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Exploring the Impact of TFA and other Counterions on Synthetic Peptides
Trifluoroacetic acid (TFA) is a volatile organic acid and a crucial cleavage reagent in solid phase peptide synthesis (SPPS). TFA is also used as an additive in the following chromatographic purification and analysis of peptides to improve separation on reversed phase HPLC columns. Various publications indicate that the secondary structure of synthetic peptides is not only given by the sequence but also by the solvent, counterions, solubilizers and others.1,2 However, a detailed examination of the influence of TFA and other anionic counterions on different secondary structures of synthetic peptides is still missing.
Trifluoroacetic acid (TFA) plays a crucial role as a cleavage reagent in solid-phase peptide synthesis (SPPS), contributing to the successful generation of synthetic peptides. Beyond its role in synthesis, TFA is widely employed as an additive in chromatographic purification and analysis, particularly in enhancing separation on reversed-phase High-Performance Liquid Chromatography (HPLC) columns. While existing literature acknowledges the multifaceted factors influencing the secondary structure of synthetic peptides, such as solvent properties, counterions, and solubilizers, a comprehensive exploration of how anionic counterions, particularly TFA, affect distinct secondary structures remains not well described.
This project aims to delve into the nuanced influence of TFA in comparison to other anionic counterions, including chloride and acetate, on both physicochemical and biological properties of synthetic peptides. Recognizing that secondary structure determination involves a complex interplay of various factors, this research seeks to bridge the existing gap in understanding the specific contributions of TFA and other anionic counterions to the secondary structure of synthetic peptides.
The primary focus of the investigation will involve optimizing and validating a previously established High-Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD) method. This method serves as a robust tool for monitoring the efficiency of different counterion exchange protocols. The project's methodology includes an examination of the influence of counterion exchange on model peptides, as well as on authentic samples.
To gain deeper insights into the structural alterations induced by counterion exchange, complementary analytical techniques such as Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), and Infrared (IR) spectroscopy will be employed. These techniques offer a comprehensive view of the secondary structure changes in synthetic peptides, allowing for a detailed characterization of the impact of various counterions. This project not only addresses an existing knowledge gap in peptide chemistry but also contributes valuable insights to the broader field of biochemistry and molecular biology and thereby advances the precision and reliability of peptide-based applications in various scientific domains.
Techniques / practices you learn during the project:
• HPLC-ELSD analysis and analytical method validation
• Fmoc solid phase peptide synthesis
• Peptide purification and peptide analysis (LC-MS/MS and LC-UV)
• Counter-ion exchange of peptides
• CD-spectra, IR/Raman-spectra and (2D)-NMR spectra measurement and evaluation
Trifluoroacetic acid (TFA) plays a crucial role as a cleavage reagent in solid-phase peptide synthesis (SPPS), contributing to the successful generation of synthetic peptides. Beyond its role in synthesis, TFA is widely employed as an additive in chromatographic purification and analysis, particularly in enhancing separation on reversed-phase High-Performance Liquid Chromatography (HPLC) columns. While existing literature acknowledges the multifaceted factors influencing the secondary structure of synthetic peptides, such as solvent properties, counterions, and solubilizers, a comprehensive exploration of how anionic counterions, particularly TFA, affect distinct secondary structures remains not well described.
This project aims to delve into the nuanced influence of TFA in comparison to other anionic counterions, including chloride and acetate, on both physicochemical and biological properties of synthetic peptides. Recognizing that secondary structure determination involves a complex interplay of various factors, this research seeks to bridge the existing gap in understanding the specific contributions of TFA and other anionic counterions to the secondary structure of synthetic peptides.
The primary focus of the investigation will involve optimizing and validating a previously established High-Performance Liquid Chromatography with Evaporative Light Scattering Detector (HPLC-ELSD) method. This method serves as a robust tool for monitoring the efficiency of different counterion exchange protocols. The project's methodology includes an examination of the influence of counterion exchange on model peptides, as well as on authentic samples.
To gain deeper insights into the structural alterations induced by counterion exchange, complementary analytical techniques such as Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), and Infrared (IR) spectroscopy will be employed. These techniques offer a comprehensive view of the secondary structure changes in synthetic peptides, allowing for a detailed characterization of the impact of various counterions. This project not only addresses an existing knowledge gap in peptide chemistry but also contributes valuable insights to the broader field of biochemistry and molecular biology and thereby advances the precision and reliability of peptide-based applications in various scientific domains.
Techniques / practices you learn during the project:
• HPLC-ELSD analysis and analytical method validation
• Fmoc solid phase peptide synthesis
• Peptide purification and peptide analysis (LC-MS/MS and LC-UV)
• Counter-ion exchange of peptides
• CD-spectra, IR/Raman-spectra and (2D)-NMR spectra measurement and evaluation
A closer look should be taken on the influence of TFA in comparison to other counter-ions e.g. chloride, acetate on physicochemical as well as biological properties of synthetic peptides. The project will include the optimization and validation of a previously established HPLC-evaporative light scattering detector (ELSD).3 This method will be used to monitor the efficiency of different counterion exchange protocols. The influence of the exchange on model peptides as well as authentic samples will be analysed with CD, NMR and IR measurements.
A closer look should be taken on the influence of TFA in comparison to other counter-ions e.g. chloride, acetate on physicochemical as well as biological properties of synthetic peptides. The project will include the optimization and validation of a previously established HPLC-evaporative light scattering detector (ELSD).3 This method will be used to monitor the efficiency of different counterion exchange protocols. The influence of the exchange on model peptides as well as authentic samples will be analysed with CD, NMR and IR measurements.
Final_Display_Masterthesis_TFA_4.docx
