PROCESSO PARA DESSULFURIZAR UM ANÁLOGO DE CISTEÍNA DA TIRZEPATIDA

Description

1 / 83 PROCESS FOR DESULFURIZING A CYSTEINE ANALOG OF TIRZEPATIDE Separated from BR112021012368-7, filed on 01 / 28 / 2020.

[001] The present invention relates to processes and intermediates for the preparation of a dual GIP / GLP1 agonist peptide, tirzepatide, or a pharmaceutically acceptable salt thereof.

[002] Diabetes mellitus is a chronic disorder characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. In type 2 diabetes mellitus (T2D), the combined effects of impaired insulin secretion and insulin resistance are associated with elevated blood glucose levels. The dual GIP / GLP1 agonist, tirzepatide, is described and claimed in U.S. Patent 9,474,780 (Patent 780). Tirzepatide may be useful in the treatment of T2D.

[003] US9474780 documents generally describe peptides and a method for preparing a dual GIP / GLP1 agonist.

[004] There is a need for processes and intermediates to enable an improved technology for the production of tirzepatide with a combination of advantages, including commercially desirable purity. Similarly, there is a need for efficient and environmentally green processes, including stable intermediates, to provide tirzepatide with fewer purification steps. An improved technology is also needed to provide tirzepatide manufacturing processes that produce minimal waste streams for enhanced operator and environmental safety. The large-scale preparation of pharmaceutically acceptable tirzepatide presents a number of technical challenges that can affect overall yield and purity.There is a need for processes to avoid the use of transition metals and / or adverse reaction conditions that are incompatible with peptide synthesis. Petition 870260063821, dated 06 / 29 / 2026, page 11 / 253 2 / 83

[005] The present invention seeks to meet these needs by providing new intermediates and processes useful in the manufacture of tirzepatide (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof. The improved tirzepatide manufacturing processes of the present invention provide intermediates and process reactions that incorporate a combination of advancements, including an efficient pathway with fewer steps while at the same time maintaining high quality and purity. Importantly, the improved processes and intermediates decrease resource intensity and minimize waste streams.

[006] The improved processes described in this document provide several forms of useful intermediates for the production of tirzepatide.

[007] The present invention provides a compound with SEQ ID NO: The present invention provides a compound with SEQ ID NO: 11 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 21 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 20 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 4 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 7 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 33 or a pharmaceutically acceptable salt thereof. The present. Petition 870260063821, dated 06 / 29 / 2026, page 12 / 253 The present invention provides a compound with SEQ ID NO: 32 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 34 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 35 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 36 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 38 or a pharmaceutically acceptable salt thereof. The present invention provides a compound with SEQ ID NO: 39 or a pharmaceutically acceptable salt thereof.

[008] A compound with the following formula is provided: or a pharmaceutically acceptable salt thereof.

[009] A compound with the following formula is provided: or a pharmaceutically acceptable salt thereof.

[0010] The present invention provides a process in which tirzepatide is prepared using nanofiltration.

[0011] The present invention provides a process for preparing tirzepatide comprising deprotecting a pharmaceutically acceptable compound or salt from a compound of SEQ ID NO: 22.

[0012] A process is provided for selectively acylating a lysine amino acid in which the lysine amino acid and the N-terminus are protected. A process is provided for selectively acylating a Petition 870260063821, dated 06 / 29 / 2026, page 13 / 253 4 / 83 amino acid lysine in a peptide comprising coupling a resin-linked peptide-Lysine-NH2 with t-butyl-eicosanedioyl-Glu(O-tert-butyl)-(8-amino-3,6-dioxaoctanoic acid)-(8-amino-3,6-dioxaoctanoic acid)-OH. A process is provided for preparing tirzepatide comprising deprotecting a compound of SEQ ID NO: 22 or a pharmaceutically acceptable salt thereof.

[0013] A process is provided for deprotecting tirzepatide, wherein the deprotecting solution comprises dithiothreitol, tri-isopropylsilane and trifluoroacetic acid.

[0014] A process is provided for selectively acylating the amino acid lysine, wherein the resin-bound peptide-Lysine-NH2 is a compound of the formula: or a pharmaceutically acceptable salt thereof.

[0015] A process is provided for converting the depsi peptide isomer to the desired peptide comprising: adjusting the depsi peptide isomer to a pH between about 7 and about 10; and incubating the depsi peptide isomer at a pH of 7 to 10 for at least one hour.

[0016] A process is provided for converting the depsi peptide isomer, wherein the depsi peptide isomer is adjusted to a pH of about 8.5 to a pH of about 9.5.

[0017] A process is provided for converting the depsi peptide isomer, wherein the depsi peptide isomer is a compound of SEQ ID NO: 40 or a pharmaceutically acceptable salt thereof.

[0018] A radical-based desulfurization is provided comprising contacting a peptide with a radical initiator. In one embodiment, the desulfurization comprises contacting a peptide Petition 870260063821, dated 06 / 29 / 2026, page 14 / 253 5 / 83 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 the group consisting of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (VA-050).

[0019] The radical-based desulfurization method provided herein is environmentally desirable, transition metal-free, and conditions compatible with peptide synthesis.

[0020] As used in this document, the following abbreviations have the meanings set forth in this document: SPPS means Solid Phase Peptide Synthesis, Fmoc means fluorenylmethyloxycarbonyl chloride, Pip means piperidine, DIC means diisopropylcarbodiimide, Oxyma means ethyl cyanohydroxyiminoacetate, DCM means dichloromethane, IPA means isopropanol, MTBE means methyl tert-butyl ether, TFA means trifluoroacetic acid, TIPS means triisopropylsilane, DTT means dithiothreitol, UPLC means ultra-high performance liquid chromatography, HFIP means hexafluoroisopropanol, CTC means chlorotritile, HATU means 3-oxide hexafluorophosphate of (1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium, TFET DIEA stands for N,N-diisopropylethylamine, AEEA stands for 17-amino-10-oxo-3,6,12,15tetraoxa-9-aza-heptadecanoic acid, and TCEP stands for tris(2-carboxyethyl)phosphine.DCU stands for dicyclohexylurea, DCC stands for dicyclohexylcarbodiimide, TMSA stands for trimethylsylamide, HOBt stands for hydroxybenzotriazole, HRMS stands for high-resolution mass spectrometry, LPPS stands for liquid-phase peptide synthesis, MSMPR stands for mixed-product mixed suspension reactor, MPA stands for mobile phase A, MPB stands for mobile phase B, L-GSH stands for reduced L-glutathione solution, TZP stands for tirzepatide, AP, Petition 870260063821, dated 06 / 29 / 2026, page 15 / 253 6 / 83 stands for active pharmaceutical product and API stands for active pharmaceutical ingredient, PyBOP stands for (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, DEA stands for diethylamine, TBTU stands for 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate, TNTU stands for 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate, PyOxim stands for cyano-2-ethoxy-2-oxoethylideneamino-oxy-tripyrrolidinephosphonium 1-hexafluorophosphate, PyClock stands for 6-chlorobenzotriazol-1-yloxy-tris-pyrrolidinephosphonium hexafluorophosphate. As presented in this document, one-letter abbreviations for amino acids are shown in bold, while atoms are shown as non-bold text and generally in lowercase to distinguish them from one-letter amino acid abbreviations.As used in this document, when an amino acid abbreviation appears with a number above the amino acid, the number refers to the corresponding amino acid position in the final tirzepatide product. The numbers are provided for convenience, and the appearance or absence of such numbers in a sequence does not influence the amino acid sequence or the peptide indicated in that sequence. As used in this document, the term protected means that a protecting group is attached at the indicated position. Those skilled in the art will recognize that a variety of protecting groups are well known, and alternative protecting groups may be suitable for a particular process.

[0021] Those skilled in the art will recognize that there are alternative resins for constructing the peptides presented herein. For example, Sieber and Rink amide resins are well known to those skilled in the art for the preparation of the peptides described herein; however, alternative resins may be selected for the preparation of the peptides. Petition 870260063821, dated 06 / 29 / 2026, page 16 / 253 7 / 83 described in this document. For example, but without limitation, 2-CTC and related resins can be used to prepare a target peptide, followed by a C-terminus amidation step.

[0022] Solid-phase peptide synthesis (SPPS) constructs are performed using fluorenylmethyloxycarbonyl chloride (Fmoc) peptide chemistry techniques using sequential couplings with an automated peptide synthesizer. The resin is swollen with DMF and then deprotected using piperidine (Pip) at 20% / DMF (3 x 30 min). Subsequent Fmoc deprotections use 3 x 30 min treatments with Pip at 20% / DMF, and 4 x 30 min treatments are used for more difficult couplings. After deprotection, the resin is washed with 5 x 2 min washes with 10 volumes of DMF. Amino acid pre-activation uses di-isopropylcarbodi-imide (DIC) / cyanohydroxyiminoacetate (Oxyma) / DMF solutions at room temperature for 30 min. The coupling of the activated amino acid to the resin-bound peptide occurs over a specified time for each individual amino acid. Solvent washing with 5 x 2 min 10 volumes of DMF is performed after each coupling.For isolation of the final product, the resin-bound product is washed 5 x 2 min with 10 volumes of DCM to remove DMF. The resin is washed 2 x 2 min with 10 volumes of IPA to remove DCM, then washed 5 x 2 min with 10 volumes of methyl tert-butyl ether (MTBE), and finally dried at 40°C under vacuum. The resin-bound product is stored chilled (-20°C). For analysis, the peptide is cleaved from the resin with an acid cocktail consisting of trifluoroacetic acid (TFA) / H2O / TIPS (triisopropylsilane) / DTT (dithiothreitol) in the following ratio: (0.93v / 0.04v / 0.03v / 0.03v). The resin is swollen with DCM (4-5 mL, 3 x 30 min) and drained. The cleavage cocktail (4-5 mL) is added to the pre-swelled resin and the suspension is stirred for 2 hours at room temperature. The solution is filtered, then the resin is washed with a small amount of DCM e. Petition 870260063821, dated 06 / 29 / 2026, page 17 / 253 8 / 83 combined with the cleavage solution. The resulting solution is poured into 7-10 volumes of ice-cold methyl tert-butyl ether (MTBE) (0 °C). The suspension is aged for 30 min at 0 °C, then the resulting precipitate is centrifuged and the clear solution is decanted. The residue is suspended in the same volume of MTBE and the resulting suspension is again centrifuged and decanted. After decantation, the clear MTBE solution of the precipitated peptide is dried under vacuum at 40 °C overnight. Summary of Preparation 1: SEQ ID NO: 2

[0023] The synthesis uses Sieber-Fmoc amide resin with a loading of 0.71 mmol / g. The general SPPS procedure is used with the following modifications: Amino Acid Cycle SPPS Conditions Solvent for couplings: DMF 1 Fmoc-L-Ser(t-Bu)-OH 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 2 Fmoc-L-Pro-OH 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 6 h, ta. 3 Fmoc-L-Pro-OH 4 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 6 h, ta. 4. Fmoc-L-Pro-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 6 h, ta. 5. Fmoc-L-Ala-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 6. Fmoc-Gly-OH cycles of 3 x 30 minutes of De-Fmoc, Petition 870260063821, dated 06 / 29 / 2026, p. 18 / 253 9 / 83 Post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 7 Fmoc-L-Ser(t-Bu)-OH: 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 8 Fmoc-L-Ser(t-Bu)-OH: 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 9 Fmoc-L-Pro-OH: 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta 10 Fmoc-Gly-OH cycles of 4 x 30 minutes of De-Fmoc, post-deprotection washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta.

[0024] Gentle Cleavage of Preparation 1: Ten identical deprotection reactions are performed in parallel, each on a scale of ~0.5 mmol of resin-bound Preparation 1 using the following protocol: 1) To a 40 mL frit reactor, add 1.55 g (~0.5 mmol) of resin-bound Preparation 1. 2) Swell with 3 x 15 mL of DMF (15 min each). 3) Treat with 3 x 15 mL (30 min each) of 20% Pip / DMF. 4) Wash with 4 x 15 mL of DMF, followed by 4 x 15 mL of DCM. 5) Add 1.5 mL of TFA and 28.5 mL of DCM to each of the five 40 mL reaction flasks. 6) Add one-fifth of the resin-bound Preparation 1 (2.75 g) to each of the TFA solution bottles, stopper the bottles, and mix on the rotary wheel for 5 minutes. 7) Filter the mixtures 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 into a light yellow oil, then grind with 200 mL. Petition 870260063821, dated 06 / 29 / 2026, p. 19 / 253 10 / 83 of MTBE and cool in an ice bath for 30 minutes. 10) Filter the solid, wash with 50 mL of chilled MTBE and dry in a vacuum oven at 33 °C overnight to produce 5.35 g (91% yield) of a white solid. Analysis of the isolated solid using UPLC (98.57% area, with 0.99% of t-Bu deprotection byproducts combined). Synthesis of Preparation 2: SEQ ID NO: 3

[0025] The synthesis uses Fmoc-Gly-OH2-CTC resin with a loading of 0.61 mmol / g. The general SPPS procedure is used with the following modifications: Amino Acid Cycle SPPS Conditions Solvent for couplings: DMF 1 Fmoc-L-Ala-OH 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 2 Fmoc-L-Ile-OH 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 3 Fmoc-L-Leu-OH 4 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 6 h, ta. 4. Fmoc-L-Trp(Boc)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 6 h, at room temperature. 5. Fmoc-L-Gln(Trt)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, at room temperature. 6. Fmoc-L-Val-OH: 4 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 6 h, at room temperature. 7 Fmoc-L-Phe- OH cycles of 4 x 30 minutes of De-Fmoc, Petition 870260063821, dated 06 / 29 / 2026, p. 20 / 253 11 / 83 Post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 8 h, ta. 8 Fmoc-L-Ala-OH: 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 9 Fmoc-Lys(Alloc)-OH: 4 x 30 minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 8 h, ta. 10 Fmoc-L-Gln(Trt)-OH: 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 11 Fmoc-L-Ala-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 12 Fmoc-L-Ile-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 13 Fmoc-L-Lys(Boc)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 14 Fmoc-L-Asp(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta.

[0026] Gentle Cleavage of Preparation 2: To a 40 mL glass scintillation vial, add the resin-bound Preparation 2 (3.06 g, 1.12 mmol) and 30 mL of 30% HFIP / DCM solution where the change to red color is observed. Shake the vial by rotating it on a wheel at room temperature for 1 hour. Filter the resin and wash with 3 x 10 mL of DCM. Remove the solvent under vacuum to form a glassy foam (bath at 35°C, 10 torr, 2.34 g) and replace with a Petition 870260063821, dated 06 / 29 / 2026, p. 21 / 253 Take a small portion of IPA (24 mL) and then add water (24 mL) dropwise over 25 min at room temperature. Shake the resulting solution for 30 min and then filter. Wash the washed cake with 3 x 10 mL of H2O and then dry in a vacuum oven at 25 torr and 35°C overnight. This yields Preparation 2 as a white solid (1.81 g). Summary of Preparation 4: SEQ ID NO: 4

[0027] The synthesis uses Fmoc-Leu-OH 2-CTC resin with a loading of 0.68 mmol / g. The general SPPS procedure is used with the following modifications: Amino Acid Cycle SPPS Conditions Solvent for couplings: DMF 1 Fmoc-Aib-OH 4 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 8 h, ta. 2 Fmoc-L-Ile-OH 4 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 18 h, ta. 3 Fmoc-L-Ser(tBu)-OH 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 4. Fmoc-L-Tyr(tBu)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 5. Fmoc-L-Asp(tBu)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 6. Fmoc-L-Ser(tBu)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 7 Fmoc-L-Thr(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deprotection washes 6 x 2 minutes, Petition 870260063821, dated 06 / 29 / 2026, p. 22 / 253 13 / 83 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 8 Fmoc-L-Phe-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 9 Fmoc-L-Thr(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 10 Fmoc-Gly-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 6 h, ta. 11 Fmoc-L-Glu(tBu)-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 6 h, ta. 12 Fmoc-Aib-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 8 h, ta. 13 Boc-L-Tyr(tBu)-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 18 h, ta.

