Synthesis and site-specific incorporation of a simple fluorescent pyrimidine

Abstract

We describe procedures for the synthesis of a fluorescent pyrimidine analog and its site-specific incorporation into a DNA oligomer. The 5¢-protected and 3¢-activated nucleoside 4 is synthesized in three steps with an overall yield of 40%. Site-specific incorporation into a DNA oligomer occurs with greater than 88% coupling efficiency. This isosteric fluorescent DNA analog can be used to monitor denaturation of DNA duplexes via fluorescence and can positively detect the presence of abasic sites in DNA duplexes. The total time for synthesis of the phosphoramidite 4 is about 75 h, whereas the total time for site-specific incorporation of nucleoside 2 into an oligonucleotide and purification of the corresponding oligonucleotide is about 114 hours.

A Novel Fluorescent Probe for Retaining Galactosyltransferases

“…Recently, fluorescent derivatives of uracil nucleotides have been generated, as probes for nucleic acid chemistry, by installation of a compact, heteroaromatic substituent in position 5 of the uracil base. [21] We reasoned that this phenomenon could also be harnessed for the development of autofluorescent UDP-Gal derivatives, as a novel type of fluorophore for GalT bioassays. We anticipated that such base-modified UDP-Gal derivatives might be broadly recognised by a range of different GalTs, due to the minimal steric demand of the fluorogenic substituent and the strongly conserved architecture of the nucleotide binding domain in different GalTs.…”

Structural and mechanistic basis for a new mode of glycosyltransferase inhibition

“…Our initial interest in 5-substituted UDP-Gal derivatives was prompted by the analysis of different GalT structures [ 14 , 15 ], which suggested that these enzymes might be able to accommodate donor analogues with an additional substituent in this position ( Supplementary Figure S1 ). We reasoned that such novel UDP-Gal derivatives might be useful as GalT inhibitor candidates or, in view of the strong fluorescence emission reported for structurally related, 5-substituted uridine nucleosides [ 16 ], as fluorescent probes for assay development. For the synthesis of the representative UDP-Gal derivative 2 we used Suzuki-Miyaura chemistry previously developed in our group for the direct modification of unprotected sugar-nucleotides [ 17 , 18 ].…”

Polarity of Major Grooves Explored by Using an Isosteric Emissive Nucleoside

“…The ground-state absorption spectrum of 1, which has a maximum at 316 nm when isolated from the native nucleobases, is virtually insensitive to changes in polarity, while its emission spectra are significantly impacted by the environment. [18][19][20] In addition to these attractive photophysical features, nucleoside 1 is prepared in only one step from available precursors, and can be sequence-specifically incorporated into oligonucleotides by using standard solid-phase phosphoramidite-based chemistry. [18][19][20] Regardless of the probe's identity, estimating the polarity of DNA grooves under native conditions is always referenced to values determined for the isolated chromophore in solvent mixtures of known polarity.…”

“…[18][19][20] In addition to these attractive photophysical features, nucleoside 1 is prepared in only one step from available precursors, and can be sequence-specifically incorporated into oligonucleotides by using standard solid-phase phosphoramidite-based chemistry. [18][19][20] Regardless of the probe's identity, estimating the polarity of DNA grooves under native conditions is always referenced to values determined for the isolated chromophore in solvent mixtures of known polarity. To generate an expanded polarity scale for nucleoside 1, its absorption and emission spectra were measured in methylcyclohexane/isopropanol and dioxane/water mixtures-two solvent systems that cover a wide, yet considerably overlapping polarity range (Figure 2 A).…”