Phosphoramidite Monomers Research Articles

Application of Iodine-Amine Oxidation Approach in the Synthesis of Various N-Alkyl Phosphoramidate Oligonucleotide Derivatives

Introduction: Nowadays, use of phosphate modifications in oligonucleotide backbone has become a common approach for imbuing its structure with the desired beneficial properties. The recent advances in successful application of different classes of phosphate modifications in the design of therapeutic oligonucleotides have led to a renewed interest in the development of approaches for introducing diverse classes of phosphate modifications. Method: This study aims to investigate the efficiency and optimize protocols for the application of the iodine-amine oxidation reaction to produce various N-alkyl phosphoramidate oligonucleotide derivatives during the conventional solid-phase phosphoramidite synthesis method. Result: Various solvents and drying reagents were tested, and it was evaluated that even minor traces of water in a reaction mixture had a significant impact on yield. Using set of commercially available amines, it was shown that steric accessibility is a more critical parameter than nucleophilicity of the amino group in oxidative amination reaction. It was demonstrated that through use of amino alcohols and diamines during iodine-amine oxidation step various branched oligonucleotide structures can be synthesized. Conclusion: The obtained data indicates that the oxidative amination approach can be a promising tool for preparing various oligonucleotide derivatives during solid-phase synthesis without the use of specialized phosphoramidite monomers.

Synthesis of N3-Methyluridine- and 2'-O-Alkyl/2'-Fluoro-N3-Methyluridine-Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides.

In this article, we describe the synthesis of N3-methyluridine (m3U) and 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites as well as their incorporation into a 14-mer DNA and RNA oligonucleotide sequence. Synthesis of the 2'-O-alkyl-m3U phosphoramidite starts with commercially available uridine to achieve a tritylated m3U intermediate, followed by 2'-O-alkylation and finally phosphitylation. Synthesis of the 2'-F-m3U phosphoramidite is obtained from a commercially available 2'-F-uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2'-position of m3U that can be employed in numerous nucleic acid-based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR-Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2'-O-alkyl-N3-methyluridine analogs and their corresponding phosphoramidites Alternate Protocol 1: Synthesis of 2'-O-TBDMS-N3-methyluridine and its phosphoramidite Alternate Protocol 2: Synthesis of 2'-fluoro-N3-methyluridine and its phosphoramidite Basic Protocol 2: Solid-phase synthesis of N3-methyluridine-modified DNA and RNA oligonucleotides.

Conception and Evaluation of a Library of Cleavable Mass Tags for Digital Polymers Sequencing

AbstractA library of phosphoramidite monomers containing a main‐chain cleavable alkoxyamine and a side‐chain substituent of variable molar mass (i.e. mass tag) was prepared in this work. These monomers can be used in automated solid‐phase phosphoramidite chemistry and therefore incorporated periodically as spacers inside digitally‐encoded poly(phosphodiester) chains. Consequently, the formed polymers contain tagged cleavable sites that guide their fragmentation in mass spectrometry sequencing and enhance their digital readability. The spacers were all prepared via a seven steps synthetic procedure. They were afterwards tested for the synthesis and sequencing of model digital polymers. Uniform digitally‐encoded polymers were obtained as major species in all cases, even though some minor defects were sometimes detected. Furthermore, the polymers were decoded in pseudo‐MS3 conditions, thus confirming the reliability and versatility of the spacers library.

Conception and Evaluation of a Library of Cleavable Mass Tags for Digital Polymers Sequencing.

A library of phosphoramidite monomers containing a main-chain cleavable alkoxyamine and a side-chain substituent of variable molar mass (i.e. mass tag) was prepared in this work. These monomers can be used in automated solid-phase phosphoramidite chemistry and therefore incorporated periodically as spacers inside digitally-encoded poly(phosphodiester) chains. Consequently, the formed polymers contain tagged cleavable sites that guide their fragmentation in mass spectrometry sequencing and enhance their digital readability. The spacers were all prepared via a seven steps synthetic procedure. They were afterwards tested for the synthesis and sequencing of model digital polymers. Uniform digitally-encoded polymers were obtained as major species in all cases, even though some minor defects were sometimes detected. Furthermore, the polymers were decoded in pseudo-MS3 conditions, thus confirming the reliability and versatility of the spacers library.

