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1 Lysine salt and method of manufacturing a lysine derivative Summary 5 The disclosure relates to a Bsmoc-protected lysine salt of formula 1X (1X) , e.g., a hydrochloric acid salt (n = 1, HY = hydrochloric acid). It further relates to a method of manufacturing Bsmoc-protected lysine derivatives, which employs the Bsmoc-protected lysine salt of formula 1X and comprises the step of reacting the epsilon amino group of lysine with an 10 activated carboxylic acid derivative. Some of the obtainable Bsmoc-protected lysine derivatives with their side chain modification are useful as starting materials in solid phase peptide synthesis. Article 15 The present disclosure relates to a Bsmoc-protected lysine salt, which is protected at its alpha nitrogen with 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc). It relates further to a method of manufacturing a Bsmoc-protected lysine derivative, which employs the Bsmoc- protected lysine salt as a starting material and comprises a reaction of its epsilon nitrogen atom 20 with an activated carboxylic acid derivative under formation of an amide group. It relates also to the use of the Bsmoc-protected lysine salt in the synthesis of a peptide. Semaglutide (CAS-No. 910463-68-2) is an active pharmaceutical ingredient and is known as a glucagon-like peptide-1 receptor agonist. Semaglutide, when it is written in the three-letter-code 25 of peptides, has the following formula: H-His1-Aib-Glu-Gly-Thr5-Phe-Thr-Ser-Asp-Val10-Ser-Ser-Tyr-Leu-Glu15-Gly-Gln-Ala-Ala- Lys20(HO-CO-(CH2)16-CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl)-Glu-Phe-Ile-Ala-Trp25-Leu-Val-Arg-Gly-Arg30-Gly-OH (SEQ ID NO:5) 30 Accordingly, Semaglutide has a linear 31-mer peptide backbone and carries at the epsilon nitrogen atom of its lysine20 a fatty acid side chain, the epsilon nitrogen atom of lysine20 is 2 substituted with HO-CO-(CH2)16-CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl]-2-[2-(2- aminoethoxy)ethoxy]acetyl. WO 2006-097537 A2 discloses in its example 4 the peptide Semaglutide. Semaglutide is 5 synthesized by an acylation of the eplison-nitrogen atom of lysine20 at an already complete linear peptide backbone. Accordingly, the fatty acid side chain at lysine20 is introduced after the complete linear peptide backbone of Semaglutide has been synthesized. CN 104356224 A discloses a synthesis of Semaglutide, which employs as a building block a 10 Fmoc-protected lysine derivative, which carries already the fatty acid side chain. Accordingly, lysine20 carrying already a fatty acid side chain is introduced during the synthesis of the linear peptide backbone of Semaglutide. The employed Fmoc-protected lysine derivative (CAS-No. 1662688-20-1) is depicted below . 15 CN 113461801 A discloses at its example 1 a solid phase peptide synthesis of the aforementioned Fmoc-protected lysine derivative (CAS-No. 1662688-20-1) via a solid-phase conjugated Alloc-protected lysine derivative (CAS-No. 2721349-46-6), which is depicted below 3 . CN 115677827 A discloses at its example 4 a synthesis of Boc-Lys(tert-BuO-CO-(CH2)16-CO- gamma-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl)-Ala-OAll 5 (CAS-No. 2921565-74-2) via a Boc-protected lysine derivative (CAS-No. 2921565-73-1), which is depicted below . WO 96-25394 discloses 2-(4-nitrophenylsulfonyl)ethoxycarbonyl respectively Nsc-group as an 10 amino-protecting group in solid phase peptide chemistry and states on its page 18 that Nsc- group is perfectly resistant to the action of acidic reagents usually used for the cleavage of protective groups of tert-butyl type. 4 Protein and Peptide Letters (1997), vol. 4, No. 5, p. 307-312 discloses a comparative study of Fmoc- and Nsc-groups in automated solid phase peptide synthesis. It is concluded that comparative synthesis of three test peptides result in similarly looking HPLC-UV chromatograms. On page 312, a retention time of Nsc-Phe-OH with 16 minutes versus a 5 retention of Fmoc-Phe-OH with 24 minutes is stated for an analytical HPLC with a C18 column and 0.1 % trifluoroacetic acid in a water / acetonitrile gradient. A decomposition seems not