Convenient Synthesis of Nucleoside‐5′‐Diphosphates from the Corresponding Ribonucleoside‐5′‐phosphoroimidazole

The reaction of ribonucleoside‐5′‐phosphoroimidazolide with a tributylammonium orthophosphate in anhydrous dimethylformamide at room temperature provides a general method for the synthesis of nucleoside‐5′‐diphosphates. The novelty of the approach is to use the triethylammonium salt of 5′‐monophosphate nucleoside derivative prior to the imidazolate reaction with imidazole, triphenylphosphine, and 2,2′‐dithiodipyridine. Deprotection, followed by displacement of the imidazole moiety using tributylammonium orthophosphate and a catalytic amount of zinc chloride in dimethylformamide gave the desired 5′‐diphosphate products. The triethyl ammonium salt of 5′‐diphosphate nucleosides was purified by flash chromatography using DEAE (diethylaminoethyl weak anion exchange resin) Sepharosa fast flow packed in an XK 50/60 column on an Akta FPLC (Fast Protein Liquid Chromatography). Synthesis procedures are reported for adenosine‐5′‐diphosphate, uridine‐5′‐diphosphate, cytidine‐5′‐diphosphate, and guanosine‐5′‐diphosphate. Yields for the displacement reactions ranged from 95 to 97%. Thus, this method offers the advantages of shorter reaction time, greater product yield, and a more cost‐effective synthetic route.     

Comprehensive Analysis of DNA Strand Breaks at the Guanosine Site Induced by Low‐Energy Electron Attachment

To elucidate the role of guanosine in DNA strand breaks caused by low‐energy electrons (LEEs), theoretical investigations of the LEE attachment‐induced C? O σ‐bonds and N‐glycosidic bond breaking of 2′‐deoxyguanosine‐3′,5′‐diphosphate (3′,5′‐dGMP) were performed using the B3LYP/DZP++ approach. The results reveal possible reaction pathways in the gas phase and in aqueous solutions. In the gas phase LEEs could attach to the phosphate group adjacent to the guanosine to form a radical anion. However, the small vertical detachment energy (VDE) of the radical anion of guanosine 3′,5′‐diphosphate in the gas phase excludes either C? O bond cleavage or N‐glycosidic bond breaking. In the presence of the polarizable surroundings, the solvent effects dramatically increase the electron affinities of the 3′,5′‐dGDP and the VDE of 3′,5′‐dGDP^?. Furthermore, the solvent–solute interactions greatly reduce the activation barriers of the C? O bond cleavage to 1.06–3.56 kcal mol^?1. These low‐energy barriers ensure that either C_5′? O_5′ or C_3′? O_3′ bond rupture takes place at the guanosine site in DNA single strands. On the other hand, the comparatively high energy barrier of the N‐glycosidic bond rupture implies that this reaction pathway is inferior to C? O bond cleavage. Qualitative agreement was found between the theoretical sequence of the bond breaking reaction pathways in the PCM model and the ratio for the corresponding bond breaks observed in the experiment of LEE‐induced damage in oligonucleotide tetramer CGTA. This concord suggests that the influence of the surroundings in the thin solid film on the LEE‐induced DNA damage resembles that of the solvent.     

A Practical Synthesis of 3′-Thioguanosine and of Its 3′-Phosphoramidothioite (a Thiophosphoramidite)

Starting from guanosine, an efficient method for the synthesis of 3′-thioguanosine (see 13 ) and of its 3′-phosphoramidothioite (see 23 ), suitable for automated incorporation into oligonucleotides, was developed. Reaction of 5′-N²-protected guanosine with 2-acetoxyisobutyryl bromide afforded stereoselectively the 2′-O-acetyl-3′-bromo-β-D -xylofuranosyl derivative 3 , which was converted to a 7 : 3 mixture of the S-acyl ribofuranosyl intermediates 5 or 6 and the 3′,4′-unsaturated by-product 4 . The S-acylated nucleosides 5 and 6 were then converted in three steps to 5′-O-(4,4′-dimethoxytrityl)-3′-S-(pyridin-2-ylthio)-3′-thioguanosine ( 11 ), which served as a common intermediate for the preparation of free 3′-thionucleoside 13 and 3′-thionucleoside 3′-phosphoramidothioite 23 .     

