SYNTHESIS OF (S)-6-HYDROXYTRYPTOPHAN AND DERIVATIVES THEREOF

Abstract

The present invention relates to novel processes and compounds for synthesizing amanitin derivatives. The invention relates in particular to processes for synthesizing (S)-hydroxytryptophan derivatives that can be used as building blocks in the synthesis of amanitin derivatives or amatoxin-drug conjugates. The invention also relates to intermediate compounds of said synthetic routes for use in the synthesis of amanitin derivatives and amatoxin-drug conjugates, as well as to the use of suitable catalysts for mediating said synthetic routes.

Description

SYNTHESIS OF (S)-6-HYDROXYTRYPTOPHAN AND DERIVATIVES THEREOF Field of invention The present invention relates to novel processes and compounds for synthesizing amanitin derivatives. The invention relates in particular to processes for synthesizing (S)-6-hydroxytryptophan derivatives that can be used as building blocks in the synthesis of amanitin derivatives or amatoxin-drug conjugates. The invention also relates to intermediate compounds of these synthetic routes for use in the synthesis of amanitin derivatives and amatoxin-drug conjugates, as well as to the use of suitable catalysts for mediating these synthetic routes. Background Amatoxins are cyclic peptides composed of eight amino acids found in the Amanita phalloides mushroom (see Fig. 1). Amatoxins specifically inhibit DNA-dependent RNA polymerase II in mammalian cells and, therefore, also inhibit transcription and protein biosynthesis in affected cells. Inhibition of transcription in a cell leads to the arrest of growth and proliferation. Although not covalently bound, the complex between amanitin and RNA polymerase II is very strong (KD = 3 nM). Dissociation of amanitin from the enzyme is a very slow process, making recovery of an affected cell unlikely. When the inhibition of transcription lasts long enough, the cell undergoes programmed cell death (apoptosis). Amatoxins can be isolated from the fruiting bodies of the Amanita phalloides mushroom, either harvested or from pure cultures (Zhang P, et al., FEMS Microbiol Lett. 15 Nov 2005; 252(2): 223-8. Epub 15 Sep 2005). However, the amounts of amatoxins that can be obtained are rather low (in the range of approximately 0.3-3 mg / g dry matter from natural fruiting bodies, and approximately 10% from a pure culture), and the flexibility to further modify natural amatoxin variants is limited. Alternatively, amatoxins can be obtained by fermentation using a basidiomycete (Muraoka S, and Shinozawa T., J Biosci Bioeng. 2000; 89(1): 73-6) or A. fissa (Guo XW, et al., Jun 2006; 46(3): 373-8). Yields are also low, and the flexibility to further modify the natural amatoxin variants is likewise limited.Finally, amatoxins have been prepared by total or partial synthesis (e.g., Zanotti G, Mahringer C, and Wieland T., Int J. Pept Protein Res. Oct 1987; 30(4): 450-9; Zanotti G, Wieland T, Benedetti E, Di Blasio B, Pavone V, and Pedone C., Int J. Pept Protein Res. Sep 1989; 34(3): 222-8). Alternatively, the use of fully synthetic routes to produce amatoxins can provide the larger quantities of amatoxins required for therapeutic uses and can enable the construction of a number of new amatoxin variants by using appropriate starting materials as building blocks. Naturally occurring amanitins such as α-amanitin, β-amanitin, and γ-amanitin comprise a phenolic hydroxyl (-OH) group at the 6' position of tryptophan, which represents amino acid 4 in the cyclic octapeptide of amanitin. This group allows the attachment of a linker to amatoxin (see Fig. 1). Target-binding macromolecules, such as antibodies or aptamers, can then be attached via this linker to generate conjugates, for example, antibody-drug conjugates. The use of amatoxins as cytotoxic debris for tumor therapy had already been explored in 1981 by coupling an anti-Thy 1.2 antibody to α-amanitin using a connector attached to the indole ring of tryptophan (Trp, amino acid 4; see figure 1) by diazotization (Davis & Preston, Science 1981,213, 1385-1388). With the fully synthetic amanitin compounds currently under investigation, the incorporation of a functionalized tryptophan for coupling elements has only been achieved very recently. Prior to this, the coupling elements were predominantly coupled to fully synthetic amanitins at the amino acid 1 (aspartic acid) position or at the indole nitrogen (N1) of amino acid 4 (tryptophan). Since the biological activity profiles of amanitins coupled at different anchoring positions in the amanitin structure have been shown to be very different, it is of great interest to be able to provide synthetic tryptophan hydroxylated at the 6' position—that is, corresponding to natural α-amanitin, β-amanitin, or γ-amanitin—as a building block that can be incorporated during amanitin synthesis. The synthetic introduction of tryptophan into the amanitin structure can be carried out by the Savige-Fontana reaction (Savige & Fontana, rccQnn / Lznz / e / YiAi 1980, Int. J. Pept. Protein. phies. 15(3): 285-97). According to this reaction, tryptophan is converted into a mixture of cis-2-carboxy-3a-hydroxy-1,2,3,3a,8,8ahexahydropyrrolo[2,3-b]indole and trans-2-carboxy-3a-hydroxy-1,2,3,3a,8,8ahexahydropyrrolo[2,3-b]indole protected with Boc and is subsequently incorporated into the amino acid sequence of the linear precursor of amanitin. For the fully synthetic production of amanitin, the synthesis of (S)-6-hydroxytryptophan and the synthesis of its respective building blocks are of essential importance. In particular, one of these essential building blocks is (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550). CO, H / \iHBoc AcO' HDP 30,2550 To date, no efficient and satisfactory prior art synthetic route for (S)-6-hydroxythiptophan and its building blocks has been described. In particular, no synthetic route has been available that provides the necessary purity of the (L or S) enantiomers of this amino acid derivative. Several basic synthetic options could be considered. One option could be crystallization of the racemic form with chiral acids or bases. However, only a maximum yield of 50% could be obtained with this procedure. A second option is enzymatic production; however, this method is time-consuming, the results are uncertain, and reproducibility is low. An