to occur at Nsc-Phe-OH in view of the statement of one retention time. Tetrahedron Letters (1994), vol. 35, no. 42, p. 7821-7824 discloses on its page 7822 the 10 preparation of Nsc-Lys(Boc)-OH via its trimethylsilyl derivative. A yield of 83% is reported for a homogeneous product after recrystallization. During the work-up, an exposure to an aqueous 5% sodium hydrogencarbonate solution at an extraction has taken place. Organic Letters (2001), vol. 3, no. 5, p. 781-783 discloses solid phase peptide synthesis with 15 alpha-azide-protected amino acids and states at its table 2 that alpha-azido acids outperform comparative Fmoc-amino acid at several tested peptide sequences. Journal American Chemical Society (1997), vol. 119, p. 9915-9916 discloses Bsmoc group, which is an abbreviation for 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl, as an amino- 20 protecting group in peptide synthesis. Bulletin of Korean Chemical Society (1998), vol. 19, no. 6, p. 696-698 discloses 2-(phenyl- sulfonyl)ethoxycarbonyl respectively Psc group for the orthogonal protection of the epsilon amino group of lysine in conjunction with the Boc group, i.e. Boc-Lys(Psc)-OH, and its usage in 25 liquid phase peptide synthesis. RU 2196144 C1 discloses at its example 11 a peptide Psc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)- Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2 (SEQ ID NO:12) with Psc meaning 2-(phenylsulfonyl)- ethoxycarbonyl. 30 EP 3819308 A1 discloses a process for the manufacture of protected amino acids substituted at their sidechains. At step 1f of its example 1, Fmoc-Lys-OH is persilylated with N-methyl-N- trimethylsilylacetamide and afterwards reacted with an activated tBuOOC-(CH2)16-CO- Glu(AEEA-AEEA)-OtBu to obtain Fmoc-Lys([tBuOOC-(CH2)16-CO-gamma-Glu-OtBu)-AEEA- 35 AEEA]-OH. At step 3b of its example 3, Fmoc-Lys-OH is persilylated with N-methyl-N- trimethylsilylacetamide and afterwards reacted with Pal-Glu(Osu)-OtBu to obtain Fmoc-Lys(Pal- Glu-OtBu)-OH. 5 WO 2024-079043 A1 discloses a method of manufacturing a peptide P, which comprises a step of condensing of an alpha amino acid derivative S-am, which has one unprotected alpha amino group or one unprotected alpha imino group, with a protected alpha amino acid derivative of formula Pr-L (Pr-L) , 5 wherein the amino protecting group RL-N-1 is 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc). A method of manufacturing the alpha amino acid derivative of formula Pr-L is also disclosed. 10 In general, an increase of the yield of a condensing reaction is desirable. Especially, if the condensing reaction is a part of a multi-step reaction scheme for a larger molecule, for example a molecule with a molecular weight above 1000 g / mol - which is often the case for a (poly-) peptide. A reason is that a removal of those one or more by-products, which are generated due to an ineffective condensing reaction and are comparatively large molecules, from the targeted 15 molecule turns often out to be difficult. This is often due to an overall still similar physical behavior and necessitates in the case of a (poly-) peptide often a purification by a preparative high performance liquid chromatography. Thus, an initial purity of the crude material containing the targeted larger molecule is of relevance and contributes to an effectiveness of the condensing reaction. Furthermore, it is beneficial, if an applied starting material has a sufficient, 20 at least temporary stability against degradation at a contact with a base or an acid, for example at a contact with trifluoroacetic acid in the technical area of peptide synthesis. The meaning of ‘at least temporary’ refers to a sufficient stability under common conditions like room temperature, exposure times of for example up to an hour and for example a presence of water. On the one hand, this allows for example typically a benign synthetic accessibility of the applied 25 starting material, for example during an aqueous work-up. Here again, an initial purity of a crude 6 material containing the starting material and thus an avoidance of a preparative liquid chromatography is of relevance. On the other