5-Aminoallyl-2'-fluoro-dUTP, 5-aminoallyl-UTP, and N(6)-([6-aminohexyl]carbamoylmethyl)-ATP were systematically tested for their suitability for the systematic evolution of ligands by exponential enrichment (SELEX) process with the aim of introducing additional functionalities to RNA libraries. All three aminomodified nucleoside triphosphates proved to be compatible with the enzymatic steps required for SELEX and maintained strict Watson-Crick basepairing. Complementary RNA molecules modified with the two uridine analogues show a significantly increased melting temperature, whereas the introduction of N(6)-([6-aminohexyl]carbamoylmethyl)-ATP leads to a decreased T(m) and thus less stable basepairing. The chemical synthesis of 5-aminoallyl-2'-fluoro-dUTP is reported in detail.

A convenient and time-saving solid phase synthesis of different 2'and 3'-amino-functionalized nucleoside-5'-triphosphates using polymer bounded

The synthesis of the novel class of 3¢-C-modified thymidine nucleotides is described. The aldehyde 1 was converted into the derivatives 5, 7, and 10 that differ in the length of the carbon chain. One-pot phosphorylation gave the corresponding triphosphates 11, 12, 13. The 3¢-C-(2-azidoethyl)-3¢-deoxythymidine 5¢-triphosphate 13 was reduced by triphenylphosphine to the amino nucleotide 14. All amino nucleotides have been dye-labelled at the aliphatic amino group with the oxazine dye JA242. All triphosphates were tested as terminators in DNA-sequencing reactions.The use of labelled 2¢,3¢-dideoxynucleoside 5¢-triphosphates 1 as terminators in DNA-sequencing has found wide acceptance, and is the method of choice for the determination of single-stranded DNA sequences. In recent years, much effort has been made in developing sequencing protocols, which are faster and deliver results with improved accuracy. The labelling of each of the four 2¢,3¢-dideoxynucleoside 5¢-triphosphates with different dyes and the use of capillary electrophoresis methods offers the possibility to measure the fluorescence decay time 2 with pulsed lasers and therefore allow the identification of each nucleotide in short time. The formation of 3¢-dye-labelled 2¢,3¢-dideoxynucleoside 5¢-triphosphates via heteroatoms has been reported. 3 However, the problem of cleavage of 3¢-ester and -amide modified nucleoside 5¢-triphosphates by DNA polymerases 4 cannot be excluded during sequencing reaction and thus a class of terminators with more suitable properties is required. Following our concept 5 of 3¢-modified sequencing terminators derived from sugar substitutions the synthesis of 3¢-C-modified nucleoside 5¢-triphosphates labelled with oxazine derivative JA242, as fluorescent dye, will be described (Scheme 1 and 2).The aldehyde 1 as starting material was synthesized from thymidine via a radical reaction involving regioselective addition of a C-centered radical to allyltributyltin as described in the literature. 6 The overall yield over four steps was 28%.For elongation of the carbon chain we used the Wittig reaction for the C=C bond. The aldehyde 1 reacted with 4-carboxybutyltriphenylphosphonium bromide in THF resulting in the formation of 2. The protection of the base on N3-position is not necessary in this reaction step. The ylide which reacted with 1 was generated from the phosphonium salt and the base NaN(TMS) 2 . With an excess of the 4-carboxybutyltriphenylphosphonium bromide (3 equivalents) and the NaN(TMS) 2 (6 equivalents) to the nucleoside 1, only the desired product was obtained in good yield (68%). NOESY-NMR spectra show a cross peak between both of the newly formed olefinic protons. The coupling constant is 10.9 Hz, indicating the formation of the (Z)-isomer of 2.For further carbon-chain elongation, the TSTU-method 7 was used. Therefore the free carboxyl group of 2 was activated with N, N,N¢,N¢-tetramethyl-O-succinimidouronium tetrafluoroborate (TSTU) in DMF/dioxane. The activation was monitored by TLC and was completed after 20 min. T...

Treatment of 5Ј-O-TBDMS-2Ј,3Ј-dideoxyadenosine 2 with phenoxycarbonyltetrazole followed by N-trifluoroacetyl-1,6-diaminohexane yielded 6-N-carbamoyl derivative 4, which was further converted into the deprotected nucleoside 5. The latter compound was transformed into its triphosphate 6, which has been dye-labelled at the aliphatic amino group with the fluorescent dye JA242. The conjugate was shown to terminate DNA synthesis catalysed by terminal transferase and different DNA polymerases, allowing DNA detection by time-resolved fluorescence.

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