Preparation of Thiol-Reactive Cy5 Derivatives from Commercial Cy5 Succinimidyl Ester
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- 作者：Hermann J. Gruber ; Gerald Kada ; Bernt Pragl ; Christian Riener ; Christoph D. Hahn ; Gregory S. Harms ; Werner Ahrer ; Thomas G. Dax ; Karin Hohenthanner ; and Hans-Gü ; nther Knaus
- 刊名：Bioconjugate Chemistry
- 出版年：2000
- 出版时间：March 2000
- 年：2000
- 卷：11
- 期：2
- 页码：161 - 166
- 全文大小：75K
- 年卷期：v.11,no.2(March 2000)
- ISSN：1520-4812
文摘
The present study offers reliable protocols for the preparation of new thiol-reactive Cy5 derivativeswhich are urgently needed for single molecule fluorescence microscopy. In a systematic approach,two alternate strategies were found for the extension of commercial amine-reactive Cy5 with thiol-reactive end groups. In the two-step method, Cy5 succinimidyl ester was first reacted withethylenediamine under conditions which gave ~99% asymmetric "Cy5-amine" and only ~1% symmetricproduct with two Cy5 residues. Subsequently, "Cy5-amine" was derivatized with commercialheterobifunctional cross-linkers to introduce thiol-reactive end groups (maleimide or pyridyldithio).Alternatively, commercial Cy5 succinimidyl ester was reacted with a primary amine (MTSEA,methanethiosulfonylethylamine, or PDEA, pyridyldithioethylamine) or a secondary amine (PEM,piperazinylethylmaleimide) to give the corresponding thiol-reactive derivatives in a single step. Resultswere good for MTSEA, moderate for PEM, and poor for PDEA. An additional drawback of the one-step method was the need for rigorous removal of unreacted Cy5 succinimidyl ester, which wouldlabel lysine residues on probe molecules. It is concluded that, except for the Cy5-MTSEA conjugate,the two-step method is much more general, reliable, and easier to follow by the typical biophysicist,biologist, etc., for whose benefit, these procedures are being published. All thiol-reactive Cy5 derivativesshowed similar absorption and fluorescence properties as Cy5 succinimidyl ester, and fluorescencewas fully retained after binding to thiols on proteins. The kinetics of protein labeling was also examinedin order to get an idea of proper labeling conditions.