Collect. Czech. Chem. Commun. 2009, 74, 515-534
Published online 2009-03-18 11:47:05

Synthesis of 3′-O-(2-cyanoethyl)-2′-deoxythymidine-5′-phosphate as a model compound for evaluation of cyanoethyl cleavage

Angelika C. Kellera,*, Saulius Servab, Diana C. Knappa, Marek Kwiatkowskic and Joachim W. Engelsa

a Institut für Organische Chemie und Chemische Biologie, J. W. Goethe Universität Frankfurt am Main, Max-von-Laue Str. 7, 60438 Frankfurt am Main, Germany
b Fermentas UAB, V. Graiciuno 8, 02241 Vilnius, Lithuania
c Oligovation AB, Dag Hammarskjölds väg 32B, Uppsala Science Park, 751 83 Uppsala, Sweden


An essential and challenging task during the development of our sequencing-by-synthesis (SBS) technique is the evaluation of efficient cyanoethyl (CE) cleavage conditions. For this purpose 3′-O-(2-cyanoethyl)-2′-deoxythymidine-5′-phosphate as a model compound as well as a short DNA oligomer bearing the CE function as terminal group were synthesized and used for various deprotection experiments. As it is already known for 2′-O-CE-protected RNA oligonucleotides, the CE function can be cleaved with tetrabutylammonium fluoride (TBAF) in THF. Indeed, by using 3′-O-(2-cyanoethyl)-2′-deoxythymidine-5′-phosphate as a simple model compound for cleavage tests, we found out that the 3′-O-CE function is quantitatively cleaved with 1 M TBAF in THF. However, the CE group is also cleaved by other small bases like hydroxy groups under alkaline conditions. The CE cleavage with TBAF in THF gives the fastest and quantitative removal of the CE group under mild conditions for our sequencing-by-synthesis (SBS) application. The efficient removal of the 3′-CE group is crucial for the proof-of-principle of our SBS approach using dye-labeled 3′-CE-blocked dNTPs, which is currently under investigation. Herein we describe the application of 3′-O-(2-cyanoethyl)-2′-deoxythymidine-5′-phosphate as model compound for the development of reversible terminators for the SBS technique. Furthermore we suggest that nucleoside phosphates bearing any removable 3′-modification might be suitable model compounds for cleavage studies in a heterogeneous environment comparable to an oligonucleotide/ aprotic solvent system.

Keywords: DNA; Sequencing-by-synthesis; Polymerase incorporation; Phosphorylations; Cyanoethyl protecting group; 3′-O-Modified nucleotides; Cleavage reactions; Oligonucleotides; Phosphoramidites; Nucleoside triphosphates.

References: 14 live references.