The raw materials for Retatrutide synthesis

Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-OH

α-aminoisobutyric acid (Aib) is a non-natural amino acid with α-position dimethylation and rigid molecular structure. Its steric hindrance at carboxyl and amino reaction sites increases sharply, causing the coupling speed of the two amino acids to slow down significantly. Especially as the peptide chain continues to extend, the resin contracts, making the active N-terminus easily encapsulated, making coupling increasingly difficult. Ritaglutide molecules contain two Aib non-natural amino acids (located at positions 2 and 20 of the sequence, respectively). Using the tetrapeptide Boc-Tyr1(tBu)-Aib2-Gln3(Trt)-Gly4-OH (reaction site glycine), which has a lower carboxyl terminal block, can replace Fmoc-Aib2-OH or the dipeptide Boc-Tyr1(tBu)-Aib2-OH, solving the problem of difficult conjugation and avoiding the rearrangement side reaction of Gln.

Fmoc-Gln(Trt)-Aib-Ala-OH

Replacing Fmoc-Aib-OH with the tripeptide Fmoc-Gln(Trt)-Aib-Ala-OH (reaction site alanine), which has relatively low carboxyl terminal resistance, can effectively solve the problem of abnormal coupling difficulties.

Fmoc-Ile-α-Me-Leu-Leu-OH

Similar to α -aminoisobutyric acid (Aib), α -methyl-leucine (α-Me-Leu) is a non-natural amino acid methylated at the α -position of the carbonyl group. Its steric hindrance in the reaction space is greater than that of Aib amino acids. The coupling speed of the amino acids before and after it is very slow, sometimes taking several days, and it also significantly increases the risk of amino acid racemization. Therefore, the α -ME-LEU at the 13 position of retalutide was made into a tripeptide Fmoc-Ile-α -ME-LEU-LEU-OH (to reduce the large steric hindrasis effect caused by α -ME-LEU). Replacing the dipeptide FMOC-ILE-α -ME-LEU-OH, Fmoc-α -ME-LEU-LEU-OH or substituting Fmoc-α -ME-LEU-OH can effectively solve the problem of difficult coupling of α -ME-LEU in solid-phase synthesis.

Fmoc-Pro-Pro-Pro-OH

Proline (Pro) has a unique cyclic structure that brings about ‌ steric hindrance ‌ and rigid ‌ conformations, which can lead to extremely difficult coupling or even chain termination. The amino acids at positions 36-38 of Retalutide are three consecutive prolines (Pro). Replacing Fmoc-Pro-OH with the tripeptide Fmoc-Pro-Pro-Pro-OH can significantly enhance the condensation efficiency while avoiding various issues of deletion and insertion of Pro peptides.

Fmoc-Ser(tBu)-Gly-OH

Serine (Ser) and threonine (Thr) are high-risk and easily racemic amino acids (due to the presence of β -hydroxyl groups, their α -hydrogen acidity is enhanced, making them more prone to deproteration and racemization), and their side chains have a relatively large steric hindrance of tert-butyl protection, which slows down the coupling rate and further accelerates racemization. Therefore, the dipeptide Fmoc-Ser(tBu)-Gly-OH is used to replace Fmoc-Ser(tBu)-OH. Replacing Fmoc-Thr(tBu)-OH with the dipeptide Fmoc-Thr(tBu)-Phe-OH can easily solve the problems of difficult coupling and racemization of serine (Ser) and threonine (Thr).

Pseudopeptides

During the solid-phase peptide synthesis process, as sequences extend, certain sequences easily form stable β-folded secondary structures, leading to peptide chains aggregating and tightly packing, which can result in incomplete coupling or even synthesis failure. By utilizing the special structures of serine (Ser) or threonine (Thr), we designed them as pseudo-prochlor dipeptides, which fundamentally alter the conformational preferences of local peptides, prevent β-folding, and effectively accelerate coupling; Additionally, pseudo-protripeptide amide N lacks active H and cannot enol to form the oxazoline intermediate, thus perfectly avoiding racemic Ser/Thr (Ser/Thr is a high-risk racemic amino acid). The following fragments of ritaglutide pseudopro-prochlorodipeptide we have developed and recommend are: Fmoc-Gly-Thr (Psi(Me,Me)pro)-OH, Fmoc-Thr(tBu)-Ser (Psi(Me,Me)pro)-OH, Fmoc-Tyr(tBu)-Ser (Psi(Me,Me)pro)-OH, Fmoc-Ser(tBu)-Ser (Psi(Me,Me)). pro)-OH.

Side chain

As semaglutide and tirzepatide APIs gradually scale up globally, their side chain synthesis processes have matured. Solid-phase synthesis directly uses mature large fragments (Lys/AEEA/Glu/C20) for conjugation, offering significant advantages in quality and cost control. For ritaglutide, it is also recommended to directly use the side-chain fragment Fmoc-L-Lys [C20-OtBu-γ-Glu(OtBu)-AEEA]-OH for solid-phase synthesis, offering both cost and quality advantages.

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