The synthesis of deuterionucleosides

The synthesis of deuterionucleosides for site-specific incorporation into oligo-DNA or -RAA 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     

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.     

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.     

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.     

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 7-(β-D-Glucopyranosyl)-6-Thiotheophylline and 7-(α-D-Arabinopyranosyl)-6-Thiotheopwlline. Conformational Study of the Peracetyl Derivatives.

Abstract

The synthesis of two 7-glycosyl-6-thiotheophylline nucleosides where the sugar moieties are β-D-glucose (1b) and α-D-arabinose (2b) is reported. The syn-anti equilibrium of the peracetyl derivatives was studied by the line-shape and the ¹H-NMR nOe methods, and molecular mechanics analysis.     

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.     

An Efficient Synthesis of 2-[(4-Amino∼1,2-dihyro-2-oxo-1- pyrimidinyl)methoxyi-l,3-propanediyl-di-L-valinate an Anti-cytomegalovirus Agent

Abstract

A novel synthesis of 1-(β-D-pentopyranosyl)pyridinethione nucleosides utilizing pyridine-2(1H)-thiones and α-bromoxylose or β-bromoarabinose triacetate as starting components is described. The free nucleosides were tested for their potential activity against HIV and different types of tumor virus.     

Synthesis and Antiviral Activity of Some N-Pentopyranosyl-2-Pyridinethiones

Abstract

A novel synthesis of 1-(β-D-pentopyranosyl)pyridinethione nucleosides utilizing pyridine-2(1H)-thiones and α-bromoxylose or β-bromoarabinose triacetate as starting components is described. The free nucleosides were tested for their potential activity against HIV and different types of tumor virus.     

Derivatives of 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-phenyluracil and 5-Benzyluracil. Synthesis and Biological Properties

Abstract

A number of 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil and -cytosine nucleosides substituted at the 5 position with a nitrophenyl or nitrobenzyl group were synthesized from 5-phenyl- and 5-benzyluracil via condensation of the fluorinated sugar, followed by nitration. The corresponding amino analogues were also prepared by reduction of the nitro nucleosides. The uracil nucleosides were converted into the corresponding cytosine nucleosides by way of the triazole intermediates. None of these nucleosides exhibited significant activity against herpes simplex virus type 1 in Vero cells. However, cytosine nucleosides containing the o-nitrophenyl, p-nitrophenyl, p-nitrobenzyl or p-aminobenzyl substituent were found to be toxic (even at 1 μM) to uninfected Vero cells, although they were essentially nontoxic in HL-60 cells. The 5′-monophosphates of the uracil nucleosides were inhibitors of the reaction catalyzed by purified Ehrlich ascites carcinoma thymidylate synthase, the 5-phenyluracil nucleotides causing a strong inhibition, competitive vs dUMP, described by the K_i value of 0.01 μM.     

Synthesis and Biological Evaluation of Some Acyclic 4,6-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidine Nucleosides

Abstract

The chemical synthesis and biological evaluation of some acyclic α-[6-(1′-carbamoylalkylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylamide nucleosides are described.     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 4′-C-HYDROXYMETHYL-2′-FLUORO- D-ARABINOFURANOSYLPURINE NUCLEOSIDES

A series of 4′-C-hydroxymethyl-2′-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity. The details of a convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-α-D-arabinofuranosyl bromide (13) are presented. Proof of the structure and configuration at all chiral centers of the sugars and the nucleosides were obtained by proton NMR. All five target nucleosides were evaluated for cytotoxicity in human tumor cell lines. The 4′-C-hydroxymethyl clofarabine analogue (16β) showed slight cytotoxicity in CCRF-CEM leukemia cells.     

SYNTHESIS OF 5-(2,2′-BIPYRIDINYL AND 2,2′-BIPYRIDINEDIIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: PRECURSORS TO METALLO-DNA CONJUGATES

ABSTRACT

The synthesis of 2,2′-bipyridinyl-2′-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2′-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2′-deoxyuridine nucleoside (Bipy²⁺-dU) with an ethylenyl linker. The Bipy²⁺-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.     

Selective Deuteration Labelling of 2′-Deoxyguanosine at the Carbon C-4’ Position

Abstract

Selective incorporation of deuterium within the sugar moiety of nucleosides and oligonucleotides can be used for different purposes including isotopic effect determination in mechanistic studies, massspectrometry fragmentation investigations, nuclear magnetic resonance analyses. We wish to report a simple method which allows the selective deuteration labelling of 2'-deoxyguanosine at the C-4'position through the intermediary of 9-(2-deoxy-B-D-erythropento-1,5-dialdo-114-furanosyllquanine. Heating of aqueous pyridine solution [1:11 of 2′-deoxyguanosine-5′-aldehyde for 1 hr at 60°C leads to a partial epimerisation of carbon C-4' with subsequent formation of 9-(2-deoxy-α-L-threopento-1,5-dialdo-1,4-furanosyl)guanine in 40% yield. A likely intermediate of this reaction appears to be a 5'-enol derivative. Similar treatment of 2′-deoxyguanosine-5′-aldehyde in D₂0-pyridine [1-1] gives after NaBH₄ reduction 60% of 2′-deoxyguanosine which is selectively deuterated at the C-4′ position. The extend of the isotopic labelling was up to 95% as determined by high resolution electron impact mass spectrometry and ¹H NMR analyses. Heating of the aqueous pyridine solution of 2′-deoxyguanosine-5′-aldehyde for a longer period (3–4 hrs) gave rise to two other nucleosides which where assigned as 9-(2-deoxy-α-D-threo-pentofuranosy1)guanine and 9-(2-deoxy-n-L-erythro-pentofuranosyl)guanine. A retro-aldol mechanism appears to be involved in the epimerization reaction which takes place at carbon C-3′.     

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.     

Facile Synthesis of 3′-C-Branched 1,5-Anhydrohexitol Nucleosides

Abstract

The 3′-β-C-branched anhydrohexitol nucleosides have been conveniently synthesised starting from commercially available D-ribose following the reaction sequence: (i) conversion of protected pentofuranose sugar to the corresponding hexopyranosyl nitrosugar (ii) addition of the conjugate base of nitrosugar to formaldehyde to obtain C-branched nitro sugar (iii) removal of nitro group by n-tributyltin hydride treatment and (iv) Mitsunobu type alkylation to build up the nucleobase.     

A Versatile Synthesis of Dihydropyrimidinone C‐Nucleosides

A versatile synthesis of N‐substituted dihydropyrimidinone C‐nucleosides (20–29) is described. Glycosyl amino esters (3–9), obtained by reductive alkylation of glycosyl amino esters 1 and 2, on condensation with different isocyanates afforded respective ureido derivatives (10–19) in good to quantitative yields. The latter on cyclative amidation with a combination of DBU/TBAB (tetrabutylammonium bromide)/4Å molecular sieve gave the corresponding nucleosides (20–29) in good yields.     

Synthesis and Biological Activity of 5-Fluoro-2-thiocytosine Nucleosides

Abstract

Two pathways are described for the synthesis of the 2′-deoxynucleosides of 2-thiocytosine and 5-fluoro-2-thiocytosine: (a) by nucleoside condensation, (b) by amination of the corresponding nucleosides of 2,4-dithiouracil. Biological activities vs two cell systems are described. The nucleosides are moderate to weak substrates of deoxycytidine kinase and, partly as a result of this, reasonable good inhibitors of the enzyme