Oligonucleotide interactions. IV. Conformational differences between deoxy- and ribodinucleoside phosphates

The circular dichroism spectra of all 16 ribodinudeoside phosphates containing the bases adenine, uracil, cytosine, and guanine have been measured at room temperature and neutral pH. These results are compared with the circular dichroism spectra of the corresponding deoxy compounds. From the optical properties it is clear that the geometry of the base-stacked conformation of ribo compounds must differ substantially from that of deoxy compounds. Because of this, it is not possible to draw firm conclusions about the relative extent of stacking in most ribo and deoxy compounds. The optical rotatory dispersion of about a dozen deoxy and ribodinucleoside phosphates has been studied as a function of temperature. These results confirmed the conclusions drawn earlier from measurements at a single temperature. Several dinucleoside phosphates containing a 2′ → 5′ phosphodiester bond have also been examined. These compounds have a substantial degree of base stacking at room temperature. The geometry of the stacked conformation is different from that of either the normal ribo dimer or the deoxy dimer. The role of the 2′-hydroxyl group in stabilizing base stacked geometries has been examined by studies on C-2′-O-methyl-pC. This compound has optical properties almost identical to those of CpC. This suggests that the effect of the 2′ hydroxyl is felt indirectly through its perturbation of the geometry of the sugar ring rather than directly by hydrogen bonding. It is not possible at present to identify precise conformational differences among deoxy-, ribo-and 2′ → 5′ ribodinucleoside phosphates.     

Circular dichroism study of 2'-5' dinucleoside monophosphates modified with N-2-acetylaminofluorene.

The conformational properties of four 2′ – 5′ dinucleoside monophosphates modified with $\text{N}$-2-acetylaminofluorene have been studied by circular dichroism spectroscopy. Covalent binding of this chemical carcinogen at the C₈ position of guanosine in the 2′ – 5′ dinucleoside monophosphates induces striking changes in their circular dichroic spectra depending on their base sequence and composition. The changes in CD spectra, redshift of the extrema and change of their polarity, not observed in the spectra of corresponding 3′ – 5′ derivatives modified with $\text{N}$-2-acetylaminofluorene are correlated with the difference in the configuration of 2′ – 5′ and 3′ – 5′ dinucleoside monophosphates and discussed in respect to the intramolecular stacking interactions.     

Improved Synthesis of Trinucleotide Phosphoramidites and Generation of Randomized Oligonucleotide Libraries

A new method to produce a set of 20 high quality trinucleotide phosphoramidites on a 5–10 g scale each was developed. The procedure starts with condensation reactions of P-components with N-acyl nucleosides, bearing the 3 ′-hydroxyl function protected with 2-azidomethylbenzoyl, to give fully protected dinucleoside phosphates 13. Upon cleavage of dimethoxytrityl group from 13, dinucleoside phosphates 16 are initially transformed into trinucleoside diphosphates 19 and then the 2-azidomethylbenzoyl is selectively removed under neutral conditions to generate trinucleoside diphosphates 5 in excellent yield. Subsequent 3 ′-phosphitylation affords target trinucleotide phosphoramidites 7. When mutagenic oligonucleotides are synthesized employing mixtures of building blocks 7 as well as following the new synthetic protocol, representative oligonucleotide libraries are generated in good yields.     

Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside‐2′,5′‐Monophosphate Prodrug Model of 2‐5A

Protected dinucleoside‐2′,5′‐monophosphate has been prepared to develop a prodrug strategy for 2‐5A. The removal of enzymatically and thermally labile 4‐(acetylthio)‐2‐(ethoxycarbonyl)‐3‐oxo‐2‐methylbutyl phosphate protecting group and enzymatically labile 3′‐O‐pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine‐2′,5′‐monophosphate 1 used as a model compound for 2‐5A. The desired unprotected 2′,3′‐O‐isopropylideneadenosine‐2′,5′‐monophosphate ( 9 ) was observed to accumulate as a major product. Neither the competitive isomerization of 2′,5′‐ to a 3′,5′‐linkage nor the P–O5′ bond cleavage was detected. The phosphate protecting group was removed faster than the 3′‐O‐protection and, hence, the attack of the neighbouring 3′‐OH on phosphotriester moiety did not take place.     

Proton magnetic resonance investigation of interactions between L-tryptophan and dinucleoside phosphates at low pD

Interactions between mononucleoside and dinucleoside phosphates containing adenine and/or cytosine and L -tryptophan have been studied at low pD by proton magnetic resonance (pmr) spectroscopy. The results of those studies indicate that, despite extensive protonation of ring positions, and resulting electrostatic repulsion, ring stacking does occur between both like and unlike molecules. Geometries for stacked complexes are proposed and the extent of complex formation between L -tryptophan and adenosine or cytidine in 3′ or 5′ esterified positions is discussed qualitatively.     