[0028] Gentle Cleavage of Preparation 4: To a 20 mL glass scintillation flask, add the resin-bound Preparation 4 (2.0 g, 0.62 mmol) and 10 mL of 30% HFIP / DCM solution, where a color change is observed. Shake the flask by rotating it on a wheel at room temperature, then filter the resin, wash with 3 x 2 mL of DCM, and remove the solvent under vacuum to form a glassy, ​​sticky foam. Dissolve the foam in 5.2 mL of DMSO. Add this solution to 6 mL of water at equal flow rates (T ~ 15°C) over 45 min with 1 mL of water. Once the peptide solution is fully added, add another 6 mL of water over 45 min. White solids precipitate upon addition. Shake the resulting suspension at 15°C for 30 min. Filter out the solids, wash with 6 mL of water, and then... Petition 870260063821, dated 06 / 29 / 2026, p. 23 / 253 14 / 83 then transfer to a vacuum oven at 35 °C and 25 torr. This results in Preparation 4 (Boc-1-14-OH, 1.0763 g) as a fluffy white solid. Synthesis of Preparation 3 via LPPS: SEQ ID NO: 5

[0029] To a 20 mL glass scintillation vial, add the Preparation 2 (500 mg, 0.183 mmol), Preparation 1 (179 mg, 0.175 mmol) and DMSO (10 mL). Add DIEA (46 pL, 0.265 mmol) to this solution, followed by PyBOP (hexafluorophosphate of (benzotriazol-1-yloxy)tripyrrolidinophosphonium) (123 mg, 0.230 mmol). Stir the reaction for 2 hours, then supplement with diethylamine (DEA) (183 microliters, 1.77 mmol) and stir the resulting solution for 2 hours. Extract the reaction contents with a syringe and add to a shaken 50 mL flask with simultaneous dropwise addition of water (12 mL) over 1 hour. After the additions are complete, collect the precipitated product by filtration and subsequently wash with water (2 x 4 mL). Dry the wet cake under vacuum at 35°C for 18 hours to obtain Preparation 3 as a white solid (yield of 88% 0.6003 g, HRMS calc. for C184H261N31O38 expected 3512.9444, actual 3512.9430). Synthesis of Preparation 5 Through LPPS: SEQ ID NO: 6 Petition 870260063821, dated 06 / 29 / 2026, p. 24 / 253 15 / 83HOHO Bm-YN^J*-E*GTF*T'8-D'Y*SjI—N ^JL-OH fflu Z\. rtlLi íBu ÍBlíQuíBuÍBliiBu A.. M& MS Mβ MS Preparation 4 °y°' NH hDKI-AQ-ν γ II I H II íBu Boc Trt Q AFV Q WLIAGGPSSGAPPP S-nhsTrl Boc iBulBu ífiu H í? Bog-YN-Jf011Mh Ws NH Preparation 3 AFVQ WLlAGGPSSGAPPPS-NHZ TrtSoD IBidBu Su Preparation 5

[0030] To a 20 mL glass scintillation flask, add Preparation 3 (338.8 mg, 0.09 1 mmol), Preparation 4 (192.1 mg, 0.09 1 mmol), and DMSO (10 mL). To this solution, add PyBOP (63.5 mg, 0.118 mmol), followed by DIEA (79 microliters, 0.454 mmol). Shake the reaction solution for 2.5 hours. Extract the reaction contents with a syringe and add the contents to a shaken 50 mL flask with simultaneous dropwise addition of water (12 mL) over 1 hour. After the additions are complete, collect the precipitated product by filtration and subsequently wash with water (2 x 4 mL). Dry the moist cake under vacuum at 35 °C for 18 hours to obtain Preparation 5 as a white solid (0.3568 g, 70% yield, HRMS calc. for C293H435N45O64 expected 5608.2168, actual 5608.2066). Synthesis of Preparation 6 via Method 1 (LPPS) Petition 870260063821, dated 06 / 29 / 2026, page 25 / 253 16 / 83

[0031] Dissolve eicosanedioic acid, mono(1,1-dimethylethyl) ester (15.0 kg, limiting reagent) and N-hydroxysuccinimide (1.2 eq.) in ethyl acetate at 27 °C. Add a DCC solution (1.25 eq.) in ethyl acetate and stir the reaction for 24 h at 22 °C. Filter the resulting DCU byproduct and then extract the organic phase three times with 5% aqueous NaCl solution. After extraction, concentrate the organic phase, coevaporate with isopropanol, and then crystallize by adding heptane. After filtration, rinse the filter cake with heptane and dry at 25 °C to obtain 17.0 kg of INT1 with 87% yield and 99% purity.

[0032] 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), then stir the reaction mixture for 1 h at 40°C. Add INT1 solution (17.0 kg) to DCM at room temperature and stir for 8 h. After the reaction is complete, DCM is exchanged for ethyl acetate by distillation. Wash the organic phase three times with 2% aqueous KHSO4 / NaCl solution, then wash four times with 2% aqueous NaCl solution. After separation and removal of the aqueous phase, concentrate the organic phase, coevaporate with propanol, dilute with isopropanol, and then crystallize by adding water. After filtration, wash the filter cake with a mixture of Petition 870260063821, dated 06 / 29 / 2026, page 26 / 253 17 / 83 water / isopropanol and then dry at 30°C to produce 17.3 kg of INT2 with 86% yield and 99% purity.

[0033] Dissolve INT2 (17.3 kg) and N-hydroxy-succinimide (4.1 kg, Add a solution of DCC (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 for 24 hours at 22°C. Filter the resulting DCU byproduct. Concentrate the organic phase, coevaporate with isopropanol, and then crystallize by cooling the isopropanol solution (~125 L). Then, rinse the filter cake with chilled isopropanol and dry at 25°C to yield 16.3 kg of INT3 with 81% yield and 96% purity.

[0034] Suspend 17-amino-10-oxo-3,6,12,15-tetraoxa-9aza-heptadecanoic acid (AEEA 2) (8.1 kg, 26.3 mol) in DCM (54 L) at 22°C, add TMSA (7.68 kg, 59.9 mol) in DCM (6.2 L), and then stir the reaction mixture for one hour at 40°C. Suspend INT3 (16 kg) in DCM (31 L) at 35°C and add to the TMS-protected mixture (AEEA 2) at 22°C. Stir the reaction for 12 hours, and after the reaction is complete, concentrate the mixture and then replace it with ethyl acetate. Wash the organic phase three times with aqueous KHSO4 / NaCl solution (~200 L) and then wash four times with 2% aqueous NaCl solution (~200 L) to a target pH of 4.5. Concentrate the organic phase and replace it with acetonitrile. Cool the acetonitrile solution to -20°C and then age the resulting suspension for 15 hours at -20°C. Filter the mixture, wash the filter cake with chilled acetonitrile, and dry at <0°C to obtain 40 pounds of Preparation 6 (88% yield) with 96% purity. Overall yield = 53%. Synthesis of Preparation 6 via Method 2 (SPPS)

[0035] Alternatively, Preparation 6 can be prepared using solid-phase peptide synthesis using a peptide synthesizer. Petition 870260063821, dated 06 / 29 / 2026, page 27 / 253 18 / 83

[0036] Standard coupling procedures are used. Standard Coupling Conditions:

[0037] 2.0 equiv. of HATU at 0.133 M, 5.0 equiv. of DIEA, room temperature, 3 hours, unprotected for 3 x 15 min with piperidine at 20% / DMF. Resin Loading:

[0038] FmocNH -AEEA on 2-CTC resin (0.99 mmol / g): 1.01 g in each of the parallel reactions.

[0039] An automatic program using swelling in DMF, followed by Pip / DMF; washing with DMF; and amino acid, DIEA, HATU mixture; and DMF washing cycles followed by drying.

[0040] The resin is cleaved by agitating the combined batches in HFIP at 30% / DCM (240 mL) for 1.5 hours. The resin is filtered, washed, and the solvent is removed from the filtrate under vacuum. The resulting oil is dissolved in acetonitrile and the solvent is removed again. This operation provides 30.47 g (146% of the theoretical yield) of a viscous yellow oil containing 52.3% of the desired product area by UPLC analysis. The crude product is purified by fast chromatography (500 grams of silica gel, eluted with 85% DCM / 10% methanol / 5% acetic acid, 38 x 100 mL fractions collected). The previously chromatographed concentrate (17.94 g) is crystallized to provide 13.4 g (74.7% yield), with a UPLC purity of 91.65% area. Example 1 Petition 870260063821, dated 06 / 29 / 2026, page 28 / 253 19 / 83 3S .......: » o ...... 'K NII L:·0 for-V-FL. ^ü-EQTFT-5-DY SlH.. ,^<-Ο-ΚΉΑ···ΰ'^ γ A -FVq W Ld-A 3 GP' S SGAPPP A4^: iBu iSt? fen .'BufBtL'ELi&Hfiu <BÜ Búc tfl d tn&oc feitf&jfell Preparation 5 ® IK t hH?&c-¥-Uví-E 6 TF TSD YS ·ί· fflu kj / 'f. / . ?Βυ ifcu lEníBufeiíFinfiu “v rfp? Preparation 7 AFV OW LlA GGPSSGAPPP -i-hH: Triéoc íBíiÍBu Feb Preparationfl Preparation fl |g Pre for fan ο B Example 1 Example of Synthesis 1 SEQ ID NO: 1

[0041] To a first HPLC vial, add Preparation 5 (10.5 mg, 0.00187 mmol) and DCM (200 µl, 20 L / kg). To this solution, add 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). Shake the solution at 24°C for one hour to obtain Petition 870260063821, dated 06 / 29 / 2026, p. 29 / 253 20 / 83 a non-isolated solution of Preparation 7 (SEQ ID NO: 7). To a second HPLC vial, add DCM (150 μL), followed by Preparation 6 (0.118 M in DCM, 16 pL, 0.00189 mmol), PyBOP (0.186 M in DCM, 16 pL, 0.00298 mmol) and DIEA (0.573 M in DCM, 5 equiv.). Add the contents of the second vial to the first vial and stir the reaction for 1 hour to obtain a non-isolated solution of Preparation 8 (SEQ ID NO: 8). Concentrate the solution of Preparation 8 under vacuum and, to the resulting solid, add 50 μL of a solution of trifluoroacetic acid (4.65 mL), tri-isopropylsilane (20 μL) and DTT (20 mg). Shake the suspension for 18 hours and monitor by HPLC to confirm the formation of Example 1 (HRMS calc. for C225H348N48O68 expected 4810.5249, actual 4810.5257). Summary of Preparation 9 SEQ ID NO: 9

[0042] Suspend Sieber amide resin (13.42 g, 0.75 mmol / g, 10.1 mmol) in DMF (130 mL, 10 vols.) for approximately 20 min, then drain. Wash the resulting resin with DMF (80 mL, 6 vols.) for approximately 5 min. Remove the Fmoc group by treating the Fmoc amino acid resin with 5% by volume piperidine, 1.25% by volume DBU, 1.0% by weight HOBt / DMF solution (80 mL, 6 vols.) twice, for 10 min and 20 min, respectively. Wash twice with DMF (80 mL, 6 vols.), twice with MTBE (80 mL, 6 vols.), and again twice with DMF (80 mL, 6 vols.) after draining the de-Fmoc solution.

[0043] Using standard Fmoc chemistry, assemble the amino acid chain. In general, 1.5 equiv. of Fmoc-amino acid and HOBt (2.47 g, 20% wet water, 14.6 mmol, 1.46 equiv.) are dissolved in DMF (60 mL, 4.5 vols.), followed by the addition of DIEA (1.94 mL, 11.1 mmol, 1.11 equiv.). Cool the resulting solution to < 5 °C with an ice bath and activate by adding TBTU (4.83 g, 15.0 mmol, 1.5 equiv.). Petition 870260063821, dated 06 / 29 / 2026, page 30 / 253 21 / 83 Allow to stand for approximately 5 minutes at 0°C - 5°C. Add DCM (60 mL, 1.5 vols.) to the resin, followed by the addition of the activated Fmoc amino acid solution. Stir the resulting mixture at near room temperature for 2 hours. Repeat the deFmoc procedure and coupling with the remaining amino acids sequentially. After completing the last deFmoc procedure, wash the resin with 2-propanol (130 mL, 10 vols.) for 5 min twice, followed by washing with MTBE (130 mL, 10 vols.) six times. The resin is dried at 35°C in vacuo, resulting in Preparation 9-Seiber (21.21 g, 0.435 mmol / g theoretical, 91.7% yield based on mass increase).

[0044] A portion of the Preparation 9-resin complex (10.15 g, A fraction (0.435 mmol / g, 4.41 mmol) is treated with 5% by volume of TFA in DCM solution (101 mL, 10 vols.) and washed with DCM. The cleavage and washing fractions are neutralized with DIEA (26.29 g, 35.5 mL, molar ratio of 1.01:1 to TFA). The fractions are combined and concentrated under vacuum to 50% of the original volume. Wash the DCM solution with a saturated aqueous solution of NaHCO3 (2 x 94 mL). Dry the resulting solution over anhydrous MgSO4 and concentrate to dryness to produce a sticky solid. Resuspend this sticky solid in MTBE at < 5°C (100 mL) to decompose the gum, resulting in a white suspension product. Filter, wash and dry the white powder suspension, resulting in Preparation 9 (3.84 g, 92.3% area, 37.8% by weight DIEA^TFA, 57.4% by weight, 2.29 mmol, 51.9% yield, HRMS calc. for C46H78N10O12 expected 962.5801, actual 962.5806) as a white powder. Summary of Preparation 10 SEQ ID NO: 10

[0045] Suspend the Fmoc-Gly-Gly-O-2CTC resin complex (18.09 g, 0.57 mmol / g, 10.3 mmol) in DMF (180 mL, 10 vols.) for Petition 870260063821, dated 06 / 29 / 2026, page 31 / 253 22 / 83 min and then drain. Wash the resulting resin with DMF (108 mL, 6 vols.) for 5 min. Remove the Fmoc group by treating the amino acid-Fmoc resin with 5% by volume piperidine, 1.25% by volume DBU, 1.0% by weight HOBt / DMF solution (108 mL, 6 vols.) twice, for 10 min and 20 min, respectively. Drain the de-Fmoc solution and wash the resin twice with DMF (110 mL, 6 vols.), twice with MTBE (110 mL, 6 vols.), and again twice with DMF (110 mL, 6 vols.). Chain assembly is conducted with standard Fmoc chemistry.

[0046] For amino acid coupling, generally 1.5 equiv. Fmoc-amino acids and HOBt (2.54 g, 20% water, 15.0 mmol, 1.5 equiv.) were dissolved in DMF (80 mL, 4.4 vols.), followed by the addition of DIEA (1.94 g, 15.0 mmol, 1.5 equiv.) to allow the amino acids to bind. Cool the resulting solution to 0–5 °C with an ice bath and activate by adding 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU) (4.84 g, 15.1 mmol, 1.5 equiv.). Let stand for 5 min at 0–5 °C. DCM (35 g, 1.5 vol.) and then add 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. The peptide resin is washed after completion of the synthetic steps with 2-propanol (180 mL, 10 vols.) for 5 min twice and then with MTBE (180 mL, 10 vols. each, 6 times), followed by drying at 35 °C, resulting in the 10-resin preparation complex (25.52 g, 0.216 mmol / g, 53.6% yield).

