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Multiple sequential labeling

Multiple sequential labeling with up to three different marker-azides:

For the attachment of up to three different labels, phosphoramidites 5 and 6 have been developed. The alkyne groups are protected respectively with TIPS and TMS protecting groups.[5]

In order to modify oligonucleotides with two sensitive molecules, nucleosides 1 and 5 are incorporated into dna-strands using standard phosphoramidite chemistry. The first click reaction yields the singly modified oligonucleotide with full retention of the TIPS protecting group. For the second click 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 oligonucleotides in excellent yields (60–90% over three steps, see picture below).

For the introduction of three different labels, nucleosides 1, 5 and 6 are introduced into oligonucleotides. The first click reaction is performed directly on the resin. The singly modified oligonucleotide is subsequently cleaved from the support under 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 oligonucleotides in excellent overall yields (see picture).

Sequential introduction of two different labels:

Sequential introduction of three different labels: