Method for preparing GIP / GLP1 dual agonist

Abstract

The present invention provides novel intermediates and methods useful in the manufacture of tirzepatide or a pharmaceutically acceptable salt thereof.

Description

[0001] The present invention provides methods and intermediates for preparing the GIP / GLP1 dual agonist peptide tirzepatide or a pharmaceutically acceptable salt thereof.

[0002] Diabetes is a chronic disease characterized by hyperglycemia caused by defects in insulin secretion, insulin action, or both. In type 2 diabetes (“T2D”), the combined effects of impaired insulin secretion and insulin resistance are associated with elevated blood glucose levels. The GIP / GLP1 dual agonist tirzepatide is described and claimed in U.S. Patent 9,474,780 (“the '780 patent”). Tirzepatide can be used to treat T2D.

[0003] US 9,474,780 generally describes peptides and methods for preparing GIP / GLP1 dual agonists.

[0004] There is a need for methods and intermediates to enable improvement of the technology for producing tirzepatide such that the technology has various advantages including the purity required commercially. Similarly, there is a need for efficient and environmentally friendly “green” methods, including stable intermediates, to provide tirzepatide with fewer purification steps. For environmental and operator safety improvements, there is also a need for improved technology to provide a method for preparing tirzepatide that generates minimal waste streams. The preparation of large-scale, pharmaceutically elegant tirzepatide presents many technical challenges that can affect the overall yield and purity. There is a need for methods that avoid the use of transition metals and / or harsh reaction conditions incompatible with peptide synthesis.

[0005] The present invention seeks to meet these needs by providing novel intermediates and methods that can be used to prepare tirzepatide (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof. The improved methods for preparing tirzepatide of the present invention provide intermediates and method reactions that embody a combination of advantages, including an efficient route with fewer steps while maintaining high quality and purity. Importantly, the improved methods and intermediates reduce resource intensity and minimize waste streams.

[0006] The improved methods described herein provide various embodiments of intermediates that can be used to produce tirzepatide.

[0007] The present invention provides a compound of SEQ ID NO: 17 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 11 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 21 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 20 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 4 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 7 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 33 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 32 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 34 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 36 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 38 or a pharmaceutically acceptable salt thereof. The present invention provides a compound of SEQ ID NO: 39 or a pharmaceutically acceptable salt thereof.

[0008] The present invention provides a compound of the following formula:

[0009]

[0010] or a pharmaceutically acceptable salt thereof.

[0011] The present invention provides a compound of the following formula:

[0012]

[0013] or a pharmaceutically acceptable salt thereof.

[0014] The present invention provides a method in which tirzepatide is prepared using nanofiltration.

[0015] The present invention provides a method for preparing tirzepatide, comprising deprotecting a compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof.

[0016] The present invention provides a method for selectively acylating a lysine amino acid, wherein the lysine amino acid and the N-terminus are protected. The present invention provides a method for selectively acylating a lysine amino acid in a peptide, which comprises coupling a resin-bound peptide-lysine-NH 2With tert-butyl-eicosanedioyl-Glu-(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH. The present invention provides a method for preparing tirzepatide, which includes deprotecting the compound of SEQ ID NO: 22 or its pharmaceutically acceptable salt.

[0017] The present invention provides a method for deprotecting tirzepatide, wherein the deprotection solution contains dithiothreitol, triisopropylsilane, and trifluoroacetic acid.

[0018] The present invention provides a method for selectively acylating lysine amino acids, wherein the resin-bound peptide-lysine-NH 2 is a compound of the following formula:

[0019]

[0020] or its pharmaceutically acceptable salt.

[0021] The present invention provides a method for converting a depsipeptide isomer into a desired peptide, which includes: adjusting the pH of the depsipeptide isomer to about pH 7 to about pH 10; and incubating the depsipeptide isomer at pH 7 to pH 10 for at least one hour.

[0022] The present invention provides a method for converting a depsipeptide isomer, wherein the depsipeptide isomer is adjusted to about pH 8.5 to about pH 9.5.

[0023] The present invention provides a method for converting a depsipeptide isomer, wherein the depsipeptide isomer is the compound of SEQ ID NO: 40 or its pharmaceutically acceptable salt.

[0024] The present invention provides radical-based desulfurization, which includes contacting a peptide with a radical initiator. In one embodiment, the desulfurization includes contacting a peptide suitable for desulfurization with a water-soluble radical initiator. In one embodiment, the radical initiator is an azo initiator. In one embodiment, the radical initiator is selected from 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (VA-050).

[0025] The radical-based desulfurization method provided herein is environmentally desirable, free of transition metals, and compatible with peptide synthesis conditions.

[0026] As used herein, the following abbreviations have the meanings given herein: "SPPS" means solid phase peptide synthesis, "Fmoc" means fluorenylmethyloxycarbonyl chloride, "Pip" means piperidine, "DIC" means diisopropylcarbodiimide, "Oxyma" means ethyl cyano(hydroxyimino)acetate, "DCM" means dichloromethane, "IPA" means isopropyl alcohol, "MTBE" means methyl tert-butyl ether, "TFA" means trifluoroacetic acid, "TIPS" means triisopropylsilane, "DTT" means dithiothreitol, "UPLC" means ultra performance liquid chromatography, "HFIP" means hexafluoroisopropanol, "CTC" means trityl chloride, "HATU" means (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), "TFET" means 2,2,2-trifluoroethylthiol, "DIEA" means N,N-diisopropylethylamine, "AEEA" means 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid, "TCEP" means tris(2-carboxyethyl)phosphine, "DCU" means dicyclohexylurea, "DCC" means dicyclohexylcarbodiimide, "TMSA" means trimethylsilyl azide, "HOBt" means hydroxybenzotriazole, "HRMS" means high resolution mass spectrometry, "LPPS" means liquid phase peptide synthesis, "MSMPR" means mixed product mixed suspension reactor, "MPA" means mobile phase A, "MPB" means mobile phase B, "L-GSH" means L-glutathione reduced solution, "TZP" means tirzepatide, "AP" means active pharmaceutical, and "API" means active pharmaceutical ingredient, "PyBOP" means (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate), "DEA" means diethylamine, "TBTU" means 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate, "TNTU" means 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate, "PyOxim" means 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tripyrrolidinophosphonium hexafluorophosphate, "PyClock" means 6-chloro-benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate. As shown herein, one-letter amino acid abbreviations are shown in bold, while atoms are shown in non-bold text and are generally smaller in font to distinguish them from one-letter amino acid abbreviations. As used herein, when an amino acid abbreviation appears with a number above the amino acid, the number refers to the position of the corresponding amino acid in the final tirzepatide product. These numbers are provided for convenience, and the presence or absence of these numbers in the sequence does not affect the amino acid sequence or the peptide indicated in such a sequence.As used herein, the term "protected" means that a protecting group is attached at the indicated position. Those skilled in the art will recognize that various protecting groups are well known and alternative protecting groups may be suitable for a particular method.

[0027] Those skilled in the art will understand that there are alternative resins for constructing the peptides shown herein. For example, Sieber and Rink amide resins are well known to those skilled in the art for the preparation of the peptides disclosed herein; however, alternative resins may be selected for the preparation of the peptides described herein. For example but not limited to, 2-CTC and related resins can be used for the preparation of the target peptides, followed by a C-terminal amidation step.

[0028] Solid-phase peptide synthesis (SPPS) construction is accomplished using standard fluorenylmethyloxycarbonyl chloride (Fmoc) peptide chemistry techniques that employ sequential coupling with an automated peptide synthesizer. The resin is swollen with DMF and then deprotected with 20% piperidine (Pip) / DMF (3×30 min). Subsequent Fmoc deprotection is carried out using 20% Pip / DMF 3×30 min treatment, and 4×30 min treatment is used for more difficult couplings. After deprotection, the resin is washed with 5×2 min, 10 volumes of DMF wash solution. Amino acid pre-activation is carried out using a diisopropylcarbodiimide (DIC) / ethyl cyano(hydroxyimino)acetate (Oxyma) DMF solution held at room temperature for 30 min. For each individual amino acid, the coupling of the activated amino acid to the resin-bound peptide occurs at a specific time. After each coupling, the solvent is washed with 5×2 min 10 volumes of DMF. To isolate the final product, the resin-bound product is washed with 10 volumes of DCM 5×2 min to remove DMF. The resin is washed with 2×2 min 10 volumes of IPA to remove DCM, washed with 5×2 min 10 volumes of methyl tert-butyl ether (MTBE), and then the product is dried under vacuum at 40 °C. The resin-bound product is refrigerated (-20 °C). For analysis, use trifluoroacetic acid (TFA) / H 2An acidic mixture composed of O / TIPS (triisopropylsilane) / DTT (dithiothreitol) in the following ratio cleaves peptides from the resin: (0.93 v / 0.04 v / 0.03 v / 0.03 w). The resin was swollen and drained with DCM (4 - 5 mL, 3×30 min). The cleavage mixture (4 - 5 mL) was added to the pre-swollen resin, and the suspension was stirred at room temperature for 2 hours. The solution was filtered, then the resin was washed with a small amount of DCM and combined with the cleavage solution. The resulting solution was poured into 7 - 10 volumes of cold (0 °C) methyl tert-butyl ether (MTBE). The suspension was aged at 0 °C for 30 min, then the resulting precipitate was centrifuged and the clear solution was decanted. The residue was suspended in the same volume of MTBE, and the resulting suspension was centrifuged and decanted again. After decantation, the clear MTBE solution of the precipitated peptide was dried under vacuum at 40 °C overnight.

[0029] Synthesis of Preparation 1

[0030] SEQ ID NO: 2

[0031] Synthesis was carried out using Fmoc-Sieber amide resin with a loading of 0.71 mmol / g. The general SPPS procedure was used with the following modifications:

[0032]

[0033] Preparation 1 Soft Cleavage: Ten identical deprotection reactions were carried out in parallel, each with a resin-bound Preparation 1 on a scale of ~0.5 mmol, using the following protocol: 1) Add 1.55 g (~0.5 mmol) of resin-bound Preparation 1 to a 40 mL sintered reactor. 2) Swell with 3 × 15 mL of DMF (15 min each). 3) Treat with 3 × 15 mL (30 min each) of 20% Pip / DMF. 4) Wash with 4 × 15 mL of DMF, followed by 4 × 15 mL of DCM. 5) Add 1.5 mL of TFA and 28.5 mL of DCM to each of five 40 mL reaction vials. 6) Add one-fifth of the Preparation 1 resin-bound (2.75 g) to each of the TFA solution vials, cap the vials, and mix on a rotator for 5 minutes. 7) Filter the mixture and wash with 100 mL of DCM to give a total filtrate volume of 500 mL. 8) Combine the filtrates and transfer to a round-bottom flask containing 1000 mL of MTBE. 9) Concentrate the resulting suspension to a pale yellow oil, triturate with 200 mL of MTBE, and cool in an ice bath for 30 minutes. 10) Filter the solid, wash with 50 mL of cold MTBE, and dry overnight in a vacuum oven at 33 °C to yield 5.35 g (91% yield) of a white solid. The isolated solid was analyzed by UPLC (98.57 area %, with 0.99% of combined t-Bu deprotection by-products).

[0034] Synthesis of Preparation 2

[0035] SEQ ID NO: 3

[0036] Synthesis was carried out using Fmoc-Gly-OH 2-CTC resin with a loading of 0.61 mmol / g. The general SPPS procedure was modified as follows:

[0037]

[0038]

[0039] Preparation 2 Soft Cleavage: Add resin-bound Preparation 2 (3.06 g, 1.12 mmol) and 30 mL of 30% HFIP in DCM solution to a 40 mL glass scintillation vial, where a red change was observed. Agitate the vial by rotating on a wheel at room temperature for 1 hour. Filter out the resin and wash with 3 × 10 mL of DCM. Remove the solvent in vacuo to form a glassy foam (35 °C bath, 10 Torr, 2.34 g), and replace with a small portion of IPA (24 mL), then add water (24 mL) dropwise over 25 min at room temperature. Stir the resulting solution for 30 min, then filter. Wash with 3 × 10 mL of H2 Wash the filter cake and then dry it overnight in a vacuum oven at 25 Torr and 35 °C. This yielded Preparation 2, a white solid (1.81 g).

[0040] Synthesis of Preparation 4

[0041] SEQ ID NO: 4

[0042] Synthesis was carried out using Fmoc-Leu-OH 2-CTC resin with a loading of 0.68 mmol / g. The general SPPS procedure was modified as follows:

[0043]

[0044]

[0045] Soft cleavage of Preparation 4: To a 20 mL glass scintillation vial was added resin-bound Preparation 4 (2.0 g, 0.62 mmol) and 10 mL of a 30% HFIP in DCM solution, where a red color change was observed. The vial was agitated by rotation on a wheel at ambient temperature, then the resin was filtered off, washed with 3 × 2 mL of DCM, and the solvent was removed in vacuo to form a glassy, viscous foam. The foam was dissolved in 5.2 mL of DMSO. A solution of 6 mL of water was added to this solution at a constant flow rate (T ~ 15 °C) over 45 min with 1 mL of water. Once the peptide solution was completely added, an additional 6 mL of water was added over 45 min. A white solid precipitated upon addition. The resulting slurry was stirred at 15 °C for 30 min. The solid was filtered, washed with 6 mL of water, and then transferred to a vacuum oven at 35 °C and 25 Torr. This yielded Preparation 4 (Boc-1-14-OH, 1.0763 g), a white fluffy solid.

[0046] Synthesis of Preparation 3 using LPPS

[0047] SEQ ID NO: 5

[0048] To a 20 mL glass scintillation vial, add Preparation 2 (500 mg, 0.183 mmol), Preparation 1 (179 mg, 0.175 mmol), and DMSO (10 mL). Add DIEA (46 µL, 0.265 mmol) to the solution, followed by PyBOP (benzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate) (123 mg, 0.230 mmol). Then stir the reaction for 2 h, add diethylamine (DEA) (183 µL, 1.77 mmol), and stir the resulting solution for 2 h. Withdraw the contents of the reaction into a syringe and add it dropwise over 1 h to a stirred 50 mL flask while simultaneously adding water (12 mL). After addition is complete, collect the precipitated product by filtration and wash it successively with water (2 × 4 mL). Dry the wet filter cake under vacuum at 35 °C for 18 h to obtain Preparation 3 as a white solid (0.6003 g, 88% yield, HRMS calcd for C 184 H 261 N 31 O 38 expected 3512.9444, found 3512.9430).

[0049] Synthesis of Preparation 5 using LPPS

[0050] SEQ ID NO: 6

[0051]

[0052] To a 20 mL glass scintillation vial, add Preparation 3 (338.8 mg, 0.091 mmol), Preparation 4 (192.1 mg, 0.091 mmol), and DMSO (10 mL). Add PyBOP (63.5 mg, 0.118 mmol) to the solution, followed by DIEA (79 µL, 0.454 mmol). Stir the reaction solution for 2.5 h. Withdraw the contents of the reaction into a syringe and add it dropwise over 1 h to a stirred 50 mL flask while simultaneously adding water (12 mL). After addition is complete, collect the precipitated product by filtration and wash it successively with water (2 × 4 mL). Dry the wet filter cake under vacuum at 35 °C for 18 h to obtain Preparation 5 as a white solid (0.3568 g, 70% yield, HRMS calcd for C 293 H 435 N 45 O 64 expected 5608.2168, found 5608.2066).