[0047] Treat a portion of the 10-resin Preparation complex (10.075 g, 0.216 mmol / g, 2.18 mmol) three times with 1% by volume of TFA in DCM solution (100 mL, 10 vols.) and wash with DCM (75 mL, 7.5 vols.). Neutralize the cleavage fractions and wash with pyridine (3.18 g, molar ratio of 1.01:1 to TFA). Combine and concentrate the fractions under vacuum until dry at < 35 °C. Reconstitute. Petition 870260063821, dated 06 / 29 / 2026, p. 32 / 253 23 / 83 with ethanol (40 mL, 10% vol. of the combined filtrates), followed by concentration until dry. Finally, triturate the peptide with stirring in deionized water (150 mL, 40% vol. of the combined filtrates). Collect the crude solid peptide precipitate by centrifugation and wash with deionized water twice (150 mL each time). Wash the solid twice with n-heptane (100 ml each time), isolate and dry in vacuo at 40 °C to yield Preparation 10 (SEQ ID NO: 10) as a light yellow solid (4.10 g, 72.4 % area, 3.0 % by weight of pyridine^TFA, 70.2 % by weight, 1.85 mmol, 85.1 % yield, HRMS calc. for C88H103N11O15 expected 1553.7635, actual 1553.7656). Preparation Summary 11 SEQ ID NO: 11

[0048] Suspend the H-Alanine-O-2CTC resin complex (40.39 g, 0.5 mmol / g, 20.20 mmol) in DMF (400 mL, 10 vols.) for about 20 minutes and then drain. Wash the resulting resin with DMF (400 mL, 10 vols.) for 5 min twice. Assemble the amino acid chain using standard Fmoc chemistry. In general, dissolve 1.5 equiv. Mix Fmoc-amino acid and HOBt (5.51 g, 80 wt%, 32.6 mmol, 1.6 equiv.) in DMF (150 mL, 3.7 vols.), followed by the addition of DIEA (4.22 g, 32.7 mmol, 1.6 equiv.). Cool the resulting solution to approximately < 5°C with an ice bath and activate by adding TBTU (10.39 g, 32.4 mmol, 1.6 equiv.). Stir for about 5 minutes at 0-5°C. Add DCM (80 mL, 2 vols.) to the resin, followed by the addition of the activated Fmoc-amino acid solution. Stir the resulting mixture at near room temperature for 2 hours.

[0049] Remove the Fmoc group by treating the Fmoc amino acid resin with 5% by volume piperidine, 1.25% by volume DBU, 1.0% by weight HOBt / DMF solution (240 mL, 6 vols.) twice, for 10 min and 20 min, respectively. Drain the Fmoc solution, wash the resin twice with DMF (240 mL, 6 vols.), twice Petition 870260063821, dated 06 / 29 / 2026, page 33 / 253 24 / 83 with MTBE (240 mL, 6 vols.) and again twice with DMF (240 mL, 6 vols.). The peptide resin is carefully washed with 2-propanol (400 mL, 10 vols.) twice and MTBE (400 mL, 10 vols. each, 6 times) after completion of the synthetic steps, followed by vacuum drying at 35°C to produce resin loaded with the last amino acid (74.82 g, 0.159 mmol / g, 11.90 mmol, 58.9% yield). Add the last amino acid, Fmoc-Leu-OH, separately to a portion of the resin (13.61 g, 0.159 mmol / g, 2.16 mmol). Swell this resin with DMF (130 mL, 10 vols., 3 times) for > 5 min each time, then deprotect (130 mL of deprotection mixture prepared from 5.6 g piperidine, 1.67 g DBU, 1.3 g HOBt in 120 mL of DMF, 10 vols. twice) for 10 min and 20 min. Wash the resin with DMF (80 mL, 6 vols., twice), followed by MTBE (80 mL, 6 vols., twice) and then DMF (80 mL, 6 vols., twice) for 5 min each time. Dissolve in DMF (50 mL, 3.7 vols.), followed by the addition of DIEA (0.54 g, 4.2 mmol, 1.9 equiv. for Fmoc-Leu-OH coupling, Fmoc-Leu-OH (1.47 g, 4.16 mmol, 1.9 equiv.) and HOBt (0.704 g, 80% by weight, 4.17 mmol, 1.9 equiv.)). Cool the resulting solution to < 5°C with an ice bath and activate by adding TBTU (1.34 g, 4.17 mmol, 1.9 equiv.) and stir for 5 min at 0-5°C. Add DCM (20 mL, 1.5 vols.) to the resin, followed by the addition of the activated Fmoc-amino acid solution. Stir the resulting mixture at near room temperature for 2 hours. Wash this resin with DMF (180 mL, 13 vols., twice), MTBE (180 mL, 13 vols., twice) and DMF (180 mL, 13 vols., twice) for 5 min each. Wash the resin with DCM (130 mL, 10 vols., 6 times, 5 minutes each), before drying the resin in vacuo at 35°C, resulting in a loaded resin (12.90 g, 0.203 mmol / g, 2.62 mmol, 121% yield).

[0050] Treat a part of the resin (7.09 g, 0.203 mmol / g, 1.44 mmol) Petition 870260063821, dated 06 / 29 / 2026, p. 34 / 253 25 / 83 three times with 1% by volume of TFA in DCM solution (70 mL, 10 vols.) for 10 minutes each at around room temperature, followed by washing with DCM (55 mL, 7.5 vols.). Neutralize the cleavage fractions and wash with pyridine (3.02 g, molar ratio of 1.02:1 to TFA). Combine and concentrate the fractions under vacuum until dry to < 35°C. Reconstitute with ethanol (28 mL, 11% vol. of the combined filtrates), followed by concentration until dry. Finally, stir the peptide in deionized water (105 mL, 40% vol. of the combined filtrates). Collect the crude solid peptide precipitate by filtration and wash with deionized water (4 x 50 mL). Wash the solid with n-heptane (3 x 100 mL), isolate and dry in vacuo at 40 °C, resulting in Preparation 11 as a white powder (4.54 g, 87.6% area, 44.4% by weight of pyridine^TFA, 48.7% by weight, 0.936 mmol, yield 65.0%, HRMS calc. for C127H192N14O28 expected 2361.4031, actual 2361.4021). The overall yield for the preparation of Preparation 11 in resin is 71.3%. Summary of Preparation 12 SEQ ID NO: 12

[0051] Suspend the Fmoc-Aib-O-CTC resin complex (19.16 g, Mix 0.54 mmol / g, 10.35 mmol) in DMF (190 mL, 10 vols.) for 20 min and then drain. Wash the resulting resin with DMF (190 mL, 10 vols.) for 5 min and then drain. Combine piperidine (77.82 g), DBU (23.16 g), HOBt (18.09 g, 80% by weight) and DMF (1800 mL) to provide a 5% piperidine, 1.25% DBU, 1.0% HOBt / DMF solution as a deprotecting solution. Remove the Fmoc group by treating the amino acid-Fmoc resin with deprotecting solution (190 mL, 10 vols.) twice, for 10 min and 20 min, respectively. Drain the de-Fmoc solution and wash the resin twice with DMF, twice with MTBE, and again twice with DMF (190 mL, 10 vols. for each wash). Petition 870260063821, dated 06 / 29 / 2026, p. 35 / 253 26 / 83

[0052] Add DIEA (2.62 g, 20.3 mmol, 2.0 equiv.) to a solution of Fmoc-Ile-OH (7.11 g, 10.1 mmol, 2.0 equiv.) in DMF (85 mL). Cool the resulting solution to 0–5°C, add 6-chlorobenzotriazol-1-yloxy-tris-pyrrolidinephosphonium hexafluorophosphate (PyClock) (11.36 g, 20.06 mmol, 2.0 equiv.) and dissolve completely. Add the activated solution to the pre-swelled H-Aib-O-CTC resin complex in DCM (30 mL, 1.5 vols.) after standing for 3–5 minutes. Allow the reaction to warm to room temperature and stir for 2 hours. The unreacted material is approximately 18%, as indicated by the assay evaluation. Wash with DMF twice, MTBE twice, and DMF twice (190 mL, 10 vols. each). Add a solution of Fmoc-Ile-OH (10.63 g, 30.08 mmol, 6 equiv.) in DMF (165 mL) to Oxyma (50 mL, 0.6 M in DMF, 30 mmol, 6 equiv.) and DIC (50 mL, 0.66 M in DMF, 33 mmol, 6.6 equiv.).Stir for 5 minutes at around room temperature, 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 hours. Wash the resin with DMF (5 x 140 mL, 7 vols.), more DMF (2 x 180 mL, 9 vols.), MTBE (2 x 180 mL, 9 vols.), and then DMF (2 x 180 mL, 9 vols.).

[0053] Conduct the remainder of the chain assembly with standard Fmoc chemistry sequentially for the remaining amino acids. In general, dissolve Fmoc-amino acid (2.0 equiv.), HOBt (3.42 g, 80 wt%, 2.0 equiv.) in DMF (85 mL), followed by the addition of DIEA (2.64 g, 2.0 equiv.). Cool the resulting solution to 0–5°C with an ice bath and activate by adding TBTU (6.45 g, 2.0 equiv.) and let stand for 3–5 min at 0–5°C. Add DCM (30 mL) to the resin followed by the addition of the activated Fmoc-amino acid solution. Stir the resulting mixture at room temperature for 2 hours. Wash the resulting resin twice with DMF, twice with MTBE, and again twice with DMF (190 mL, 10 vols. for each wash). Petition 870260063821, dated 06 / 29 / 2026, p. 36 / 253 27 / 83 Remove the Fmoc group by treating the Fmoc amino acid resin with a deprotecting solution (190 mL, 10 vols.) twice, for 10 min and 20 min, respectively. Wash the resin twice with DMF, twice with MTBE, and again twice with DMF (190 mL, 10 vols. for each wash) after draining the de-Fmoc solution.

[0054] Activate the Boc-Y-Aib-E(tBu)G-OH tetramer (12.25 g, 2.0 equiv.) in DMF (50 mL) with Oxyma (30 mL of 0.6 M in DMF, 20 mmol, 2 equiv.) and DIC (33 mL of 0.66 M, 22 mmol, 2.1 equiv.) for 5 min to add the last four amino acids as a tetramer. Add this mixture to the resin and couple for 18 hours. Drain the mixture after 18 hours and wash the resin with DMF (190 mL for 5 min each time, 5 times). Add more tetramer (6.21 g, 1.0 equiv.) to DMF (40 mL), activate with PyBOP (5.77 g, 1.1 equiv.) and DIEA (3.32 g, 2.6 equiv.) for 5 min before adding this mixture to the resin and stir for 4 hours. Drain the mixture after 4 hours, wash with DMF (190 mL, 5 min each, 5 times). 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 stir for 1 h.Wash the peptide resin after chain assembly is complete, for 5 min each time, five times with DMF (190 mL each time), six times with DCM (190 mL each time), and then dry under vacuum at 35°C, resulting in the 12-resin Preparation complex (31.03 g, 0.2595 g / mmol theoretical, 8.05 mmol, 77.8% yield). Treat a portion of the 12-resin Preparation complex (15.975 g, 0.2595 mmol / g, 4.146 mmol) three times with 1% by volume of TFA in DCM solution (160 mL, 10 vols.) for 10 min each time at room temperature, followed by washing with DCM (120 mL, 7.5 vols.). Neutralize the cleavage fractions and wash with pyridine (4.74 g, molar ratio of 0.94:1 for TFA). Combine and concentrate the fractions under vacuum until dry to < Petition 870260063821, dated 06 / 29 / 2026, page 37 / 253 28 / 83 35°C. Reconstitute with ethanol (30 mL, 5% vol. of the combined filtrates), followed by concentration until dry. Mechanically agitate the peptide in deionized water (242 mL, 40% vol. of the combined filtrates) for 10 minutes. Collect the crude solid peptide by filtration and wash with deionized water (4 x 100 mL). Wash the solid with n-heptane (4 x 100 mL), isolate and dry in vacuo at 35°C, resulting in Preparation 12 as a white powder (9.38 g, 82.2 % area, 0.2% by weight of pyridine^TFA, 82.1 % by weight, 3.85 mmol, yield of 92.8%, HRMS calc. for C103H165N13O26 expected 2000,1989, actual 2000,1968). Summary of Preparation 13 SEQ ID NO: 13

[0055] In a vial under N2, Preparation 9 (2.887 g) is added. 70.2% by weight, 1.30 mmol), Preparation 10 (3.576 g, 57.4% by weight, 2.13 mmol, 1.63 equiv.), DMSO (18.1 g, 16.4 mL), DMF (15.8 g, 16.7 mL) and DIEA (655 mg, 5.07 mmol, 3.89 equiv.) with stirring until a golden solution is obtained. The solution is cooled in ice water before PyBOP (1.414 g, 2.72 mmol, 2.08 equiv.) is added. Remove the ice bath and allow the mixture to warm to room temperature. Monitor the reaction for approximately 5 hours to ensure adequate conversion. An aliquot of diethylamine (2.116 g, 28.9 mmol, 22.2 equiv.) is added to the reaction mixture at room temperature. The mixture is stirred for about one hour to allow a conversion of approximately >99% of Preparation 13. The product is precipitated by adding a mixture at <4°C containing aqueous saturated NaHCO3 (50 ml) and deionized water (50 ml) to the reaction mixture. The mixture is stirred under chilled conditions for at least about 15 minutes. A muddy white paste is filtered out.The wet cake is washed with deionized water (3 x 50 mL), followed by MTBE (6 x 50 mL) and drying at 40 °C in vacuum with N2 purging for approximately 62 h. Petition 870260063821, dated 06 / 29 / 2026, p. 38 / 253 Process 29 / 83 results in Preparation 13 (4.45 g, 60.4 % area, 16.4 % area of ​​dibenzofulvene, 1.18 mmol, 90.5 % yield, HRMS calc. for C119H169N21O24 expected 2276.2649, actual 2276.2550) as a light yellow solid. Summary of Preparation 14 SEQ ID NO: 14

[0056] An aliquot of Preparation 11 (3.012 g, 48.7 % by weight, A 0.621 mmol, 1.00 equiv.) solution is added to a flask under N2 containing 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.). The mixture is stirred until a golden solution appears. Cool the mixture with ice water to <10°C. Add an aliquot of PyBOP (742 mg, 1.42 mmol, 2.30 equiv.) to the mixture. Remove the ice bath and allow the mixture to warm to approximately room temperature. Monitor the conversion reaction to Preparation 14 for approximately 22 hours. This yields a conversion of approximately >96%. Piperidine (530 mg, 6.22 mmol, 10.0 equiv.) is added to the chilled reaction mixture when the temperature is < 10°C. The mixture is stirred at room temperature for about 2 hours to provide a conversion of > about 99% to Preparation 14. The reaction mixture is added to another flask containing 0.5 N aqueous HCl at < 4°C (12.72 g, 6.23 mmol, 10.0 equiv.).) and deionized water (16.71 g) to allow precipitation of Preparation 14. The chilled suspension is stirred for about 15 minutes and the white suspension is filtered. The wet cake is washed with deionized water (2 x 30 mL), aqueous saturated NaHCO3 (2 x 30 mL), deionized water (3 x 30 mL) and MTBE (4 x 45 mL) and dried at 40°C in vacuo with N2 purging for about 17 h. The product, Preparation 14 (5.418 g, 48.9% area, 0.603 mmol, 97.0% yield, HRMS calc. for C231H349N35O49 expected 4397.5893, actual 4397.6057) is obtained as a white powder. Petition 870260063821, dated 06 / 29 / 2026, page 39 / 253 30 / 83 Summary of Preparation 15 SEQ ID NO: 15

[0057] An aliquot of Preparation 12 (671 mg, 82.1% by weight, Preparation 14 (2.009 g, 48.9% area, 10.7% area of ​​isomers, 0.223 mmol, 1.00 equiv.), DMSO (11.1 g, 10.0 mL), DMF (19.0 g, 20.1 mL) and DIEA (76 mg, 0.588 mmol, 2.63 equiv.) are added to the flask with stirring, resulting in a golden-colored solution. Add an aliquot of 0.6 M HOAt (619 mg, 0.384 mmol, 1.72 equiv.) before cooling to -5°C. Add a sample of PyClock (220 mg, 0.397 mmol, 1.78 equiv.). Allow the mixture to warm to approximately room temperature to permit a conversion of approximately 84% to Preparation 15. Isolate the product by adding the reaction mixture to ice-cold deionized water (548 mL) over 10 min, resulting in precipitation of the product. Rinse the reaction flask with DMF (5 mL) and add it to the suspension. The suspension is stirred for approximately 15 minutes, allowed to warm to approximately room temperature, and filtered.The moist cake is washed with deionized water (3 x 80 mL) and the waxy white solid is dried at 35°C for 3.5 days in vacuo, resulting in Preparation 15 (2.506 g, 41.6% area, 0.163 mmol, 73.1% yield, HRMS calc. for C334H512N48O74 expected 6379.7777, actual 6379.8652) as a white powder. Example 2 Summary of Example 2 SEQ ID NO: 1