Stacked and continuous helical self-assemblies of guanosine monophosphates detected by vibrational circular dichroism

The aim of this study was to characterize self-assembled structures of guanosine derivatives in aqueous solutions by vibrational circular dichroism (VCD) and electronic circular dichroism (ECD). Three guanosine derivatives were studied [5'-guanosine monophosphate (GMP), diphosphate (GDP), and triphosphate (GTP)] using a broad range of concentrations and various metal/guanosine ratios. VCD was used for the first time in this field and showed itself to be a powerful method for obtaining specific structural information in solution. It can also help to determine the impact that the cations have, when added to the solution, on the versatile structures of guanine derivatives in terms of their association and disassociation. Based on the markedly different intensities and signs of the VCD signals observed for different concentrations of guanosine derivatives, we propose various structures based on guanine quartets for high guanosine concentrations and high K(+)/guanosine ratios (i.e., columnar helical organization of the quartets, which are rearranged into a continuous helix). We performed a degenerate coupled oscillator (DCO) calculation to interpret the VCD spectra obtained and how they vary during the assembly of guanosine derivatives. The calculations correctly predicted the VCD spectra and enabled us to identify the structures of the metal cation/guanosine monophosphate aggregates. ECD in the ultraviolet region was used as a diagnostic tool to characterize the studied systems and as a contact point between the previously defined structures of the guanine derivative assemblies and the molecular systems studied here. These studies revealed that the VCD technique is a powerful new method for determining the structures of optically active guanosine motifs.     

Reliable Synthesis of Various Nucleoside Diphosphate Glycopyranoses

A reliable and high yielding synthetic pathway for the synthesis of the biologically highly important class of nucleoside diphosphate sugars (NDP‐sugars) was developed by using various cycloSal‐nucleotides 1 and 9 as active ester building blocks. The reaction with anomerically pure pyranosyl‐1‐phosphates 2 led to the target NDP‐sugars 20 – 45 in a nucleophilic displacement reaction, which cleaves the cycloSal moiety in anomerically pure forms. As nucleosides cytidine, uridine, thymidine, adenosine, 2′‐deoxy‐guanosine and 2′,3′‐dideoxy‐2′,3′‐didehydrothymidine were used while the phosphates of D ‐glucose, D ‐galactose, D ‐mannose, D ‐NAc‐glucosamine, D ‐NAc‐galactosamine, D ‐fucose, L ‐fucose as well as 6‐deoxy‐D ‐gulose were introduced.     

Conformation-sensitive Raman lines of mononucleotides and their use in a structure analysis of polynucleotides: guanine and cytosine nucleotides

Raman spectra are presented for nine crystals containing the guanosine residue and ten crystals containing the cytidine residue whose conformations are known from their X-ray crystallographic analyses. A nearly complete set of assignments of all the observed Raman lines in the 1700—150 cm⁻¹ range is proposed on the basis of a previous normal coordinate treatment of guanine and cytosine with a set of force constants determined by an ab initio MO method, and on the basis of a mutual comparison of the observed spectra. A number of conformation sensitive Raman lines are found here, and several rules on the structure—spectrum correlations are proposed. Raman spectral features in the 1400—1300 cm⁻¹ and 700—600 cm⁻¹ ranges seem to reflect sensitively and regularly the conformation of the guanosine residue, namely its ribose-ring puckering state at the torsion angle around its glycosidic bond. A spectral feature in the 1300—1200 cm⁻¹ range is found to be sensitive to the cytidine conformation. The position of a strong Raman line in the 900—750 cm⁻¹ region, on the other hand, seems to indicate a particular set of torsion angles along the PO5′C5′C4′C3′O3′ backbone. In the light of these proposed rules, the so-called B-form poly [d(GC)].· poly[d(GC)] in solution must have an O4′endo-anti guanosine, a C2′endo-anti cytidine, and an “alternating B” backbone as proposed by Klug [7] while its Z-form should have a C3′ endo-syn guanosine, a form of cytidine in between C2′endoC1′exo-anti cytidine, and a Z_I form backbone, as defined by Wang [41].     