hand, a possibility for an analytical monitoring of the starting material with a typically acidic analytical reverse phase high performance liquid chromatography in case of a (poly-) peptide allows a better insight into and thus steering of the 5 condensing reaction. Another desire in case of a (poly-) peptide is that a condensing reaction allows to remain in the scheme of a base-catalyzed deprotection of an amino-protecting group for a further condensing reaction. Thus, a new reactive amino group respectively a new reactive imino group for the further condensing reaction to form an amide bond can be generated without endangering an acid-sensitive linking group to a resin or endangering an acid-sensitive 10 protection group at a side chain of a respective, already condensed amino acid in the pre- existing peptide. Furthermore, a condensing reaction, which does not lead to a presence of a heavy metal during a following deprotection reaction of an amino-protecting group, is sometimes desirable. 15 For a benign synthetic accessibility of the applied starting material, it is also of interest that synthetic precursors of the applied starting material have a sufficient, at least temporary stability against degradation. The meaning of ‘at least temporary’ refers in case of a synthetic precursor to a stability against degradation adapted to the position of the synthetic precursor in a synthesis pathway to the finally applied starting material. A synthetic precursor has a hold 20 position if the synthetic precursor is stored in isolated form until the beginning of a chemical reaction or is worked-up to a storable isolated form at the end of a chemical reaction. In contrast, a synthetic precursor has a transient position in the synthesis pathway to the finally applied starting material if the synthetic precursor is not isolated from the reaction mixture of the chemical reaction. A synthetic precursor with a hold position has preferably a storage stability, 25 which allows a storage in isolated form at room temperature over a prolonged period. This allows a transport with less temperature control and a stockpiling at the production facility, where the chemical reaction will take place. An additional advantage is also a stability of the synthetic precursor with a hold position once it is dissolved in a solvent or suspended in a suspension medium. For example, this allows a preparation of a larger amount of a solution of 30 the synthetic precursor with a hold position. The larger amount can be stored in a tank and used in portions for several different chemical reactions, which are conducted at different times during a production campaign. The synthetic precursor with a hold position should preferably be usable in a chemical reaction of the synthesis pathway without a need for a previous chemical transformation or in case of a need, only a simple chemical transformation immediately before 35 the desired chemical reaction. A lysine derivative, whose alpha nitrogen atom is protected by an Fmoc group and carries no other base-sensitive protecting groups, often allows an exchange of the protecting group at the 7 alpha nitrogen atom by a base-induced Fmoc-deprotection and a subsequent introduction of a different protecting group. However, a synthesis pathway, which avoids a deprotection and reprotection circle by using already initially the finally desired protecting group for the alpha nitrogen atom of the lysine, is more desirable. 5 It has now surprisingly been discovered that Bsmoc-Lys-OH is significantly less stable than Fmoc-Lys-OH. This applies inter alia for Bsmoc-Lys-OH in an isolated solid form and for Bsmoc- Lys-OH dissolved as a solution. 