object of the present invention, therefore, is to provide an efficient, simple, and reproducible process for synthesizing (S)-6-hydroxytryptophan, its derivatives, and building blocks. Preferably, an object of the present invention is to provide an efficient process for synthesizing (S)-6-acetyloxy-N-tert-butoxycarbonyltryptophan (HDP 30.2550) with a sufficiently high enantiomeric purity. As a further object of the present invention, the use of said (S)-6-hydroxytryptophan and its derivatives, preferably (S)-6-acetyloxyN-tert-butoxycarbonyl-tryptophan, as building blocks for the fully synthetic production of amatoxins is to be provided. These and other objects are obtained by the procedures and means according to the independent claims of the present invention. The dependent claims relate to specific embodiments. Summary of the invention The present invention provides processes for the synthesis of (S)-6-hydroxytryptophan, (S)-6-acetyloxy-N-tert-butoxycarbonyltryptophan, and their derivatives, and building blocks for their synthesis. The present invention further provides compounds and building blocks for use in the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates. The invention and the general advantages of its features will be discussed in more detail below. Description of the figures Figure 1 shows the structural formulas of different amatoxins. The numbers in bold (1 to 8) designate the conventional numbering of the eight amino acids that make up the amatoxin. The conventional designations of the atoms in amino acids 1, 3, and 4 are also shown (Greek letters aay, Greek letters aa δ, and numbers 1' to 7, respectively). Fig. 2 illustrates the synthesis of compound HDP 30.2822. Fig. 3 illustrates the synthesis of compound HDP 30.2758. Fig. 4 illustrates the synthesis of compounds HDP 30.2550 and HDP 30.2555. Fig. 5 shows the structural compositions of the catalysts used for the conversion of compound HDP 30.2824 to HDP 30.2826. Fig. 6 shows the results of the 1H NMR spectroscopy of compound HDP 30.2826 (400 MHz, CDCIs, δ = ppm). Fig. 7 shows the results of the 13C NMR spectroscopy of compound HDP 30.2826 (100 MHz, CDCh, δ = ppm). Detailed description of the invention Before describing the invention in detail, it should be understood that the present invention is not limited to the particular elements of the described means or the process steps of the described procedures, since such means and procedures may vary. It should also be understood that the terminology used in the present invention is intended to describe particular embodiments only and is not intended to be limiting. It should be noted that, as used in the specification and in the appended claims, the singular forms a, an, an, the, and a include singular and / or plural referents unless the context clearly indicates otherwise. Furthermore, it should be understood that where parameter ranges are defined by numerical values, those ranges are considered to include the limiting values. It should also be understood that the embodiments disclosed herein are not intended to be considered as unrelated, isolated embodiments. The characteristics discussed with respect to one embodiment are intended to be disclosed also with respect to other embodiments shown herein. If, in one instance, a specific characteristic is not disclosed with respect to one embodiment but is disclosed with respect to another, a person skilled in the art will understand that this does not necessarily mean that the characteristic is not intended to be disclosed with respect to the other embodiment. A person skilled in the art will understand that the key to this application is to disclose the characteristic for the other embodiment as well, even though this has not been done for the sake of clarity and to keep the application to a manageable length. Furthermore, the content of prior art documents referenced herein is incorporated herein by reference. This applies in particular to prior art documents that disclose conventional or routine procedures. In such cases, the primary purpose of incorporation by reference is to provide sufficient enabling disclosure and avoid tedious repetition. Large-scale, industrially feasible production of (S)-6-hydroxytryptophan, as comprised in natural α-, β-, and γ-amanitin, and / or chemically protected forms thereof, requires an efficient, simple, and reproducible synthesis procedure. For the first time, the inventors were able to provide such a simple and efficient production procedure by employing enantioselective hydrogenation of olefinic amino acid precursors. Using specific chiral catalysts, they surprisingly discovered that this type of reaction could yield very high enantiomeric purity. rccQnn / Lznz / e / YiAi The disclosed simple synthetic route yields an (S)-6-hydroxytryptophan that is entirely identical to the corresponding natural structure. The ability to use this compound and protected forms thereof for large-scale industrial production of amanitin will considerably reduce costs and increase the efficiency of, for example, manufacturing synthetic α-, β-, or γ-amanitin as well as amatoxin-based antibody-drug conjugates for therapeutic applications. According to a first aspect, the present invention relates to a process for synthesizing (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550) or (S)-6-hydroxytryptophan, said process comprising at least one enantioselective hydrogenation step of an olefinic precursor compound using at least one chiral catalyst. In an embodiment of the claimed process, the olefinic precursor used for the asymmetric hydrogenation is an unsaturated amino acid olefinic precursor. Preferably, it is compound HDP 30.2824. Gbz cbz rccQnn / Lznz / e / YiAi HDP 38.2824 HDP 30.282$ In one embodiment of the claimed process, said olefinic precursor is synthesized using the following compound: HDP 30.2822 This olefinic precursor can be further synthesized using any of compounds A, B, or C. In a further embodiment of the claimed process, said process comprises at least the following steps: HDP 30.2803 HDP 30.2824 HDP 30.2550 HDP 30.2832 HDP 30.2826 In another further embodiment of the claimed process, said process comprises at least the following steps: (S)-6 - h id ro xi -ir ip tófa no rccQnn / Lznz / e / YiAi In the context of the present invention, the term “amatoxin” includes all cyclic peptides composed of 8 amino acids isolated from the genus Amanita and described in Wieland, T. and Faulstich H. (Wieland T, Faulstich H.