The synthesis of tirzepatide mainly adopts the strategy of combining solid-phase synthesis (SPPS) and liquid-phase synthesis (LPPS), taking into account both efficiency and purity.

Basic Info
Name：Tirzepatide
Sequence：H-Tyr-{Aib}-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{Aib}-Leu-Asp-Lys-Ile-Ala-Gln-{diacid-C20-gamma-Glu-(AEEA)2-Lys}-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
Molecular Formula：C225H348N48O68
Mol. wt.：4813.45

Synthesis method

The synthesis of tirpotide mainly adopts the strategy of combining solid-phase synthesis (SPPS) and liquid-phase synthesis (LPPS), taking into account efficiency and purity.
Solid-phase/liquid-phase combined synthesis method
Eli Lilly: The dual agonist of glucose-dependent insulinotropic peptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor developed by Eli Lilly is to synthesize multiple short peptide fragments (AA1-14, AA15-21, AA22-29 and AA30-39 fragments) by solid-phase synthesis, as shown in Figure 1 below. The final product is obtained by combining the above four fragments by liquid phase synthesis method. In the process of solid-phase synthesis of these short peptide fragments, multiple long fragments are obtained by coupling dipeptides or tetrapeptides with Fmoc or Boc protection. This reduces the amount of amino acids used while reducing the formation of impurities, improving production efficiency and product quality.

The segmented synthesis strategy of fully protected peptides was used to synthesize fully protected peptides (e.g., 1-17 and 18-39) in solid phase, and then deprotected and conjugated through liquid phase deprotection, which significantly shortened the synthesis cycle and improved the yield.
Solid-phase synthesis method
Amino acids are stepwise coupled using Rinkamide resin as a solid-phase carrier. For example, Fmoc-Ser(tBu)-OH is first fixed, then unprotected (piperidine/DMF) and then conjugated (HOBt/DIC activated carboxyl groups) until the target sequence is completed. Solid-phase synthesis method: After the synthesis, the resin is lysed with trifluoroacetic acid (TFA) and the side-chain protection group is removed to obtain crude peptides. Fragment condensation and natural chemical linkage: For long peptide chains, pre-synthesized short peptide fragments can be used for liquid phase coupling, such as by thioester bonds (e.g., NCL technology).

Key Synthesis Steps

1. Amino acid activation and conjugation Use activation reagents such as HOBt, HATU, and DIC to activate carboxyl groups and condense with free amino groups to form peptide bonds. For example, complex amino acids such as Fmoc-Lys (aeea-aeea-γGlu(α-OtBu)-eicosanedioic tBu ester)-OH need to be activated step by step. Control the reaction conditions (temperature, pH, time) to reduce racemic and side reactions7.
2. Protective base strategy Fmoc/tBu strategy: the amino group is protected by Fmoc, and the side chain (such as the hydroxyl group of Ser and Thr) is protected by tert-butyl (tBu)7. BOC/BZL Strategy: Applies to side-chain protection for specific amino acids.
3. Lysis and crude peptide preparation The lysate is usually a DCM solution containing TFA (20% TFE), which is preliminarily purified by ether precipitation after lysis.

Purification process

Optimizing impurity control in tirpotide synthesis is a challenge and requires a combination of advanced purification technologies:
Functionalized silica gel separation column: 3-chloropropyltriethoxysilane and dimeraminocyanocyano/acetazolamide modified silica gel to enhance adsorption selectivity for target peptides and improve separation efficiency.
Combined with modified diatomaceous earth (levulinic acid treatment) to further remove hydrophobic impurities.
Chromatographic purification: Reversed-phase high-performance liquid chromatography (RP-HPLC, C18 and C8 columns) and monodisperse polymer reverse packing Seplife RP LXMS 15 (300) are used for fine purification of the end product, ensuring a purity > 98%.

