocin was confirmed by HPLC-MS, and so was the absence of psilocybin at this stage. Solid reaction components were then removed by centrifugation at 21 .000 x g for 5 min and the supernatant was collected.

[0302] For subsequently converting psilocin to psilocybin, the supernatant was mixed with

[0303] 182 pL / mL of an E. coli cell-free extract preparation containing a heterologously expressed 4-hydroxytryptamine kinase from Psilocybe cubensis (pcu_PsiK), or 182 pL / mL of an E. coli cell-free extract preparation containing a heterologously expressed 4-hydroxytryptamine kinase from Psilocybe cyanescens (pcy_PsiK), creatine kinase from rabbit muscle (238 U / mL),

[0304] 1 mM ATP,

[0305] 50 mM creatine phosphate.

[0306] The mixture was reacted for 2 h at 30 °C.

[0307] The formation of psilocybin was confirmed by HPLC-MS and so was the absence of psilocin. Thus, psilocin was completely converted to psilocybin.

[0308] Example 5.2: One-step approach

[0309] For converting 4-hydroxyindole (1 e in Example 4) to psilocybin, a reaction mixture was provided comprising 7 mM 4-hydroxyindole (i.e. compound according to formula (V)),

[0310] 9.8 mM L-serine,

[0311] 1 .75 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed tryptophan synthase from Salmonella typhimurium (StTrpS) (133 mU / mL),

[0312] 3.5 mg / mL of a lyophilized E. coli whole-cell preparation containing a heterologously expressed L-amino-acid deaminase from Proteus myxofaciens (PmaLAAD) (525 mU / mL),

[0313] 1 .75 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phenylpyruvate decarboxylase from Saccharomyces cerevisae (ScPPDC) (8.75 U / mL),

[0314] 7 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed imine reductase from Pyxidicoccus fallax (PfIRED) (2.362 U / mL),

[0315] 14 mM dimethylamine,

[0316] 0.35 mM NADP+,

[0317] 3.5 mg / mL of a lyophilized E. coli cell-free extract preparation containing a heterologously expressed phosphite dehydrogenase from Pseudomonas stutzeri (PDH) (1142 U / mL),

[0318] 14 mM Na2HPO3, and furthermore, 50 mM KPi-buffer pH 7.5, DMSO (3.5% v / v), 0.53 mM MgCI2, and 0.35 mM Thiamine diphosphate.

[0319] Moreover, the mixture comprised

[0320] 200 pL / mL of an E. coli cell-free extract preparation containing a heterologously expressed 4-hydroxytryptamine kinase from Psilocybe cyanescens (pcy_PsiK), creatine kinase from rabbit muscle (119 U / mL),

[0321] 0.7 mM ATP,

[0322] 69.5 mM creatine phosphate.

[0323] The mixture was reacted for 17.5 h at 30 °C and pH 7.5 and was shaken at 800 rpm.

[0324] As a control, a blank, which did not include 4-hydroxytryptamine kinase was performed in parallel. The obtained, reacted mixture was then analysed by HPLC-MS. While the blank only yielded psilocin but no psilocybin, psilocybin was successfully produced in the full approach.

Claims

Claims1 . Method for producing a tryptamine derivative according to formula (I),formula (I), wherein R1is selected from the group consisting of hydrogen, deuterium, COOH, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, wherein R2is selected from the group consisting of hydrogen, -H2C-CH=CH2, and linear or branched Ci-Cs-alkyl groups, preferably -CH3, -CD3 (D: deuterium), -CH2CH3, -CH(CH3)2, -CH2CH2CH3, or -CH2CH2CH2CH3, wherein R3is selected from the group consisting of hydrogen, -(C=O)-CH3, -H2C- CH=-CH2, and linear or branched Ci-Cs-alkyl groups, preferably -CH3, -CD3 (D: deuterium), -CH2CH3, -CH(CH3)2, -CH2CH2CH3, or -CH2CH2CH2CH3, and / or wherein R2and R3together with the nitrogen atom, to which they are bound, form a 5-, 6- or 7-membered, mono- or polyunsaturated heterocyclic ring, wherein R4is selected from the group consisting of hydrogen, deuterium, -OH, - OCH3, -O(C2H5), -CN, -OPO3H2, O-(C=O)-CH3, halogens, preferably -F, -Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, wherein R5is selected from the group consisting of hydrogen, deuterium, -OH, - OCH3, -O(C2H5), -CN, -C(=O)NHCH3, -CH2SO2NHCH3, halogens, preferably -F, - Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or-CH2CH3, wherein R6is selected from the group consisting of hydrogen, deuterium, -OH, - OCH3, -O(C2H5), -CN, -C(=O)NHCH3, -CH2SO2NHCH3, halogens, preferably -F, - Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or-CH2CH3,and / or wherein R5and R6together with the carbon atoms, to which they are bound, form a 5-, 6- or 7-membered, mono- or polyunsaturated heterocyclic ring, such as R5and R6both being oxygen and being connected by -(CH2)n- to form a corresponding ring, with n=1 , 2, 3, or 4, wherein R7is selected from the group consisting of hydrogen, deuterium, -OH, - OCH3, -O(C2H5), -CN, -CH2SO2NHCH3, halogens, preferably -F, -Cl, -Br, or -I, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, wherein R8is selected from the group consisting of hydrogen, deuterium, and linear or branched Ci-Cs-alkyl groups, preferably -CH3 or -CH2CH3, the method comprising the stepsI) providing one or more compound(s) according to formula (II),wherein X is selected from the group consisting of oxygen and N(R9)(R10), wherein R9and R10are independently selected from the group consisting of hydrogen, -H2C-CH=CH2, and linear or branched Ci-Cs-alkyl groups, preferably -CH3, -CD3(D: deuterium), -CH2CH3, -CH(CH3)2, -CH2CH2CH3, or - CH2CH2CH2CH3, and / or wherein R9and R10together with the nitrogen atom, to which they are bound, form a 5-, 6- or 7-membered, mono- or polyunsaturated heterocyclic ring, wherein R1, R4, R5, R6, R7and R8are selected as described above,II) providing a protein with reductive aminase- or imine reductase activity, and a nicotinamide cofactor, preferably NADPH, NADP+, NADH, NAD+or a mixture thereof, wherein in case X in formula (II) is oxygen, an amine nucleophile is further provided,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),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,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, 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.