„ CRC Crit Rev Biochem. 5 (1978) 185-260), plus all chemical derivatives thereof; plus all semisynthetic analogues thereof; plus all synthetic analogues thereof constructed from building blocks according to the original structure of the natural compounds (cyclic, 8 amino acids); plus all synthetic or semisynthetic analogues containing non-hydroxylated amino acids instead of hydroxylated amino acids; plus all synthetic or semisynthetic analogues in which the thioether sulfoxide moiety is substituted by a sulfide, sulfone, thioether, or by atoms other than sulfur, for example, a carbon atom as in a carbon analogue of the amanitin. Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferential amatoxins are those with a functional group (e.g., a carboxyl group, an amino group, a hydroxyl group, or a thiol or thiol-capturing group) that can react with linker molecules or target-binding molecules, as defined above. In the context of the present invention, the term amanitins refers particularly to the bicyclic structure which is based on an aspartic acid or asparagine residue in position 1, a proline residue, particularly a hydroxyproline residue, in position 2, an isoleucine, hydroxyisoleucine or dihydroxyisoleucine in position 3, a tryptophan or hydroxytryptophan residue in position 4, glycine residues in positions 5 and 7, an isoleucine residue in position 6 and a cysteine ​​residue in position 8, in particular a cysteine ​​derivative which is oxidized to a sulfoxide or sulfone derivative (for numbering and representative examples of amanitins see Figure 1) and also includes all chemical derivatives thereof; furthermore all semisynthetic analogues thereof;Furthermore, all synthetic analogues thereof constructed from building blocks according to the original structure of the natural compounds (cyclic, 8 amino acids), furthermore all synthetic or semisynthetic analogues containing non-hydroxylated amino acids instead of hydroxylated amino acids, furthermore all synthetic or semisynthetic analogues in which, in each case, said derivative or analogue is functionally active by inhibiting mammalian RNA polymerase II. The term target-binding residue, as used herein, refers to any molecule or part of a molecule that can specifically bind to a target molecule or target epitope. Preferred target-binding residues in the context of the present invention are (i) antibodies or antibody-binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers. Target-binding residues suitable for use in the present invention typically have a molecular mass of 40,000 Da (40 kDa) or higher. A connector in the context of the present invention refers to a molecule that increases the distance between two components, for example, to alleviate spherical interference between the target-binding residue and amatoxin, which might otherwise decrease the ability of amatoxin to interact with RNA polymerase II. The connector may serve another purpose by facilitating the release of amatoxin specifically from the cell targeted by the target-binding residue. Preferably, the connector, and preferably the linkage between the connector and amatoxin on one side and the linkage between the connector and the antibody on the other, is stable under physiological conditions outside the cell, for example, in the blood, while being cleaved inside the cell, particularly within the target cell, for example, a cancer cell or an immune cell.To provide this selective stability, the connector may comprise functionalities that are preferentially pH-sensitive or protease-sensitive. Alternatively, the link connecting the connector to the target-binding moiety may provide selective stability. Preferably, a connector has a length of at least 1, and preferably 1–30 atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms), in which one side of the connector has reacted with amatoxin and the other side with a target-binding moiety. In the context of the present invention, a connector is preferably a C1-30 alkyl group, C1-30 heteroalkyl, C2-30 alkenyl, C2-30 heteroalkenyl, C2-30 alkynyl, C2-30 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl, optionally substituted.The connector may contain one or more structural elements such as amide, ester, ether, thioether, disulfide, hydrocarbon, and similar moieties. The connector may also contain combinations of two or more of these structural elements. Each of these structural elements may be present in the connector more than once, for example, two, three, four, five, or six times. In some embodiments, the connector may comprise a disulfide bond. It is understood that the connector is to be attached in a single step or in two or more subsequent steps to the amatoxin and the target-binding moiety. To this end, the connector shall have two groups, preferably at the proximal and distal ends, which (i) can form a covalent bond with a group, preferably an activated group of an amatoxin or the target-binding peptide, or (ii) are activated or can be activated to form a covalent bond with a group of an amatoxin.Accordingly, if the connector is present, it is preferable that the chemical groups be at a distal and proximal end of the connector, resulting from such a coupling reaction, for example, an ester, an ether, a urethane, a peptide bond, etc. The presence of a connector is optional; that is, the toxin may be directly linked to a residue of the target-binding residue in some embodiments of the target-binding residue-toxin conjugate. According to a second aspect, the present invention relates to (S)6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550), to (S)-6-hydroxy5 tryptophan or to any of the precursor compounds according to the present invention for use in the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates. In one embodiment, the present invention relates to a dehydroamino acid compound selected from the group consisting of compounds I, II, III, IV and V, rccQnn / Lznz / e / YiAi COjBn NHBoc in which R1 is selected from: H, alkyl, alkenyl, optionally substituted arylalkyl R2 is selected from: the N-protective groups Boc, Cbz, R3 is selected from: the N-protective groups Boc, Cbz, R4 is an amino acid residue