Shiyue Fang

Scalable Synthetic Oligodeoxynucleotide Purification with Use of a Catching by Polymerization, Washing, and Releasing Approach

Synthetic oligodeoxynucleotides are purified with use of a catching by polymerization, washing, and releasing approach. The method does not require any chromatography, and purification is achieved by simple operations such as shaking, washing, and extraction. It is therefore useful for large-scale purification of synthetic oligonucleotide drugs. In addition to purification of oligonucleotides, this catching by polymerization concept is expected to be equally useful for purification of other synthetic oligomers such as peptides and oligosaccharides.

Purification of synthetic peptides using a catching full-length sequence by polymerization approach.

During automated solid-phase peptide synthesis, failure sequences were capped with acetic anhydride. After synthesis, a polymerizable methacrylamide tag was attached to the full-length sequences. Peptide purification was then achieved by polymerizing the full-length sequences, washing away impurities, and cleaving the peptide product from the polymer.

Polymerizable phosphoramidites with an acid-cleavable linker for eco-friendly synthetic oligodeoxynucleotide purification

Methacrylation phosphoramidites containing a linker cleavable with acetic acid were synthesized, and used for synthetic oligodeoxynucleotide (ODN) purification. During automated synthesis, the full-length ODN was tagged with the phosphoramidite. The failure sequences were not. In purification, the full-length ODN was co-polymerized into a polyacrylamide gel, and the failure sequences and other impurities were removed by washing. Pure ODN was cleaved from the gel with 80% acetic acid. Using this method, purification of sequences as long as a 197-mer, which are from the phi29 DNA polymerase gene, and at scales as large as 50 μmol was demonstrated. The products have high purity and the recovery yields are high. The method does not use any type of chromatography and purification is achieved through simple manipulations such as shaking and filtration. Compared with gel electrophoresis and HPLC purification methods, the new technology is less labor-demanding and more amendable for automation, consumes a smaller amount of environmentally harmful organic solvents and requires little energy for solvent evaporation. Therefore, it is ideal for high throughput purification and large scale ODN-based drug purification as well as small scale purification.

Synthetic 5′-phosphorylated oligodeoxynucleotide purification through catching full-length sequences by polymerization

The readily scalable catching by polymerization purification technology has been further advanced to purify 5′-phosphorylated synthetic oligodeoxynucleotides (ODNs). The new technology utilizes a phosphoramidite that contains a fluoride-cleavable diisopropylsilyl acetal linker and a polymerizable methacrylamide group, and is capable of phosphorylation of ODN. For purification, the phosphoramidite was coupled to the 5′-end of full-length ODN on a synthesizer. Because failure sequences were capped in each synthetic cycle, only the full-length sequences were phosphinylated and acrylated. After cleavage and deprotection, the crude ODN was subjected to polymerization under typical acrylamide gel formation conditions. The full-length ODN was incorporated into polymer. The failure sequences and other impurities were simply removed by washing with water. Pure full-length ODN that contained a 5′-phosphate group was cleaved from the polymer with HF–pyridine. Reversed-phase (RP) HPLC showed that the ODN was pure, and the recovery yield was higher than that of typical preparative HPLC purification.

Synthetic oligodeoxynucleotide purification by capping failure sequences with a methacrylamide phosphoramidite followed by polymerization

Oligodeoxynucleotide (ODN) purification was achieved by capping failure sequences with a polymerizable methacrylamide phosphoramidite during automated synthesis, polymerizing the failure sequences into an acrylamide gel after cleavage and deprotection, and extraction of full-length sequences with water. The details regarding the technology including the capping efficiency of four polymerizable phosphoramidites, optimal capping time, diffusion speeds of ODN from gels with different cross-linking ratios to solution, and the efficiency of ODN extraction from gel were investigated. In addition, the technology was tested for purification of a long sequence and purification on larger scales. We also found that polymerization of failure sequences in a centrifuge tube in air did not affect purification results. Finally, we provided additional evidence that ODNs are stable under radical polymerization conditions by complete digestion of ODN followed by reversed-phase HPLC analysis of nucleosides.

Simple and Efficient Synthesis of 4,7‐Dimethoxy‐1(H)‐indene

Abstract Stirring 4,7‐dimethoxy‐1‐indanol in chloroform at room temperature in the presence of a catalytic amount of p‐toluenesulfonic acid gave 4,7‐dimethoxy‐1(H)‐indene in quantitative yield. Other solvents, including benzene, which was the most frequently used one for this reaction, gave mostly polymeric materials. The new method was also effective for dehydration of other electron‐rich benzylic alcohols.

Evidence of Splitting 1,2,3‐Triazole into an Alkyne and Azide by Low Mechanical Force in the Presence of Other Covalent Bonds

The cycloaddition reaction of an alkyne and azide to form a 1,2,3-triazole is widely used in many areas. However, the stability of the triazole moiety under mechanical stress is unclear. To see if a triazole could be selectively split into an alkyne and azide in the presence of other typical covalent bonds, a mica surface functionalized with a molecule containing a triazole moiety in the middle and an activated ester at the end was prepared. An atomic force microscope (AFM) tip with amino groups on its surface was ramped over the mica surface at predefined locations, which could temporarily link the tip to the surface through amide bond formation. During retraction, the triazole or another bond in the linkage broke, and a force was recorded. The forces varied widely at different ramps from close to 0 pN to 860 pN due to nonspecific adhesions and to the inherent inconsistency of single bond rupture. If some of the forces were from triazole cycloreversion, there would be alkynes at the predefined ramping locations. The surface was reacted with an azide carboxylic acid followed by labeling with amino Au nanoparticles (AuNPs). AFM imaging revealed AuNPs at the predicted locations, which provided evidence that under certain conditions triazole could be split selectively in the presence of other bonds at forces below 860 pN.

Conjugation of (E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine to hydrophilic microspheres: development of a mobile microscale UV light actinometer.

( E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine was biotinylated through a diisopropylsilylacetal linkage and attached to the surface of hydrophilic streptavidin-coated microspheres through the high-affinity noncovalent interaction between biotin and streptavidin. The functionalized microspheres form a stable suspension in water. Upon UV irradiation, the nonfluorescent ( E)-5-[2-(methoxycarbonyl)ethenyl]cytidine on the microspheres undergoes photocyclization to produce highly fluorescent 3-beta-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine. The fluorescence intensity of the microspheres can be correlated to the particle-specific UV doses applied at different suspension concentrations. The microspheres allow one to measure the UV dose (fluence) distribution in high-throughput water disinfection systems.

Synthesis of Oligodeoxynucleotides Containing Electrophilic Groups.

By use of 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) as a protecting group and linker for oligodeoxynucleotide (ODN) synthesis, deprotection and cleavage are achieved under non-nucleophilic oxidative conditions. The nucleophile-sensitive thioester and α-chloroacetyl groups are conveniently incorporated into ODN sequences. The technology could be universally useful for electrophilic ODN synthesis.

Denaturing Reversed-Phase HPLC Using a Mobile Phase Containing Urea for Oligodeoxynucleotide Analysis

Denaturing reversed-phase (RP) high performance liquid chromatography (HPLC) is usually achieved by elevating column temperature. In this article, an alternative method involving using a mobile phase that contains urea and performing HPLC at room temperature is described. The efficacy of the new method was demonstrated by analyzing a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs. The multiple peaks of the 61-mer ODN under normal conditions merged into one under the denaturing conditions. The broad single peaks of dsODNs under normal conditions were split into two sharp peaks.