Accurate quantification of modified cyclic peptides without the need for authentic standards

Area of Science:

• Biochemistry
• Analytical Chemistry
• Medicinal Chemistry

Background:

• Cyclic peptides are promising therapeutics, but their efficient synthesis and quantification pose challenges.
• Cyanobactin-derived enzymes offer a novel route for synthesizing azole-containing cyclic peptides.
• Accurate quantification is essential for determining reaction yields and for downstream biological assessments.

Purpose of the Study:

• To develop and validate robust methods for quantifying azole-containing cyclic peptides.
• To facilitate the production and development of cyclic peptide therapeutics.
• To overcome the limitation of requiring authentic standards for quantification.

Main Methods:

• High-Performance Liquid Chromatography (HPLC) coupled with Electrospray Ionization Mass Spectrometry (ESMS) and Inductively Coupled Plasma Mass Spectrometry (ICPMS) to correlate sulfur content with peptide amount.
• Nuclear Magnetic Resonance (NMR) using the Electronic Reference to in situ Concentration (ERETIC) method for solution concentration determination.
• Validation of both methods for accurate quantification in reaction mixtures and purified samples.

Main Results:

• Successful development and validation of two distinct quantitative methods for azole-containing cyclic peptides.
• Demonstrated correlation between sulfur content (via ICPMS) and cyclic peptide concentration (via HPLC-ESMS).
• Established the utility of the NMR ERETIC method for precise solution concentration measurements.

Conclusions:

• The developed HPLC-ESMS/ICPMS and NMR ERETIC methods provide accurate and reliable quantification of azole-containing cyclic peptides.
• These methods eliminate the need for authentic standards, simplifying the analysis of novel compounds.
• The validated techniques will accelerate the development pipeline for cyclic peptide therapeutics.