[0053] Synthesis of Preparation 6 using Method 1 (LPPS)

[0054]

[0055] Dissolve monoisopropyl dodecanedioate (15.0 kg, limiting reagent) and N-hydroxy-succinimide (1.2 eq) in ethyl acetate at 27 °C. Add a solution of DCC (1.25 eq.) in ethyl acetate and stir the reaction mixture at 22 °C for 24 hours. Filter off the resulting DCU by-product, then extract the organic phase 3 times with 5% aqueous NaCl solution. After extraction, concentrate the organic phase, co-evaporate with isopropanol, and then crystallize by adding heptane. After filtration, wash the filter cake with heptane and dry at 25 °C to afford 17.0 kg of INT1, in 87% yield and 99% purity.

[0056] Dissolve H-Glu-OtBu (7.7 kg, 1.1 eq) in DCM (54 L) at 20 °C, then add a solution of TMSA (11.3 kg) dissolved in DCM (7 L), and then stir the reaction mixture at 40 °C for 1 hour. Add a solution of INT1 (17.0 kg) in DCM at room temperature and stir for 8 hours. After the reaction is complete, exchange DCM for ethyl acetate by distillation. Wash the organic phase 3 times with 2% KHSO 4 / aqueous NaCl solution and then 4 times with 2% aqueous NaCl solution. After separating and removing the aqueous phase, concentrate the organic phase with isopropanol, dilute with isopropanol, and then crystallize by adding water. After filtration, wash the filter cake with a mixture of water / isopropanol and then dry at 30 °C to yield 17.3 kg of INT 2, in 86% yield and 99% purity.

[0057] Dissolve INT 2 (17.3 kg) and N-hydroxy-succinimide (4.1 kg, 1.2 eq) in ethyl acetate (336 kg) at 27 °C. Add a solution of DCC (8.33 kg, 1.25 eq) in ethyl acetate and stir the reaction mixture at 22 °C for 24 hours. Filter off the resulting DCU by-product. Concentrate the organic phase, co-evaporate with isopropanol, and then crystallize by cooling the isopropanol solution (~125 L). Subsequently, wash the filter cake with cold isopropanol and dry at 25 °C to afford 16.3 kg of INT 3, in 81% yield and 96% purity.

[0058] Suspend 17 - amino - 10 - oxo - 3,6,12,15 - tetraoxa - 9 - azadecanoic acid (AEEA2) (8.1 kg, 26.3 mol) in DCM (54 L) at 22 °C. Add a solution of TMSA (7.68 kg, 59.9 mol) in DCM (6.2 L), and then stir the reaction mixture at 40 °C for 1 hour. Suspend INT 3 (16 kg) in DCM (31 L) at 35 °C and add it to the TMS - protected (AEEA2) mixture at 22 °C. Stir the reaction for 12 hours, and after the reaction is complete, concentrate the mixture and then exchange it to ethyl acetate. Wash the organic phase three times with 2% KHSO 4 / NaCl aqueous solution (~200 L), and then wash it 4 times with 2% NaCl aqueous solution (~200 L) to a target pH of 4.5. Concentrate the organic phase and exchange it to acetonitrile. Cool the acetonitrile solution to -20 °C, and then age the resulting suspension at -20 °C for 15 hours. Filter the mixture, wash the filter cake with cold acetonitrile, and then dry it at <0 °C to provide 18.4 kg of Preparation 6 (88% yield), with a purity of 96%. Total yield = 53%.

[0059] Synthesis of Preparation 6 using Method 2 (SPPS)

[0060] Alternatively, Preparation 6 can be prepared using solid - phase peptide synthesis with a peptide synthesizer.

[0061] Adopt the standard coupling procedure.

[0062] Standard coupling conditions:

[0063] 0.133 M, 2.0 equivalents of HATU, 5.0 equivalents of DIEA, ambient temperature, 3 hours, deprotect with 20% piperidine / DMF for 3×15 min.

[0064] Resin charge:

[0065] FmocNH - AEEA (0.99 mmol / g) on 2 - CTC resin: 1.01 g in each parallel reaction.

[0066] The automated program uses DMF swelling, followed by Pip / DMF; DMF washing; and a mixture of amino acid, DIEA, and HATU; and a DMF washing cycle, followed by drying.

[0067] The resin was cleaved by stirring the combined batches in 30% HFIP / DCM (240 mL) for 1.5 h. The resin was filtered, washed, and the solvent was removed from the filtrate in vacuo. The resulting oil was dissolved in acetonitrile and the solvent was removed again. This operation provided 30.47 g (146% of the theoretical yield) of a viscous yellow oil which contained 52.3 area % of the desired product as analyzed by UPLC. The crude product was purified by flash chromatography (500 g of silica gel, eluting with 85% DCM / 10% methanol / 5% acetic acid, 38×100 mL fractions were collected). The previous chromatographic concentrate (17.94 g) was crystallized to yield 13.4 g (74.7% yield) which had a UPLC purity of 91.65 area %.

[0068] Example 1

[0069]

[0070]

[0071] Synthesis Example 1

[0072] SEQ ID NO: 1

[0073] To the first HPLC vial was added Preparation 5 (10.5 mg, 0.00187 mmol) and DCM (200 µL, 20 L / kg). To this solution was added a solution of phenylsilane (0.81 M in DCM, 22.1 µL, 0.0178 mmol) and tetrakis(triphenylphosphine)-palladium(0) (0.8 M in DCM, 22.1 µL, 0.00064 mmol). The solution was stirred at 24 °C for one hour to obtain a non-separated solution of Preparation 7 (SEQ ID NO: 7). To the second HPLC vial was added DCM (150 µL), followed by Preparation 6 (0.118 M in DCM, 16 µL, 0.00189 mmol), PyBOP (0.186 M in DCM, 16 µL, 0.00298 mmol) and DIEA (0.573 M in DCM, 5 eq). The contents of the second vial were added to the first vial and the reaction was stirred for 1 h to obtain a non-separated solution of Preparation 8 (SEQ ID NO: 8). The solution of Preparation 8 was concentrated under vacuum and the resulting solid was added to a solution of 50 µL of trifluoroacetic acid (4.65 mL), triisopropylsilane (20 µL) and DTT (20 mg). The slurry was stirred for 18 h and monitored by HPLC to confirm the formation of Example 1 (HRMS calculated C 225 H 348 N 48 O 68The expected value is 4810.5249, and the measured value is 4810.5257).

[0074] Synthesis of Preparation 9

[0075] SEQ ID NO: 9

[0076] Suspend Sieber amide resin (13.42 g, 0.75 mmol / g, 10.1 mmol) in DMF (130 mL, 10 volumes) for about 20 min and then drain. Wash the resulting resin with DMF (80 mL, 6 volumes) for about 5 min. Remove the Fmoc group by treating the Fmoc-amino acid resin twice (for 10 min and 20 min respectively) with a 5 volume % piperidine, 1.25 volume % DBU, 1.0 wt.% HOBt / DMF solution (80 mL, 6 volumes). After draining the de-Fmoc solution, wash twice with DMF (80 mL, 6 volumes), twice with MTBE (80 mL, 6 volumes), and then twice again with DMF (80 mL, 6 volumes).

[0077] Assemble the amino acid chain using standard Fmoc chemistry. Generally, dissolve 1.5 equivalents of Fmoc-amino acid and HOBt (2.47 g, 20% water-wetted, 14.6 mmol, 1.46 equivalents) in DMF (60 mL, 4.5 volumes), then add DIEA (1.94 mL, 11.1 mmol, 1.11 equivalents). Cool the resulting solution to < 5 °C with an ice bath and activate by adding TBTU (4.83 g, 15.0 mmol, 1.5 equivalents). Allow to stand at 0 °C - 5 °C for about 5 minutes. Add DCM (60 mL, 1.5 volumes) to the resin, then add the activated Fmoc-amino acid solution. Stir the resulting mixture at about ambient temperature for 2 hours. Repeat the de-Fmoc process and couple with the remaining amino acids in sequence. After completing the final de-Fmoc process, wash the resin twice with 2-propanol (130 mL, 10 volumes) for 5 min, then wash six times with MTBE (130 mL, 10 volumes). Dry the resin in vacuo at 35 °C to obtain Preparation 9 - Seiber (21.21 g, 0.435 mmol / g theoretical, 91.7% yield based on mass increase).

[0078] A portion of the Preparation 9-resin complex (10.15 g, 0.435 mmol / g, 4.41 mmol) was treated with a solution of 5 vol% TFA in DCM (101 mL, 10 volumes) and a DCM wash step. The cleavage fractions and washes were neutralized with DIEA (26.29 g, 35.5 mL, 1.01:1 molar ratio to TFA). The fractions were combined and concentrated in vacuo to 50% of the original volume. The DCM solution was washed with saturated NaHCO 3 aqueous solution (2 × 94 mL). The resulting solution was dried over anhydrous MgSO 4 and concentrated to dryness to yield a viscous solid. The viscous solid was reslurried in MTBE (100 mL) at < 5 °C to break up the gum, giving a white slurry product. The white powder slurry was filtered, washed, and dried to give Preparation 9 (3.84 g, 92.3 area%, 37.8 wt% DIEA•TFA, 57.4 wt%, 2.29 mmol, 51.9% yield, HRMS calculated C 46 H 78 N 10 O 12 expected value 962.5801, found 962.5806), as a white powder.

[0079] Synthesis of Preparation 10

[0080] SEQ ID NO: 10

[0081] The Fmoc-Gly-Gly-O-2CTC resin complex (18.09 g, 0.57 mmol / g, 10.3 mmol) was suspended in DMF (180 mL, 10 volumes) for 20 min and then drained. The resulting resin was washed with DMF (108 mL, 6 volumes) for 5 min. The Fmoc group was removed by treating the Fmoc-amino acid resin twice (10 min and 20 min respectively) with a 5 vol% piperidine, 1.25 vol% DBU, 1.0 wt.% HOBt / DMF solution (108 mL, 6 volumes). The de-Fmoc solution was drained and the resin was washed twice with DMF (110 mL, 6 volumes), twice with MTBE (110 mL, 6 volumes), and twice again with DMF (110 mL, 6 volumes). Chain assembly was carried out using standard Fmoc chemistry.

[0082] For the coupling of amino acids, generally 1.5 equivalents of Fmoc - amino acid and HOBt (2.54 g, 20% water - moistened, 15.0 mmol, 1.5 equivalents) are dissolved in DMF (80 mL, 4.4 volumes), followed by the addition of DIEA (1.94 g, 15.0 mmol, 1.5 equivalents) to provide the coupling of amino acids. The resulting solution is cooled to 0 - 5 °C in an ice bath and activated by the addition of 2 - (1H - benzotriazol - 1 - yl) - 1,1,3,3 - tetramethylammonium tetrafluoroborate (TBTU) (4.84 g, 15.1 mmol, 1.5 equivalents). It is allowed to stand at 0 - 5 °C for 5 min. Then DCM (35 g, 1.5 volumes) is added to the resin, followed by the addition of the activated Fmoc - amino acid solution. The resulting mixture is stirred at room temperature for 2 h. After completion of the synthesis step, the peptide resin is washed twice with 2 - propanol (180 mL, 10 volumes) for 5 min, then washed with MTBE (180 mL, 10 volumes each, 6 times), and then dried at 35 °C to obtain the Preparation 10 resin complex (25.52 g, 0.216 mmol / g, 53.6% yield).

[0083] A portion of the Preparation 10 resin complex (10.075 g, 0.216 mmol / g, 2.18 mmol) is treated three times with a 1 volume% solution of TFA in DCM (100 mL, 10 volumes) and washed with DCM (75 mL, 7.5 volumes). The cleavage fractions and washings are neutralized with pyridine (3.18 g, 1.01:1 molar ratio to TFA). The fractions are combined and concentrated to dryness in vacuo at ≤ 35 °C. Reconstitution is carried out with ethanol (40 mL, 10% volume of the combined filtrate), followed by concentration to dryness. Finally, the peptide is triturated with stirring in deionized water (150 mL, 40% volume of the combined filtrate). The solid crude peptide precipitate is collected by centrifugation and washed twice with deionized water (150 mL each time). The solid is washed twice with n - heptane (100 mL each time), separated, and dried in vacuo at 40 °C to yield Preparation 10 (SEQ ID NO: 10) as a friable pale - yellow solid (4.10 g, 72.4 area%, 3.0 wt% pyridine•TFA, 70.2 wt%, 1.85 mmol, 85.1% yield, HRMS calculated for C 88 H 103 N 11 O 15 expected value of 1553.7635, found value of 1553.7656).

[0084] Synthesis of Preparation 11

[0085] SEQ ID NO: 11

[0086] The H-alanine-O-2CTC resin complex (40.39 g, 0.5 mmol / g, 20.20 mmol) was suspended in DMF (400 mL, 10 volumes) for about 20 minutes and then drained. The resulting resin was washed twice with DMF (400 mL, 10 volumes) for 5 min. The amino acid chain was assembled using standard Fmoc chemistry. Generally, 1.5 equivalents of Fmoc-amino acid and HOBt (5.51 g, 80 wt.%, 32.6 mmol, 1.6 equivalents) were dissolved in DMF (150 mL, 3.7 volumes), followed by the addition of DIEA (4.22 g, 32.7 mmol, 1.6 equivalents). The resulting solution was cooled to about < 5 °C in an ice bath and activated by the addition of TBTU (10.39 g, 32.4 mmol, 1.6 equivalents). It was stirred at 0 - 5 °C for about 5 minutes. DCM (80 mL, 2 volumes) was added to the resin, followed by the addition of the activated Fmoc-amino acid solution. The resulting mixture was stirred at about ambient temperature for 2 hours.

[0087] The Fmoc group was removed by treating the Fmoc-amino acid resin twice (10 min and 20 min each) with 5 vol% piperidine, 1.25 vol% DBU, 1.0 wt% HOBt / DMF solution (240 mL, 6 vol). The de-Fmoc solution was drained, and the resin was washed twice with DMF (240 mL, 6 vol), twice with MTBE (240 mL, 6 vol), and then twice again with DMF (240 mL, 6 vol). After completion of the synthesis step, the peptide resin was washed thoroughly twice with 2-propanol (400 mL, 10 vol) and six times with MTBE (400 mL, 10 vol each time), and then dried in vacuo at 35 °C to yield the loaded resin that was not the final amino acid (74.82 g, 0.159 mmol / g, 11.90 mmol, 58.9% yield). The final amino acid Fmoc-Leu-OH was added separately to a portion of the resin (13.61 g, 0.159 mmol / g, 2.16 mmol). The resin was swollen with DMF (130 mL, 10 vol, 3 times) for >5 min each time, and then deprotected for 10 min and 20 min (130 mL of the deprotection mixture was prepared from 5.6 g of piperidine, 1.67 g of DBU, 1.3 g of HOBt in 120 mL of DMF, 10 vol twice). The resin was washed with DMF (80 mL, 6 vol, twice), followed by MTBE (80 mL, 6 vol, twice), and then DMF (80 mL, 6 vol, twice) for 5 min each. It was dissolved in DMF (50 mL, 3.7 vol), and then DIEA (0.54 g, 4.2 mmol, 1.9 equiv), Fmoc-Leu-OH (1.47 g, 4.16 mmol, 1.9 equiv), and HOBt (0.704 g, 80 wt.%, 4.17 mmol, 1.9 equiv) for the coupling of Fmoc-Leu-OH were added. The resulting solution was cooled to <5 °C in an ice bath and activated by adding TBTU (1.34 g, 4.17 mmol, 1.9 equiv) and stirring it at 0 - 5 °C for 5 min. DCM (20 mL, 1.5 vol) was added to the resin, followed by the activated Fmoc-amino acid solution. The resulting mixture was stirred at approximately ambient temperature for 2 h. The resin was washed with DMF (180 mL, 13 vol, twice), MTBE (180 mL, 13 vol, twice), and DMF (180 mL, 13 vol, twice) for 5 min each. Before drying the resin in vacuo at 35 °C, the resin was washed with DCM (130 mL, 10 vol, 6 times, 5 min each time) to give the loaded resin (12.90 g, 0.203 mmol / g, 2.62 mmol, 121% yield).

