Synthesis of Protected 8-Substituted Deoxyribonucleosides and Its Helix Stability in Oligodeoxyribonucleotides Containing the Eco RI Recognition Site

Abstract

Octadeoxyribonucleotides with the sequences d(GGA?ATTCC), d(GGAA?TTCC), and d(GG?AATTCC) have been prepared by solid phase synthesis using the H-phosphonate units containing modified base moieties. These oligomers which have an isosterically altered recognition sequence of the restriction endodeoxyribonuclease Eco RI. The oligomers, with replacement to deoxy-7,8-dihyroadenosine-8-one (dA^OH), 8-methoxydeoxyadenosine (dA_OMe) and 8-methoxydeoxyguanosine (dG^OMe) from deoxyadenosine or deoxyguanosine were used for studying recognition phenomena at the functional group level. From thermodyamic data of these alternating octamers it was shown that the oligomer containing 8-methoxydeoxyadenosine in the center of the recognition sequence destabilizes such duplexes less strongly than the oligomers containing other 8-substituted nucleosides in the 5′-side of the recognition sequences. Further, the hydrolysis by Eco RI of the modified oligomers perfectly resisted compared to d(GGAATTCC).     

Isopoly-L-ornithine Derivatives of Thymine and Thymidine†

Abstract

L-Ornithine derivatives of thymine, and thymidine gave oligomers by solid phase elongation reactions. These oligomers 2 and 3, however, hardly interact with the complementary polynucleotide. Conformational studies of the oligomers with CD and NMR revealed that stable intramolecular hydrogen bonding was formed between thymine base and ornithine unit.

     

Synthesis and Evaluation of Oligodeoxynucleotides Containing 3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine: Introduction of a Novel Class of Phosphodiester Internucleoside Linkages

Abstract

3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine (5) was synthesized and converted into the phosphoramidite building block 8. Novel oligodeoxynucleotide analogues containing 4′-C-hydroxymethyl phosphodiester internucleoside linkages were synthesized on an automated DNA-synthesizer. The hybridization properties and enzymatic stability were studied on oligomers with one to four modifications. The 3′-end modified oligodeoxynucleotides were resistent towards 3′-exonuclease degradation and showed only moderate lowered affinity towards complementary DNA compared with oligodeoxynucleotides bearing modifications in the middle.     

Oligonucleotides Containing Pyrazolo[3,4-d]Pyrimidines: 8-Aza-7-deazaadenines With Bulky Substituents in the 2- or 7-Position

The synthesis of the 2′-deoxyadenosine analogues 1b, 2b, and 3c modified at the 7- and/or 2-position is described. The effect of 7-chloro and 2-methylthio groups on the duplex stability is evaluated. For that, the nucleosides 1b, 2b, and 3c were converted to the corresponding phosphoramidites 15, 19, and 22, which were employed in the solid-phase oligonucleotide synthesis. In oligonucleotide duplexes, compound 1b forms stable base pairs with dT, of which the separated 1b- dT base pairs contribute stronger than that of the consecutive base pairs. Compound 2b shows universal base pairing properties while its N8 isomer 3c forms duplexes with lower stability.     

Synthesis,Biophysical and Biological Evaluations of Novel Antisense Oligonucleosides Containing Dephosphono-Internucleosidic Linkages

Abstract

Efficient large-scale syntheses of methylene(methylimino) (MMI) linked mixed base nucleosidic dimers have been accomplished. These dimers were successfully incorporated into deoxyoligonucleotides by automated solid-support synthesis. The hybridization properties, nuclease stability, RNase H mediated cleavage and in vitro biological activity of novel chimeric oligomers have been studied. The biophysical and biological evaluation of these chimeric oligomers containing MMI linkages suggests that MMI is a promising chemical modification of the backbone linkage for the construction of antisense molecules useful as therapeutics.     

SYNTHESIS AND HYBRIDIZATION PROPERTIES OF RNA CONTAINING 8-CHLOROADENOSINE

ABSTRACT

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3′- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by ～5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.     

Static and Dynamic Conformational Properties of AT sequences in B-DNA

Abstract

A theoretical study of the optimal conformations of nucleic acid oligomers containing tracts of AT base pairs is presented. The oligomers are studied in isolation and complexed with netropsin, a minor groove binding ligand. The flexibility of the oligomers and of their complexes is calculated by adiabatic mapping with respect to the total winding angle. The results of this study show that in uncomplexed oligomers the dinucleotide junctions AA, AT and TA have very different structural parameters and different responses to winding stress. The TA junction is clearly the most flexible and is the principal site for accommodating the imposed overwinding. Complexation by netropsin leads to two important effects: firstly, the three junctions adopt more uniform structures, the largest changes again being observed for TA, secondly, the differences in flexibility as a function of sequence are strongly attenuated.     