[0058] A sample of TFA (19.656 g, 13.03 mL) is added to a flask under a N2 atmosphere with DCM (815 mg, 0.62 mL), DTT (434 mg), and TIPS (362 mg, 0.47 mL). Cool the mixture in ice water before adding water (468 mg, 0.47 mL). A sample of Preparation 15 Petition 870260063821, dated 06 / 29 / 2026, page 40 / 253 31 / 83 (1016 mg, 39.0% area, 0.0620 mmol) is added to this mixture at 2°C to provide a solution. Heat the mixture to approximately room temperature and stir for about 2 hours. Add the reaction mixture to MTBE at -15°C (150 mL), rinsing the reactor with MTBE (3 mL). Centrifuge the suspension after about 10 min and decant the supernatant. The wet cake is resuspended in MTBE (3 x 50 mL), centrifuging for each wash and decanting the supernatant. The moist cake is dried at 35°C in vacuum, resulting in Example 2 (784 mg, 26.5% area, 0.0432 mmol, 69.7% yield, HRMS calc. for C225H348N48O68 expected 4810.5249, actual 4810.5642) as a white solid. Summary of Preparation 16 SEQ ID NO: 16

[0059] The synthesis uses Fmoc-Gly-OH 2-chlorotrityl resin with a loading of 0.61 mmol / g. The general SPPS procedure is substantially as described herein. Preparation 16 results from gentle cleavage of the peptide in the resin, as described herein, using methods known to those skilled in the art. Reconstruction of the concentrated material is carried out with ethanol (5% vol. of the combined filtrates) and concentration to dryness. The peptide is triturated with stirring in water (40% vol. of the combined filtrates). The solid is isolated and dried under vacuum at 40°C to a constant weight to give 5.24 g (99%) of Preparation 16 as a white powder. Summary of Preparation 18 SEQ ID NO: 17

[0060] The synthesis uses Fmoc-Ala-OH 2-chlorotrityl resin with a loading of 0.50 mmol / g. The general SPPS procedure is used substantially as described in this document with the following modifications: Petition 870260063821, dated 06 / 29 / 2026, p. 41 / 253 32 / 83 Amino Acid Cycle SPPS Conditions Coupling Solvent: DMF Comments 1 (52S)-52-((((9Hfluoren-9yl)methoxy)carbonyl)amino)25-(tert-butoxycarbonyl)2,2-dimethyl-4,23,28,37,46pentaoxo-3,32,35,41,44pentaoxa-24,29,38,47tetra-azatripentacontan53-oic Acid De-Fmoc cycles of 10 and 20 min with DBU / HOBt, post-deproteinization washes 6 x 2 minutes, 1.7 AA / 3.0 HOBt / 3.0 TBTU / 3.0 DIEA 2 h, ta. Preparation 17 2 Fmoc-L-Gln(Trt)-OH De-Fmoc cycles of 10 and 20 min, post-deproteinization washes 6 x 2 minutes, 1.3.0 AA / 3.0 HOBt / 3.0 TBTU / 3.0 DIEA, 2 h, ta 2. Recoupling 1.5 AA / 1.5 HOBt / 1.5 TBTU / 1.5 DIEA, 4 h, ta Capping performed at the end using: Ac2O / Pyr mixture at room temperature 3 Fmoc-L-Ala-OH De-Fmoc cycles of 10 and 20 min, post-deproteinization washes 6 x 2 minutes, 2.0 AA / 2.0 HOBt / 2.0 TBTU / 2.0 DIEA, 2 h, ta 4 Fmoc-L-Ile-OH De-Fmoc cycles of 10 and 20 min, post-deproteinization washes. 6 x 2 minutes, 2.0 AA / 2.0 HOBt / 2.0 TBTU / 2.0 DIEA, 2 h, ta5 Fmoc-L-Lys(Boc)-OH De-Fmoc cycles of 10 and 20 min, post-deproteinization washes 6 x 2 minutes, 2.0 AA / 2.0 HOBt / 2.0 TBTU / 2.0 DIEA, 2 h, ta 6 Fmoc-L-Asp(tBu)-OH De-Fmoc cycles of 10 and 20 min, post-deproteinization washes 6 x 2 minutes, 2.0 AA / 2.0 HOBt / 2.0 TBTU / 2.0 DIEA, 2 h, ta Gentle Cleavage Preparation 18:

[0061] A 42.13 g sample of peptide in the intermediate of Petition 870260063821, dated 06 / 29 / 2026, p. 42 / 253 33 / 83 resin is placed in a flask and treated 3 times with 10 volumes (400 mL) of 1% TFA / DCM for 10 min each, followed by washing with DCM. Each treatment is quenched by the addition of 4.4 mL of pyridine. The resulting solutions are combined and concentrated in vacuo. Reconstitution is performed with ethanol (25 mL), followed by concentration to dryness to provide 56.6 g of foamy semisolid. A volume of 400 mL of water is added 10 times to provide a suspension. The suspension is filtered and washed with water. The solid is isolated and dried under vacuum at 40°C to a constant weight to yield 23.3 g of Preparation 18 as a white powder. Summary of Preparation 17

[0062] ((52S)-52-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)25-(tert-butoxycarbonyl)-2,2-dimethyl- acid 4,23,28,37,46-pentaoxo3,32,35,41,44-pentaoxa-24,29,38,47-tetra-azatripentacontan-53-oico)

[0063] Preparation 6 (80 g, 92 mmol), DIEA (17.53 m L, 101 mmol), TSTU (30.3 g, 101 mmol) and acetonitrile (1 L) are loaded into a container and stirred at 23°C for 17 h. The solution is concentrated, then the resulting orange-colored residue is redissolved in EtOAc (1.6 L), then washed with 0.1 M HCl (2 x 1 L). The organic layer is washed with water (2 x 1 L), then dried over MgSO4, filtered, and concentrated in vacuo to leave an orange-colored oil (83 g). A second batch is run on the same scale and combined to provide 123 g of crude oil. The intermediate ester (123 g, 110 g active, 113 mmol) is dissolved in EtOH (700 mL), then Fmoc-lysine (45.9 g, 125 mmol) and DIEA (21.70 mL, 125 mmol) are added and the reaction is stirred for 17 hours. After completion of the reaction, the EtOH is removed in vacuo to leave an orange oil (201 g). The residue is dissolved in EtOAc (1.1 L) and washed with 0.1 M HCl solution (3 x 400 mL), then aqueous NaHCO3 solution (400 mL).The layers are separated, and then the organic layer is washed with one. Petition 870260063821, dated 06 / 29 / 2026, page 43 / 253 34 / 83 Saturated aqueous solution of sodium chloride (1 x 400 mL). The organics are concentrated to produce an orange oil (~190 g). Acetone (400 mL) is added, then the resulting suspension is filtered to remove the inorganics. The mixture is concentrated, then purified by normal phase chromatography (1.1 kg of silica prepared with heptane / acetone at 60 / 40) and eluted with increasing eluent polarity (collecting fractions of ~3 L). Fractions of at least 95% HPLC area are combined and concentrated to provide thick yellow oil (70 g) of Preparation P17. Summary of Preparation 19A SEQ ID NO: 18

[0064] The synthesis uses Fmoc-Leu-OH 2-chlorotrityl resin with a loading of 0.65 mmol / g. The general SPPS procedure is used with the following modifications: Amino Acid Cycle SPPS Conditions Solvent for couplings: DMF 1 Fmoc-Aib-OH 4 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 8 h, ta. 2 Fmoc-L-Ile-OH 4 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 18 h, ta. 3 Fmoc-L-Ser(tBu)-OH 3 x 30 minute cycles of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 4. Fmoc-L-Tyr(tBu)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 5. Fmoc-L-Asp(tBu)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. 6. Fmoc-L-Ser(tBu)-OH: 3 x 30-minute cycles of De-Fmoc, post-deproteinization washes: 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma, 4 h, ta. Petition 870260063821, dated 06 / 29 / 2026, p. 44 / 253 35 / 83 7 Fmoc-L-Thr(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 8 Fmoc-L-Phe-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 9 Fmoc-Gly-Thr(Me, MePro)-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 18 h, ta. 10 Fmoc-L-Glu(tBu)-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 8 h, ta. 11 Fmoc-Aib-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 18 h, ta. 12 Boc-L-Tyr(tBu)-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 18 h, ta.

[0065] Pseudoproline-derived Preparation 19A can be processed in Example 3 in a manner analogous to Preparation 19B, as described in this document. Summary of Preparation 19B SEQ ID NO: 19

[0066] The synthesis uses Fmoc-Leu-OH 2-chlorotrityl resin with a loading of 0.65 mmol / g. Preparation 19B is prepared using the SPPS procedure substantially as described in this document. Amino Acid Cycle SPPS Conditions Solvent for couplings: DMF Comments 1 Fmoc-Aib-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 8 h, ta. Petition 870260063821, dated 06 / 29 / 2026, p. 45 / 253 36 / 83 2 Fmoc-L-Ile-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 18 h, ta. 3 Fmoc-LSer(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 4 Fmoc-LTyr(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 5 Fmoc-LAsp(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 6 Fmoc-LSer(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 7 Fmoc-LThr(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta. 8 Fmoc-L-Phe-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes 6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta 9 Fmoc-LThr(tBu)-OH cycles of 3 x 30 minutes of De-Fmoc, post-deproteinization washes.6 x 2 minutes, 3.0 AA / 3.3 DIC / 3.0 Oxyma 4 h, ta 10 Boc-Tyr(tBu)- Aib-Glu(tBu)- Gly-OH cycles of 4 x 30 minutes of De-Fmoc, post-deproteinization washes. 6 x 2 minutes, 1.2.0 AA / 2.2 DIC / 2.0 Oxyma 6 h, ta. 2. coupling, 1.0 AA / 1.1 PyBOP / 2.5 DIPEA, 4 h, ta Capping performed at the end using: Ac2O / Pyr mixture. Gentle Cleavage Preparation 19B:

[0067] Preparation 19B is prepared using gentle cleavage of 19B bonded to the resin substantially as described in this document using methods known to those skilled in the art. See, for example, Preparation method 18. The solid Petition 870260063821, dated 06 / 29 / 2026, p. 46 / 253 The resulting 37 / 83 is isolated and dried under vacuum at 30-40°C until a constant weight is achieved to yield 2.94 g of the product as a light yellow powder. Summary of Preparation 20 SEQ ID NO: 20

[0068] 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) is added to PyBOP (2.60 g, 5.00 mmol) and DIEA (1.75 mL, 10.0 mmol) at room temperature. The solution is stirred for 18 hours and then quenched by the addition of an excess of diethylamine (10.0 mL). The quenched solution is stirred for 2 hours and then slowly added to a saturated aqueous solution of sodium bicarbonate / water (1:1, 300 mL) at 0°C. The resulting precipitate is stirred for 10 minutes and then collected by filtration. The filtrate is washed successively with water (3 x 150 mL), followed by methyl tert-butyl ether (3 x 150 mL). The solid is dried under vacuum at 40°C to give Preparation 20 as a white solid (5.30 g, 69% yield, HRMS calc. for C119H169N21O24 expected 2276.2649, actual 2276.2652). Summary of Preparation 21 SEQ ID NO: 21

[0069] 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) is added to PyBOP (314 mg, 0.30 mmol) and DIEA (0.21 mL, 1.20 mmol) at room temperature. The solution is stirred for 18 hours and then quenched with piperidine (0.79 mL, 4.00 mmol). The quenched solution is stirred for 2 hours and then cooled to 0 °C and quenched with a dilute HCl solution (50 mL). The resulting suspension is stirred for 10 minutes and the solid is collected by filtration. The filtrate is washed successively with saturated aqueous sodium bicarbonate solution (2 x 50 mL), water (3 x 50 mL), followed by methyl tert-butyl ether (3 x 50 mL). The solid is dried under vacuum at 40 °C for 18 hours. Petition 870260063821, dated 06 / 29 / 2026, page 47 / 253 38 / 83 to provide Preparation 21 as a white solid (1.80 g, 106% yield, HRMS calc. for C225H338N34O48 expected 4284.5053, actual 4284.5062). Summary of Preparation 22 SEQ ID NO: 22

[0070] 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) is added to PyBOP (57 mg, 0.11 mmol) and DIEA (58 μL, 0.33 mmol) at room temperature. The solution is stirred for 18 hours and then quenched with a 1:1 mixture of saturated aqueous sodium bicarbonate and water (10 mL). The resulting suspension is stirred for 10 minutes and the resulting solid is collected by filtration. The solid is washed with water (3 x 10 mL) and the solid is dried under vacuum at 40°C to provide Preparation 22 (285 mg, 89% yield, HRMS calc. for C334H512N48O74 expected 6379.777, actual 6379.7730). Example 3 Summary of Example 3 SEQ ID NO: 1

[0071] A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (0.1 mL) and DTT (75 mg) is cooled to 0°C, added to the solution of Preparation 22 (100 mg, 0.015 mmol) and the reaction mixture is allowed to warm to room temperature and stirred for 2 hours. The resulting mixture is poured into a pre-cooled (20°C) solution of methyl tert-butyl ether (25 mL). The resulting precipitate is held for 15 minutes at -20°C and the suspension is centrifuged and washed with methyl tert-butyl ether (2 x 25 mL). The solid is dried under vacuum at 35°C for 18 hours to obtain Example 3 as a white solid (71 mg, 93% yield, HRMS calc. for C225H348N46O68 expected 4810.5249, actual 4810.5036). Step 1 Petition 870260063821, dated 06 / 29 / 2026, p. 48 / 253 39 / 83

[0072] Step 1: A stream solution of Preparation 25 (1.05 equiv.) is prepared in 5 vols. of DMSO / ACN (90:10 v / v). A second stream solution of Preparation 26 is prepared in 20 vols. of DMSO / ACN (90:10 v / v). A third stream solution is prepared of PyOxim (1.5 equiv.) in 3 vols. of ACN. A fourth stream of DIEA (4 equiv.) in ACN is prepared. The first three streams are pumped into a mixer and, at the mixer outlet, the DIEA is combined and the mixture is pumped through another mixer and through a plug flow reactor for a residence time of 2 h in a constant temperature bath of 20°C. At the reactor outlet, acetic acid can be added to consume the remaining PyOxim (1-cyano-2-ethoxy-2-oxoethylideneaminooxytris-pyrrolidinephosphonium hexafluorophosphate). After > 2 h, pure diethylamine (10 equiv.) is added and mixed using a mixer.This stream progressed to a second plug flow reactor with a residence time of 1 h in a constant temperature bath of 20°C. The product solution from Preparation 27 is collected and sent via nanofiltration with a 70 / 30 DMSO / ACN solution for reagent removal to 10-20 diavolumes.