[0088] Treat a portion of the resin (7.09 g, 0.203 mmol / g, 1.44 mmol) three times for 10 min each with a 1% by volume solution of TFA in DCM (70 mL, 10 volumes) at approximately ambient temperature, followed by washing with DCM (55 mL, 7.5 volumes). Neutralize and cleave the fractions and washes with pyridine (3.02 g, 1.02:1 molar ratio to TFA). Combine and concentrate the fractions to dryness under vacuum at ≤ 35 °C. Reconstitute with ethanol (28 mL, 11% by volume of the combined filtrate), followed by concentration to dryness. Finally, stir the peptide in deionized water (105 mL, 40% by volume of the combined filtrate). Collect the solid crude peptide precipitate by filtration and wash with deionized water (4 × 50 mL). Wash the solid with n - heptane (3 × 100 mL), separate and dry in vacuo at 40 °C to give Preparation 11 as a white powder (4.54 g, 87.6 area%, 44.4 wt% pyridine•TFA, 48.7 wt%, 0.936 mmol, 65.0% yield, HRMS calculated for C 127 H 192 N 14 O 28 expected value 2361.4031, found 2361.4021). The overall yield of Preparation 11 on the resin was 71.3%.

[0089] Synthesis of Preparation 12

[0090] SEQ ID NO: 12

[0091] Suspend the Fmoc - Aib - O - CTC resin complex (19.16 g, 0.54 mmol / g, 10.35 mmol) in DMF (190 mL, 10 volumes) for 20 min, then drain. Wash the resulting resin with DMF (190 mL, 10 volumes) for 5 min, then drain. Combine piperidine (77.82 g), DBU (23.16 g), HOBt (18.09 g, 80 wt%) and DMF (1800 mL) to provide a solution of 5% piperidine, 1.25% DBU, 1.0% HOBt / DMF as the deprotection solution. Remove the Fmoc group by treating the Fmoc - amino acid resin twice (10 min and 20 min each) with the deprotection solution (190 mL, 10 volumes). Drain the de - Fmoc solution, then wash the resin twice with DMF, twice with MTBE, and twice again with DMF (190 mL, 10 volumes for each wash).

[0092] Add DIEA (2.62 g, 20.3 mmol, 2.0 eq) to a solution of Fmoc-Ile-OH (7.11 g, 10.1 mmol, 2.0 eq) in DMF (85 mL). Cool the resulting solution to 0 - 5 °C, add 6-chloro-benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock) (11.36 g, 20.06 mmol, 2.0 eq) and dissolve completely. After standing for 3 to 5 minutes, add the activated solution to the pre-swollen H-Aib-O-CTC resin complex in DCM (30 mL, 1.5 volumes). Allow the reaction to warm to ambient temperature and stir for 2 hours. As determined by analysis, the unreacted material was approximately 18%. Wash with DMF twice, MTBE twice, and DMF twice (190 mL, 10 volumes each). Add a solution of Fmoc-Ile-OH (10.63 g, 30.08 mmol, 6 eq) in DMF (165 mL) to Oxyma (50 mL, 0.6 M in DMF, 30 mmol, 6 eq) and DIC (50 mL, 0.66 M in DMF, 33 mmol, 6.6 eq). Stir at approximately ambient temperature for 5 min, then add to the resin and stir for 18 hours. Add a mixture of pyridine, acetic anhydride, and DMF to the resin and stir for 0.5 hour. Wash the resin with DMF (5 × 140 mL, 7 volumes), more DMF (2 × 180 mL, 9 volumes), MTBE (2 × 180 mL, 9 volumes), then DMF (2 × 180 mL, 9 volumes).

[0093] For the remaining amino acids, perform the remaining chain assembly in sequence using standard Fmoc chemistry. Generally, dissolve Fmoc-amino acid (2.0 eq), HOBt (3.42 g, 80 wt%, 2.0 eq) in DMF (85 mL), then add DIEA (2.64 g, 2.0 eq). Cool the resulting solution to 0 - 5 °C with an ice bath and activate by adding TBTU (6.45 g, 2.0 eq) and standing at 0 - 5 °C for 3 - 5 min. Add DCM (30 mL) to the resin, then add the activated Fmoc-amino acid solution. Stir the resulting mixture at room temperature for 2 hours. Wash the resulting resin with DMF twice, MTBE twice, and DMF twice again (190 mL, 10 volumes each wash). Remove the Fmoc group by treating the Fmoc-amino acid resin twice with the deprotection solution (190 mL, 10 volumes) (10 min and 20 min each). After draining the de-Fmoc solution, wash the resin with DMF twice, MTBE twice, and DMF twice again (190 mL, 10 volumes each wash).

[0094] Activate the tetramer Boc-Y-Aib-E(tBu)G-OH (12.25 g, 2.0 equiv) with Oxyma (30 mL, 0.6 M in DMF, 20 mmol, 2 equiv) and DIC (33 mL, 0.66 M, 22 mmol, 2.1 equiv) in DMF (50 mL) for 5 min to add the final four amino acids as a tetramer. Add the mixture to the resin and couple for 18 h. Drain the mixture at the end of 18 h and wash the resin with DMF (190 mL, 5 times for 5 min each). Add more tetramer (6.21 g, 1.0 equiv) in DMF (40 mL), activate with PyBOP (5.77 g, 1.1 equiv) and DIEA (3.32 g, 2.6 equiv) for 5 min and stir for 4 h before adding the mixture to the resin. Drain the mixture at the end of 4 h and wash with DMF (190 mL, 5 times for 5 min each). Cap the resin by adding a mixture of DMF (105 mL), pyridine (13.48 g, 17 equiv) and acetic anhydride (14.27 g, 14 equiv) to the resin and stirring for 1 h. After completion of chain assembly, wash the peptide resin with DMF five times (190 mL each), DCM six times (190 mL each) for 5 min each, then dry under vacuum at 35 °C to give the Preparation 12 resin complex (31.03 g, 0.2595 g / mmol theoretically, 8.05 mmol, 77.8% yield). Treat a portion of the Preparation 12 resin complex (15.975 g, 0.2595 mmol / g, 4.146 mmol) with a 1 vol% solution of TFA in DCM (160 mL, 10 vol) three times for 10 min each at ambient temperature, then wash with DCM (120 mL, 7.5 vol). Neutralize the cleavage fractions and washes with pyridine (4.74 g, 0.94:1 molar ratio to TFA). Combine and concentrate the fractions to dryness under vacuum at ≤ 35 °C. Perform reconstitution with ethanol (30 mL, 5% volume of the combined filtrate), then concentrate to dryness. Mechanically stir the peptide in deionized water (242 mL, 40% volume of the combined filtrate) for 10 min. Collect the solid crude peptide by filtration and wash with deionized water (4 × 100 mL). Wash the solid with n-heptane (4 × 100 mL), separate, and dry under vacuum at 35 °C to give Preparation 12 as a white powder (9.38 g, 82.2 area%, 0.2 wt% pyridine•TFA, 82.1 wt%, 3.85 mmol, 92.8% yield, HRMS calcd C 103 H 165 N 13 O 26The expected value is 2000.1989, and the measured value is 2000.1968).

[0095] Synthesis of Preparation 13

[0096] SEQ ID NO: 13

[0097] In N 2 While stirring, add Preparation 9 (2.887 g, 70.2 wt%, 1.30 mmol), Preparation 10 (3.576 g, 57.4 wt%, 2.13 mmol, 1.63 equivalents), DMSO (18.1 g, 16.4 mL), DMF (15.8 g, 16.7 mL), and DIEA (655 mg, 5.07 mmol, 3.89 equivalents) to the flask until a golden solution is obtained. Cool the solution in an ice bath, then add PyBOP (1.414 g, 2.72 mmol, 2.08 equivalents). Remove the ice bath and allow the mixture to warm to ambient temperature. Monitor the reaction for about 5 hours to ensure complete conversion. Add an aliquot of diethylamine (2.116 g, 28.9 mmol, 22.2 equivalents) to the reaction mixture at ambient temperature. Stir the mixture for about one hour to provide a conversion of > 99% to Preparation 13. Precipitate the product by adding a mixture of saturated NaHCO 3 aqueous solution (50 mL) and deionized water (50 mL) at < 4 °C to the reaction mixture. Stir the mixture for at least about 15 minutes under cold conditions. Filter the pasty white slurry. Wash the wet filter cake with deionized water (3 × 50 mL), followed by MTBE (6 × 50 mL), and dry in a N 2 purified vacuum at 40 °C for about 62 hours. This method gives Preparation 13 (4.45 g, 60.4 area%, 16.4 area% dibenzofulvene, 1.18 mmol, 90.5% yield, HRMS calculated C 119 H 169 N 21 O 24 The expected value is 2276.2649, and the measured value is 2276.2550), a pale yellow solid.

[0098] Synthesis of Preparation 14

[0099] SEQ ID NO: 14

[0100] In N 2An aliquot of Preparation 11 (3.012 g, 48.7 wt%, 0.621 mmol, 1.00 equiv) was added to a flask with Preparation 13 (3.951 g, 60.4 wt%, 1.05 mmol, 1.69 equiv), DMSO (9.8 g, 8.9 mL), DMF (52.0 g, 55.0 mL) and DIEA (372 mg, 2.88 mmol, 4.63 equiv) at 4 °C. The mixture was stirred until a golden solution was produced. Ice water was used to cool the mixture to < 10 °C. An aliquot of PyBOP (742 mg, 1.42 mmol, 2.30 equiv) was added to the mixture. The ice bath was removed and the mixture was allowed to warm to about ambient temperature. The reaction was monitored for about 22 hours to convert to Preparation 14. This resulted in a conversion of about > 96%. At a temperature of <10°C, piperidine (530 mg, 6.22 mmol, 10.0 equiv) was added to the cooled reaction mixture. The mixture was stirred at ambient temperature for about 2 hours to provide a > about 99% conversion to Preparation 14. The reaction mixture was added to another flask containing <4°C 0.5N aqueous HCl (12.72 g, 6.23 mmol, 10.0 equiv) and deionized water (16.71 g) to provide precipitation of Preparation 14. The cold slurry was stirred for about 15 minutes, and the white slurry was filtered. The wet cake was washed with deionized water (2×30 mL), saturated NaHCO 3 The mixture was washed with aqueous solution (2 × 30 mL), deionized water (3 × 30 mL), and MTBE (4 × 45 mL) and stirred at 40 °C under N 2 The product was dried in a purified vacuum for about 17 hours to obtain Preparation 14 (5.418 g, 48.9 area %, 0.603 mmol, 97.0% yield, HRMS calculated C 231 H 349 N 35 O 49 The expected value is 4397.5893, the measured value is 4397.6057), and it is a white powder.

[0101] Synthesis of Preparation 15

[0102] SEQ ID NO: 15

[0103] In N 2Next, the preparation 12 of the aliquot (671 mg, 82.1 wt%, 0.275 mmol, 1.23 equivalents) was added to a flask. With stirring, preparation 14 (2.009 g, 48.9 area%, 10.7 area% isomer, 0.223 mmol, 1.00 equivalent), DMSO (11.1 g, 10.0 mL), DMF (19.0 g, 20.1 mL), and DIEA (76 mg, 0.588 mmol, 2.63 equivalents) were added to the flask to obtain a golden solution. Before cooling to -5 °C, an aliquot of 0.6 M HOAt (619 mg, 0.384 mmol, 1.72 equivalents) was added. The PyClock sample (220 mg, 0.397 mmol, 1.78 equivalents) was added. The mixture was warmed to about ambient temperature to provide a conversion of about 84% to preparation 15. The product was isolated by adding the reaction mixture dropwise over 10 min to ice-cold deionized water (548 mL), giving a precipitate of the product. The reaction flask was rinsed with DMF (5 mL) and added to the slurry. The slurry was stirred for about 15 minutes, warmed to about ambient temperature, and filtered. The wet filter cake was washed with deionized water (3 x 80 mL), and the white waxy solid was dried in vacuo at 35 °C for 3.5 days to give preparation 15, (2.506 g, 41.6 area%, 0.163 mmol, 73.1% yield, HRMS calculated for C 334 H 512 N 48 O 74 expected value 6379.7777, found 6379.8652), as a white powder.

[0104] Example 2

[0105] Synthesis of Example 2

[0106] SEQ ID NO: 1

[0107] In N 2Next, a sample of TFA (19.656 g, 13.03 mL) was added to a flask containing DCM (815 mg, 0.62 mL), DTT (434 mg), and TIPS (362 mg, 0.47 mL). The mixture was cooled in ice water before adding water (468 mg, 0.47 mL). A sample of Preparation 15 (1016 mg, 39.0 area%, 0.0620 mmol) was added to the mixture at 2 °C to provide a solution. The mixture was warmed to approximately ambient temperature and stirred for about 2 hours. The reaction mixture was added to MTBE (150 mL) at -15 °C, and the reactor was rinsed with MTBE (3 mL). The slurry was centrifuged after about 10 minutes, and the supernatant was decanted. The wet filter cake was reslurried in MTBE (3 × 50 mL), centrifuged for each wash, and the supernatant was decanted. The wet filter cake was dried in vacuo at 35 °C to give Example 2 (784 mg, 26.5 area%, 0.0432 mmol, 69.7% yield, HRMS calculated for C 225 H 348 N 48 O 68 expected value 4810.5249, found 4810.5642), as a white solid.

[0108] Synthesis of Preparation 16

[0109] SEQ ID NO: 16

[0110] Synthesis was performed using Fmoc-Gly-OH-2-chlorotrityl resin with a loading of 0.61 mmol / g. The general SPPS procedure was essentially the same as described herein. Preparation 16 was obtained from the soft cleavage of the peptide on the resin using methods known to those skilled in the art as described herein. The concentrated material was reconstituted with ethanol (5% volume of the combined filtrates) and concentrated to dryness. The peptide was triturated in water (40% volume of the combined filtrates) with stirring. The solid was isolated and dried in vacuo at 40 °C to constant weight to yield 5.24 g (99%) of Preparation 16 as a white powder.

