Oligodeoxynucleotide Synthesis Under Non-Nucleophilic Deprotection Conditions.

This protocol describes a method for the incorporation of sensitive functional groups into oligodeoxynucleotides (ODNs). The nucleophile-sensitive epigenetic N4-acetyldeoxycytosine (4acC) DNA modification is used as an example, but other sensitive groups can also be incorporated, e.g., alkyl halide, α-haloamide, alkyl ester, aryl ester, thioester, and chloropurine groups, all of which are unstable under the basic and nucleophilic deprotection and cleavage conditions used in standard ODN synthesis methods. The method uses a 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) group that carries a methyl group at the carbon of the methoxy moiety (meDmoc) for the protection of exo-amines of nucleobases. The growing ODN is anchored to a solid support via a Dmoc linker. With these protecting and linking strategies, ODN deprotection and cleavage are achieved without using any strong bases and nucleophiles. Instead, they can be carried out under nearly neutral non-nucleophilic oxidative conditions. To increase the length of ODNs that can be synthesized using the meDmoc method, the protocol also describes the synthesis of a PEGylated Dmoc (pDmoc) phosphoramidite. With some of the nucleotides being incorporated with pDmoc-CE phosphoramidite, the growing ODN on the solid support carries PEG moieties and becomes more soluble, thus enabling longer ODN synthesis. The ODN synthesis method described in this protocol is expected to make many sensitive ODNs that are difficult to synthesize accessible to researchers in multiple areas, such as epigenetics, nanopore sequencing, nucleic acid-protein interactions, antisense drug development, DNA alkylation carcinogenesis, and DNA nanotechnology. © 2024 Wiley Periodicals LLC. Basic Protocol: Sensitive ODN synthesis Support Protocol 1: Synthesis of meDmoc-CE phosphoramidites Support Protocol 2: Synthesis of a pDmoc-CE phosphoramidite.

Synthesis of 4'-Thiomodified c-di-AMP Analogs.

Cyclic diadenosine monophosphate (c-di-AMP) is a bacterial cyclic dinucleotide (CDN) comprising two adenosine monophosphates covalently linked by two 3',5'-phosphodiester bonds. c-di-AMP works as a second messenger, regulating many biological processes in bacteria such as cell wall homeostasis, DNA integrity, and sporulation via specific protein and/or RNA receptors. Moreover, c-di-AMP can function as an immunomodulatory agent in eukaryote cells via the stimulator of interferon genes (STING) signaling pathway. This protocol describes the chemical synthesis of two c-di-AMP analogs with a sulfur atom at the 4'-position of the furanose ring instead of an oxygen atom: c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2). Analogs 1 and 2 have resistance to phosphodiesterase-mediated degradation and are therefore useful for understanding the diverse biological phenomena regulated by c-di-AMP. In this protocol, two 4'-thioadenosine monomers are initially prepared via a Pummerer-like reaction assisted by hypervalent iodine. The CDN skeleton is then constructed through two key reactions based on phosphoramidite chemistry: dimerization of two appropriately protected nucleoside monomers to produce a linear dinucleotide, followed by macrocyclization of the resulting linear dinucleotide to form the CDN skeleton. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4'-thioadenosine monomers 13 and 14 Basic Protocol 2: Preparation of c-di-4'-thioAMP (1) and cAMP-4'-thioAMP (2).

A DNA Cleavage Assay Using Synthetic Oligonucleotide Containing a Single Site-Directed Lesion for In Vitro Base Excision Repair Study.

Many chemicals cause mutation or cancer in animals and humans by forming DNA lesions, including base adducts, which play a critical role in mutagenesis and carcinogenesis. A large number of such adducts are repaired by the DNA glycosylase-mediated base excision repair (BER) pathway, and some are processed by nucleotide excision repair (NER) and nucleotide incision repair (NIR). To understand what structural features determine repair enzyme specificity and mechanism in chemically modified DNA in vitro, we developed and optimized a DNA cleavage assay using defined oligonucleotides containing a single, site specifically placed lesion. This assay can be used to investigate novel activities against any newly identified derivatives from chemical compounds, substrate specificity and cleavage efficiency of repair enzymes, and quantitative structure-function relationships. Overall, the methodology is highly sensitive and can also be modified to explore whether a lesion is processed by NER or NIR activity, as well as to study its miscoding properties in translesion DNA synthesis (TLS).

