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Introduction

Peptides, which possess the characteristic of remarkable potency, selectivity and low toxicity, has attracted many researcher’ and industrial group’s attention. At present, there are approximately 80 peptide drugs on the global market, more than 150 peptides in clinical development and another 400~600 peptides undergoing preclinical studies. However, peptide synthesis, especially hydrophobic peptides remain challenging. This article aims to provide an overview of hydrophobic peptide production challenges and solutions.

What are hydrophobic peptides?

Peptides are formed by arranging different amino acids in sequence. According to the difference in polarity, amino acids can be divided into polarity (such as Asp, Glu and Arg) and non-polarity amino acids (such as Leu, Val, and Ile). Hydrophobic peptides are peptides sequences that contain high number of non-polarity amino acids with hydrophobic side chains.

Why are hydrophobic peptides challenging to produce?

Hydrophobic peptides are mainly obtained via chemistry synthesis, and there are three key procedures to be considered for a successful production, including peptide synthesis, analytical methods, and purification.

• 1) Peptide synthesis. The main challenge lies in the high aggregation potential and low solubility in aqueous or organic solvents of those peptides and its original amino acids. Besides, the internal nature for those peptides that is readily to form α-helix or β-sheet in their secondary structure further exacerbates the difficulty of peptide coupling. This is promoted by the inter- or intra molecular, non-covalent interactions induced by those amino acids. Among them, Gly is known to induce β-sheet packing in combination with non-polarity amino acids. Although multiple coupling reagents has been developed, such as HATU/DIEA, PyBOP/HOBt/DIEA, DIC/HOBt and HATU/HOAT/DIEA, the peptide elongation for those amino acid is still a big matter. Moreover, Kaiser test, which is used for process control during peptides coupling, is no more applicable if the synthesis sequence reaching to a certain of length or the aimed peptide is very hydrophobic, which further complicates the synthesis of hydrophobic peptides.
• 2) Analytics methods.Purity identification by HPLC and molecular weight identification by MS are two major methods used for structure identification of peptides. However, the solubility of hydrophobic peptides significantly affects the accuracy of these assessments. In some cases, formic acid, acetic acid, HFIP or else need to be added in order to enhance solubility.
• 3) Purification.This is also related to the solubility of hydrophobic peptides, as RP-HPLC is normally used to purify peptides. The purification procedure may fail if the peptides can’t be fully dissolved. Especially for hydrophobic peptides, as they can often form high inter-molecular interactions with stationary phase of the column and be adsorbed to the column, this frequently leads to failed product generation.

Strategies on effective hydrophobic peptides production

To effectively synthesize hydrophobic peptides, many new methods and techniques were developed, like non-polar resins and cleavable tags. TFE, DMSO and NMP are sometimes used to inhibit peptide aggregation during peptide synthesis, thus improving the success rate of hydrophobic peptides. Chaotropic agents (like SCN- and ClO4-), surfactants (like POPC and OG), and ionic liquids (like [C2mim][OAc]) are additional solvents to break the unfavorable secondary structure or enhance solubility during SPPS. O-acyl isopeptides and pseudoprolines strategy are another two useful ways to obtain hydrophilic peptides. However, among those methods, temporary internal modifications of those peptides with hydrophilic tags have attracted the attention of many researchers, as it can not only resolve the synthesis issue, but also give some useful instruction for peptide purification.

Synthesis of hydrophobic peptides with the help of hydrophilic tags

This method is generated by adding hydrophilic tags to the aimed hydrophobic peptides during SPPS. These hydrophilic tags are usually made of multiple hydrophilic amino acids (usual K and R), and are attached to the hydrophobic sequence by a special linker. This method is designed towards facilitating purification via HPLC, peptide condensation or NCL.

Hydrophilic tags can be classified into two categories: (1) modifications, removable during cleavage from the resin. (2) modifications, removable after cleavage from the resin. The first group of modifications only facilitates the SPPS by preventing on-resin aggregation of “difficult sequences”, while the latter is stable during final cleavage from solid support, which can additionally improve the handling and purification of poor soluble peptides. Once the purification procedure has been finished, another procedure will be conducted to remove the hydrophilic tags.

Hydrophilic tags can be designed to attach to the N-terminal, C-terminal or side chain of amino acids. The application and characteristics of hydrophilic tags

1. C-terminal hydrophilic tags

C-terminal tags is the most applied strategy to increase the solubility of poorly soluble peptides. In 1996, Englebretsen and Alewood (1996) first tested a C-terminal polyglycine-arginine tail to increase the solubility of the peptide CP10 (42–55). All these first approaches were mainly used to aid the purification of hydrophobic peptides via HPLC (Method 1). Especially the use of C-terminal tag in combination with thioester peptides. Therefore, the solubilizing tag can be directly attached and removed during NCL (Method 2). Also, researches use 4-Hmb and Mmsb as linker to aid the synthesis and purification of hydrophobic peptides. The former depends on ester bond to accomplish the introduction of tags and remove under 0.1 M NaOH conditions, while the latter is removed under NH4I/TFA conditions. Both of them are stable under peptide synthesis and cleavage procedure, thus solving the purification issue of hydrophobic peptides.

2. N-terminal hydrophilic tags

N-terminal hydrophilic tags are mostly used to increase the solubility of hydrophobic peptides for purification. Below are two methods used to incorporate hydrophilic tags.

3. Other hydrophilic tags

There are other hydrophilic tags that are closely related to the side chain group of different amino acids, like Gly, Cys, Asp. Phacm linker can be attached to the thiol group of Cys and selectively removed by MgCl2/PdCl2 in 6 M Gdn HCl, while the Trt-K linker linked to the thiol group of Cys by a liquid modification procedure with purified peptides, and removed under TFA/TIS conditions, but the latter giving a low removal efficiency with about 41%. Another type of hydrophilic tag linkers including Ddae linker for Lys, Nitrobenzene linker for Gln, and Dbz-Trt-Kn linker for Asp/Gln/Glu/Asn.

The inner nature of hydrophobic peptides determined its complexity in production. Although using methods mentioned above, hydrophobic peptides synthesis and purification can be improved, but not in all cases. For example, some hydrophobic peptides can’t be purified by the usual RP-HPLC method, as they can form high interaction with C18 column, thus induing no product eluted out. Therefore, eliminating the use of HPLC in the final purification step is another way to obtain hydrophobic peptides.

At GenScript, we optimized the synthesis process and purification methods, also developed a new method to obtain hydrophobic peptides by adding hydrophilic tags to the aimed peptides. After peptides synthesis, cleavage, and purification of prepeptides, the aimed hydrophobic peptides can be produced by removing hydrophilic tags. Then, washing and centrifugation to obtain target product, avoiding the use of RP-HPLC.

Interested in learning more about our hydrophobic peptide synthesis capabilities, please contact us at peptide@genscript.com.