for use in the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates. Compounds III, IV, and V are preferred. Furthermore, the inventors surprisingly discovered that from a larger panel of catalytic compounds tested for asymmetric hydrogenation (see Table 1, Fig. 5), the cyclooctadiene-1,5 [(R,R)-DIPAMP] rhodium tetrafluoroborate catalyst, HDP 30.2758, provided the highest enantiomeric purity, which was > 98%. HDP 30.2758 rccQnn / Lznz / e / YiAi Only the HDP 30.2758 catalyst was found to yield a very high purity of over 98% (S) enantiomers. The remaining catalysts, with the exception of (R,R)-Et-DUPHOS (BF4), resulted in considerably lower purities of 50–70% (S) enantiomers. Furthermore, the overall absolute compound yield and performance rates were much worse than with HDP 30.2758. The catalysts tested for asymmetric hydrogenation and the respective (S) enantiomer purity levels are listed in Table 1. Table 1: Comparative evaluation of various catalysts used for enantioselective hydrogenation Catalyst # Identity Purity level 1 HDP 30.2758 > 98% chiral purity 2 (R,R)-Et-DUPHOS (CF3SO3) without conversion 3 (R,R)-Et-DUPHOS (BF4 ) 90-95% chiral purity 4 (R,R)-DuPhos-ferrocene (BF4') 65% chiral purity 5 (R,R)-DuPhos-ferrocene-Et2 (BF4') 55% chiral purity 6 (R,R)-DuPhos-alkyl (CF3SO3·) 73% chiral purity 7 (R,R)-phenyl-DuPhos-alkyl (BF4 ) < 60% chiral purity pccQnn / Lznz / e / YiAi Thus, according to a third aspect, the present invention relates to the use of a compound selected from the group consisting of compound HDP 30.2758, (R,R)-Et-DUPHOS (BF4), (R,R)-DuPhos-ferrocene (BF4'), (R,R)-DuPhos5 ferrocene-Et2 (BF4·), (R,R)-DuPhos-alkyl (CF3SO3) and (R,R)-phenyl-DuPhos-alkyl (BF4-) as a catalyst for hydrogenation in the following reaction: HDP 30,2824 HDP 30,2828 Accordingly, the present invention preferably relates to the use of chiral catalysts cyclooctadiene-1,5 tetrafluoroborate [(R,R)-DIPAMP] rhodium (HDP 30.2758) or (R,R)-Et-DUPHOS (BF4-), more preferably using the chiral catalyst cyclooctadiene-1,5 tetrafluoroborate [(R,R)-DIPAMP] rhodium (HDP 30.2758) as a catalyst for hydrogenation in the following reaction: HDP 39.2824 HDP 30826 The incorporation of tryptophan derivatives as building blocks in amanitin precursors has been previously described in the document PCT / EP2018 / 071268, the content of which is incorporated herein by reference. As such, HDP 30.2115 can be synthesized by incorporating an unsubstituted Hpi building block into an amanitin peptide precursor molecule to give a synthetic amanitin; the 6-hydroxy-substituted building block HDP 30.2555 can be used for the synthesis of 6-hydroxy-substituted amanitins according to the present invention. Examples Although the invention is illustrated and described in detail in the figures and the preceding description, such illustration and description are to be considered illustrative or by way of example and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments may be understood and realized by those skilled in the art in implementing the claimed invention, based on a study of the figures, the description, and the appended claims. In the claims, the expression "comprising" does not exclude other elements or steps, and the indefinite articles "a," "an," or "a" do not exclude a plurality. The mere fact that certain measurements are enumerated in different, dependent claims does not indicate that a combination of these measurements cannot be advantageously used. Any reference sign in the claims shall not be construed as limiting their scope. Example 1: Synthesis of the phosphonium precursor (building block), HDP 30.2822. The synthesis of the phosphonium precursor (building block), HDP 30.2822, was carried out as described (CHEMISTRY European Journal, 2018, Vol. 24, n.a7, pp. 1544-1553): P(OXOMe)., MeO2C' ' NHCbz 1. PcíC H. 2. Boc2O P(OXOMe)2 MeO2C' NHBoc HDP 30.2819 KOH P(O)(OMe)2 HOjC' LHBoc HDP 30,2021 rccQnn / Lznz / e / YiAi PhCHjOH DCC / DMAP PíOXOMe), SnO2C'X'NHBoc HDP 30.2822 Example 1.1: Preparation of the trimethyl ester of the (R,S)-Boc-a15 ω NCN phosphonoglycine, HDP 30.2819 CC σ uuu P(O)(OMe)2 MeO2C^NHCbz Pd / C. H2 2. BoCjO P(OXOfVle), MeO2C' ^NHBoc HDP 30.2819 Five hundred milligrams (15.1 mmol) of the trimethyl ester of (R,S)-N-Cbzphosphonoglycine (CAS No. 88568-95-0) were hydrogenated with 1.4 g of 10% Pd / C in 100 mL of methanol at 1 atm until reaction completion was determined by TLC (chloroform / methanol 15:1). The reaction was complete in 3 hours. The catalyst was removed by filtration over a bed of Celite® (diatomaceous earth), and the methanolic solution of the free amine was concentrated under vacuum to give a colorless oil (2.9 g). The crude oil was used for the next step without purification. 2.9 g of the crude hydrogenation product were dissolved in 20 mL of dichloromethane and treated with 3.23 mL (15.1 mmol) of di-tert-butyl dicarbonate (BoczO). After 17 hours of stirring at room temperature under an argon atmosphere, the reaction mixture was concentrated to dryness. The remaining colorless oil crystallized to give a white solid (4.3 g). The crude HDP 30.2819 was used for the next step without purification. Example 1.2: Preparation of the trimethyl ester of (R,S)-N-Boc-aphosphonoglycine HDP 30.2821 P(O)(OMe)2 MeO2C^^NHBoc KOH P(O)(OMe)2 HO2C^^NHBoc HDP 30.2819 HDP 30.2821 4.3 g (assumed 14.3 mmol) of crude HDP 30.2819 were dissolved in 10 mL of 1,4-dioxane and rapidly treated under argon atmosphere at room temperature with 14.5 mL of 1 N KOH. After 85 minutes, the reaction mixture was diluted with 36 mL of water and extracted with 35 mL of ethyl acetate. The ethyl acetate extract was discarded, and the aqueous solution was acidified to pH 3 by dropwise addition of 1 N HCl. The reaction mixture was extracted with 60 mL of ethyl acetate (2x) and dried over MgSO4. The resulting white solid, 2.1 g of HDP 30.2821, was vacuum dried and used directly without purification for the next reaction step. Pe: 148-150 °C (Lit. JACS 111.6244, 1989, mp: 154-155 °C) MS (ESI ) set: 282.00 [MH]-; cal.: 283.08 (C9H18NO7P) MS (ESI-) encountered: 238.17 [M-CO2] Example 1.3: Preparation of the benclic ester of (R,S)-N-Boc-adimethylphosphono)glycine HDP 30.2822 P(O)(OMe), PhCH2OH P(O)(OMe)2 HO2C^ ^NHBoc DCC / DMAP8nO2C^ ^'NHBac HDP 30.2821 HDP 30.2822 2.0 g (7.1 mmol) of HDP 30.2821 were