Alpha melanocyte stimulating hormone (α - MSH) is responsible for triggering the production of melatonin, which is the natural protection of the human body against ultraviolet (UV) radiation. Exposure of the skin to ultraviolet radiation promotes the production of alpha MSH; However, stimulating the biosynthesis of melatonin before UV exposure may prevent UV induced skin cancer.

Considering that the natural form of alpha MSH is too unstable in vivo to be used as a therapeutic agent, different analogues have been synthesized, one of which is MT-II (Figure 1), a cyclic peptide with high resistance and extraordinary efficacy against enzymatic degradation.

Figure 1

　　Due to the fact that the production of MI-II often needs to be scaled up to the KG level to meet clinical needs, the optimization of process conditions at the laboratory scale must take into account the scalability of the process.

　　

　　Gyros Protein Technologies provides peptide synthesizers from laboratory to pilot level, helping to smoothly scale up from process development to commercial production.

This article will discuss the optimization and scaling up of fully automated synthesis pathways, namely the synthesis of linear sequences and resin intramolecular amidation of final peptides. In this work, the optimization of MT-II large-scale synthesis is carried out in three stages:

　　1Find an appropriate protection scheme for resin cyclization;

　　2Using PurePep Chorus to optimize synthesis conditions on a small scale and complete cyclization, the instrument runs fully automatically;

　　3On the pilot scale synthesizer PurePep Sonata+, MT-II was synthesized by amplifying the previously optimized conditions.

　　01Finding a suitable resin cyclization protection scheme

The structure of MT-II is completed after the formation of a lactam bridge between aspartic acid and lysine, and these residues need to be selectively deprotected before the cyclization step on the resin. Therefore, it is ideal to find side chain protecting groups that can be removed to minimize the difficulty of deprotection. In the first stage of this experiment, MT-II was synthesized using different combinations of side chain protecting groups:

1Asp(ODmab)/Lys(ivDde)

2Asp(O-2-PhiPr)/Lys(Mtt)

3Asp(OAlly)/Lys(Alloc)

It can be removed simultaneously and selectively using 2% hydrazine in DMF solution, 1% TFA in DCM solution, and palladium tetrakis (triphenylphosphine) (Pd (PPh3) 4) and phenylsilane (PhSiH) in DCM solution. The crude purity of the final peptide is shown in Table 1.

Table 1 Purity of MT-II crude peptides synthesized by different combinations of side chain protecting groups

When comparing different protection schemes, combination 2 obtained the highest purity of crude peptide. However, Fmoc Asp (O-2-PhiPr) - OH is relatively expensive, which will increase the cost of subsequent scale-up production. The crude peptide obtained from combination 1 has the lowest purity, which is attributed to the poor deprotection effect on ODmab and ivDde by treatment with 2% hydrazine. In addition, although combination 3 exhibits much lower crude purity than combination 2, this is mainly related to the formation of asparagine after analysis. By minimizing this side reaction, the purity of crude peptides can be significantly improved. Therefore, Asp (OAlly)/Lys (Alloc) was selected as the monomer for synthesizing MT-II and optimized for the next step.

　　02 Small scale optimization of condensation conditions

We first studied the use of DIC and OxymAPure for the coupling step, without pre activation (4) and pre activation for 5 minutes (5). Like combination 3, these methods resulted in a significant decrease in crude peptide purity due to the formation of asparagine (Table 2). Therefore, MT-II is synthesized in a stepwise manner: the peptide is synthesized to His (His-D-Phe-Arg-Trp-Lys (Alloc) - resin) and condensed with Asp (OAlly) using HCTU/NMM (6) or DIC/OxymaPure (7). After Asp (OAlly) condensation, microcutting was performed, and no formation of aspartic imide was observed. The crude purity of these fragments is 88.9%.

Table 2 Purity of MT-II synthesized under different conditions

　　Synthesis of Linear Peptides

Linear MT-II was synthesized on PurePep Chorus at a scale of 100 μ mol on Rink Amide MBHA resin (0.35 mmol/g). Before synthesis, swell the resin in DMF for 2 × 10 minutes. The deprotection step is carried out using a 20% piperidine DMF solution for 2 × 5 minutes, and the condensation step is carried out using 5 equivalents of AA, 5 equivalents of DIC, and 5 equivalents of OxymaPure DMF solution (without preactivation-4, or 5-minute preactivation-5), at room temperature for 30 minutes. For fragments 7 and 9, Asp (OAlly) and Nle were condensed under the same conditions as 5. For fragments 6 and 8, Asp (OAlly) and Nle were condensed with HCTU (5 equivalents) and NMM (10 equivalents) in DMF at room temperature, with a pre activation time of 5 minutes and a single condensation time of 30 minutes. The terminal amine of MT-II was acetylated with a 10% Ac2O DMF solution at room temperature for 30 minutes.

Selective removal of protective groups from Asp and Lys side chains: Asp (OAlly) and Lys (Alloc) were deprotected by washing twice with 0.2 equivalents of Pd (PPh3) 4 in 24 equivalents of PhSiH in DCM solution at room temperature for 60 minutes, followed by washing three times with DCM and DMC respectively.

　　Resin cyclization

At room temperature, cyclize the resin with 5 equivalents of oxime and 5 equivalents of DIPEA in DMF solution for 60 minutes.

　　cutting

Wash the resin with DMF 3 times, DCM 6 times, and then dry for 20 minutes. Each cutting was performed using a mixture of TFA/EDT/H2O/TIS (94:2.5:2.5:1) at room temperature for 60 minutes, followed by precipitation in ether.

　　03在 PurePep Sonata+ 上放大 MT-II 的合成

After small-scale optimization synthesis on PurePep Chorus, Scheme 8 was found to be the optimal solution. Large scale (5 mmol) synthesis of MT-II on PurePep Sonata+.

According to the synthesis method and conditions of Scheme 8, MT-II was synthesized on PurePep Sonata+until Asp (OAlly) [Fmoc Asp (OAlly) - His D-Phe-Arg-Trp-Lys (Alloc) - resin] was formed, followed by selective removal of side chain protecting groups for subsequent resin cyclization. After cyclization, the sequence is completed by adding Nle and N-terminal acetylation. According to this strategy, MT-II with a crude purity of 80.2% was obtained, which is equivalent to the purity of the crude peptide synthesized on PurePep Chorus at a scale of 0.1 mmol.

　　Conclusion

In the PurePep Chorus multi-channel multifunctional fully automatic synthesizer, small-scale synthesis of MT-II can be achieved and different synthesis conditions can be explored and optimized. Subsequently, the optimal synthesis conditions can be smoothly applied to PurePep Sonata+, and the synthesis scale can be enlarged from 100 µ mol to 5mmol (increased by 50 times), resulting in a crude peptide purity of almost 80.1%.

●This study benefits from the flexibility of PurePep Chorus, and MT-II is fully automated from start to finish: from linear peptides to selective side chain deprotection and resin cyclization, different condensation conditions are selected for each step, and online micro cutting extraction samples can be tested during the synthesis process to adjust conditions based on experimental results. The process on PurePep Chorus can be smoothly transferred and scaled up to PurePep Sonata+, and the same results can be obtained.