Tandem Oligonucleotide Synthesis on Solid-Phase Supports for the Production of Multiple Oligonucleotides

More than one oligonucleotide can be synthesized at a time by linking multiple oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5‘-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two oligonucleotides are linked via a cleavable 3‘-O-hydroquinone-O,O‘-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5‘-OH position of immobilized oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3‘- or 5‘-end of oligonucleotides. A single NH4OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5‘- and 3‘-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucleotides is useful for producing sets of primers for PCR, DNA sequencing, and other diagnostic applications as well as double-stranded oligonucleotides. Tandem synthesis of the same sequence multiple times increases the yield of material from any single synthesis column for maximum economy in large-scale synthesis. This method can also be combined with reusable solid-phase supports to further reduce the cost of oligonucleotide production

Synthesis and Structural Studies of Nucleobase Functionalized Hydrogels for Controlled Release of Vitamins

The development of biomolecule-derived biocompatible scaffolds for drug delivery applications is an emerging research area. Herein, we have synthesized a series of nucleobase guanine (G) functionalized amino acid conjugates having different chain lengths to study their molecular self-assembly in the hydrogel state. The gelation properties have been induced by the correct choice of chain lengths of fatty acids present in nucleobase functionalized molecules. The effect of alkali metal cations, pH, and the concentration of nucleobase functionalized amino acid conjugates in the molecular self-assembly process has been explored. The presence of Hoogsteen hydrogen bonding interaction drives the formation of a G-quadruplex functionalized hydrogel. The DOSY nuclear magnetic resonance is also performed to evaluate the self-assembling behavior of the newly formed nucleobase functionalized hydrogel. The nanofibrillar morphology is responsible for the formation of a hydrogel, which has been confirmed by various microscopic experiments. The mechanical behaviors of the hydrogel were evaluated by rheological experiments. The in vitro biostability of the synthesized nucleobase amino acid conjugate is also investigated in the presence of hydrolytic enzymes proteinase K and chymotrypsin. Finally, the nucleobase functionalized hydrogel has been used as a drug delivery platform for the control and sustained pH-responsive release of vitamins B2 and B12. This synthesized nucleobase functionalized hydrogel also exhibits noncytotoxic behavior, which has been evaluated by their in vitro cell viability experiment using HEK 293 and MCF-7 cell lines

Purification of DMT-On Oligonucleotide by Simulated Moving-Bed (SMB) Chromatography

Recent clinical advances in the development of oligonucleotides as therapeutics have triggered a significant demand for these molecules in large quantities. To facilitate their development, purification of full-length oligonucleotide from various synthesis related byproducts is an important endeavour. Herein, we report for the first time an application of simulated moving-bed (SMB) chromatography as a purification tool for DMT-protected oligonucleotides. An efficient separation has been accomplished with polystyrene/divinyl benzene copolymer as the stationary phase and 0.05 M NH4HCO3 buffer (pH 8)/MeOH (6:4, v/v) as the mobile phase. A DMT-protected product purity of >99% with 77% yield for a 20-mer phosphorothioate oligonucleotide is reported

Synthesis of 2‘-<i>O</i>-Methoxyethylguanosine Using a Novel Silicon-Based Protecting Group

A short and efficient synthesis of 2‘-O-methoxyethylguanosine (8) is described. Central to this strategy is the development of a novel silicon-based protecting group (MDPSCl2, 2) used to protect the 3‘,5‘-hydroxyl groups of the ribose. Silylation of guanosine with 2 proceeded with excellent regioselectivity and in 79% yield. Alkylation of the 2‘-hydroxyl group of 6 proceeded with methoxyethyl bromide and NaHMDS and afforded compound 7 in 85% yield, without any noticeable cleavage of the silyl protecting group and without the need to protect the guanine base moiety. Finally, deprotection of 7 was achieved using TBAF and produced 8 in 97% yield

Synthesis of 2‘-<i>O</i>-Methoxyethylguanosine Using a Novel Silicon-Based Protecting Group