Large scale synthesis of dinucleoside monophosphates catalyzed by ribonuclease from Aspergillus clavatus

A gram scale enzymatic synthesis of eight, dinucleoside monophosphates (ApC, ApU, CpC, CpU, GpC, GpU, UpC, and UpU) is described. The synthesis involves a reaction between the appropriate ribonucleoside-2′,3′-cyclie phosphates and cytidine or uridine in the presence of ribonuelease from Aspergillus clavatus at 30°C. The enzyme is removed from the reaction mixture by chromatography on Bio-Gel P–4, and the dinucleoside monophosphate is further purified by chromatography on a DEAE-Sephadex A–25, column. A procedure for the large scale preparation of the ribonuclease from Aspergillus clavatus is also described.     

DNA methylases of Hemophilus influenzae Rd. II. Partial recognition site base sequences

A small percentage of the adenine bases in Hemophilus influenzae strain Rd DNA are methylated in the 6-amino position. The methyl groups are introduced specifically by at least four different DNA methylases (I, II, III and IV). A method is described for determining the 3′ and 5′ nearest-neighbor bases to methylated adenine so as to reveal the specificity of each methylase. Tritium-labeled methyl groups are introduced into the DNA. The DNA is then digested to dinucleotides using the Bacillus subtilis phage SP3 DNase, followed by removal of the terminal 5′-phosphoryl group with phosphatase to produce dinucleoside monophosphates. These are analyzed by Aminex A25 (Bio-Rad) chromatography. Dinucleoside monophosphate species containing the 3′ neighbor or the 5′ neighbor are resolved so that a trinucleotide is determined that contains the centrally placed methylated adenine. H. influenzae Rd DNA contains seven dinucleoside monophosphate species, about 80% representing GpmA and mApT in approximately equal amount. DNA methylases I, II, III and IV introduce methyl groups into sequences containing the trinucleotides CpmApC, PupmApC, NpmApA and GpmApT, respectively. The sequence methylated by NDA methylase II is consistent with the recognition site determined by Kelly and Smith (1970) for the H. influenzae restriction enzyme, endonuclease R.     

Stereochemistry of 2′-5′ Linked Nucleic Acids: Crystal and Molecular Structure of Ammonium Adenylyl-2′, 5′-Adenosine Tetrahydrate: A Core Fragment of 2′-5′ Oligo A′s Produced by Interferon Induced Adenylate Synthetased

Abstract

The preponderance of 3′-5′ phosphodiester links in nucleic acids is well known. Albeit less prevalent, the 2′-5′ links are specifically utilised in the formation of ‘lariat’ in group II introns and in the msDNA-RNA junction in myxobacterium. As a sequel to our earlier study on cytidylyl-2′,5′-adenosine we have now obtained the crystal structure of adenylyl-2′,5′-adenosine (A2′p5′A) at atomic resolution. This dinucleoside monophosphate crystallises in the orthorhombic space group P2₁2₁2₁ with a= 7.956(3)Å, b = 12.212(3)Å and c = 36.654 (3) Å. CuKα intensity data were collected on a diffractometer. The structure was sloved by direct methods and refined by full matrix least squares methods to R = 10.8 %. The 2′ terminal adenine is in the commonly observed anti (χ₂ =?161°) conformation and the 5′ terminal base has a syn (χ₁ = 55°) conformation more often seen in purine nucleotides. A noteworthy feature of A2′p5′ A is the intranucleotide hydrogen bond between N3 and 05′ atoms of the 5′ adenine base. The two furanose rings in A2′ p5′ A show different conformations-C2′ endo, C3′ endo puckering for the 5′ and 2′ ends respectively. In this structure too there is a stacking of the purine base on the ribose 04′ just as in other 2′-5′ dinucleoside structures, a feature characteristically seen in the left handed ZDNA. In having syn, anti conformation about the glycosyl bonds, C2′ endo, C3′ endo mixed sugar puckering and N3–05′ intramolecular hydrogen bond A2′p5′ A resembles its 3′-5′ analogue and several other 2′-5′ dinucleoside monophosphate structures solved so far. Striking similarities between the 2′-5′ dinucleoside monophosphate structures suggest that the conformation of the 5′-end nucleoside dictates the conformation of the 2′ end nucleoside. Also, the 2′-5′ dimers do not favour formation of miniature classical double helical structures like the 3′-5′ dimers. It is conceivable, 2–5(A) could be using the stereochemical features of A2′p5′ A which accounts for its higher activity.     

Vibrational study of a nucleoside analogue with antiviral activity, 5-chloro-2'-deoxyuridine, CDU.

