A Study on 5′-Nucleotides in D2O Solution by Proton Magnetic Resonance Spectroscopy

Nucleotides, 5′-AMP, 5′-GMP, 5′-UMP, 5′-CMP and 5′-TMP, in D₂O solution have been investigated by proton magnetic resonance spectroscopy. The concentration and the pD dependences of the proton chemical shifts of the nucleotides have been examined in detail. These results indicate that intermolecular association of vertical stacking of the base rings and intramolecular association between base protons and ionized phosphate group occur in solution. The effects of the temperature and lithium ion on 5′-AMP and 5′-UMP have been also investigated. The increase of temperature causes to reduce the intramolecular association for 5′-UMP and the both intra- and intermolecular association for 5′-AMP. Lithium ion reduces the intramolecular association for both 5′-AMP and 5′-UMP, and at the same time promotes the intermolecular one for the former. This can be interpreted by the ion-pair formation of lithium ion with the ionized phosphate group.     

NMR SOLUTION STRUCTURE AND CONFORMATIONAL ANALYSIS OF THE CALCIUM RELEASE AGENT CYCLIC ADENOSINE 5′-DIPHOSPHATE RIBOSE (cADPR)

A high-resolution NMR study of the solution structure of the calcium release agent cADPR has been performed. Pseudorotationals analysis reveals that in solution both sugar rings in cADPR adopt predominantly (～75%) South conformations, with the A and N rings adopting approximately ²T₃ (C2′-endo(major)-C3′-exo(minor) and ⁴ ₃T (C3′-exo-C4′-endo) conformations, respectively. The backbone torsion angles β and γ have also been determined. While the minor North conformers were not observed in the crystal structure of cADPR, the solution values of the major South conformers compare well to those found in crystal structure.     

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.     

Ordered forms of 5′-8-aminoguanylic acid

5′-8NH₂GMP forms an ordered structure in moderately acid (pD 4.7) solution. We propose for this ordered form a novel hemiprotonated G·G structure with a twofold rotation axis and three hydrogen bonds between each pair of guanine residues. Gel formation does not occur with this nucleotide in either neutral or acid solution. In neutral solution 5′-8NH₂GMP also forms a regular, ordered structure, quite different from the acid form and similar to that formed by 5′-GMP under the same neutral conditions. We suggest that this ordered structure consists of a regularly stacked array of planar tetramers, similar to that proposed for 3′-GMP at pH 5.²     

COMPARATIVE CONFORMATIONAL ANALYSIS OF NUCLEOSIDES BY NMR,X-RAY,AND SEMI-EMPIRICAL (PM3 VS. AM1) METHODS

The 2′-deoxy-2′-fluoro-β-D-arabinofuranosyl ortho-aza-purine and -pyrimidine nucleosides manifest an unusually rigid sugar N conformation in solution.     

Preparation of ligation intermediates and related polynucleotide pyrophosphates

Unprotected oligonucleotides and oligodeoxynucleotides terminated with an unhindered 5′-phosphate group react with nucleoside 5′-phosphorimidazolides in aqueous solution to give ‘capped’ pyrophosphates in at least 70% yield. If adenosine 5′-phosphorimidazolide is used as a substrate in the reaction, ligase intermediates are obtained as products.     

Synthesis and properties of 3′-C-methylnucleosides and their phosphoric esters

3′-C-Methyluridine and 3′-C-methylcytidine were synthesized in 11 steps starting from d-glucose. The conformation of 3′-C-methylnucleosides was studied in solution and in the crystal by using the techniques of c.d., ¹H-n.m.r. spectroscopy, and X-ray diffraction analysis. 3′-C-Methyluridine 2′,3′-cyclophosphate was prepared, and its hydrolysis with nucleases was studied. 3′-C-Methyluridine 5′- mono- and 5′-tri-phosphate were also synthesized.     

Partial Protection of Carbohydrate Derivatives. Part 19. Highly Regioselective 5′-O-Aroylation of 2′-Deoxyribonucleosides in Terms of Dilution - Drop-by-Drop - Addition Procedure

Abstract

Treatment of a 2′-deoxyribonucleoside in pyridine with an aroyl chloride, which was added dropwise as a dilute solution in pyridine, gave the corresponding 5′-O-aroyl-2′-deoxyribonucleoside in 84 - 93% yields.     

An NMR Study of the Conformation and Thermodynamics of the Circular Dumbbell d<pCGC-TT-GCG-TT>

Abstract

The circular DNA decamer 5′-d<pCGC-TT-GCG-TT>3′ is studied in solution by means of NMR spectroscopy. At low temperature the molecule adopts a dumbbell structure with three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature another conformer appears, in which the closing C-G base pair in the 5′-GTTC-3′ loop is disrupted, whereas the opposite 5′-CTTG-3′ loop remains stable. The two conformers are in slow equilibrium over a limited temperature range.     

