Hydrophobic Tag-Assisted Liquid-Phase Synthesis of Tirzepatide

Today, we share an important research article from the team led by Lin Dong, published in Organic Letters. This work addresses the bottlenecks of high cost and cumbersome procedures in solid-phase peptide synthesis (SPPS) for the peptide drug Tirzepatide (a novel dual-target agonist for treating type 2 diabetes and obesity) by developing a novel hydrophobic tag-assisted liquid-phase peptide synthesis (LPPS) strategy. This method introduces soluble hydrophobic tags (TAGa and TAGb), leveraging their unique solubility properties to achieve efficient separation, significantly reducing amino acid and solvent waste. It also employs a dual-protection (Cbz/Fmoc) strategy on lysine to precisely control side-chain assembly, ultimately successfully synthesizing the 39-amino-acid Tirzepatide with high yield. This work provides a new approach for the green and economical large-scale production of complex peptide drugs.

Figure 1. Structure of tirzepatide.

01 Research Background

As the first dual GIP/GLP-1 receptor agonist, Tirzepatide has demonstrated outstanding efficacy in the treatment of diabetes and obesity. However, its synthesis heavily relies on traditional solid-phase peptide synthesis (SPPS). Although SPPS is a mature technology, it has inherent drawbacks such as high resin cost, decreasing coupling efficiency as the peptide chain lengthens, and complex purification steps, especially for long peptides like Tirzepatide (39 amino acids), where the overall yield is often below 10%. Liquid-phase peptide synthesis (LPPS) can avoid resin use but faces challenges like difficult control of product solubility and tedious intermediate isolation. Although hybrid SPPS/LPPS strategies have been reported in recent years, production costs remain high. Hydrophobic tag-assisted LPPS introduces soluble tags that keep the peptide chain soluble in the reaction system, enabling simple precipitation for separation, combining the homogeneous reaction advantages of liquid-phase synthesis with the separation convenience of solid-phase synthesis. This study is the first to apply this strategy to the total synthesis of Tirzepatide, aiming to establish an efficient, economical, and scalable synthetic route.

02 Innovative Highlights

Dual-Function Hydrophobic Tags Enable Flexible Guidance for C-terminal Acid/Amide Synthesis:​ The research team designed and synthesized two structurally tunable hydrophobic tags, TAGa (alkoxybenzyl alcohol type) and TAGb (Rinker linker type), suitable for C-terminal carboxylic acid and C-terminal amide synthesis, respectively. TAGa allows product recovery via simple acetonitrile precipitation, while TAGb can directly connect to the C-terminal serine amide of Tirzepatide, avoiding additional activation steps. The hydrophobic nature of the tags (based on a docosyl chain) ensures good solubility in polar solvents and quantitative precipitation in non-polar solvents, achieving a "homogeneous reaction-heterogeneous separation" cycle.

Segmental Assembly and Lysine Dual-Protection Strategy Overcomes Side-Chain Modification Challenges:​ The 39-mer peptide chain was rationally divided into four fully protected intermediates (7-14 amino acids each), reducing single-step reaction complexity through iterative coupling. For the critical step of Lys20 side-chain modification, an innovative dual Cbz/Fmoc protection strategy was employed: Fmoc served for temporary deprotection to connect the side-chain fragment, while Cbz was removed in the final stage to allow coupling with the main chain. This orthogonal protection design avoided issues like high cost and numerous side reactions associated with the traditional Alloc protecting group, significantly improving side-chain assembly precision.

One-Pot Deprotection-Precipitation Enables Direct Acquisition of High-Purity Final Product:​ At the synthesis endpoint, the fully protected peptide-tag conjugate was treated directly with a TFA mixture containing 2.5% TIS and 2.5% H₂O, achieving side-chain deprotection and tag cleavage in one step and promoting precipitation of the crude peptide. This one-pot method avoided lengthy chromatographic purification steps. The crude product, after simple reversed-phase high-performance liquid chromatography (RP-HPLC), yielded Tirzepatide with over 90% purity, greatly improving synthesis efficiency.

03 Results and Discussion

3.1 Synthesis and Property Validation of Hydrophobic Tags

The study first synthesized the two core hydrophobic tags TAGa and TAGb. TAGa started from benzaldehyde, undergoing reductive alkylation to attach a docosyl chain, yielding an alkoxybenzyl alcohol structure; TAGb was formed by condensing benzaldehyde with 2,4-dimethoxybenzylamine, producing an amine tag containing a Rinker linker. Key physicochemical property characterization showed that both TAGa and TAGb have solubility greater than 50 mg/mL in polar solvents like DMF and THF, but achieve quantitative precipitation (recovery >95%) upon adding 4 volumes of acetonitrile. This significant solubility switching behavior is the foundation for the subsequent "homogeneous reaction-heterogeneous separation" cycle.

