Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

Area of Science:

• Biochemistry and Biotechnology
• Chemical Synthesis and Materials Science
• Chromatography and Separation Science

Background:

• Purification of therapeutic glycoproteins via affinity chromatography faces challenges in developing specific glycan-binding ligands.
• Current methods utilize immobilized lectins or boronates, but biomimetic ligands offer a promising alternative.
• The Ugi multi-component reaction provides a scaffold for combinatorial synthesis of novel affinity ligands.

Purpose of the Study:

• To rationally design and combinatorially synthesize novel carbohydrate-binding ligands using the Ugi reaction.
• To identify and characterize a ligand with affinity for glycoprotein glycan moieties.
• To evaluate the efficacy of the developed ligand for glycoprotein purification.

Main Methods:

• Solid-phase multi-component Ugi reaction was employed using an aldehyde-functionalized Sepharose support.
• A library of 48 ligands was synthesized by varying amine, carboxylic acid, and isocyanide components.
• Screening against glucose oxidase (GOx) identified the lead ligand A13C24I8, followed by binding capacity and specificity studies.

Main Results:

• Ligand A13C24I8 exhibited significant affinity for GOx, with a static binding capacity of 16.7 mg GOx/ml resin and Kd of 1.45 × 10⁻⁶ M.
• The ligand demonstrated specificity for various sugars, with a binding sequence prioritizing sorbitol and fructose.
• Purification of GOx from spiked E. coli samples achieved 92% purity (w/w) with 69% protein specific activity.

Conclusions:

• The Ugi scaffold provides a versatile strategy for developing cost-effective, pseudo-biospecific ligands for glycoprotein purification.
• The developed ligand A13C24I8 shows potential for efficient purification of glycoproteins based on their glycan structures.
• This approach offers an alternative to traditional lectin or boronate affinity chromatography.