The experimental FTIR and FT-Raman spectra of 5-chloro-2'-deoxyuridine have been assigned on the basis of normal coordinate analyses, in the light of observed and calculated wavenumbers and isotopic shifts. The results indicate that virtually all normal modes of IDU involve some degree of vibrational coupling between the chlorouracil base and the deoxyribose moiety.     

Preference for Ribose Over Deoxyribose in Loop-Closing Base Pairs of Extra Stable Nucleic Acid Hairpins

We have investigated the effect of switching ribose to deoxyribose at the closing base-pair of an extra-stable RNA hairpin. Specifically, we studied the sequence 5′-GGAC(UUCG)GUCC, a dodecanucleotide that folds into a well-characterized, “extra stable” RNA hairpin structure. Recently, we showed that hairpins containing a 2′,5′-linked (UUCG) loop instead of the native 3′,5′-linked loop also exhibit extra-stability (Hannoush and Damha, J. Am. Chem. Soc., 2001, 123, 12368–12374). In this article, we show that the ribose units located at the loop-closing positions (i.e., rC ₄ and rG ₉ ) contribute significantly to the stabilization of RNA hairpins, particularly those containing the 3′,5′-UUCG loop. Interestingly, the requirement of rC4 and rG9 is more relaxed for DNA hairpins containing the 2′,5′-UUCG loop and, in fact, they may be replaced altogether (ribose → deoxyribose) without affecting stability. The results broaden our understanding of the behavior of highly stable (UUCG) hairpin loops and how they respond to structural perturbation of the loop-closing base pairs.     

Effects of 3′-C-Methylation on the Hydrolytic Stability and Hydroxyl pK a Values of Dinucleoside 2′,5′- and 3′,5′-Monophosphates

Abstract

The first-order rate constants for hydrolysis of 3′-C-methyluridylyl(2′,5′)- and -(3′,5′)adenosine and the corresponding native dinucleoside monophosphates (2′,5′- and 3′,5′-UpA) have been determined as a function of hydroxide-ion concentration (0.025 - 7 M) at 25°C. In addition to the effects on the hydrolytic stability of the compounds, the effects of the 3′-C-methyl substitution on the kinetically determined pK _a values for the sugar hydroxyls of the undine moiety are discussed.     

The structure of drug-deoxydinucleoside phosphate complex; generalized conformational behavior of intercalation complexes with RNA and DNA fragments.

A 2:2 complex of proflavine and deoxycytidylyl-3', 5'-guanosine has been crystallized and its structure determined by x-ray crystallography. The two dinucleoside phosphate strands form self complementary duplexes with Watson Crick hydrogen bonds. One proflavin is asymmetrically intercalated between the base pairs and the other is stacked above them. The conformations of the nucleotides are unusual in that one strand has C3',C2'endomixed sugar puckering and the other has C3',C3' endo deoxyribose sugars. These results show that the conformation of the 3'sugar is of secondary importance to the intercalated geometry.     

Vibrational Study of a Nucleoside Analogue with Antiviral Activity, 5-Chloro-2′-deoxyuridine,CDU

Abstract

The experimental FTIR and FT-Raman spectra of 5-chloro-2′-deoxyuridine have been assigned on the basis of normal coordinate analyses, in the light of observed and calculated wavenumbers and isotopic shifts. The results indicate that virtually all normal modes of IDU involve some degree of vibrational coupling between the chlorouracil base and the deoxyribose moiety.     

Conformational Analysis of Canonical 2-Deoxyribonucleotides. 1. Pyrimidine Nucleotides

Abstract

The molecular structure and relative stability of north and south conformers of 2′-deoxyribonucleotides containing pyrimidine nucleic acid bases (2′-deoxythymidilic (pdT), 2′- deoxycytidilic (pdC) acids and their mono- and dianions) have been obtained and analyzed at the DFT/B3LYP level using the standard 6–31G(d) basis set. We have revealed that, when the nucleobase moiety is incorporated into the nucleotides, it maintains a nonplanar and nonrigid conformation due to out-of-plane deformation of the amino group and pyrimidine ring. It has been demonstrated that an increase of negative charge of the phosphate group results in increase of amino group pyramidalization, discrimination between conformers with syn and anti orientation of base with respect to sugar, strengthening of intramolecular C-H…O hydrogen bonds leading to deformation and fixation of geometry of nucleotides, and weakening of phosphodiester bond. These results allow to make suggestions about sources of twist and buckle deformations of base pairs, mechanisms of repaire of DNA via change of base orientation, and conditions for breakage of the P-O bonds during hydrolysis.     

Dinucleoside monophosphates. I. Optical properties and conformation in solution with one base charged.