Atomic Absorption Spectrophotometric Analysis of Condensed Phosphates by a Flow Detector Connected with a Gel Chromatographic Column

Abstract

An atomic absorption flow detector combined with a gel chromatographic column (Sephadex G-25) gives a sensitive and quantitative method of determining various condensed phosphates such as diphosphate, tri-phosphate, tetraphosphate and Kurrol's salt. This method is based on the automatic recording of atomic absorption at the resonance line of magnesium due to magnesium complexes of condensed phosphates which are produced during the elution of condensed phosphate anions through the column pre-equilibrated with a magnesium chloride solution.     

A homogeneous fluorescence resonance energy transfer system for monitoring the activation of a protein switch in real time

A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.     

A Facile and Cost‐effective Electroanalytical Strategy for the Quantification of Deoxyguanosine and Deoxyadenosine in Oligonucleotides Using Screen‐printed Graphite Electrodes

The development of electroanalytical methods for the detection and quantification of nucleotides in DNA offers vital implications in assessing the degree of oxidation or epigenetic modification in DNA. Unfortunately, the electrochemical response of oligonucleotides is strongly influenced by the size, composition and nucleic base sequence. In this article, an optimized analytical procedure for the enzymatically breakdown of the oligonucleotides to their corresponding nucleotides for the evaluation of the electrochemical response through the use of square wave voltammetry (SWV) is presented. Enzymatic digestion of oligonucleotides has been optimized in terms of buffer composition, digestion time, strategy for stopping the enzymatic reaction and filtration requirement for enzyme removal, and then compared to an established protocol. Under the optimized protocol SWV response of a number of untreated and enzymatically digested six‐mer oligonucleotides, namely 5′‐GGGGGG‐3′, 5′‐AAAAAA‐3′, 5′‐CGCGCG‐3′ and 5′‐AAACGC‐3′ have been analysed, providing a higher sensitivity for the determination of guanosine and adenosine monophosphate species under digestion conditions with a more facile and cost effective procedure. The novel strategy for the enzymatically treated oligonucleotides in combination with the SWV response provides a proof of principle for feasible applications in the diagnosis of methylated guanosine in DNA as a potential biomarker due to its relation with cancer.     

无机层状复合氢氧化物中顺铂-DNA模型分子的选择性插入

药物分子的选择性包裹和控制释放是药物研究领域中具有挑战性的研究方向。本文研究表明:顺铂-DNA模型分子cis-[Pt(NH₃)₂(5′-GMP)₂](5′-GMP 5′-单磷酸鸟苷)可插入无机层状复合氢氧化物[Zn_0.68Al_0.32(OH)₂](NO₃)_0.32·mH₂O。但另一种层状复合氢氧化物[LiAl₂(OH)₆]Cl·H₂O由于其阳离子层中正电荷密度较高、阳离子层与层间阴离子之间静电作用较强,因而顺铂-DNA模型分子不能通过离子交换方式插入其层间。光谱数据证实插入层间的顺铂-DNA模型分子结构不变。这可能为铂-DNA分子的传递提供新的方法。     

Mass spectra of nucleoside derivatives

The mass spectra of derivatives of uridine, adenosine, cytidine and guanosine are recorded. Derivatization techniques include permethylation, acetylation, trifluoroacetylation, trimethylsilylation and the synthesis of 2′,3′-O-isopropylidenes and 2′,3′-O-phenylboronic esters. Sequential derivatization by a selective combination of some of these procedures results in nucleosides which are blocked with a characteristic group at the cis 1,2 diol position, and, which contain other substituent groups that enhance the volatility of the compound. The specific substitution at the cis-glycol region has been shown to be particularly useful in asymmetrically derivatizing dinucleoside phosphates since certain fragment ions from their mass spectra indicate the sequence of the two nucleoside components. Sequence isomers such as adenylyl-(3′-5′)-uridine and uridylyl-(3′-5′)-adenosine can be unambiguously distinguished.     

