Synthesis and thermodynamics of oligonucleotides containing chirally pure R(P) methylphosphonate linkages.

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.     

Chemical modification of pyrimidine TFOs: effect on i-motif and triple helix formation

In order to form more stable triple helical structures or to prevent their degradation in cells, oligonucleotide analogs are routinely used, either in the backbone or among the bases. The target sequence chosen for this study is a 16-base-long oligopurine-oligopyrimidine region present in the human neurotrophin 4/5 gene. Seven different chemical modifications were tested for their effect on (i) triple helix formation and (ii) i-DNA stability. i-DNA is a tetrameric structure involving hemiprotonated C x C+ base pairs, which may act as a competing structure for triplex formation, especially in the case of a cytosine-rich third strand. At acid pH, oligophosphoramidates formed the most stable triple helix, whereas oligonucleotides including 5-propynyl-dU formed a stable i-motif which precluded triplex formation. Only two candidates stabilized triple helices at neutral pH: oligonucleotides with phosphoramidate linkage and phosphodiester oligonucleotides containing 5-methyl-dC and 5-propynyl-dU.     

Fluorescent pteridine nucleoside analogs

Pteridine nucleoside analog probes are highly fluorescent and offer different approaches to monitor subtle DNA interactions with other molecules. Similarities in structure and size to native nucleosides make it possible to incorporate these probes into oligonucleotides through the standard deoxyribose linkage. These probes are formulated as phosphoramidites and incorporated into oligonucleotides using automated DNA synthesis. Their position within the oligonucleotide renders them exquisitely sensitive to changes in structure as the oligonucleotide meets and reacts with other molecules. Changes are measured through fluorescence intensity, anisotropy, lifetimes, spectral shifts, and energy transfer. The fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into single and double stranded oligonucleotides are reviewed. The two guanosine analogs, 3MI and 6MI, and two adenosine analogs, 6MAP and DMAP, are reviewed in detail along with applications utilizing them.     

Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets.

Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.     

Synthesis and Cellular Uptake of Fluorescently Labeled Oligodeoxynucleotide Prodrugs

Abstract

In this paper we described the synthesis on solid support of 5′-fluorescein labeled dodecathymidylates having interucleoside phosphodiester or phosphorothioate linkages temporary masked with enzymolabile S-pivaloyl-2-thioethyl (tBuSATE) groups.     

A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes.

A photoaffinity labelling study of the messenger RNA-binding region of E. coli ribosomes has been made, using oligoadenylic acids as mRNA analogs. The oligonucleotides, of chain length 6 to 8 and thus several nucleotides longer than oligonucleotides previously employed for this purpose, carried a radioactive photolabile aromatic azide reagent bound covalently to the 3'-terminal ribose moiety. The synthesis of the reagent, p-azidobenzoyl-(3H)-glycylhydrazide, is described. The derivatized oligonucleotides were shown to be functional messengers. They stimulated the binding of the cognate aminoacyl-tRNA, lysyl-tRNA: their binding was reciprocally stimulated by lysyl-tRNA; and they competed with underivatized oligoadenylates for ribosomal binding sites. When the 70 S ribosomal binding complex was irradiated, the photolabile reagent reacted covalently with both RNA and proteins of the 30 S subunit and with tRNA, but not with the 50 S subunit. The 16 S RNA appeared to be labelled at more than one site. Of the proteins, S3 and S5 reacted with the reagent with high specificity; and the possibility was not eliminated that S4 may have been labelled to a minor degree. Functional studies in other laboratories have implicated S3 and S5 in the decoding process, but these proteins were not labelled by any of the previously reported mRNA affinity labelling analogs. The results reported here therefore indicate that S3 and S5 not only affect the decoding process, but are located in the mRNA-binding region of the ribosome, presumably to the 3' side of the decoding site.     

Quantitative analysis of two dinor urinary metabolites of prostaglandin I2

The synthesis of deuterium- and tritium-labeled analogs of 2,3-dinor-6-keto-prostaglandin F_1α and of 6,15-diketo-13,14-dihydro-2,3-dinor-prostaglandin F_1α is described. These analogs were used as internal standards in the assay of the corresponding unlabeled metabolites in human urine by stable isotope dilution and combined gas chromatography-mass spectrometry. In male subjects the 24-h urinary excretion of the two metabolites was found to be 719 ± 264 and 314 ± 115 ng, respectively. The method offers a noninvasive approach to the study of prostaglandin I₂ synthesis in man.     

Reagents for the preparation of two oligonucleotides per synthesis (TOPS).