A least squares analysis of the titration properties of several dinucleoside monophosphates enables calculation of the pK's for protonation. These pK's are used to resolve the spectral properties of dinucleoside monophosphates with one base charged from the apparent spectral properties of a dinucleoside monophosphate in aqueous solution. This method is applied to dinucleoside monophosphates containing adenosine and/or cytidine. Results of CD, nmr, and CD-temperature dependence measurements are presented. The results indicate that singly protonated dimers of these nucleosides stack as do their unprotonated analogs. It is suggested that this is true for all dimers with one base charged.     

Minor nucleosides in RNA: Optical studies of dinucleoside phosphates containing dihydrouridine

Photoreduction with NaBH₄ was used to reduce the dinucleoside phosphates ApU, UpA, and GpU to the corresponding molecules containing dihydrouridine (H). Also obtained from this reaction are dinucleoside phosphates containing (β-N-ribosyl) ureido-propanol, an open ring form of dihydrouridine. The results of CD and ultraviolet absorption studies with these compounds imply that HpA is strongly stacked, but that ApH and GpH are only slightly stacked. The temperature dependence of the CD suggests that the stacking in HpA is unusual. The results with compounds containing open ring forms of dihydrouridine indicate that there is more intramolecular interaction when the ring is open than when it is closed.     

Backbone conformations in deoxyribonucleic acids. Conformational role of the 2′-hydroxyl group on the internucleotide phosphodiester conformations

The conformations accessible to the internucleotide phosphodiester group in deoxydinucleoside monophosphates, deoxydinucleoside triphosphates, and deoxypolynucleotides have been explored in detail by potential energy calculations. The two most predominant conformations for the nucleotide moiety (³E and ²E) and their possible combinations (³E^?3E, ³E^?2E, ²E^?2E, ²E^?3E) have been employed, similar to our earlier studies on polyribonucleotides. The internucleotide P-O bond torsions are very sensitive to the sugar pucker (³E and ²E) and sugar type (ribose and 2′-deoxyribose) on the 3′-residue of dinucleoside phosphates. The preferred phosphodiester conformations found for the deoxydinucleoside monophosphates and triphosphates, in general, follow the same pattern as those obtained for ribose sugars when the sugar on the 3′-side of the molecule has the ³E sugar-ring conformation. The internucleotide P-O bonds show a greater degree of conformational freedom when the 3′-sugar has the ²E pucker. The double gauche g^?g^? conformation for the phosphodiester, which leads to the overlap of the adjacent bases, is shown to be one of the energetically most favored conformations for all the sequence of sugar puckers. It is found that the ²E^?2E sequence of sugar puckers shows a greater energetic preference for the stacked helical conformation (g^?g^?) than the (³E^?3E) and the mixed sugar-pucker combinations. This effect becomes more pronounced in going from a dinucleoside monophosphate to a dinucleoside triphosphate suggesting that the 2′-deoxy sugars favor the ²E sugar pucker in di-, oligo-, and polydeoxyribonucleotide structures. In addition to g^?g^?, the conformations g⁺g^?, tg^?, g^?t, tg⁺, and g⁺t are also found to be possible for the phosphodiester in a polydeoxyribonucleotide and their populations depend to some extent on the sugar-pucker sequence. It is shown that the short-range intramolecular interactions involving the sugar and the phosphate groups dictate to a large extent the backbone conformations of nucleic acids and polynucleotides.     

Recognition of Ribose Moiety by Adenosine (Phosphate) Deaminase from Squid Liver

An adenosine (phosphate) deaminase from the squid liver had much lower activity for 5′-deoxyadenosine than that for adenosine, 2′-, or 3′-deoxyadenosine. 3′-IMP and inosine as well as purine riboside and adenine competitively inhibited the deamination of adenosine 3′ phenylphosphonate by the enzyme, but 5′-AMP and 5′-IMP did not. The enzyme deaminated the 5′-hydroxyl terminal adenosine residue in dinucleotides and trinucleotide, but not the 3′-hydroxyl terminal one in dinucleotides. The 5′-hydroxyl group of the ribose moiety was necessary for the substrate binding and catalytic activity of the squid enzyme. These results indicated that the recognition of ribose moiety in the substrate by the squid enzyme might be intermediate between those by adenosine deaminase and adenosine (phosphate) deaminase from microorganisms.     

Ab-inito Quantum Mechanical Calculations of NMR Chemical Shifts in Nucleic Acids Constituents II Conformational Dependence of the lH and 13C Chemical Shifts in the Ribose

Abstract

The magnetic shielding constant of the different ¹³C and ¹³H nuclei of a deoxyribose are calculated for the C2′ endo and C3′ endo puckerings of the furanose ring as a function of the conformation about the C4′C5′ bond. For the carbons the calculated variations are of several ppm, the C3′ endo puckering corresponding in most cases to a larger shielding than the C2′ endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose ?3′ and 5′ phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides.

The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.