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 >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.

5'-Chalcogen-Substituted Nucleoside Pyrophosphate and Phosphate Monoester Analogues: Preparation and Hydrolysis Studies.

Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using 31P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.

Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography.

Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michaelis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5'-deoxythymidine-5'-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then chain extended using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 to -3.1 °C per phosphoroselenolate) when introduced at the 5'-termini of A-form or mixed duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.

Mechanochemistry of nucleosides, nucleotides and related materials.

The application of mechanical force to induce the formation and cleavage of covalent bonds is a rapidly developing field within organic chemistry which has particular value in reducing or eliminating solvent usage, enhancing reaction rates and also in enabling the preparation of products which are otherwise inaccessible under solution-phase conditions. Mechanochemistry has also found recent attention in materials chemistry and API formulation during which rearrangement of non-covalent interactions give rise to functional products. However, this has been known to nucleic acids science almost since its inception in the late nineteenth century when Miescher exploited grinding to facilitate disaggregation of DNA from tightly bound proteins through selective denaturation of the latter. Despite the wide application of ball milling to amino acid chemistry, there have been limited reports of mechanochemical transformations involving nucleoside or nucleotide substrates on preparative scales. A survey of these reactions is provided, the majority of which have used a mixer ball mill and display an almost universal requirement for liquid to be present within the grinding vessel. Mechanochemistry of charged nucleotide substrates, in particular, provides considerable benefits both in terms of efficiency (reducing total processing times from weeks to hours) and by minimising exposure to aqueous conditions, access to previously elusive materials. In the absence of large quantities of solvent and heating, side-reactions can be reduced or eliminated. The central contribution of mechanochemistry (and specifically, ball milling) to the isolation of biologically active materials derived from nuclei by grinding will also be outlined. Finally non-covalent associative processes involving nucleic acids and related materials using mechanochemistry will be described: specifically, solid solutions, cocrystals, polymorph transitions, carbon nanotube dissolution and inclusion complex formation.

Nucleophilic displacement reactions of 5'-derivatised nucleosides in a vibration ball mill.

Vibration ball-milling in a zirconia-lined vessel afforded clean and quantitative nucleophilic displacement reactions between 4-methoxybenzylthiolate salts and nucleoside 5'-halides or 5'-tosylates in five to 60 minutes. Under these conditions, commonly-encountered nucleoside cyclisation byproducts (especially of purine nucleosides) were not observed. Liquid-assisted grinding of the same 5'-iodide and 5'-tosylate substrates with potassium selenocyanate in the presence of DMF produced the corresponding 5'-selenocyanates in variable yields over the course of between one and eleven hours thereby avoiding the preparation and use of hygroscopic tetrabutylammonium salts.