Semiempirical studies on the interactions of dialkyldi(aquo)tin cations, [R2Sn(H2O)2]2+, with selected nucleotides

Semiempirical calculations have been carried out on the interactions of [R₂Sn(H₂O)₂]²⁺, [R = H(CH₂)_n: n = 1–8], mainly with five nucleotides, 5′‐adenosine monophosphate (5′‐AMP), but also with guanosine 5′‐monophosphate (5′‐GMP), cytidine 5′‐monophosphate (5′‐CMP), uridine‐5′‐monophosphate (5′‐UMP) and inosine 5′‐monophosphate (5′‐IMP). The preferred sites of interaction were calculated to be the ribose O2 and O3 hydroxyl oxygens and/or the phosphate oxygens, with the nitrogen sites in the bases the least attractive to the tin compounds. This is in general agreement with experimental findings. Structures of the 1:1 coordination complexes vary from distorted tetrahedral, to distorted trigonal pyramidal to distorted octahedral geometries. Copyright © 2007 John Wiley & Sons, Ltd.     

Drawing with Iron on a Gel Containing a Supramolecular Siderophore

Guanosine‐5′‐hydroxamic acid ( 3 ) forms hydrogels when mixed with guanosine ( 1 ) and KCl. The 5′‐hydroxamic acid (HA) unit is pH‐responsive and also chelates Fe³⁺. When gels are prepared under basic conditions, the 5′‐HA groups are deprotonated and the anionic hydrogel binds cationic thiazole orange (TO), signaled by enhanced fluorescence. The HA nucleoside 3 , when immobilized in the G‐quartet gel, acts as a supramolecular siderophore to form red complexes with Fe³⁺. We patterned the hydrogel's surface with FeCl₃, by hand and by using a 3D printer. Patterns form instantly, are visible by eye, and can be erased using vitamin C. This hydrogel, combining self‐assembled G‐quartet and siderophore–Fe³⁺ motifs, is strong, can be molded into different shapes, and is stable on the bench or under salt water.     

芳环缩合核苷衍生物的合成

A novel efficient synthetic route to 1,3-dihydrobenzo[c]furan glycone was developed and the corresponding 5-fluoro, 5-iodo uracil and guanosine derivatives, the aromatic analogues of the well known antiviral 2＇,3＇-dideoxy-2＇,3＇-dihydronucleosides （d4N）, were synthesized.     

The reaction of propane sultone with cuanosine,adenosine, and related compounds

Propane sultone (3-hydroxypropanesulfonic acid-γ-lactone) which is a monofunctional directacting alkylating carcinogen, has been shown to react irreversibly with DNA from various sources in vitro. It reacts with guanosine in DMSO to yield the N-7 alkyl nucleoside, while reaction with 2′-deoxyguanosine yields the corresponding N-7 alkyl purine. Evidence for the structure of these products and of one formed when adenosine and propane sultone react is presented.     

Macrocyclic amines as catalysts of the hydrolysis of the triphosphate bridge of the mRNA 5'-cap structure

The reactions of a 5'-cap model compound P1-(7-methylguanosine) P3-guanosine 5',5'-triphosphate, m7GpppG, were studied in the presence of three different macrocyclic amines (2-4) under neutral conditions. The only products observed in the absence of the macrocycles resulted from the base-catalysed imidazole ring-opening and the acid-catalysed cleavage of the N7-methylguanosine base, whereas in the presence of these catalysts hydrolysis of the triphosphate bridge predominated. The latter reaction yielded guanosine 5'-monophosphate, guanosine 5'-diphosphate, 7-methylguanosine 5'-monophosphate and 7-methylguanosine 5'-diphosphate as the initial products, indicating that both of the phosphoric anhydride bonds were cleaved. The overall catalytic activity of all three macrocycles was comparable. The hydrolysis to guanosine 5'-diphosphate and 7-methylguanosine 5'-monophosphate was slightly more favoured than the cleavage to yield guanosine 5'-monophosphate and 7-methylguanosine diphosphate. All the macrocycles also enhanced the subsequent hydrolysis of the nucleoside diphosphates, 2 being more efficient than 3 and 4.     

