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Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition reaction

Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition reaction

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WEISBROD, Samuel Hieronymus, 2010. Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition reaction [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Weisbrod2010Catal-19211, title={Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition reaction}, year={2010}, author={Weisbrod, Samuel Hieronymus}, address={Konstanz}, school={Universität Konstanz} }

terms-of-use The defined labelling of DNA with functional moieties is a difficult task. An interesting approach is the postsynthetic labelling method, in which small reactive anchors are incorporated into DNA for the subsequent attachment of functional moieties by a chemoselective reaction. Truly chemoselective reactions are rarely represented. The Staudinger ligation proofed to be a very chemoselective reaction by numerous in vivo studies. For site-specific labelling of DNA the Staudinger ligation is an interesting candidate and was further investigated in this work.<br /><br />Base modified 2’-deoxyadenosine and thymidine 5’ triphosphates linked to an azido moiety by different linkers were used to synthesize single azidofunctionalized DNA enzymatically by DNA polymerases. The employed nucleotides allow also the generation of DNA carrying multiple azido functions using PCR. Taq, Vent (exo-) and Pwo DNA polymerases can completely replace the natural substrate by the azido base modified analogue yielding site-specific incorporation into DNA.<br /><br />The single azido modified DNA was investigated regarding the ability to be a substrate in Staudinger ligations with specific substituted triarylphosphines. For a better watersolubility the phosphine was connected to a triethyleneglycol linker. In Staudinger ligations with this unlabelled phosphine optimized reaction conditions led to addition of an organic co-solvent (DMF) and use of elevated reaction temperature (60 °C) during the ligation. For further labelling of the DNA the phosphines were equipped with biotin as affinity tag, fluorescein and an polyamidoamine (PAMAM) dendrimer (second generation).<br />The biotinylation of DNA by Staudinger ligation occured with 70 % conversion determined by radioactive polyacrylamide gel electrophoresis without the formation of byproducts. The biotinylated DNA could then quantitatively conjugated to streptavidin. The synthesis of fluorescein and dendrimer linked phosphines showed an increased susceptibility to oxidation. While the unlabelled and biotinylated phosphine were stable to oxidation over month, fluorescein and dendrimer linked phosphines were oxidized within hours by air. Although the subsequent Staudinger ligation was performed under argon, only low conversion could be observed for these types of phosphines (37 % and traces, respectively).<br />For the optimization of the Staudinger ligation a phosphine was synthesized which combines the ability to mediate the traceless Staudinger ligation as well as support an increased water-solubility. Based upon the phosphinophenol developed by Bertozzi et al. trimethylamine should be attached to the unsubstituted phenyl rings, which would provide water-solubility since the amines will be protonated and charged at physiological pH.<br /><br />The synthesis of the water-soluble phosphine succeeded in 5 steps as well as a traceless Staudinger ligation in water, in which a acetyl group was transfered to 2-azidoacetamide. The reactivity of this phosphine was reduced, thus no conjugation of biotin was feasible to azidofunctionalized DNA.<br />In the second part of this work, the Diels Alder cycloaddition was used for labelling DNA. Based upon the publication of Saito et al. reporting the good properties of 7-vinyl-7-deaza-dG in Diels Alder reaction with maleimides, 7-vinyl-7-deaza-dA was found to be a similiar reactive diene. Due to the small modification incorporation by the 5’ triphosphate in PCR and primer extension reactions suceeded with several DNA polymerases. Vinyl modified DNA was also amenable by solid phase DNA synthesis. While the Diels Alder reaction using N-methylmaleimide occured quantitatively using an excess of maleimide, the conjugation of fluorescein yielded only 60 %. In contrast to Saito et al., longer DNA oligonucleotides were used for the Diels Alder reaction carrying the modification in the middle of the strand.<br /><br />To decrease steric demand at the reaction site, the reactive vinyl moiety should be put apart from the DNA by connecting it with a linker to the base. Since vinyl-dA or vinyl-dG derivatives could disturb base pairing, 3-vinylindole was used showing a similar reaction rate. Due to increased instability of this vinylindole in particular towards acids the synthesis was hampered. After some unsuccessful synthesis routes, the final vinylindole linked nucleoside could be prepared in 7 steps showing the same fast reaction rate in test Diels Alder reactions with N-methylmaleimide compared to vinyl-dA. With this building block in hand the way is paved for further investigations regarding DNA conjugation by Diels Alder chemistry under mild conditions.<br /><br />In conclusion, several reactive anchors for subsequent post-synthetic labelling of DNA could incorporated into DNA by DNA polymerases or DNA solid phase synthesis. Subsequent Staudinger ligation yielded good yields for biotin labelling. In other cases only moderate to bad yields were obtained. A synthesized water-soluble phosphine mediated traceless Staudinger ligation with small organic molecules. The labelling of DNA failed in this case due to the insufficient reactivity of the phosphine. The Diels Alder reaction allows higher yields under milder conditions using the small vinyl modification. To improve the reactivity in Diels Alder mediated DNA conjugation reactions a nucleoside was synthesized with a similar reactive diene connected by a linker to the base. Further investigations should show a higher reactivity and lead to mild DNA conjugation with high yields without the need of additional catalysts. eng Weisbrod, Samuel Hieronymus Catalyst-free chemoselective DNA conjugation by the Staudinger ligation and Diels Alder cycloaddition reaction 2012-05-04T14:52:57Z Weisbrod, Samuel Hieronymus 2012-05-04T14:52:57Z 2010

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