[0073] A current solution of Preparation 25, 2.40 kg, 96.7% by weight, 2.274 mol) is prepared by dissolving the solid in DMSO (13.88 kg, 12.62 L) and diluting the solution with ACN (1.09 kg, 1.39 L), creating a solution of Preparation 25 (114.3 mg / mL, 0.112 M) in DMSO:ACN at 90:10 v / v. A second running solution of Preparation 26 (SEQ ID NO: 26, 2.79 kg, 98.6% by weight, 1.836 mol) is prepared by dissolving the solid in DMSO (54.8 kg, 49.8 L) and diluting with ACN (4.3 kg, 5.47 L), creating a solution of Preparation 26 (45.5 mg / mL, 0.030 M) in DMSO:ACN at 90:10 v / v. A third running solution is prepared of PyOxim (4.5 kg, 8.53 mol) in ACN (47.33 kg, 60.22 L) creating a 0.132 M solution. DIEA is added as a pure liquid. The solution Petition 870260063821, dated 06 / 29 / 2026, page 49 / 253 40 / 83 of Preparation 25 (14.91 L, 1.704 kg, 1.670 mol, 0.95 equiv., 5.9 g / min) and Preparation 26 (58.46 L, 2.660 kg, 1.750 mol, 1.00 equiv., 22.5 g / min) and PyOxim (1.4 equiv., 5.4 g / min) is pumped into a mixer combined with pure DIEA (4.0 equiv., 0.446 mL / min) at 20°C. The mixture is pumped through another mixer and through a plug flow reactor for a residence time of 3 h in a bath at 20°C and collected over 42.9 h, resulting in 88.6 kg of product solution.

[0074] Nanofiltration is a membrane-based filtration process used to separate chemical species based on their differences in size and molecular weight. The product solution of Preparation 27 contains reagents (diethylamine, PyOxim, DIEA, etc.) and unwanted byproducts (e.g., dibenzofulvene) that it is desirable to remove before proceeding to the next step. Nanofiltration is applied to remove unwanted species (molecular weight < 500 Da).

[0075] The product solution from Preparation 27 is loaded into an NF stream tank and pumped through a recirculation circuit via a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymeric) to cause the desired separation. Undesirable species are removed from the permeate and collected separately or discarded. In order to maintain a constant volume in the NF tank, fresh solvent, i.e., DMSO / ACN at 70:30 v / v, is continuously pumped to match the rate of permeate extracted. The product solution from Preparation 27 is purified by nanofiltration and transported directly to Step 2.

[0076] The solution of Preparation 27 protected with Fmoc in DMSO / ACN (88.6 kg) and diethylamine (1.34 kg) are added to a reactor. The mixture is stirred at 20°C for 2 h, resulting in Preparation 27. Petition 870260063821, dated 06 / 29 / 2026, page 50 / 253 41 / 83 (87.6 L, 38.45 mg / mL, 3.37 kg, 1.48 mol). The product solution from Preparation 27 is loaded into a nanofiltration stream tank and then pumped in a recirculation circuit through a heat exchanger and a nanofiltration unit containing a suitable membrane (ceramic or polymeric) to cause the desired separation. Undesirable species are removed on the permeate side and collected separately or discarded. The operation is continued until sufficient removal of unwanted impurities is achieved. In order to maintain a constant volume in the nanofiltration tank, fresh 70:30 v / v DMSO / ACN is continuously pumped in to match the rate of permeate extracted. This results in Preparation 27 in DMSO / ACN (72.4 L, 40.8 mg / mL, 2.95 kg, 1.30 mol, 78.1% yield through coupling, deFmoc and nanofiltration). Step 1: Example - Analytical Results.

[0077] HPLC confirms the conversion of Preparation 25 and Preparation 27 to Preparation 27. The analytical method uses a phenylhexyl stationary phase column at 65°C (2.1 mm id x 150 mm x 1.7 micron particle size) with a gradient of 2-98% B in TFA to 0.1% in water and acetonitrile over 12 minutes. UV detection at 214 nm is used for this material.

[0078] Table A.1 shows high-resolution mass spectrometry data collected for the coupling reaction product of Step 1 (Preparation 27 protected with Fmoc) and the deprotection reaction product of Step 1 (Preparation 27). Mass precision is the metric used to confirm the correspondence of the measured species with the predicted species. Petition 870260063821, dated 06 / 29 / 2026, page 51 / 253 42 / 83 Compounds Chemical Formula Theoretical Monoisotropic Mass (neutral) Observed Ions m / z Charge State Calculated Monoisotropic Mass (neutral) Mass Precision (ppm) Preparation - tion 27 Protected with Fmoc C134H179N21O26 2498.33 3 2499.340 2 1 2498.33 29 0.05 Preparation - tion 27 C119H169N21O24 2276.26 49 2277.274 3 1 2276.26 7 0.9 Table A.1 Confirmation of Preparation 27 and Fmoc-protected Preparation 27 measured by mass accuracy calculated using high-resolution mass spectrometry data. Step 2 SEQ ID NO:28 SEQ ID NO:27 SEQ ID NO:29 Scheme A.2 Synthesis of Preparation 29 (SEQ ID NO: 29) from fragments of Preparation 27 (SEQ ID NO: 27) and Preparation 28 (SEQ ID NO: 28). Petition 870260063821, dated 06 / 29 / 2026, page 52 / 253 43 / 83 Step 2 Chemistry Step 2 hFVQ WLlA GGPSSGA -PPPS-NHs frt áoc cBucBu iBu Preparafão 27

[0079] Step 2: A stream solution of Preparation 28 (1.15 equiv.) is prepared in 10 vols. of DMSO / ACN (90:10 v / v). A second stream solution is prepared of PyOxim (2 equiv.) in 1 vol. of ACN. A third stream of DIEA (3 equiv.) in ACN is prepared (5% w / w solution). The solution of Preparation 27 from Step 1 and the streams of Preparation 28 and PyOxim are pumped into a mixer and, at the mixer outlet, DIEA is combined and the mixture is pumped through another mixer and through a plug flow reactor for a residence time of 2 h in a constant temperature bath of 20°C. At the reactor outlet, acetic acid may be added to consume residual PyOxim. After > 2 h, pure diethylamine (10 equiv.) is added and mixed through a mixer. This stream progresses to a second plug flow reactor with a residence time of 1 h in a bath with a constant temperature of 20 °C.The product solution from Preparation 29 is collected and sent through nanofiltration with DMF solution as a diafilter to remove reagents to 10-20 diavolumes.

[0080] A stream solution of Preparation 28 (4.68 kg, 98.9% by weight, 2.058 mol) is 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), creating a Petition 870260063821, dated 06 / 29 / 2026, page 53 / 253 44 / 83 solution of Preparation 28 (94.7 mg / mL, 0.0421 M) in DMSO:ACN at 90:10 v / v. A second stream solution is prepared of PyOxim (3.0 kg, 5.69 mol) in ACN (11.81 kg, 15.03 L) creating a 0.327 M solution. DIEA is added as a pure liquid. The solution streams from Preparation 27 of Step 1 (73.86 L, 41.2 mg / mL, 3.04 kg, 1.336 mol, 0.0181 M, 1.0 equiv., 29.9 g / min) and Preparation 28 (1.3 equiv., 17.7 g / min) and PyOxim (2.1 equiv., 2.9 g / min) are pumped into a mixer and, at the mixer outlet, the stream is adjusted to 20 °C and combined with pure DIEA (4.0 equiv., 0.374 mL / min) at 20 °C. The mixture is pumped through another mixer and through a plug flow reactor for a residence time of 3 h in a bath at a temperature of 20°C and collected over 43.2 h, resulting in 129.35 kg of solution of the product from Preparation 29.

[0081] A nanofiltration process substantially as described in the present document above uses the solution resulting from Preparation 29 instead of the solution resulting from Preparation 27.

[0082] A nanofiltration process that uses a solution of Preparation 29 protected by Fmoc in DMSO / ACN is conducted substantially as described in this document. Preparation 29 protected with Fmoc (129.35 kg) and diethylamine (2.0 kg) are added to a reactor for nanofiltration. The nanofiltration process results in Preparation 29 in DMF (98.85 g, 42.24 mg / ml, 4.18 kg, 0.974 mol, yield 73.1% between coupling, Fmoc and nanofiltration).

[0083] HPLC confirms the synthesis of Preparation 29 from Preparation 28 and Preparation 27. The analytical method uses a C4 stationary phase column at 65 °C (2.1 mm id x 150 mm x 1.7 micron particle size) with a gradient of 25-98% B in TFA to 0.1% in water and acetonitrile over 12 minutes. UV detection at 214 nm is used for this material. Petition 870260063821, dated 06 / 29 / 2026, page 54 / 253 45 / 83

[0084] Table A.3 shows high-resolution mass spectrometry data collected for the coupling reaction product of Step 2 (Preparation 29 protected with Fmoc) and the deprotection reaction product of Step 2 (Preparation 29). The mass accuracy confirms the measured species product. Monoisotopic mass of the neutral species. Compound Chemical Formula Theoretical Monoisotropic Mass (neutral) Observed Ions m / z Charge State Calculated Monoisotropic Mass (neutral) Mass Precision (PPm) Preparation 29 Protected with Fmoc C240H348 N34O50 4506.573 4 2254.29 4 2 4506.573 5 0.0 2 Preparation 29 C225H338 N34O48 4284.505 3 2143.25 96 2 4284.504 6 0.17 0085] Table A.3 Confirmation of Preparation 29 and Preparation 29 protected by Fmoc measured through mass accuracy calculated using high-resolution mass spectrometry data. Step 3 Scheme A.3 Synthesis of Preparation 31 (SEQ ID NO: 31) from fragments of Preparation 30 (SEQ ID NO: 30) and Preparation 29 (SEQ ID NO: 29). h 5? H 9 Boc-Y-3 EGTFT - SDY - 5—I —N L-OH tSU tfiL íBll ϊΒυϊΒυ tfiL tEL tBU .XZ\. W Mi Mi Preparafão 30

[0086] Step 3. Description of the Batch Process: A Petition 870260063821, dated 06 / 29 / 2026, p. 55 / 253 46 / 83 Nanofiltered solution in DMF 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) at -5°C is added a 5.0% by weight DIEA solution in DMF (114.0 mg, 5.70 mg DIEA, 0.0441 mmol, 1.8 equivalents) and a 10.1% by weight solution of 3-oxide hexafluorophosphate of (1-[bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium (HATU) in DMF (154.9 mg, 15.59 mg HATU, 0.0410 mmol, 1.7 equivalents). The solution is stirred for 4 hours at -5°C and then quenched with 5% by weight aqueous sodium bicarbonate (5.295 g, 4.8 mL) at room temperature, which is added over 15 minutes. The resulting suspension is stirred at 0°C for 15 minutes and the resulting solid is collected by filtration. The solid is washed with water (4 x 2 mL) and then washed with MTBE (4 x 2 mL).The gummy solid is dried under vacuum at 35°C to generate Preparation 31 (219.7 mg, 47.6% surface area, 0.0164 mmol, 67.1% yield).

[0087] A stream solution of Preparation 30 is prepared in DMF vols. A second stream solution is prepared from HATU (1.8 equiv.) with a 10 wt% ACN solution. A third stream of DIEA (2.5 equiv.) in DMF is prepared (5 wt% solution). The solution of Preparation 29 from Step 2 and the streams of Preparation 30 and DIEA are pumped into a mixer and, at the mixer outlet, the stream is cooled and combined with chilled HATU solution. The mixture is pumped through another mixer and through a plug flow reactor for a residence time of 3 h in a constant temperature bath of -5°C and collected. A brine / bicarbonate solution (aqueous solution, 17% by weight aqueous sodium chloride, 0.5% by weight aqueous sodium bicarbonate) with a salt load of 19% by weight is prepared. The resulting solution from Preparation 31 in DMF is then pumped to a Petition 870260063821, dated 06 / 29 / 2026, page 56 / 253 47 / 83 Mixed suspension mixed products reactor (MSMPR) together with the saline solution to form a precipitate. The tau in the mixed suspension mixed products reactor is 1 hour. The second MSMPR is run colder for a tau of 1 hour and this suspension is fed to a filter intermittently. The suspension is washed with water and dried under vacuum at 35°C.

[0088] A stream of solution of Preparation 30 (9.42 kg, 83.6% by weight, 3.72 mol) is prepared in DMF (54.12 kg), creating a second stream of solution at 0.0563 M. DIEA is prepared from HATU (1.15 kg, 3.02 mol) in ACN (10.4 kg), creating a stream at 0.215 M. DIEA is added as a pure liquid. The solution streams from Preparation 29 of Step 2 (98.85 L, 42.24 mg / mL, 4.18 kg, 0.975 mol, 0.0099 M, 1.0 equiv., 40.2 g / min) and Preparation 30 (1.3 equiv., 9.2 g / min) and DIEA (2.1 equiv., 0.152 mL / min) are pumped into a mixer. At the mixer outlet, the stream is cooled to 0°C and combined with chilled HATU solution (2.0 equiv., 3.2 g / min) at 0°C. The mixture is pumped through another mixer and through a plug flow reactor for a residence time of 4 hours in a 0°C water bath and collected over 42 hours, resulting in 131.8 kg of product solution. The product solution is precipitated in two sections. One is a 17% by weight aqueous NaCl solution.0.5 wt% of aqueous NaHCO3 (29 kg) is combined with DMF (13.2 kg) in an inert reactor and cooled to no more than 20°C. The product solution in DMF (66.6 kg) is then co-added with 17 wt% of aqueous NaCl / 0.5 wt% of aqueous NaHCO3 (34.4 kg) to the reactor over 1 h, maintaining 20°C, resulting in the precipitation of the product, Preparation 31. The suspension is cooled to 5°C over 1 h before water (32.2 kg total) is added in two portions. The 5°C suspension is stirred for 0.5 h before the suspension is filtered. The wet cake is resuspended in water (63.9 kg) for 0.5 h and... Petition 870260063821, dated 06 / 29 / 2026, p. 57 / 253 48 / 83 filtered. The second section of the product solution is precipitated in a comparable manner. A solution of 17 wt% aq. NaCl / 0.5 wt% aq. NaHCO3 (29 kg) and DMF (13.55 kg) are combined in a reactor and cooled to no more than 20 °C. The second section of the product solution in DMF (65.2 kg) is co-added to 17 wt. NaCl / 0.5 wt. NaHCO3 (36.8 kg) in the reactor over 1 h, maintaining 20 °C, resulting in the precipitation of the product, Preparation 31. The suspension is cooled to 5 °C over 1.25 h before water (32.1 kg total) is added in two portions. The suspension is stirred for 0.5 h before being filtered over the first wet cake. The combined wet cake is resuspended with water twice (64 kg each time) for 0.5 h each time and filtered. This is followed by two displacement washes with water (64 kg each). The combined washed wet cake is purged with N2 and then dried under vacuum at 38°C until KFto be < 4% by weight, resulting in Preparation 31 (10.34 kg, assumed potency of 60%, 0.972 mol, assumed yield of 100%). Example 3 Tirzepatide (SEQ ID NO: 1) H OHO ®«OíCNQΆ·17O COjiBu oC L Preparation 311H0H0Xew-Y-HILE-GTFTSQYSIn ULDKlA QN^pA-FV QW LlAGGP-$-$-GAPPPS-nhi(SliuA. Jôu !&1 tBuíBu]&ui&Uffiu Λ ΛίΙ (Ut + <l O +rt έdc tButBu rSu Me Me Me Me

[0089] A solution of TFA (2.3 mL), water (0.1 mL), triisopropylsilane (TIPS, 0.1 mL) and dithiothreitol (DTT, 75 mg) is cooled to 0°C. Add to the solution of Preparation 31 (100 mg, 0.015 mmol) and the Petition 870260063821, dated 06 / 29 / 2026, p. 58 / 253 49 / 83 The reaction mixture is allowed to warm to room temperature and stirred for 2 hours. The resulting mixture is poured into a pre-cooled (-20°C) MTBE solution (25 mL). The resulting precipitate is held for 15 minutes at -20°C and the suspension is centrifuged and washed with MTBE (2 x 25 mL). The solid is dried under vacuum at 35°C for 18 hours to obtain Example 3 as a white solid (71 mg, 93% yield, HRMS calc. for C225H348N46O68 expected 4810.5249, actual 4810.5036).