2'-Modified Thymidines with Bioorthogonal Cyclopropene or Sydnone as Building Blocks for Copper-Free Postsynthetic Functionalization of Chemically Synthesized Oligonucleotides.

The development of facile methods for conjugating relevant probes, ligands, or delivery agents onto oligonucleotides (ONs) is highly desirable both for fundamental studies in chemical biology and for improving the pharmacology of ONs in medicinal chemistry. Numerous efforts have been focused on the introduction of bioorthogonal groups onto phosphoramidite building blocks, allowing the controlled chemical synthesis of reactive ONs for postsynthetic modifications. Among these building blocks, alkyne, cyclooctynes, trans-cyclooctene, and norbornene have been proved to be compatible with automated solid-phase chemistry. Herein, we present the development of novel 2'-functionalized nucleoside phosphoramidite monomers comprising bioorthogonal methylcyclopropene or sydnone moieties and their introduction for the first time to ON solid-phase synthesis. Traceless ON postsynthetic modifications with reactive complementary probes were successfully achieved through either inverse electron-demand Diels-Alder (iEDDA) reactions or strain-promoted sydnone-alkyne cycloaddition (SPSAC). These results expand the set of bioorthogonal phosphoramidite building blocks to generate ONs for postsynthetic labeling.

Syntheses of Base-Labile Pseudo-Complementary SNA and l-aTNA Phosphoramidite Monomers

We previously synthesized phosphoramidite monomers bearing Boc-protected 2,6-diaminopurine (D) and 2-methyl-4-methoxybenzyl-protected 2-thiouracil (sU) as building blocks for the preparation of pseudo-complementary serinol nucleic acids (SNAs). Since SNA is stable under acidic conditions, an acid-deprotection step could be inserted into the work-up. However, as the 4,4'-dimethoxytrityl group was concurrently removed at this step, purification of SNA by reversed-phase HPLC was difficult. Here, we report the syntheses of SNA and acyclic l-threoninol nucleic acid (l-aTNA) phosphoramidite monomers with bis(phenoxyacetyl)-protected D and 4-acetoxybenzyl-protected sU, both of which can be deprotected under mild basic conditions. Using these monomers, we prepared pseudo-complementary SNA and l-aTNA in high yield using conventional oligonucleotide synthesis protocols. These monomers can be used for large-scale syntheses of SNAs and l-aTNAs.

Synthesis of 4'-C-(Aminoethyl)thymidine and 4'-C-[(N-Methyl)aminoethyl] Thymidine Nucleosides to Enhance DNA Stability.

Antisense oligonucleotide (ASO) therapeutics target the pathogenic mRNA directly and modulate protein expression. Novel chemical modifications help to improve the action of ASOs with better thermal stability and resistance against nucleases. Oligodeoxynucleotides (ODNs) containing 4'-C-(aminoethyl)thymidine modifications exhibit efficient and stable hybridization with complementary DNA as well as RNA strands showing remarkably improved resistance against nucleolytic hydrolysis, which makes them promising candidates for antisense therapeutics. This article describes the synthesis of a novel nucleoside analog, 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T), 3, and previously reported 4'-C-aminoethyl-thymidine (4'-AE-T), 2, through a newly designed synthetic route to obtain a high overall yield. This has been established by changing the starting material from thymidine to diacetone-D-glucofuranose and synthesizing the known 4-C-hydroxyethyl pentofuranose. Conversion of the hydroxy group to an azide functional group through Mitsunobu azidation and performing acetolysis, provide the common intermediate 4-C-(2-azidoethyl)-ribofuranose. Subsequent coupling of the thymine nucleobase with the common intermediate under Vorbrüggen glycosylation conditions provides the corresponding modified nucleoside in high yield. It was subjected for conversion of the azide to an amine by Staudinger reaction and 2'-deoxygenation using Barton-McCombie conditions. Debenzylation with Lewis acid and mono-dimethoxytritylation of the 5'-OH afforded a fully protected 3'-OH intermediate for phosphitylation to give the corresponding phosphoramidites. In the case of 4'-MAE-T, benzyloxymethyl protection of the N3 -position and methylation were carried out prior to debenzylation. These phosphoramidite monomers were suitable with conventional oligonucleotide synthesis, and imparted ameliorated nuclease resistance, and competent RNase H activity, suggesting its potential utilization in ASO drugs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4-C-(2-azidoethyl)-ribofuranose (6) Basic Protocol 2: Synthesis of 4'-C-aminoethyl thymidine phosphoramidite (15) Basic Protocol 3: Synthesis of 4'-C-(N-methyl)aminoethyl thymidine phosphoramidite (20).

