What Is Solution Phase Peptide Synthesis 

Peptides are amino acid chains consisting of 30-50 amino acids that function as chemical messengers in an organism and are building blocks for proteins. Like other gene products (DNA, RNA, and proteins), peptide expression occurs naturally in living cells.  

Therefore, in vitro peptide expression systems generate peptides at an industrial scale for various research and industrial applications. However, such peptide expression systems have disadvantages regarding labor intensiveness, peptide modification, and the making of novel peptides. 

Consequently, researchers developed solution or liquid-phase peptide synthesis to overcome the shortcomings of expression systems. Therefore, learn more about the process below or click solution phase peptide synthesis

Solution-Phase Peptide Synthesis Overview 

Also called liquid-phase peptide synthesis (LPPS), solution-phase peptide synthesis (SPS)was the first cell-free peptide production method. According to one historical account, a German chemist, Emil Fischer, made the first chemically synthesized peptide in 1882 using a less advanced form of solution-phase peptide synthesis. 

Although groundbreaking, Fischer’s pioneering peptide synthesis project had one significant deficit, he was yet to discover a viable amino acid polymerization prevention strategy. However, Max Bergmann, Fischer’s student and later assistant, built on the chemist’s work and created the benzyloxycarbonyl (Z) group as the first amino acid reversible protection group. 

Reversible protection groups prevent amino acid polymerization during the condensation reactions involved in peptide synthesis. Polymerization alters the amino acid’s molecular structure and physical characteristics like temperature resistance, leading to a low yield of bioactive peptides. Moreover, protection groups also prevent side reactions during peptide synthesis. 

Solution-phase peptide synthesis was the standard synthetic peptide production method from the 1920s to the 1980s. However, biochemists started adopting solid-phase peptide synthesis in the early 1980s.  

Solid-phase peptide synthesis (SPPS) builds on the core SPS principles. The only difference between the two strategies is solid polymeric support in SPPS. 

SPS may be a classic peptide synthesis strategy but remains viable. Although most labs today use SPPS, SPS has several advantages over SPPS. Below is an overview of the steps used in solid-phase peptide synthesis. 

Steps In Solution-Phase Peptide Synthesis 

Solution-phase peptide synthesis is a stepwise process that uses reagents and solvents in a solution medium to generate target peptides. Below is a breakdown of the steps involved in SPS. 


Coupling reactions in SPS entail forming an amide or peptide bond between two amino acids in a solution medium. The coupling reaction begins with the reversible protection of the first amino acid. Reversible protection means that subsequent deprotection to facilitate peptide bond formation is possible. 

Protection Group Strategy Deployment 

Protection strategies are vital in preventing racemization or side reactions. Although different protection strategies exist, orthogonal protection of the carboxyl group is ideal for solution-phase peptide synthesis. 

The coupling process in SPS entails a condensation reaction between the carboxyl groups of two amino acids. Therefore, the reaction moves from the amino acid’s carboxyl (C-terminus) terminal towards the amine (N-terminus terminal). 

Orthogonal protection’s primary principle is facilitating the deprotection of a select segment from a whole entity without affecting the non-selected portions of the entity. Therefore, it allows for the deprotection of the carboxyl-terminal without affecting the N-terminal or the amino acids. The primary deprotection group used in SPS is the Fmoc protection group. 

SPS is the ideal peptide protection method for short-chain peptides because the amide bonds form between two amino acids. Regardless, it is possible to make longer peptide chains using the process by linking multiple nascent peptides in a solution medium. However, the process will take significantly longer and has a low probability of yielding a high target peptide volume. 


The nascent peptide formed undergoes purification with each cyclical reaction. While repetitive purification is labor-intensive and time-consuming, it produces peptides with high purity quality. Moreover, multiple purification methods help detect possible side reactions, facilitating timely interventions that prevent time and resource wastage. 

Difference Between Solution-phase Peptide Synthesis (SPS) and Solid-phase Peptide Synthesis (SPPS) 

As stated above, SPPS utilizes SPS principles but adds some modifications for enhanced efficiency. The primary improvement distinguishing SPS from SPPS is using insoluble polymeric support in the latter technique. 

Therefore, the coupling reaction in SPPS begins with a covalent attachment step linking the first amino acid to the non-soluble polymeric support. Second, SPPS utilizes a chain reaction that forms the desired nascent peptide, while SPS utilizes cyclical reactions between two amino acid carboxyl terminals. 

Third, the chain reaction utilized in SPPS means that only one purification step is necessary at the end of the process. Lastly, SPS’s use of a solution medium eliminates the need for additional steps like capping cleaving used in SPPS. 

Solid-Phase Peptide Synthesis Advantages 

Although chemists classify SPS as a classical peptide synthesis method, it has the following advantages over contemporary peptide synthesis techniques. 

  • SPS is economically scalable for large-scale peptide production. 
  • It is a better strategy for short peptide production 
  • Multiple purification steps used in SPS produce purer peptides and help in early side reaction detection 
  • The technique helps synthesize peptides with structures that are challenging to synthesize via SPPS. 

Solution-phase peptide Synthesis Improvements 

Although SPS is a classical peptide production method, modified versions of the technology help improve its efficiency. Such modifications include group-assisted purification (GAP) and a rapid, continuous solution phase; both improvements reduce SPS time requirements.   


Solution-phase peptide synthesis is a classic technique, but its principles still apply in contemporary peptide production. Therefore, the method remains helpful despite the ubiquity of new-age peptide synthesis techniques.  

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