Olefinic Peptide Nucleic Acid (OPA)

Abstract

The olefinic peptide nucleic acid analogues (OPA) monomers containing the bases thymine and adenine were synthesised in 11 steps. Fully modified oligomers containing these units were prepared and their pairing properties assessed by means of UV-melting experiments.     

Post-synthetic and site-specific modification of endocyclic nitrogen atoms of purines in DNA and its potential for biological and structural studies

Site-specific modification of the N1-position of purine was explored at the nucleoside and oligomer levels. 2′-Deoxyinosine was converted into an N1-2,4-dinitrophenyl derivative 2 that was readily transformed to the desired N1-substituted 2′-deoxyinosine analogues. This approach was used to develop a post-synthetic method for the modification of the endocyclic N1-position of purine at the oligomer level. The phosphoramidite monomer of N1-(2,4-dinitrophenyl)-2′-deoxyinosine 9 was prepared from 2′-deoxyinosine in four steps and incorporated into oligomers using an automated DNA synthesizer. The modified base, N1-(2,4-dinitrophenyl)-hypoxanthine, in synthesized oligomers, upon treatment with respective agents, was converted into corresponding N1-substituted hypoxanthines, including N1-¹⁵N-hypoxanthine, N1-methylhypoxanthine and N1-(2-aminoethyl)-hypoxanthine. These modified oligomers can be easily separated and high purity oligomers obtained. Melting curve studies show the oligomer containing N1-methylhypoxanthine or N1-(2-aminoethyl)-hypoxanthine has a reduced thermostability with no particular pairing preference to either cytosine or thymine. The developed method could be adapted for the preparation of oligomers containing mutagenic N1-β-hydroxyalkyl-hypoxanthines and the availability of the rare base-modified oligomers should offer novel tools for biological and structural studies.     

Oligonucleotides with 2,6-Diaminopurine Base Replacing for Adenine: Synthesis and Properties

Abstract

Synthesis of three nucleoside building blocks with a benzoyl protected diaminopurine (DAP) base and their incorporation at different positions of DNA, RNA and hexitol oligomers (HNA) have been accomplished. DNA hairpins with a DAP substituted for one (or more) adenine in the loop structure were not found to be more stable. But the stability of RNA-hexitol as well as hexitol-hexitol duplexes improved when the adenine base was replaced with DAP.     

Dinucleoside Monophosphate Analogues Containing Disulfide Linkages

Abstract

Two dinucleoside monophosphate analogues containing disulfide linkages (1 and 2) have been prepared for incorporation into oligonucleotides. The modified oligomers will be tested for their potential as antisense agents.     

Synthesis of Triple Helix Forming Oligonucleotides with a Stretched Phosphodiester Backbone

Abstract

Total synthesis of novel DMT-phosphoramidites of thymidine (11 and 15) and 2′-deoxyguanosine (8 and 20) have been accomplished. The utility of these modified building blocks in the preparation of triple helix forming oligodeoxyribonucleotides with a stretched phosphodiester backbone has been evaluated. It was found that the oligonucleotides with extended backbones were unable to enhance the binding to duplex targets containing CG or TA base pairs.     

Xylose-DNA: Sequence-Dependent Duplex Stability as Function of Backbone Configuration

Abstract

The base pairing of a series of homooligomeric or telomeric oligonucleotides built-up from 2′-deoxy-β]-D-xyloadenosine and/or -thymidine were synthesized and studied with respect to their thermodynamics of duplex formation. Oligo (2′-deoxyxylonucleotides) are more stable than the corresponding regular oligomers the more d(xA-xT) elements they contain.     

A New Phosphoramidite Reagent for the Incorporation of Diazaphenoxazinone Nucleoside With Enhanced Base-Pairing Properties into Oligodeoxynucleotides

Abstract

New phosphoramidite reagent 7 suitable for incorporation of dC analogue was synthesized. ODNs containing diazaphenoxaziione residues in defined positions were prepared. The stability of duplexes formed was increased up to 3-5°C per modified base. Preliminary results of molecular biological testing were reported.