Optimization direction of synthesis process

Fragment design and connection efficiency: Optimize the fragment length (e.g., 1-17 and 18-39) and coupling conditions for solid-phase synthesis to reduce side reactions.
Biosynthesis technology: Using genetically engineered strains to express tirpotide, combined with chemical synthesis and modification, reduce costs.
Separation Technology Upgrade: Introduce nanofiltration, ultrafiltration and other technologies to simplify the post-processing process and increase production capacity.

Challenges and prospects

Impurity control: Impurities (such as missing peptides and oxidation products) in the solid-liquid mixing method are difficult to completely remove, and the purification process needs to be continuously improved. Market competition: Eli Lilly's tirpotide is 20% lower than semaglutide due to its flexible chemical synthesis path and sufficient production capacity, which puts pressure on domestic GLP-1 drug companies. Indication expansion: Tirpotide has been approved for the treatment of obstructive sleep apnea, and may be expanded to heart failure and other fields in the future, promoting further optimization of the synthesis process.

Summary

The synthesis process of tirpotide is based on the solid-liquid combination method, combined with high-efficiency activation reagents and functionalized purification technology, balancing cost and purity. In the future, through the integration of biosynthesis and chemical synthesis and the development of new separation materials, its process efficiency and product quality are expected to be further improved, supporting its continued expansion in the global diabetes and obesity treatment market.

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Linear cell-penetrating peptides can improve and enhance the delivery of oligonucleotide drugs, but linear peptides are still unstable to proteolytic enzymes and may limit oligonucleotide delivery and efficacy and safety. Cyclization of cellular transmembrane peptides can improve their stability and improve the efficiency of oligonucleotide drug delivery.
The method of preparing cyclic peptide-oligonucleotide conjugates is mainly by liquid phase connection. That is, linear peptides are synthesized first, then the linear peptides are cyclized to form cyclic peptide fragments, and then the cyclic peptide fragments and oligonucleotide fragments are connected to form cyclic peptide-oligonucleotide conjugates through chemoselective reactions. In addition, cyclized cell-osmotic peptides often contain multiple positively charged arginine or lysine, which will aggregate or precipitate with negatively charged oligonucleotides during reaction. The solid-phase method can avoid this problem of aggregation and precipitation.
As with the preparation of linear peptide-oligonucleotide conjugates by solid-phase synthesis, the destruction of oligonucleotides by trifluoroacetic acid also needs to be considered when preparing cyclic peptide-oligonucleotide conjugates by solid-phase synthesis, so it is also necessary to avoid the use of trifluoroacetic acid. Another important issue considered in the preparation of cyclic peptide-oligonucleotide conjugates by solid-phase synthesis method is the cyclization of peptide fragments. The simplest way to cyclize polypeptides is to ring with dibrominated small molecules by sulfhydryl groups, which is a fairly mature reaction at the peptide level, but cyclic peptide-oligonucleotide conjugates include not only polypeptides, but also oligonucleotides, and it is best to cyclize with the presence of sulfhydryl groups, so it is necessary to prepare cyclic peptide-oligonucleotide conjugates containing sulfhydryl groups. The sulfhydryl protection group of hemiphotoine is generally triphenylmethyl (Trt), but triphenylmethyl (Trt) finally needs to be removed from trifluoroacetic acid, which is definitely incompatible with solid-phase synthesis systems. Therefore, a suitable cysteine sulfhydryl protection group needs to be found to prepare cyclic peptide-oligonucleotide conjugates. StBu can form a disulfide bond with cysteine to protect cysteine, and only a reducing agent can remove this protective group. There is no reducing agent involved in the synthesis of solid-phase peptides and solid-phase oligonucleotides, so in theory, StBu exists stably in these two solid-phase synthesis processes, and Fmoc-Cys(StBu)OH can be used to synthesize amino acids.
[Solid-phase condensation of cyclic peptide-oligonucleotide conjugates, liquid-phase ringing]
The operation steps of cyclic peptide-oligonucleotide conjugates before ring formation are the same as those of linear peptide-oligonucleotide conjugates, which are first carried out for peptide synthesis and then for the synthesis of solid-phase oligonucleotides. First, the resin needs to be connected to a small C7-NH2 molecule, the N-terminus of this small molecule is protected by Fmoc, where peptide synthesis can be carried out, hydroxyl group is protected by DMTr, and solid-phase oligonucleotide synthesis can be carried out here. Then, the Fmoc protective group on the small molecule was removed with 20% piperidine, and the remaining amino acids were sequentially coupled to the C7-NH2 molecule. The amino groups of tryptophan, histidine, arginine and lysine side chains need to be protected with Boc, the hydroxyl groups of serine, tyrosine, threonine side chains are protected with TBS, and the sulfhydryl groups of cysteine are protected by StBu. The end of peptide synthesis requires the drying of the solid-phase carrier and then the synthesis of solid-phase oligonucleotides. After the synthesis, the polypeptide-oligonucleotide conjugate containing cysteine is cut off from the resin directly with ammonia. At this time, according to the design of the experiment, the protective group should continue to retain on the peptide-oligonucleotide conjugate, without purification, the StBu protective group of cysteine acid can be completely removed by dissolving the reaction with TCEP solution for 1 hour, purifying and lyophilizing and 1,3-di(bromomethyl)benzene in the solution for ring formation, and monitoring the reaction process by high-performance liquid chromatography (HPLC), generally one hour can be successfully cyclized.

