Synthesis of fully protected trinucleotide building blocks on a disulphide-linked soluble support

Suchsland, Ruth; Appel, Bettina; Virta, Pasi; Müller, Sabine

doi:10.1039/d0ra10941j

Cited by 5 publications

(7 citation statements)

References 32 publications

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“…The precipitation efficiency and/or solubility of the tetrapodal ON constructs may need an adjusted nucleobase protecting group scheme . An orthogonal linker chemistry would allow preparation of protected blockmers , or fragments, which with an appropriate ligation chemistry may be used for the assembly of full-length ON products. It may be emphasized that recent improvements in liquid phase utilize tetra- and pentameric fragments for the convergent assembly of ONs in a kg scale .…”

Section: Discussionmentioning

confidence: 99%

“…23 In the present study, 5′-O-(2-methoxyisopropyl) (MIP)protected 2′-deoxynucleosides as chiral limonene-based oxathiaphospholane sulfide [named as (+) and (−)-Ψ] building blocks (1−4 Rp/Sp , Scheme 1) were synthesized and their applicability for the stereo-controlled LPOS of di-, tri-, and tetranucleotide phosphorothioates using a tetrapodal precipitative soluble support was demonstrated (Scheme 2). This is a preliminary study, which will guide to find procedures for stereo-controlled LPOS of longer ON sequences, but especially of short ON fragments 24 or blockmers 25,26 that can be ligated to gain full length ON products.…”

Section: ■ Introductionmentioning

confidence: 99%

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Stereo-Controlled Liquid Phase Synthesis of Phosphorothioate Oligonucleotides on a Soluble Support

Rosenqvist,

Saari,

Pajuniemi

et al. 2023

J. Org. Chem.

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5′-O-(2-Methoxyisopropyl) (MIP)-protected 2′-deoxynucleosides as chiral P(V)-building blocks, based on the limonenederived oxathiaphospholane sulfide, were synthesized and used for the assembly of di-, tri-, and tetranucleotide phosphorothioates on a tetrapodal pentaerythritol-derived soluble support. The synthesis cycle consisted of two reactions and two precipitations: (1) the coupling under basic conditions, followed by neutralization and precipitation and (2) an acid catalyzed 5′-O-deacetalization, followed by neutralization and precipitation. The simple P(V) chemistry together with the facile 5′-O-MIP deprotection proved efficient in the liquid phase oligonucleotide synthesis (LPOS). Ammonolysis released nearly homogeneous Rp or Sp phosphorothioate diastereomers in ca. 80% yield/synthesis cycle.

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Section: Discussionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Stereo-Controlled Liquid Phase Synthesis of Phosphorothioate Oligonucleotides on a Soluble Support

Rosenqvist,

Saari,

Pajuniemi

et al. 2023

J. Org. Chem.

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“…12, 13 A more stable, controlled, and stereoselective reagent platform may allow for improved synthetic approaches, such as synthesis via blockmers (short dimers or trimers made through a standard small molecule methods, prior to fragment coupling on solid phase -significantly improving efficiency and product quality). 14 The ability to easily make single isomer species from stable species/fragments will aid our control over product quality, and has the potential to enable commercialization of these novel therapeutics sustainably (i.e. through enhanced regulatory control of these complex molecules).…”

Section: Introductionmentioning

confidence: 99%

A P(V)-Platform for Oligonucleotide Synthesis

Baran¹,

Knouse²,

Huang³

et al. 2021

Preprint

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<div><div><div><p>The early promise of gene-based therapies is currently being realized at an accelerated pace with over 155 active clinical trials for antisense compounds and multiple FDA-approved oligonucleotide therapeutics. Fundamental advances in this area are vital and present an unprecedented opportunity to both address disease states that have been resistant to other common modalities and improve the significant sustainability challenges associated with production of these complex molecules on a commercial scale. The advent of phosphoramidite coupling chemistry and solid-phase synthesis 40 years ago democratized oligonucleotide synthesis to the scientific community, paving the way for many of these stunning developments. The reliability and generality of this approach for the preparation of native phosphate-diesters is attributed to the high reactivity of phosphorus when in the P(III)-oxidation state versus the desired P(V), as it enables rapid P-heteroatom bond formation. However, the growing demand for more diverse phosphorus-based linkages has challenged the limits of this technology. For example, the phosphorothioate (PS) linkage, which stabilizes oligonucleotides towards nuclease cleavage, is universally employed in current oligonucleotide therapeutics but is generally incorporated in racemic form. Stereodefined PS oligonucleotides may have desirable biological and physical properties but are accessed with difficulty using phosphoramidite chemistry. Here we report a flexible and efficient [P(V)]-based platform that can install a wide variety of phosphate linkages at will into oligonucleotides. This approach uses readily accessible reagents and can efficiently install not only stereodefined or racemic thiophosphates, but can install any combination of (S, R or rac)-PS with native phosphodiester (PO2) and phosphorodithioate (PS2) linkages into DNA and other modified nucleotides. Importantly this platform easily accesses this diversity under a standardized coupling protocol with sustainably prepared, stable, P(V) reagents.</p></div></div></div>

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“…The opportunity to install broader combinations and variations at will and in any order thus presents an important step in the evolution of oligonucleotide therapeutics and requires the invention of enabling science. The commercialization of oligonucleotides and, more specifically, phosphorothioate antisense oligonucleotides (PS-ASOs) faces major challenges (8)(9)(10)(11)(12)(13)(14). The ability to easily make single-isomer species from stable species and/or fragments will aid control over product quality and has the potential to enable commercialization of these new therapeutics sustainably (i.e., through enhanced regulatory control of these complex molecules).…”

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confidence: 99%

“…By contrast, using y 2 , dimer (10) was cleanly accessed in two steps through P(V) adduct (11) in >99% purity. To field test and contrast the synthesis of mixed PO-PS backbones by the two platforms, PS dimer (13) was subjected to a standard phosphoramidite coupling with (18) to yield protected trimer (14), which, upon oxidation of P(III) to the requisite P(V), resulted in rapid desulfurization [see ratio of (15)/(16) over time]. Conversely, the redox-neutral P(V) approach using P(V) adduct (17) cleanly delivered the unprotected mixed PS-PO trimer (19) without any loss of sulfur.…”

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confidence: 99%

A P(V) platform for oligonucleotide synthesis

Huang

Knouse

Qiu

et al. 2021

Science

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Platform for the synthesis of diverse oligos DNA is primarily viewed as a carrier of information encoded in the sequence of bases, but the chemistry of the phosphodiester backbone is crucial to oligonucleotide stability and structure. Building on previous work in synthetic P(V) phosphorothioate coupling chemistry, Huang et al . developed two new reagents for making phosphorodithioate- and phosphate-based linkages (see the Perspective by Virta). The authors incorporated all of these reagents into a unified P(V)-based synthesis platform capable of running at high efficiency on a commercial automated solid-phase synthesizer. They demonstrate the flexibility of this system by producing oligonucleotides with all three linkage types in specific positions. Access to such precisely constructed molecules opens new approaches to therapeutic oligonucleotide design. —MAF

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Synthesis of fully protected trinucleotide building blocks on a disulphide-linked soluble support

Abstract: Fully protected trinucleotides are synthesized on a tetrapodal soluble support using a disulphide linkage that upon reductive cleavage allows release of the trinucleotide with free 3′-OH group for further conversion to a phosphoramidite.

Cited by 5 publications

References 32 publications

Stereo-Controlled Liquid Phase Synthesis of Phosphorothioate Oligonucleotides on a Soluble Support

Stereo-Controlled Liquid Phase Synthesis of Phosphorothioate Oligonucleotides on a Soluble Support

A P(V)-Platform for Oligonucleotide Synthesis

A P(V) platform for oligonucleotide synthesis

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