Formation of <i>N</i>-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

Cazenave, Christian; Bathany, Katell; Rayner, Bernard

doi:10.1089/oli.2006.16.181

Cited by 5 publications

(5 citation statements)

References 14 publications

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“…For synthesis of novel oligonucleotides, a continuously growing number of supports carrying other modified nucleosides and 3′-terminal modifiers are required. Universal supports are also expected to reduce side reactions that take place on the preattached nucleoside of standard supports because universal supports lack any nucleosides attached to them. , …”

Section: Introductionmentioning

confidence: 99%

UnyLinker: An Efficient and Scaleable Synthesis of Oligonucleotides Utilizing a Universal Linker Molecule: A Novel Approach To Enhance the Purity of Drugs

Ravikumar¹,

Kumar²,

Olsen³

et al. 2008

Org. Process Res. Dev.

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A novel universal linker (UnyLinker) molecule which has a conformationally rigid and chemically stable bridge head ring oxygen atom carrying a conventional 4,4′-dimethoxytrityl (DMT) and succinyl groups locked in a syn orientation has been developed to carry out oligonucleotide synthesis efficiently and smoothly. The geometry of the vicinal syn oxygen functionalized group allows fast and clean cleavage under standard aqueous ammonia deprotection conditions to afford high-quality oligonucleotides. No base modification is observed, based on the ion-pair HPLC−UV−MS (IP-HPLC−UV−MS) method with detection limit of <0.1%. A class of impurities formed by branching from the exocyclic amino group of nucleosides loaded onto a solid support has been eliminated by the use of this method. Examples demonstrating the versatile nature of this molecule are shown by syntheses of different chemistries such as 2′-deoxy, 2′-O-methyl, 2′-O-methoxyethyl, Lock nucleic acids (LNA), 2′-α-fluoro nucleic acids (FANA), conjugates such as 5′-phosphate monoester and biotin, and phosphate diester and phosphorothioate backbone modifications. This molecule was loaded onto several commercial solid supports and used in both gas-sparged and packed-bed automated DNA/RNA synthesizers. Large-scale syntheses (up to 700 mmol) of multiple phosphorothioate first- and second-generation antisense drugs on GE-Amersham’s OligoProcess synthesizer are demonstrated further, showing that this chemistry could be used for efficient synthesis of multiple oligonucleotide drugs using a single raw material, thereby eliminating a difficult to characterize nucleoside-loaded polymer matrix used as a starting material. A mechanism for deprotection and cleavage of the linker molecule to liberate the free oligonucleotide is proposed. Characterization of the cyclic byproduct formed during release of the oligonucleotide is presented. The exo-syn configuration of the dihydroxy structure of the UnyLinker molecule is conclusively established by X-ray crystallography studies. A novel method to remove the last traces of osmium used during the synthesis of the UnyLinker molecule to reach undetectable levels (<1 ppm) is also described.

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Section: Introductionmentioning

confidence: 99%

UnyLinker: An Efficient and Scaleable Synthesis of Oligonucleotides Utilizing a Universal Linker Molecule: A Novel Approach To Enhance the Purity of Drugs

Ravikumar¹,

Kumar²,

Olsen³

et al. 2008

Org. Process Res. Dev.

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“…The loss of nucleobase protecting groups during storage or synthesis results in the formation of branched impurities. The unprotected exocyclic amino groups will react with the incoming phosphoramidite to form two branched chains (Cazenave, Bathany, & Rayner, 2006; Kurata et al, 2006). If this happens for the residue attached to the support, the resulting product will have a mass about twice the expected mass.…”

Section: Oligonucleotide Synthesis and Related Impuritiesmentioning

confidence: 99%

“…In RNA synthesis, post‐synthesis treatment of the branched oligonucleotide with triethylamine trihydrofloride (TEA‐3HF), a reagent commonly used for the removal of tert‐butyldimethylsilyl (TBDMS, Fig. 3) deprotection, selectively cleaves the branchmer oligonucleotide into two pieces, the desired FLP and an ( n −1) carrying a 3′‐phosphate group (Cazenave, Bathany, & Rayner, 2006).…”

Section: Oligonucleotide Synthesis and Related Impuritiesmentioning

confidence: 99%

Therapeutic Oligonucleotides, Impurities, Degradants, and Their Characterization by Mass Spectrometry

Pourshahian

2019

Mass Spectrometry Reviews

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Oligonucleotides are an emerging class of drugs that are manufactured by solid‐phase synthesis. As a chemical class, they have unique product‐related impurities and degradants, characterization of which is an essential step in drug development. The synthesis cycle, impurities produced during the synthesis and degradation products are presented and discussed. The use of liquid chromatography combined with mass spectrometry for characterization and quantification of product‐related impurities and degradants is reviewed. In addition, sequence determination of oligonucleotides by gas‐phase fragmentation and indirect mass spectrometric methods is discussed. © 2019 John Wiley & Sons Ltd. Mass Spec Rev

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“…Incorrect loading results in an oligomer with a 3‘-error that has reduced or no biological functionality − Universal supports are also expected to reduce side reactions that take place on the preattached nucleoside of standard supports because the universal support does not have a preattached nucleoside. …”

Section: Introductionmentioning

confidence: 99%

“…11 A universal support is particularly important for use in highthroughput oligonucleotide synthesis where manually loading supports in high-density titer plates is time-consuming, errorprone, and cumbersome. [12][13][14] Universal supports are also expected to reduce side reactions that take place on the preattached nucleoside of standard supports 15 because the universal support does not have a preattached nucleoside.…”

Section: Introductionmentioning

confidence: 99%

A Novel Catechol-Based Universal Support for Oligonucleotide Synthesis

et al. 2007

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A novel universal support for deoxyribo- and ribonucleic acid synthesis has been developed. The support, constructed from 1,4-dimethoxycatechol, represents an improvement over existing universal supports because of its ability to cleave and deprotect under mild conditions in standard reagents. Because no nonvolatile additives are required for cleavage and deprotection, the synthesized oligonucleotides do not require purification prior to use in biochemical assays. Using reverse phase HPLC and electrospray mass spectroscopy, it was determined that oligonucleotides synthesized on the universal support (UL1) 3'-dephosphorylate quickly (9 h in 28-30% ammonium hydroxide (NH4OH) at 55 degrees C, 2 h in 28-30% NH4OH at 80 degrees C, or <1 h in ammonium hydroxide/methylamine (1:1) (AMA) at 80 degrees C). Oligonucleotides used as primers for the polymerase chain reaction (PCR) assay were found to perform identically to control primers, demonstrating full biological compatibility. In addition, a method was developed for sintering the universal support directly into a filter plug which can be pressure fit into the synthesis column of a commercial synthesizer. The universal support plugs allow the synthesis of high-quality oligonucleotides at least 120 nucleotides in length, with purity comparable to non-universal commercial supports and approximately 50% lower reagent consumption. The universal support plugs are routinely used to synthesize deoxyribo-, ribo-, 3'-modified, 5'-modified, and thioated oligonucleotides. The flexibility of the universal support and the efficiency of 3'-dephosphorylation are expected to increase the use of universal supports in oligonucleotide synthesis.

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Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

Cited by 5 publications

References 14 publications

UnyLinker: An Efficient and Scaleable Synthesis of Oligonucleotides Utilizing a Universal Linker Molecule: A Novel Approach To Enhance the Purity of Drugs

UnyLinker: An Efficient and Scaleable Synthesis of Oligonucleotides Utilizing a Universal Linker Molecule: A Novel Approach To Enhance the Purity of Drugs

Therapeutic Oligonucleotides, Impurities, Degradants, and Their Characterization by Mass Spectrometry

A Novel Catechol-Based Universal Support for Oligonucleotide Synthesis

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