treated in 90 mL of dry dichloromethane with 4.6 mL (44.1 mmol) of benzyl alcohol, 230 mg of DMAP, and 2.2 g (10.6 mmol) of DCC dissolved in 7 mL of dichloromethane. The reaction mixture was stirred under an argon atmosphere at room temperature for 24 hours. The urea was then separated by filtration, and the organic phase was washed with 5% citric acid and dried over MgSO4. After evaporation of the dichloromethane, the remaining semisolid was collected in ethyl acetate and filtered again to remove further urea. The crude product was purified by flash chromatography on a column with 330 g of silica gel (detection wavelength: 254 mm) with a gradient from n-hexane to n-hexane / ethyl acetate (1:2) and gave upon evaporation 1.94 g (73%) of HDP 30.2822 as a white solid. MS (ESI+) found: 373.92 [MH]+; heat.: 373.13 (C16H242O7P) MS (ESI+) found: 396.17 [M+Na]+ Example 2: Synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan, HDP 30.2550, as a precursor of HDP 30.2555 (Hydroxy-Hpi) The synthesis route is summarized in the following synthesis scheme. rccQnn / Lznz / e / YiAi rccQnn / Lznz / e / YiAi CO,H HDP 30.2655 ( cisArans ) Example 2.1: Preparation of N-Cbz-6-benzyloxy-indol-3-aldehyde, HDP 30.2803 The starting material 6-benzyloxy-indole-3-aldehyde for the synthesis is commercially available or can be produced by the Vilsmeier reaction with high yields starting from 6-benzyloxy-indole. either.. VH Chromatography is not necessary for its purification. HDP 38.2803 rccQnn / Lznz / e / YiAi Triethylamine (1.66 mL, 11.94 mmol, 1.50 eq) was added with a syringe to a solution of 6-benzyloxy-3-formylindole (2.00 g, 7.96 mmol, 1 eq) and (DMAP)-4-dimethylaminopyridine (97.23 mg, 796 pmol) in dichloromethane (20 mL) at 23 °C. Benzyl chloroformate (1.45 mL, 10.35 mmol, 1.30 eq) was added dropwise with a syringe to the solution. After 1 h, another portion of benzyl chloroformate (223 mL, 1.59 mmol, 0.20 eq) was added with the syringe. After 95 min, the reaction mixture was diluted with dichloromethane (85 mL) and washed with a saturated aqueous solution of sodium bicarbonate (85 mL). The aqueous layer was further extracted with dichloromethane (2 x 20 mL). The combined organic layers were washed with aqueous hydrogen chloride (1 N, 85 mL), and the resulting aqueous layer was extracted with dichloromethane (2 x 20 mL). The combined organic layers were dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure.The crude product was purified by flash chromatography on a column with 330 g of silica gel (detection wavelength: 254 mm) with a gradient from n-hexane / ethyl acetate (4:1) to n-hexane / ethyl acetate (1:1) and gave upon evaporation 2.33 g (76%) of HDP 30.2803 as a solid. white. 1H NMR (400 MHz, CDCIs, δ = ppm) δ = 5.05 (s, 2H, OCH2); 5.47 (s, 2H, COOCH2); 7.06 - 8.14 (m, Ar-H, 14H); 10.01 (s, 1H, CHO) Example 2.2: Preparation of the benzyl ester of [6-benzyloxy-1H(benzyloxycarbonyl)-3-indole]-2-(tert-butyloxycarbonylamino)-acrylic acid, HDP 30.2824 CHO HDP 30.2803 HDP 30.2824 rccQnn / Lznz / e / YiAi 1.90 g (5.09 mmol) of the benzyl ester of (R,S)-N-Boc-adimethylphosphono)-glycine, HDP 30.2822, was suspended in an argon atmosphere in 8 mL of dichloromethane. 0.705 mL (4.73 mmol) of DBU was added. After 10 min of stirring, 1.66 g (4.31 mmol) of N-Cbz-6-benzyloxy-indole-3-aldehyde, HDP 30.2803, was slowly added in 4.7 mL of dichloromethane. The reaction mixture was stirred for 5 hours and the solvent was evaporated under reduced pressure. The residue was dissolved in 120 mL of ethyl acetate, and the organic solution was washed twice with 50 mL of 1N HCl and 50 mL of brine, dried over MgSO4, and concentrated under reduced pressure to give 2.70 g of crude material. The crude product was purified by ultrafast chromatography on a column with 330 g of silica gel (detection wavelength: 254 nm) with an n-hexane to n-hexane / ethyl acetate (1:1) gradient and, after evaporation, gave 2.00 g (73%) of HDP 30.2824 as a white solid. MS (ESI+) found: 632.92 [MH]+; cal.: 632.25 (C38H36N2O7) MS (ESI+) found: 655.25 [M+Na]+ Example 2.3: Preparation of the benzyl ester of (S)-6-benzyloxy-N-tercbutoxycarbonyl-1-Cbz-L-tryptophan, HDP 30.2826 CO2Bn CO2Bn HDP 30.2824 HDP 30.2826 Example 2.3.1: Synthesis of the cyclooctadiene-1,5 [(R,R)-DIPAMP] rhodium tetrafluoroborate catalyst, HDP 30.2758 HDP 30.2758 rccQnn / Lznz / e / YiAi 97.0 mg (0.20 mmol) of bis(cyclooctadiene-1,5)-dichlorodirodium [Rh(COD)Cl]2 (CAS No. 12092-47-6, Alfa Aesar) was added to a suspension of 180.0 mg (0.39 mmol) of (R,R)-DIPAMP (CAS No. 55739-58-7, Alfa Aesar) in 2.0 mL of methanol / water (1.5 mL / 0.5 mL). The orange suspension, stirred for 1 hour under an argon atmosphere, yielded an orange solution. The complex was precipitated by slowly adding (over 30 minutes) a solution of 65.0 mg (0.6 mmol) of sodium tetrafluoroborate in 0.5 mL of water. After 2.5 hours of stirring at room temperature, the orange crystals were separated by filtration, washed twice with small portions of water, and dried under high vacuum. 240 mg (81%) of the catalyst rhodium-1,5-cyclooctadiene-(R,R)-DIPAMP) tetrafluoroborate, HDP 30.2758, was obtained as a bright yellow powder. The catalyst was used without further purification. Example 2.3.2: Synthesis of the benzyl ester of (S)-6-benzyloxy-N-tert-butoxycarbonyl-1-Cbz-L-tryptophan, HDP 30.2826 A 250 mL stainless steel autoclave was charged with 35.0 mg (0.08 mmol) of rhodium-1,5-cyclooctadiene-(R,R)-DIPAMP) tetrafluoroborate, HDP 30.2758, and 1000 mg (1.8 mmol) of [6-benzyloxy-1H-(benzyloxycarbonyl)3-indole]-2-(tert-butyloxycarbonylamino)-acrylic acid benzyl ester, HDP 30.2824, in 40 mL of dry methanol / 15 mL of dichloromethane. After four vacuum / Ar and H2 cycles, the reaction was pressurized to an initial pressure of 1.2 MPa (12 bar). The reaction was allowed to proceed for 4 days at room temperature. After solvent evaporation, the crude product was purified by ultrafast chromatography on a column with 220 g of silica gel (detection wavelength: 254 mm) with n-hexane / ethyl acetate (3:1) and after evaporation gave 0.79 g (79%) of HDP 30.2826 in the form of a white powder. MS (ESI+) heat: 634.26 (C38H38N2O7) MS (ESI+) found: 657.33 [M+Na]+; Example 2.4: Preparation of N-tert-butoxycarbonyl-(S)-tryptophan, HDP 30.2832 rccQnn / Lznz / e / YiAi HDP 30.2826 HDP 30.2832 700 mg (1.10 mmol) of (S)-6-benzyloxy-Ntert-butoxycarbonyl-1-Cbz-L-tryptophan benzyl ester, HDP 30.2826, was hydrogenated with 100 mg of 10% Pd / C in a mixture of 7 mL of ethyl acetate and 4 mL of ethanol. After 3 hours of hydrogenation (controlled by TLC with chloroform / methanol 19:1 + 1% AcOH) at room temperature and 1 atm, the catalyst was removed by filtration over a Celite® bed. The solvent was discarded, and the remaining crude residue, 378 mg of HDP 30.2832, was used for the next step without purification. Example 2.5: Preparation of (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan, HDP 30.2550 CO,H CO,H HDP 30.2832 HDP 30.2550 378 mg of crude HDP 30.2832 (1.10 mmol assumed) were dissolved in 2.21 mL of NaOH. Under an argon atmosphere and at room temperature, 208.5 mL (2.20 mmol) of acetic anhydride were added all at once. The mixture was stirred for 3.5 hours and acidified with 5% citric acid. The reaction mixture was extracted three times with ethyl acetate, and the combined organic phases were washed with 5% sodium chloride and dried over MgSO4. Filtration and evaporation to dryness yielded 380 mg of crude material. The crude product was purified by ultrafast chromatography on a column with 120 g of silica gel (detection wavelength: 254 mm) with a gradient of dichloromethane + 2% AcOH / dichloromethane / methanol (15:1) + 2% AcOH and after evaporation gave 270 mg (68%) of HDP 30.2550 in the form of a white solid. MS (ESI') found: 361.17 [MH] cale.: 362.15 (C18H22N2O6) MS (ESI') found: 723.08 [2M-H]Example 3: Preparation of (S)-6-hydroxy-tryptophan by asymmetric hydrogenation of the dehydro-amino acid The synthesis route is summarized in the following synthesis scheme. HDP30.2738 HDP30.273§ HDP30.2760 deprotection (S)-6-hydroxytryptophan Example 3.1: Preparation of 6-benzyloxy-1H-indole-3-carbaldehyde A stirred solution of phosphorus oxychloride (10.0 mL, 107.0 mmol) in DMF (35 mL) was mixed with a solution of 6-benzyloxyindole (22.3 g, 100.0 mmol) in DMF (25 mL) at room temperature. After 45 min, the reaction mixture was poured onto ice water (200 mL). Solid NaOH (19.0 g, 475.0 mmol) and water (100 mL) were added to this mixture. After 30 min, more water (200 mL) was added, and the entire mixture was refluxed for 3 min. The precipitate was collected, washed with five 50 mL portions of cold water, and dried to yield 24.8 g (98.8%) of 6-benzyloxy-1H-indole-3-carbaldehyde as a white powder. The compound was identical to the reference material and pure enough for the following reaction. Example 3.2: Preparation of 6-benzyloxy-IH-l-tert-butoxycarbonyl-indolecarbaldehyde, HDP 30.2738 rccQnn / Lznz / e / YiAi HDP 30.2738 10.0 g (39.8 mmol) of 6-benzyloxy-1H-indol-3-carbaldehyde was suspended in 100 mL of dichloromethane and treated with 0.56 g (4.5 mmol) of 4-dimethylaminopyridine (DMAP) and 10.5 g (47.3 mmol) of di-tert-butyl dicarbonate (BOC2O) dissolved in 10 mL of dichloromethane. After stirring for 2 hours, 100 mL of 1 N KHSO4 was added and the dichloromethane was evaporated. The aqueous layer was extracted with several portions of diethyl ether (2 x 200 mL) and the combined organic extracts were washed with 250 mL of 1 N KHSO4, 250 mL of 1 N NaHCO3, and 250 mL of brine. The organic layer was dried over MgSO4 and concentrated under reduced pressure to give 12.0 g (86%) of a reddish-brown powder. The compound was sufficiently pure for the next reaction step. Example 3.3: Preparation of the methyl ester of 3-[6-benzyloxy-1H-(1-tert-butoxycarbonyl)-3-indole]-2-(benzyloxycarbonylamino)-acrylic acid, HDP 30.2739 pccQnn / Lznz / e / YiAi HDP 30.2738 HDP 30.2739 5.12 g (15.44 mmol) of the trimethyl ester of (R,S)benzyloxycarbonyl-o-phosphonoglycine (CAS No. 88568-95-0, Alfa Aesar) were dissolved in an argon atmosphere in 18 mL of dichloromethane. 2.14 mL (14.31 mmol) of DBU were added. After 10 min of stirring, 4.60 g (13.07 mmol) of 6-benzyloxy-1H-1-tert-butyloxycarbonyl-indole-3-carbaldehyde, HDP 30.2738, were slowly added in 14 mL of dichloromethane. The reaction mixture was stirred for 6 hours and the solvent was evaporated under reduced pressure. The residue was dissolved in 300 mL of ethyl acetate, then the organic solution was washed twice with 120 mL of 1N HCl and 120 mL of brine, dried over MgSCU, and concentrated under reduced pressure to give 7.43 g of crude material. The crude product was purified by ultrafast chromatography on a column with 330 g of silica gel (detection wavelength: 254 nm) with an n-hexane to n-hexane / ethyl acetate gradient (2:1) and after evaporation gave 5.23 g (72%) of HDP 30.2739 in the form of a white solid. MS (ESI+) found: 557.1 7 [MH]+; cale.: 557.22 (C32H32N2O7) MS (ESI+) found: 579.25 [M+Na]+ Example 3.4: Preparation of the methyl ester of 6-benzyloxy-N-carbobenzyloxy-1-tert-butoxycarbonyl-L-tryptophan, HDP 30.2760 HDP 30.2739 HDP 30.2760 Example 3.4.1: Synthesis of rhodium-1,5-cyclooctadiene tetrafluoroborate [(R,R)DIPAMP], HDP 30.2758 The rhodium cyclooctadiene-1,5 [(R,R)-DIPAMP] tetrafluoroborate catalyst, HDP 30.2758, was synthesized as described in Example 2.3.1. Example 3.4.2: Synthesis of the methyl ester of (S)-6-benzyloxy-N-carbobenzyloxy-1-tert-butoxycarbonyl-tryptophan, HDP 30.2760 A 250 mL stainless steel autoclave was charged with 60.0 mg (0.08 mmol) of rhodium-1,5-cyclooctadiene-(R,R)-DIPAMP) tetrafluoroborate, HDP 30.2758, and 1000 mg (1.8 mmol) of [6-benzyloxy-1H-(1-tert-butoxycarbonyl)-3-indole]-2-(benzyloxycarbonylamino)-acrylic acid methyl ester, HDP 30.2739, in 40 mL of dry methanol. After four vacuum / Ar and H2 cycles, the reaction was pressurized to an initial pressure of 3 MPa (30 bar). The reaction was allowed to proceed for 4 days at room temperature. After evaporation of the solvent, the crude product was purified by flash chromatography on a column with 120 g of silica gel (detection wavelength: 254 mm) with a gradient from n-hexane to n-hexane / ethyl acetate (2:1) and gave after evaporation 0.85 g (86%) of HDP 30.2760 as a white solid. MS (ESI+) heat: 558.23 (C33H34N2O7) MS (ESl+) found: 581.17 [M+Na]+; 1138.83 [2M+Na]+ Example 3.5: Preparation of the methyl ester of 6-benzyloxy-N-carbobenzyloxyL-tryptophan, HDP 30.2790 HDP 30.2760 HDP 30.2790 pccQnn / Lznz / e / YiAi 100.0 mg (0.18 mmol) of the methyl ester of (S)-6-benzyloxy-N-carbobenzyloxy-1-tert-butoxycarbonyl-tryptophan, HDP 30.2760, was