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

The present study offers reliable protocols for the preparation of new thio l-reactive Cy5 derivatives which are urgently needed for single molecule fl uorescence microscopy. In a systematic approach, two alternate strategies w ere found for the extension of commercial amine-reactive Cy5 with thiol-rea ctive end groups. In the two-step method, Cy5 succinimidyl ester was first reacted with ethylenediamine under conditions which gave similar to 99% asy mmetric "Cy5-amine" and only similar to 1% symmetric product with two Cy5 r esidues. Subsequently, "Cy5-amine" was derivatized with commercial heterobi functional cross-linkers to introduce thiol-reactive end groups (maleimide or pyridyldithio). Alternatively, commercial Cy5 succinimidyl ester was rea cted with a primary amine (MTSEA, methanethiosulfonylethylamine, or PDEA, p yridyldithioethylamine) or a secondary amine (PEM, piperazinylethylmaleimid e) to give the corresponding thiol-reactive derivatives in a single step. R esults were good for MTSEA, moderate for PEM, and poor for PDEA. An additio nal drawback of the onestep method was the need for rigorous removal of unr eacted Cy5 succinimidyl ester, which would label lysine residues on probe m olecules. 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 ty pical biophysicist, biologist, etc., for whose benefit, these procedures ar e being published. All thiol-reactive Cy5 derivatives showed similar absorp tion and fluorescence properties as Cy5 succinimidyl ester, and fluorescenc e was fully retained after binding to thiols on proteins. The kinetics of p rotein labeling was also examined in order to get an idea of proper labelin g conditions.