[0090] DCM (27.3 kg, 20.6 is added to an inert reactor at 15°C L), water (4.1 kg), Preparation 31 (10.34 kg, assumed potency of 60%, 0.972 mol) and DTT (3.10 kg). In a separate inert reactor, TFA (154.1 kg, 103.4 L) and TIPS (3.2 kg, 4.2 L) are added. The TFA / TIPS solution is added to the suspension of Preparation 31, DCM, water and DTT over 0.25 h, forming a colorless solution, and heated and maintained at 20°C for 3 h. After 3 h at 20°C, the reactor is cooled to -10°C. In a separate reactor, MTBE (382.4 kg, 516.8 L) is added, which is cooled to -20°C. A portion of this chilled MTBE (91.8 kg, 124.1 L) is added to the chilled reaction solution over 2 h, maintaining it at -5°C to -18°C. The remaining chilled MTBE (294.3 kg, 397.7 L) is added over 1.5 h, maintaining it at -5°C to -18°C, resulting in the precipitation of Example 3. The suspension is adjusted to 0°C and maintained for > 0.5 h before being filtered in three sections.The combined wet cake is resuspended with MTBE (114.7 kg, 155 L) twice and filtered before a final displacement wash with MTBE (114.7 kg, 155 L). The wet cake is dried at 28°C until < 4.5 wt% of MTBE is measured. This resulted in Example 3 (7.77 kg, 46.8 wt%, 0.755 mol, 77.7% yield). Example 4A (Linear SPPS) Tirzepatide (SEQ ID NO: 1) Petition 870260063821, dated 06 / 29 / 2026, p. 59 / 253 50 / 83 FmocHN Sieber resin Preparation 24 Depsi peptides, not shown, are present at all Serine and Threonine residues. *8 Depsi Peptides Serine Example 4A

[0091] Sieber Fmoc resin (17 kg, 0.76 mmol / g) is loaded into a reactor. The resin is swollen with DMF, stirred for 2 hours, and then the DMF is removed by filtration of the resin. The resin is then washed with DMF a total of two times. The Fmoc-protected resin is then deprotected using treatments with Pip at 20% / MPN. Sampling to verify Fmoc removal is performed after the last PIP / MPN treatment to confirm >99% Fmoc removal by UV analysis (target IPC < 1%). Petition 870260063821, dated 06 / 29 / 2026, page 60 / 253 51 / 83 of remaining Fmoc). After final treatment with 20% w / w / NMP PIP, the resin bed is washed several times with DMF. The peptide backbone is then constructed using the following general conditions for each amino acid coupling and deprotection: Process Step Solvent / Reagent Volume Equivalence Deprotection of Fmoc Piperidine 20% (v / v) / NMP 9 ml / g resin Post-deprotection washes DMF 9 ml / g resin Coupling reaction solution NMP 7.25 ml / g resin Amino acid 3.0 equiv. Oxyma Pure 3.0 equiv. DIC 3.3 equiv. Post-coupling washes DMF 9 ml / g resin Removal of ivDde Hydrazine 8% / DMF 9 ml / g resin Post-ivDde removal washes DMF 9 ml / g resin Post-swelling washes IPA 1.8 mL / g resin

[0092] Fmoc Deprotection: The resin in the peptide reactor is treated with three or four charges of 20% v / v / MPN PIP solution. Each treatment is stirred into the resin for 30 min, followed by filtration to complete the removal of the Fmoc protective group. After the final treatment with 20% v / v / MPN PIP, the resin bed is washed a minimum of six times with DMF at the pre-specified charge and volume.

[0093] Amino Acid Activation: A pre-prepared solution of Oxyma Pure / NMP at 12% w / w is charged into a reactor. The selected amino acid Fmoc is then added. The mixture is stirred at 20 ± 5 °C until the Fmoc amino acid is completely dissolved. The Fmoc-AA / Oxyma Pure / NMP solutions are then cooled to 15 ± 3 °C before Petition 870260063821, dated 06 / 29 / 2026, page 61 / 253 52 / 83 of the activation to ensure that the secondary exothermic activation reaction is controlled and the temperature of the resulting solution is maintained within the specified range of 20 ± 5°C. The amino acid solution is then activated by the addition of DIC. The activated ester solution is then stirred for 20-30 min before transferring the solution to the reactor containing the peptide over the resin intermediate.

[0094] Coupling: After completion of the pre-activation step, the activated ester solution is transferred to the reactor containing the unprotected peptide on the resin to initiate the coupling reaction. The peptide coupling reaction is stirred at 20 ± 5°C for at least 4 hours. After the required stirring time, the resin suspension is sampled for coupling completion (IPC). Sampling is repeated at specific intervals as needed until an approved IPC result is obtained. Recoupling operations are performed if necessary. When coupling is complete, the contents of the peptide reactor solution are filtered, then the peptide on the resin intermediate is washed several times with DMF to prepare for the next coupling.

[0095] Ile(12) to Aib(13) Coupling: The coupling of FmocIle(12) to Aib(13) is performed using a symmetric anhydride approach that uses six equivalents of Fmoc-AA, three equivalents of DIC. The activation time is extended to 40-60 min for this sequence to ensure the formation of the activated symmetric anhydride species. An extended coupling stirring time (18 h) is required to achieve reaction completion (< 1 % uncoupled) as determined by HPLC analysis.

[0096] Deprotection of Lys(20)ivDe (Preparation 23): A selective deprotection of the Lys(20)ivDde group from the 39 fully protected amino acids in the peptide backbone on Boc resin. Petition 870260063821, dated 06 / 29 / 2026, page 62 / 253 53 / 83 The Tyr(1)-Ser(39) reaction is performed. Deprotection is achieved using 8% w / w hydrazine hydrate in DMF solution with stirring for 4 h at room temperature. The deprotection reaction is monitored by HPLC aiming for an IPC limit of < 1% of the remaining Lys(ivDde) ​​component after deprotection. The resulting peptide fragment (Preparation 23) is repeatedly washed (8x) with DMF to completely remove residual hydrazine. The fully constructed Preparation 23 fragment is washed four times with IPA and then dried at < 40°C until a LOD of < 1% is achieved. Preparation 23 is packaged and stored chilled (-20°C) before coupling with Preparation 6.

[0097] Coupling of Preparation 6 to Preparation 23: Preparation (1.5 equiv.) and PyBOP (1.5 equiv.) solids are loaded into a reactor, followed by DMF, and the mixture is stirred until dissolution occurs. Colidine is then loaded to initiate the formation of the active ester species. The activated ester solution is stirred for 60 min before being transferred to the reactor containing the intermediate from Preparation 23. The reaction suspension is stirred for 18 ha at 25°C. The suspension is sampled for coupling completion (IPC), and sampling is repeated, if necessary, at specific intervals as needed to obtain IPC-approved results (< 1% Preparation 23). When coupling is complete, the solution contents are filtered and discarded. The fully constructed Preparation 24 intermediate is washed several times with DMF and then IPA. Preparation 24 is dried at < 40°C until a LOD < 1% is achieved. Preparation 24 is packaged and stored chilled (-20°C) before resin cleaving.

[0098] Resin Cleavage and Isolation of Example 4A (Raw): A cleavage cocktail is prepared consisting of trifluoroacetic acid. Petition 870260063821, dated 06 / 29 / 2026, page 63 / 253 54 / 83 (TFA), triisopropylsilane (TIPS), dithioteritol (DTT), DCM, and water. The cleavage cocktail is cooled to 15 ± 5°C. Reagent loadings are shown in the following table: Process Step Solvent / Reagent Volume (per Loaded Bonded Resin) Cleavage Cocktail TFA 7.16 mL / g Water 0.34 mL / g TIPS 0.24 mL / g DTT 0.24 g / g DCM 0.75 mL / g Liquid Cocktail Loading n / d ~ 8.50 mL / g Washing Consumed Resin DCM 3 mL / g Antisolvent MTBE 14 g / g Washing Container and Cake MTBE 3 g / g

[0099] Preparation 24 is charged to a reactor, followed by the cleavage cocktail. The mixture is stirred and maintained at 23°C for 3 h. The mixture is filtered, then the consumed resin is washed with DCM. The DCM wash filtrate is combined with the bulk deprotection solution and the contents are cooled to < -10°C. MTBE is cooled to < -13°C. Then, chilled MTBE is fed to the chilled filtrate in two portions. The MTBE feed rate is controlled to maintain the internal temperature of the raw solution at < 5°C. The initial MTBE charge constituted ~ 45% of the total MTBE charge. A soft precipitate forms near the end of the MTBE addition, but is readily redissolved in the solution. The precipitate solution is then cooled again to an internal temperature of -15 ± 5°C. The second addition of MTBE is fed at a rate of approximately 5-10 times the initial MTBE feed rate and constituted ~55% of the total MTBE loading.The internal temperature of the precipitation suspension is maintained at < 0°C. Petition 870260063821, dated 06 / 29 / 2026, page 64 / 253 55 / 83 during addition. The resulting suspension is aged at -8 ± 3°C for a minimum of 6 h, then heated to 0 ± 3°C and aged for a further 2 h before insulation.

[00100] The chilled crude peptide suspension is filtered, then the resulting wet cake is washed with MTBE. The crude wet cake of Example 4A is then dried to a target LOD value by IPC of < 1%. The crude product, Example 4A, is packaged and stored. The crude intermediate is stored chilled (-20 °C) until purification. Overall, 45.39 kg of crude Example 4A are produced at 45% by weight and a purity of 64% of the area by HPLC. Content yield based on Sieber resin = 47%. Purification of Example 4A: Moving phases: Mobile Phase A (MPA) % water, TFA at 0.1% and 10% ACN Mobile Phase B (MPB) % water, TFA at 0.1% and 90% ACN

[00101] Reverse Phase Purification 1 (RP1): Crude Example 4A is dissolved in 90 wt% MPA and 10 wt% MPB. The solution is stirred for at least 7 h to complete the tryptophan decarboxylation. The aged crude solution is filtered and loaded onto a pre-equilibrated packed Kromasil 100-10-C8 column. The column is washed with a mixture of buffers A (90% water, 0.1% TFA and 10% ACN) and B (10% water, 0.1% TFA and 90% ACN) resulting in 30% ACN for two column volumes and prepared for elution by increasing the mixed ACN concentration from 30% to 35% in one column volume. Tirezepatide is eluted from the column using an ACN increase of 1.5% by column volume until elution is complete. The eluent is fractionated and tested for purity using RP-HPLC. The column is regenerated by increasing the ACN from 47% to 65% by a Petition 870260063821, dated 06 / 29 / 2026, page 65 / 253 56 / 83 column volume and continuing to flow 65% ACN for three column volumes. The column is rebalanced using 30% ACN for two column volumes before the next injection sequence.

[00102] Fractions that qualify for inclusion in the mainstream are pooled. Fractions that do not meet the purity criteria but have a purity greater than 50% may be pooled for recycling injections after all primary injections are completed. Recycling fractions are separated into forward and backward fractions, the fractions diluted with buffer A and stored chilled. Recycling injections are processed and pooled using the primary injection criteria; however, only the main peak fractions are forwarded and no further recycling is performed. After completion of the main pooling, the intermediate is tested for concentration and purity. The material is diluted and the pH adjusted to a pH of 8 before RP2 processing. RP1 processing yields 37 kg of crude product and 14277 g of contained product with an average pooling purity of 90.9%.

[00103] Reverse Phase Purification 2: The RP1 solution from Example 4 is loaded onto a pre-equilibrated packed Kromasil 100-10-C8 column. The column is washed with a mixture of Buffer C (90% aqueous NH4OAc, pH 8.0, 10% ACN) and Buffer D (10% aqueous NH4OAc, pH 8.0, 90% ACN) resulting in a 20% ACN solution for two column volumes. Tirzepatide is eluted from the column using a 3.5% ACN increase per column until elution is complete. The eluent is fractionated and tested for purity using RP-HPLC. After elution, the column is regenerated by increasing the ACN to 80% in one column volume and continuing to flow 80% ACN for three column volumes. The column is rebalanced using 20% ​​ACN for two column volumes before the next injection sequence. Petition 870260063821, dated 06 / 29 / 2026, page 66 / 253 57 / 83

[00104] Fractions that qualify for inclusion in the mainstream are pooled. Fractions that do not meet the purity criteria but have a purity greater than 60% may be pooled for recycling injections after all primary injections are completed. Recycling fractions are separated into forward and backward fractions, diluted with buffer C, and stored chilled. Recycling injections are processed and pooled using the primary injection criteria; however, only the main peak fractions will be forwarded, and no further recycling will be performed. After completion of the main pooling, the intermediate is tested for concentration and purity. The material may have its pH adjusted to 8.0 in preparation for the TFF step. RP2 processing, starting with 14.2 kg, yields 10.9 kg of contained product with a yield of 76.7%.

[00105] Ion Exchange Chromatography (IEX): The RP2 solution from Example 4A is filtered and loaded onto an Amberchrom GC 300M column. Two fractions are eluted using mobile phase E (10% aqueous ammonium acetate, 5% IPA, pH 8) and mobile phase F (isopropanol). The fractions are analyzed for peptide content, and those < 3 mg / mL are discarded. The pooled fractions are concentrated and stored at 20°C before precipitation. The IEX process starts with 10.9 kg of RP2 to yield 14.3 kg of the material from Example 4A with a purity of 97.8% of the pool.

[00106] Precipitation: The IEX solution from Example 4A (333 kg) is filtered, and then isopropanol (850 L) is added to reduce the water content to < 10% w / w water. The diluted solution is cooled to 0 ± 3 °C in preparation for loading and precipitation of MTBE. MTBE (2304 L, 1708 kg) is cooled to 0 ± 3 °C. The chilled MTBE is fed to the IEX solution at a rate of ~ 0.69 kg / min during the first ~ 37% of MTBE loading. The feed rate is, Petition 870260063821, dated 06 / 29 / 2026, p. 67 / 253 58 / 83 then increased to an average of ~2.3 kg / min to complete the remaining ~63% of the MTBE loading. The temperature during feeding is maintained at < 5°C. The resulting precipitation suspension is filtered ice-cold (< -10°C). Then, the filter cake is washed with MTBE. The filter cake is dried to a LOD < 2%.

[00107] Humidification of Example 4A: Example 4A is humidified by passing wet nitrogen through the dryer filter. The humidity of the gas stream exiting the filter outlet is monitored every 60 min. Humidification is continued until <0.5% MTBE and <0.2% IPA remain in the wet cake. After completion of the humidification process, the nitrogen flow is changed to a dry nitrogen flow through the pure product cake of Example 4A. The material is sampled for residual water and solvents against specific IPC targets, and drying using dry nitrogen is continued until the desired target water content of 5-7% w / w is achieved. A total of 12.9 kg of Example 4A is isolated with >95% purity in peptide content. Overall yield based on Sieber resin loading = 31%. Example 4B (SPPS of linear) Tirzepatide (SEQ ID NO: 1) Petition 870260063821, dated 06 / 29 / 2026, page 68 / 253 59 / 83 Example 4B Preparation 23

[00108] The process for producing Preparation 23 is substantially as provided by Example 4A, except that NMP is globally replaced by DMF for all couplings and deprotections. Furthermore, the amino acid:oxyma:DIC stoichiometry is reduced to 2.5:2.5:2.7 molar equivalents based on Sieber resin. The only exception related to the use of DMF is the coupling of Ile12 to Aib13, where the NMP is retained. Petition 870260063821, dated 06 / 29 / 2026, p. 69 / 253 60 / 83 In this example, 17.6 kg of Sieber resin is processed to produce 92.2 kg of peptides on the resin intermediate. Preparation 23 Preparation 24

[00109] The process substantially as provided by Example 4A prepares 92.1 kg of Preparation 23; subsequently processed to 97.3 kg of Preparation 24 of peptides on resin intermediate. Preparation 24 is packaged and stored chilled (-20 °C) before cleavage of the resin.

[00110] Resin Cleavage and Isolation of Example 4B Crude: Two batches are run in Preparation 24 on a 32 kg scale using conditions substantially as described in Example 4A to yield 24.4 kg of Example 4B, 69.5% Purity by HPLC and 52.6% yield, and 21.3 kg of Example 4B, 88.3% purity by HPLC and 45.2% yield. The crude intermediate is stored chilled (20°C) until purification.