1H NMR Structural Analysis of the Interactions of Proflavine with Self-Complementary Deoxytetranucleosides of Different Base Sequence

Abstract

The molecular associations and structures of the complexes between the acridine dye, proflavine, and self-complementary deoxytetraribonucleoside triphosphates 5′-d(GpCpGpC), 5′-d(CpGpCpG), 5′-d(ApCpGpT), 5′-d(ApGpCpT) in aqueous solution have been investigated using one-dimensional and two-dimensional 500 MHz ¹H NMR spectroscopy.     

Conformational analysis of the 2'-deoxyribofuranose ring from proton-proton coupling constants: analysis of a nucleoside-carcinogen adduct formed from 2-acetylaminofluorene utilizing a three-state model

The conformation of the sugar moiety of 8-(N-fluoren-2-ylamino)-2′-deoxyguanosine in solution has been examined as a function of temperature by ¹H-nmr spectroscopy. Analysis of coupling constants shows that lowering the temperature to ?50°C in methanol shifts the conformational equilibrium of the sugar ring resulting in a C2′-endo conformation at a mole fraction of 0.97. The computed phase angle of pseudorotation and amplitude of pucker are 154° and 36°, respectively, with very little discrepancy between the five calculated coupling constants and coupling constants extrapolated from the temperature profiles. A computer program has been written enabling a three-state best-fit analysis. The three-state analysis indicates an equilibrium between C2′-endo, C3′-endo, and 04′-endo conformations. In aqueous solution, the computed mole fraction of the 04′-endo form is 0.18 at 30°C. The conformation associated with the sugar ring and the C4′? C5′ bond is compared to that of 2′-deoxyguanosine.     

On the Conformation of UDPG,a Sugar Nucleotide

Abstract

UDPG [uridine 5′-(∝-D-glucopyranosyl pyrophosphate)] has a folded conformation in solution in which the glucose and uracil rings lie, approximately, in the same plane with the 2′- & 3′- hydroxyl groups facing the 5- & 6- hydrogens of the uracil ring.     

Gramicidin S analogs with a D-Ala, Gly, or L-Ala residue in place of the D-Phe residue: molecular conformations and interactions with phospholipid membrane

The proton nmr and CD spectra of gramicidin S (GS) cyclic-(Val^1,1′-Orn^2,2′-Leu^3,3′-D-Phe^4,4′-Pro^5,5′)₂ and of GS analogs—namely, [D-Ala^4,4′]-GS, [Gly^4,4′]-GS, and [L-Ala^4,4′]-GS—were analyzed. The molecular conformation of [D-Ala^4,4′]-GS is similar to that of GS, with the trans form about the D-Ala-Pro peptide bond. The molecular conformation of [Gly^4,4′]-GS depends on the solvent composition of dimethylsulfoxide-d₆/trifluoroethanol (DMSO)-d₆/TFE and DMSO-d₆/H₂O as well as the solute concentration. In DMSO-d₆ solution, [Gly^4,4′]-GS forms the GS-type conformation of the monomer at lower concentration. At higher concentration, the GS-type conformer is converted to the other one that forms molecular aggregates. The cis form about the X-Pro peptide bonds is found for [Gly^4,4′]-GS and [L-Ala^4,4′]-GS in DMSO-d₆ and for [L-Ala^4,4′]-GS in TFE solution. The large temperature dependences of α-proton chemical shifts of [L-Ala^4,4′]-GS in DMSO-d₆ solution indicate that the conformer equilibrium changes with temperature. The GS-type conformation is not formed in [L-Ala^4,4′]-GS. The two active peptide analogs, [D-Ala^4,4′]-GS and [Gly^4,4′]-GS, interact with the phospholipid membrane, taking the GS-type conformation. By contrast, an inactive analog, [L-Ala^4,4′]-GS, does not interact with phospholipid membrane. The activities of GS analogs are found to correlate to the formation of the GS-type conformation upon binding with phospholipid membrane.     

Synthesis,Solution Conformation and Biological properties of 2′,3′-Dideoxy-3′-fluoro-D-erythro-pentofuranosides of 2-Thiouracil and 2-Thiothymine

Abstract

The synthesis of the α- and β-anomers of 2′,3′-dideoxy-3′-fluoro-2-thiouridine and 2′,3′-dideoxy-3′-fluoro-2-thiothymidine via Lewis acid catalysed nucleoside condensation is described. High resolution ¹H NMR data, solution conformations and biological properties are also presented.     

Oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their derivatives: III. 5′-Mono- and 5′-bispyrenyl derivatives of oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their 3′-modified analogues: Synthesis and properties

5′-Pyrenylmethylphosphamide and 5′-bispyrenylmethylphosphordiamide derivatives of oligo(2′-O-methylribonucleotides) and their analogues with thymidine attached at their 3′-termini by a 3′-3′-phosphodiester internucleotide bond (“inverted” thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5′-mono- and 5′-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5′-Pyrenylphosphamide derivatives of oligo(2′-O-methylribonucleotides) and their inverted analogues were shown to be used as sensitive probes for the detection of single nucleotide polymorphisms in RNA and DNA by the method of thermal duplex denaturation with fluorescence change registration.     

