Analysis of the Synthesis Process for Eli Lilly's Key Drug: Tirzepatide

To achieve dual activity, Tirzepatide not only incorporates amino acid residues derived from GLP-1 and GIP but also utilizes several unique amino acid residues. Its peptide structure is primarily derived from GLP-1, GIP, exenatide, and semaglutide, with a few residues being distinctive.

In the research on the synthesis process of Tirzepatide, numerous methods have been employed for producing synthetic peptides. The selection of an appropriate synthesis method is typically based on molecular characteristics, such as the presence of non-coding amino acids, non-peptide side chains, and the number of amino acids involved.

Biosynthesis：The recombinant techniques commonly used for macromolecules (such as monoclonal antibodies) can also be applied to smaller peptides. In this approach, external DNA is introduced into Escherichia coli or mammalian (China hamster ovary, CHO) cell lines, where the peptides are synthesized by the cells. Although the development timeline is typically longer than that of conventional synthesis methods, this approach has been successfully employed for semisynthetic peptides (e.g., semaglutide, insulin degludec, and liraglutide). Two drawbacks of this method are: (1) difficulty in introducing amino acids not encoded by DNA sequences; and (2) the need for post-recombinant synthetic modifications to incorporate non-peptide side chains.

Chemical Synthesis：

Solid-phase peptide synthesis (SPPS) has proven effective for many medium-sized peptides (e.g., Lisinaptide and Exenatide), including those synthesized using non-coding amino acids. Unfortunately, for larger peptides (typically>30 amino acids), SPPS often suffers from low process yields and poor purity. Moreover, due to the requirement for numerous consecutive unit operations to be performed without error (with an estimated failure rate of 20% for peptides exceeding 30 amino acids), it carries significant production risks.

Liquid-phase peptide synthesis (LPPS). This method is similar to SPPS, with the exception that the growing peptides do not bind to the resin and their C-termini are non-reactive amides or protected esters. It is well-suited for synthesizing smaller peptides (≤10 amino acids), but yield and purity rapidly decline as peptide length increases.

The SPPS/LPPS hybrid method. This approach utilizes SPPS to produce high-purity short peptide fragments and well-characterized precipitation intermediates, thereby avoiding many manufacturing risks and purity issues inherent in longer SPPS constructs. Subsequently, the protected fragments are coupled in liquid phase.

The SPPS/LPPS hybrid method, which combines the advantages of both techniques, represents an excellent approach. To implement this hybrid strategy, appropriate fragments synthesized via LPPS must first be selected. Key considerations in fragment selection include peptide length, total number of fragments, amino acid identity at cleavage sites, and side chain characteristics. Fragment size and quantity are critical; while smaller fragments may achieve high purity, they necessitate more complex manufacturing processes involving additional LPPS steps. Conversely, fewer large fragments impose greater production challenges and pose higher risks for achieving high purity. After evaluating these factors, Eli Lilly researchers selected four fragments for tilitropine synthesis. Regarding cleavage sites, a pivotal consideration was the diastereomerism of the C-terminal amino acids in each fragment, as the amide backbone facilitates diastereomerization of the α-stereocenter more readily compared to Fmoc-protected amino acids used in SPPS constructs. Following extensive evaluation of various fragments, four specific fragments were ultimately chosen (Figures 2–5). All four fragments were synthesized via SPPS, exhibit easy separability as solid products, and achieve exceptionally high purity levels (97.5–99.5%).

Next, fragment 2–5 are coupled together through a four-step LPPS process.

Step 1: Connect the first two fragments, 2 and 3, representing amino acids 30–39 and 22–29, respectively. Preparate separate solutions of fragments 2 and 3 in dimethyl sulfoxide (DMSO) and acetonitrile (ACN), as well as PyOxim and pure diisopropylethylamine in ACN, and flow them together before entering the plug flow reactor (PFR) 27 for online mixing to ensure uniformity；

Step 2: The obtained amino acids 22–39 (Compound 6) were then coupled with Fragment 4 to yield amino acids 15–39 (Compound 7)；

Step 3: The obtained amino acid sequence 15–39 (Compound 7) was coupled with Fragment 5 to yield the Boc-protected tildropptide；

Step 4: Remove the protective coating to obtain Tirzepatide.

The intermediates for tirzepatide synthesis

Fmoc-Lys[AEEA-AEEA-γ-Glu(OtBu)-C20-OtBu]-OH  Cas:CAS:2915356-76-0

Boc-Tyr(tBu)-Aib-OH  CAS:2639221-78-4

Fmoc-Ile-Aib-OH  CAS:2171139-20-9

Fmoc-Ser(tBu)-Ser(tBu)-OH  CAS:110098-50-5

Fmoc-Pro-Pro-OH  CAS:129223-22-9

Fmoc-Thr(tBu)-Phe-OH  CAS:1962160-86-6

Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH  CAS:2682040-93-1

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