【Monacyclic peptide-oligonucleotide conjugates】
There is only one cysteine, which cannot be cyclized and generally needs to contain 2 cysteines. The synthesis steps are the same as those described above. After removing the two StBu protective groups on the peptide-oligonucleotide conjugate, cyclic peptide-oligonucleotide conjugates can be synthesized by using the principle of 1,3-dis(bromomethyl)benzene and two sulfhydryl groups in solution.

In addition to di(bromomethyl) benzene, ring-forming linkers can also be other types of linkers. Monacyclic peptide-oligonucleotide conjugates can also be synthesized from alkyl olefins and biphenyls, the difference is mainly that in the last step of ring formation, alkyl olefins and biphenyls are used to replace di(bromomethyl)benzene, and the reaction time is also 1 hour.
[Synthesis of bicyclic peptide-oligonucleotide conjugates]
In addition to monocyclic peptides, there are also bicyclic peptides, which react with two cysteines and 1,3-dis(bromomethyl)benzene to form a monocyclic structure, and if you want to form a bicyclic structure, you must have three cysteines, and then react with 1,3,5-tris(bromomethyl)benzene and three mercaptogroups to form a bicyclic peptide.

[Solid-phase condensation of cyclic peptide-oligonucleotide conjugates, solid-phase ringing]
The above-mentioned method of solid-phase coupling liquid phase ring formation is used to prepare cyclic peptides, but this method still has certain shortcomings. We need to consider the stability of cysteine in ammonia, which can produce a large number of β elimination by-products if not paid attention to. The cyclization of peptides can be directly realized on the resin, on the resin is attached to a small C7-NH2 molecule, the N-terminal of this small molecule is protected by Fmoc, where peptide synthesis can be carried out, hydroxyl group is protected by DMTr, and solid-phase oligonucleotide synthesis can be carried out here. Then use 20% piperidine to remove the Fmoc protective group on the small molecule, and conjugate the amino acids in order, and then use a reducing agent such as DTT or dimercaptoethanol on the resin to remove the StBu protective group on the peptide cysteine first, so that the sulfhydride group on the peptide will leak out. The resin is then ring-forming with 1,3-dis(bromomethyl)benzene and exposed sulfhydryl groups. If the cyclization is successful, the solvent in the resin is drained, and then the solid-phase oligonucleotides are synthesized. Then the cyclic peptide-oligonucleotide conjugate is cut off from the resin with ammonia, and at this time, because the peptide has been successfully cyclocycized, the original cysteine has now become a sulfide bond, so there is no need to worry about the damage of ammonia to it, so as to avoid the elimination side reaction of the sulfhydryl group always encountered by the previous solid-phase synthesis of cyclic peptide-oligonucleotide conjugates.