In order to increase the efficiency of use of automated DNA synthesizers (i.e. the number of oligomers prepared per day), we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotides sequence upon the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over.     

Prostacyclin stimulates the adenylate cyclase system of human thyroid tissue

Since Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in the human thyroid, we have studied the effects of PGI2 on cAMP accumulation in human thyroid slices and cultured thyrocytes. In both systems, PGI2 caused a dose- and time-dependent increase of cAMP accumulation with higher potency and efficacy than PGE2. Two optically active isomers of 5,6-dihydro-PGI2, i.e. stable synthetic analogs of PGI2, had qualitatively similar effects to PGI2. The relative potency ratio between the alpha- and beta- isomer as well as their potency compared to PGI2 were substantially similar to their potency in inhibiting human platelet aggregation. In thyroid slices, PGI2 and its stable analogs had a greater effect than TSH in causing cAMP accumulation; however, in contrast to TSH, this effect was not associated with increased iodothyronine release except at maximal PGI2 concentrations. TSH had no detectable effect on thyroidal PGI2 synthesis and release. In cultured thyrocytes the effects of PGI2 and its stable analogs were considerably less than those obtained with TSH and required higher concentrations. Such a discrepancy was not found in the case of PGE2. These findings suggest the existence of a specific PGI2-responsive adenylate cyclase system in human thyroid cells other than thyrocytes, of possible physiologic significance.     

Molecular modeling of the interactions of trichosanthin with four substrate analogs

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) that possesses N-glycosidase activity. It inactivates ribosomes and arrests protein synthesis by removing a specific adenine from 28S rRNA. A molecular dynamics simulated annealing method was applied to study the binding modes of TCS with substrate analogs, three oligonucleotides GAG, GAGA, and CGAGAG, based on the crystal structures of the stable complexes of TCS with NADPH and with the reaction product adenine. A water molecule proposed to be responsible for hydrolyzing the N-glycosidic bond was included in the model. All the oligoribonucleotides can dock into the active cleft of TCS without unfavorable contacts. The interaction energies between TCS and the three oligonucleotides were calculated. The interactions of TCS with NADH were also studied by a molecular dynamics simulated annealing method. The interaction energy between NADH and TCS was compared with that between NADPH and TCS, showing that the lack of 2-phosphate group leads to an energy rise of 20 kcal/mol.     

Incorporation of two modified nucleosides allows selective platination of an oligonucleotide making it suitable for duplex cross-linking

Platinated oligonucleotides are promising tools for the control of gene expression, since they may target and cross-link nucleic acid chains. Here we describe a method for the preparation of platinated oligonucleotides that has proved able to selectively cross-link complementary sequences, making use of 5-methylcytidine analogs with thioether or imidazole groups attached to the 4-position. These nucleoside analogs were derivatized as phosphoramidites and introduced in oligonucleotide chains using standard phosphite triester chemistry. Different oligonucleotide sequences containing either one or two analogs appending from the 5′-end were synthesized and used in preliminary platination studies. The reaction of transplatin with oligonucleotides containing the thioether-modified nucleobase was fast, but generally afforded unstable adducts and complex reaction mixtures. The imidazole-containing oligonucleotides reacted with transplatin much more slowly, in particular at slightly basic pH, and it was found that the imidazole-modified cytosine was less reactive than the natural nucleobases. In contrast, transplatin selectively reacted with the thioether and imidazole groups of oligonucleotides containing the two cytosine analogs in neighboring positions, even in the presence of the four nucleobases and particularly three guanines, affording platinated oligonucleotides suitable for cross-linking. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS

Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain. Three of them were also modified with methyl groups at the 2'-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K(AS)) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the beta-substituted analog m(7)Gpp(S)pG) was characterized by a K(AS) that was more than fourfold higher than that of its unmodified counterpart (m(7)GpppG). All analogs modified in the gamma position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the alpha position (i.e., m(7)Gppp(S)G and m(2) (7,2'-O )Gppp(S)G) were established as S(P) and R(P) , respectively, using enzymatic digestion and correlation with the S(P) and R(P) diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.     

Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.     

