Protocols for Efficient Chemical Synthesis of N2 -Modified Guanosine Phosphate Derivatives: Versatile Probes for mRNA Labeling.

A facile, reliable, and efficient method for the synthesis of N2 -modified guanosine nucleotides such as N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-monophosphate, N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-diphosphate, N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-triphosphate, and N2 -[benzyl-N-(propyl)carbamate]-N7 -methyl-guanosine-5'-O-diphosphate, starting from the corresponding nucleotide is described. The general process entails condensation between the exocyclic amine of guanosine nucleotide and 3-[(benzyloxycarbonyl)amino]propionaldehyde in aqueous methanol, followed by reduction using sodium cyanoborohydride to furnish the corresponding N2 -modified guanosine nucleotide in moderate yield with high purity (>99.5%). © 2023 Wiley Periodicals LLC. Basic Protocol: Synthesis of N2 -modified guanosine derivatives.

Efficient chemical synthesis of N2-modified guanosine derivatives: a versatile probes for mRNA labeling.

An efficient method for the synthesis of N2-modified guanosine nucleotides such as N2-[benzyl-N-(propyl)carbamate]-guanosine-5'-O-monophosphate, N2-[benzyl-N-(propyl)carbamate]-guanosine-5'-O-diphosphate, N2-[benzyl-N-(propyl)carbamate]-guanosine-5'-O-triphosphate, and N2-[benzyl-N-(propyl)carbamate]-N7-methyl-guanosine-5'-O-diphosphate, starting from the corresponding nucleotide is described. The overall reaction involves the condensation between the exocyclic amine of guanosine nucleotide with 3-[(benzyloxycarbonyl)amino]propionaldehyde in aqueous methanol, followed by reduction using sodium cyanoborohydride to furnish the corresponding N2-modified guanosine nucleotide in moderate yield with high purity (>99.5%).

An Efficient Gram-Scale Chemical Synthesis of UNA-Nucleoside-5'-O-Triphosphates.

This unit describes a facile, reliable, and efficient method for the gram-scale chemical synthesis of unlocked nucleic acid- (UNA) nucleoside-5'-O-triphosphates such as UNA-guanosine-5'-O-triphosphate (UNA-GTP), UNA-adenosine-5'-O-triphosphate (UNA-ATP), UNA-cytidine-5'-O-triphosphate (UNA-CTP), and UNA-uridine-5'-O-triphosphate (UNA-UTP), starting from the commercially available corresponding nucleoside-5'-O-triphosphate. The present process involves a "one-pot, two-step" strategy that utilizes green chemistry principles. The overall reaction involves the oxidation of nucleoside-5'-O-triphosphate using sodium periodate under aqueous conditions, followed by reduction using sodium borohydride to furnish the corresponding UNA-nucleoside-5'-O-triphosphate in good yields with high purity (>99.5%). © 2023 Wiley Periodicals LLC. Basic Protocol: Synthesis of UNA-nucleoside-5'-O-triphosphates.

Click-iT trinucleotide cap analog: Synthesis, mRNA translation, and detection.

The first example of the synthesis of a new trinucleotide cap analog containing propargyl group such as m7,3'-O-propargylG(5')PPP(5')AmpG is reported. The effect of the propargyl group in trinucleotide analog with a standard trinucleotide cap analog (GAG), m7G(5')ppp(5')AmpG was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured A549 lung carcinoma epithelial cells. The new propargyl cap analog is a substrate for T7 RNA polymerase. Notably, the mRNA capped with the propargyl cap is translated âˆ¼ 1.3 times more efficiently than the mRNA capped with the GAG cap. The most characteristic feature of the new propargyl cap analog is that the presence of the propargyl group allows further modification of the mRNA by chemical ligation of an azide-containing fluorescent-labeled substrate to the mRNA via click chemistry.

Solution-Phase Chemical Synthesis of Modified RNA Dinucleotides.

This article describes a simple, reliable, efficient, and improved solution-phase method for the gram-scale chemical synthesis of RNA dinucleotides such as pAm pA, pAm pG, and pAm pU that utilizes phosphoramidite chemistry. The overall synthetic strategy involves three steps. The first step involves the coupling reaction between 5'-O-MMT protected nucleoside-3'-O-phosphoramidite and a protected nucleoside containing a free 5'-OH group in the presence of tetrazole, followed by the oxidation of phosphite triester using tert-butyl hydroperoxide to give the corresponding protected Nm pN. Next, the 5'-O-MMT is cleaved under 3% trichloroacetic acid in dichloromethane conditions. Finally, the 5'-hydroxyl group is phosphorylated by the use of an activated bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite using tetrazole, followed by the oxidation of trivalent to pentavalent phosphorus using tert-butyl hydroperoxide and subsequent deprotection using ammonium hydroxide to afford the corresponding RNA dinucleotide, pNm pN, in good yields with high purity (>99.5%). © 2022 Wiley Periodicals LLC.

An Efficient Gram-Scale Chemical Synthesis of Purine Locked Nucleic Acid Nucleoside-5'-O-Triphosphates.

This article presents a simple, reliable, straight-forward, general, and efficient chemical method for the gram-scale synthesis of purine locked nucleic acid (LNA) nucleotides, such as LNA guanosine-5'-O-triphosphate (LNA-GTP) and LNA adenosine-5'-O-triphosphate (LNA-ATP), starting from the corresponding nucleoside. The reaction pathway employs an improved protection-free "one-pot, three-step" Ludwig synthetic strategy. The first step involves monophosphorylation of nucleoside with phosphorus oxychloride followed by reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate to furnish the corresponding LNA nucleotide in good yields. It is noteworthy that the reaction affords high-purity (>99.5%) LNA nucleotide after diethylaminoethyl Sepharose column purification. © 2022 Wiley Periodicals LLC. Basic Protocol: Synthesis of LNA nucleoside-5'-O-triphosphates.

Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines.

The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS-CoV-2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m7 G[5']ppp[5']N (where N=G, A, C or U), present at the 5'-end of many eukaryotic cellular and viral RNAs and several non-coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.

An improved protection-free one-pot chemical synthesis of purine locked nucleic acid nucleoside-5'-triphosphates.

A simple, reliable, straightforward, and efficient method for the gram-scale chemical synthesis of purine locked nucleic acid (LNA) nucleotides such as LNA-guanosine-5'-triphosphate (LNA-GTP) and LNA-adenosine-5'-triphosphate (LNA-ATP) starting from the corresponding nucleoside is described. The overall reaction utilizes an improved "one-pot, three-step" Ludwig synthetic strategy that involves the monophosphorylation of LNA nucleoside, followed by the reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate using water to furnish the corresponding purine LNA nucleotide in good yield with high purity (>99.5%).

Trinucleotide Cap Analogue Bearing a Locked Nucleic Acid Moiety: Synthesis, mRNA Modification, and Translation for Therapeutic Applications.

The synthesis of a new trinucleotide cap analogue containing a locked nucleic acid (LNA) moiety such as m7(LNA)G(5')ppp(5')AmpG and its molecular biology applications are described. The most appealing feature is that this new cap analogue outperforms the standard trinucleotide cap m7G(5')ppp(5')AmpG and the anti-reverse cap analogue m27,3'-OG(5')ppp(5')G by a factor of 5 in terms of translational efficiency.

Chemical Synthesis of a Locked Nucleic Acid-Substituted Dinucleotide Cap Analog.

This article describes a reliable and efficient method for synthesis of the dinucleotide cap analog m7(LNA) G[5']ppp[5']G containing a locked nucleic acid moiety. The required LNA intermediate for the final coupling reaction, m7(LNA) GDP, is prepared in six steps starting from 5'-DMTr-N-DMF LNA guanosine. The overall reaction involves removal of DMTr and DMF groups, 5' monophosphorylation, imidazolide formation, diphosphorylation, and regioselective m7 methylation. The final coupling reaction of m7(LNA) GDP with ImGMP in the presence of zinc chloride as a catalyst affords m7(LNA) G[5']ppp[5']G in 59% yield. © 2021 Wiley Periodicals LLC. Basic Protocol: Synthesis of an LNA-substituted dinucleotide cap analog Support Protocol: Preparation of the tris(tributylammonium) phosphate linker.

Highly regioselective 1,3-dipolar cycloaddition of 3'-O-propargyl guanosine with nitrile oxide: An efficient method for the synthesis of guanosine containing isoxazole moiety.

The 1,3-dipolar cycloaddition reaction of 3'-O-propargyl guanosine with various in-situ generated nitrile oxides in the presence of DMF as a solvent is described. It is noteworthy that the reaction is highly regioselective that affords biologically important guanosine containing isoxazole moiety in good yields with high purities.

Highly regioselective methylation of inosine nucleotide: an efficient synthesis of 7-methylinosine nucleotide.

A facile, straightforward, reliable, and an efficient chemical synthesis of inosine nucleotides such as 7-methylinosine 5'-O-monophosphate, 7-methylinosine 5'-O-diphosphate, and 7-methylinosine 5'-O-triphosphate, starting from the corresponding inosine nucleotide is delineated. The present methylation reaction of inosine nucleotide utilizes dimethyl sulfate as a methylating agent and water as a solvent at room temperature. It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylinosine nucleotide in good yields with high purity (>99.5%).

An efficient protection-free chemical synthesis of inosine 5'-nucleotides.

A facile, straightforward, reliable, and efficient chemical synthesis of inosine nucleotides such as inosine-5'-monophosphate, inosine-5'-diphosphate, and inosine-5'-triphosphate, starting from inosine is delineated. The inosine-5'-monophosphate is achieved by the highly regioselective monophosphorylation of inosine using the Yoshikawa procedure. The inosine-5'-diphosphate is obtained by the coupling reaction of tributylammonium phosphate with an activated inosine-5'-monophosphate using zinc chloride as a catalyst. The inosine-5'-triphosphate is efficiently achieved by the improved "one-pot, three-step" Ludwig synthetic strategy. In all the cases, the resulting final product is isolated in good yields with high purity (>99.5%).

Highly Regioselective Methylation of Guanosine Nucleotides: An Efficient Synthesis of 7-Methylguanosine Nucleotides.

This article describes a simple, reliable, efficient, and general method for the synthesis of 7-methylguanosine nucleotides such as 7-methylguanosine 5'-O-monophosphate (m7 GMP), 7-methylguanosine 5'-O-diphosphate (m7 GDP), 7-methyl-2'-deoxyguanosine 5'-O-triphosphate (m7 2'dGTP), and 7-methylguanosine 5'-O-triphosphate (m7 GTP) starting from the corresponding guanosine nucleotide is described. The present protocol involves methylation reaction of guanosine nucleotide using dimethyl sulfate as a methylating agent and water as a solvent at room temperature to provide the corresponding 7-methylguanosine nucleotide in good yields with high purity (>99.5%). It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylguanosine nucleotide. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 7-methylguanosine nucleotides.

An efficient synthesis of 3'-O-triazole modified guanosine-5'-O-monophosphate using click chemistry.

First chemical synthesis of 3'-O-1,2,3-triazolyl-guanosine-5'-O-monophosphate by copper catalyzed click chemistry is described. The present cycloaddition reaction involves, in situ generation of azide from the corresponding bromide followed by copper catalyst cycloaddition with 3'-O-propargyl guanosine monophosphate in water, in the presence of catalytic amount of ß-cyclodextrin. The CuAAC reaction is highly regioselective forming 1,4-cycloadduct with good yield and high purity. The final compound, 3'-O -triazole substituted guanosine monophosphate has the potential to use in various biomolecules such as labeled nucleic acids, mRNA dinucleotide cap analogs for molecular biology and their applications in the therapeutic field.

An Efficient Synthesis of 5-Aminopropargyl-Pyrimidine-5'-O-Triphosphates Through Palladium-Catalyzed Sonogashira Coupling.

The utilization of 5-aminopropargyl nucleotide serves as a versatile molecular biology tool for the introduction of functional groups into a nucleic acid target of interest by using in-vitro enzymatic incorporation method. This article describes a simple, reliable, general, and efficient two-step chemical method for the synthesis of 5-(3-aminopropargyl)-2'-deoxycytidine-5'-O-triphosphate, 5-(3-aminopropargyl)-cytidine-5'-O-triphosphate, 5-(3-aminopropargyl)-2'-deoxyuridine-5'-O-triphosphate, and 5-(3-aminopropargyl)-uridine-5'-O-triphosphate, starting from the corresponding pyrimidine triphosphate. The first step involves regioselective C-5 iodination of pyrimidine triphosphate using N-iodosuccinimide and sodium azide. In the second step, propargylamine is coupled to the iodo-pyrimidine using the palladium-catalyzed Sonogashira reaction, producing good yields of highly pure (>99.5% HPLC) 5-aminopropargyl-pyrimidine-5'-O-triphosphate. In this approach, the palladium-catalyzed Sonogashira coupling reaction is highly chemoselective and does not involve protection and deprotection. © 2019 by John Wiley & Sons, Inc.

Palladium-Catalyzed Synthesis of (E)-5-(3-Aminoallyl)-Uridine-5'-O-Triphosphates.

This unit describes a simple, reliable, and efficient chemical method for the synthesis of 5-(3-aminoallyl)-2'-deoxyuridine-5'-O-triphosphate (AA-dUTP) and 5-(3-aminoallyl)-uridine-5'-O-triphosphate (AA-UTP), starting from the corresponding nucleoside triphosphate. The presented strategy involves regioselective iodination of nucleoside triphosphate using N-iodosuccinimide followed by the palladium-catalyzed Heck coupling with allylamine to provide the corresponding (E)-5-aminoallyl-uridine-5'-O-triphosphate in good yields. It is noteworthy that the protocol not only provides a high-purity product but also eliminates the use of toxic mercuric reagents. © 2017 by John Wiley & Sons, Inc.

Gram-Scale Chemical Synthesis of Base-Modified Ribonucleoside-5'-O-Triphosphates.

This unit delineates a simple, reliable, straight-forward, general, and efficient chemical method for the synthesis of modified nucleoside-5'-O-triphosphates such as 5-methylcytidine-5'-O-triphosphate (5-Me-CTP), pseudouridine-5'-O-triphosphate (pseudo-UTP), and N1 -methylpseudouridine-5'-O-triphosphate (N1 -methylpseudo-UTP), starting from the corresponding nucleoside. The reaction utilizes an improved protection-free "one-pot, three-step" Ludwig synthetic strategy that involves the monophosphorylation of the nucleoside with phosphorous oxychloride followed by reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate to furnish the corresponding ribonucleoside triphosphate (NTP) in moderate yields. It is noteworthy that the reaction affords high purity (>99.5%) NTPs after DEAE Sepharose column purification. © 2016 by John Wiley & Sons, Inc.

An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5'-Triphosphates.

A simple, reliable, and an efficient "one-pot, three step" chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5'-triphosphate (5-MeCTP), pseudouridine-5'-triphosphate (pseudoUTP) and N(1)-methylpseudouridine-5'-triphosphate (N(1)-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%).

Design, synthesis and biological evaluation of dinucleotide mRNA cap analog containing propargyl moiety.

The first example of the synthesis of new dinucleotide cap analog containing propargyl group such as m(7,3'-O-propargyl)G[5']ppp[5']G is reported. The effect of propargyl cap analog with standard cap was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. It is noteworthy that propargyl cap analog outperforms standard cap by 3.1 fold in terms of translational properties. The propargyl cap analog forms a more stable complex with translation initiation factor eIF4E based on the molecular modeling studies.