2. Method according to claim 1 , wherein 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),formula (III) wherein X is oxygen, and R1is COOH, and wherein R4, R5, R6, R7and R8are 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 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 (II).

3. Method according to claim 2, wherein providing one or more compound(s) according to formula (III) in step a) comprises the steps1) providing one or more compound(s) according to formula (IV),formula (IV) wherein R1is COOH and R2and R3are hydrogen, andwherein R4, R5, R6, R7and R8are selected as described above,2) providing 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,3) mixing the compound(s) provided in step 1) with the protein provided in step 2),4) reacting the mixture obtained in step 3) 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 (III).

4. Method according to claim 3, wherein providing one or more compound(s) according to formula (IV) in step 1) comprises the stepsA) providing one or more compound(s) according to formula (V),formula (V), wherein R4, R5, R6and R7are selected as described above,B) providing 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,C) providing a protein with tryptophan synthase activity,D) mixing the compound(s) provided in step A) with the compound(s) provided in step B) and with the protein provided in step C),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).

5. Method according to any of the preceding claims, wherein the reacting step4) 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,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, orVI) 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.

6. Method according to claim 1 , wherein the, one, two, three or more or all tryptamine derivative(s) according to formula (I) is / are selected from the group consisting of7. Method according to any one of the preceding claims, wherein the, or one or two or all protein(s) with tryptophan synthase activity provided in step C) or iii) is a tryptophan synthase beta-subunit, preferably from Thermotoga maritima (TmaTrpB_2F3) or a tryptophan synthase from Salmonella typhimurium (StT rpS) or a tryptophan synthase 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 TmaTrpB_2F3 or StTrpS.

8. Method according to any one of the preceding claims, wherein the, one, two, three or all protein(s) catalysing the transformation of an alpha-amino acid into a 2-oxoacid provided in step 2) or iv) is / are selected from the group consisting of L-amino-acid deaminase, L-amino-acid dehydrogenase and L-amino-acid transaminase, preferably wherein the, one, two, three or all protein(s) catalysing the transformation of an alpha-amino acid into a 2-oxoacid provided in step 2) or iv) is / are selected from the group consisting of L-amino-acid deaminases from Proteus myxofaciens (PmaLAAD or PmaLAAD-W2) or from Proteus mirabilis (PmiLAAD), and L-amino- acid deaminases 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 PmaLAAD or PmaLAAD-W2 or PmiLAAD.

9. Method according to any one of the preceding claims, wherein the or one protein with 2-oxoacid decarboxylase activity provided in step b) is a phenylpyruvate decarboxylase or an indolepyruvate decarboxylase, preferably wherein the, one, two, three or more or all protein(s) with 2-oxoacid decarboxylase activity provided in step b) or v) is / are selected from phenylpyruvate decarboxylases and / or indolepyruvate decarboxylases from Saccharomyces cere- visae, Azospirilium brasiliense, Enterobacter cloacae or Klyuvera intermedia.

10. Method according to any one of the preceding claims, wherein 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 IRED023.11 . Method according to any one of the preceding claims, wherein the or one 4-hydrox- yindole kinase provided in step VI) is 4-hydroxytryptamine kinase, preferably wherein the, one, two, three or all 4-hydroxytryptamine kinases is / are selected from the group consisting of 4-hydroxytryptamine kinase from Psilocybe cyanescens (pcy_PsiK), 4- hydroxytryptamine kinase from Psilocybe cubensis (pcu_PsiK) or 4-hydroxytrypta- mine kinases 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 pcy_PsiK or pcu_PsiK.

12. Method according to any one of the preceding claims, wherein the, one, two, three or more or all amine nucleophile(s) provided in step vi) is I are selected from the group consisting of methylamine, dimethylamine, diethylamine, methylethylamine, cyclopropylamine, piperidine, and propargylamine.

13. Method according to any one of the preceding claims, wherein in step II) 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, and / or wherein the method includes an electrochemical regeneration of NADH and / or NADPH from NADP+ and / or NAD+, preferably wherein the enzyme is selected from the group consisting of phosphite dehydrogenase (PDH), glucose dehydrogenase, alcohol dehydrogenase, formate dehydrogenase, transhydrogenase, and mixtures thereof, and / or wherein in step VI) 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.

14. Mixture comprising one or more tryptamine derivative(s) according to formula (I), preferably obtainable or obtained by a method according to one of the preceding claims, wherein the mixture, preferably the tryptamine derivative(s) according to formula (I) in the mixture contain at least 5 %, preferably at least 10 %, particularly preferably at least 12.5 %, further preferably at least 15 % carbon of renewable sources, based on the total carbon content of the mixture or, respectively, tryptamine derivative(s) according to formula (I) in the mixture.

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