A Novel Ultraviolet Assay for Testing Side Reactions of Carbodiimides

Carbodiimides possess considerable absorbance in the ultraviolet region; the extinction coefficient of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in water is ϵ214 = 6.3 · 103 L mol−1 cm−1. It provides a very simple method for testing possible side reactions of carbodiimides. This technique was used to study effects of pH, different buffers, and other components typically present in biological samples on EDC stability. It was shown that the hydrolysis rate in pure water increased from 1.5 · 10−5 to 5 · 10−4 s−1 by pH decreasing from 7 to 4. A strong increase of the rate of EDC loss was observed in the presence of different components. This effect can be described by the following row of hydrolysis rates: citrate ⪢ acetate ≈ phosphate ⪢ SDS. The results can be used to optimize carbodiimide-mediated reactions of peptide bond formation in organic chemistry or peptide synthesis or during immobilization or cross-linking of biological molecules.