Fluorescence Quenching of Xanthene Dyes during Amide Bond Formation Using DMTMM.
Fluorescence Quenching of Xanthene Dyes during Amide Bond Formation Using DMTMM.
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DOI：
10.1021/acsomega.2c03085
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发表时间：
2022-09-20
期刊：
影响因子：
4.1
通讯作者：
Heemstra, Jennifer M.
中科院分区：
文献类型：
--
作者：
Patterson, Kristen N.;Romero-Reyes, Misael A.;Heemstra, Jennifer M.
荧光团与蛋白质、核酸和其他重要分子的生物缀合可以为传感、成像和量化化学和生物过程提供有力的方法。用于荧光团连接的最普遍的方法之一是通过酰胺键的形成，这通常通过偶联剂来促进以活化羧酸部分用于随后的胺的亲核攻击。4-（4，6-二甲氧基-1，3，5-三嗪-2-基）-4-甲基-吗啉鎓氯化物（DMTMM）是这些用于生物缀合的偶联剂中最受欢迎的一种，因为其能够促进酰胺键在水中形成。在DMTMM存在下观察到5-荧光素胺（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程度和范围。一致地观察到较高的猝灭效果的氧杂蒽染料相比，花青染料。DMTMM对FAM的影响的进一步分析表明，猝灭的发生与染料在溶液中是否游离或连接到寡核苷酸或抗体无关。此外，我们发现，FAM结合的DNA是无法恢复其荧光后，去除DMTMM，和UV-vis和NMR分析表明，形成新的产品，如FAM和DMTMM的二甲氧基三嗪之间形成的加合物。因此，不推荐高浓度的DMTMM用于靶被荧光标记的偶联反应。这项研究为那些在涉及DMTMM的生物缀合反应中利用含咕吨荧光团的人提供了警告。
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.
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DOI：
10.1039/f29726801479
发表时间：
1972-01-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY-FARADAY TRANSACTIONS II
影响因子：
--
作者：
DEMPSTER, DN;THOMPSON, GF;RANKIN, R
通讯作者：
RANKIN, R
影响因子：
1.8
作者：
Raw, Steven A.
通讯作者：
Raw, Steven A.
影响因子：
3.7
作者：
Song HO;Lee B;Bhusal RP;Park B;Yu K;Chong CK;Cho P;Kim SY;Kim HS;Park H
通讯作者：
Park H
影响因子：
4.7
作者：
Marmé, N;Knemeyer, JP;Wolfrum, J
通讯作者：
Wolfrum, J
影响因子：
4.3
作者：
Zhang M;Nilsson L;Lee S;Choi J
通讯作者：
Choi J