Solid-phase Peptide Synthesis Services

Peptides play a pivotal role in biological, medical and pharmaceutical research. Synthetic peptides have very diverse uses in different fields, including as biologics, in epitope mapping, in peptide microarrays, and in vaccine development. Among the peptide synthesis techniques, solid-phase synthesis has developed into a efficient techniques for the preparation of numerous peptides and even small molecule proteins.

Amerigo Scientific provides solid-phase peptide synthesis services for a wide range of applications at a highly competitive price. Our service can provide a wide range of modification solutions to meet customer customization requirements. Get an instant quote by filling out the form, or order online to speed up your project.

What is Solid-phase Peptide Synthesis

Solid-phase peptide synthesis (SPPS) is a method to synthesize peptides by attaching peptide chains to polymeric solid support, which has become a mature industrial process and the first choice for peptide production due to its flexibility and cost-effectiveness. SPPS can be used to synthesize peptides with different structures ranging from 5 to 150 amino acids, and can also be used to assemble peptides with non-native or modified residues.

SPPS technology requires Nα-protecting groups, side chain protecting groups, coupling reagents, linkers, and solid supports. The main features of SPPS are:

• The first building block is attached (anchored) to a matrix that can be filtered;
• Repeated cycles of chemical transformation are performed, also by automation;
• The end product is released from the matrix and deprotected in the same step.

Advantages of Solid-phase Peptide Synthesis

SPPS is a scalable, economical, convenient, and efficient method for peptide synthesis. SPPS has a distinct advantage over the synthesis in solution because the coupling reaction can be performed more rapidly using an excess of activated amino acid derivatives, and these amino acid derivatives are removed by simple washing operations at the end of the reaction.

Since new chains are anchored to a solid support, a large excess of reagents can be used to ensure proper coupling of new amino acids or segments to growing peptide chains. Due to the stability of the solid support, excess reagents can also be easily removed by washing and filtration, significantly simplifying the purification process. In addition, all steps of SPPS can be completed in a single reactor without the need to transfer materials, effectively reducing production costs and operation time. Alternatively, solid supports can be packaged in columns and used in continuous flow mode. Therefore, excess reagents and washing chemicals are pumped through the column to speed up the synthesis process.

The Process of Solid-Phase Peptide Synthesis

The SPPS process involves repetitive cycles, each adding one amino acid. It begins by attaching the C-terminal amino acid to the solid support. Then, the N-terminal protecting group is removed, revealing a reactive amine. The next protected amino acid is then coupled using an activating agent, forming a new amide bond. Diverse coupling reagents are used, such as carbodiimide (such as DCC or DIC), or in situ coupling reagents (such as PyBOP, TBTU, HATU, COMU). After each deprotection and coupling, excess reagents are removed thorough washing. In addition, capping can prevent the formation of deletion peptides by blocking unreacted sites.

Due to the cyclic nature of SPPS, deprotection commonly occurs several times during the synthesis process. Therefore, deprotection should be rapid and preferentially take place under mild conditions to avoid removal of side chain protecting groups, and the by-products should be easily eliminated. Activation of the carboxylic acid moiety of the amino acid is necessary to achieve its reaction with the Nα-amino group in the forming peptide chain.

During the synthesis process, it is critical to cleave the peptide from the solid support and remove all residual side-chain protecting groups while minimizing alterations to the peptide’s composition. In standard SPPS process, the cleavage and deprotection are conducted in the presence of 1-5% trifluoroacetic acid (TFA). However, using this reagent is prone to generating highly reactive cationic species that can alter amino acids in the peptide backbone, particularly those containing nucleophilic groups. To mitigate this undesirable effect, nucleophilic scavengers are added to neutralize the reactive cations. Common scavengers include triethylsilane (TES), ethanedithiol (EDT), and phenol.

Protecting Groups

The terminal protection of amino acids is a challenging in peptide synthesis. Most peptide synthesis processes proceed in the C-to-N direction, where the α-amino protecting group is removed in each synthesis cycle. These groups need to ensure solubility and prevent or minimize isomerization, particularly during the coupling stage. The most common α-amino protecting groups in SPPS are the 9-fluorenylmethyloxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc) groups.

Solid Supports

The solid support of the SPPS consists of a stable resin and reversible linkers. A number of resins have been developed for use in SPPS, including polyethylene glycol polyacrylamide (PEGA), cross-linked acrylate ethoxy resin (CLEAR), and augmented surface polyethylene (ASPECT) resins. CLEAR resin is a commonly used material for SPPS due to its high crosslink ability, stability, and good solubility in a variety of solvents. PEGA resin is unique in being permeable to proteins up to 35-70 kDa, which makes it well suited for biochemical studies of peptides immobilized to the support. ASPECT is a chemically modified PEGA resin that has been optimized with improved properties, including the ability to accommodate large numbers of peptide chains, greater stability, and responsiveness to different solvents and conditions.

Linkers

Efficient SPPS depend on the selection of suitable linker. The ideal linker should facilitate attachment of the starting amino acid to the solid support, be sufficiently stable under a wide range of reaction conditions, and allow selective cleavage at the end of synthesis without degradation of the final product.

The following table lists common linkers: