Spacer Phosphoramidite 9 - CAS 146668-73-7

The ability to manipulate and fuse nano-compartmentalized volumes addresses a demand for spatiotemporal control in the field of synthetic biology, for example in the bottom-up construction of (bio)chemical nanoreactors and for the interrogation of enzymatic reactions in confined space. Herein, we mix entrapped sub-attoliter volumes of liposomes (~135 nm diameter) via lipid bilayer fusion, facilitated by the hybridization of membrane-anchored lipidated oligonucleotides. We report on an improved synthesis of the membrane-anchor phosphoramidites that allows for a flexible choice of lipophilic moiety. Lipid-nucleic acid conjugates (LiNAs) with and without triethylene glycol spacers between anchor and the 17 nt binding sequence were synthesized and their fusogenic potential evaluated. A fluorescence-based content mixing assay was employed for kinetic monitoring of fusion of the bulk liposome populations at different temperatures. Data obtained at 50 °C indicated a quantitative conversion of the limiting liposome population into fused liposomes and an unprecedently high initial fusion rate was observed. For most conditions and designs only low leakage during fusion was observed. These results consolidate LiNA-mediated membrane fusion as a robust platform for programming compartmentalized chemical and enzymatic reactions.

Riboflavin (RF) is an essential vitamin for cellular metabolism. Recent studies have shown that RF is internalized through RF transporters, which are highly overexpressed by prostate and breast cancer cells, as well as by angiogenic endothelium. Here, we present an optimized synthesis protocol for preparing tailor-made amphiphilic phospholipid-based RF derivatives using phosphoramidite chemistry. The prepared RF amphiphile-RfdiC14-can be inserted into liposome formulations for targeted drug delivery. The obtained liposomes had a hydrodynamic size of 115 ± 5 nm with narrow size distribution (PDI 0.06) and a zeta potential of -52 ± 3 mV. In vitro uptake studies showed that RfdiC14-containing liposomes were strongly internalized in HUVEC, PC3, and A431 cells, in a specific and transporter-mediated manner. To assess the RF targeting potential in vivo, an amphiphile containing PEG spacer between RF and a lipid was prepared-DSPE-PEG-RF. The latter was successfully incorporated into long-circulating near-infrared-labeled liposomes (141 ± 1 nm in diameter, PDI 0.07, zeta potential of -33 ± 1 mV). The longitudinal μCT/FMT biodistribution studies in PC3 xenograft bearing mice demonstrated similar pharmacokinetics profile of DSPE-PEG-RF-functionalized liposomes compared to control. The subsequent histological evaluation of resected tumors revealed higher degree of tumor retention as well as colocalization of targeted liposomes with endothelial cells emphasizing the targeting potential of RF amphiphiles and their utility for the lipid-containing drug delivery systems.

Super-paramagnetic beads (SPMB)s used for a variety of molecular diagnostic assays are prepared by attaching pre-synthesized oligonucleotides to the surface via a cumbersome and low efficient method of carbodiimide-mediated amide bond formation. To mainstream the process, we describe a novel procedure of direct oligonucleotide synthesis onto the surface of SPMBs (e.g. MyOne Dynabeads). With the many challenges surrounding containment of paramagnetic beads (≤1 μm) during automated oligonucleotide synthesis, we show that by applying a magnetic force directly to the SPMBs we prevent their loss caused by high-pressure drain steps during synthesis. To date we have synthesized 40 mers using a Spacer 9 phosphoramidite (triethylene glycol) coupled to the surface of hydroxylated SPMBs. HPLC analysis shows successful product generation with an average yield of 200 pmol per sample. Furthermore, because of the versatility of this powerful research tool, we envision its use in any laboratory working with conventional synthesis automation, as employed for single columns and for multi-well titer plates. In addition to direct synthesis of oligodeoxynucleotides (DNA) onto SPMBs, this platform also has the potential for RNA and peptide nucleic acid synthesis.