1. What is the basic principle of peptide synthesis?
Solid-phase peptide synthesis (SPPS) is a major breakthrough in peptide synthesis chemistry. Its most significant feature is that intermediate products do not need to be purified, allowing the synthesis process to proceed continuously, which has laid the foundation for the automation of peptide synthesis. Currently, fully automated peptide synthesis is largely based on solid-phase synthesis.
The basic process is as follows: Based on fluorenylmethyloxycarbonyl (Fmoc) chemistry, the carboxyl group of the C-terminal amino acid of the target peptide to be synthesized is first covalently bonded to an insoluble polymer resin. The amino group of this amino acid is then used as the starting point for peptide synthesis. A peptide bond is formed by reacting the activated carboxyl group of the next amino acid with the amino group of the growing peptide chain. By continuously repeating this process, the peptide is synthesized.
Depending on the amino acid composition of the peptide, different post-synthesis treatments and purification methods may be needed.
2. How is the quality of custom peptide services controlled?
At Yanfen Biotech, peptides are strictly produced in accordance with the ISO quality management system. Each peptide is assigned a unique identification number for tracking. Quality control tests, including High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC/MS) analysis, are performed on the crude product, in-process samples, and final lyophilized product to verify product identity and ensure purity.
3. What are the technical expertise and delivery times for Yanfen Biotech’s peptides?
Yanfen Biotech has accumulated numerous internationally advanced techniques in difficult peptide synthesis. We can achieve the chemical synthesis of long-chain peptides using peptide fragment synthesis and ligation technologies. Additionally, we have developed solubilization technologies for synthesizing highly hydrophobic membrane protein fragments. With these, we have fully synthesized the longest membrane protein chemically synthesized globally. Furthermore, by using unnatural amino acids to replace disulfide bonds, we have successfully achieved peptide cyclization, which enhances the stability of synthetic peptide drugs. Standard peptides can typically be delivered within one week, while most custom peptides are ready for delivery within three weeks. Quantities range from milligrams to kilograms.
4. What is peptide content, and what is its significance?
The weight of dry peptides includes not only the peptides themselves but also non-peptide components such as water, residual organic solvents, and salts. The net content of a peptide refers to the percentage by weight of the peptide within this mixture. This percentage can vary significantly, ranging from 80% to 95%, depending on factors like purity, sequence, and the methods used for synthesis and purification.
It is crucial to differentiate between net content and purity, as they represent two separate concepts. Purity, typically assessed by HPLC, is defined as the percentage of peptides that have the correct amino acid sequence. On the other hand, net content refers to the proportion of peptide substances in relation to non-peptide substances present in the sample. Net content is usually determined through amino acid composition analysis or UV spectroscopy. This information is crucial in experiments sensitive to peptide concentration, as it aids in calculating the actual concentration of the peptides.
5. How to store peptides?
Peptides should be stored in a dark environment for long-term preservation, ideally at -20°C, while short-term storage can be done at 4°C. They can be transported at room temperature for brief periods. After removing the freeze-dried peptides from the freezer, they can be stored at room temperature in a desiccator before being exposed to air. This practice effectively helps to minimize any increase in moisture content.
If freeze-drying is not an option, the most effective strategy is to store small working quantities of the peptides. For those containing Cys, Met, or Trp, it is crucial to use reducing buffers during dissolution because these peptides are susceptible to oxidation when exposed to air. Additionally, passing nitrogen or argon gas over the peptides before sealing them in a vial can further mitigate oxidation risks. Peptides that include Gln or Asn are also prone to degradation, which limits their shelf life compared to peptides without these residues.
6. Why do some peptides have lower synthesis yields or purity?
The synthesis of peptides differs significantly from oligonucleotide synthesis, where there are very few instances of unsynthesizable primers. In contrast, it is common to encounter peptides that cannot be synthesized effectively. Amino acids such as Val, Ile, Tyr, Phe, Trp, Leu, Gln, and Thr can hinder the complete unfolding and solubilization of the peptide chain during the synthesis process, resulting in decreased yield.
Certain scenarios lead to lower yield and product purity, such as the repetition of Pro, Ser-Ser, or Asp, as well as having four consecutive Gly residues. These configurations can cause structural issues that interfere with successful synthesis.
7. If my peptide purity is 95%, what constitutes the remaining 5%?
During the synthesis process, the efficiency of cross-linking between amino acids may fall short of 100%, resulting in various impurities, primarily peptides that lack one or more amino acids in their sequences. Although most of these impurities are eliminated during purification, some may exhibit chromatographic behavior similar to that of the target peptide. The remaining impurities, which consist of these amino acid-deficient peptides, account for the difference between the peptide's purity and 100%.
8. How are peptides purified?
Peptide purification generally employs reverse-phase columns, such as C8 and C18, using a detection wavelength of 214nm. The buffer system typically consists of solvents that contain trifluoroacetic acid (TFA) at a pH of 2.0. Buffer A is made up of 0.1% TFA in deionized distilled water (ddH₂O), while Buffer B consists of 1% TFA in acetonitrile (ACN) at the same pH. Prior to purification, the peptide is dissolved in Buffer A; if it does not dissolve well, Buffer B can be used first, followed by dilution with Buffer A. For peptides with high hydrophobicity, adding a small amount of formic acid or acetic acid may be beneficial.
HPLC is used to analyze the peptide product. Peptides with fewer than 15 amino acids usually exhibit a distinct main peak, which typically indicates the full-length product. For longer peptides (greater than 20 amino acids), if multiple peaks are observed, it is advisable to use mass spectrometry in conjunction with HPLC to ascertain the molecular weight and determine which peak corresponds to the desired peptide.
9. How to evaluate the solubility of a peptide from its sequence?
If a peptide contains a high proportion of strongly hydrophobic amino acids such as Leu, Val, Ile, Met, Phe, and Trp, it is likely to be difficult to dissolve in aqueous solutions. These amino acids can pose challenges during both purification and synthesis processes.
In general, if the proportion of hydrophobic amino acids in a peptide is less than 50% and there are no stretches of five consecutive hydrophobic amino acids, along with approximately 20% charged amino acids (such as Lys, Arg, His, Glu, and Asp), the solubility of the peptide may improve. Additionally, increasing the number of polar amino acids at either the N- or C-terminus can further enhance solubility.
10. What factors should be considered when conjugating peptides with other compounds or carriers?
Peptides often need to be connected to certain drugs or functional compounds, or they may need to be linked to specific carriers using chemical bonds. Generally, peptides possess amino or carboxylic acid functional groups, which can react with corresponding carboxylic acids or amines for condensation. Another option is to connect via thiol groups with maleimide under pH 8 conditions. Modifications in peptides can be made using cysteine or maleimide functional groups. Maleimide modifications are often achieved with the compound 3-Maleimidopropionic Acid (CAS 7423-55-4).
