Formation of nucleoside 5′-polyphosphates under potentially prebiological conditions

Summary Aqueous solutions of linear inorganic polyphosphates incubated in presence of Mg ions depolymerize to give trimetaphosphate. The presence of a nucleoside 5-phosphate has little influence upon the reaction. Drying the products obtained by incubating a linear polyphosphate with Mg ions in the presence of a nucleoside 5-phosphate yields nucleoside 5-polyphos-phates. The prebiological relevance of the reactions is discussed.Abbreviations P_n(n=1,2,3,) linear polyphosphate containing n phosphate residues - P₃! trimetaphosphate - A adenosine - p_nN nucleoside 5-polyphosphate containing n phosphate residues, e.g. with N = A, n = 4 - p₄N adenosine 5-tetraphosphate - _P ^* _lp_mA p_nA, (n = 1 + m); adenosine 5-polyphosphate containing n phosphate units with³³p-label on terminal 1 phosphate groups     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇     

Formation of 2′-5′ Phosphodiester Linkages in Polyribonucleotides

Unlike technical grade yeast RNA, which was confirmed to contain several per cent of 2′–5′ phosphodiester linkages, RNA prepared from different kinds of commercial yeast in a cold room consisted exclusively of 3′–5′ phosphodiester linkages. Heat treatment of the 3′–5′ linked RNA solution resulted in partial isomerization of the internucleotide linkage of the polynucleotide chain (C₃′-C₅′->C₂′-C₅′). The isomerization of RNA occurred in the presence of water, at high temperature, and under acidic conditions. Treatment of dry RNA at 100°C for 2hr did not result in any detectable isomerization. The isomerization was actually observed in yeast RNA when yeast cells suspended in sodium chloride solution were heated. It is concluded therefore that 2′-5′ phosphodiester linkages found in technical grade RNA had been formed neither at a step of precipitating RNA with acid nor at a step of drying RNA, but had been formed at a step of heat extraction of RNA from yeast. When 0.1 % poly (A) solution, pH 4.8, was heated for 20 hr in a boiling water bath, the isomerization proceeded during the first 6hr, and finally reached about 37%, irrespective of chain length.     

Chemical Synthesis of Cap Analogues: P1,P2-Dinucleoside-5′-diphosphates

Abstract

A procedure was developed for the chemical synthesis of P¹,P²-dinucleoside-5′-diphosphates (N₁(5′)pp(5′)N₂) on a nanomolar scale Reaction conditions for activating purine-5′-monophosphates (pA, pG, and pm⁷G) by 1,1′-carbonyldiimidazole were studied and optimized in respect to solvents and amount of activating reagent used. Various dinucleoside-5′-diphosphates were synthesized in 62-98% yield by incubating activated and non-activated purine-5′-monophosphates. Two unexpected by-products were formed by competition reactions: the imidazolidate of the non-activated nucleotide and the corresponding symmetrically substituted dinucleoside-5′-diphosphate. A mechanism is proposed to explain the observed side reactions.     

Comparative Studies of Duplex and Triplex Formation of 2′-5′ and 3′-5′ Linked Oligoribonucleotides

Abstract

We have studied double and triple helix formation between 2′–5′ or 3–5′ linked oligoriboadenylates and oligoribouridylates with chain length 7 or 10 by CD spectrometry. The complex formation depends on the type of linkage of oligoribonucleotides, chain length, concentration and molar ratio of the strands, temperature and the cationic concentration. Mixture of any linkage isomers of oligo(rA) and oligo(rU) in 1:1 molar ratio form duplex at 0.1 M NaCl. The duplex stability largely depends on the type of the linkages and is in the following order; [35′] oligo(rA)·[3′-5′] oligo(rU) > [2′-5′] oligo(rA)'[3′-5′] oligo(rU) > [3′-5′] oligo(rA)·[2′-5′] oligo(rU) > [2–5′] oligo(rA)*[2′-5′] oligo(rU). The higher cationic concentrations, 0.5 M MgCl₂, stabilize the complex and either duplex or triplex is formed depending on the input strand ratio and the type of linkage. Thermodynamic parameters, DH and DS, for the complex formation between linkage isomers of oligo(rA) and oligo(rU) showed a linear relationship indicating an enthalpy-entropy compensation phenomena. The duplex and triplex composed of [2′-5′] oligo(rA) and [2′-5′] oligo(rU) exhibit different CD spectra compared to those of any others containing 3–5′ linkage, suggesting that the fully 2–5′ duplex and triplex may possess a unique conformation. We describe prebiological significance of the linkage isomers of RNA and selection of the 3–5′ linkage against 2′-5 linkage.     

Synthesis and Properties of P1, P2-, P1, P3- and P1, P4-Dinucleoside Di-, Tri- and Tetraphosphate mRNA 5′-Cap Analogues

Abstract

Chemically synthesized dinucleoside P¹, P²-di-, P¹, P³-tri- and P¹, P⁴-tetraphosphates, derivatives of 5′-linked 7-methylguanosine and guanosine were characterized with respect to their structural properties and functional effect on eukaryotic translation inhibition.     

2′-Deoxy-1-methylpseudocytidine,a stable analog of 2′-deoxy-5-methylisocytidine

2′-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2′-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2′-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2′-deoxycytidine and 2′-deoxyguanosine pair.     

Antiplatelet Activity,P2Y1 and P2Y12 Inhibition,and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background

Diadenosine tetraphosphate (Ap₄A), a constituent of platelet dense granules, and its P¹,P⁴-dithio and/or P²,P³-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y₁ and P2Y₁₂. The most active of those analogs, diadenosine 5′,5″″-P¹,P⁴-dithio-P²,P³-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

Objective

To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y₁ and P2Y₁₂ receptor antagonism, and their metabolism in human plasma.

Methods

We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y₁-mediated changes in cytosolic Ca²⁺, P2Y₁₂-mediated changes in VASP phosphorylation, and metabolism in human plasma.

Results

The inhibition of ADP-induced human platelet aggregation and human platelet P2Y₁₂ receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P¹- and P⁴-phosphorothioate groups, the S_PS_P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R_PR_P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S_PR_P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P²,P³-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S_PS_P and the R_PR_P diastereomers.

Conclusions

The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y₁ and P2Y₁₂ receptors.     

Formation of nucleoside 5′-polyphosphates from nucleotides and trimetaphosphate

Summary When solutions of nucleoside 5-phosphates and trimetaphosphate are dried out at room temperature, nucleoside 5-polyphosphates are formed. The Mg⁺⁺ ion shows a superior catalytic function in this reaction when compared with other divalent metal ions. Starting with nucleoside 5-phosphates, Mg⁺⁺ and trimetaphosphate, the predominant products in the nucleoside 5-polyphosphate series p_nN are p₄N, p₇N and P₁₀N. Nucleoside 5-diphosphates yield p₅N and p₈N, nucleoside 5-triphosphates give p₆N and p₉N. The prebiological relevance of these reactions is discussed.Abbreviations P_n (n = 1,2,3,) linear polyphosphate containing n phosphate residues - P₃! trimetaphosphate - A adenosine - U uridine - dA 2-dexyadenosine - T thymidine - P_nN nucleoside 5-polyphosphate containing n phosphate residues, e.g. with N = A and n = 4 - p₄A adenosine 5-tetraphosphate     

Design,synthesis, and X-ray structural studies of BACE-1 inhibitors containing substituted 2-oxopiperazines as P1′-P2′ ligands

We report the design and synthesis of a series of BACE1 inhibitors incorporating mono- and bicyclic 6-substituted 2-oxopiperazines as novel P1′ and P2′ ligands and isophthalamide derivative as P2-P3 ligands. Among mono-substituted 2-oxopiperazines, inhibitor 5a with N-benzyl-2-oxopiperazine and isophthalamide showed potent BACE1 inhibitory activity (K_i = 2 nM). Inhibitor 5g, with N-benzyl-2-oxopiperazine and substituted indole-derived P2-ligand showed a reduction in potency. The X-ray crystal structure of 5g-bound BACE1 was determined and used to design a set of disubstituted 2-oxopiperazines and bicyclic derivatives that were subsequently investigated. Inhibitor 6j with an oxazolidinone derivative showed a BACE1 inhibitory activity of 23 nM and cellular EC₅₀ of 80 nM.     

Efficient Synthesis of 2′-Amino-2′-deoxypyrimidine 5′-Triphosphates

Abstract

The synthesis of 2′-amino-2′-deoxypyrimidine 5′-triphosphates is described. The 2′-amino-2′-deoxyuridine 5′-triphosphate is obtained from uridine in four steps with 25% overall yield. The 2′-amino-2′-deoxycytidine 5′-triphosphate is obtained from uridine in seven steps with 13% overall yield.     

Formation of an Unexpected 2-Deoxy-α-D-Threo-Pentofuranosyl Azide by Reaction of O2,3′-Anhydro-5′-O-trityl-2′-deoxycytidine with Lithium Azide

Abstract

Reaction of 0²,3′-anhydro-5′-0-trityl-2′-deoxycytidine (1) with LiN₃s in DMF resulted in the formation of 1-(3-azido-2,3-dideoxy-5-0-trityl-β-D-erythro-pentofuranosyl) cytosine (2) and 3-0-(4-amino-1,3-pyrimidin-2-yl)-5-0-trityl-2-deoxy-α-D-threo-pentofuranosyl azide (3) (2:3 = 1:1) in 88% yield. Compound 3 was deprotected with 80% aqueous AcOH yielding 4     

Facile synthesis of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N 6-ethenoadenosine nucleosides

Facile synthetic methods of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N ⁶-ethenoadenosine nucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction were proposed. The synthetic products were isolated and purified by preparative HPLC and their structures were confirmed by¹H NMR (500 MHz) and FAB-MS including high resolution mass measurement. These modified nucleoside analogs have not been reported yet. Therefore, these modified nucleoside analogs are of potential value to be studied further for biological activity such as anticancer or antiviral.     

Synthesis,Cytostatic Activity and Inhibition of Ribonucleotide Reductase by 5′-Phosphoramidates and 5′-Diphosphates,of 2′-O-Allyl-arabinofuranosyl Nucleosides

Abstract

The diphosphates of a series of 2′-O-allyl-1-β-D-arabinofuranosyl derivatives, previously obtained by us, have been prepared and tested for their inhibitory activity in an in vitro assay using R1 and R2 subunits of the purified recombinant mouse ribonucleotide reductase (RNR). 2′-O-Allyl-araU diphosphate proved to be inhibitory, with an IC₅₀ of 100 μM. The 5′-phosphoramidate pronucleotide of 2′-O-allyl-araU was also prepared and tested for inhibition of tumor cell proliferation.     

A 31P NMR Study of the Reaction of Adenosine 3′ 5′-Cyclic Monophosphate with 2, 4, 6-Triisopropylbenzenesulfonyl Chloride

Abstract

Detection limits for the minor component in binary mixtures of Ado/AraA, Ado/XyloA, and Urd/dUrd depend strongly on the combined concentration of analytes. Limiting concentrations (in which ≤1% of the minor component was detected) were about two orders of magnitude lower with HPLC (UV detection) than with ¹H NMR and TLC (UV detection) with these nucleosides (ε_max 10 000–15 000). Minimum molar percentages of minor components detected in the 0.1–10 mM range were 0.25–1% with HPLC (UV), 1–2% with ¹H NMR, and ～2% with TLC (UV).     

The 5′-untranslated region of the maize alcohol dehydrogenase gene provides efficient translation of mRNA in plants under stress conditions

The reduced level of expression of most cell proteins under stress conditions is determined by the low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is a gene whose mRNA is efficiently translated in hypoxia. The reporter gene assay showed that the leader sequence of the adh1 mRNA provided for efficient translation of the reporter gfp gene in Nicotiana benthamiana cells in hypoxia or heat shock. The presence of this sequence in the 5′-UTR of mRNA did not change the level of expression under aerobic conditions, but the levels of gfp expression in hypoxia or heat shock were reduced five-to tenfold in the absence of this leader and remained unaffected when the adh leader sequence was present in the 5′-UTR. The adh1 leader sequence did not change the mRNA stability nor exhibited a promoter activity. Thus, the adh leader sequence acted as a translational enhancer, providing for efficient mRNA translation in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes was proposed for the development of new systems to efficiently express the target proteins in plants under stress conditions.     

Distinct 5′ UTRs regulate XIAP expression under normal growth conditions and during cellular stress

X-chromosome linked inhibitor of apoptosis, XIAP, is cellular caspase inhibitor and a key regulator of apoptosis. We and others have previously shown that XIAP expression is regulated primarily at the level of protein synthesis; the 5′ untranslated region (UTR) of XIAP mRNA contains an Internal Ribosome Entry Site (IRES) that supports cap-independent expression of XIAP protein during conditions of pathophysiological stress, such as serum deprivation or gamma irradiation. Here, we show that XIAP is encoded by two distinct mRNAs that differ in their 5′ UTRs. We further show that the dominant, shorter, 5′ UTR promotes a basal level of XIAP expression under normal growth conditions. In contrast, the less abundant longer 5′ UTR contains an IRES and supports cap-independent translation during stress. Our data suggest that the combination of alternate regulatory regions and distinct translational initiation modes is critical in maintaining XIAP levels in response to cellular stress and may represent a general mechanism of cellular adaptation.     

Synthesis and Biological Evaluation of Some 5-Nitro- and 5-Amino Derivatives of 2′-Deoxycytidine, 2′,3′-Dideoxyuridine,and 2′,3′-Dideoxycytidine

Abstract

In this article, we describe the synthesis of 5-nitro-1-(2-deoxy-α-D-erythro-pentofuranosyl)cytosine (4α), 5-nitro-1-(2-deoxy-β-D-erythro-pentofuranosyl)cytosine (4β), 5-amino-1-(2-deoxy-α-D-erythro-pentofuranosyl)cytosine (5α), 5-nitro-1- (2-deoxy-β-D-erythro-pentofuranosyl)cytosine (5β), 5-nitro-1-(2,3-dideoxy-β- D-ribofuranosyl)uracil (6β), 5-amino-1-(2,3-dideoxy-α,β-D-ribofuranosyl)uracil (7), 5-nitro-1-(2,3-dideoxy-α,β-D-ribofuranosyl)cytosine (8) and 5-amino-1-(2,3-dideoxy-β-D-ribofuranosyl)cytosine (9β). The prepared compounds were tested for their activity against HIV and HBV viruses, but they did not show significant activity.     

Differences in biochemical properties of two 5′-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions

Deep-sea Shewanella violacea 5′-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions.

Abbreviations: NTase: 5′-nucleotidase; SANTase: Shewanella amazonensis 5′-nucleotidase; SVNTase: Shewanella violacea 5′-nucleotidase; CD: circular dichroism