Supporting Information: Fast, Copper-Free Click Chemistry, A Convenient Solid-Phase Approach to Oligonucleotide Conjugation

General experimental Analytical TLC was performed on precoated (250 μm) silica gel 60 F-254 plates from Merck. All plates were visualized by UV irradiation, and/or staining with 5% H2SO4 in ethanol followed by heating. Flash chromatography grade silica gel 60 (230-400 mesh) was obtained from Merck. Mass analysis was performed on an Ettan MALDI-TOF Pro from Amersham Biosciences or LASER-TOF LT3 from Scientific Analytical Instruments with 3- hydroxypicolinic acid or 2,’ 4’, 6’-trihydroxyacetophenone as matrix. The NMR spectra were obtained at 1H (300 MHz), 13C (75 MHz) and 31P (121 MHz) on a Bruker instrument at 25 ºC. Chemical shifts are reported in ppm downfield from TMS as standard. HPLC was carried out using a Gilson instrument equipped with a UV detector and a Nucleosil C18 column (4.0 × 250 mm) or Phenomenex Clarity. Fluorescence spectra were recorded on a Varian Cary Eclipse instrument. All other chemical agents were purchased from Aldrich Chemical Company unless otherwise noted

Metal free click chemistry on nucleosides and oligonucleotides

Chemoselective ligation of biologically significant moieties through azide alkyne Click Chemistry has recently received much attention1. The reaction is attractive in that it regioselectively affords stable triazole linked bioconjugated products under mild conditions. However, from the view point of the synthetic oligonucleotide chemist, a significant disadvantage is that the non-thermal reaction requires an in situ generated Cu (I) catalyst. Unwanted Cu (I) mediated chemistry, specifically oxidative degradation etc

Synthesis and characterisation of a nucleotide based pro-drug formulated with a peptide into a nano-chemotherapy for colorectal cancer

Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P1, P4-di(2',5'-dideoxy-5'-selenouridinyl)-tetraphosphate (P4-SedU2), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P4-SedU2 and the corresponding selenothymidine analogue P4-SeT2 were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P4-SedU2 intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma Cmax than the equivalent injection of free P4-SedU2, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by &gt;2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC. </p