[00111] Purification of Example 4B: Crude Dissolution: The crude Tirzepatide of Example 4B is loaded into a dissolution vessel and dissolved in a 1:1 acetonitrile:water solution to a final concentration of 25 g solids / L solution. The pH of the resulting solution is adjusted to 8.5–9.5 with ammonium hydroxide to initiate the conversion of the Depsi peptide isomers (10–15%) to the Tirzepatide of Example 4B. The pH-adjusted mixture is stirred for at least one hour to allow the Depsi conversion to occur. The pH is then adjusted to 1.5–2.5 by the addition of trifluoroacetic acid and diluted to an acetonitrile content of 30% in preparation for chromatography. In total, the crude solution is stirred for at least 7 hours to convert the TrpCO2 salt to the Tirzepatide of Example 4B. Conversion of tirzepatide (TZP) to depsipeptide: Petition 870260063821, dated 06 / 29 / 2026, page 70 / 253 61 / 83 Depsipepfideo Conversion of depsi peptide to API: Q □ H II H II R1N CH—C hr CH—C NHR I 1 CH, OH OH Triflu-acetic acid V o II O3N--CH—C NUR HEY H II / CH' Rr---N--CH—CO CH; OH Neutralization to pH >6 H II HII R--N--CH—C--N--CH—CNUR II CHjCH OHOH

[00112] Reverse Phase Purification 1 (RP1)-. The RP1 process, substantially as presented in Example 4A, can be used to convert the depsi peptide into API. The RP1 purification process is substantially the same as described in Example 4A; however, the crude dissolution step described above increases the capacity of the RP1 chromatographic step. This allows a loading of g of resin per L of Tirzepatide and decreases the number Petition 870260063821, dated 06 / 29 / 2026, p. 71 / 253 62 / 83 of injections are required to purify crude Tirzepatide under the conditions described in Example 4. In this example, the corrected 23.7 kg of crude Example 4B yields 25.4 kg of Example 4B (107%) after RP1. Total solution volume = 2910 L @ 8.72 g / L and once all cluster fractions are collected, the mixture is stirred, sampled, and held before Reverse Phase Purification 2 (RP2).

[00113] Reverse Phase Purification 2 (RP2): Substantially the same purification process as described in Example 4A, and using methods known to those skilled in the art, is used for Reverse Phase Purification 2 (RP2). In this example, 15.2 kg contained in Example 4B of RP1 are purified after RP2 to 13.8 kg of Example 4B at ~98% purity. Total solution volume = 808 L @ 17.0 g / L. The mixture is stored before tangential flow filtration.

[00114] Tangential Flow Filtration (TFF): TFF membranes are installed and washed with water. Ammonium acetate buffer is prepared using Low Endotoxin Purified Water, acetic acid, and ammonium hydroxide. Isopropanol is then charged to provide a 100 mM NH4OAc buffer, pH 8.0:IPA 5:95. The 17 g / L RP2 solution from Example 4B is concentrated via TFF to ~125 g / L. The RP2 solution is recirculated, allowing the solvent to penetrate through the membrane while retaining the peptide solution on the retentate side of the membrane in solution. After concentration, diafiltration buffer is fed to the retentate retention tank while the permeate is continuously collected. Buffer exchange is continued until the desired solvent composition and peptide concentration are achieved. The solution is emptied from the system, and the resulting polarization layer is rinsed from the membrane and combined with the peptide concentrate.Two sections of RP2 solution (403.9 L @ 17 g / L, 9.87 kg API). Petition 870260063821, dated 06 / 29 / 2026, p. 72 / 253 63 / 83 were processed through TFF.

[00115] Co-feed Precipitation: The TFF sections are combined (138.2 kg, 78.3 g / L) and the measured KF (8.9%) is found to contain <10% water. MTBE (243 kg) is loaded into a separate vessel and cooled to 0°C. IPA (48 kg), water (6 kg), and MTBE (100 kg) are added to the precipitation vessel, and the solution is cooled to 0°C. The TFF and MTBE process streams are co-fed to the precipitation vessel at rates of 1.6–1.8 and 2.9–3.1 kg / min, respectively. The resulting suspension is aged for a further 0.7 hours at 0°C and then heated to 15°C. The suspension is aged at 15°C for 1 h, followed by the addition of MTBE (118 kg). The suspension is aged at 15 °C for 1 hour and then cooled to 2.5 °C. The suspension is filtered cold and the filter cake washed with MTBE (573 kg). The filter cake is dried until LOD < 2%.

[00116] Humidification: Humidification, substantially as presented in Example 4A, and using methods known to those skilled in the art, is applied to the material of Example 4B. A total of 14.5 kg of Example 4B (SEQ ID NO: 1) is isolated with > 97.7% purity by HPLC and 88.4% peptide content. Overall yield based on Sieber resin loading = 46%. Example 5 Continuous Synthesis of Preparation 31 Using Convergent Flow Chemistry

[00117] The synthesis of Preparation 31 from peptide fragments is carried out using both discontinuous approaches to the chemistry as well as the sequential addition of fragments in a tubular flow reactor. The generalized approach to the synthesis involves coupling the two fragments by mixing two solutions along with the coupling agent in a tubular reactor, which is then followed by a base addition and a residence time. Petition 870260063821, dated 06 / 29 / 2026, page 73 / 253 64 / 83 additional in a tubular reactor to perform FMOC protection group removal. This prepares the coupled species for the addition of the next fragment. Excess reagents, base, and solvent are removed between consecutive coupling reactions using a nanofilter with a membrane that is sized to retain the peptide and permeate low molecular weight impurities. Diafiltration is used to completely remove low molecular weight impurities before subsequent coupling steps. A description and analytical results exemplifying these transformations are included below.

[00118] HPLC is used to confirm the synthesis of Preparation 31 from Preparation 29 and Preparation 30. The analytical method uses a C4 stationary phase column at 65 °C (2.1 mm id x 150 mm x 1.7 micron particle size) with a gradient of 60-98% B in TFA to TFA in water and acetonitrile over 12 minutes. UV detection at 214 nm is used for this material.

[00119] Table A.5 shows high-resolution mass spectrometry data collected for the coupling reaction product of Step 3 (Preparation 31) performed in flow. The mass precision confirms the desired species. Compound Chemical Formula Theoretical Monoisotopic Mass (neutral) Observed Ions m / z Charging State Calculated Typical Monoisotopic Mass (neutral) Mass Precision (ppm) Preparation 31 C334H512N48O74 6379.77 77 1595.95 12 4 6379.775 694 0.3 00120] Table A.5 Confirmation of Preparation 31 measured by means of mass precision calculated using high-resolution mass spectrometry data.

[00121] Native chemical bonding is a useful process for preparing Petition 870260063821, dated 06 / 29 / 2026, page 74 / 253 65 / 83 full-length peptides comprising a cysteine ​​or alanine in sequence. The process employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The thioester-linked intermediate rearranges to provide a full-length linkage product with a native peptide linkage at the linkage site. Those skilled in the art will recognize that the native chemical linkage technique can be useful in the chemical synthesis of full-length peptides containing cysteine ​​or alanine. Example 6 Native Chemical Bonding Process Tirzepatide (SEQ ID NO: 1) 2-Chlorotritile chloride resin (2-CTC) Synthesis of Fmoc-hydrazine-CTC Resin (Preparation 32)

[00122] 2-CTC resin (10.7 g, 17.7 mmol) is swollen in 100 mL of DCM for 20 min at 0°C. 9-Fluorenylmethyl carbazate (15.6 g, 61.4 mmol, 3.5 equiv.) is dissolved in 210 mL of DMF:DCM at 2:1. DIEA (31 mL, 178 mmol, 10.1 equiv.) is added to the 9-fluorenylmethyl carbazate solution. This solution is then slowly added to the resin at 0°C. It is stirred at 0°C for about one hour and allowed to warm to room temperature. The reaction mixture is stirred for 16 hours at room temperature. Methanol (10 mL) is then added to quench the remaining 2-CTC resin and stirred for 15 min. The resin is rinsed with 200 mL of DMF, followed by DMF (2 x 100 mL), water (3 x 100 mL), DMF (3 x 100 mL), methanol (3 x 100 mL). Petition 870260063821, dated 06 / 29 / 2026, page 75 / 253 66 / 83 and finally with DCM (3 x 100 mL). The resin is dried in a vacuum oven at 27 °C for 16 hours. The resin loading is measured at 0.74 mmol / g using quantitative NMR. Synthesis of Hydrazide Peptide (17-mers) (Preparation 33) SEQ ID NO: 321H í H 9 NH2 HYN^Xe-GTFTSDYSI-N_Ll-DKI N·2 Me Me Me Me Preparation 33

[00123] Hydrazine-CTC resin (1.01 g, loading value: 0.65 mmol / g) is taken in a 40 mL reactor vessel and swollen with 3 x 4 mL of DCM (30 s each), followed by 2 x 10 mL of DMF (20 min each) in a peptide synthesizer. Fmoc-lle-OH (0.919 g, 2.60 mmol, 4 equiv.) and HBTU (0.99 g, 2.61 mmol, 4 equiv.) are dissolved in 7 mL of DMF. DIPEA (0.91 mL, 5.22 mmol, 8 equiv.) is added to the amino acid solution and the volume is made up to 10 mL with DMF. The activated amino acid solution is added to the resin. The suspension is allowed to mix with nitrogen for 8 hours. After 8 hours, the resin is washed with 5 x 10 mL of DMF, 5 x 10 mL of DCM and dried for 12 hours. The loading of the resulting resin is measured as 0.54 mmol / g by quantitative NMR. 0.91 g of this resin is used for the synthesis of Preparation 33 (SEQ ID NO: 32).

[00124] Deprotection: 4 x 9 mL of 20% v / v piperidine in DMF, 30 minutes each.

[00125] Coupling: 3 amino acid equivalents, 3 OXYMA equivalents, and 3.3 DIC equivalents are used for amino acid coupling. The resin is washed with 5 x 9 mL of DMF with 1 min of N2 mixing after each coupling and the final deprotection iteration. At the end of peptide hydrazide synthesis, the resin is washed with DCM with N2 mixing. The resin is dried in the synthesizer.

[00126] Deprotection and Cleavage: 25 mL of cleavage cocktail Petition 870260063821, dated 06 / 29 / 2026, page 76 / 253 67 / 83 prepared with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) are added to the dry resin (2.37 g) and mixed for 3 hours in a rotary mixer. The resin is filtered and washed with 2 x 2.5 mL of TFA. The filtrate is poured into 175 mL of ice-cold MTBE, and the peptide is immediately precipitated. The filtration flask is washed with 2 x 2.0 mL of TFA and poured into ice-cold MTBE. It is cooled to -20 °C for half an hour and then centrifuged. The peptide precipitate is then washed twice with 150 mL of MTBE and centrifuged. The peptide precipitate is dried in a vacuum oven at 27 °C for 16 hours. 1.25 g of sample of the crude product, Preparation 33, is obtained after drying [Expected (mass + 2H+) / 2 = 968.4883, observed (mass + 2H+) / 2 = 968.4879].

[00127] Approximately 0.62 mmol of Preparation 34 are synthesized on Sieber amide resin using standard SPPS protocols. Fmoc-Lys(ivDde)-OH is used for orthogonal deprotection and lysine acylation.

[00128] Deprotection of ivDde: Hydrazine monohydrate (64% w / w) (1.98 g, 25.3 mmol) is diluted to 24.4 g with DMF and 20 g are added to the resin. The suspension is allowed to be stirred with a stream of nitrogen and washed with 5 x 9 mL of DMF after about two hours. This is repeated once more.

[00129] 2-[2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy20-oxo-icosanoyl)amino]-5-oxopentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (1094.4 mg, 1.252 mmol, 2 equiv.) is dissolved in 10 mL of anhydrous DMF. TNTU (506.9 mg, 1.360 mmol, 2.2 equiv.) and DIEA (0.24 mL, 1.4 mmol, 2.2 Petition 870260063821, dated 06 / 29 / 2026, p. 77 / 253 68 / 83 equiv.) are added to the same. The volume is made up to 15 mL with anhydrous DMF. It is left to mix for 30 minutes in a rotary mixer. The activated ester from Preparation 6 is then added to the resin and left to mix for 12 hours with a stream of nitrogen. After 12 hours, the solution is drained and the resin is washed with 5 x 10 mL of DMF and 7 x 10 mL of DCM with 1 min of mixing with N2. The resin is dried for 8 hours in the synthesizer.

[00130] Deprotection and Cleavage: 20 mL of the cleavage mixture made with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) are added to the dry resin (2.42 g) and mixed for 3 hours in a rotary mixer. The resin is filtered and washed with 2 x 2.0 mL of TFA. The filtrate is poured into 200 mL of ice-cold MTBE, and the peptide is immediately precipitated. The filtration flask is washed with 2 x 2 mL of TFA and poured into ice-cold MTBE. It is cooled to -20°C for 30 min and then centrifuged. The peptide precipitate is washed twice with 240 mL of MTBE and centrifuged. The peptide precipitate is dried in a vacuum oven at 27°C for 14 hours. 1.853 g of the crude product, Preparation 34, are obtained after drying.It is purified by RP-HPLC on a 10 μM (30 mm x 250 mm) Kromasil 100-10-C8 column at room temperature with a linear gradient of 30-55% acetonitrile in water for 25 min, then 15% acetonitrile in water for the first 5 min and a constant TFA of 0.1% over 30 min. 1.28 g of purified Preparation 34 (SEQ ID NO: 33) are obtained [Expected (mass + 2H+) / 2 = 1470.7929, observed (mass + 2H+) / 2 = 1470.7885]. Synthesis of Thioester (Conversion of Preparation 33 to Preparation 35

[00131] ) Crude peptide hydrazide (Preparation 33, 2.422 g, 1.251 mmol) is dissolved in 50 mL of binding buffer (6 M guanidine hydrochloride and 0.2 M monobasic sodium hydrogen phosphate, pH Petition 870260063821, dated 06 / 29 / 2026, page 78 / 253 69 / 83 of 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 equiv.) are added to the crude peptide hydrazide solution and stirred for 20 min at -15°C. Meanwhile, 1 mL of 2,2,2-trifluoroethanethiol (TFET) is made up to 10 mL with binding buffer (6 M guanidine hydrochloride and 0.2 M monobasic sodium hydrogen phosphate, pH 7.0). After 20 min, 10 mL of the TFET mixture are added to the peptide hydrazide solution to cause in situ thiolysis of the peptidyl azide generated from Preparation 33. Me Me EGTFTSDYSl Preparation 33 9 ld-kn-n H „NH2lí ιι Ke-gtftsdysin ο17? Kl-dki> <s^cf3 Me Me Me Me Preparation 35

[00132] The pH of the reaction mixture is adjusted to approximately 6.95 with 5N sodium hydroxide solution. Thiolysis of peptidyl azide is left to stand for 45 min and the volume is brought up to 100 ml with binding buffer (pH 7.0). The crude thioester mixture is purified by RP-HPLC on a 10 μM (10 mm x 250 mm) Waters X-Bridge C18 column at room temperature with a linear gradient of 25-42% acetonitrile in water for 25 min, then 10% acetonitrile in water for the first 2.8 min, and a TFA constant of 0.1% for the 28 min of purification. This yields 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].