[0111] Synthesis of Preparation 18

[0112] SEQ ID NO: 17

[0113] Synthesis was performed using Fmoc-Ala-OH-2-chlorotrityl resin with a loading of 0.50 mmol / g. The general SPPS procedure used was essentially the same as described herein, with the following modifications:

[0114]

[0115] Soft cleavage of Preparation 18

[0116] A peptide intermediate sample on 42.13 g of resin was placed in a flask and treated 3 times with 10 volumes (400 mL) of 1% TFA / DCM for 10 min each time, then washed with DCM. Each treatment was quenched by adding 4.4 mL of pyridine. The resulting solutions were combined and concentrated in vacuo. It was reconstituted with ethanol (25 mL) and then concentrated to dryness to give 56.6 g of a foamy semi-solid. Water was added 10 times in a volume of 400 mL to provide a slurry. The slurry was filtered and washed with water. The solid was separated and dried in vacuo at 40 °C to constant weight to yield 23.3 g of Preparation 18 as a white powder.

[0117] Synthesis of Preparation 17

[0118] ((52S)-52-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-25-(tert-butoxycarbonyl)-2,2-dimethyl-4,23,28,37,46-pentaoxo-3,32,35,41,44-pentaoxa-24,29,38,47-tetraazapentatriacontan-53-oic acid)

[0119] Preparation 6 (80 g, 92 mmol), DIEA (17.53 mL, 101 mmol), TSTU (30.3 g, 101 mmol) and acetonitrile (1 L) were added to a container and stirred at 23 °C for 17 h. The solution was concentrated and the resulting orange residue was redissolved in EtOAC (1.6 L) and then washed with 0.1 M HCl (2 × 1 L). The organic layer was washed with water (2 × 1 L) and then dried over MgSO 4 dried, filtered and concentrated in vacuo to leave an orange oil (83 g). A second batch was run on the same scale and combined to give 123 g of a crude oil. The intermediate ester (123 g, 110 g active, 113 mmol) was dissolved in EtOH (700 mL), then Fmoc-lysine (45.9 g, 125 mmol) and DIEA (21.70 mL, 125 mmol) were added and the reaction was stirred for 17 h. After completion of the reaction, EtOH was removed in vacuo and an orange oil (201 g) was left. The residue was dissolved in EtOAc (1.1 L) and washed with 0.1 M HCl solution (3 × 400 mL) and then with NaHCO 3Wash with aqueous solution (400 mL). Separate the layers, and then wash the organic layer with saturated aqueous sodium chloride solution (1 × 400 mL). The organic matter was concentrated to give an orange oil (~190 g). Acetone (400 ml) was added, and the resulting suspension was filtered to remove inorganic matter. The mixture was concentrated and then purified by normal phase chromatography (1.1 kg silica gel preconditioned with 60 / 40 heptane / acetone), and eluted with an eluent of increasing polarity (collect ~3 L fractions). The fractions with at least 95% HPLC area were combined and concentrated to give a viscous yellow oil (70 g) of Preparation 17.

[0120] Synthesis of Preparation 19A

[0121] SEQ ID NO: 18

[0122] Synthesis was carried out using Fmoc-Leu-OH 2-chlorotrityl resin with a loading of 0.65 mmol / g. The general SPPS procedure was used with the following modifications:

[0123]

[0124]

[0125] The pseudo-proline-derived Preparation 19A can be processed into Example 3 in a manner similar to Preparation 19B described herein.

[0126] Synthesis of Preparation 19B

[0127] SEQ ID NO: 19

[0128] Synthesis was carried out using Fmoc-Leu-OH-2-chlorotrityl resin with a loading of 0.65 mmol / g. Preparation 19B was prepared using a SPPS procedure substantially the same as described herein.

[0129]

[0130] Soft cleavage of Preparation 19B

[0131] Preparation 19B was prepared using soft cleavage of resin-bound 19B substantially as described herein using methods known to those skilled in the art. See, for example, the method of Preparation 18. The resulting solid was isolated and dried to constant weight under vacuum at 30 - 40 °C to give 2.94 g of product as a pale yellow powder.

[0132] Synthesis of Preparation 20

[0133] SEQ ID NO: 20

[0134] At ambient temperature, PyBOP (2.60 g, 5.00 mmol) and DIEA (1.75 mL, 10.0 mmol) were added to a solution of Preparation 1 (4.25 g, 4.166 mmol) and Preparation 16 (5.00 g, 3.340 mmol) in DMSO / DMF (1:1, 200 mL). The solution was stirred for 18 h and then quenched by addition of excess diethylamine (10.0 mL). The quenched solution was stirred for 2 h and then slowly added to a saturated sodium bicarbonate / water (1:1, 300 mL) solution at 0 °C. The resulting precipitate was stirred for 10 min and then collected by filtration. The filtrate was washed successively with water (3 × 150 mL) and then methyl tert-butyl ether (3 × 150 mL). The solid was dried under vacuum at 40 °C to afford Preparation 20 as a white solid (5.30 g, 69% yield, HRMS calcd for C 119 H 169 N 21 O 24 2276.2649, found 2276.2652).

[0135] Synthesis of Preparation 21

[0136] SEQ ID NO: 21

[0137] At ambient temperature, PyBOP (314 mg, 0.30 mmol) and DIEA (0.21 mL, 1.20 mmol) were added to a solution of Preparation 20 (1.00 g, 0.44 mmol) and Preparation 18 (0.90 g, 0.40 mmol) in DMSO / DMF (1:1, 20 mL). The solution was stirred for 18 h and then quenched with piperidine (0.79 mL, 4.00 mmol). The quenched solution was stirred for 2 h and then cooled to 0 °C and quenched with dilute HCl solution (50 mL). The resulting slurry was stirred for 10 min and the solid was collected by filtration. The filtrate was washed successively with saturated aqueous sodium bicarbonate (2 × 50 mL), water (3 × 50 mL), and then methyl tert-butyl ether (3 × 50 mL). The solid was dried under vacuum at 40 °C for 18 h to afford Preparation 21 as a white solid (1.80 g, 106% yield, HRMS calcd for C 225 H 338 N 34 O 48 4284.5053, found 4284.5062).

[0138] Synthesis of Preparation 22

[0139] SEQ ID NO: 22

[0140] At ambient temperature, PyBOP (57 mg, 0.11 mmol) and DIEA (58 μL, 0.33 mmol) were added to a solution of Preparation 21 (214 mg, 0.05 mmol) and Preparation 19B (116 mg, 0.055 mmol) in DMSO / DMF (1:1, 3 mL). The solution was stirred for 18 h and then quenched with a 1:1 mixture of saturated aqueous sodium bicarbonate and water (10 mL). The resulting slurry was stirred for 10 min and the resulting solid was collected by filtration. The solid was washed with water (3 × 10 mL) and dried in vacuo at 40 °C to afford Preparation 22 (285 mg, 89% yield, HRMS calcd for C 334 H 512 N 48 O 74 expected 6379.7777, found 6379.7730).

[0141] Example 3

[0142] Synthesis of Example 3

[0143] SEQ ID NO: 1

[0144] A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (0.1 mL) and DTT (75 mg) was cooled to 0 °C. Preparation 22 (100 mg, 0.015 mmol) was added to the solution and the reaction mixture was warmed to ambient temperature and stirred for 2 h. The resulting mixture was poured into a pre-cooled (–20 °C) solution of methyl tert-butyl ether (25 mL). The resulting precipitate was kept at –20 °C for 15 min, centrifuged, and the slurry was washed with methyl tert-butyl ether (2 × 25 mL). The solid was dried in vacuo at 35 °C for 18 h to afford Example 3 as a white solid (71 mg, 93% yield, HRMS calcd for C 225 H 348 N 46 O 68 expected 4810.5249, found 4810.5036).

[0145] Step 1

[0146] Step 1: Prepare a feed solution of Preparation 25 (1.05 equivalents) in 5 volumes of DMSO / ACN (90:10 volume / volume). Prepare a second feed solution of Preparation 26 in 20 volumes of DMSO / ACN (90:10 volume / volume). Prepare a third feed solution, which is PyOxim (1.5 equivalents) in 3 volumes of ACN. Prepare a fourth stream of DIEA (4 equivalents) in ACN. Pump the first three streams into a mixer, and mix DIEA at the mixer outlet, and pump the mixture through another mixer and through a plug flow reactor, with a residence time of 2 hours in a 20 °C constant temperature bath. At the reactor outlet, acetic acid can be added to consume the remaining PyOxim (1-cyano-2-ethoxy-2-oxoethylideneaminooxy-tris-pyrrolidinophosphonium hexafluorophosphate). After > 2 hours, add pure diethylamine (10 equivalents), and mix through a mixer. This stream enters a second plug flow reactor, with a residence time of 1 hour in a 20 °C constant temperature bath. Collect the product solution of Preparation 27, and deliver it together with a 70 / 30 DMSO / ACN solution through nanofiltration to remove 10 - 20 diavolumes of reagents.

[0147] The feed solution of Preparation 25 (2.40 kg, 96.7 wt%, 2.274 mol) was prepared as follows: The solid was dissolved in DMSO (13.88 kg, 12.62 L) and the solution was diluted with ACN (1.09 kg, 1.39 L), resulting in a solution of Preparation 25 in 90:10 DMSO:ACN volume / volume (114.3 mg / mL, 0.112 M). The second feed solution of Preparation 26 (SEQ ID NO: 26, 2.79 kg, 98.6 wt.%, 1.836 mol) was prepared as follows: The solid was dissolved in DMSO (54.8 kg, 49.8 L) and diluted with ACN (4.3 kg, 5.47 L), resulting in a solution of Preparation 26 in 90:10 DMSO:ACN volume / volume (45.5 mg / mL, 0.030 M). The third feed solution was prepared from PyOxim (4.5 kg, 8.53 mol) in ACN (47.33 kg, 60.22 L), resulting in a 0.132 M solution. DIEA was added as a pure liquid. Solutions of Preparation 25 (14.91 L, 1.704 kg, 1.670 mol, 0.95 equivalent, 5.9 g / min), Preparation 26 (58.46 L, 2.660 kg, 1.750 mol, 1.00 equivalent, 22.5 g / min), and PyOxim (1.4 equivalents, 5.4 g / min) were pumped into a mixer at 20 °C and mixed with pure DIEA (4.0 equivalents, 0.446 mL / min). The mixture was pumped through another mixer and through a plug flow reactor, held in a 20 °C constant temperature bath for a residence time of 3 hours, and collected over 42.9 hours to give 88.6 kg of product solution.

[0148] Nanofiltration is a membrane-based filtration method used to separate chemical substances based on differences in their size and molecular weight. The product solution of Preparation 27 contains reagents (diethylamine, PyOxim, DIEA, etc.) and unwanted by-products (such as dibenzofulvene), which need to be removed before proceeding to the next step. Nanofiltration is used to remove unwanted substances (molecular weight < 500 Da).

[0149] The product solution of Preparation 27 is loaded into the NF feed tank and pumped through a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymer) in a circulation loop to achieve the desired separation. Unwanted substances are removed in the permeate and collected separately or discarded as waste. To maintain a constant volume in the NF tank, fresh solvent, i.e., 70:30 volume / volume DMSO / ACN, is continuously pumped in to match the resulting permeation rate. The product solution of Preparation 27 purified by nanofiltration directly enters Step 2.

[0150] To the reactor is added the solution of Fmoc-protected Preparation 27 in DMSO / ACN (88.6 kg) and diethylamine (1.34 kg). The mixture is stirred at 20 °C for 2 hours to obtain Preparation 27 (87.6 L, 38.45 mg / mL, 3.37 kg, 1.48 mol). The product solution of Preparation 27 is loaded into the nanofiltration feed tank and then pumped through a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymer) in a circulation loop to achieve the desired separation. Unwanted substances are removed on the permeate side and collected separately or discarded as waste. This operation is continued until sufficient removal of unwanted impurities is obtained. To maintain a constant volume in the nanofiltration tank, fresh solvent, i.e., 70:30 volume / volume DMSO / ACN, is continuously pumped in to match the resulting permeation rate. This gives Preparation 27 in DMSO / ACN (72.4 L, 40.8 mg / mL, 2.95 kg, 1.30 mol, yield of 78.1% by coupling, de-Fmoc, and nanofiltration).

[0151] Step 1 Illustration - Analysis Results

[0152] HPLC confirmed the conversion of Preparation 25 and Preparation 26 to form Preparation 27. The analytical method used a phenylhexyl stationary phase column (2.1 mm inner diameter x 150 mm × 1.7 micron particle size) at 65 °C, with a 2 - 98% B gradient over 12 minutes, 0.1% TFA in water, and acetonitrile. The material was detected by UV at 214 nm.

[0153] Table A.1 shows the high-resolution mass spectrometry data collected for the product of the Step 1 coupling reaction (Fmoc-protected Preparation 27) and the product of the Step 1 deprotection reaction (Preparation 27). Mass accuracy is a measure used to confirm the match between the measured substance and the predicted substance.

[0154] 。

[0155] Table A.1 confirmed the measured Fmoc-protected Preparation 27 and Preparation 27 using the mass accuracy calculated from the high-resolution mass spectrometry data.

[0156] Step 2

[0157]

[0158] Scheme A.2 Synthesis of Preparation 29 (SEQ ID NO: 29) from fragment Preparations 27 (SEQ ID NO: 27) and 28 (SEQ ID NO: 28). Preparation of Preparation 29 (SEQ ID NO: 29).

[0159]

[0160] Step 2: Prepare a feed solution of Preparation 28 (1.15 equivalents) in 10 volumes of DMSO / ACN (90:10 v / v). Prepare a second feed solution which is PyOxim (2 equivalents) in 1 volume of ACN. Prepare a third stream which is DIEA (3 equivalents) (5 wt% solution) in ACN. Pump the solution of Preparation 27 from Step 1, and the Preparation 28 and PyOxim streams into a mixer, and mix DIEA at the outlet of the mixer, pump the mixture through another mixer, and through a plug flow reactor with a residence time of 2 hours in a 20 °C constant temperature bath. At the reactor outlet, acetic acid can be added to consume residual PyOxim. After > 2 hours, add pure diethylamine (10 equivalents) and mix through a mixer. This stream enters a second plug flow reactor with a residence time of 1 hour in a 20 °C constant temperature bath. Collect the product solution of Preparation 29 and deliver it by nanofiltration with a DMF solution as the diafiltrant to remove 10 - 20 diafiltration volumes of reagents.

[0161] The feed solution of Preparation 28 (4.68 kg, 98.9 wt.%, 2.058 mol) was prepared by dissolving the solid in DMSO (41.75 kg, 37.95 L) and diluting the solution with ACN (3.3 kg, 4.20 L), resulting in a solution of Preparation 28 in 90:10 DMSO:ACN volume / volume (94.7 mg / mL, 0.0421 M). The second feed solution was prepared as PyOxim (3.0 kg, 5.69 mol) in ACN (11.81 kg, 15.03 L), resulting in a 0.327 M solution. DIEA was added as a pure liquid. The solution of Preparation 27 from Step 1 (73.86 L, 41.2 mg / mL, 3.04 kg, 1.336 mol, 0.0181 M, 1.0 equivalent, 29.9 g / min) and the feeds of Preparation 28 (1.3 equiv, 17.7 g / min) and PyOxim (2.1 equiv, 2.9 g / min) were pumped into a mixer, and the feeds were adjusted to 20 °C at the mixer outlet and combined with pure DIEA (4.0 equiv, 0.374 mL / min). The mixture was pumped through another mixer and through a plug flow reactor, with a residence time of 3 hours in a bath at 20 °C and collected over 43.2 hours to give 129.35 kg of the Preparation 29 product solution.