Enzymatic and Hybridization Properties of Oligonucleotide Analogues Containing Novel Phosphotriester Internucleotide Linkage

Abstract

Enhanced cellular uptake, stable and discriminating hybridization and increased stability in biological media are of particular interest for oligonucleotides of potential therapeutic application. Additionally, toxicity or immunogenicity of the oligonucleotide analogues and their biodegradation products should be minimized by minimal alteration of the biological structure and effort and cost of bulk production should be as low as possible by using a standard automated synthesis protocol. Oligonucleotide phosphotriesters with oligoethyleneglycol substituents show promise to ideally combine all these advantages. Here we describe the hybridization properties and the stability of modified oligonucleotides containing triester internucleotide linkages substituted with α,ω-dihydroxy-(3,6-dioxa)-octan-1-yl group (“triethyleneglycol triester linkages”) towards enzymatic degradation. The triester linkages are stable towards exo- and endonucleases. Regardless of number and position of triester linkages, the modified oligonucleotides showed practically no decrease of T_m in hybridization studies with complementary biological oligonucleotides. In further enzymatic studies the modified oligonucleotides were highly stable towards nucleases in human blood serum.     

Nucleotide pyrophosphatase/phosphodiesterase 1 is responsible for degradation of antisense phosphorothioate oligonucleotides

The rapid degradation of unmodified phosphodiester oligodeoxynucleotides (PO-oligos) by exo -and endonucleases limits their application as antisense constructs and requires the synthesis and use of modified oligonucleotides. Phosphorothioate analogs of oligonucleotides (PS-oligos) are much more stable against nucleolytic degradation than their unmodified counterparts, and this is one of the reasons for which they are a promising class of antisense oligonucleotides. However, PS-oligos also undergo slow hydrolysis by enzymes present in plasma. The oligonucleotide degradation proceeds mainly from the 3' -end, resulting in the formation of a typical ladder of shorter products and the release of the mononucleoside 5' -phosphorothioates. So far, little has been known concerning the molecular identity of the enzymes involved in the degradation of PS-oligos. We now identify the human plasma 3' -exonuclease responsible for their degradation as a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9), also known as the plasma cell differentiation antigen PC-1. We also show that adenosine or deoxyadenosine (alpha-thio)triphosphates can act as potent inhibitors of NPPs.     

Mononucleoside SATE glucosyl phosphorothiolates as a new series of pronucleotides

The synthesis and the study of two phosphorothiolate derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and glucosyl residues associated to the phosphorus atom by a 2-oxyethyl link, are reported. These derivatives could be considered as prototypes of a new series of nucleotide prodrugs (pronucleotides).     

Prostacyclin stimulates the adenylate cyclase system of human thyroid tissue

Since Prostacyclin (PGI₂) is a major product of arachidonic acid metabolism in the human thyroid, we have studied the effects of PGI₂ on cAMP accumulation in human thyroid slices and cultured thyrocytes. In both systems, PGI₂ caused a dose- and time-dependent increase of cAMP accumulation with higher potency and efficacy than PGE₂. Two optically active isomers of 5,6-dihydro-PGI₂, i.e. stable synthetic analogs of PGI₂, had qualitatively similar effects to PGI₂. The relative potency ratio between the α- and β- isomer as well as their potency compared to PGI₂ were substantially similar to their potency in inhibiting human platelet aggregation. In thyroid slices, PGI₂ and its stable analogs had a greater than TSH in causing cAMP accumulation; however, in contrast to TSH, this effect was not associated with increased iodothyronine release except at maximal PGI₂ concentrations. TSH had no detectable effect on thyroidal PGI₂ synthesis and release. In cultured thyrocytes the effects of PGI₂ and its stable analogs were considerably less than those obtained with TSH and required higher concentrations. Such a discrepancy was not found in the case of PGE₂. These findings suggest the existence of a specific PGI₂-responsive adenylate cyclase system in human significance.     

Benzimidazole 2'-isonucleosides: design, synthesis, and antiviral activity of 2-substituted-5,6-dichlorobenzimidazole 2'-isonucleosides

2,5,6-Trihalogenated benzimidazole-beta-D-ribofuranosyl nucleosides and 2-substituted amino-5,6-dichlorobenzimidazole-beta-L-ribofuranosyl nucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV). The D-ribofuranosyl analogs are metabolized rapidly in vivo rendering them unsuitable as drug candidates. The primary source of instability is thought to be the anomeric bond. The synthesis of a series of chemically stable benzimidazole-2'-isonucleosides is presented. The synthetic schemes employed are based on nucleophilic displacements of a 2'-tosylate from carbohydrate intermediates with 2-bromo-5,6-dichlorobenzidazole. 2-Bromo and 2-isopropyl amino analogs with 3'- and 5'-oxo and deoxy substitutions were prepared. The benzimidazole-2-'isonucleosides presented here demonstrated reduced activity against HCMV when compared to other D-ribofuranosyl benzimidazole analogs. In addition, they were not found to be inhibitors of HIV.