Synthesis of Ortho-Functionalized Tetrachlorinated Azobenzene Phosphoramidites for Incorporation Into siRNAzos for Photocontrolled Gene Silencing.

This paper contains the detailed synthesis and characterization protocols of ortho-functionalized tetrachlorinated azobenzene-containing small interfering RNAs (siRNAs), which have photoswitchable properties effectively controlled with visible light. To design this tetrachlorinated azobenzene scaffold, a late-stage chlorination with N-chlorosuccinimide and palladium is used. Next, a single hydroxyl group from the tetrachlorinated azobenzene is protected with a 4,4'-dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate tetrachlorinated azobenzene-containing oligonucleotides. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of ortho-functionalized chlorinated azobenzene-containing siRNAs (Cl-siRNAzos), which have photoswitchable properties that can be controlled with visible light. First, the Cl-siRNAzos are characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (Tm ) of the duplexes. Secondly, the biological testing of the Cl-siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to red light in order to inactivate the Cl-siRNAzo, and then either violet light or infrared thermal relaxation is deployed, which re-activates the Cl-siRNAzo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxyethyl) ortho-functionalized tetrachlorinated azobenzene phosphoramidite (5) Basic Protocol 2: Synthesis, purification, and characterization of siRNAs containing ortho-functionalized tetrachlorinated azobenzene Basic Protocol 3: Gene-silencing evaluation of ortho-functionalized tetrachlorinated azobenzene using firefly luciferase.

Synthesis of Nucleobase-Modified Oligonucleotides by Post-Synthetic Modification in Solution.

Oligonucleotides containing modified nucleobases have applications in various technologies. In general, to synthesize oligonucleotides with different nucleobase structures, each modified phosphoramidite monomer needs to be prepared over multiple steps and then introduced onto the oligonucleotides, which is time-consuming and inefficient. Post-synthetic modification is a powerful strategy for preparing many types of modified oligonucleotides, especially nucleobase-modified ones. Depending on the stage of modification, post-synthetic modification can be divided into two stages: "solid-phase modification," wherein an oligonucleotide attaches to the resin, and "solution-phase modification," wherein an oligonucleotide detaches itself from the resin. In this review, we focus on post-synthetic modification in solution for the synthesis of nucleobase-modified oligonucleotides, except the modifications to linkers for conjugation. Moreover, the reactions are summarized for each modified position of the nucleobases.

Use of Arabinonucleosides for Development and Implementation of a Novel 2'-O-Protecting Group for Efficient Solid-Phase Synthesis and 2'-O-Deprotection of RNA Sequences.

The implementation of protecting groups for 2'-hydroxyl function of ribonucleosides is very demanding in that synthetic RNA sequences must be highly pure to ensure the safety and efficacy of nucleic acid-based drugs for treatment of human diseases. A synthetic approach consisting of a condensation reaction between 2'-O-aminoribonucleosides with ethyl pyruvate has been employed to provide stable 2'-O-imino-2-methyl propanoic acid ethyl esters. Conversion of these esters to fully protected ribonucleoside phosphoramidite monomers has allowed rapid and efficient incorporation of 2'-O-protected ribonucleosides into RNA sequences while minimizing the formation of process-related impurities during solid-phase synthesis. Two chimeric 20-mer RNA sequences have been synthesized and then exposed to a solution of sodium hydroxide to saponify the 2'-O-imino-2-methyl propanoic acid ethyl ester protecting groups to their sodium salts. When subjected to ion-exchange conditions at 65°C and near neutral pH, fully deprotected RNA sequences are isolated without production of alkylating side-products and/or formation of mutagenic nucleobase adducts. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Synthesis of uridine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 2: Synthesis of N6 -protected adenosine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 3: Synthesis of N4 -protected cytidine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 4: Synthesis of N2 -protected guanosine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 5: Automated solid-phase RNA synthesis and deprotection using 2'-O-imino-2-proponate-protected phosphoramidites.

Innovative 2'-O-Imino-2-propanoate-Protecting Group for Effective Solid-Phase Synthesis and 2'-O-Deprotection of RNA Sequences.

The implementation of protecting groups for the 2'-hydroxyl function of ribonucleosides is still challenging, particularly when RNA sequences must be of the highest purity for therapeutic applications as nucleic acid-based drugs. A 2'-hydroxyl-protecting group should optimally (i) be easy to install; (ii) allow rapid and efficient incorporation of the 2'-O-protected ribonucleosides into RNA sequences to minimize, to the greatest extent possible, the formation of process-related impurities (e.g., shorter than full-length sequences) during solid-phase synthesis; and (iii) be completely cleaved from RNA sequences without the production of alkylating side products and/or formation of mutagenic nucleobase adducts. The reaction of 2'-O-aminoribonucleosides with ethyl pyruvate results in the formation of stable 2'-O-imino-2-methyl propanoic acid ethyl esters and, subsequently, of the fully protected ribonucleoside phosphoramidite monomers, which are required for the solid-phase synthesis of two chimeric RNA sequences (20-mers) containing the four canonical ribonucleosides. Upon treatment of the RNA sequences with a solution of sodium hydroxide, the 2'-O-imino-2-methyl propanoic acid ethyl ester-protecting groups are saponified to their sodium salts, which after ion exchange underwent quantitative intramolecular decarboxylation under neutral conditions at 65 °C to provide fully deprotected RNA sequences in marginally better yields than those obtained from commercial 2'-O-tert-butyldimethylsilyl ribonucleoside phosphoramidites under highly similar conditions.

Observation of Z-DNA Structure via the Synthesis of Oligonucleotide DNA Containing 8-Trifluoromethyl-2-Deoxyguanosine.

This article contains detailed synthetic protocols for the preparation of DNA oligonucleotides containing 8-trifluoromethyl-2'-deoxyguanosine (CF3 dG) and their application to observe Z-DNA structure in vitro and in living HeLa cells. First, using a catalytic system consisting of FeSO4 , H2 SO4 , and H2 O2 in DMSO, we achieved a one-step synthesis of CF3 dG through a radical reaction between deoxyguanosine (dG) and CF3 I, with a yield of 45%. We then obtained the 3'-phosphoramidite of CF3 dG through a routine three-step procedure. Next, we employed the CF3 dG phosphoramidite monomer in the synthesis of oligonucleotides on a solid-phase DNA synthesizer. Finally, we used the CF3 dG-modified DNA oligonucleotides to observe Z-DNA structure in vitro and in living HeLa cells through 19 F NMR spectroscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of CF3 dG phosphoramidites Basic Protocol 2: Preparation of CF3 dG-modified DNA oligonucleotides Basic Protocol 3: Evaluation of CF3 dG stabilization of Z-DNA structure by CD spectroscopy Basic Protocol 4: Investigation of Z-DNA structure in vitro and in HeLa cells with CF3 dG-modified DNA oligonucleotides and 19 F NMR spectroscopy.

A novel class of polymeric fluorescent dyes assembled using a DNA synthesizer.

In the pursuit of a novel class of fluorescent dyes we have developed a programmable polymer system that enables the rational design and control of macromolecular constructs through simple control of polymer primary sequence. These polymers are assembled using standard phosphoramidite chemistry on a DNA synthesizer which allows for extremely rapid prototyping and enables many permutations due to the large selection of phosphoramidite monomers presently available on the market. This programmability to some extent allows us to control the interactions/spacing of payload molecules distributed along the designed polymeric backbone. Control of molecular architecture using this technology has allowed us to address the long-standing technical issue of contact quenching between fluorescent dyes offering new possibilities in the life sciences arena. Much like peptidic sequences coding for enzymes, cofactors, and receptors (all needing control of tertiary structure for proper function via primary sequence) our programmable system approaches a similar endpoint using a phosphate based polymeric backbone assembled in a completely automated fashion. Using this novel technology, we have efficiently synthesized several types of fluorescent dyes and demonstrated the programmability in molecule design, including the increases in brightness of the fluorescence emission.

Mechanochemical Synthesis of Short DNA Fragments.

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxytrityl (DMTr) protecting group in situ in good yields (up to 60 % over three steps) to produce DNA dimers in a one-pot manner. H-Phosphonate chemistry under milling conditions enabled coupling and protection of the H-phosphonate linkage, as well as removal of the 5'-DMTr protecting group in situ, enabling a one-pot process with good yields (up to 65 % over three steps, or ca. 87 % per step). Sulfurization of the internucleotide linkage was possible using elemental sulfur (S8) or sulfur transfer reagents, yielding the target DNA phosphorothioate dimers in good yield (up to 80 % over two steps). This work opens the door to creation of solvent-free synthesis methodologies for DNA and RNA therapeutics.

Synthesis of Azobenzene Derivative Phosphoramidites for Incorporation into Oligonucleotides.

This article contains the detailed synthesis and characterization protocols of azobenzene containing siRNAs, which have photoswitchable properties effectively controlled with light. First, the azobenzene scaffolds are synthesized via reduction of nitrophenyl alcohols in the presence of zinc. Next, the hydroxyl group of azobenzene derivatives are protected with a dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate azobenzene-containing oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxymethyl)-azobenzene phosphoramidite Basic Protocol 2: Synthesis of 4,4'-bis(hydroxyethyl)-azobenzene phosphoramidite Basic Protocol 3: Synthesis, purification and characterization of oligonucleotides containing azobenzene derivatives.

High-Capacity Digital Polymers: Storing Images in Single Molecules

High-capacity digital poly(phosphodiester)s were synthesized by stepwise automated phosphoramidite chemistry. Coding libraries containing either 4 or 8 phosphoramidite monomers of different mass we...

Synthesis of Phosphoramidite Monomers Equipped with Complementary Bases for Solid-Phase DNA Oligomerization.

We describe the preparation of two monomers that bear complementary nucleobases at the edges (guanine-2'-deoxycytidine and 2-aminoadenine-2'-deoxyuridine) and that are conveniently protected and activated for solid-phase automated DNA synthesis. We report the optimized synthetic routes leading to the four nucleobase derivatives involved, their cross-coupling reactions into dinucleobase-containing monomers, and their oligomerization in the DNA synthesizer.

Photo-editable macromolecular information

Light-induced alteration of macromolecular information plays a central role in biology and is known to influence health, aging and Darwinian evolution. Here, we report that light can also trigger sequence variations in abiotic information-containing polymers. Sequence-coded poly(phosphodiester)s were synthesized using four phosphoramidite monomers containing either photo-sensitive or photo-inert substituents. These monomers allow different sequence manipulations. For instance, using two light-cleavable monomers containing o-nitrobenzyl ether and o-nitroveratryl ether motifs, photo-erasable digital polymers were prepared. These polymers can be decoded by tandem mass spectrometry but become unreadable after UVA exposure. The opposite behavior, i.e. photo-revealable sequences, was obtained with polymers made of two isobaric monomers containing light-cleavable o-nitrobenzyl ether and light-inert p-nitrobenzyl ether substituents. Furthermore, when the latter two monomers were used in conjunction with a third monomer bearing a light-inert OH group, site-directed photo-mutations were induced in synthetic polymers. This was used herein to change the meaning of binary sequences.

(Invited) DNA Quadruplex Hydrogels: An Application of Synthetic DNA As Bulk Material

DNA is a water-soluble and biodegradable polymer, which forms right-handed double helices and carries genetic information in life. Because of its high structure regularity and sequence programmability, DNA has been used as a key component in development of functional nanomaterial. Despite such success in nanoscience, reports on application of DNA to typical material, i.e. hydrogels, are very few, except for some excellent achievements, because preparation of DNA in gram scales usually requires unreasonable costs. One solution for this problem is liquid-phase DNA synthesis method developed by Bonora et al. They used monofunctional MeO-PEG as a soluble polymeric support, which enables coupling reaction of phosphoramidite monomers in liquid phase and can be easily precipitated by ether addition for product separation. We have applied this system to synthesize DNA-PEG-DNA tri-block copolymer in grams, and successfully prepared biodegradable PEG hydrogels by using higher-order DNA structures, such as G-quadruplexes, for cross-linking points. Aqueous solution of a tri-block copolymer bearing multiple guanosines at the ends, for example, immediately turns into gel upon addition of Na+ or K+ ions at reasonable concentration. Various body-related fluids such as PBS, artificial saliva, sweat, or tear can trigger gelation. The resulting hydrogels are self-healable since the crosslinking points are stable but still reversible structure. Moreover, introduction of toehold sequence realized complete control of such sol-gel transition by the addition of complementary DNA strand.

Novel Cluster and Monomer-Based GalNAc Structures Induce Effective Uptake of siRNAs in Vitro and in Vivo.

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.