[0162] A nanofiltration method substantially as described above, using the product solution of Preparation 29 instead of the product solution of Preparation 27.

[0163] A nanofiltration method using the Fmoc-protected Preparation 29 solution in DMSO / ACN was carried out substantially as described herein. The Fmoc-protected Preparation 29 (129.35 kg) and diethylamine (2.0 kg) were added to a reactor for nanofiltration. The nanofiltration method yielded Preparation 29 in DMF (98.85 L, 42.24 mg / mL, 4.18 kg, 0.974 mol, 73.1% yield by coupling, de-Fmoc, and nanofiltration).

[0164] HPLC confirmed the synthesis of Preparation 29 from Preparation 28 and Preparation 27. The analytical method used a C4 stationary phase column (2.1 mm inner diameter × 150 mm × 1.7 micron particle size) at 65 °C, with a 25 - 98% B gradient over 12 minutes, 0.1% TFA in water, and acetonitrile. UV detection at 214 nm was used for the material.

[0165] The 25 - 98% B gradient was 0.1% TFA in water and acetonitrile for 12 minutes. The material was detected by 214 nm UV.

[0166] Table A.3 shows the high-resolution mass spectrometry data collected for the product of the coupling reaction in Step 2 (Fmoc-protected Preparation 29) and the product of the deprotection reaction in Step 2 (Preparation 29). The mass accuracy confirmed the measured substance product. The single isotope mass of the neutral substance.

[0167] 。

[0168] Table A.3 The measured Fmoc-protected Preparation 29 and Preparation 29 were confirmed by mass accuracy calculated using high-resolution mass spectrometry data.

[0169] Step 3

[0170] Scheme A.3 Synthesis of Preparation 31 (SEQ ID NO: 31) from fragment Preparations 30 (SEQ ID NO: 30) and 29 (SEQ ID NO: 29). Preparation of Preparation 31 (SEQ ID NO: 31).

[0171]

[0172] Step 3 Batch method description: At -5 °C, a solution of 5.0 wt% DIEA in DMF (114.0 mg, 5.70 mg DIEA, 0.0441 mmol, 1.8 equivalents) and a solution of 10.1 wt% (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) in DMF (154.9 mg, 15.59 mg HATU, 0.0410 mmol, 1.7 equivalents) were added to a nanofiltration DMF solution of Preparation 29 (2.249 g, 46.5 mg / g, 104.6 bmg, 0.0244 mmol) and Preparation 30 (71.5 mg, 90.6 area%, 0.0306 mmol) in DMF (0.3068 g, 0.325 mL). The solution was stirred at -5 °C for 4 hours and then quenched at ambient temperature with 5 wt% aqueous sodium bicarbonate solution (5.295 g, 4.8 mL) added over 15 minutes. The resulting slurry was stirred at 0 °C for 15 minutes and the resulting solid was collected by filtration. The solid was washed with water (4 × 2 mL) and then with MTBE (4 × 2 mL). The viscous solid was dried under vacuum at 35 °C to give Preparation 31 (219.7 mg, 47.6 area%, 0.0164 mmol, 67.1% yield).

[0173] Prepare the feed solution of Preparation 30 in 10 volumes of DMF. The second feed solution is prepared as 10 wt% HATU (1.8 equivalents) in ACN. Prepare the third feed stream as DIEA (2.5 equivalents) in DMF (5 wt% solution). Pump the solution of Preparation 29 from Step 2, along with the Preparation 30 and DIEA feed streams, into a mixer, and cool the stream at the outlet of the mixer and combine it with the cooled HATU solution. Pump the mixture through another mixer and through a plug flow reactor, hold it in a constant temperature bath at -5 °C for 3 hours and collect. Prepare a 19 wt% salt-loaded brine / bicarbonate solution (17 wt% aqueous sodium chloride solution, 0.5 wt% aqueous sodium bicarbonate solution). Then pump the product solution of Preparation 31 in DMF together with the salt solution into a mixed product mixed suspension reactor (MSMPR) to form a precipitate. The τ (tau) in the mixed product mixed suspension reactor is 1 hour. A second MSMPR runs for 1 hour of τ under colder conditions, and then the slurry is intermittently added to a filter. Wash the slurry with water and dry it under vacuum at 35 °C.

[0174] The feed solution of Preparation 30 (9.42 kg, 83.6 wt%, 3.72 mol) is prepared in DMF (54.12 kg) to yield a 0.0563 M feed. Prepare the second feed solution as HATU (1.15 kg, 3.02 mol) in ACN (10.4 kg), yielding a 0.215 M feed. Add DIEA as a pure liquid. Pump the solution of Preparation 29 from Step 2 (98.85 L, 42.24 mg / mL, 4.18 kg, 0.975 mol, 0.0099 M, 1.0 equivalent, 40.2 g / min) and Preparation 30 (1.3 equivalents, 9.2 g / min) and the DIEA (2.1 equivalents, 0.152 mL / min) feed stream into a mixer. At the outlet of the mixer, cool the stream to 0 °C and combine it at 0 °C with the cooled HATU solution (2.0 equivalents, 3.2 g / min). Pump the mixture through another mixer and through a plug flow reactor, hold it in a bath at 0 °C for 4 hours and collect over 42 hours to obtain 131.8 kg of product solution. The product solution is precipitated in two parts. Combine a 17 wt% aqueous NaCl solution / 0.5 wt% NaHCO 3 aqueous solution (29 kg) with DMF (13.2 kg) in an inert reactor and cool to no more than 20 °C. Then combine the product solution in DMF (66.6 kg) with the 17 wt% aqueous NaCl solution / 0.5 wt% NaHCO 3An aqueous solution (34.4 kg) was co-added to the reactor over 1 hour while maintaining at 20 °C to obtain a precipitate of Preparation 31. The slurry was cooled to 5 °C over 1 hour before adding water in two portions (total 32.2 kg). The 5 °C slurry was stirred for 0.5 hour before filtering the slurry. The wet cake was re-slurried in water (63.9 kg) for 0.5 hour and filtered. The second portion of the product solution was precipitated in a comparable manner. A 17 wt% aqueous NaCl solution / 0.5 wt% NaHCO 3 An aqueous solution (29 kg) and DMF (13.55 kg) were combined in a reactor and cooled to no more than 20 °C. The second portion (65.2 kg) of the product solution in DMF was combined with a 17 wt% aqueous NaCl solution / 0.5 wt% NaHCO 3 An aqueous solution (36.8 kg) was co-added to the reactor over 1 hour while maintaining at 20 °C to obtain a precipitate of Preparation 31. The slurry was cooled to 5 °C over 1.25 hours and then water was added in two portions (total 32.1 kg). The slurry was stirred for 0.5 hour and then the slurry was filtered over the first wet cake. The combined wet cake was re-slurried twice with water (64 kg each time) for 0.5 hour each time and filtered. Subsequently, two displacement water washes were carried out (64 kg each time). The combined washed wet cake was purged with N 2 and then dried under vacuum at 38 °C until K.F. < 4 wt% to obtain Preparation 31 (10.34 kg, assuming 60% potency, 0.972 mol, assuming 100% yield).

[0175] Example 3

[0176] Tirzepatide (SEQ ID NO: 1)

[0177]

[0178] A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (TIPS, 0.1 mL) and dithiothreitol (DTT, 75 mg) was cooled to 0 °C. Preparation 31 (100 mg, 0.015 mmol) was added to the solution and the reaction mixture was warmed to ambient temperature and stirred for 2 hours. The resulting mixture was poured into a pre-cooled (-20 °C) MTBE solution (25 mL). The resulting precipitate was held at -20 °C for 15 minutes, held for 15 minutes, and the slurry was centrifuged and washed with MTBE (2 × 25 mL). The solid was dried under vacuum at 35 °C for 18 hours to obtain Example 3 as a white solid (71 mg, 93% yield, HRMS calculated C 225 H 348 N46 O 68 The expected value is 4810.5249, and the measured value is 4810.5036).

[0179] Add DCM (27.3 kg, 20.6 L), water (4.1 kg), Preparation 31 (10.34 kg, assuming 60% potency, 0.972 mol), and DTT (3.10 kg) to an inerted reactor at 15 °C. Add TFA (154.1 kg, 103.4 L) and TIPS (3.2 kg, 4.2 L) to a separate inerted reactor. Add the TFA / TIPS solution to the slurry of Preparation 31, DCM, water, and DTT within 0.25 hours to form a colorless solution, and heat up and hold at 20 °C for 3 hours. After 3 hours at 20 °C, cool the reactor to -10 °C. In a separate reactor, add MTBE (382.4 kg, 516.8 L) and cool it to -20 °C. Add a portion of this cold MTBE (91.8 kg, 124.1 L) to the cold reaction solution over 2 hours, maintaining -5 °C to -18 °C. Add the remaining cold MTBE (294.3 kg, 397.7 L) over 1.5 hours, maintaining -5 °C to -18 °C, to obtain the precipitate of Example 3. Adjust the slurry to 0 °C and hold for > 0.5 hours, then filter in three portions. Redisperse the combined wet cake twice with MTBE (114.7 kg, 155 L) and filter before the final MTBE displacement wash (114.7 kg, 155 L). Dry the wet cake at 28 °C until < 4.5 wt% MTBE is measured. This gives Example 3 (7.77 kg, 46.8 wt.%, 0.755 mol, 77.7% yield).

[0180] Example 4A (Linear SPPS)

[0181] Tirzepatide (SEQ ID NO: 1)

[0182]

[0183]

[0184] Add Fmoc Sieber resin (17 kg, 0.76 mmol / g) to the reactor. The resin is swollen with DMF, stirred for 2 hours, and then the DMF is filtered out from the resin. Then the resin is washed twice in total with DMF. Then deprotection of the Fmoc-protected resin is carried out using 20% PIP / NMP treatment. Sampling is carried out after the last PIP / NMP treatment to verify Fmoc removal, and > 99% Fmoc removal is confirmed by UV analysis (IPC target < 1% Fmoc remaining). After the final 20% w / w PIP / NMP treatment, the resin bed is washed several times with DMF. Then, for each amino acid coupling and deprotection, the following general conditions are used to construct the peptide backbone.

[0185]

[0186] Fmoc deprotection: The resin in the peptide reactor is treated with a 20% v / v PIP / NMP solution in three or four portions. Each treatment is stirred on the resin for 30 min and then filtered to complete the removal of the Fmoc protecting group. After the last 20% v / v PIP / NMP treatment, the resin bed is washed at least six times with DMF in a pre-specified DMF volume addition amount.

[0187] Amino acid activation: Add the pre-prepared 12% w / w Oxyma Pure / NMP solution to the reactor. Then add the selected Fmoc amino acid. Stir the mixture at 20 ± 5 °C until the Fmoc amino acid is completely dissolved. Before activation, cool the Fmoc-AA / Oxyma Pure / NMP solution to 15 ± 3 °C to ensure control of the slightly exothermic activation reaction and keep the temperature of the resulting solution within the specified 20 ± 5 °C range. Then activate the amino acid solution by adding DIC. Then stir the activated ester solution for 20 - 30 min before transferring the solution to the reactor containing the peptide intermediate on the resin.

[0188] Coupling: After the pre-activation step is completed, transfer the activated ester solution to the reactor containing the deprotected peptide on the resin to initiate the coupling reaction. Stir the peptide coupling reaction at 20 ± 5 °C for at least 4 hours. After the required stirring time, sample the resin slurry to complete the coupling (IPC). Repeat sampling at specific time intervals as needed until a qualified IPC result is obtained. If necessary, perform a re-coupling operation. When the coupling is completed, filter the contents of the peptide reactor solution, and then wash the peptide intermediate on the resin several times with DMF to prepare for the next coupling.

[0189] Coupling of Ile (12) with Aib (13): The coupling of Fmoc-Ile(12) with Aib (13) was carried out using the symmetric anhydride method, with six equivalents of Fmoc-AA and three equivalents of DIC. For this sequence, the activation time was extended to 40 - 60 min to ensure the formation of the activated symmetric anhydride species. As determined by HPLC analysis, an extended coupling stirring time (18 hours) was required to achieve reaction completion (< 1% de-coupling).

[0190] Deprotection of Lys (20) ivDe (Preparation 23): Selective deprotection of the fully protected Lys(20) ivDde group on the 39 - amino acid resin Boc-Tyr(1)-Ser(39) peptide backbone was performed. Deprotection was achieved by stirring with an 8% w / w hydrazine hydrate solution in DMF at ambient temperature for 4 hours. The deprotection reaction was monitored by HPLC, targeting an IPC limit of < 1% for the remaining Lys(ivDde) component after deprotection. The resulting peptide fragment (Preparation 23) was washed repeatedly with DMF (8x) to completely remove residual hydrazine. The fully constructed Preparation 23 fragment was washed four times with IPA and then dried at ≤40 °C until an LOD ≤1% was achieved. Before coupling with Preparation 6, Preparation 23 was packaged and refrigerated (-20 °C).

[0191] Coupling of Preparation 6 with Preparation 23: Solid Preparation 6 (1.5 equivalents) and PyBOP (1.5 equivalents) were charged into a reactor, followed by the addition of DMF, and the mixture was stirred until dissolution occurred. Then, collidine was added to initiate the formation of the active ester species. The activated ester solution was stirred for 60 min and then transferred to a reactor containing the Preparation 23 intermediate. The reaction slurry was stirred at 25 °C for 18 hours. Samples of the slurry were taken to complete the coupling (IPC), and sampling was repeated at specific time intervals as necessary to achieve a satisfactory IPC (<1% Preparation 23) result. After coupling was complete, the solution contents were filtered off and discarded. The fully constructed Preparation 24 intermediate was washed several times with DMF and then with IPA. Preparation 24 was dried at ≤40 °C until an LOD ≤1% was reached. Before cleavage from the resin, Preparation 24 was packaged and refrigerated (-20 °C).

[0192] Resin cleavage and isolation of the crude product of Example 4A: A cleavage mixture was prepared, consisting of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), dithiothreitol (DTT), DCM, and water. The cleavage mixture was cooled to 15 ± 5 °C. The reagent feed is shown in the following table:

[0193]

[0194] Add Preparation 24 to the reactor, followed by the cleavage mixture. Stir the mixture and maintain it at 23 °C for 3 hours. Filter the mixture and then wash the waste resin with DCM. Combine the DCM wash filtrate with the bulk deprotection solution and cool the contents to ≤ -10 °C. Cool MTBE to ≤ -13 °C. Then add the cold MTBE to the cold filtrate in two portions. Control the MTBE feed rate such that the internal temperature of the crude solution remains at ≤ 5 °C. The initial MTBE charge accounts for ~45% of the total MTBE charge. A soft precipitate forms near the end of the MTBE addition but readily redissolves into the solution. Then cool the precipitate solution to an internal temperature of -15 ± 5 °C. The feed rate of the second MTBE addition is about 5 - 10 times the initial MTBE feed rate and accounts for ~55% of the total MTBE charge. During the addition, the internal temperature of the precipitate slurry remains at ≤ 0 °C. The resulting slurry is aged at -8 ± 3 °C for at least 6 hours and then warmed to 0 ± 3 °C and aged for another 2 hours before separation.