Although linear cell transmembrane peptides can improve and enhance the delivery of oligonucleotide drugs, the presence of ubiquitous proteolytic enzymes makes linear cell-penetrating peptides unstable and limits the efficacy of oligonucleotides. Cyclization of peptides can improve their stability and improve oligonucleotide drug delivery. The current method of preparing cyclic peptide-oligonucleotide conjugates is mainly by liquid phase connection. However, cyclized cell transmembrane peptides usually contain multiple positively charged arginine or lysine, which will aggregate or precipitate with negatively charged oligonucleotides when connected by liquid phase method. The solid-phase method can avoid this problem of aggregation and precipitation.

Valprotide acetate is an artificially synthetic octapeptide somatostatin drug, which is clinically used for gastrointestinal bleeding, acromegaly, pancreatitis and other diseases, and also has a certain tumor inhibitory effect on neuroendocrine tumors, prostate cancer, etc. Therefore, using valprotide acetate as a template can improve the biocompatibility of precious metal nanomaterials. Valprotide acetate was approved for marketing in 2004 with the chemical name D-phenylalanyl-cysteine-tyrodyl-D-chromatoyl-lysazyl-valinel-cysteine-tryptamide-cyclo[2-7]-disulfide monoacetate.

Basic information:
Chinese name: valprotide acetate
English name: Vapreotide
Company number: GT-B016
CAS Number: 103222-11-3
Sequence: D-Phe-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH2
Formula: C57H70N12O9S2
Molecular weight: 1131.4

Physical and chemical properties
When valprotide acetate is dissolved in an acidic aqueous solution, it has a large positive charge on the surface because it is below its isoelectric point (pI≈10.0). In addition, because it is a small molecule compound composed of amino acids, the acidic environment will change the spatial conformation of the template, mainly manifested in α-helix and β-folding, etc., so that it can maintain a stable secondary and tertiary structure in aqueous solution for a long time. Similarly, when the valproptide acetate template is heat-treated at high temperature (≥70°C), its supersecondary structure (αα, βαβ, ββ) will change significantly, and the peptide chain forms a three-structure local fold area on the basis of the secondary structure or supersecondary structure, which is a relatively independent tight sphere, that is, the domain, which will also change due to changes in the environment. Of course, there are other means of processing peptide chains, such as heavy metal salts, ultrasound, etc. After the above acid and heat treatments, valprotide acetate will form a relatively stable three-dimensional structure, and the specific groups will be regularly exposed on the surface of the template. Therefore, when the metal salt solution is co-incubated with the pretreated Peptide acetate solution, the metal ions will bind to the surface of the template under the action of electrostatic forces and specific groups, and due to biomineralization, these attached ions will be reduced into trace small crystals, which we call "crystal seeds", and then under the action of suitable reducing agents, a large number of free metal ions are continuously reduced, and grow along one or several crystal planes of the "crystal seed", and finally form nanomaterials with the expected morphology.

