Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester

The present study offers reliable protocols for the preparation of new thiol-reactive Cy5 derivatives which 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 with ethylenediamine under conditions which gave approximately 99% asymmetric "Cy5-amine" and only approximately 1% symmetric product with two Cy5 residues. Subsequently, "Cy5-amine" was derivatized with commercial heterobifunctional 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. Results were 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 would label 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 derivatives showed similar absorption and fluorescence properties as Cy5 succinimidyl ester, and fluorescence was fully retained after binding to thiols on proteins. The kinetics of protein labeling was also examined in order to get an idea of proper labeling conditions.