Rigid, Conjugated, Fluoresceinated Thymidine Triphosphates: Syntheses and Polymerase Mediated Incorporation into DNA Analogues

Abstract

Syntheses of a unique set of energy transfer dye labeled nucleoside triphosphates, compounds 1-3, are described. Attempts to prepare these compounds were only successful if the triphosphorylation reaction was performed before coupling the dye to the nucleobase, and not the other way around. Compounds were prepared as both the 2'-deoxy (a) and 2',3'-dideoxy- (b) forms. They feature progressively longer rigid conjugated linkers connecting the nucleobase and the hydroxyxanthone moiety. UV spectra of the parent nucleosides 12-14 show that as the length of the linker increases so does the absorption of the donor in the 320-330 nm region, but with relatively little red-shift of the maxima. Fluorescence spectra of the same compounds show that radiation in the 320-330 nm region results in predominant emission from the fluorescein. When the linker is irradiated at 320 nm, the only significant emission observed corresponds to the hydroxyxanthone part of the molecules at 520 nm; this corresponds to an effective Stokes' shift of 200 nm. As the absorption at 320-330 nm by the linker increases with length, so does the intensity of the fluorescein emission. A gel assay was used to gauge relative incorporation efficiencies of compounds 1-3, dTTP, ddTTP, and 6-TAMRA-ddTTP. Throughout, the thermostable polymerase TaqFS was used, as it is the one most widely applied in high throughput DNA sequencing. This assay showed that only compounds 3 were incorporated efficiently; these have the longest linkers. Of these, the 2'-deoxy nucleoside 3 a was incorporated and did not prevent the polymerase from extending the chain further. The 2',3'-dideoxy nucleoside 3 b was incorporated only about 430 times less efficiently than ddTTP under the same conditions, and caused chain termination. The implications of these studies on modified sequencing protocols are discussed.

An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross‐Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR

“…Keywords: cross coupling · DNA · nucleosides · nucleotides · poly merase chain reaction ple of a Sonogashira reaction of 5 iodo dUTP had been known previously, [11] this was the first example of direct cross coupling modification of a purine dNTP. Soon after, other groups reported similar Suzuki Miyaura reactions be tween 8 bromoGTP and boronic acids [14] and coupling of a chloromercury derivative of dUTP with carbohydrate conju gated acrylamide derivatives.…”

“…Recently, several types of modified DNA bearing diverse groups, for example, aminoalkynyl or aminoalkyl [4 6] and several types of attached functional molecules, for example, biotin, [6] acridones, [7] fer rocene, [8] amino acids, [9] carbohydrates, [10] or fluorescein labels, [11] have been prepared from the corresponding modi fied dNTPs by this methodology. The dNTP building blocks are usually prepared [4 9] by troublesome and laborious tri phosphorylation of the corresponding modified nucleosides, in which the functional groups usually have to be protected and deprotected.…”

1‐(Phenylethynyl)pyrene and 9,10‐Bis(phenylethynyl)anthracene, Useful Fluorescent Dyes for DNA Labeling: Excimer Formation and Energy Transfer

“…This has been shown for naphthalene, [15] anthracene and tetracene, [16] pyrene, [17] perylene, [18] 2-phenylbenzoxazole, [19] bimanes, [20] Eu III chelates, [21] coumarins, [22] BODIPY, [23] and fluorescein. [24] Suitably functionalized phenylethynyl derivatives of pyrene and anthracene could be promising tools for the development of fluorescent probes for structural studies of nucleic acids. These compounds exhibit fluorescence within a range of 400Ϫ550 nm, have high quantum yields, and are chemically stable and capable of various non-covalent interactions.…”

Aminophenyl‐ and Nitrophenyl‐Labeled Nucleoside Triphosphates: Synthesis, Enzymatic Incorporation, and Electrochemical Detection

“…[8,9] The Suzuki-Miyaura reaction of 7-iodo-7-deaza-2'-deoxyadenosine 5'-triphosphate (7-I-7-deaza-dATP), 5-iodo-2'-deoxyuridine 5'-triphosphate (5-I-dUTP), or 5-iodo-2'-deoxycytidine 5'-triphosphate (5-I-dCTP) with either 3-aminophenyl-or 3-nitrophenylboronic acid (Scheme 1) gave the corresponding 3-aminophenyl or 3-nitrophenyl derivatives of dATP (dA NH 2 TP, dA NO 2 TP), dUTP (dU NH 2 TP, dU NO 2 TP), or dCTP (dC NH 2 TP, dC NO 2 TP) in acceptable yields after isolation by reversed-phase HPLC (40-43 % for NH 2 , 26-28 % for NO 2 ). This straightforward and efficient approach gives directly the desired functionalized dNTPs, which are suitable as substrates for polymerase incorporation.…”