[0195] Filter the cold crude peptide slurry and then wash the resulting wet filter cake with MTBE. Then dry the crude wet cake of Example 4A to an IPC target LOD value of < 1%. Package and store the crude product of Example 4A. Refrigerate the crude intermediate (-20 °C) until purification. Overall, 45.39 kg of crude Example 4A was produced with an HPLC area percentage purity of 45 wt% and 64%. The contained yield based on Sieber resin = 47%.

[0196] Purification of Example 4A:

[0197] Mobile phase:

[0198] Mobile phase A (MPA)

[0199] 90% water, 0.1% TFA, and 10% ACN

[0200] Mobile phase B (MPB)

[0201] 10% water, 0.1% TFA, and 90% ACN

[0202] Reverse Phase Purification 1 (RP1): The crude product of Example 4A was dissolved in 90 wt% MPA and 10 wt% MPB. The solution was stirred for at least 7 hours to complete the tryptophan decarboxylation reaction. The aged crude solution was filtered and loaded onto a pre-equilibrated Kromasil 100-10-C8 packed column. The column was washed with a mixture of buffer A (90% water, 0.1% TFA, and 10% ACN) and buffer B (10% water, 0.1% TFA, and 90% ACN), which achieved 30% ACN for two column volumes and prepared for elution by increasing the mixed concentration of ACN from 30% to 35% over one column volume. Tirezepatide was eluted from the column using 1.5% ACN per column volume until elution was complete. The eluate was fractionated and the purity was determined using RP-HPLC. The column was regenerated by increasing ACN from 47% to 65% over one column volume and flowing 65% ACN for three column volumes. Prior to the next injection sequence, the column was re-equilibrated with 30% ACN for two column volumes.

[0203] Fractions meeting the main stream inclusion criteria were pooled. For fractions that did not meet the purity criteria but had a purity greater than 50%, they could be combined for recycle injection after all primary injections were completed. The recycle fractions were divided into a frontside fraction and a backside fraction, diluted with buffer A, and refrigerated. The recycle injection was processed and pooled using the primary injection criteria; however, only the main peak fraction was continued to be processed and no further recycling was performed. After the main stream pooling was completed, the concentration and purity of the intermediate were determined. Before RP2 treatment, the material was diluted and the pH was adjusted to pH 8. The RP1 method produced 37 kg of crude product and 14,277 g of contained product, with an average pool purity of 90.9%.

[0204] Reverse Phase Purification 2: The Example 4A RP1 solution was loaded onto a pre-equilibrated Kromasil 100-10-C8 packed column. Buffer C (90% aqueous NH 4 OAC at pH 8.0, 10% ACN) and buffer D (10% NH 4Wash the column with a mixture of OAC aqueous solution at pH 8.0 and 90% ACN, which achieves a 20% ACN solution for two column volumes. Use 3.5% ACN per column to increase the elution of Tirzepatide from the column until the elution is complete. Fractionate the eluate and determine the purity using RP-HPLC. After elution, regenerate the column by increasing the ACN to 80% for one column volume and flowing 80% ACN for three column volumes. Before the next injection sequence, re-equilibrate the column with 20% ACN for two column volumes.

[0205] Pool the fractions that meet the main stream inclusion criteria. For fractions that do not meet the purity standard but have a purity greater than 60%, they can be combined for recycle injection after all the first injections are completed. The recycled fractions are divided into a front pool and a back pool, diluted with buffer C, and refrigerated. Process and pool the recycle injections using the first injection standard; however, only the main peak fractions will be further processed and no further recycling will be performed. After completing the main stream pooling, determine the concentration and purity of the intermediate. The pH of the material can be adjusted to 8.0 to prepare for the TFF step. The RP2 method starts with 14.2 kg and gives 10.9 kg of the contained product with a yield of 76.7%.

[0206] Ion Exchange Chromatography (IEX): Filter and load the Example 4A RP2 solution into an Amberchrom CG300M column. Elute the two components using mobile phase E (10% aqueous ammonium acetate, 5% IPA, pH 8) and mobile phase F (isopropanol). Analyze the peptide content of the components and discard those with < 3 mg / mL. Before precipitation, store the concentrated pooled components at 20 °C. The IEX method starts with 10.9 kg of RP2 to provide 14.3 kg of the contained Example 4A material with an overall purity of 97.8%.

[0207] Precipitation: Filter the Example 4A IEX solution (333 kg), then add isopropanol (850 L) to reduce the water content to < 10% w / w water. Cool the diluted solution to 0 ± 3 °C in preparation for the addition of MTBE and precipitation. Cool MTBE (2304 L, 1708 kg) to 0 ± 3 °C. In the first ~ 37% of the MTBE charge, feed the cold MTBE to the IEX solution at a rate of ~ 0.69 kg / min. Then increase the feed rate to an average of ~ 2.3 kg / min to complete the remaining ~ 63% of the MTBE charge. The temperature during feeding is maintained at < 5 °C. Cold filter the resulting precipitate slurry (≤ - 10 °C). Then wash the filter cake with MTBE. Dry the filter cake to a LOD < 2%.

[0208] Humidification 4A: Example 4A was humidified by passing wet nitrogen gas through a filter dryer. The humidity of the exhaust gas stream at the filter outlet was monitored every 60 minutes. Humidification was continued until < 0.5% MTBE and < 0.2% IPA remained in the wet cake. After the humidification process was completed, the nitrogen gas flow was switched so that dry nitrogen gas flowed over the pure product cake of Example 4A. Samples of water and residual solvents in the material were taken according to specific IPC targets, and drying was continued using dry nitrogen gas until the desired target water content of 5 - 7% w / w was reached. A total of 12.9 kg of Example 4A was isolated, with a peptide content purity > 95%. The overall yield based on Sieber resin loading = 31%.

[0209] Example 4B (linear SPPS)

[0210] Tirzepatide (SEQ ID NO: 1)

[0211]

[0212]

[0213] Preparation 23

[0214] The method for generating Preparation 23 was substantially as described in Example 4A, except that NMP was completely replaced by DMF in all couplings and deprotections. Additionally, based on Siber resin, the stoichiometric ratio of amino acid: Oxyma to DIC was reduced to 2.5: 2.5: 2.7 molar equivalents. The only exception related to the use of DMF was the coupling of Ile 12 with Aib 13 where NMP was retained. In this example, 17.6 kg of Sieber resin was coupled with the Preparation 23 peptide intermediate on 92.2 kg of resin in the method.

[0215] Preparation 24

[0216] 92.1 kg of Preparation 23 was prepared by a method substantially as described in Example 4A; further processing yielded 97.3 kg of the Preparation 24 peptide intermediate on resin. Before cleavage from the resin, Preparation 24 was packaged and refrigerated (-20 °C).

[0217] Resin cleavage and isolation of crude Example 4B: Two batches were run on 32 kg of Preparation 24 under conditions substantially the same as described in Example 4A to provide 24.4 kg of Example 4B with 69.5% HPLC purity and 52.6% yield; and 21.3 kg of Example 4B with 88.3% HPLC purity and 45.2% yield. The crude intermediate was refrigerated (-20 °C) until purification.

[0218] Example 4B Purification: Crude Dissolution: The crude Tirzepatide Example 4B was charged into a dissolution vessel and dissolved in a 1:1 acetonitrile:aqueous solution at a final concentration of 25 g solid / L solution. The pH of the resulting solution was adjusted to 8.5 - 9.5 with ammonium hydroxide to initiate the conversion of the desamido peptide isomers (10 - 15%) to Tirzepatide Example 4B. The pH-adjusted mixture was stirred for at least one hour to allow the desamido conversion to occur. The pH was then adjusted to 1.5 - 2.5 by adding trifluoroacetic acid and diluted to 30% acetonitrile content in preparation for chromatography. In total, the crude solution was stirred for at least 7 hours to convert the Trp CO 2 salt to Tirzepatide Example 4B.

[0219] Conversion of tirzepatide (TZP) to desamido peptide:

[0220]

[0221] Conversion of desamido peptide to API:

[0222]

[0223] Reverse Phase Purification 1 (RP1): The RP1 method was substantially as described in Example 4A and was used to convert the desamido peptide to the API. The RP1 purification method was essentially the same as that described in Example 4A; however, the above crude dissolution step enhanced the capacity of the RP1 chromatography step. This allowed for a higher g of Tirzepatide per L of resin load and reduced the number of injections required to purify the crude Tirzepatide under the conditions described in Example 4. In this example, 23.7 kg of content-corrected crude Example 4B yielded 25.4 kg of Example 4B (107%) after RP1. Total solution volume = 2910 L @ 8.72 g / L, and once all the pooled fractions were collected, the mixture was stirred, sampled, and allowed to stand before Reverse Phase Purification 2 (RP2).

[0224] Reverse Phase Purification 2 (RP2): Reverse Phase Purification 2 (RP2) used a purification method substantially the same as that described in Example 4A and methods known to those skilled in the art. In this example, 15.2 kg of the Example 4B-containing material from RP1 was purified to 13.8 kg of Example 4B with a purity of ~98% after RP2. Total solution volume = 808 L @ 17.0 g / L. The mixture was stored before tangential flow filtration.

[0225] Tangential Flow Filtration (TFF): Install the TFF membrane and rinse with water. Prepare ammonium acetate buffer using low endotoxin pure water, acetic acid, and ammonium hydroxide. Then add isopropanol to provide a 5:95 100 mM NH 4 OAc pH 8.0: IPA buffer. Concentrate the 17 g / L RP2 solution of Example 4B to ~125 g / L by TFF. Recirculate the RP2 solution so that the solvent permeates through the membrane while retaining the peptide solution on the retentate side of the membrane in the solution. After concentration, while continuously collecting the permeate, feed the diafiltration buffer into the retentate storage tank. Continue to exchange the buffer until the desired solvent composition and peptide concentration are reached. Empty the solution from the system, rinse the resulting polarization layer from the membrane, and pool it with the peptide concentrate. Process two portions of the RP2 solution (403.9 L @ 17 g / L, 9.87 kg API) by TFF.

[0226] Co-feed precipitation: Combine the TFF portions (138.2 kg, 78.3 g / L) and measure KF (8.9%) to verify < 10% water. Add MTBE (243 kg) to a separate container and cool to 0 °C. Add IPA (48 kg), water (6 kg), and MTBE (100 kg) to the precipitation container and cool the solution to 0 °C. Co-feed the TFF and MTBE feed streams into the precipitation container at rates of 1.6 - 1.8 kg / min and 2.9 - 3.1 kg / min, respectively. The resulting slurry is aged at 0 °C for an additional 0.7 hours and then warmed to 15 °C. The slurry is aged at 15 °C for 1 hour and then MTBE (118 kg) is added. The slurry is aged at 15 °C for 1 hour and then cooled to 2.5 °C. Filter the cold slurry and wash the filter cake with MTBE (573 kg). Dry the filter cake to LOD < 2%.

[0227] Humidification: Humidify substantially as described in Example 4A and apply to the Example 4B material using methods known to those skilled in the art. Isolate a total of 14.5 kg of Example 4B (SEQ ID NO: 1) with > 97.7% HPLC purity and 88.4% peptide content. Total yield based on Sieber resin loading = 46%.

[0228] Example 5

[0229] Continuous synthesis of Preparation 31 using convergent chemistry in flow

[0230] The preparation of Peptide Fragment Synthesis Preparation 31 was carried out using both a chemical batch method and a method of sequential addition of fragments in a tubular flow reactor. The general method of synthesis involves coupling two fragments by mixing two solutions together with a coupling agent into a tubular reactor, followed by the addition of a base, and an additional residence time in the tubular reactor for the removal of the Fmoc protecting group. This prepares the coupled material for the addition of the next fragment. A nanofiltration filter with a membrane was used to remove excess reagents, base, and solvent between consecutive coupling reactions. The membrane was sized to retain the peptide and permit the permeation of lower molecular weight impurities. Diafiltration was used to completely remove lower molecular weight impurities prior to subsequent coupling steps. The following includes a description and analysis results of examples obtained from these transformations.

[0231] HPLC was used to confirm the synthesis of Preparation 31 from Preparations 29 and 30. The analytical method used a C4 stationary phase column (2.1 mm inner diameter × 150 mm × 1.7 micron particle size) at 65 °C, with a 60 - 98% B gradient of 0.1% TFA in water and acetonitrile over 12 minutes. UV detection at 214 nm was used for this material.

[0232] Table A.5 shows the high - resolution mass spectrometry data for the collection of the product (Preparation 31) of the Step 3 coupling reaction carried out in flow. The mass accuracy confirmed the desired substance.

[0233] 。

[0234] Table A.5 confirmed the measured Preparation 31 by mass accuracy calculated using high - resolution mass spectrometry data.

[0235] Native chemical ligation is a method for preparing full - length peptides containing cysteine or alanine in the sequence. This method employs a chemoselective reaction of two unprotected peptide segments to generate an intermediate with a transient thioester linkage. The thioester - linked intermediate rearranges to provide a full - length ligation product with a native peptide bond at the ligation site. Those skilled in the art will understand that native chemical ligation techniques can be used for the chemical synthesis of full - length peptides containing cysteine or alanine.

[0236] Example 6

[0237] Native Chemical Ligation Method

[0238] Tirzepatide (SEQ ID NO: 1)

[0239]

[0240] Synthesis of Fmoc-hydrazine-CTC resin (Preparation 32)

[0241] The 2-CTC resin (10.7 g, 17.7 mmol) was swollen in 100 mL of DCM at 0 °C for 20 min. The 9-fluorenylmethyl hydrazinecarboxylate (15.6 g, 61.4 mmol, 3.5 equiv) was dissolved in 210 mL of 2:1 DMF:DCM. DIEA (31 mL, 178 mmol, 10.1 equiv) was added to the solution of 9-fluorenylmethyl hydrazinecarboxylate. Then this solution was slowly added to the resin at 0 °C. It was stirred at 0 °C for about one hour and then warmed to room temperature. The reaction mixture was stirred at room temperature for 16 h. Then methanol (10 mL) was added to quench the remaining 2-CTC resin and stirred for 15 min. The resin was rinsed with 200 mL of DMF and then rinsed with DMF (2 × 100 mL), water (3 × 100 mL), DMF (3 × 100 mL), methanol (3 × 100 mL), and finally DCM (3 × 100 mL). The resin was dried in a vacuum oven at 27 °C for 16 h. The resin loading was measured by quantitative NMR to be 0.74 mmol / g.