A general and efficient approach to synthesize the phosphoramidites of 5'-18O labeled purine nucleosides.

5'-18O labeled RNA oligos are important probes to investigate the mechanism of 2'-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5'-18O labeled nucleosides starting from the corresponding commercially available 5'-O-DMT protected nucleosides. Using this method, we prepared 5'-18O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5'-18O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5'-18O-2'-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5'-18O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2'-O-transphosphorylation reactions.

Preparation of Lipid-Conjugated siRNA Oligonucleotides for Enhanced Gene Inhibition in Mammalian Cells.

Nucleic acid conjugates are promising drugs for treating gene-related diseases. Conjugating specific units like lipids, cell-penetrating peptides, polymers, antibodies, and aptamers either at the 3'- or 5'-termini of a siRNA duplex molecule has resulted in a plethora of siRNA bioconjugates with improved stabilities in bloodstream and better pharmacokinetic values than unmodified siRNAs. In this sense, lipid-siRNA conjugates have attracted a remarkable interest for their potential value in facilitating cellular uptake. In this chapter, we describe a series of protocols involving the synthesis of siRNA oligonucleotides carrying either neutral or cationic lipids at the 3'- and 5'-termini. The resulting lipid-siRNA conjugates are aimed to be used as exogenous effectors for inhibiting gene expression by RNA interference. A protocol for the formulation of lipid siRNA using sonication in the presence of serum is described yielding interesting transfection properties for cell culture without the use of transfecting agents.

Rapid Synthesis of Nucleoside Triphosphates and Analogues.

Nucleoside triphosphates (NTPs) are essential biomolecules involved in almost all biological processes, and their study is therefore critical to understanding cellular biology. Here, we describe a chemical synthesis suitable for obtaining both natural and highly modified NTPs, which can, for example, be used as surrogates to probe biological processes. The approach includes the preparation of a reagent that enables the facile introduction and modification of three phosphate units: cyclic pyrophosphoryl P-amidite (c-PyPA), derived from pyrophosphate (PV ) and a reactive phosphoramidite (PIII ). By using non-hydrolyzable analogues of pyrophosphate, the reagent can be readily modified to obtain a family of non-hydrolyzable analogues containing CH2 , CF2 , CCl2 , and NH that are stable in solution for several weeks if stored appropriately. They enable the synthesis of NTPs by reaction with nucleosides to give deoxycyclotriphosphate esters that are then oxidized to cyclotriphosphate (cyclo-TP) esters. The use of different oxidizing agents provides an opportunity for modification at P-α. Furthermore, terminal modifications at P-γ can be introduced by linearization of the cyclo-TP ester with various nucleophiles. © 2020 The Authors. Basic Protocol 1: Synthesis of cyclic pyrophosphoryl P-amidite (c-PyPA) and derivatives (c-PyNH PA, c-PyCH2 PA, c-PyCCl2 PA, c-PyCF2 PA) Basic Protocol 2: Synthesis of 3'-azidothymidine 5'-γ-P-propargylamido triphosphates and analogues Basic Protocol 3: Synthesis of 2'-deoxythymidine 5'-γ-P-propargylamido triphosphate (15) Basic Protocol 4: Synthesis of adenosine 5'-γ-P-amido triphosphate (19) and adenosine 5'-γ-P-propargylamido triphosphate (20) Basic Protocol 5: Synthesis of d4T 5'-γ-propargylamido ß,γ-(difluoromethylene)triphosphate Support Protocol: Synthesis of diisopropylphosphoramidous dichloride.

Synthesis of 2'-Fluorinated Northern Methanocarbacyclic (2'-F-NMC) Nucleosides and Their Incorporation Into Oligonucleotides.

This article describes chemical synthesis of 2'-fluorinated Northern methanocarbacyclic (2'-F-NMC) nucleosides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all four natural nucleobases (U, C, A, and G), and their incorporation into oligonucleotides by solid-supported synthesis. This synthesis starts from commercially available cyclopent-2-en-1-one to obtain the fluorinated carbocyclic pseudosugar intermediate (S.13), which can be converted to the uridine intermediate by condensation with isocyanate, followed by cyclization, and to adenine and guanine precursors by microwave-assisted reactions. All four 2'-F-NMC phosphoramidites are synthesized from S.13 in a convergent approach, and the monomers are used for synthesis of 2'-F-NMC-modified oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of fluorinated carbocyclic pseudosugar intermediate Basic Protocol 2: Preparation of 2'-F-NMC uridine and cytidine phosphoramidites Basic Protocol 3: Preparation of 2'-F-NMC adenosine phosphoramidite Basic Protocol 4: Preparation of 2'-F-NMC guanosine phosphoramidite Basic Protocol 5: Synthesis of oligonucleotides containing 2'-F-NMC.

Improved Synthesis of Phosphoramidite-Protected N6-Methyladenosine via BOP-Mediated SNAr Reaction.

N6-methyladenosine(m6A) is the most abundant modification in mRNA. Studies on proteins that introduce and bind m6A require the efficient synthesis of oligonucleotides containing m6A. We report an improved five-step synthesis of the m6A phosphoramidite starting from inosine, utilising a 1-H-benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP)-mediated SNAr reaction in the key step. The route manifests a substantial increase in overall yield compared to reported routes, and is useful for the synthesis of phosphoramidites of other adenosine derivatives, such as ethanoadenosine, an RNA analogue of the DNA adduct formed by the important anticancer drug Carmustine.

Synthesis of N2 -Aryl-2'-Deoxyguanosine Modified Phosphoramidites and Oligonucleotides.

The N2 -position of 2'-deoxyguanosine (N2 -position in dG) is well known for forming carcinogenic minor groove DNA adducts, which originate from environmental pollutants, chemicals, and tobacco smoke. The N2 -dG DNA adducts have strong implications on biological processes such as DNA replication and repair and may, therefore, result in genomic instability by generating mutations or even cell death. It is crucial to know the role of DNA polymerases when they encounter the N2 -dG damaged site in DNA. To get detailed insights on the in vitro DNA damage tolerance or bypass mechanism, there is a need to synthetically access N2 -dG damaged DNAs. This article describes a detailed protocol of the synthesis of N2 -aryl-dG modified nucleotides using the Buchwald-Hartwig reaction as a main step and incorporation of the modified nucleotides into DNA. In Basic Protocol 1, we focused on the synthesis of five different N2 -dG modified phosphoramidites with varying bulkiness (benzyl to pyrenyl). Basic Protocol 2 describes the details of synthesizing N2 -dG modified oligonucleotides employing the standard solid phase synthesis protocol. This strategy provides robust synthetic access to various modifications at the N2 -position of dG; the modified dGs serve as good substrates to study translesion synthesis and repair pathways. Overall data presented in this article are based on earlier published reports. © 2019 by John Wiley & Sons, Inc.

Chemical synthesis, purification, and characterization of 3'-5'-linked canonical cyclic dinucleotides (CDNs).

So far, four cyclic dinucleotides (CDNs) have been discovered as important second messengers in nature, where three canonical CDNs of c-di-GMP, c-di-AMP and c-AMP-GMP were found in bacteria containing two 3'-5' phosphodiester linkages and one non-canonical CDN 2'3'-c-GMP-AMP was identified in mammals containing mixed 2'-5' and 3'-5' phosphodiester linkages. The CDNs are produced by specific cyclases and degraded by phosphodiesterases (PDEs). All of the known CDNs could bind to the stimulator of interferon genes (STING) to induce type I interferon (IFN) responses and the three bacterial CDNs are sensed by specific riboswitches to regulate gene expression. The emerging physiological functions of bacterial CDNs lead the motivation to investigate other possible canonical CDNs. In recent years, many endeavors have been devoted to develop fast, convenient and cheap strategies for chemically synthesizing CDNs and their analogues. The phosphoramidite approach using commercial starting materials has attracted much attention. Herein, we describe an adapted one-pot strategy that enables fast synthesis of crude 3'-5'-linked canonical CDNs followed by purification of the obtained CDNs using reversed phase high-performance of liquid chromatography (HPLC). Furthermore, we report the full characterization of CDNs by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques.

A Phosphoramidite Analogue of Cyclotriphosphate Enables Iterative Polyphosphorylations.

An iterative polyphosphorylation approach is described, which is based on a phosphoramidite (P-amidite) derived reagent (c-PyPA) obtained from the cyclization of pyrophosphate with a reactive diisopropylaminodichlorophosphine. This type of reagent is unprecedented as it represents a reactive P-amidite without protecting groups. The reagent proved to be stable in solution over several weeks. Its utility is described in the context of iterative monodirectional and bidirectional polyphosphorylations. The ensuing functionalized cyclotriphosphate can be opened with a variety of nucleophiles providing ready access to diverse functionalized polyphosphate chains of defined length with several tags, including both P-N and P-O labels. Their interaction with exo- and endopolyphosphatases is described.

Studies directed toward the asialoglycoprotein receptor mediated delivery of 5-fluoro-2'-deoxyuridine for hepatocellular carcinoma.

The anticancer nucleoside 5-fluoro 2'-deoxyuridine-5'-phosphate (5-FdU-P) was attached via an amide chain linker to a triantennary GalNAc cluster as a means to deliver the drug to hepatic cells that recognize the amino sugar units.

Silibinin phosphodiester glyco-conjugates: Synthesis, redox behaviour and biological investigations.

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS+ and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40-70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC50 = 73 µM) than silibinin.

A one-pot synthesis of α-l-threofuranosyl nucleoside triphosphates (tNTPs).

TNA (α-l-threofuranosyl nucleoside) triphosphates of adenosine (tATP), guanosine (tGTP), cytidine (tCTP), and thymidine (tTTP) were synthesized from their corresponding 3'-O-phosphoramidite derivatives using a novel one-pot reaction that is less moisture sensitive than traditional methods. The chemically synthesized tNTPs, despite containing an unnatural 3'-triphosphate moiety, are similar in thermal stability to natural nucleotide triphosphates.

Highly Ordered Pyrene π-Stacks on an RNA Duplex.

The syntheses of 2'-O-(pyren-1-ylmethyl)uridine phosphoramidite, 2'-O-(pyren-1-ylmethyl)adenosine phosphoramidite, and multiple pyrene-attached oligo-RNAs are described in this unit. The 2'-O-(pyren-1-ylmethyl)nucleosides are converted into the corresponding 2'-O-(pyren-1-ylmethyl)nucleoside 3'-phosphoramidites, which can be incorporated into the specific position of oligo-RNAs by solid-phase oligonucleotide synthesis. The multiple pyrene-attached oligo-RNA forms an A-form duplex with a complementary multiple pyrene-attached oligo-RNA; the pyrenes are associated with π-stacking along the outside of the duplex.

Efficient Automated Solid-Phase Synthesis of DNA and RNA 5'-Triphosphates.

A fast, high-yielding and reliable method for the synthesis of DNA- and RNA 5'-triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite was coupled to the 5'-end. Oxidation of the formed 5'-phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5'-cycloSal-oligonucleotides. Reaction of the support-bonded 5'-cycloSal-oligonucleotide with pyrophosphate yielded the corresponding 5'-triphosphates. The 5'-triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.

New silibinin glyco-conjugates: synthesis and evaluation of antioxidant properties.

New silibinin glyco-conjugates have been synthesized by efficient method and in short time. Exploiting our solution phase strategy, several structurally diverse silibinin glyco-conjugates (gluco, manno, galacto, and lacto-) were successfully realized in very good yields and in short time. In preliminary study to evaluate their antioxidant and neuroprotective activities new derivatives were subjected to DPPH free radical scavenging assay and the Xanthine oxidase (XO) inhibition models assay. Irrespective of the sugar moiety examined, new glyco-conjugates are more than 50 times water-soluble of silibinin. In the other hand they exhibit a radical scavenging activities slightly higher than to silibinin and XO inhibition at least as silibinin.

Synthesis and in vitro cytostatic activity of 1,2- and 1,3-diacylglycerophosphates of clofarabine.

The conjugates of anticancer nucleoside clofarabine [2-chloro-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)adenine] with 1,2- and 1,3-diacylglycerophosphates have been prepared by the phosphoramidite method using a combination of 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl protecting group for the sugar moiety of the nucleoside and 2-cyanoethyl protection for the phosphate fragment. Some of the synthesized conjugates exhibited cytostatic activity against HL-60, A-549, MCF-7, and HeLa tumor cell lines.