10 It has now been found a compound of formula 1X (1X) wherein n is 1 or 0.5, when n is 1 15 HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, hydrobromic acid, hydroiodic acid, perchloric acid, triflic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid or phosphoric acid, 20 when n is 0.5 HY is sulfuric acid. A toluenesulfonic acid is for example para-toluenesulfonic acid (CAS-No. 104-15-4), meta- toluenesulfonic acid (CAS-No. 617-97-0), ortho-toluenesulfonic acid (CAS-No. 88-20-0) or a 25 mixture thereof. Preferably, toluenesulfonic acid is para-toluenesulfonic acid. Propanesulfonic acid is for example 1-propanesulfonic acid (CAS-No. 5284-66-2), 2-propanesulfonic acid (CAS- No. 14159-48-9) or a mixture thereof. Preferably, propanesulfonic acid is 1-propanesulfonic acid. Butanesulfonic acid is for example 1-butanesulfonic acid (CAS-No. 2386-47-2), 2- butanesulfonic acid (CAS-No. 16794-12-0), 2-methyl-2-propanesulfonic acid (CAS-No. 16794- 30 13-1), or a mixture thereof. Preferably, butanesulfonic acid is 1-butanesulfonic acid. Camphorsulfonic acid is for example (-)-camphorsulfonic acid (also named (1R,4S)-7,7- dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonic acid, CAS-No. 35963-20-3), (+)- 8 camphorsulfonic acid (also named (1S,4R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1- methanesulfonic acid, CAS-No. 3144-16-9) or a mixture thereof (also named 7,7-dimethyl-2- oxobicyclo[2.2.1]heptane-1-methanesulfonic acid, (+/-)-camphorsulfonic acid, CAS-No. 5872- 08-2). Preferably, camphorsulfonic acid is (+)-camphorsulfonic acid. 5 Hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, hydrobromic acid, hydroiodic acid, perchloric acid, triflic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid, difluoroacetic acid, phosphoric acid and sulfuric acid have in common a pKa value below 2.3. In case of phosphoric acid, its first 10 pKa value of 2.15 is meant, whereas the first pKa value and the second pKa value of sulfuric acid are below 2.3. An advantage of this low pka value of HY is that its related anion Y- is rather inert in a chemical reaction, where a carboxylic acid derivative reacts with a molecule, which possesses an unprotected amino group or an unprotected imino group under formation of an amide group. 15 Preferably, the compound is of formula 1X, wherein when n is 1 HY is hydrochloric acid, trifluoroacetic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, 20 hydrobromic acid, hydroiodic acid, perchloric acid, triflic acid, nitric acid, picric acid, trichloroacetic acid, camphorsulfonic acid, dichloroacetic acid or difluoroacetic acid, when n is 0.5 HY is sulfuric acid. 25 A molar excess of HY, which is added at a preparation of a compound of formula 1X, can be more easily removed by evaporation, if the boiling point of HY is comparatively low. Preferably, HY has a boiling point below 200 °C at 101,325 Pa. Preferably, HY is hydrochloric acid (- 85 °C), trifluoroacetic acid (72.4 °C), hydrobromic acid (- 67 °C), hydroiodic acid (-35 °C), nitric acid (83 °C), trichloroacetic acid (198 °C), dichloroacetic acid (194 °C) or difluoroacetic acid (133 30 °C). More preferably, HY has a boiling point below 140 °C at 101,325 Pa. More preferably, HY is hydrochloric acid (- 85 °C), trifluoroacetic acid (72.4 °C), hydrobromic acid (- 67 °C), hydroiodic acid (-35 °C), nitric acid (83 °C) or difluoroacetic acid (133 °C). Very preferably, HY has a boiling point below 90 °C at 101,325 Pa. Very preferably, HY is hydrochloric acid (- 85 °C), trifluoroacetic acid (72.4 °C), hydrobromic acid (- 67 °C), hydroiodic acid (-35 °C) or nitric acid. 35 Preferred is a compound of formula 1X, wherein when n is 1 9 HY is hydrochloric acid, trifluoroacetic acid, para-toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanesulfonic acid, 2-propane-sulfonic acid, 1-butanesulfonic acid, hydrobromic acid, hydroiodic acid, perchloric acid, triflic acid or nitric acid, when n is 0.5 5 HY is sulfuric acid. Preferred is a compound of formula 1X, wherein when n is 1 HY is hydrochloric acid, trifluoroacetic acid, para-toluenesulfonic acid or methanesulfonic 10 acid, when n is 0.5 HY is sulfuric acid. Preferably, the compound of formula 1X has been stored for at least 12 hours. More preferably, 15 the compound of formula 1X has been stored for a period between 12 hours and 1 month, very preferably for a period between 18 hours and 3 weeks, particularly for a period between 24 hours and 2 weeks, more particularly for a period of 36 hours and 7 days and very particularly for a period of 48 hours and 120 hours. 