Custom Peptide Synthesis - an overview
Peptides are short chains of amino acids that play a crucial role in various biological processes. Custom peptide synthesis is an essential tool in life sciences, enabling researchers to create peptides with specific amino acid sequences for a wide range of applications. This article explores the technology employed in custom peptide synthesis, the methods used to modify and purify peptides, and some notable applications in the field.
Technology in Custom Peptide Synthesis
Solid-Phase Peptide Synthesis (SPPS) is the primary technology utilized in custom peptide synthesis. In this method, peptides are constructed step-by-step on a solid support. Different resins are commonly employed as solid supports, including polystyrene, cross-linked polystyrene, and polyethylene glycol. These resins provide a stable matrix for attaching the growing peptide chain and facilitate efficient and selective peptide assembly.
Multi- vs. Single Channel Synthesis
Advancements in automation have revolutionized custom peptide synthesis. Automated peptide synthesizers allow for faster synthesis, higher throughput, and increased productivity. These synthesizers utilize robotic arms to accurately dispense reagents, control reaction times, and monitor reactions. Automation has made custom peptide synthesis more accessible and efficient for researchers in both academic and industrial settings.
Specifically, one can differentiate between parallel or single channel synthesis. During parallel synthesis, multiple peptides are simultaneously synthesized in different reaction chambers on different solid supports or resin beads. This approach is particularly useful for high-throughput screenings and the synthesis of peptide libraries (at Intavis, we use our self-developed MultipepTM robots). Single channel synthesis, on the other hand, involves the sequential synthesis of individual peptides, allowing for more focused and targeted modifications.
Peptide modification plays a critical role in enhancing the properties and functionality of peptides for specific applications. Several modifications can be made to peptides, broadening their utility and targeting capabilities.
Incorporation of Unnatural Amino Acids
The incorporation of unnatural amino acids expands the range of chemical functionalities available in peptides. This enables the synthesis of peptides with enhanced stability, increased bioactivity, or novel properties. Unnatural amino acids, such as D-amino acids, can be introduced during peptide synthesis. D-amino acids are mirror images of their naturally occurring L-amino acid counterparts. Incorporating D-amino acids can enhance peptide stability against enzymatic degradation and improve biological activity due to their increased resistance to proteolytic enzymes. Furthermore, non-standard amino acids can include unique functional groups, such as fluorophores or photo-crosslinking moieties, increasing the versatility of the synthesized peptide.
Conjugation with Different Molecules
Peptides can be conjugated with various molecules to enhance peptide solubility, pharmacokinetic properties, protect against enzymatic degradation, promote cellular uptake or targeting abilities. Common modifications include the addition of lipids, carbohydrates, or polyethylene glycol (PEG) moieties. For example, conjugation of peptides with fatty acids can improve membrane permeability and enhance their oral bioavailability. Several approved peptide therapeutics, such as liraglutide and exenatide, incorporate conjugated fatty acids to extend their half-life and improve therapeutic efficacy.
Isotope-Labeled Amino Acids
During synthesis also isotope-labeled amino acids can be incorporated into peptides. Isotopic labeling, such as ^13C or ^15N, allows for tracking and quantification of peptides in complex biological systems. This technique is widely used in proteomics and peptide metabolism studies, enabling researchers to gain insights into peptide distribution, turnover rates, and interactions within biological systems.
Purification of Synthesized Peptides
After synthesis, it is crucial to purify peptides to remove impurities and ensure their high quality and biological activity. Various purification techniques can be employed, depending on the complexity and characteristics of the synthesized peptides.
- Liquid-Phase Extraction (LPE): Liquid-phase extraction, also known as solvent extraction or liquid-liquid extraction, is a commonly used technique for peptide purification. In this method, the synthesized peptides are dissolved in a suitable solvent and extracted using a different immiscible organic solvent. The organic solvent selectively removes impurities, while the desired peptides remain in the aqueous layer. Multiple rounds of extraction can be performed to achieve higher purity.
- Chromatography Techniques: Chromatography methods are widely utilized for peptide purification due to their high resolution and efficiency. Depending on the specific requirements, various chromatographic techniques can be employed such as Reverse-Phase Chromatography (RPC), Size-Exclusion Chromatography (SEC), Ion-Exchange Chromatography (IEC), and Affinity Chromatography.
- RPC separates peptides based on hydrophobicity, with less hydrophobic peptides eluting first. A commonly used stationary phase in RPC is C18.
- SEC separates peptides based on size, allowing larger peptides to elute earlier, while smaller impurities are retained longer.
- IEC separates peptides based on charge, utilizing a stationary phase with charged functional groups that selectively bind peptides based on their charge properties.
- Affinity Chromatography utilizes a stationary phase with specific ligands that interact with peptides of interest. This technique allows selective purification of peptides based on their affinity for the ligand.
- High-Performance Liquid Chromatography (HPLC): HPLC is a powerful and popular technique for peptide purification. It combines high-pressure pumping systems, specialized columns, and detectors to achieve excellent resolution and separation of peptides. HPLC methods can be tailored by adjusting mobile phase composition, pH, and gradient profiles to optimize separation efficiency for specific peptides.
- Solid-Phase Extraction (SPE): Solid-phase extraction is another purification technique commonly employed for peptides. In this method, peptides are loaded onto a solid-phase sorbent, and impurities are washed away. The bound peptides are then eluted selectively using an appropriate solvent or pH conditions.
- Crystallization: Under certain conditions, peptides can form crystals, which can be utilized as a purification method. By adjusting solvent composition, temperature, and concentration, impurities may remain in solution while the desired peptides crystallize. The crystals can then be separated from the solution and washed to enhance purity.
After purification, the quality of the synthesized peptides can be assessed using various analytical techniques, such as high-performance liquid chromatography (HPLC), mass spectrometry (MS), and amino acid analysis. These analytical tools ensure the identity, purity, and integrity of the peptides before further utilization in research, diagnostics, or therapeutic applications.
Application of Customized Peptides
Customized peptides have a wide range of applications across various fields. In the pharmaceutical industry, customized peptides are utilized for drug discovery and development. They are used to study the interaction between proteins and drugs, helping to design more effective and targeted therapeutics. Custom peptides also find application in diagnostic assays, where they act as essential components in the detection of specific diseases and biomarkers. The field of agriculture benefits from customized peptides as well, as they are used in the development of biopesticides and plant growth regulators. In addition, customized peptides have applications in cosmetic formulations, where they are employed for skin repair, anti-aging, and wound healing purposes. Overall, the versatility of customized peptides makes them indispensable in various scientific and commercial sectors.
Custom peptide synthesis, driven by advancements in technology, has revolutionized the field of life sciences. The employment of solid-phase peptide synthesis, along with automation, has allowed researchers to efficiently synthesize peptides with high purity and yields. Peptide modification techniques, such as the incorporation of unnatural amino acids and conjugation with different molecules, have expanded the functional diversity of peptides. Following synthesis, purification is a critical step to obtain high-quality peptides with desired characteristics.
As technology continues to advance, custom peptide synthesis will remain a vital tool for drug discovery, targeted drug delivery, and investigating biological processes. The ability to design and modify peptides with specific characteristics will undoubtedly contribute to advancements in diverse fields, including medicine, biotechnology, and chemical biology.
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