[0242]

[0243] Synthesis of peptidyl hydrazide (17-mer) (Preparation 33)

[0244] SEQ ID NO: 32

[0245] The hydrazine-CTC resin (1.01 g, loading value: 0.65 mmol / g) was taken in a 40 mL reactor vessel and swollen with 3 × 4 mL of DCM (30 s each), and then swollen with 2 × 10 mL of DMF (20 min each) on a peptide synthesizer. Fmoc-Ile-OH (0.919 g, 2.60 mmol, 4 equiv) and HBTU (0.99 g, 2.61 mmol, 4 equiv) were dissolved in 7 mL of DMF. DIPEA (0.91 mL, 5.22 mmol, 8 equiv) was added to the amino acid solution and the volume was made up to 10 mL with DMF. The activated amino acid solution was added to the resin. The slurry was mixed with nitrogen for 8 h. After 8 h, the resin was washed with 5 × 10 mL of DMF and 5 × 10 mL of DCM and dried for 12 h. The loading of the resulting resin was measured by quantitative NMR to be 0.54 mmol / g. 0.91 g of this resin was used for the synthesis of Preparation 33 (SEQ ID NO: 32).

[0246] Deprotection: 4 × 9 mL of piperidine in 20% v / v DMF, 30 min each time.

[0247] Coupling: 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC were used for amino acid coupling. After each coupling and the last deprotection, the resin was washed with 5 × 9 mL of DMF and 1 min of N 2 mixed to wash the resin. At the end of the synthesis of peptidyl hydrazide, the resin was washed with DCM in N 2 mixed. The resin was dried on a synthesizer.

[0248] Deprotection and cleavage: 25 mL of a cleavage mixture made of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS) and 90% trifluoroacetic acid (TFA) was added to the dried resin (2.37 g) and mixed on a rotary mixer for 3 h. The resin was filtered and washed with 2 × 2.5 mL of TFA. The filtrate was poured into 175 mL of cold MTBE, and the peptide immediately precipitated. The filter flask was washed with 2 × 2.0 mL of TFA and poured into cold MTBE. It was cooled to -20 °C and kept for half an hour, then centrifuged. Then the peptide precipitate was washed twice with 150 mL of MTBE and centrifuged. The peptide precipitate was dried in a vacuum oven at 27 °C for 16 h. A sample of 1.25 g of crude preparation 33 was obtained after drying [expected (mass + 2H + ) / 2 = 968.4883, observed (mass + 2H + ) / 2 = 968.4879].

[0249]

[0250] Approximately 0.62 mmol of preparation 34 was synthesized on Sieber amide resin by a standard SPPS protocol. Fmoc-Lys(ivDde)-OH was used for orthogonal deprotection and lysine acylation.

[0251] Deprotection of ivDde: Hydrazine monohydrate (64% w / w) (1.98 g, 25.3 mmol) was diluted to 24.4 g with DMF, and 20 g was added to the resin. The slurry was stirred with a nitrogen stream. After about 2 h, it was washed with 5 × 9 mL of DMF. Repeat once more.

[0252] Dissolve 2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-eicosanoyl)amino]-5-oxo-valeryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (1094.4 mg, 1.252 mmol, 2 eq) in 10 mL of anhydrous DMF. Add TNTU (506.9 mg, 1.360 mmol, 2.2 eq) and DIEA (0.24 mL, 1.4 mmol, 2.2 eq) thereto. Make up the volume to 15 mL with anhydrous DMF. Mix it on a rotary mixer for 30 min. Then add the activated ester of Preparation 6 to the resin and mix it with a nitrogen stream for 12 hours. After 12 hours, drain the solution and wash the resin with 5 × 10 mL of DMF and 7 × 10 mL of DCM with 1 min N 2 Mix. Dry the resin on a synthesizer for 8 hours.

[0253] Deprotection and cleavage: Add 20 mL of a cleavage mixture made of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS) and 90% trifluoroacetic acid (TFA) to the dried resin (2.42 g) and mix it on a rotary mixer for 3 hours. Filter the resin and wash it with 2 × 2.0 mL of TFA. Pour the filtrate into 200 mL of cold MTBE, and the peptide precipitates immediately. Wash the filter flask with 2 × 2 mL of TFA and pour it into cold MTBE. Cool it to -20 °C and hold for 30 min, then centrifuge. Wash the peptide precipitate twice with 240 mL of MTBE and centrifuge. Dry the peptide precipitate in a vacuum oven at 27 °C for 14 hours. After drying, 1.853 g of crude Preparation 34 is obtained. Purify it by RP-HPLC on a Kromasil 100-10-C8 10 μm column (30 mm × 250 mm) at ambient temperature. The purification is a linear gradient as follows: 15% acetonitrile in water for the first 5 minutes, then 30 - 55% acetonitrile in water for 25 minutes, and a constant 0.1% TFA for 30 minutes. 1.28 g of purified Preparation 34 (SEQ ID NO: 33) is obtained [expected (mass + 2H + ) / 2 = 1470.7929, observed (mass + 2H + ) / 2 = 1470.7885].

[0254] Thioester synthesis (conversion of Preparation 33 to Preparation 35)

[0255] The crude peptide hydrazide (Preparation 33, 2.422 g, 1.251 mmol) was dissolved in 50 mL of ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate monobasic, pH 3.35) and cooled to -15 °C in an acetone-ice bath. 9.4 mL of 1 M sodium nitrite solution (9.4 mmol, 7.5 equivalents) was added to the peptide hydrazide solution and it was stirred at -15 °C for 20 min. Meanwhile, 1 mL of 2,2,2-trifluoroethylthiol (TFET) was made up to 10 mL with ligation buffer (6 M guanidine hydrochloride and 0.2 M sodium hydrogen phosphate, pH 7.0). After 20 min, 10 mL of the TFET mixture was added to the peptide hydrazide solution to thiolyze the peptide azide generated in situ from Preparation 33.

[0256]

[0257] The pH of the reaction mixture was adjusted to approximately 6.95 with 5 N sodium hydroxide solution. The thiolysis of the peptide azide was continued for 45 min and the volume was made up to 100 mL with ligation buffer (pH 7.0). The crude thioester mixture was purified by RP-HPLC at ambient temperature on a Waters X-Bridge C18 10 μm column (10 mm × 250 mm) with the following linear gradient: 10% acetonitrile in water for the first 2.8 min, then 25 - 42% acetonitrile in water for 25 min, and a constant 0.1% TFA over 28 min. This gave 1.03 g of the TFET thioester (Preparation 35 (SEQ ID NO: 34)) [Expected (mass + 2H + ) / 2 = 1010.4650, Observed (mass + 2H + ) / 2 = 1010.4620].

[0258] Native Chemical Ligation: An aqueous solution of 6 M guanidine hydrochloride and 0.3 M sodium dihydrogen phosphate (pH 7.0) is the ligation buffer used in native chemical ligation. All solutions are prepared in this ligation buffer. 350.4 mg (0.174 mmol) of peptide thioester preparation 35 (SEQ ID NO: 34) was dissolved in 50 mL of ligation buffer. 8.0 mL portions of a 0.5 M 4-mercaptobenzoic acid (MPAA) solution were added to the peptide thioester solution. The peptide containing an N-terminal cysteine (preparation 34 (SEQ ID NO: 33), 524.6 mg, 0.178 mmol, 1.03 equivalents) was dissolved in 48 mL of ligation buffer in a 50 mL centrifuge tube. The solution of preparation 34 was added to the thioester solution. The tube was rinsed with 2 x 8 mL of ligation buffer (approx. pH 7.0) and added to the reaction mixture. The pH of the reaction mixture was adjusted to approximately 7 with 5N NaOH solution. 8.0 mL portions of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.0) were added to the reaction mixture, and the pH was adjusted to 7.0 again with 0.2 mL of 5N sodium hydroxide solution. The reaction was stirred at room temperature for 24 hours and then stored in the refrigerator. Before purification, an additional 3 mL of 0.5 M TCEP solution was added. Preparation 36 (SEQ ID NO: 35) was purified by RP-HPLC on a Kromasil 100-10-C8 10 μm column (10 mm x 250 mm) at ambient temperature, with a linear gradient of purification: 10% acetonitrile in water for the first 4 minutes during 28 minutes of purification, followed by 20 - 50% acetonitrile in water (0.1% acetic acid, and titrated to pH 9.0) for the next 23 minutes. Approximately 372 mg (44.3%) of the tirzepatide cysteine analogue preparation 36 [expected (mass + 3H + ) / 3 = 1615.17263, observed (mass + 3H + ) / 3 = 1615.1686] was obtained after purification.

[0259]

[0260] Desulfurization: The buffer solution used in desulfurization is an aqueous solution of 6 M guanidine hydrochloride and 0.3 M sodium dihydrogen phosphate (pH 7.0). All solutions were prepared in this buffer. 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Preparation 37, 808.2 mg, 2.5 mmol) was dissolved in 10 mL of the buffer, and the pH was adjusted to approximately 7.0 with 5N NaOH. The volume was made up to 15 mL with the buffer. The Tirzepatide cysteine analogue Preparation 36 (105.2 mg, 0.022 mmol) was dissolved in 30 mL of the buffer, and 6 mL of the Preparation 37 solution was added thereto. 5 mL of 0.3 M reduced L-glutathione solution (L-GSH, pH 7.0) and 7.5 mL of 0.5 M TCEP solution (pH 7.0) were added thereto. The solution was heated at 44 °C for 4.5 hours, and it was found by UPLC analysis that the reaction was complete [expected (mass + 3H + ) / 3 = 1604.5153, observed (mass + 3H + ) / 3 = 1604.5122]. The desulfurization yield was calculated by UPLC using the tirzepatide (SEQ ID NO: 1) reference standard. The expected yield was 47%.

[0261]

[0262] Native chemical ligation (Method 2): Synthesis of peptidyl hydrazide Preparation 39

[0263] SEQ ID NO: 36

[0264] Take hydrazine-CTC resin (2.03 g, 1.32 mmol, loading value: 0.65 mmol / g) in a 40 mL reactor vessel and swell it on a Symphony synthesizer with 3 × 10 mL DCM (30 s each), then with 2 × 10 mL DMF (20 min each). Dissolve HBTU (1.48 g, 3.90 mmol, 3.0 equivalents) in a solution of (25S,52S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-25-(tert-butoxycarbonyl)-2,2-dimethyl-4,23,28,37,46-pentaoxo-3,32,35,41,44-pentaoxa-24,29,38,47-tetraaza-pentatriacontan-53-oic acid (Preparation 17, 365 mg / mL, in DMF) (3.91 mmol, 3.0 equivalents). Add DIPEA (1.4 mL, 8.04 mmol, 6.1 equivalents) to the above solution and make up the volume to 19 mL with DMF. Mix the solution on a rotary mixer at room temperature for 30 min. Add the activated ester solution of Preparation 17 to the resin. Mix the slurry with nitrogen for 8 h. After 8 h, wash the resin with 5 × 10 mL DMF and 5 × 10 mL DCM and dry for 12 h. The loading of the resulting resin was measured by quantitative NMR to be 0.26 mmol / g. Use 1.82 g of this resin for the synthesis of peptidyl hydrazine Preparation 39 (SEQ ID NO: 36).

[0265] Deprotection: 4 × 9 mL of 20% v / v piperidine in DMF, 30 min each time.

[0266] Coupling: Use 3 equivalents of amino acid, 3 equivalents of OXYMA and 3.3 equivalents of DIC for amino acid coupling.

[0267] After each coupling and the last deprotection, wash the resin with 5 × 9 mL DMF and 1 min N 2 Mix. At the end of the peptidyl hydrazine synthesis, wash the resin with 7 × 10 mL DCM and 1 min N 2 Mix. Then dry the resin on the synthesizer for about 12 h.

[0268] Deprotection and cleavage: 25 mL of a cleavage mixture consisting of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) was added to the dried resin and mixed on a rotary mixer. The resin was filtered, washed with TFA (2 x 2.5 mL), and the filtrate was poured into 175 mL of cold MTBE. The filter flask was washed with TFA (2 x 2.5 mL), and the washings were poured into the cold MTBE. It was cooled to -20 °C and held for 30 min, then centrifuged. The peptide precipitate was then washed twice with 150 mL MTBE and centrifuged. The peptide precipitate was dried in a vacuum oven at 27 °C for 16 h. After drying, 1.70 g of crude peptide hydrazide preparation 39 (SEQ ID NO: 36) was obtained. The crude peptide hydrazide, preparation 39, was purified by RP-HPLC on a Waters XSelect CSH C18 10 μm column (10 mm x 250 mm) at ambient temperature with a linear gradient of: 10% acetonitrile in water for the first 3 min, followed by 20 - 55% acetonitrile in water for 23 min, and 28 min at a constant 0.1% TFA. Approximately 110 mg of partially purified hydrazide preparation 39 was obtained.

[0269] Deprotection and cleavage: 25 mL of a cleavage mixture consisting of 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) was added to the dried resin (2.92 g) and mixed on a rotary mixer. The resin was filtered and washed with 2 x 2.5 mL TFA. The filtrate was poured into 200 mL of cold MTBE, and the peptide immediately precipitated. The filter flask for the filtrate was then washed with 2 x 2 mL TFA, and the washings were poured into the cold MTBE. It was cooled to -20 °C and held for 30 min, then centrifuged. The peptide precipitate was then washed twice with 240 mL MTBE and centrifuged. The peptide precipitate was then dried in a vacuum oven at 27 °C for 16 h. Approximately 1.7 g of crude 19-mer preparation 40 (SEQ ID NO: 37) was obtained.

[0270] Native chemical ligation: An aqueous solution of 6 M guanidine hydrochloride and 0.3 M sodium dihydrogen phosphate (pH 7.0) is the ligation buffer used in native chemical ligation. All solutions are prepared in this ligation buffer. Partially purified peptidyl hydrazide (Preparation 39, 56 mg, 0.019 mmol) was dissolved in 5 mL of ligation buffer (6 M guanidine hydrochloride and 0.3 M sodium dihydrogen phosphate, pH 3.35) and cooled to -15 °C in an acetone-ice bath. To the peptidyl hydrazide solution was added 0.25 mL of 1 M sodium nitrite solution (0.25 mmol, 13.2 equiv), and it was stirred at -15 °C for 10 min. After 10 min, 0.8 mL of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution was added to the peptidyl hydrazide solution to cause in situ thiolysis of the peptidyl azide generated from Preparation 39. The pH of the reaction mixture was adjusted to approximately 7.0 with 5 N sodium hydroxide solution. The thiolysis of the peptidyl azide was allowed to proceed for 30 min.

[0271]

[0272] Approximately 0.62 mmol of Preparation 40 (SEQ ID NO: 37) was synthesized on Sieber amide resin using a standard SPPS protocol. Preparation 40 containing N-terminal cysteine (26.1 mg, 0.014 mmol, 0.74 quiv) was dissolved in 1 mL of ligation buffer. The solution of Preparation 40 was added to the thioester solution. The vial containing Preparation 40 was rinsed with 1 mL of ligation buffer (pH 7.0) and added to the reaction mixture. After 15 min, 1.0 mL of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.0) was added to the reaction mixture, and the pH was adjusted to 7.0 with 5 N sodium hydroxide solution. The reaction was stirred at room temperature for one hour. The Tirzepatide cysteine analogue Preparation 42 was observed in the reaction mixture.

[0273]

[0274] Sequence

[0275]

[0276] wherein X 1 is Aib; X 2 is Aib; Lys at position 20 is replaced with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γGlu) 1 -CO-(CH 2 ) 18 -CO 2H is chemically modified by conjugating to the ε-amino group of the K side chain; and the C-terminal amino acid is amidated to a C-terminal primary amide.

