Systematic Investigation of EDC/sNHS-Mediated Bioconjugation Reactions for Carboxylated Peptide Substrates

Abstract

1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet-to-be-reported side products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show that the trends established with model peptides hold for more complex systems.

Nanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune Potentiation

“…NHS or its sulfonated from (sulfo-NHS) is efficiently coupled to carboxyl groups with the aid of EDC to form NHS esters. The NHS esters then covalently conjugate to primary amines to form an intermediate compound that is subsequently hydrolysed to the desired conjugate [195]. Many nanoparticle vaccines have been prepared with the EDC/NHS conjugation method with a high coupling yield [196,197].…”

Vectorisation of agrochemicals via amino acid carriers: influence of the spacer arm structure on the phloem mobility of phenylpyrrole conjugates in the Ricinus system

“…In order to study the influence of the triazole ring on phloem mobility, we synthesised compound L‐16 which does not have this heterocycle in its structure. In this case, the strategy was to form an amide bond using a coupling reagent . The different processes are outlined in Fig.…”

The Effect of Physicochemical Modification on the Function of Antibodies Induced by Anti-Nicotine Vaccine in Mice

“…Previous studies investigating the kinetics of EDC/sNHS conjugation using a model amine and model peptides indicate several competing side reactions. These side reactions include interactions among EDC, sNHS, and the amine, intra-peptide conjugation, and hydrolysis of EDC and sNHS all of which compete for conjugation of the amine to the peptides [20]. By analogy, we would expect the same competing reactions and similar conjugation efficiencies for the conjugation of Hapten 7 to CRM 197 and would also expect increased levels of sNHS to have a minimal impact on hapten load; however, the role of sNHS in promoting a conformational change in CRM 197 requires further study to determine the exact mechanism of action.…”

“…Unfolding of CRM 197 upon conjugation has been previously reported with other antigens [20] and conformational changes to either carrier or antigen have been shown to impact the efficacy of a number of other vaccines, highlighting the importance of physicochemical characterization [21,22,23,24]. …”