Scheme 1. Synthesis of TAGa and TAGb

3.2 Segmental Peptide Synthesis and Tag-Guided Coupling Optimization

The full-length Tirzepatide sequence was divided into four fragments (Intermediate I-IV), with Intermediate II containing the Lys20 side-chain modification site. Taking the TAGa-mediated synthesis of Intermediate I as an example (Scheme 2): First, Fmoc-Gly-OH was coupled to TAGa via DIC/DMAP catalysis. After acetonitrile precipitation, TAGa-Gly-OH was obtained with a 92% yield. Subsequently, Fmoc was removed with 1% DBU/piperidine to expose the amino group, which was then coupled with the next Fmoc-protected amino acid using the HBTU/HOBt/DIPEA system. This cycle was repeated, successfully constructing Intermediate I containing 7 amino acids, with each coupling efficiency >98% (monitored by HPLC) and crude product purity above 85%.

Figure 2. Strategies for total synthesis of tirzepatide.

Intermediate IV, with its C-terminal amide, posed a synthetic challenge. The research team initially attempted to first synthesize TAG-COOH and then couple it with the C-terminal serinamide, but failed due to steric hindrance and poor reaction site selectivity. The final successful approach involved directly connecting TAGb to the serinamide with the exposed amino group, successfully constructing TAGb-Intermediate IV with an 88% yield for this step (Scheme 2).

Scheme 2. Synthesis of TAGa-Intermediates and Intermediate IV

3.3 Precise Side-Chain Assembly: Successful Implementation of the Dual-Protection Strategy

Intermediate II, containing the Lys20 side-chain modification, was key to the synthesis. Initially, the Alloc group was used to protect the Lys side chain, but deprotection led to side reactions, with TLC showing tailing spots and product darkening. For this, the team innovatively adopted a Cbz/Fmoc dual-protection strategy (Scheme 3). First, both Cbz and Fmoc were simultaneously introduced onto the Lys of sub-fragment II-a. In the first step, Fmoc was selectively removed with DBU/piperidine, and the exposed amino group was used to connect the side-chain fragment II-b; in the second step, Cbz was removed via Pd/C-catalyzed hydrogenolysis, exposing another amino group for coupling with the main chain fragment II-c. This strategy successfully achieved precise side-chain introduction, ultimately yielding TAGa-Intermediate II with a total yield as high as 78%. The team attempted to obtain intermediate II-a' via protecting group transposition but was unsuccessful, likely due to the low deprotection reactivity of Cbz at that site.

Scheme 3. Synthesis of Intermediate II

3.4 Full-Length Assembly and Final Product Characterization

The four fragments were sequentially connected via tag-mediated coupling: as shown in Scheme 4, the four intermediates were assembled into the full-length molecule through iterative coupling. First, after deprotecting the Fmoc group from TAGb-Intermediate IV, it was coupled with de-tagged TAGa-Intermediate III to yield TAGb-Intermediate VI. Similarly, TAGa-Intermediate I and II were coupled to yield TAGa-Intermediate V. Finally, the two large fragments were connected to yield the fully protected target peptide-TAG conjugate. After deprotection with the TFA/TIS/H₂O mixture, the crude product precipitated with a 78% yield. Following RP-HPLC purification, high-purity Tirzepatide was obtained as a white powder, confirming the success of the synthetic route.

Scheme 4. Synthesis of Tirzepatide

04 Conclusion and Future Perspectives

This study successfully developed a hydrophobic tag-assisted liquid-phase synthesis strategy, achieving the efficient preparation of Tirzepatide. The advantages of this method are: ① Tag-guided precipitation purification avoids resin use and frequent chromatographic separation, reducing solvent consumption by approximately 40%; ② The dual-protection strategy ensures precise side-chain introduction, increasing the overall yield by 2.3 times compared to traditional SPPS; ③ The one-pot deprotection-precipitation simplifies final step operations. In the future, this method holds promise for further extension to the synthesis of other modified peptides (such as glycopeptides, fluorescently labeled peptides). Optimizing the tag structure (e.g., introducing photolabile groups) could enable spatiotemporally controlled release. Combined with continuous flow reactors, it has the potential to achieve kilogram-scale continuous production of Tirzepatide, providing a new paradigm for the green manufacturing of peptide drugs.

Original  Article

Pang ZJ, Mao CY, Feng TT, Dong L. Hydrophobic Tag-Assisted Liquid-Phase Synthesis of Tirzepatide. Org Lett. 2025 Sep 19;27(37):10442-10446. 

https://pubs.acs.org/doi/10.1021/acs.orglett.5c03314