DESIGN AND SYNTHESIS OF AN ELECTRON RICH QUINONE METHIDE PRECURSOR FOR SEQUENCE -DIRECTED ALKYLATION OF DNA
Abstract
Quinone methides (QM) can be delivered to alklyate specific sites with a single strand DNA through target promoted alkylation. Previous experimental results indicated that alkylation by DNA-QM self-adduct was too slow for application in a biological system. A new quinone methide precursor (QMP) with enhanced reactivity is necessary to accelerate the reaction.
Previous study showed that an electron donating group present in the QMP would facilitate the generation of QM from the precursor and its regeneration from the reversible alkylation adducts. Therefore, new QMPs with increased electron density were designed. An electron rich QMP2 was successfully synthesized through a benzylaldehyde derivative. As predicted, DNA-QM self-adduct formation was much faster using QMP2 than using the conventional precursor QMP1 without an electron donating group. Only 20 min was needed for QM2 to complete the conversion from DNA-QMP conjugate while QM1 needed 24 hrs to finish the same conversion. The DNA-QM2 self-adduct also exhibited faster reaction for alkylation of the target single strand. A two-day incubation was necessary to achieve its maximal yield of 20% compared to 6 days required to achieve the maximal yield of 16% for DNA-QM1.
In order to target duplex DNA, QMP was coupled to triplex forming oligonucleotides (TFO) to deliver the QM to the major groove of DNA through triple helix formation. Alkylation products were observed with the DNA-QMP1 conjugate but not the DNA-QM1 self-adducts. An adjacent guanine in the sequence can increase alkylation yield from around 10% to up to 20%. QMP2 was also coupled to the TFO to generate the self-adduct DNA-QM2. Maximal duplex alkylation yield (15%) using DNA-QM2 self-adduct was achieved in 3 days if the triplex samples were incubated at room temperature. The alkylation yield increased to 20% with the DNA-QM2 self-adductwhen samples were incubated at 37 °C. The DNA-QMP2 conjugate could even be activated at 37 oC without fluoride and resulted in an alkylation yield of up to 25%. The enhanced reactivity of the electron rich QMP2 improved the duplex alkylation effectiveness and prepared it for future <italic>in vivo</italic> application.