[00133] Native Chemical Bond: Aqueous solution of hydrochloride Petition 870260063821, dated 06 / 29 / 2026, p. 79 / 253 70 / 83 guanidine at 6 M and monobasic sodium hydrogen phosphate at 0.3 M (pH 7.0) is the binding buffer used in the native chemical bonding. All solutions are made in this binding buffer. Dissolve 350.4 mg (0.174 mmol) of the peptide thioester, Preparation 35 (SEQ ID NO: 34), in 50 mL of the binding buffer. An 8.0 mL portion of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution is added to the peptide thioester solution. The N-terminal cysteine-containing peptide (Preparation 34 (SEQ ID NO: 33), 524.6 mg, 0.178 mmol, 1.03 equiv.) is dissolved in 48 mL of the binding buffer in a 50 mL centrifuge tube. The solution from Preparation 34 is added to the thioester solution. The tube is rinsed with 2 x 8 mL of binding buffer (pH approximately 7.0) and added to the reaction mixture. The pH of the reaction mixture is adjusted to approximately 7 with 5N NaOH solution.An 8.0 mL portion of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.0) is added to the reaction mixture and the pH is adjusted back to 7.0 with 0.2 mL of 5N sodium hydroxide solution. The reaction is allowed to stir at room temperature for 24 hours and then stored in a freezer. A further 3 mL of 0.5 M TCEP solution is added before purification. Preparation 36 (SEQ ID NO: 35) is purified by RP-HPLC on a 10 μM (10 mm x 250 mm) Kromasil C18 column at room temperature with a linear gradient of 20–50% acetonitrile in water (0.1% acetic acid and titrated to pH 9.0) over 23 minutes, then 10% acetonitrile in water for the first 4 minutes and for the remaining 28 minutes of purification. Approximately 372 mg (44.3%) of the cysteine ​​analog, tirzepatide from Preparation 36, are obtained after purification [Expected (mass + 3H+) / 3 = 1615.17263, observed (mass + 3H+) / 3 = . 1615, 1686]. Petition 870260063821, dated 06 / 29 / 2026, p. 80 / 253 71 / 83 HUS EGTFTSD YSIN^Al-DK^I^S^CF, m / Me Preparation 35

[00134] Desulfurization. A 6 M aqueous solution of guanidine hydrochloride and 0.3 M monobasic sodium hydrogen phosphate (pH 7.0) is the buffer used in desulfurization. All solutions were made in this buffer; 2,2'-azobis[2-(2-imidazolin-2-yl)propane dihydrochloride] (Preparation 37, 808.2 mg, 2.5 mmol) is dissolved in 10 mL of the buffer and the pH is adjusted to approximately 7.0 with 5N NaOH. The volume is made up to 15 mL with buffer. The cysteine ​​analog, tirzepatide from Preparation 36 (105.2 mg, 0.022 mmol), is dissolved in 30 mL of the buffer and 6 mL of the solution from Preparation 37 is added to it. Five mL of 0.3 M reduced L-glutathione solution (LGSH, pH 7.0) and 7.5 mL of 0.5 M TCEP solution (pH 7.0) are added to the same.The solution is heated to 44 °C for 4.5 hours, after which the reaction is considered complete by UPLC analysis [Expected (mass + 3H+) / 3 = 1604.5153, observed (mass + 3H+) / 3 = 1604.5122]. The desulfurization yield is calculated by UPLC using a tirzepatide reference standard (SEQ ID NO: 1). The yield is estimated at 47%. Petition 870260063821, dated 06 / 29 / 2026, page 81 / 253 72 / 83 Preparation 36 Preparation 37 Preparation 38 Native Chemical Bonding (Approach 2): Synthesis of Peptide Hydrazide Preparation 39 SEQ ID NO: 36

[00135] Hydrazine-CTC resin (2.03 g, 1.32 mmol, loading value: 0.65 mmol / g) is taken in a 40 mL reactor vessel and swollen with 3 x 10 mL of DCM (30 s each), followed by 2 x 10 mL of DMF (20 min each) in a Symphony synthesizer. HBTU (1.48 g, 3.90 mmol, 3.0 equiv.) is dissolved in 13.1 mL 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-pentaoxa24,29,38,47-tetra-azatripentacontan-53-oic acid (Preparation 17, 365 mg / mL in DMF) (3.91 mmol, 3.0 equiv.). DIPEA (1.4 mL, 8.04 mmol, 6.1 equiv.) is added to the above solution and the volume is made up to 19 mL with DMF. The solution is allowed to mix at room temperature in a rotary mixer for 30 min. The activated ester solution from Preparation 17 is added to the resin. The suspension is allowed to mix with nitrogen for 8 hours. After 8 hours, the resin is washed with 5 x 10 mL of DMF, 5 x 10 mL of DCM and dried for 12 hours.The loading of the resulting resin is measured as 0.26 mmol / g by quantitative NMR. 1.82 g of this resin are used for the synthesis of peptide hydrazide, Preparation 39 (SEQ ID NO: 36). Petition 870260063821, dated 06 / 29 / 2026, page 82 / 253 73 / 83

[00136] Deprotection: 4 x 9 mL of 20% v / v piperidine in DMF, 30 minutes each.

[00137] Couplings: 3 amino acid equivalents, 3 OXYMA equivalents and 3.3 DIC equivalents are used for amino acid coupling.

[00138] The resin is washed with 5 x 9 mL of DMF with 1 min of N2 mixing after each coupling and the final deprotection iteration. At the end of the peptide hydrazide synthesis, the resin is washed with 7 x 10 mL of DCM with 1 min of N2 mixing. The resin is then dried for approximately 12 hours in the synthesizer.

[00139] Deprotection and Cleavage: 25 mL of the cleavage cocktail made with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) are added to the dry resin and mixed in a rotary mixer. The resin is filtered, washed with TFA (2 x 2.5 mL), and the filtrate is poured into 175 mL of ice-cold MTBE. The filtration flask is washed with TFA (2 x 2.5 mL), and the washes are poured into ice-cold MTBE. It is cooled to -20°C for 30 min and then centrifuged. The peptide precipitate is then washed twice with 150 mL of MTBE and centrifuged. The peptide precipitate is dried in a vacuum oven at 27°C for 16 hours. 1.70 g of the crude peptide hydrazide, Preparation 39 (SEQ ID NO: 36), is obtained after drying.Crude peptide hydrazide, Preparation 39, is purified by RP-HPLC on a 10 μm (10 mm x 250 mm) Waters XSelect CSH C18 column at room temperature with a linear gradient of 20–55% acetonitrile in water over 23 min, then 10% acetonitrile in water for the first 3 minutes, and a constant TFA of 0.1% for the remaining 28 minutes of purification. Approximately 110 mg of the partially purified hydrazide, Preparation 39, is obtained.

[00140] Deprotection and Cleavage: 25 mL of cleavage cocktail Petition 870260063821, dated 06 / 29 / 2026, page 83 / 253 74 / 83 prepared with 5% w / v dithiothreitol (DTT), 2.5% v / v water, 2.5% v / v triisopropylsilane (TIPS), and 90% trifluoroacetic acid (TFA) are added to the dry resin (2.92 g) and mixed in a rotary mixer. The resin is filtered and washed with 2 x 2.5 mL of TFA. The filtrate is poured into 200 mL of ice-cold MTBE, and the peptide is immediately precipitated. The filtration flask is then washed with 2 x 2 mL of TFA, and the washes are poured into ice-cold MTBE. It is cooled to -20°C for 30 min and then centrifuged. The peptide precipitate is then washed twice with 240 mL of MTBE and centrifuged. The peptide precipitate is then dried in a vacuum oven at 27°C for 16 hours. Approximately 1.7 g of the crude 19-mers, Preparation 40 (SEQ ID NO: 37), is obtained.

[00141] Native Chemical Bonding: A 6 M aqueous solution of guanidine hydrochloride and 0.3 M monobasic sodium hydrogen phosphate (pH 7.0) is the binding buffer used in native chemical bonding. All solutions are made in this binding buffer. Partially purified peptide hydrazide (Preparation 39, 56 mg, 0.019 mmol) is dissolved in 5 mL of the binding buffer (6 M guanidine hydrochloride and 0.3 M monobasic sodium hydrogen phosphate, pH 3.35) and cooled to -15°C in an ice-cold acetone bath. 0.25 mL of 1 M sodium nitrite solution (0.25 mmol, 13.2 equiv.) is added to the peptide hydrazide solution and stirred for 10 min at -15°C. After 10 min, 0.8 mL of 0.5 M 4-mercaptophenylacetic acid (MPAA) solution is added to the peptide hydrazide solution to cause in situ thiolysis of the peptidyl azide generated from Preparation 39. The pH of the reaction mixture is adjusted to approximately 7.0 with 5N sodium hydroxide solution.Peptidyl azide thiolysis is allowed to run for 30 minutes. Petition 870260063821, dated 06 / 29 / 2026, page 84 / 253 75 / 83 Preparation 39 Preparation 41

[00142] Approximately 0.62 mmol of Preparation 40 (SEQ ID NO: 37) is synthesized on Sieber amide resin using standard SPPS protocols. Preparation 40 containing N-terminal cysteine ​​(26.1 mg, 0.014 mmol, 0.74 equiv.) is dissolved in 1 mL of binding buffer. The Preparation 40 solution is added to the thioester solution. The vial containing Preparation 40 is rinsed with 1 mL of binding 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) is added to the reaction mixture and the pH is adjusted to 7.0 with 5N sodium hydroxide solution. The reaction is allowed to stir at room temperature for one hour. The cysteine ​​analogue, tirzepatide from Preparation 42, is observed in the reaction mixture. Petition 870260063821, dated 06 / 29 / 2026, page 85 / 253 76 / 83 Preparation 41 HCFVQWLI~AGGPS-SG-APPPS-NH2 Preparation 40 SEQUENCES SEQ ID NO: 1 Tirzepatide YX1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS where X1 is Aib; X2 is Aib; K at position 20 is chemically modified by conjugation to the epsilon-amino group of the K side chain with (2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(YGIu)1-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide. SEQ ID NO: 2 HGPSSGAPPPS-NH2i ii tBu tBu tBu SEQ ID NO: 3 NH Fmoc-DKIAQN i H Trt tBu Boc AFVQ WLIAG-OH _ 1st Trt Boc Petition 870260063821, dated 06 / 29 / 2026, p. 86 / 253 77 / 83 SEQ ID NO: 4 h 2 Boc-YN-ÁÍBuMeMe HH EGTFTSDYSIN^Jm.-OH fBu fBu fBu I fBu\ 'fBu MeMe fBu fBu SEQ ID NO: 5 NH A-F-V-Q W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH2Il II I TrtBoc tBufBu fBu SEQ ID NO: 6 SEQ ID NO: 7 H ° 8oc-Y-N JL mA Me Me E G-T-F-T-S-D Y-S-l rBu I ieul '«u iBuDU nh2 A-F-V-Q W L-l-A G G-P-S-S G A P P P-S-NH, J 1 fl I Trt Bq< ®li fBu (Bu SEQ ID NO: 8 A-F-V O W L-I-A G-G P-S-S G-A-P-P P-S-NH, Tn éoc i8u SEQ ID NO: 9 Fmoc—P-S-S-G-A-P-P-P-S-N f-Bu f-Bu r-Bu H2 Petição 870260063821, de 29 / 06 / 2026, pág. 87 / 253 78 / 83 SEQ ID NO: 10 Fmoc—F-V-Q—W-L-l—A—G—G—oh Trt Boc SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 H—F-V-Q W-L-I-A-GG-P-S-SG-A-P-P-P-S—NH2 TrtBoc tButBu [R11 SEQ ID NO: 14 SEQ ID NO: 15 nh2 Petition 870260063821, 06 / 29 / 2026, pág. 88 / 253 79 / 83 SEQ ID NO: 16 Fmoc—FV-QW-L—lAG-OH II Trt Boc SEQ ID NO: 17 SEQ ID NO: 18 H ° Boc—Y—N !ISli E-hn MeMe Gly-ThifJ^^^Pro) Preparação 19A OR Λ- L-oh fBU Me Me SEQ ID NO: 19 oo SEQ ID NO: 20 HFVQ WLIAGGPSSGAPPPS-NH2 The II I Trt Boc tButBu tBu SEQ ID NO: 21 Petition 870260063821, 06 / 29 / 2026, pág. 89 / 253 80 / 83 SEQ ID NO: 22 SEQ ID NO: 23 L| O HObm-Yn GTFTSD Y δ-Ι'Κ^Λί- Ο Κ-ΗΛ Q-[j !Βι / m / LI BlJ jlBili Itsi u / tae fill ίθυ rtJu jBlj AFV QW LlA G GPSSGA PP PS-<>ÍHu l«l SEQ ID NO: 24 SEQ ID NO: 25 H2N-GPSSGAPPPS-NH2tBufBu tBu SEQ ID NO: 26 FmocHN-FV-QW-LlAG-OH II Trt Boc SEQ ID NO: 27 Petition 870260063821, 06 / 29 / 2026, pág. 90 / 253 81 / 83 h2n-FVQ WLIAGGPSSGAPPPS-nh2 The II I Trt Boc tBu tBu tBu SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 H ? BocHN-YN^ J1tBu .. 7\ tBuMeMe L-OH tBu u / tBu | tBu ®u Me Me SEQ ID NO: 31 AFVQ W LlA GGF SS GAFP PS-NHt Tri Boc iBu (Bu *Bu SEQ ID NO: 32 Η Ο h 9 HYN^ JEGTFTSDYSlN^ Λ Me Me Me Me Petition 870260063821, 06 / 29 / 2026, pág. 91 / 253 82 / 83 SEQ ID NO: 33 SEQ ID NO: 34 H fl EGTFTSDYSlNk Λ Me Me SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 H-C-F-V-Q-W-L-I-A-G-G-P-S-S-G-A-P-P-P-S-NH2 SEQ ID NO: 38 Petição 870260063821, de 29 / 06 / 2026, pág. 92 / 253 83 / 83 SEQ ID NO: 39 H fl H fl Η 0 μ 0 J H-Y-N^Le_g_t_f_t_s_d_v_s_|_n ILL_D_K_|_A_q.|^ μ / Me m / Me SEQ ID NO: 40 H fl H fl Y-N^-E-G-T-F-K,,. Ko 0 „r.A JL .N-D-Y-S-l-N. Xl-d-k-i-a-q A H0 ’ HaN V X H ό m / Me pC-F-V-Q-W-L-l-A-G-G-P-S-S-G-A-P-P-P-S^NH2 ^j-A-F-V-Q-W-L-kAG-G-P-S-S-G-A-P-P-P-S-NH, Petição 870260063821, de 29 / 06 / 2026, pág. 93 / 253

Claims

1 / 2 CLAIMS 1. Process for desulfurizing a cysteine ​​analog of tirzepatide to form tirzepatide SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, characterized in that it comprises contacting the cysteine ​​analog of tirzepatide with a radical initiator.

2. Process according to claim 1, characterized in that the radical initiator is a water-soluble radical initiator.

3. Process according to claim 1, characterized in that the radical initiator is an azo initiator.

4. Process according to claim 1, characterized in that the radical initiator is selected from the group consisting of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride and 2,2'-azobis(2-methylpropionamidine) dihydrochloride.

5. Process according to claim 1, characterized in that the thiol comprises 2,2,2-trifluoroethanethiol or mercaptophenylacetic acid.

6. Process according to claim 1, characterized in that the cysteine ​​analogue of tirzepatide is either SEQ ID NO: 35 or SEQ ID NO:

39.

7. Process according to claim 6, characterized in that the cysteine ​​analogue of tirzepatide is SEQ ID NO:

35.

8. Process according to claim 7, characterized in that the process comprises: coupling the thioester peptide intermediate SEQ ID NO: 34 with the tirzepatide intermediate SEQ ID NO: 33 to form the tirzepatide cysteine ​​analog SEQ ID NO: 35; and bringing the tirzepatide cysteine ​​analog SEQ ID NO: 35 into contact with a radical initiator to form tirzepatide (SEQ ID NO: 1).

9. Process according to claim 8, characterized in that the process comprises: (a) synthesizing a tirzepatide intermediate SEQ ID NO: 32 by solid-phase peptide synthesis; (b) contacting the tirzepatide intermediate of step (a) with a thiol to form the thioester peptide intermediate SEQ ID NO: 34; (c) coupling the thioester peptide intermediate with the tirzepatide intermediate SEQ ID NO: 33 to form the cysteine ​​analog of tirzepatide SEQ ID NO: 35; and (d) contacting the cysteine ​​analog of tirzepatide (SEQ ID NO: 35) with a radical initiator to form tirzepatide (SEQ ID NO: 1). Petition 870260063821, dated 06 / 29 / 2026, p. 95 / 253