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.
What is Solid Phase Peptide Synthesis?
Solid Phase Peptide Synthesis (SPPS) is a method for synthesizing peptides on an insoluble solid-phase support. The basic principle involves covalently attaching the carboxyl group of the first amino acid of the target peptide to the solid-phase carrier. Subsequently, protected amino acids are activated and sequentially reacted with the amino acid on the solid-phase carrier to form peptide bonds, thereby extending the peptide chain step by step. After each amino acid addition, the protecting group is removed, and the next round of amino acid coupling is performed. This technique simplifies the cumbersome separation and purification steps required in traditional solution-phase synthesis. By merely filtering and washing, unreacted reagents and byproducts can be removed, significantly improving synthesis efficiency. This enables more precise and efficient peptide synthesis, providing robust technical support for custom peptide services.
History 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.
Methods for Solid Phase Peptide Synthesis
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.
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.
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.
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Raw Materials for Solid Phase Peptide Synthesis
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.
- Polymer carriers for solid phase peptide synthesis
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:
1. It must contain suitable connecting molecules (or reaction groups) so that the peptide chain can be attached to the carrier and removed later.
2. It must be stable during synthesis and does not react with amino acid molecules.
3. 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.
- Linker for solid phase peptide synthesis
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 of peptide bond
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.
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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.
- Qualitative color 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.
- Quantitative free amino group detection
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.
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Analysis of Custom Peptide Service Demand
The demand for custom peptide services is currently experiencing robust growth. With the deepening of life science research and the rapid rise of the biopharmaceutical industry, the scale of demand for custom peptide services continues to expand.
| Field | Special Requirements | Applications |
|---|
| Medical | Extremely high purity and activity requirements; strict adherence to Good Manufacturing Practices (GMP). Custom peptides must have nearly 100% sequence accuracy and ensure batch-to-batch consistency. | - Development of disease diagnostic reagents, such as tumor marker detection kits;
- Therapeutic drugs, such as insulin analogs for diabetes treatment;
- Vaccine development to stimulate immune responses.
|
| Research | Custom peptides with unique sequences to meet various experimental needs. Requires relatively fast synthesis speeds to quickly obtain peptides for experiments. | - Protein interaction studies, using custom peptides to mimic specific protein regions;
- Cell signaling research, using peptides to activate or inhibit specific pathways;
- Drug target validation, determining drug-target interaction mechanisms.
|
| Cosmetics | Focus on peptide safety and stability, complying with cosmetic regulations. Specific requirements for peptide appearance and odor. | - Anti-wrinkle and firming, using specific peptides to promote collagen production;
- Whitening and spot reduction, custom peptides that inhibit melanin generation;
- Moisturizing and repair, using peptides to enhance skin barrier function.
|
Advantages of Solid Phase Peptide Synthesis
1. Efficiency and Convenience
SPPS technology significantly enhances peptide synthesis efficiency. In traditional solution-phase synthesis, each reaction step requires cumbersome separation and purification, making the process complex and time-consuming. In contrast, SPPS immobilizes the peptide chain on a solid-phase carrier, allowing excess reagents to drive the reaction forward. Subsequent washing steps easily remove unreacted reagents and byproducts, eliminating the need for complex separation operations. This enables continuous and rapid synthesis, greatly shortening the synthesis cycle and allowing target peptides to be obtained in a shorter time, meeting urgent research and production needs.
2. High Purity and Quality
SPPS excels in ensuring peptide purity. The solid-phase carrier creates a relatively isolated reaction system, reducing impurity introduction. Additionally, multiple washing and purification steps during synthesis effectively remove unreacted amino acids and byproducts. SPPS also allows precise control over reaction conditions, ensuring the accuracy of each step and thereby improving peptide purity and quality. This meets the high-quality demands of medical and research fields.
