Solid Phase Peptide Synthesis

Synthetic peptides are a kind of special drugs, which can be regarded as a kind of drugs between small organic molecules and protein macromolecules. Liquid phase synthesis and solid phase synthesis are the main methods of peptide drug synthesis. Compared with the classical liquid phase synthesis of peptides, solid phase peptide synthesis has become a conventional method of peptide synthesis and extended to other organic fields such as nucleotide synthesis because of its outstanding advantages of time-saving, labor-saving, material-saving.

The birth of solid phase peptide synthesis

The research of peptide synthesis has gone through a brilliant course of more than one hundred years. Emil Fischer, a German chemist who won the Nobel Prize in chemistry for successfully solving the structure of sugar and his research achievements in purine derivatives and peptides in 1902, first began to pay attention to peptide synthesis. Because the knowledge of peptide synthesis was too little at that time, the progress was quite slow. It was not until 1932 that peptide synthesis began to have a certain development.

In the 1950s, organic chemists synthesized a large number of bioactive peptides, including oxytocin and insulin, and made a lot of achievements in peptide synthesis and amino acid protective groups. This provides an experimental and theoretical basis for the emergence of solid-phase synthesis methods.

In 1963, Merrifield first proposed solid phase peptide synthesis (SPPS), which is a milestone in the peptide chemistry. As soon as it appeared, it became the preferred method of peptide synthesis because of its convenience and rapidness. It also brought a revolution in peptide organic synthesis, and became an independent subject-solid phase organic synthesis (SPOS). For this reason, Merrifield won the Nobel Prize in chemistry in 1984. Merrifield invented the first peptide synthesizer in the late 1960s and synthesized biological protease, ribonuclease (124 amino acids) for the first time.


Firstly, the hydroxyl groups of the hydroxyl terminal amino acids of the synthetic peptide chain are connected with an insoluble polymer resin by a covalent bond structure. Then the amino acid bound on the solid phase carrier is deamino-protected and reacts with excess activated carboxyl to lengthen the peptide chain. Repeat (condensation→washing→deprotection→neutralization and washing→condensation) operation to achieve the length of the peptide chain to be synthesized, and finally cleave the peptide chain from the resin and purify it to obtain the desired peptide. The α-amino protected by Boc (tert-butoxycarbonyl) is called Boc solid-phase synthesis, and the α-amino protected by Fmoc (9-fluorenylmethoxycarbonyl) is called Fmoc solid-phase synthesis. Successful SPPS depends upon the choice of the solid support, linker (between the solid support and the synthesized peptide), appropriately protected amino acids, coupling method, and protocol for cleaving the peptide from the solid support.

Solid Phase Peptide Synthesis

→ Boc synthesis method

During synthesis, a Boc-protected α-amino acid was covalently crosslinked to the resin, Boc was removed by TFA, and the free amino terminal was neutralized by triethylamine, then activated by DCC and coupled to the next amino acid. Finally, the target peptide was dissociated from the resin by strong acid HF method or trifluoromethanesulfonic acid (TFMSA). In Boc synthesis, because acid is repeatedly used to deprotect for the next step of coupling, some side reactions are introduced, such as peptides are easily removed from the resin, amino acid side chains are unstable under acidic conditions and side reactions occur.

→ Fmoc synthesis method

In 1978, Meienlofer and Atherton et al developed the Fmoc method for the synthesis of peptides using Fmoc (9-fluorenylmethoxycarbonyl) group as a protective group of α-amino groups. In the Fmoc method, Fmoc, which can be removed by alkali, was used as the protective group of alpha-amino acids, and the side chain was protected by acid removal of Boc. The advantage of Fmoc as an amino protection group is that it is stable to acid, the treatment with TFA and other reagents is not affected, only mild alkali treatment is needed. Finally, the peptide was quantitatively removed from the resin with TFA/dioxane (DCM) to avoid the use of strong acid. Compared with Boc method, Fmoc method is widely used in peptide synthesis because of its mild reaction conditions, few side reactions and high yield.

Dimethylformamide (DMF) and dichloromethane (DCM) is commonly used solvents in solid phase synthesis of peptides, especially DMF has high solubility to agents and products, so it is widely used in a variety of reaction systems. Although DMF has better solubility, it has a higher boiling point and needs to be evaporated under reduced pressure. Moreover, the by-product N-acylurea is easy to be formed in high dielectric constant solvents (DMF, CH2CN, DMSO, H2O, etc.), but not easily formed in low dielectric constant solvents (CH2Cl2, CCl4, C6H6, etc.). In nonpolar solvents, N-protected amino acids can react quickly with DCC to form symmetrical anhydrides. Therefore, as long as the reactants can be dissolved, the solvent with low dielectric constant should be chosen as far as possible. In solid phase synthesis, most of them use DCM as solvent, which has two advantages: lower racemization rate and slower formation of N-acylurea than using DMF as reaction solvent. When the carboxyl components are not easy to dissolve, a few drops of re-steamed DMF can be added to help the solution.

The most important feature of separating solid phase synthesis from other peptide synthesis techniques is solid phase carriers, and polymers that can be used as solid phase carriers of peptides must meet the following conditions:
→ It must contain suitable connecting molecules (or reaction groups) so that the peptide chain can be attached to the carrier and removed later.
→ It must be stable during synthesis and does not react with amino acid molecules.
→ Sufficient junctions must be provided to meet the growing needs of peptide chains.