20 Preferred is a compound of formula 1X, wherein the compound has been stored for at least 12 hours. Preferably, the compound of formula 1X has been stored at a temperature between 4 °C and 40 °C, more preferably at a temperature between 7°C and 38 °C, very preferably at a temperature 25 between 9 °C and 36 °C, particularly at a temperature between 14 °C and 34 °C, more particularly at a temperature between 17 °C and 32 °C, very particularly at a temperature between 19 °C and 30 °C, especially at a temperature between 21 °C and 28 °C. Preferably, the compound of formula 1X is in a solid state. More preferably, the compound of 30 formula 1X is in the solid state at a temperature between 4 °C and 40 °C, very preferably at a temperature between 21 °C and 28 °C, very preferably at a temperature of 23 °C. Solid state herein means that the compound of formula 1X accounts for 50 to 100 weight-percent of a composition comprising the compound of formula 1X. Accordingly, a frozen solution of a compound of formula 1X, which has a weight percentage of a compound of formula 1X from for 35 example 10 weight-percent, is not considered herein as a compound of formula 1X in the solid state. Preferred is a compound of formula 1X, which is in a solid state. 10 Preferably, the compound of formula 1X is dissolved or suspended. More preferably, the compound of formula 1X is dissolved or suspended in a solvent composition SolvI-1, which comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2- 5 pyrrolidone, N-octyl-2-pyrrolidone, methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, gamma-butyrolactone, gamma-valerolactone, triethyl phosphate, methanol, ethanol, propanol, butanol, diethyl ether, 10 methyl tert-butyl ether, diisopropyl ether, benzene, toluene, xylene, chlorobenzene, pentane, hexane, heptane, octane, cyclohexane, water, or a mixture thereof. Propyl acetate is n-propyl acetate, isopropyl acetate or a mixture thereof, preferably isopropyl acetate. Butyl acetate is n-butyl acetate, sec-butyl acetate, iso-butyl acetate, tert-butyl acetate 15 or a mixture thereof, preferably sec-butyl acetate. Propanol is n-propanol, iso-propanol or a mixture thereof, preferably iso-propanol. Butanol is n-butanol, sec-butanol, iso-butanol, tert- butanol or a mixture thereof, preferably tert-butanol. Xylene is ortho-xylene, meta-xylene, para- xylene or a mixture thereof, preferably meta-xylene. Hexane is n-hexane, 2-methylpentane, 3- methylpentane, 2,3-dimethylbutane, 2,2-dimethylbutane or a mixture thereof, preferably n- 20 hexane. Heptane is n-heptane, 2-methylhexane, 3-methylhexane, 2,2-dimethylpentane, 2,3- dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethlypentane, 2,2,3- trimethylbutane or a mixture thereof, preferably n-heptane. Octane is for example n-octane, 2- methylheptane, 3-methylheptane, 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,2,4- trimethylpentane or a mixture thereof, preferably n-octane. 25 Preferably, the solvent composition SolvI-1 comprises N,N-dimethylformamide, N,N- dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, 30 acetonitrile, benzonitrile, acetone, methyl ethyl ketone, gamma-butyrolactone, methanol, ethanol, propanol, butanol, diethyl ether, methyl tert-butyl ether, diisopropyl ether, water or a mixture thereof. More preferably, the solvent composition SolvI-1 comprises N,N- dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl 35 acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, nitromethane, acetonitrile, benzonitrile, acetone, methyl ethyl ketone, gamma-butyrolactone, methanol, ethanol, propanol, butanol, water or a mixture thereof. Very preferably, the solvent composition SolvI-1 comprises N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2- 11 pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, ethyl acetate, propyl acetate, butyl acetate, nitromethane, acetonitrile, gamma-butyrolactone, methanol, ethanol, propanol, butanol, water or a mixture thereof. Particularly, the solvent composition SolvI-1 comprises N,N-dimethylformamide, N,N- 5 dimethylacetamide, N-methyl-2-pyrrolidone, N-butyl-2-pyrrolidone, dimethyl sulfoxide, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, acetonitrile, methanol, ethanol, propanol, but...