A short and efficient synthesis of 2‘-O-methoxyethylguanosine (8) is described. Central to this strategy is the development of a novel silicon-based protecting group (MDPSCl2, 2) used to protect the 3‘,5‘-hydroxyl groups of the ribose. Silylation of guanosine with 2 proceeded with excellent regioselectivity and in 79% yield. Alkylation of the 2‘-hydroxyl group of 6 proceeded with methoxyethyl bromide and NaHMDS and afforded compound 7 in 85% yield, without any noticeable cleavage of the silyl protecting group and without the need to protect the guanine base moiety. Finally, deprotection of 7 was achieved using TBAF and produced 8 in 97% yield

Regioselective Enzymatic Acylation of β-l-2‘-Deoxynucleosides: Application in Resolution of β-d/l-2‘-Deoxynucleosides

A practical synthesis of β-l-3‘- and β-l-5‘-O-levulinyl-2‘-deoxynucleosides has been described for the first time through enzymatic acylation and/or hydrolysis processes. It is noteworthy that the different behavior exhibited by Pseudomonas cepacia lipase in the acylation of d- and l-nucleosides allows the parallel kinetic resolution of d/l-nucleosides

Oligonucleotide Mimics for Antisense Therapeutics: Solution Phase and Automated Solid-Support Synthesis of MMI Linked Oligomers

Oligonucleotide Mimics for Antisense Therapeutics:  Solution Phase and Automated Solid-Support Synthesis of MMI Linked Oligomer

Building Blocks for the Solution Phase Synthesis of Oligonucleotides: Regioselective Hydrolysis of 3‘,5‘-Di-<i>O</i>-levulinylnucleosides Using an Enzymatic Approach

A short and convenient synthesis of 3‘- and 5‘-O-levulinyl-2‘-deoxynucleosides has been developed from the corresponding 3‘,5‘-di-O-levulinyl derivatives by regioselective enzymatic hydrolysis, avoiding several tedious chemical protection/deprotection steps. Thus, Candida antartica lipase B (CAL-B) was found to selectively hydrolyze the 5‘-levulinate esters, furnishing 3‘-O-levulinyl-2‘-deoxynucleosides 3 in >80% isolated yields. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) and Candida antarctica lipase A (CAL-A) exhibit the opposite selectivity toward the hydrolysis at the 3‘-position, affording 5‘-O-levulinyl derivatives 4 in >70% yields. A similar hydrolysis procedure was successfully extended to the synthesis of 3‘- and 5‘-O-levulinyl-protected 2‘-O-alkylribonucleosides 7 and 8. This work demonstrates for the first time application of commercial CAL-B and PSL-C toward regioselective hydrolysis of levulinyl esters with excellent selectivity and yields. It is noteworthy that protected cytidine and adenosine base derivatives were not adequate substrates for the enzymatic hydrolysis with CAL-B, whereas PSL-C was able to accommodate protected bases during selective hydrolysis. In addition, we report an improved synthesis of dilevulinyl esters using a polymer-bound carbodiimide as a replacement for dicyclohexylcarbodiimide (DCC), thus considerably simplifying the workup for esterification reactions

Enzymatic Parallel Kinetic Resolution of Mixtures of d/l 2′-Deoxy and Ribonucleosides: An Approach for the Isolation of β-l-Nucleosides

We have developed a lipase-catalyzed parallel kinetic resolution of mixtures of β-d/l-nucleosides. The opposite selectivity during acylation exhibited by Pseudomonas cepacia lipase (PSL-C) with β-d- and β-l-nucleosides furnished acylated compounds that have different Rf values. As a consequence, isolation of both products was achieved by simple column chromatography. Computer modeling of the transition-state analogues during acylation of β-d- and β-l-2′-deoxycytidine with PSL-C was carried out to explain the high selectivity. PSL-C favored the 3′-O-levulination of the β-d enantiomer, whereas the 5′-OH group was acylated in 2′-deoxy-β-l-cytidine. In both cases, the cytosine base was placed in the alternate hydrophobic pocket of PSL’s substrate-binding site, where it can form extra hydrogen bonds (in addition to the five essential catalytically relevant hydrogen bonds) that stabilize these intermediates catalyzing the selective acylation of β-d/l-nucleosides

Oligonucleotide Mimics for Antisense Therapeutics: Solution Phase and Automated Solid-Support Synthesis of MMI Linked Oligomers

Oligonucleotide Mimics for Antisense Therapeutics:  Solution Phase and Automated Solid-Support Synthesis of MMI Linked Oligomer