     

Removal of Y-37 from tRNA phe yeast alters oligomer binding to two loops

The association constants of complementary oligomers were used to monitor changes in the structure of tRNA_yeast^phe as a consequence of excision of a single base Y in the anticodon loop and of clipping the molecule at the point of excision. Significant changes were found not only in the binding constants of oligomers complementary to the anticodon loop but also in the K of an oligomer complementary to the dihydro U loop. The results suggest that either a single base change in a tRNA may alter structure elsewhere in the molecule or that the acid treatment necessary to remove Y irreversibly alters the structure of tRNA_yeast^phe.     

Double base lesions in DNA X-irradiated in the presence or absence of oxygen

Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in a deoxygenated environment.     

The Design and Synthesis of N 4-Anthraniloyl-2′-dC,the Improved Syntheses of N 4-Carbamoyl-and N4- Ureidocarbamoyl-2′-dC,Incorporation into Oligonucleotides and Triplex Formation Testing

Abstract

Three modified nucleosides were designed with the aim of achieving triplet formation with the CG base pair of duplex DNA. Direct anthraniloylation of 2′-deoxycytidine, using isatoic anhydride, afforded the novel N ⁴-anthraniloyl-2′-deoxycytidine. Much improved preparations of N ⁴-carbamoyl-2′-deoxycytidine and of N ⁴-ureidocarbonyl-2′-deoxycytidine were accomplished. The modified nucleosides were incorporated into oligonucleotides. Thermal denaturation studies and gel mobility shift analysis suggest that these nucleosides do not form base triplets with any of the four base pairs of DNA.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

A Duplex of the Oligonucleotides d(GGGGGTTTTT) and d(AAAAACCCCC) Forms an A to B Conformational Junction in Concentrated Salt Solutions

Abstract

The coexistence of both A form and B form tracts and formation of an A-B junction in the oligomer d(GGGGGTTTTT)· d(AAAAACCCCC) in saturated sodium chloride solution have been detected by Raman spectroscopy. The entire duplex adopts the familiar B-form conformation in aqueous solution at low salt concentrations (0.1M NaCl). In 6M NaCl the adoption of an A form is observed within the G,C tract while a B-form is maintained in the A,T tract. The experimental results indicate that two different helical forms can co-exist in a rather short oligonucleotide and that formation of an A-B junction can occur over a fairly small span of bases. This is in agreement with recent rules governing the relation between base sequence and secondary structure of DNA published from this laboratory.

The conformational preferences of each of the individual oligomers d(AAAAACCCCC) and d(GGGGGTTTTT) have also been investigated. The oligomer d(AAAAACCCCC) is single stranded but some evidence for base stacking is observed at 2°C. In contrast, a double stranded B-form structure characterized by wobble G-T base pairing is observed for d(GGGGGTTTTT) in 0.1M and 6M NaCl.     

Nucleotides. LXXIV Synthesis of a-D-Arabino-oligonucleotides

The 5 α-D-arabinofuranosylnucleosides α-araU (15), α-araT (18), α-araC (22), α-araA (25), and α-araG (28) have been synthesized by the modified silyl-method. The amino groups at the nucleobases and the 2′-hydroxy group at the sugar moiety were protected by the 2-(4-nitro-phenyl) ethoxycarbonyl (npeoc) group (37-40) and the amide function in α-araG was additionally blocked by the 2-(4-nitrophenyl)ethyl group (63) to improve solubility in organic solvents. Mono-and dimethoxytritylation of the 5′-OH group was performed in the usual manner to give 41-48, 64, and 65 in high yields and further substitution of the 3′-OH group led to the monomeric building blocks 66-75 as well as the 3′-O-succinoyl derivatives 76-85 functioning as starting units in solid-support oligonucleotide synthesis. A large number of oligo-α-arabinonucleotides have been prepared on modified CPG-material applying the npeoc/npe strategy as a very efficient synthetic tool for highly purified, homogenous oligomers. Hybridizations between α-arabinonucleotide strands revealed in analogy to earlier findings an antiparallel orientation whereas the combination of an oligo-α-D-arabinonucleotide with a complementary oligo-2′-deoxy-β-D-ribofuranosylnucleotide showed base-pairing only if a parallel polarity was present. The advantages in oligo-α-arabinonucleotide synthesis were furthermore demonstrated by the synthesis of the tα-ANA ^his a structural analog of the natural tRNA ^his of the phage T5.