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Disulfide Bonding: Improvements and Alternatives

May 12, 2015

 

In nature a large amount of proteins contain at least one disulfide bond. These covalent bonds provide conformational rigidity as well as structural, thermal, and biological stability. As a result, peptides containing disulfide bonds are often appealing for peptide pharmaceuticals making them an area of active research. However, regiospecific disulfide bonding based on standard post-synthetic oxidation methods present significant synthetic challenges. This process requires complete orthogonality of multiple cysteine protecting groups and highly dilute reaction conditions for avoiding intermolecular side products.  

 

Dr. Olga Avrutina has been actively researching synthetic improvements for oxidative folding through disulfide bond formation. Recently, she published an improved procedure based on an optimized solution containing 10% acetonitrile, 10% DMSO, 10% trifluoroethanol, and 1M aqueous guanidinium hydrochloride at pH 7. This procedure was found to have optimal reaction rates and control of undesirable side reactions. In addition to her work in improving the existing process, Dr. Avrutina has been involved in developing alternatives to disulfide bonds which maintain biological activity while opening new synthetic avenues. It has previously been described that the introduction of 1,4-disubstituted 1,2,3-triazoles can mimic disulfide bonds and be introduced through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Dr. Avurtina and her coworkers recently explored how 1,5-disubstituted 1,2,3-triazoles could also be used as disulfide replacements based on an ruthenium(II)-catalyzed method (RuAAC). They were able to synthesize the sunflower trypsin inhibitor (1-14SFTI-1) with a 1,5-disubstituted 1,2,3-triazole formed using Cp*RuCl(cod) on resin with microwave irradiation at 60°C for 5 hours. Interestingly, the 1,5-disubstituted 1,2,3-triazole was found to uniquely maintain biological activity compared to the natural disulfide version while the 1,4 variant had a significant loss of activity.

 

 

 

Dr. Avrutina has been using microwave peptide synthesis for the last 10 years. She initially used CEM’s first generation Liberty Peptide Synthesis System, but has more recently acquired the Liberty Blue™ Peptide Synthesizer. The microwave has enabled the high quality synthesis of longer 30-40 amino acid peptides with up to 4 disulfide bonds using multiple cysteine side chain protecting groups. Additionally, the use of microwave energy has been helpful for modifications, such as the formation of 1,5-disubstituted 1,2,3-triazoles. Dr. Avrutina commented: “The peptide quality has been very good. Additionally, the Liberty Blue software is very user friendly and flexible which has been important for us to make many non-standard peptides.”

 

 

A selection of Dr. Olga Avrutina’s current research is included below:

Effective PHIP Labeling of Bioactive Peptides Boosts the Intensity of the NMR Signal

Angewandte Chemie Internationl Edition, 2014, 53 (47), 12941–12945

DOI: 10.1002/anie.201404668

 

Oxidative Folding of Peptides with Cystine-Know Architectures: Kinetic Studies and Optimization of Folding Conditions

ChemBioChem, 2013, 14 (1), 137–146

DOI: 10.1002/cbic.201200604

 

Braces for the Peptide Backbone: Insights into Structure-Activity Relationships of Protease Inhibitor Mimics with Locked Amide Conformations

Angewandte Chemie Internationl Edition, 2012, 51 (15), 3708–3712

DOI: 10.1002/anie.201108983

 

“Triazole Bridge”: Disulfide-Bond Replacement by Ruthenium-Catalyzed Formation of 1,5-Disubstituted 1,2,3-Triazoles

Angewandte Chemie Internationl Edition, 2011, 50 (22), 5207–5211

DOI: 10.1002/anie.201008142

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