Figure 5A and B. (A) Controlled concatenation of three oligonucleotides using successive Cu(I)-free and Cu(I)-catalyzed chemical ligation by triazole formation. First the central oligonucleotide (7) is chemically ligated to (8) using the copper-free reaction of the azide group in (7) with the strained alkyne in (8), thereby preserving the terminal alkyne in (7) which is subsequently reacted in a Cu(I)-catalyzed reaction with the azide in the subsequently added third oligonucleotide (9). (B) LC time-course of Cu(I)-free conjugation of (7) with (8) using absorbance at 260 nm.