Application
Vapapritide acid can be used as a biological template, and its advantages include: (1) small molecular weight, simple structure, and can be analyzed and designed according to the morphology of the intended material; (2) In acidic solution, the surface of vapritide acetate will carry a large amount of positive charge, and the precursor metal ions will be continuously adsorbed to the surface of the template through electrostatic force and specific groups, thereby increasing the metal load of nanomaterials and conducive to better their physical and chemical properties. (3) Valprotide acetate belongs to the somatostatin class, which has high biocompatibility, and the precious metal nanomaterials made from it as templates can be used in the field of physical medicine. Examples of its applications are as follows:
1) Preparation of a silver-gold alloy nanospherical shell based on valproptide acetate, It is mainly prepared according to the ratio of 0.91~1.13mg vapritide acetate per ml of hydrochloric acid solution, treated at 65~67°C for 20~24min, according to the ratio of 1:3~9, silver nitrate solution is added, put into a water bath constant temperature shaker at 24°C for 24~26h, according to the ratio of silver nitrate: sodium borohydride is 1:1~2, and then the reducing agent sodium borohydride is added drop by drop, and the reaction is 16~18min at 24°C; and then placed in a boiling water bath at 100°C for 10min, and gold trichloride solution is added according to the ratio of gold trichloride:silver nitrate molar ratio of 1:25~45 , 100°C boiling water bath for 2~4min, silver-gold alloy nanospheres with particle size of 90~110nm were prepared. The present invention is easy to control, simple to operate, green and environmentally friendly, mild reaction conditions, and the prepared silver-gold alloy has uniform particle size and good dispersion.
2) Preparation of palladium nanocubes. Valprotide acetate was dissolved in a hydrochloric acid solution with a pH of 1~3, A concentration of 0.1~0.2mM was prepared into 300μL vaprotide acetate solution, and the palladium chloride solution was added to the molar ratio of vaprotide acetate:palladium chloride at a ratio of 1:20~60, and placed in a constant temperature metal bath at 60~80°C. According to the molar ratio of palladium chloride:ascorbic acid was 1:10~50, the newly prepared ascorbic acid solution was quickly added for reduction, and the solution gradually changed from light yellow to black-brown, so as to obtain palladium nanocubes with regular morphology and uniform particle size. The preparation process of the present invention is simple and easy to operate; And this method does not use toxic reagents, which is green and environmentally friendly; The prepared palladium nanocubes have regular morphology, good photothermal conversion performance, and rapid heating after short-term low-power laser irradiation, which can be used as hyperthermic agents for cancer treatment.
3) Silver nanospheres were prepared with valprotide acetate as a biological template, It is mainly formulated into a vaprotide acetate solution with a concentration of 0.2~0.4mM, kept warm at 70~80°C for 10~30min, and the silver nitrate solution is added to the vapritide acetate solution at a ratio of 1:6~8, put into a water bath constant temperature shaker, 80~100rpm, 25°C for 12~24h, according to the molar ratio of silver nitrate solution: sodium borohydride is added to the incubated solution, the drop rate is 2 drops/minute, 60μL per drop, 25°C reaction for 5~15min, so as to obtain silver nanospherical shells with uniform particle size and good dispersion of 80~100nm. The silver nanosphere shell prepared by the invention overcomes the shortcomings of easy aggregation and precipitation in the traditional preparation process, and has a high metal load.

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1. Introduction

Peptides are compounds formed by two or more amino acids linked by peptide bonds, which are widely present in living organisms and participate in a variety of physiological processes, including cell signaling, enzyme catalysis, immune response, and metabolic regulation. In recent years, with the development of biosynthesis and chemical synthesis technologies, peptide customization services have increasingly become a core tool in life sciences, pharmaceutical research and development, and molecular biology research.
The editor will introduce in detail the technical principles, main processes, quality control processes and typical applications of peptide customization in scientific research.

2. Technical principle of peptide customization

Peptide customization is the process of synthesizing amino acids in specific sequences to obtain the target peptide molecules with the desired structure and function. Its main synthesis techniques include:

1. Solid Phase Peptide Synthesis (SPPS)
   At present, a wide range of synthesis technologies are used. By fixing one amino acid on a solid-phase resin, then gradually adding other amino acids, using condensing agents (e.g., HBTU, DIC) to connect the amino acid residues, and finally chemically cutting the synthesized peptides from the solid-phase carrier.
   Advantages: high degree of automation; can synthesize long peptides; Easy to purify and modify.
2. Liquid Phase Synthesis
   It is suitable for short-chain peptides and large-scale industrial synthesis, with mild reaction conditions but cumbersome steps.