An Efficient Synthesis of 3′‐Amino‐3′‐deoxyguanosine from Guanosine

3′‐Amino‐3′‐deoxyguanosine was synthesized from guanosine in eight steps and 58% overall yield. The 2′,3′‐diol of 5′‐O‐[(tert‐butyl)diphenylsilyl]‐2‐N‐[(dimethylamino)methylidene]guanosine was reacted with α‐acetoxyisobutyryl bromide and treated with 0.5n NH₃ in MeOH to yield 9‐{2′‐O‐acetyl‐3′‐bromo‐5′‐O‐[(tert‐butyl)diphenylsilyl]‐3′‐deoxy‐β‐D ‐xylofuranosyl]‐2‐N‐[(dimethylamino)methylidene]guanine, which was reacted with benzyl isocyanate, NaH, and then 3.0n NaOH, and finally with Pd/C (10%) and HCO₂NH₄ in EtOH/AcOH to afford 3′‐amino‐3′‐deoxyguanosine.     

Separation of isomeric and nonisomeric monoribonucleotides by isocratic ion pair high performance liquid chromatography

A simple, isocratic, high performance liquid chromatographic procedure is described for the first time for the separation of nine monoribonucleotides using the ion-pairing technique. An aqueous mobile phase containing 100 mM KH₂PO₄ and 12.5 mM tetramethylammonium hydroxide as the solvophobic ion, pH 3.9, was used with a reverse phase RP-18 column. The nine monoribonucleotides studied were separated and eluted in the following order: cytidine-5′ -phosphate, uridine-5′ -phosphate, cytidine-3′ -phosphate, guanosine-5′ -phosphate, uridine-3′ -phosphate, uridine-2′ -phosphate, adenosine-5′ -phosphate, guanosine-3′ -phosphate, and adenosine-3′ -phosphate. Generally the 5′ nucleotides eluted faster than the 3′ and the order of elution within each series was: cytidine, uridine, guanosine, and adenosine. The only nucleotide where three isomers were studied was uridine, and the 2′ eluted later than the 3′. Baseline separation was attained for a mixture containing four 3′ nucleotides and uridine-2′ -phosphate. When the four 5′ nucleotides were chromatographed, baseline separation was also obtained except between cytidine-5′ -phosphate and uridine-5′ -phosphate. The coefficient of variation of the retention characteristics, which reflected day-to-day variation, averaged 6.4%.     

Nucleosides. Part LXI. A Simple Procedure for the Monomethylation of Protected and Unprotected Ribonucleosides in the 2′-O- and 3′-O-Position Using Diazomethane and the Catalyst Stannous Chloride

Intensive studies on the diazomethane methylation of the common ribonucleosides uridine, cytidine, adenosine, and guanosine and its derivatives were performed to obtain preferentially the 2′-O-methyl isomers. Methylation of 5′-O-(monomethoxytrityl)-N²-(4-nitrophenyl)ethoxycarbonyl-O⁶-[2-(4-nitrophenyl)ethyl]-guanosine ( 1 ) with diazomethane resulted in an almost quantitative yield of the 2′- and 3′-O-methyl isomers which could be separated by simple silica-gel flash chromatography (Scheme 1). Adenosine, cytidine, and uridine were methylated with diazomethane with and without protection of the 5′ -O-position by a mono- or dimethoxytrityl group and the aglycone moiety of adenosine and cytidine by the 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) group (Schemes 2–4). Attempts to increase the formation of the 2′-O-methyl isomer as much as possible were based upon various solvents, temperatures, catalysts, and concentration of the catalysts during the methylation reaction.     

Nucleotides. Part. XXXII. Synthesis of 2′–5′ Connected oligonucleotides. Prodrugs for antiviral and antitumoral nucleosides

The cytotoxically and antivirally active compounds bvU_d ( 1 ), flU_d ( 4 ), acyclovir ( 7 ), and A_a ( 12 ) have chemically been combined with the appropriately protected (2′–5′)diadenylate 20 by the phosphotriester approach to give the 2′–5′ oligonucleotide trimers 21 – 24 . The deprotection of the various blocking groups by chemical means afforded the 2′–5′ trimers 25 – 28 , which can be regarded as new type of a potential prodrug form delivering nucleotides to the targets inside cells. In an analogous series of reactions, 9-(3′-azido-3′-deoxy-β-D-xylofuranosyl)adenine was coupled with 7 to the 2′–5′ trimer 31 . The antiviral screening of the oligonucleotides 25–27 and 31 showed biological activities closely related to the parent nucleosides, possibly indicating their release by enzymatic cleavage of the oligomers.