dissolved in 5.0 mL of formic acid and stirred for 1 hour at 40 °C. The reaction mixture was evaporated to dryness and the residue was dissolved in ethyl acetate. The ethyl acetate solution was washed with water, saturated NaHCO3, and brine and dried over MgSO4. After evaporation of the solvent, the crude product was purified by flash chromatography on a column with 24 g of silica gel (detection wavelength: 254 mm) with a gradient from n-hexane to n-hexane / ethyl acetate (1:1) and gave after evaporation 29 mg (35%) of HDP 30.2790 as a white solid. MS (ESI+) heat: 458.52 (C27H26N2O5) MS (ESI+) found: 459.25 [M+H]+ Example 3.6: Preparation of (S)-6-benzyloxy-N-carbobenzyloxy-tryptophan, HDP 30.2782 rccQnn / Lznz / e / YiAi HDP 30.2790 HDP 30.2782 A 2 N aqueous solution of LiOH (84.7 µL) was added to a solution of 25.9 mg (0.056 mmol) of HDP 30.2790 in 1000 µL of tetrahydrofuran / water (10:1) at room temperature. The reaction mixture was stirred for 2.5 hours and partitioned between ethyl acetate and 5% citric acid. The aqueous layer was extracted with ethyl acetate, and the organic layers were combined, dried (MgSO4), and concentrated. The resulting HDP 30.2782 carboxylic acid was purified on silica gel using dichloromethane / methanol (+ 1% acetic acid) as the mobile phase. 13.7 mg (55%), white solid. MS (ESI+) cal.: 444.17.23 (C26H24N2O5) MS (ESI+) found: 445.25 [M+H]+; 467.17 [M+Na]+ Example 3.7: Preparation of (S)-6-hydroxy-N-(tert-butoxycarbonyl)-tryptophan, HDP 30.2832 HDP 30.2782 HDP 30.2832 Ten mg (10 wt%) of palladium on carbon were added to a solution of 50 mg (0.11 mmol) of HDP 30.2782 in 800 µL of methanol. The reaction mixture was purged three times with hydrogen and stirred for 2.5 h at room temperature. The suspension was filtered through a Celite® bed, washed with methanol, and concentrated to dryness. The solid residue of (S)-6-hydroxytryptophan (22.2 mg) was dissolved in 1000 mL of 1,4-dioxane / water (1:1) and treated with 101 µL (0.101 mmol) of 1 N NaOH and 21.57 µL (0.10 mmol) of di-tert-butyl dicarbonate. The reaction mixture was stirred for 16 hours and the pH was adjusted to 2 with 1 N hydrochloric acid. The aqueous solution was extracted three times with ethyl acetate, and the combined organic phases were washed with brine, dried, and evaporated to dryness. The resulting crude HDP 30.2832 was purified on silica gel using dichloromethane / methanol (+ 1% acetic acid) as the mobile phase. 9.9 mg (31%) of a white solid was obtained.The material was identical to the reference sample. MS(ES') cal.: MS.: 320.14 (C16H20N2O5) MS (ESI') found: 319.08 [MH]' Example 4: Preparation of cis,trans-1-(tert-butoxycarbonyl)-2-carboxy-3ahydroxy-6-acetoxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole, cis-HDP 30.2555, and trans-HDP 30.2555 (cis,trans-6-acetoxy-Hpi) Example 4.1: Preparation of (S)-N-(tert-butoxycarbonyl)-6-acetoxy-tryptophan, HDP 30.2550 (S)-6-hydroxy-tryptophan HDP 30.2550 rccQnn / Lznz / e / YiAi 590.0 mg (2.68 mmol) of the (S)-6-hydroxytryptophan from the hydrogenation step of Example 3.7 was suspended in a 30 mL (v / v) mixture of 1,4-dioxane / water. Under an argon atmosphere, 2.68 mL (2.68 mmol) of 1 N NaOH was added all at once at room temperature. The resulting yellow solution was then treated with 574.6 mL (2.68 mmol) of Boc anhydride (BOC₂O) and stirred for 24 hours at room temperature. The solution was acidified with 1 N hydrochloric acid to pH 2.4 and extracted three times with 25 mL of ethyl acetate. The pooled ethyl acetate extracts were washed with a saturated NaCl solution and dried over MgSCu. 785.0 mg of crude material was obtained by filtration and evaporation to dryness. The crude N-Boc-6-hydroxy-L-tryptophan was dissolved in 4.91 mL (4.91 mmol) of 1 N NaOH and treated with 463.2 mL (500.3 mg, 4.90 mmol) of acetic anhydride. The reaction mixture was stirred for 3 hours under an argon atmosphere and acidified with 5% citric acid.The aqueous phase was extracted three times with 25 mL of ethyl acetate, washed with saturated NaCl, and dried over MgSO4. Filtration and evaporation yielded 635 mg of crude solid. The crude product was purified by ultrafast chromatography on a column with 330 g of silica gel (detection wavelength: 254 nm) with a gradient from CH2Cl2 + 1% acetic acid to CH2Cl2 / MeOH (15:1) + 1% acetic acid, and after co-evaporation with toluene, 564.4 mg (56% yield) of a white powder was obtained. MS (ESI ) found: 361.08 [MH] ; cale.: 362.15 (C18H22N2O6) Example 4.2: Preparation of cis,trans-6-acetoxy-Hp¡ ® Γ V; cis-6-acetoxy-Hpi „„ 1 h'^sens* <O... p. · / YY 2. chalk-H DP 30.3555 Χ:·' 3 O i MeOH + unswiwn sens-Rosa bengalB o rccQnn / Lznz / e / YiAi trans-6-aceíoxy-Hp! trans-HDF 30.2555 Photooxygenation was carried out using a 400 W high-pressure sodium lamp (Sirius X400 lamp, 230 V, 400 W; 55,000 lumens at a distance of 1.3 m). Rose Bengal was used as the color sensitizer. The reaction took place in a 500 ml cylindrical reaction vessel with a heat exchange jacket made of borosilicate glass, with a flat bottom and a flat laboratory flange (DN) with two GL18 threaded connectors. The distance between the lamp and the reaction vessel was 15 cm, and the reaction temperature was in the range of 3–4 °C. The final product was purified on a Teledyne ISCO ultrafast chromatography system with a column containing 330 g of Redi Sept Flash silica gel (Teledyne ISCO cat. 69-2203-330). The solvents CH₂Cl₂, -CH₃OH, and CH₃COOH were of HPLC or conventional BP quality. Dry oxygen (99.5% purity) was bubbled through the reaction mixture at a rate of 2–4 liters per minute. 