The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between azides and alkynes to form 1,2,3-triazoles, as reported1 by Sharpless, was found to be so exquisitely regioselective and efficient at even the most mild conditions that Sharpless coined the term ‘Click Chemistry" to describe it.The use of this method for DNA modification has been somewhat delayed by the fact that copper ions damage DNA, typically yielding strand breaks.2 As these problems have now been overcome by the use of copper(I)-stabilizing ligands (e.g., tris(benzyltriazolylmethyl)amine, TBTA3), Carell et al. and Seela et al. discovered that the CuAAC reaction can be used to functionalize alkyne-modified DNA nucleobases with extremely high efficiency.4 Oligonucleotides bearing a single nucleosidic alkyne group can be prepared usinga C8-Alkyne-dC or dT-CE Phosphoramidite.Purified oligonucleotides are usually modified with 2-5 equivalents of the corresponding marker-azide (e.g., fluorescent-dye azides). After the addition of precomplexed Cu(I), complete conversion to the labelled oligo is observed in a time span between 30 min and 4 hours. After a simple precipitation step, labelled oligonucleotides can be recovered in near quantitative yields. Using a combination of C8-Alkyne, C8-TIPS-Alkyne and C8-TMS-Alkyne, it is possible to label oligonucleotides in up to three separate click reactions. The alkyne groups on the last two monomers are protected, respectively, with triisopropylsilyl (TIPS) and trimethylsilyl (TMS) protecting groups.5,6The first click reaction on solid phase on a C8-Alkyne yields the singly modified oligonucleotide with full retention of the TIPS and/or TMS protecting group.For double click, a C8-TIPS-Alkyne is used as the second nucleoside and the TIPS protecting group is cleaved with tetrabutylammonium fluoride (TBAF) without causing any damage to the DNA.The second click reaction in solution yields the doubly modified oligonucleotide in excellent yield. For the introduction of three different labels, all three nucleosides are introduced into oligonucleotides. The first click reaction is performed directly on the resin. The singly modified oligonucleotide is subsequently cleaved from the support with concomitant cleavage of the TMS group and retention of the TIPS protecting group. The second click reaction is performed in solution. Precipitation of the doubly modified oligonucleotide, cleavage of the TIPS group with TBAF, and a subsequent third click reaction in solution furnishes the desired triply modified oligonucleotide in excellent overall yield.