1. Solid-phase synthesis, thermal denaturation studies, nuclease resistance, and cellular uptake of (oligodeoxyribonucleoside)methylborane phosphine-DNA chimeras
Heera Krishna, Marvin H Caruthers J Am Chem Soc. 2011 Jun 29;133(25):9844-54. doi: 10.1021/ja201314q. Epub 2011 Jun 7.
The major hurdle associated with utilizing oligodeoxyribonucleotides for therapeutic purposes is their poor delivery into cells coupled with high nuclease susceptibility. In an attempt to combine the nonionic nature and high nuclease stability of the P-C bond of methylphosphonates with the high membrane permeability, low toxicity, and improved gene silencing ability of borane phosphonates, we have focused our research on the relatively unexplored methylborane phosphine (Me-P-BH(3)) modification. This Article describes the automated solid-phase synthesis of mixed-backbone oligodeoxynucleotides (ODNs) consisting of methylborane phosphine and phosphate or thiophosphate linkages (16-mers). Nuclease stability assays show that methylborane phosphine ODNs are highly resistant to 5' and 3' exonucleases. When hybridized to a complementary strand, the ODN:RNA duplex was more stable than its corresponding ODN:DNA duplex. The binding affinity of ODN:RNA duplex increased at lower salt concentration and approached that of a native DNA:RNA duplex under conditions close to physiological saline, indicating that the Me-P-BH(3) linkage is positively charged. Cellular uptake measurements indicate that these ODNs are efficiently taken up by cells even when the strand is 13% modified. Treatment of HeLa cells and WM-239A cells with fluorescently labeled ODNs shows significant cytoplasmic fluorescence when viewed under a microscope. Our results suggest that methylborane phosphine ODNs may prove very valuable as potential candidates in antisense research and RNAi.
2. Synthesis of novel cationic spermine-conjugated phosphotriester oligonucleotide for improvement of cell membrane permeability
Junsuke Hayashi, Tomoko Hamada, Ikumi Sasaki, Osamu Nakagawa, Shun-ichi Wada, Hidehito Urata Bioorg Med Chem Lett. 2015 Sep 1;25(17):3610-5. doi: 10.1016/j.bmcl.2015.06.071. Epub 2015 Jun 27.
A spermine-conjugated ethyl phosphotriester oligonucleotide was obtained by solid-phase synthesis based on phosphoramidite chemistry. The ethyl phosphotriester linkage was robust to exonuclease digestion and stable in fetal bovine serum. Cell membrane permeability of the spermine-conjugated ethyl phosphotriester oligonucleotide was studied by fluorescence experiments. The effective cell penetrating potency of the spermine-conjugated ethyl phosphotriester oligonucleotide was determined by confocal laser scanning microscopy and measurement of intracellular fluorescence intensity.
3. Alkynyl phosphonate DNA: a versatile "click"able backbone for DNA-based biological applications
Heera Krishna, Marvin H Caruthers J Am Chem Soc. 2012 Jul 18;134(28):11618-31. doi: 10.1021/ja3026714. Epub 2012 Jul 6.
Major hurdles associated with DNA-based biological applications include, among others, targeted cell delivery, undesirable nonspecific effects, toxicity associated with various analogues or the reagents used to deliver oligonucleotides to cells, and stability toward intracellular enzymes. Although a plethora of diverse analogues have been investigated, a versatile methodology that can systematically address these challenges has not been developed. In this contribution, we present a new, Clickable, and versatile chemistry that can be used to rapidly introduce diverse functionality for studying these various problems. As a demonstration of the approach, we synthesized the core analogue, which is useful for introducing additional functionality, the triazolylphosphonate, and present preliminary data on its biological properties. We have developed a new phosphoramidite synthon--the alkynyl phosphinoamidite, which is compatible with conventional solid-phase oligonucleotide synthesis. Postsynthesis, the alkynylphosphonate can be functionalized via "Click" chemistry to generate the 1,2,3-triazolyl or substituted 1,2,3-triazolyl phosphonate-2'-deoxyribonucleotide internucleotide linkage. This manuscript describes the automated, solid-phase synthesis of mixed backbone oligodeoxyribonucleotides (ODNs) having 1,2,3-triazolylphosphonate (TP) as well as phosphate or thiophosphate internucleotide linkages and also 2'-OMe ribonucleotides and locked nucleic acids (LNAs) at selected sites. Nuclease stability assays demonstrate that the TP linkage is highly resistant toward 5'- and 3'-exonucleases, whereas melting studies indicate a slight destabilization when a TP-modified ODN is hybridized to its complementary RNA. A fluorescently labeled 16-mer ODN modified with two TP linkages shows efficient cellular uptake during passive transfection. Of particular interest, the subcellular distribution of TP-modified ODNs is highly dependent on cell type; a significant nuclear uptake is observed in HeLa cells, whereas diffuse cytoplasmic fluorescence is found in the WM-239A cell line. Cytoplasmic distribution is also present in human neuroblastoma cells (SK-N-F1), but Jurkat cells show both diffuse and punctate cytoplasmic uptake. Our results demonstrate that triazolylphosphonate ODNs are versatile additions to the oligonucleotide chemist's toolbox relative to designing new biological research reagents.