At present, there are three main types of polymer carriers for solid phase synthesis: polystyrene-phenylene diethylene crosslinked resin, polyacrylamide, polyethylene-ethylene glycol resin and derivatives. Only when the corresponding connecting molecules are introduced into these polymer carriers can they be connected with amino acids. According to the different connecting molecules, resins are divided into several types: chloromethyl resin, carboxyl resin, amino resin or hydrazide resin.

An ideal linker must be very stable in the whole synthesis process, and can be cut off quantitatively without destroying the synthesized target molecule after synthesis. At the same time, linker to be selected according to the C-terminal structure of the peptide connected to the resin, such as carboxylic acid, amide or amino alcohol. Linker used in solid phase peptide synthesis are bifunctional compounds containing chloromethyl, mercapto, acyl chloride, p-benzoyl, aryl sulfonyl chloride, allyl, succinyl, o-nitrobenzyl and diphenylchlorosilane.

The formation principle of peptide bond in solid phase is basically the same as that in liquid phase, and the main methods used are condensation agent method, mixed anhydride method, acyl chloride method, activated ester method and so on. DCC, HOBT or HOBT/DCC symmetrical anhydride method and activated ester method are widely used because they can reduce side reactions and inhibit racemization in the process of peptide bond formation.

After the peptides have been synthesized in the established order, the target peptides should be cut off from the resin and further purified. Because there are two kinds of peptides synthesis: Boc method and Fmoc method, so their cutting methods are not exactly the same. In the Boc method, TFA + HF is mainly used for cleavage and side chain protection, and in the Fmoc method, TFA is directly used for cutting. The further purification, separation and purification of synthetic peptide chains are usually carried out by high performance liquid chromatography, affinity chromatography, capillary electrophoresis and so on. At present, high performance liquid chromatography is the most widely used.

Detection of solid phase synthetic peptides

Even the efficient coupling technology can not guarantee that the acylation reaction can be carried out at 100%. Moreover, the efficiency of the coupling reaction is greatly reduced when the sequences such as stereo barrier or bilayer are encountered. There are always missing or truncated peptide chains on the polymer carrier, and when they are released, they also enter into the product, which brings great difficulties to separation. Therefore, when solid-phase synthesis of peptides, especially longer peptides, the condensation rate of each amino acid should reach 99.9%, otherwise the product will be very impure. Therefore, it is particularly important to monitor the progress of each reaction.

Ninhydrin chromogenic method (Kaiser method) is a rapid determination of amino groups on the resin by ninhydrin color reaction, so as to determine whether the acylation reaction is complete. The sensitivity of ninhydrin method for the determination of amino group of polystyrene resin can reach 5μmol/g. This sensitivity can detect whether the condensation reaction has been carried out by more than 99%. In the detection of ninhydrin, the color intensity is different due to the difference of terminal amino acid residues and sequences. Aspartic acid (Asp) and asparagine (Asn) produce very weak blue or light brown. The reaction of the chromogenic reagent 2, 4, 4, 6-trinitrobenzenesulfonic acid with the amino group on the resin showed orange-red, and the sensitivity was 5μmol/g resin.

The salicylaldehyde method can be used to determine the amount of residual amino groups on the resin after receiving peptides and the total amino content after the removal of protective groups, which can quantitatively detect whether the condensation reaction and the removal of protective groups are complete. If not completely, it can be dealt with repeatedly in time. The amino group on the resin was reacted with 2% salicylaldehyde + 6% pyridine ethanol solution (60℃, 30min). After washing, the salicylaldehyde was replaced by 5% benzylamine ethanol solution (60℃, 30min). After the ethanol solution with benzylamine was diluted, the light absorption value of 315nm was read, and the amount of amino group was calculated.

The partially protected intermediate peptides were in the middle of peptide synthesis. A small amount of peptide resin (3~10mg) was cleaved, precipitated with ether, dissolved in appropriate solvents and directly analyzed by HPLC. During cleavage, the N-terminal protective group of the peptide was retained or removed as needed. When the synthesized peptide is a short polar peptide, the protective group can be retained, otherwise the retention time in HPLC is too short, which is not conducive to analysis. Although this method is troublesome, it takes a short time and has high accuracy.

Advantage of solid phase peptide synthesis


With the increasing demand for peptide products in the market, especially in medical and health, the peptide synthesis industry is developing rapidly. With the continuous improvement of peptide synthesis technology and the continuous improvement of synthesis instruments, rare amino acids can be introduced into the solid phase synthesis of peptides to study the structure and function of new proteins. Solid-phase synthesis of peptides has incomparable advantages in drug research, protein structure research, immunology research and so on.


  1. Amblard, M., Fehrentz, J. A., Martinez, J., & Subra, G. (2006). Methods and protocols of modern solid phase peptide synthesis. Molecular biotechnology, 33(3), 239-254.
  2. Behrendt, R., White, P., & Offer, J. (2016). Advances in Fmoc solid‐phase peptide synthesis. Journal of Peptide Science, 22(1), 4-27.
  3. Varnava, K. G., & Sarojini, V. (2019). Making solid‐phase peptide synthesis greener: a review of the literature. Chemistry–An Asian Journal, 14(8), 1088-1097.
  4. Palomo, J. M. (2014). Solid-phase peptide synthesis: an overview focused on the preparation of biologically relevant peptides. Rsc Advances, 4(62), 32658-32672.
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