Different strategies for the fluorescence labeling of nucleosides and oligonucleotides and techniques for their detection are discussed. Pre-and post-synthetic routes as well as automated solid phase-based syntheses and enzymatic methods are compared. Tailor-made chemical syntheses are presented that meet the requirements of a broad spectrum of applications for oligonucleotides as probes.

Two 3′‐modified and three base‐modified ddNTPs were synthesized and tested with several DNA polymerases for incorporation activity. Starting from 3′‐azido‐3′‐deoxythymidine (AZT; 1), we were able to produce 3′‐deoxy‐3′‐isocyanato‐thymidine and 3′‐deoxy‐3′‐isothiocyanatothymidine (3 and 4, resp.) in a rapid synthesis based on the solid‐support approach (Scheme 1). These 3′‐functionalities could be used to attach a spacer molecule via urea and thiourea groups, respectively. Since the thus‐obtained tethered nucleotides 7 and 8 can be used to label with fluorescent dyes (cf. Scheme 5), they are convenient building blocks for practical applications in DNA sequencing. Furthermore, we synthesized, via 14 (Scheme 2), 17 (Scheme 3), and 19 (Scheme 4), the N4‐modified dideoxycytidine 5′‐triphosphate dye derivatives 22, 23, and 24, respectively, with different lengths of linkers between the base residue and the dye (Scheme 5). Base‐specific termination for the derivatives 22 and 24 was demonstrated (Fig. 2a and 2b).

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