Fluorescence Quenching of Xanthene Dyes during Amide Bond Formation using DMTMM

Fluorophore bioconjugation to proteins, nucleic acids, and other important molecules can provide a powerful approach to sensing, imaging, and quantifying chemical and biological processes. One of the most prevalent methods for fluorophore attachment is through the formation of amide bonds, which are often facilitated by coupling agents to activate carboxylic acid moieties for subsequent nucleophilic attack by amines. DMTMM is among the most popular of these coupling agents for bioconjugation due to its ability to facilitate amide bond formation in water. After observing quenching of 5-fluoresceinamine (FAM)-conjugated oligonucleotides in the presence of DMTMM, we sought to evaluate the magnitude and scope of this challenge by surveying the effect of DMTMM on a range of fluorescent dyes. A higher quenching effect was consistently observed for xanthene dyes compared to cyanine dyes. Further analysis of the impact of DMTMM on FAM shows that quenching occurs independently of whether the dye is free in solution or attached to a DNA oligonucleotide or antibody. Furthermore, we found that FAM-conjugated DNA was unable to recover its fluorescence after the removal of DMTMM and UV-Vis and NMR analysis suggests the formation of new products. However, further studies are needed to elucidate the specific analogues being formed. This research serves as a word of caution to those utilizing xanthene-containing fluorophores in bioconjugation reactions involving DMTMM.