N-2′-Acetoxybenzoyl derivatives of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose

N-2′-Acetoxybenzoyl (aspirin) derivatives (degree of substitution 0·35–1·00) of chitosan, N-desulphated heparin and 2-amino-2-deoxy-d-glucose were prepared by methods that gave yields in the range 65–86%. The salicylate of chitosan was isolated with a 98% yeild. Aspirin or salicylic acid was released much more slowly from N-(2′-acetoxybenzoyl)-chitosan than from the salicylate of chitosan, and much faster at 37°C in 0·1 m NaOH solution than in 2% aqueous acetic acid solution. Salicylic acid was isolated from the dialysate (0·1 m NaOH solution) of N-(2′-acetoxybenzoyl)-chitosan.     

Fluorometric determination of trioses with o-phenylphenol.

A spectrophotometric method for determining ascorbic acid based on the redox reaction between a copper (II)-2,2′-biquinoline solution and ascorbic acid was developed. The purple color of the copper (I)-2,2′-biquinoline complex formed in a buffered acetone-water solution was measured at 540 nm. Minerals, sugars, and other vitamins do not interfere when present in quantities usually found in pharmaceutical preparations. Ferrous interference was eliminated by treating the sample solution with the cation-exchange resin Dowex 50W-X12. Several single component and multivitamin formulations were satisfactorily analyzed by this method. Its high sensitivity permits measurements of quantities of ascorbic acid to 3.4 μg/ml of sample solution. The procedure is simple, rapid, and suitable for routine control.     

Effects of Chloride,Bromide, and Iodide Upon Decomposition of Nucleosides Induced by Ultrasound in Neutral Solution

When the ultrasound of 42 kHz was irradiated on a neutral mixture of 2′-deoxycytidine, 2′-deoxyguanosine, 2′-deoxythymidine, and 2′-deoxyadenosine, concentrations of all the nucleosides decreased. Addition of NaCl to the system had no effect. NaBr suppressed the reactions for all the nucleosides, but the efficiency of 2′-deoxyguanosine was low. NaI suppressed the reactions for all the nucleosides more effectively. A comparison with the results of the effects of halides on the reaction of nucleosides by a Fenton system suggested that only half of the nucleoside damage in the ultrasound-irradiated solution was caused by hydroxyl radicals formed from water by the sonication.     

Studies on the hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution A synthesis of 3-methyl-2′-deoxyuridine

The hydrolysis of 3-methyl-2′-deoxycytidine in aqueous solution has been investigated. Varying proportions of 3-methylcytosine, 3-methyluracil and 3-methyl-2′-deoxyuridine are formed depending upon conditions of pH and temperature. All three hydrolytic products are formed at pH 6.8 and 90°C. At pH 2, depyrimidination of 3-methylcytosine occurs as the only hydrolysis product. When the pH is increased to 12, 3-methyl-2′-deoxycytidine on heating at 90°C is completely deaminated to 3-methyl-2′-deoxyuridine with few side products formed. This reaction serves as the basis for a convenient synthesis of 3-methyl-2′-deoxyuridine. The 300 MHz spectra of 3-methyl-2′-deoxycytidine and 3-methyl-2′-deoxyuridine indicate that the sugar ring in these compounds is predominantly in ²E conformation.     

Conformational and stacking properties of 3′–5′ and 2′–5′ linked oligoribonucleotides studied by CD

Comparative CD studies have been carried out to characterize the properties of 2′–5′ and 3′–5′ oligoriboadenylates and oligoribouridylates from dimer to decamer. The CD band of the 3′–5′ oligoribonucleotides was larger than that of the 2′–5′ oligoribonucleotides and increased with the increase in chain length, while the CD band of the 2′–5′ oligoribonucleotides increased little beyond the dimer level. The CD analysis of the chain length dependency revealed that the 3′–5′ oligoribonucleotides adopt mainly the base-base stacking interaction, while the base-sugar interaction is predominant in the 2′–5′ oligoribonucleotides. The CD intensity of 3′–5′ oligoribonucleotides decreased to a larger extent at elevated temperatures or in the presence of ethanol compared to that of the 2′–5′ counterparts. Mg²⁺ or Mn²⁺ ion enhanced the magnitude of the CD of 3′–5′ octariboadenylate, while a small decrease in the CD was observed by the presence of Mg²⁺ or Mn²⁺ ion to the 2′–5′ octariboadenylate. The 3′–5′ oligoribonucleotide is likely conformationally flexible and can form helical ordered structure with strong base-base stacking depending on changes in the environment such as temperature, the presence of Mg²⁺ ion, or hydrophobicity of the solution. © 1996 John Wiley & Sons, Inc.