[0277]

[0278]

[0279]

[0280]

[0281] SEQ ID NO: 6

[0282]

[0283] SEQ ID NO: 7

[0284]

[0285] SEQ ID NO: 8

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292] SEQ ID NO: 14

[0293]

[0294] SEQ ID NO: 15

[0295]

[0296] SEQ ID NO: 16

[0297]

[0298] SEQ ID NO: 17

[0299]

[0300]

[0301] SEQ ID NO: 19

[0302]

[0303]

[0304] SEQ ID NO: 21

[0305]

[0306] SEQ ID NO: 22

[0307]

[0308] SEQ ID NO: 23

[0309]

[0310] SEQ ID NO: 24

[0311]

[0312]

[0313]

[0314]

[0315]

[0316] SEQ ID NO: 29

[0317]

[0318]

[0319] SEQ ID NO: 31

[0320]

[0321]

[0322] SEQ ID NO: 33

[0323]

[0324]

[0325] SEQ ID NO: 35

[0326]

[0327]

[0328]

[0329]

[0330] SEQ ID NO: 39

[0331]

[0332] SEQ ID NO: 40

[0333] Sequence Listing <110> Eli Lilly and Company <120> Method for Preparing GIP / GLP1 Dual Agonist <130> X22015 <150> US 62 / 797,963 <151> January 29, 2019 <150> US 62 / 815,053 <151> March 7, 2019 <150> US 62 / 818,342 <151> March 14, 2019 <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa at position 2 is the non-naturally occurring amino acid 2-aminoisobutyric acid <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa at position 13 is the non-naturally occurring amino acid 2-aminoisobutyric acid <220> <221> MOD_RES <222> (20)..(20) <223> The Lys at position 20 is chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu)1-CO-(CH2)18-CO2H <220> <221> MOD_RES <222> (39)..(39) <223> The Ser at position 39 is amidated to the C-terminal primary amide <400> 1 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of serine is protected by tert-butyl <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of serine is protected by tert-butyl <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of serine is protected by tert-butyl <220> <221> MOD_RES <222> (10)..(10) <223> The serine at position 10 is amidated to a C-terminal primary amide. <400> 2 Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 5 10 <210> 3 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected with Fmoc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of aspartic acid is protected with tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of lysine is protected with a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of glutamine is protected with a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (6)..(6) <223> The ε-amino group of K is modified with -CO-O-CH2-CH=CH2 <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of glutamine is protected with a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <400> 3 Asp Lys Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly 1 5 10 15 <210> 4 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamate is protected with a tert-butyl group. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected with a tert-butyl group. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected with a tert-butyl group. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected with tert-butyl group. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected with tert-butyl group. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected with tert-butyl group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected with tert-butyl group. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <400> 4 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu 1 5 10 <210> 5 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of aspartic acid is protected by tert-butyl group. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of lysine is protected with tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (6)..(6) <223> Lys at position 6 is chemically modified by conjugating the ε-amino group of the K side chain with C(O)-O-CH2-CH=CH2 <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of tryptophan is protected by Boc. <220> <221> MOD_RES <222> (18)..(18) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (25)..(25) <223> Serine at position 25 is amidated to a C-terminal primary amide. <400> 5 Asp Lys Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly 1 5 10 15 Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 <210> 6 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamate is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 6 is chemically modified by conjugating the ε-amino group of the K side chain with C(O)-O-CH2-CH=CH2. <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (39)..(39) <223> Serine at position 39 is amidated to the C-terminal primary amide. <400> 6 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 7 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (13)..(13) <223> The 13 - residue Xaa is 2 - amino - isobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by a tert - butyl group. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by a tert - butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert - butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by a tert - butyl group. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by a tert - butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by a tert - butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The serine at position 39 is amidated to a C-terminal primary amide. <400> 7 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 8 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamate is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl group. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl group. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert-butyl group. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl group. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by tert-butyl group. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugating the ε-amino of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> Serine at position 39 is amidated to a C-terminal primary amide. <400> 8 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by Fmoc. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The serine at position 9 is amidated to a C-terminal primary amide. <400> 9 Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by Fmoc. <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <400> 10 Phe Val Gln Trp Leu Ile Ala Gly Gly 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by Fmoc. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (6)..(6) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (7)..(7) The Lys at position 7 is chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu(tert-butyl))1-CO-(CH2)18-CO2-(tert-butyl). <400> 11 Leu Asp Lys Ile Ala Gln Lys Ala 1 5 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamate is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <400> 12 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa 1 5 10 <210> 13 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (12)..(12) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (18)..(18) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (18)..(18) <223> Serine at position 18 is amidated to the C-terminal primary amide. <400> 13 Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser Ser Gly Ala Pro Pro 1 5 10 15 Pro Ser <210> 14 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (6)..(6) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (7)..(7) <223> The Lys at position 7 is chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (12)..(12) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (20)..(20) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (26)..(26) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (26)..(26) <223> The serine at position 26 is amidated to a C-terminal primary amide. <400> 14 Leu Asp Lys Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly 1 5 10 15 Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 <210> 15 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugating the ε-amino of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> Serine at position 39 is amidated to a C-terminal primary amide. <400> 15 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 16 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by Fmoc. <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <400> 16 Phe Val Gln Trp Leu Ile Ala Gly 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by Fmoc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (6)..(6) The Lys at position 6 was chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <400> 17 Asp Lys Ile Ala Gln Lys Ala 1 5 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamate is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> Glycine at position 4 is bound to position 5 using pseudoproline to generate Gly-Thr (psiMe,Me-Pro). <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <400> 18 Tyr Xaa Glu Gly Xaa Phe Thr Ser Asp Tyr Ser Ile Xaa Leu 1 5 10 <210> 19 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> The Xaa at position 2 is 2 - aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamic acid is protected by tert - butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert - butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert - butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert - butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert - butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert - butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert - butyl. <220> <221> MOD_RES <222> (13)..(13) <223> The Xaa at position 13 is 2 - aminoisobutyric acid (Aib). <400> 19 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu 1 5 10 <210> 20 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (12)..(12) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (18)..(18) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (18)..(18) <223> The serine at position 18 is amidated to a C-terminal primary amide. <400> 20 Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser Ser Gly Ala Pro Pro 1 5 10 15 Pro Ser <210> 21 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (6)..(6) <223> Lys at position 6 is chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (18)..(18) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (25)..(25) <223> Serine at position 25 is amidated to the C-terminal primary amide. <400> 21 Asp Lys Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly 1 5 10 15 Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 <210> 22 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamate is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugating the ε-amino group of its side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> Serine at position 39 is amidated to a C-terminal primary amide. <400> 22 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 23 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (20)..(20) <223> The ε-amino group of K is modified with 5,5-dimethyl-2-(3-methylbutylidene)cyclohexane-1,3-dione <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The C-terminal serine is amidated and bound to Sieber resin through this amine group. <400> 23 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 24 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (39)..(39) <223> The C-terminal serine is amidated and bound to Sieber resin through this amine group. <400> 24 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 25 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) The serine at position 10 is amidated to a C-terminal primary amide. <400> 25 Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 5 10 <210> 26 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by Fmoc. <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <400> 26 Phe Val Gln Trp Leu Ile Ala Gly 1 5 <210> 27 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (4)..(4) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (12)..(12) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (18)..(18) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (18)..(18) <223> Serine at position 18 is amidated to a C-terminal primary amide. <400> 27 Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser Ser Gly Ala Pro Pro 1 5 10 15 Pro Ser <210> 28 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by Fmoc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (6)..(6) <223> Lys at position 6 is chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <400> 28 Asp Lys Ile Ala Gln Lys Ala 1 5 <210> 29 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of aspartic acid is protected by a tert-butyl group. <220> <221> MOD_RES <222> (2)..(2) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (6)..(6) <223> The Lys at position 6 is chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu(tert-butyl))1-CO-(CH2)18-CO2-(tert-butyl). <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (18)..(18) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (25)..(25) <223> The serine at position 25 is amidated to a C-terminal primary amide. <400> 29 Asp Lys Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly 1 5 10 15 Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 <210> 30 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected with (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected with tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamic acid is protected with tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected with tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected with tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected with tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected with tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <400> 30 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu 1 5 10 <210> 31 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (1)..(1) <223> The N-terminus is protected by (tert-butoxycarbonyl) Boc. <220> <221> MOD_RES <222> (1)..(1) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa at position 2 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (3)..(3) <223> The side chain of glutamic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (5)..(5) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (7)..(7) <223> The side chain of threonine is protected by tert-butyl. <220> <221> MOD_RES <222> (8)..(8) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (9)..(9) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (10)..(10) <223> The side chain of tyrosine is protected by tert-butyl. <220> <221> MOD_RES <222> (11)..(11) <223> The side chain of serine is protected by tert-butyl. <220> <221> MOD_RES <222> (13)..(13) <223> Xaa at position 13 is 2-aminoisobutyric acid (Aib). <220> <221> MOD_RES <222> (15)..(15) <223> The side chain of aspartic acid is protected by tert-butyl. <220> <221> MOD_RES <222> (16)..(16) <223> The side chain of lysine is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (19)..(19) <223> The side chain of glutamine is protected by a triphenylmethyl (Trt) protecting group. <220> <221> MOD_RES <222> (20)..(20) <223> The Lys at position 20 is chemically modified by conjugating the ε-amino of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu(tert-butyl)) 1-CO-(CH2)18-CO2-(tert-butyl). <220> <221> MOD_RES <222> (24)..(24) <223> The side chain of glutamine is protected by a trityl (Trt) protecting group. <220> <221> MOD_RES <222> (25)..(25) <223> The side chain of tryptophan is protected by a tert-butoxycarbonyl (Boc) protecting group. <220> <221> MOD_RES <222> (32)..(32) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (33)..(33) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The side chain of serine is protected by a tert-butyl group. <220> <221> MOD_RES <222> (39)..(39) <223> The serine at position 39 is amidated to a C-terminal primary amide. <400> 31 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 32 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa at position 2 is Aib <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa at position 13 is Aib <220> <221> MOD_RES <222> (17)..(17) <223> The C-terminal hydroxyl of isoleucine is modified with hydrazide (N-NH2) <400> 32 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile <210> 33 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MOD_RES <222> (3)..(3) <223> Lys at position 3 is chemically modified by conjugating the ε-amino group of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu)1-CO-(CH2)18-CO2H. <220> <221> MOD_RES <222> (22)..(22) <223> Ser at position 22 is amidated to the C-terminal primary amide <400> 33 Cys Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser Ser 1 5 10 15 Gly Ala Pro Pro Pro Ser 20 <210> 34 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa at position 2 is Aib <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa at position 13 is Aib <220> <221> MOD_RES <222> (17)..(17) <223> The C-terminal hydroxyl group is modified with S-CH2-CF3 <400> 34 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile <210> 35 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa at position 2 is Aib <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa at position 13 is Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugation of the ε-amino of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu)1-CO-(CH2)18-CO2H <220> <221> MOD_RES <222> (39)..(39) <223> Ser at position 39 is amidated to the C-terminal primary amide <400> 35 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Cys Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 36 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> The Xaa at position 2 is Aib <220> <221> MISC_FEATURE <222> (13)..(13) <223> The Xaa at position 13 is Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugating the ε - amino group of the K side chain with (2 - [2 - (2 - amino - ethoxy) - ethoxy] - acetyl) 2 - (γ - Glu) 1 - CO - (CH2) 18 - CO2H <220> <221> MOD_RES <222> (20)..(20) <223> The C - terminal hydroxyl group is modified with hydrazide (-N - NH2) <400> 36 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys 20 <210> 37 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MOD_RES <222> (19)..(19) <223> Ser at position 19 is amidated to a C - terminal primary amide <400> 37 Cys Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser Ser Gly Ala Pro 1 5 10 15 Pro Pro Ser <210> 38 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa at position 2 is Aib <220> <221> MISC_FEATURE <222> (13)..(13) <223> Xaa at position 13 is Aib <220> <221> MOD_RES <222> (20)..(20) <223> Lys at position 20 is chemically modified by conjugation of the ε - amino group of the K side chain with (2 - [2 - (2 - amino - ethoxy) - ethoxy] - acetyl) 2 - (γ - Glu) 1 - CO - (CH2) 18 - CO2H. <220> <221> MOD_RES <222> (20)..(20) <223> The C - terminal hydroxyl group is modified with 4 - mercaptophenylacetic acid <400> 38 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys 20 <210> 39 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> The Xaa at position 2 is the non-naturally occurring amino acid 2-aminoisobutyric acid <220> <221> MISC_FEATURE <222> (13)..(13) <223> The Xaa at position 13 is the non-naturally occurring amino acid 2-aminoisobutyric acid <220> <221> MOD_RES <222> (20)..(20) <223> The Lys at position 20 is chemically modified by conjugation of the ε-amino of the K side chain with (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2-(γ-Glu)1-CO-(CH2)18-CO2H <220> <221> MOD_RES <222> (39)..(39) <223> The Ser at position 39 is amidated to the C-terminal primary amide <400> 39 Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Cys Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 40 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Synthetic structure <220> <221> MISC_FEATURE <222> (2)..(2) <223> The Xaa at position 2 is Aib <220> <221> MOD_RES <222> (7)..(8) <223> The Thr at position 7 and the Ser at position 8 form a desmosine <220> <221> MISC_FEATURE <222> (13)..(13) <223> The Xaa at position 13 is Aib <220> <221> MOD_RES <222> (20)..(20) <223> The Lys at position 20 is chemically modified by conjugating the ε - amino group of the K side chain with (2 - [2 - (2 - amino - ethoxy) - ethoxy] - acetyl)2 - (γ - Glu)1 - CO - (CH2)18 - CO2H. <220> <221> MOD_RES <222> (39)..(39) <223> The Ser at position 39 is amidated to a C - terminal primary amide <400> 40 Tyr Xaa Glu Gly Thr Phe Xaa Xaa Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Lys Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35

Claims

1. A method for preparing tirzepatide or a pharmaceutically acceptable salt thereof, comprising: at ambient temperature, adding PyBOP and DIEA to a solution of Preparation 21 and Preparation 19B in 3 mL of 1:1 DMSO / DMF; stirring the solution for 18 hours, then quenching with a 1:1 mixture of 10 mL of saturated aqueous sodium bicarbonate and water; stirring the resulting slurry for 10 minutes, and collecting the resulting solid by filtration; washing the solid with 3 x 10 mL of water, and drying the solid under vacuum at 40 °C to provide Preparation 22; deprotecting the compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof; wherein the amount of Preparation 21 in the above solution is 214 mg, 0.05 mmol; the amount of Preparation 19B in the above solution is 116 mg, 0.055 mmol; the amount of PyBOP added is 57 mg, 0.11 mmol; the amount of DIEA added is 58 μL, 0.33 mmol; wherein Preparation 21 is the sequence SEQ ID NO: 21: Preparation 19B is the sequence SEQ ID NO: 19: and Preparation 22 is the sequence SEQ ID NO: 22:

2. The method according to claim 1, wherein the deprotection solution comprises dithiothreitol, triisopropylsilane and trifluoroacetic acid.

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