943.0 mg (2.60 mmol) of N-(tert-butoxycarbonyl)-L-6-acetoxytryptophan, HDP 30.2550, and 100 mg of rose bengal were dissolved in 500 mL of methanol and cooled to 3°C using a Hubert cryostat with glycol / water as the cooling medium. The reaction solution was irradiated with a 400 W high-pressure sodium lamp. During irradiation, a slow stream of oxygen was bubbled through the reaction solution. After 5 hours of irradiation, oxygenation and cooling were stopped, and the reaction medium was treated with 10 mL of dimethyl sulfide. The mixture was stirred for 2 hours and evaporated to dryness using a rotary evaporator with a water bath temperature of 35°C. The dark red residue was further dried under high vacuum to give a crystalline solid of 1.20 g.The crude product was purified on a column with 330 g of silica gel (detection wavelength: 254 nm) using a gradient from CH₂Cl₂ + 5% acetic acid to CH₂Cl₂ / MeOH (30:1) + 5% acetic acid. 380 mg of cis-HDP 30.2555 and 290 mg of trans-HDP 30.2555 were eluted and co-evaporated with toluene. After lyophilization in tert-butanol, both isomers were obtained as whitish powders. cis-1-(tert-butoxycarbonyl)-2-carboxy-3a-hydroxy-6-acetoxy-1,2,3,3a, 8,8ahexahydroDyrrolo[2,3-b1indol (cis-HDP 30.2555) 380 mg of cis-HDP 30.2555; yield: 39% 1H NMR (400 MHz, CD3OD, δ = ppm) δ = 1.22, 1.44, 1.54 [s, 9H, C(CH3)3]; 2.23 (s, 3H, OCOCH3); 2.46-2.63 (m, 2H, CH2); 4.14-4.29 (m, 1H, 2-H); 5.35 (s, 1H, 8a-H); 6.39-6.46 (m, 2H, 7-H, 5-H); 7,207.24 (m, 1H, 4-H) RMN13C (100 MHz, CD3OD, δ = ppm) δ = 20.93, 28.45, 31.12, 42.80, 61.12, 69.44, 82.21, 85.82, 87.93, 104.97, 112.98, 124.84, 129.42, 151.51, 154.04, 155.97, 171.34, 175.79 MS (ESI+) encountered: 378.92 [MH]+; cal.: 378.14 (C18H22N2O7) MS (ESI+) found: 401.17 [M+Na]+; cal.: 401.14 (Ci8H22N2NaO7) UV / VIS(CH3OH): Amax = 296 nm, 239 nm, 215 nm Xmin = 266 nm, 227 nm rccQnn / Lznz / e / YiAi trans-1 -(terc-butox¡carbonil)-2-carbox¡-3a-h¡drox¡-6-acetox¡-1,2,3,3a,8,8ahexahidrop¡rrolo[2,3-bl¡ndol (trans-HDP 30.2555) 290 mg of trans-HDP 30.2555; yield: 30% RMN1H (400 MHz, CD3OD, δ = ppm) δ = 1.22, 1.45, 1.54 [s, 9H, C(CH3)3]; 2.22 (s, 3H, OCOCH3); 2.55-2.73 (m, 2H, CH2); 4.51 -4.57 (m, 1H, 2-H); 5.21 -5.24 (s, 1 H, 8a-H); 6.36-6.41 (m, 2H, 7-H, 5-H); 7.17-7.18 (m, 1H, 4-H) 13C NMR (100 MHz, CD3OD, δ = ppm) δ = 20.95, 28.50, 31.12, 42.47, 60.97, 69.44, 82.06, 84.84, 87.54, 104.74, 112.67, 125.03, 128.70, 152.31, 154.22, 156.00, 171.23, 174.67 MS (ESI+) found: 379.00 [MH]+; heating: 378.14 (C18H22N2O7) MS (ESI+) found: 401.17 [M+Na]+; heat.: 401.14 (Ci8H22N2NaO7) MS (ESI+) found: 779.00 [2M+Na]+; heating: 779.28 (C36H44N4Na2Oi4) UV / VIS(CH3OH): Amax = 299 nm, 241 nm, 215 nm Amin = 268 nm, 228 nm Example 4.3: Introduction of cis,trans-6-acetoxy-Hpi into an amanitin precursor The synthesis of amanitin using cis,trans-6-acetoxy-Hp1 was carried out as described in document PCT / EP2018 / 071268, the content of which is incorporated herein by reference, primarily for the purpose of enabling the product. References: Muraoka S, and Shinozawa T., J Biosci Bioeng. 2000; 89(1):73-6 Wieland T, Faulstich H. 1978 CRC Crit Rev Biochem. Vol. 5 185-260. Zanotti G, Máhringer C, and Wieland T., IntJ. Pept Protein Res. Oct 1987; 30 (4): 450-9; Zanotti G, Wieland T, Benedetti E, Di Blasio 8, Pavone V, and Pedone C. Int J. Pept Protein Res. Sep 1989; 34(3): 222-8 Zhang P, et al., FEMS Microbio! Lett. 15 Nov 2005; 252(2): 223-8. Epub 15 Sep 2005.

Claims

1. Process for the synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyltryptophan (HDP 30.2550) or (S)-6-hydroxytryptophan, said process comprising at least one enantioselective hydrogenation step of an olefinic precursor compound using at least one chiral catalyst.

2. The process according to claim 1, wherein said olefinic precursor is an olefinic amino acid precursor, preferably wherein said olefinic precursor is compound HDP 30.2824.

3. The process according to any of claims 1 or 2, wherein said chiral catalyst is a compound selected from the group consisting of compound HDP 30.2758, (R,R)-Et-DUPHOS (BF4 ), (R,R)-DuPhosferrocene (BF4 ), (R,R)-DuPhos-ferrocene-Et2 (BF4' ), (R,R)-DuPhos-alkyl (CF3SO3·) and (R,R)-phenyl-DuPhos-alkyl (BF4·).

4. The process according to claim 3, wherein said chiral catalyst is the compound HDP 30.2758 or (R,R)-Et-DUPHOS (BF4 ).

5. The process according to any of claims 1-4, wherein said olefinic precursor is synthesized using compound HDP 30.2822.

6. The process according to claim 5, wherein said olefinic precursor is further synthesized using compound B. O, K- rccQnn / Lznz / e / YiAi Compound Β Ί. The process according to any of claims 1-6, wherein said process comprises the use of at least one starting compound or intermediate compound selected from the group consisting of CHO HDP 30.2303 CO.Sn HDP 30.2824 CO.,Bn HDP 30.2826 8. The process for the synthesis of (S)-6-acetyloxy-N-tert-butoxycarbonyl tryptophan (HDP 30.2550) according to any one of claims 1-7, wherein said process comprises at least the following steps: HDP 30.2803 HDP 30.2822 HDP 30 2824 rccQnn / Lznz / e / YiAi HDP 30.2550 HDF 30.2332 HDP 30.2826 9. The process for the synthesis of (S)-6-hydroxytryptophan according to any of claims 1-7, wherein said process comprises at least the following steps: HDP30.2760 pccQnn / Lznz / e / YiAi (S)-6-hydroxytryptophan 10. (S)-6-acetyloxy-N-tert-butoxycarbon¡lN-tryptophan (HDP 30.2550), CQ.H / v / NHBoc HDP 30.2550 or any of the precursor compounds or derivatives selected from the group consisting of CO,Bn CO,Bn CHO, / '. · · \ NHBoc NHBoc Cbz HDP 30.2803 BnO' N Cbz HDP 30.2824 HDP 30.2826 NHBoc HDP 30.2550 HDP30.2739 HDP30.2760 ppconn / Lznz / e / YiAi 11. (s)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550), (s)-65-hydroxytryptophan, or any of the precursor compounds according to claims 7, 8, and 9, respectively, for use in the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates, said amatoxin-drug conjugate optionally comprising a connector. 10 12. A dehydroamino acid compound selected from the group consisting of compounds I, II, III, IV and V rccQnn / Lznz / e / YiAi wherein R1 is selected from: H, alkyl, alkenyl, optionally substituted arylalkyl, R2 is selected from: the N-protecting groups Boc, Cbz, R3 is selected from: the N-protecting groups Boc, Cbz, R4 is an amino acid residue, for use in the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates.

13. Use of (S)-6-acetyloxy-N-tert-butoxycarbonyl-tryptophan (HDP 30.2550) or (S)-6-hydroxytryptophan for the synthesis of amanitin or amanitin derivatives or amatoxin-drug conjugates, preferably comprising a connector.

14. Use of a compound selected from the group consisting of compound HDP 30.2758, (R,R)-Et-DUPHOS (BF4), (R,R)-DuPhos-ferrocene (BF4 ), (R,R)-DuPhos-ferrocene-Et2 (BF4_), (R,R)-DuPhos-alkyl (CFsSO3') and (R,R)-phenyl- 5 DuPhos-alkyl (BF4) as a catalyst for hydrogenation in the following reaction: HDP 30.2824 HW 30.2826 pccQnn / Lznz / e / YiAi 15. Use of rhodium-1,5-cyclooctadiene-(R,R)-DIPAMP tetrafluoroborate (HDP 30.2758) as a catalyst for hydrogenation in the following reaction: