The Synthesis of Deuterionucleosides

Abstract

The synthesis of deuterionucleosides for site-specific incorporation into oligo-DNA or -RNA is herein reviewed for NMR or biological studies. The review covers the following aspects: (i) deuteration of the aglycone; (ii) single-site chemical deuteration of the sugar residues; (iii) multiple-site chemical deuteration of the sugar residues; (iv) enzymatic synthesis of deuterated nucleosides or nucleotides; and (v) synthesis of labelled nucleosides with multiple isotopes.     

Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure.

The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2'-deoxy-nucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.     

Synthesis and anti-HIV activity of beta-D-3'-azido-2',3'-unsaturated nucleosides and beta-D-3'-azido-3'-deoxyribofuranosylnucleosides

Since the discovery of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2',3'-didehydro-2',3'dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Synthesis and Anti-HIV Activity of β-D-3′-Azido-2′,3′-unsaturated Nucleosides and β-D-3′-Azido-3′-deoxyribofuranosylnucleosides

Since the discovery of 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2′,3′-didehydro-2′,3′-dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T. The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

The deoxygenations of tosylated adenosine derivatives with Grignard reagents

The reactions of 2'-O- or 3'-O-tosylated adenosines with Grignard reagents resulted in the formation of various products, which were deoxy or branched-chain deoxy sugar nucleosides, 1',2'-unsaturated nucleosides, 3'-deoxy-2'-keto sugar nucleosides, and so on. The convenient method for the synthesis of the 3'-deoxy-2'-keto adenine nucleoside is described.     

Cell-free protein synthesis of perdeuterated proteins for NMR studies

Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form.     

A new method of synthesis of fluorescently labelled oligonucleotides and their application in DNA sequencing.

A new approach to the chemical synthesis of oligodeoxynucleotides bearing reporter functional groups at base residues of 3'-end nucleosides is reported. Applications of the 3'-end fluorescently labelled primers for automated DNA sequencing are shown.     

The occurrence of the syn-C3′ endo conformation and the distorted backbone conformations for C4′-C5′ and P-O5′ in oligo and polynucleotides

Abstract

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2′endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and ribo- guanosine residues in nucleosides and nucleotides prefer the syn-C2′endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5′- H and the N3 of the base and, a few syn-C3′endo conformations are also observed. Evidence is presented for the occurrence of the C3′endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4′-C5′ and P-O5′ bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3′endo conformation and the distorted backbone sugar-phosphate bonds (C4′-C5′ and P- O5′) as in the earlier right-handed case.     

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phosphatidylcholine (PC) is a major component of eukaryotic cell membranes and one of the most commonly used phospholipids for reconstitution of membrane proteins into carrier systems such as lipid vesicles, micelles and nanodiscs. Selectively deuterated versions of this lipid have many applications, especially in structural studies using techniques such as NMR, neutron reflectivity and small-angle neutron scattering. Here we present a comprehensive study of selective deuteration of phosphatidylcholine through biosynthesis in a genetically modified strain of Escherichia coli. By carefully tuning the deuteration level in E. coli growth media and varying the deuteration of supplemented carbon sources, we show that it is possible to achieve a controlled deuteration for three distinct parts of the PC lipid molecule, namely the (a) lipid head group, (b) glycerol backbone and (c) fatty acyl tail. This biosynthetic approach paves the way for the synthesis of specifically deuterated, physiologically relevant phospholipid species which remain difficult to obtain through standard chemical synthesis.     

Possibility for the Existence of a General Conformational Motif in the Active Sites of Enzymes Which are Involved in Nucleic Acids Metabolism

Abstract

Many different modified nucleosides and nucleotides with conformationally restricted partly flattened sugar residues are analyzed as substrates or inhibitors of several groups of enzymes of nucleic acid metabolism. A detailed examination of the sugar moiety of large group of modified nucleosides showed that there is a striking conformational similarity, i.e., they are flattened. We propose herein a hypothesis which can represent a general conformational elements in the structure of the active sites of several different groups of enzymes. This proposal envisions that during the enzymatic process natural substrates should reflect these flattened conformations. This hypothesis allows computation of conformational analyses of the enzyme actives centers as well as the design of new actively metabolized modified nucleosides.     

Synthesis of novel purine nucleosides towards a selective inhibition of human butyrylcholinesterase

The search for new and potent cholinesterase inhibitors is an ongoing quest mobilizing many organic chemistry groups around the world as these molecules have been shown to treat the late symptoms of Alzheimer’s disease as well as to act as neuroprotecting agents. In this work, we disclose the synthesis of novel 2-acetamidopurine nucleosides and, for the first time, regioselective N⁷-glycosylation with 2-acetamido-6-chloropurine, promoted by trimethylsilyl triflate, was accomplished by tuning the reaction conditions (acetonitrile as solvent, 65 °C, 5 h) starting from 1-acetoxy bicyclic glycosyl donors, or by direct coupling of a methyl glucopyranoside with the nucleobase to obtain only N⁷ nucleosides in reasonable yield (55–60%). The nucleosides as well as their sugar precursors were screened for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition. While none of the compounds tested inhibited AChE, remarkably, some of the N⁷ nucleosides and sugar bicyclic derivatives showed potent inhibition towards BChE. Nanomolar inhibition was obtained for one compound competing well with rivastigmine, a drug currently in use for the treatment of Alzheimer’s disease. Experimental results showed that the presence of benzyl groups on the carbohydrate scaffold and the N⁷-linked purine nucleobase were necessary for strong BChE inactivation. A preliminary evaluation of the acute cytotoxicity of the elongated bicyclic sugar precursors and nucleosides was performed indicating low values, in the same order of magnitude as those of rivastigmine.     

The enzymatic synthesis of antiviral agents.

The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of bio-transformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.     

The occurence of the syn-C3' endo conformation and the distorted backbone conformations for C4'-C5' and P-O5' in oligo and polynucleotides.

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2'endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and riboguanosine residues in nucleosides and nucleotides prefer the syn-C2'endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5'-H and the N3 of the base and, a few syn-C3'endo conformations are also observed. Evidence is presented for the occurrence of the C3'endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4'-C5' and P-O5' bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3'endo conformation and the distorted backbone sugar-phosphate bonds (C4'-C5' and P-O5') as in the earlier right-handed case.     

Microbial Synthesis of Antiviral Nucleosides Using Escherichia coli BL21 as Biocatalyst

Experimental conditions such as shaking (aeration) rate, concentration of reagents and extent of culture growth for the optimal synthesis of adenosine using Escherichia coli BL21 as biocatalyst were assessed, achieving 95% yield in 30 min of reaction using microorganisms harvested from late exponential phase. The ability of E. coli BL21 to synthesise purine nucleosides containing sugar residues such as 2'-deoxyribose, 2',3'-dideoxyribose and arabinose was also verified. 2'-Deoxyribo- and arabinonucleosides could be prepared in high yield, while the results obtained with 2',3'-dideoxyribonucleosides were not satisfactory. In the case of 2'-deoxyadenosine, using thymidine as a starting material, a yield of 94% was achieved at 45°C.     

Synthesis of Branched-Chain and Bicyclic Thiosugar Nucleosides

Abstract

The synthesis of strategically protected nucleosides bearing β-mercaptoethyl chains at the α-C-3′ position from 1,2-di-O-acetyl-2′-S-acetyl-5-t?butyldiphenylsilyl-3-deoxy-3-C-(2′-mercaptoethyl)-α-D-ribofuranose 1 is described. It was found that treatment of the 5-O-methanesulfonyl sugar 19 or nucleoside 5 with either benzylmercaptan or methoxide resulted in rapid cleavage of the thiolester followed by intramolecular cyclization. This was used to prepare the novel trans?fused oxathiahydrindane nucleosides 7 and 27 as well as the cAMP analogue 29.     

Stereospecific deuteration of alpha-furanosyl azomycin nucleosides: a model reaction for tritium radiolabeling

Stereospecific synthesis of 1-alpha-d-(2-deuteroribofuranosyl)-2-nitroimidazole (2'-[(2)H]-alpha-AZR) is reported. This, deuteration was independent of the configuration of C-2' -OH group (arabinose or ribose) in sugar moiety of starting molecules. Slightly better yield (>37%) of the deuterated product, 6, from arabinosyl precursor in comparison to corresponding ribose precursor (29%) was obtained which may reflect better stereochemical availability of C-2' -OH in arabinose during oxidation.     

An efficient and economic site-specific deuteration strategy for NMR studies of homologous oligonucleotide repeat sequences.

We describe herein the use of a 2H-labeling strategy to achieve specific assignments of considerably overlapped cross peaks in the 1H-NMR spectrum of a DNA trinucleotide repeat sequence. Our strategy focuses on site-specific 2H-labeling of base moieties to simplify the NMR spectral regions which contain the major portion of the structural information. To achieve efficient preparation of 2H8- or 2H6-labeled DNA and RNA nucleosides and nucleotides, the existing synthetic and purification procedures were significantly improved. Our experiments demonstrate that pyrimidine H6 deuteration reactions may be carried out using non-deuterated base reagents with DMSO-d6 as a 2H donor. These reactions are simple and economic to perform and produce base deuterated nucleosides and nucleotides in high yield. The 2H-labeled residues have been incorporated into oligonucleotides with minor modifications of the existing reaction conditions. Using the homologous CGG repeat sequence, d(CGG)5, as an example, the effectiveness of the site-specific base deuteration strategy is demonstrated. In the otherwise extensively overlapped spectra of d(CGG)5, 2H-labeling has permitted unambiguous identification of a sequential connectivity at a central CG step and confirmation of several other NOE assignments. This information is critical for elucidation of the structure and the folding of the CGG repeat sequences and will contribute to the intensive effort to understand the mechanisms of triplet expansion, which has been implicated in the development of a number of hereditary neurodegenerative diseases. In addition to the two dimensional spectral simplification in a key spectral region using site-specific 2H8/2H6-labeling, the potential applications of the prescribed strategy in homonuclear three dimensional experiments are also discussed.     

2‐Azapurine Nucleosides: Synthesis,Properties, and Base Pairing of Oligonucleotides

This review deals with 2‐azapurine (imidazo[4,5‐d] [1,2,3]triazine) nucleosides and closely related analogs. Different routes are described to yield the desired target compounds, including a sequence of ring‐opening and ring‐closure reactions performed on purine nucleosides or direct glycosylation of a 2‐azapurine nucleobase with a sugar halide. Further, physical and spectroscopic properties of 2‐azapurine nucleosides are discussed, including fluorescence, ¹³C‐NMR data, single‐crystal X‐ray analyses, and conformation studies on selected compounds; new biological data are presented. The second part of this review is dedicated to oligonucleotides containing 2‐azapurines, including building‐block (phosphoramidite) preparation and their use in solid‐phase oligonucleotide synthesis. Base‐pairing properties of 2‐azapurine nucleosides as surrogates of canonical constituents of DNA were evaluated.     

Rapid communication capturing the destabilizing effect of dihydrouridine through molecular simulations

The structural effects of the commonly occurring modified nucleoside dihydrouridine (D) observed experimentally in model oligonucleotides include a strong destabilization of the C3′‐endo sugar conformation of D, the disruption of stacking interactions of neighboring residues with D and a possible destabilization of the C3′‐endo sugar pucker of the 5′‐neighboring nucleoside. Our simulations with a combination of a set of parameters for modified RNA residues with the recently developed AMBER FF99χ force field having reoptimized glycosidic torsion angle parameters for standard nucleosides was found to reproduce the destabilizing effect of dihydrouridine better than with the AMBER FF99 force field for nucleic acids for which the parameters for the modified residues were originally developed. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 985–991, 2014.     

Deuterated detergents for structural and functional studies of membrane proteins: Properties,chemical synthesis and applications

Abstract

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.