Using a "handle" to master peptide synthesis

I. Background Introduction

In recent years, with the rapid development of the biopharmaceutical field, peptide drugs have become "star molecules" between small molecule drugs and large molecule drugs due to their unique advantages. However, the synthesis of peptide drugs has always faced enormous challenges. How to efficiently and accurately connect different molecular fragments and simplify the purification process is a core challenge for chemists.

Currently, peptide synthesis mainly relies on classical liquid-phase peptide synthesis (LPPS) and solid-phase peptide synthesis (SPPS), but both methods have significant limitations. While LPPS offers high reagent utilization efficiency, low cost, and ease of scale-up, it is limited to short peptide preparation and requires cumbersome separation and purification after each reaction step. SPPS, although simple to operate and easily automated, consumes a large amount of solvent and tends to accumulate on resins, posing challenges such as high cost and environmental impact in large-scale production. Furthermore, the physicochemical properties of peptide intermediates are highly dependent on the amino acid sequence; highly lipophilic or poorly soluble intermediates can complicate process development. To address these synthetic challenges, developing a new method that simplifies purification and is easy to scale up is imperative. Tag-assisted peptide synthesis (TAPS) has emerged as a clever strategy to address this need.

The table below compares three methods for synthesizing polypeptides:

II . Technical Principles

The basic idea is to temporarily attach "handles"—that is, soluble tags (or anchoring molecules)—to the raw material molecules participating in the reaction, so that the peptide chain remains completely soluble in certain solvents, thus facilitating peptide chain growth. Then, by taking advantage of the poor solubility of the tags in specific unsuitable solvents, the tags are precipitated out. After filtration and simple washing, excess reagents and byproducts can be efficiently removed.

This post-processing method greatly simplifies the separation process, improves the efficiency of synthesis and the purity of the product, while avoiding the dependence of solid-phase synthesis on special automated equipment and can be successfully operated in traditional batch reactors.

So, what conditions must the introduced tags meet? Mainly the following three points:

1. It can protect the C-terminus and is orthogonal to the N-terminal protecting base;

2. It can improve the solubility of molecules in organic solvents, facilitating peptide chain growth;

3. It can precipitate in poor solvents, simplifying the purification process.

Its workflow typically follows these three steps:

1. Tag connection : At the start of synthesis, the tag is connected to the starting material.

2. Chain extension and reaction : In solution, tagged molecules undergo a series of chemical reactions to facilitate peptide chain extension. Specially designed tagged molecules have unique solubility and can be easily separated from by-products/raw materials without the need for complex purification.

3. Tag Removal : After synthesis, the tag is cleanly and efficiently removed under specific chemical conditions (such as acid, base, light, catalyst, etc.) to obtain the target molecule.

III . Technology Types

The common types of tags are introduced below:

IV . Application

Small molecule hydrophobic tags mainly include benzyl, diphenylmethyl, and fluorene -derived tags, which are currently used in the production of commercial peptide drugs. For example, when using a diphenylmethyl tag to synthesize eptifibatide (Figure 1), a cyclic process of "coupling-precipitation" and "deprotection-precipitation" yields an eptifibatide intermediate with a protecting group in 86% yield. Finally, after detaglation and cyclization, the target product eptifibatide is obtained with an overall yield of 63% and no obvious racemization . However, when the peptide is prepared using SPPS, the reactivity of coupling and deprotection is significantly reduced, and deletion peptides are easily generated during peptide chain elongation.

Synthesis of eptifibatide using a diphenylmethyl tag.

V. Outlook

In summary, tag-assisted peptide synthesis technology offers a highly efficient, sustainable, and easily scalable alternative to traditional peptide synthesis. It fully combines the advantages of solid-phase and liquid-phase synthesis, achieving greater flexibility, lower costs, and better environmental sustainability. Compared to traditional solid-phase synthesis, this method significantly reduces solvent and reagent consumption, greatly simplifies post-processing steps, and is easily scaled up from laboratory scale to ton-scale production. With its successful validation in peptide drug synthesis, this technology holds promise for driving the development of peptide drugs towards greater efficiency, greener practices, and greater economic benefits.

VI. Company Introduction

Suzhou Haofan Biotech Co., Ltd. (Stock Code: 301393.SZ), founded in 2003 and headquartered in Suzhou High-tech Zone, is a national high-tech enterprise providing specialty raw materials to pharmaceutical R&D and manufacturing companies worldwide. Its products are mainly used in the synthesis of peptides, nucleotides, and pharmaceuticals, covering a wide range of products including condensing agents for specialty amide bonds, protective agents, linking agents, protein cross-linking agents for antibody-drug conjugates, molecular building blocks, liposomes, and phosphorus reagents. To date, it has cumulatively developed and produced over 1,500 different products.

After more than two decades of unremitting efforts and accumulation, Haofan Biotech has continuously cultivated its expertise in the global peptide synthesis reagent field. It has now developed into a leading enterprise with extensive customized product coverage and significant advantages in large-scale production, capable of meeting the specific needs of various customers. We sincerely invite customers interested in this product to contact us to learn more about product details and explore cooperation opportunities.

参考文献：

[1] Liquid-Phase Peptide Synthesis (LPPS): A Third Wave for the Preparation of Peptides

DOI: 10.1021/acs.chemrev.2c00132

[2] Novel diphenylmethyl-Derived Amide Protecting Group for Efficient Liquid-Phase Peptide Synthesis: AJIPHASE.

DOI: 10.1021/ol302002g