3.Detailed explanation of the customization process

A standard peptide customization process generally includes the following key steps:

1. Design and sequence confirmation
   Customers provide amino acid sequence or functional requirements;
   The service party analyzes the sequence (whether it contains special modifications, whether it contains disulfide bonds, whether it is water-soluble, etc.);
   Bioinformatics tools were used to predict secondary structure, hydrophilicity and other parameters to assist in design.
2. Synthesis stage
   Choose the appropriate solid-phase carrier (e.g., Rink amide resin, Wang resin);
   Precise control of the coupling reaction of each amino acid;
   Add protective groups (e.g., Fmoc, Boc) to avoid side effects.
3. Purification and analysis
   High Performance Liquid Chromatography (HPLC) for separation and purification, the purity can reach more than 95%;
   Molecular weight was confirmed using mass spectrometry (MALDI-TOF, ESI-MS);
   Nuclear magnetic resonance (NMR) and circular dichroism (CD) assisted analysis are performed if necessary.
4. Packaging and quality control
   Provide lyophilized powder according to customer needs;
   Evaluation of storage conditions by stability tests (high temperature, light, hydrolysis);
   Provide Quality Analysis Report (COA) and MSDS documentation.

4. Types of peptide modifications

Common functional touch-ups in the customization process include:
N-terminal modifications: acetylation, biotin, biotinylation;
C-terminal modification: amideation, esterification;
Fluorescent labeling: FITC, TAMRA;
phosphorylation/glycosylation: mimicking naturally modified peptides;
Disulfide bond bridging: stabilize the spatial structure.

5. Application of peptide customization in scientific research

1. Antibody preparation and epitope analysis
   Researchers can synthesize specific epitope peptides as antigens for the preparation of polyclonal or monoclonal antibodies.
2. Cell signaling pathway research
   Synthesis of phosphorylated or mutant site peptides for protein kinase activity screening, pathway validation, etc.
3. Pharmacological screening and functional verification
   peptide antagonists and agonist mimics;
   Combination studies with GPCR and other targets.
4. Immunology research
   Vaccine research and development, immunogenicity assessment, peptide library construction, etc.
5. Protein interaction experiments (e.g., pull-down)
   Protein function and interaction analysis are achieved by capturing protein complexes with tags (e.g., His, FLAG, Biotin) with peptides.

6. Technical challenges and development trends and Challenge:

Low synthesis efficiency of long-chain (>50aa) peptides;
Hydrophobic polypeptides are easy to aggregate and affect solubility;
The synthesis steps of special modifications (such as glycosylation) are complex and costly.
Trend:
Accelerate the promotion of intelligent and peptide synthesis automation equipment;
Peptide drug development drives breakthroughs in functional modification technology;
Custom processes are integrated with bioinformatics to improve design accuracy.

7. Summary

Peptide customization is a highly integrated biosynthesis and chemical engineering technology that requires not only precise equipment and materials, but also precise design logic and analytical capabilities. In the field of scientific research, it has become an indispensable technical support for experimental design and biological validation. With the upgrading of technology and the deepening of application, it will release greater potential in the future in basic scientific research, precision medicine, biopharmaceuticals and other directions.

Beinaglutide is a human GLP-1 polypeptide with CAS number 123475-27-4 and almost 100% homology to human GLP-1 (7-36). Benaglutide has shown dose-dependent effects in glycemic control, inhibition of food intake and gastric emptying, and promotion of weight loss. Benaglutide has the potential to study overweight/obesity and nonalcoholic steatohepatitis (NASH).

Basic information
Chinese name: Benaglutide
English name: Beinaglutide
Company number: GT-M19402
CAS Number: 123475-27-4
Sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR
Formula: C149H225N39O46
Molecular weight: 3298.61

This high degree of structural similarity allows benaglutide to mimic GLP-1 in the human body, thereby acting in multiple physiological processes. First, benaglutide has shown significant results in blood sugar control. It helps regulate blood sugar levels and keeps them within a relatively stable range, which is especially important for diabetics.
In addition, benaglutide also has a significant inhibitory effect on food intake and gastric emptying. This means that when the body ingests benaglutide, it can slow down the emptying of the stomach, which can make people feel fuller, help reduce food intake, and help with weight management. In addition, benaglutide is also able to promote weight loss, which has been confirmed in several studies.

It is important to note that the effects of benaglutide are dose-dependent, i.e., its effects are enhanced with increasing doses. This allows doctors to adjust the dosage of benaglutide according to the patient's specific situation to achieve the best treatment effect.

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