Assessment of Sulfur Impurities in GMP-Grade Diisopropylcarbodiimide and Their Impact on Coupling Reagent-Induced Side Reactions in Peptide Synthesis
Abstract
Diisopropylcarbodiimide (DIC) constitutes one of the most widely used coupling reagents for amide bond formation in peptide synthesis. We have previously reported that DIC contains a varying amount of sulfur (61–2030 ppm), and that the main S-containing impurity in DIC is the thiourea DITU, which exhibits an excellent ability to suppress side reactions caused by N-oxyl radicals formed from common N-hydroxyl-coupling additives such as HOBt and Oxyma (Green Chem. 2019, 21, 5990). Aiming to understand how the quality of DIC impacts peptide synthesis, here, we report an evaluation of 14 batches of GMP-grade DIC from seven different vendors. With the evaluation by elemental analysis (S), gas chromatography–mass spectrometry, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance, we determined that the main S-containing impurity in DIC currently in use is not DITU, but rather the diazetidine-2-thione [1,3-diisopropyl-4-(isopropylimino)-1,3-diazetidine-2-thione, DIDT], formed by a 2 + 2 cycloaddition between DIC and iPr-NCS. The identity of DIDT was confirmed by a synthesis from DIC and iPr-NCS, followed by spiking a batch of DIC with the synthesized DIDT material. In a comparative evaluation of DITU and DIDT aimed at determining their ability to suppress breakdown of Fmoc-Cys(Trt)-OH caused by N-oxyl radicals formed from Oxyma, DITU completely prevented the Cys decomposition, whereas DIDT had no effect on the suppression of the breakdown of the amino acid. Finally, catalytic amounts of DITU as well as thiols DTT and NAC had a positive effect on minimization of the HOBt-induced breakdown of Fmoc-Trp(Boc)-OH, whereas the urea DIU, formed in every DIC-mediated peptide coupling reaction, accelerated the breakdown of the Trp substrate.
