使用 DMTMM 在酰胺键形成过程中对呫吨染料进行荧光淬灭,ACS Omega

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使用 DMTMM 在酰胺键形成过程中对呫吨染料进行荧光淬灭
ACS Omega ( IF 3.7 ) Pub Date : 2022-09-07 , DOI: 10.1021/acsomega.2c03085
Kristen N Patterson ₁ , Misael A Romero-Reyes _{1,

2} , Jennifer M Heemstra _{1,

3}

Affiliation

荧光团与蛋白质、核酸和其他重要分子的生物偶联可以提供一种强大的方法来感测、成像和量化化学和生物过程。最普遍的荧光团连接方法之一是通过形成酰胺键，这通常由偶联剂促进，以激活羧酸部分，随后被胺进行亲核攻击。4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) 是最受欢迎的生物偶联剂之一，因为它能够促进酰胺键在水中形成。在 DMTMM 存在下观察到 5-荧光胺 (5-FAM) 偶联寡核苷酸的淬灭后，我们试图通过调查 DMTMM 对一系列荧光染料的影响来评估这一挑战的规模和范围。与花青染料相比，呫吨染料始终观察到更高的淬灭效果。进一步分析 DMTMM 对 FAM 的影响表明，猝灭的发生与染料是游离在溶液中还是附着在寡核苷酸或抗体上无关。此外，我们发现在去除 DMTMM 后，FAM 缀合的 DNA 无法恢复其荧光，并且 UV-vis 和 NMR 分析表明新产物的形成，例如 FAM 与 DMTMM 的二甲氧基三嗪之间形成的加合物。因此，不建议将高浓度的 DMTMM 用于目标被荧光标记的偶联反应。

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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. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (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 (5-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 that for 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 an 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 analyses suggest the formation of new products, such as an adduct formed between FAM and the dimethoxytriazine of DMTMM. As such, DMTMM at high concentrations is not recommended for coupling reactions where targets are fluorescently labeled. This research serves as a word of caution to those utilizing xanthene-containing fluorophores in bioconjugation reactions involving DMTMM.

更新日期：2022-09-07

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