3. Flexibility and Scalability
SPPS offers strong flexibility. It can easily adjust peptide sequences, lengths, and modifications based on different needs. Whether synthesizing simple short peptides or complex long peptides, SPPS can achieve the desired results through rational design. Moreover, the technology is highly scalable, enabling both small-scale laboratory synthesis and large-scale industrial production by adjusting reaction scales and equipment. This provides robust support for the industrial development of custom peptide services.
4. Ease of Automation
SPPS is easily automated. Due to its relatively fixed reaction steps, automated instruments can precisely control various parameters such as reagent quantities, reaction time, and temperature. Automation not only improves synthesis accuracy and repeatability, reducing human error, but also significantly lowers labor costs. This allows custom peptide services to operate more efficiently and stably, meeting the demands of large-scale production and diverse market needs.
Key Steps of SPPS in Custom Peptide Synthesis
1. Amino Acid Selection and Protection
In custom peptide synthesis, amino acid selection is crucial. Suitable amino acids must be chosen based on the target peptide sequence. Different amino acids have unique chemical properties, such as polarity, charge, and hydrophobicity, which influence peptide structure and function. For example, peptides rich in polar amino acids may be more water-soluble, suitable for aqueous environments, while peptides with more non-polar amino acids may be useful in membrane protein studies.
To ensure synthesis accuracy and efficiency, amino acids must be protected. Common protection methods involve introducing protecting groups to active sites. For instance, the amino group is often protected with tert-butoxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc), while the carboxyl group can be protected via esterification. This prevents unreacted active groups from participating in unwanted side reactions, ensuring precise peptide bond formation at specific locations. Protecting amino acids acts as a "navigation system" for synthesis, guiding the peptide chain to grow in the desired sequence and direction, improving synthesis success rates and product purity.
2. Stepwise Peptide Chain Synthesis
Stepwise peptide chain synthesis on a solid-phase carrier is a meticulous and orderly process. First, the carboxyl group of the first amino acid is covalently attached to the solid-phase carrier, laying the foundation for subsequent chain extension. Next, the protected second amino acid is activated to enhance its reactivity. The activated amino acid, with the help of a coupling agent, reacts with the amino group of the amino acid on the solid-phase carrier to form a peptide bond.
Reaction conditions must be carefully controlled. Temperature should be precisely regulated, typically at a low level to minimize side reactions and ensure selectivity. Reaction time must also be strictly managed; too short a time may result in incomplete reactions, while too long may cause unwanted side reactions. Additionally, the pH of the reaction system affects the process, with an optimal pH promoting peptide bond formation.
After each amino acid coupling, the protecting group is removed to prepare for the next round of coupling. Through this cyclic process, the peptide chain extends step by step on the solid-phase carrier according to the predetermined sequence, akin to constructing a molecular building with precision at every step.
3. Post-Synthesis Processing and Purification
Post-synthesis processing and purification are critical for ensuring peptide quality. First, the synthesized peptide must be cleaved from the solid-phase carrier using specific cleavage reagents under appropriate conditions.
Purification typically involves high-performance liquid chromatography (HPLC). HPLC separates and purifies peptides based on differences in distribution coefficients between the stationary and mobile phases. By selecting suitable columns and mobile phase conditions, impurities, unreacted amino acids, and byproducts can be effectively removed, enhancing peptide purity. Other methods, such as ion-exchange chromatography and gel filtration chromatography, can also be used for auxiliary purification.
Quality control is essential during purification. Mass spectrometry determines the peptide's molecular weight to verify sequence accuracy. Nuclear magnetic resonance (NMR) analyzes peptide structure to ensure correct folding and conformation. HPLC combined with UV detection measures peptide purity to meet application standards. Only through rigorous processing, purification, and quality control can custom peptides meet the high-quality demands of various fields.
SPPS Workflow
1. Preparation Phase
Adequate preparation is essential before starting SPPS. Suitable amino acid raw materials must be carefully selected based on the target peptide sequence. This is akin to building a grand structure, where each "building block"—amino acid—must be of high quality and appropriate characteristics. The solid-phase carrier, such as polystyrene resin, must also be prepared, serving as the "solid platform" for peptide synthesis. Additionally, protecting groups, coupling agents, activators, and other chemical reagents must be ready, as these "magic potions" play key roles in synthesis. Laboratory equipment, such as reaction vessels, stirring devices, and temperature control systems, must be checked to ensure proper operation, creating optimal hardware conditions for synthesis.
