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Issue 18, 2009
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Synthesis and enzymatic incorporation of modified deoxyuridine triphosphates

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Abstract

We describe the synthesis of 2′-deoxyuridine-5′-triphosphate derivatives bearing linkers of varying length, bulk and flexibility, at position 5 of the pyrimidine base. Nucleotide analogues with terminal functional groups are of interest due to their application potential for the functional labelling of DNA strands. In the course of the synthesis of the nucleotide analogues, the methodology for the Yoshikawa phosphorylation procedure was optimised, resulting in an approach which reduces the amount of side-products and is compatible with labile functional groups attached to the base. The effect of linker composition on the enzymatic incorporation into DNA was systematically investigated using two different DNA polymerases. Deep VentR exo from the B-polymerase family accepted most nucleotide analogues as substrates, while Taq from the A-family was slightly less proficient. Both polymerases had difficulties incorporating 5-(3-amino-prop-1-ynyl)-2′-deoxyuridine triphosphate. A molecular model of the active site of the polymerase was used to rationalise why this nucleotide was not accepted as a substrate.

Graphical abstract: Synthesis and enzymatic incorporation of modified deoxyuridine triphosphates

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Supplementary files

Article information


Submitted
06 Apr 2009
Accepted
24 Jun 2009
First published
21 Jul 2009

Org. Biomol. Chem., 2009,7, 3826-3835
Article type
Paper

Synthesis and enzymatic incorporation of modified deoxyuridine triphosphates

V. Borsenberger, M. Kukwikila and S. Howorka, Org. Biomol. Chem., 2009, 7, 3826
DOI: 10.1039/B906956A

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