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Quantitative and Nondestructive Colorimetric Amine Detection Method for the Solid-Phase Peptide Synthesis as an
Tomohiro Umeno1, Moeka Fujihara1, Shota Matsumoto1
1Faculty of Pharmaceutical Sciences, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida 194-8543, Japan.
Analytical Chemistry
|October 13, 2023
Summary
A new quantitative, nondestructive colorimetric method detects amine groups during solid-phase peptide synthesis (SPPS). This method offers improved monitoring for peptide synthesis, yielding purer peptides compared to traditional techniques.
Area of Science:
- Organic Chemistry
- Analytical Chemistry
- Biochemistry
Background:
- Solid-phase peptide synthesis (SPPS) is crucial for peptide production.
- Existing amine detection methods for SPPS monitoring are often destructive or lack quantitation.
- A need exists for a nondestructive, quantitative method to monitor SPPS coupling reactions.
Purpose of the Study:
- To develop the first quantitative and nondestructive colorimetric method for amine detection in SPPS.
- To enable precise monitoring of coupling reactions during peptide synthesis.
- To improve the purity of synthesized peptides.
Main Methods:
- Development of a colorimetric assay based on the acid-base reaction between HCl salt of electron donor-acceptor dyes and resin-bound amino groups.
- Tuning the pKBH+ value of the dyes for optimal color change.
- Quantitative analysis of amine groups using spectrophotometry and validation with various amino acids.
Main Results:
- A quantitative, nondestructive colorimetric amine detection method was successfully developed.
- The method demonstrated a strong linear relationship (R² = 0.999) between color intensity and amine quantity.
- Quantitative analysis of proteinogenic and nonproteinogenic amino acids showed low relative standard deviation (RSD < 3.6%).
- The method enabled detailed monitoring of coupling reactions, even for amino acids undetectable by the Kaiser test, leading to higher crude peptide purity.
Conclusions:
- The developed colorimetric method provides a robust, quantitative, and nondestructive approach for monitoring amine groups in SPPS.
- This technique offers significant advantages over existing methods, particularly for complex peptide synthesis.
- It facilitates improved control over coupling reactions, ultimately enhancing the purity of the final peptide product.