2. Initial Amino Acid Attachment
This is the critical starting point of the synthesis process. The carboxyl group of the first amino acid is covalently attached to the solid-phase carrier through a specific chemical reaction. Reaction conditions, including temperature, time, and pH, must be precisely controlled. Only by ensuring the stable attachment of the initial amino acid can the foundation for subsequent peptide chain extension be laid, much like laying a solid foundation for a tall building.
3. Cyclic Synthesis Steps
This is the "relay race" of peptide chain growth. Each cycle involves multiple key steps. First, the amino acid to be added is activated, making it more reactive. The activated amino acid, with the help of a coupling agent, forms a peptide bond with the amino acid on the solid-phase carrier. After this step, the protecting group on the newly added amino acid is removed to prepare for the next round of coupling. This cycle repeats, extending the peptide chain on the solid-phase carrier according to the predetermined sequence, much like weaving a complex yet orderly molecular chain.
4. Post-Synthesis Processing
Once the peptide chain reaches the desired length, the synthesis phase concludes, but post-synthesis processing is equally important. First, specific cleavage reagents are used under appropriate conditions to release the synthesized peptide from the solid-phase carrier. Purification follows, a critical step to refine the peptide. Depending on the peptide's properties, methods such as HPLC, ion-exchange chromatography, or gel filtration chromatography can be used to remove impurities, unreacted amino acids, and byproducts, achieving high purity.
5. Quality Control Phase
This is the "checkpoint" for ensuring peptide quality. Mass spectrometry precisely measures the peptide's molecular weight to verify sequence accuracy. NMR analyzes the peptide's structure to ensure correct folding and conformation. HPLC combined with UV detection accurately measures peptide purity. Only when the peptide passes all quality control tests can it be deemed a qualified product, meeting the stringent demands of various fields.
Case Studies of Successful Custom Peptide Services
Medical Field
In a cancer diagnostics project, SPPS technology played a key role in developing highly sensitive and specific tumor marker detection reagents for early cancer screening. Custom peptides mimicking specific tumor-associated antigen epitopes were synthesized using SPPS.
These custom peptides were used to prepare key components of diagnostic kits. In clinical testing, the kits demonstrated exceptional performance, accurately identifying trace amounts of tumor markers in early-stage cancer patients, significantly improving diagnostic accuracy. For example, in lung cancer screening, the misdiagnosis rate dropped from 20% with traditional methods to less than 5% with the custom peptide-based kit.
In cancer treatment, a custom peptide drug targeting a rare blood disorder was developed using SPPS. The peptide's sequence and structure were precisely controlled to specifically bind abnormal receptors on diseased cells. Clinical trials showed significant reduction in diseased cells and symptom relief in patients, with some achieving long-term stability. This highlights the immense value of SPPS in disease diagnosis and treatment.
Research Field Case
In a cell signaling study, SPPS technology enabled significant breakthroughs. Researchers aimed to map the interaction network of key proteins in a specific signaling pathway.
Custom peptides identical to specific protein regions in the pathway were synthesized using SPPS. These peptides were used to mimic protein interactions in cells. For example, one custom peptide specifically blocked the binding of two key proteins, interrupting signal transduction. By observing cellular responses and gene expression changes, researchers successfully mapped the signaling pathway, clarifying each protein's role. This provided critical insights into cell signaling mechanisms and laid the foundation for developing new drugs targeting this pathway, showcasing SPPS's power in advancing research.
References
- 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.
- Behrendt, R., White, P., & Offer, J. (2016). Advances in Fmoc solid‐phase peptide synthesis. Journal of Peptide Science, 22(1), 4-27.
- 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.
- 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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