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.     

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.     

SYNTHESIS AND PROPERTIES OF A NOVEL BRIDGED NUCLEIC ACID ANALOGUE, 5′-AMINO-3′,5′-BNA

An oligonucleotide P3′?N5′ phosphoramidate (5′-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5′-amino-DNA, we have designed and successfully synthesized a novel nucleic acid analogue having a P3′?N5′ phosphoramidate linkage and a constrained sugar moiety, 5′-amino-3′-C,5′-N-methylene bridged nucleic acid (5′-amino-3′,5′-BNA). The binding affinity of the 5′-amino-3′,5′-BNA towards complementary DNA and RNA strands was investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5′-amino-3′,5′-BNA and its complementary strand was much higher than that of the duplex containing the corresponding 5′-amino-DNA.     

The Crystal and Molecular Structure of the Complex of 2′3′-Didehydro-2′3′-Dideoxyguanosine with Pyridine

Abstract

The structure of 2′,3′-didehydro-2′,3′-dideoxyguanosine was determined by X-ray crystallographic analysis of the complex with pyridine. The two independent nucleoside molecules have similar, commonly observed glycosyl link (x = -102.3° and -94.2°) and 5′-hydroxyl (y = 54.0° and 47.6°) conformations. The five-membered rings are very planar with r.m.s. deviations from planarity of less than 0.015 A. 2′,3′-Didehydro-2′,3′-dideoxyadenosine has a similar glycosyl link conformation but a different 5′-hydroxyl group orientation and a slightly less planar 5-membered ring.     

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.     

Bi- and Tricyclic Nucleoside Derivatives Restricted in S-Type Conformations and Obtained by RCM-Reactions

Abstract

Ring-closing metathesis (RCM) is applied as a new and powerful technology in the construction of nucleoside analogues that are conformationally restricted in S-type conformations due to additional 3′,4′- and/or 3′,5′-linkages.     

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity

The molecular structures of 3′-azido-2′,3′-dideoxyribosylthymine 5′-triphosphate (AZTTP), 2′,3′-dideoxyribosylinosine 5′-triphosphate (ddITP), 3′-azido-2′,3′-dideoxyribosylthymine 5′-monophosphate (AZTMP) and 2′,3′-dideoxyribosyladenine 5′-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3′-endo (∼75%) and C2′-endo (∼25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3′-azido-2′,3′-dideoxyribo-sylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2′-endo (∼75%) conformers, like those observed for natural 2′-deoxynucleosides and nucleotides. The major conformers around C5′-O5′, C4′-C5′ and the glycosidic bonds are the β^t, γ⁺ and anti, respectively.     

Studies Towards Synthesis of Dinucleoside Arylphosphonates with Metal Complexing Properties

Abstract

An efficient and stereospecific synthesis of dinucleoside 4′-(2,2′:6′,2″-terpyridyl)phosphonate 2 and 5-(2,2′-bipyridyl)phosphonate 3 via a palladium(0) cross coupling strategy has been developed.     

The crystal structure of a major metabolite of thyroid hormone: 3,3′,5′-triiodo-L-thyronine

Two independent conformations of the thyroinactive thyroid hormone metabolite, 3,3′,5′-triido-L-thyronine (rT₃) were determined by X-ray diffraction methods. The conformations show significant difference in the lettering geometry when compared with those of the thyroactive thyroxine (T₄) and 3,5,3′-triido-L-thyronine (T₃). The diphenyl ether conformation of the two conformers of rT₃ is an anti-skewed one, in which the torsion angels, φ (C5-C4-O4-Cl′) are 8° and ?6°, and φ′ are 86° and 87°. This conformation is in contrast to a twist-skewed one of T₄ and T₃. The difference in the binding abilities between T₄, T₃ and rT₃ to thyroxine binding carrier proteins in serum or to a nuclear receptor protein may be explained by the characteristics solid-state conformations of these metabolites.     

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.     

Conformational stability in dinucleoside phosphate crystals. Semiempirical potential energy calculations for uridylyl-3'-5'-adenosine monophosphate (UpA) and guanylyl-3',5'-cytidine monophosphate (GpC)

Classical potential energy calculations were performed for the dinucleoside phosphates UpA and GpC. Two widely accessible low-energy regions of conformation space were found for the ω′, ω pair. That of lowest energy contains conformations similar to helical RNA, with ω′ and ω in the vicinity of 300° and 280°, respectively. All five experimental observations of crystalline GpC, two of ApU, and the helical fragment of ApApA fall in this range. The second lowest region has ω′ and ω at about 20° and 80°, respectively, which is in the general region of one experimentally observed crystalline conformer of UpA, and the nonhelical region of ApApA. It is concluded that GpC and ApU, which were crystallized as either sodium or calcium salts, are shielded from each other in the crystal by the water of hydration and are therefore free to adopt their predicted in vacuo minimum energy helical conformations. By contrast, crystalline UpA had only 1/2 water per molecule, and was forced into higher energy conformations in order to maximize intermolecular hydrogen bonding.     

Oligonucleotide analogues containing internucleotide C3′-CH<Subscript>2</Subscript>-C(O)-NH-C5′ bonds

A dinucleoside bearing an amide internucleotide C3′-CH₂-C(O)-NH-C5′ bond was synthesized by the interaction of 3′-deoxy-3′-carboxylmethylribothymidine-2′,3′-lactone obtained by hydrolysis of 2′-O-acetyl-5′-O-benzoyl-3′-deoxy-3′-ethoxycarboxylmethylribothymidine with 5′-deoxy-5′-amino-3′-O-(tert-butyldimethylsilyl)thymidine. After standard manipulations with protective groups, the dinucleoside was converted into 3′-O-(2-cyanoethyl-N,N′-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an automatic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting temperatures compared with the nonmodified ones.     

Conformational Studies on Nucleosides with Furanose Ring Modifications. 1.

Abstract

Conformational energy calculations have been presented on adenine and thymine nucleosides in which the furanose ring is replaced by 2′,3′-dideoxy-2′,3′-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (χ) and C4′-C5′ (γ) bonds torsions. In general, the χ-γ interrelationships exhibit similarities with the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the X-ray data is sensitive to the force field employed.     

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.     

Oligonucleotides Containing Acyclic Nucleoside Analogues with Carbamate Internucleoside Linkages

Abstract

Synthesis of 2′-deoxy-2′,3′-secothymidine t and its dimer t?t, where the two 2′-deoxy-2′,3′-secothymidine t units are connected via a carbamate, ?=3′-NH-CO-O-5′, internucleoside linkage has been achieved. These building blocks were protected in the 5′-position, converted into their phosphoramidites, or attached onto CPG, and then used for “chimeric oligonucleotide” synthesis.     

Parallel nucleic acid helices with hoogsteen base pairing: Symmetry and structure

Molecular structures for parallel DNA and RNA double helices with Hoogsteen pairing are proposed for the first time. The DNA helices have sugars in the C2′-endo region and the phosphodiester conformations are (trans, gauche^?), and the RNA helices have sugars in the C3′-endo region and the phosphodiester conformations are (gauche^?, gauche^?). A pseudorotational symmetry relates the two parallel strands of DNA helices and a screw symmetry relates the two strands of RNA helices, which have an associated tilt of the The conformational space of parallel helices with Hoogsteen base pairing, unlike the Watson-Crick duplex, is highly restricted due to the unique positioning of the symmetry axis in the former case. The features of the parallel double helix with Hoogsteen pairing are compared with the Watson-Crick duplex and the corresponding triple helix. © 1994 John Wiley & Sons, Inc.     

Synthesis and reactivity of guanosine 3′,5′-phosphoric acid alkyl esters

Guanosine 3′,5′-phosphoric acid alkyl esters were synthesized by reaction of the free acid of guanosine 3′,5′-phosphate (cGMP) with the appropriate diazoalkane. The resulting pair of diastereoisomers was separated chromatographically, and the conformations were assigned on the basis of ³¹P-nmr. Hydrolysis of the compounds in water was measured, and their half-lives were determined. The ability of the compounds to act as substrates for phosphodiesterase from beef heart was tested, and their inhibition of this enzyme was measured.     

Microinjection of cyclic nucleotides provides evidence for a diffusional mechanism of intraneuronal control

Cyclic 3′,5′-AMP and cyclic 3′,5′-GMP injected into large neurons of the snail Helix lucorum altered neuron activity. The effect of cAMP is usually depolarizing and that of cGMP hyperpolarizing. The results are specific for 3′,5′-cyclic nucleotides. The experiments support the hypothesis that reaction-diffusion processes involving cyclic nucleotides from the basis of an intraneuronal system of information processing.     

Eusiderins and other neolignans from an Aniba species

A previous report disclosed the presence of benzodioxan and bicyclo[3.2.1]octanoid neolignans in the benzene extract of the trunk wood of an Amazonian Aniba (Lauraceae) species. The chloroform extract of the same material contains additionally two new benzodioxan neolignans [rel-(7S,8R)-Δ^8′-7-hydroxy-3,4,5,5′-tetramethoxy-7.0.3′,8.0.4′-neolignan; rel-(7R,8R)-Δ^7′-3,4,5,5′-tetramethoxy-9′-oxo-7.0.3′,8.0.4′-neolignan], two new bicyclo[3.2.1]-octanoid neolignans [(7R,8S,1′S,2′S,3′S,4′R)-Δ^8′-2′,4′-dihydroxy-3,3′-dimethoxy-4,5-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan; (7R,8S,1′R,2′S,3′S)-Δ^8′-2′-hydroxy-3,3′,5′-trimethoxy-4,5-methylenedioxy-1′,2′,3′,4′-tetrahydro-4′-oxo-7.3′,8.1′-neolignan] and a hydrobenzofuranoid neolignan [(7S,8R,1′S,5′S)-Δ^8′-3,3′,5′-tri-methoxy-4,5-methylenedioxy-1′,4′,5′,6′-tetrahydro-4′-oxo-7.0.2′,8.1-neolignan].     

DNA-Triesters - The Synthesis and Absolute Configuration Assignments at P-Stereogenic Centres

Abstract

Decadeoxyribonucleotide GGGAATTCCC and nine diastereomeric pairs of its mono-O-ethyl ester analogues were synthesized via phosphoramidite approach using the combination of 5′-DMT-base protected (except T) nucleoside 3′-(2-cyanoethyl N,N-diisopropyl phosphoramidites) and 3′-(0-ethyl N,N-diisopropyl phosphoramidites). Under conditions of release from solid support and removal of base-protecting groups (25% NH₄OH, 25°C, 48 h) 2-cyanoethyl groups were removed while O-ethyl phosphate triester functions were practically intact. Isolation of products and separation of diastereomers were performed by means of RP-HPLC. Absolute configuration at P-stereogenic centres was established via degradation of decamers into corresponding dinucleoside O-ethyl phosphates and stereochemical correlation with dinucleoside phosphorothioates of known configuration at phosphorus. Decadeoxyribonucleotide mono-O-ethyl esters were used for mapping the contact points between DNA and Eco RI endonuclease - the restriction enzyme which recognizes canonical sequence. GAATTC and cleaves unmodified DNA strands giving G and p AATTC.