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Sulfurization of protected [i.e., 2-cyanoethyl, methyl and 2-(4-nitrophenyl)ethyl] internucleosidic phosphite triesters can be conveniently executed with the easily accessible reagent, phenylacetyl disulfide (1). High-quality oligodeoxynucleotides containing predetermined combinations of natural and phosphorothioate linkages can be obtained via a solid-phase approach by using reagent 1 and 2-(4-nitrophenyl)ethyl-protected phosphoramidites.

A solid-phase synthesis of an aminohexyl-containing hexameric fragment of the H. influenzae type b capsular polysaccharide, using a phosphoramidite approach and controlled-pore glass as the solid support, is reported.

Silylation of a H-phosphonate diester with N, O-bis(trimethylsilyl)acetamide or tert-butyldimethylsilyl chloride affords 3′,5′-internucleotidic phosphite triester intermediates. Arbusow reaction of the latter compounds with appropriate halogenides gives the corresponding phosphonate derivatives.

Immobilized and fully protected oligodeoxynucleotides containing a 4-(1,2,4-triazolyl)-thymidine residue at a predetermined position were prepared according to a well-established phosphite triester methodology using 2-cyanoethyl phosphoramidites of a 4-(1,2,4-triazolyl)-substituted thymidine and standard protected nucleosides. Treatment of the immobilized oligomer with methanol, ethanol or n-propanol in the presence of DBU at 50°C gave the corresponding oligonucleotides containing 4-methoxy, 4-ethoxy- or 4-n-propoxythymidine residue.

Ribonucleoside phosphoramidites have been prepared of which the exocyclic amino functions and 2′-hydroxyl functions are protected with 2-[(tert-butyldiphenylsiloxy)methyl]benzoyl groups and tert-butyldimethylsilyl groups, respectively. The N-SiOMB/O-TBDMS-blocked nucleosides proved to be suitable building units in a solid-phase synthesis of the RNA-fragment 5′-AGAGUACCU-3′ via a phosphite triester approach.

High-quality oligodeoxynucleotides having an 6-O-alkyl-2′-deoxyguanosine (alkyl = methyl, ethyl, n-propyl or n-hexyl) residue at a predetermined position can be obtained via a solid-phase approach using the respective 2-cyanoethyl N,N-diisopropylphosphoramidites of 5′-O-(4.4′-dimethoxytrityl)-protected 6-O-alkyl-2′-deoxyguanosines having a free exocyclic amino group, and 5′-O-(4-4′-dimethoxytrityl) N-acyl-protected (i.e., benzoyl and isobutyryl for dC/dA and dG, respectively) 2′-deoxynucleosides.

Condensation of N3-benzoyl-5′-O-dimethoxytrityl-3′-O-(4-penten-1-oxymethyl)-thymidine with N3-benzoyl-3′-O-methoxyacctyl-thymidine, in the presence of N-iodosuccinimide, afforded a fully-protected thymidine dimer having an internucleosidic-(3′-5′)-methylene linkage. The latter was utilized in the preparation, in solution and on a solid support, of DNA-fragments containing one or more T-CH2-T dimer(s).

Electrostatic interactions appear to be important in the conformation and dynamics of DNA four-way junctions. Particularly, the Coulomb repulsion between two approaching phosphate groups at the site of strand exchange is commonly supposed to have a negative influence on the thermodynamic stability of the fully stacked conformation. We synthesized a unimolecular DNA four-way junction containing an uncharged methylene-acetal linkage, -O3'-CH2-O5'-, instead of a regular phosphodiester linkage, -O3'-PO2-O5'-, between two specific residues at the branch-point to examine the effect of charge removal. Complete sequential 1H-NMR assignments of the non-exchangeable base protons and H1'/H2'/H2" sugar protons were obtained with the aid of NOESY and TOCSY experiments. On the basis of the NMR data it is concluded that the stacking arrangement at the branch-point of the modified oligonucleotide is similar to that of the previously studied parent four-way junction. Surprisingly, this is not the stacking arrangement in which the close phosphate-phosphate contact at the site of strand exchange would be absent. Some implications of this novel information regarding the role of the phosphate-phosphate repulsion in four-way junctions are discussed.

O4-Alkylthymines have been implicated as potential carcinogenic DNA lesions. We have studied the effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine in a model system in which a single lesion was located at a defined position on a SV40-based shuttle vector and have found large differences in the effects of these lesions in repair-proficient and nucleotide excision repair-deficient cells. In repair-competent human HeLa cells, normal fibroblasts, and XP-A (2OS) revertant cells, all 3 residues were highly mutagenic; a mutation frequency of approximately 20% was found for both O4-methylthymine and O4-ethylthymine, whereas that of O4-n-propylthymine was approximately 12%. These frequencies were independent of the activity of the O6-alkylguanine DNA alkyltransferase. All three O4-alkylthymines induced T-->C transitions exclusively. In nucleotide excision repair-deficient XP-A cells, however, these lesions were not mutagenic but strongly inhibited plasmid replication (> 90%). These results indicate that O4-alkylthymines are efficiently recognized by the nucleotide excision repair system and cause a complete cessation of plasmid replication if this system is deficient. Nevertheless, proficiency in the nucleotide excision repair pathway correlates with a high frequency of mutation induction by these lesions.

One-pot coupling of 2′,3′-di-O-benzoyl-uridine and 4,5,6,8-tetra-O-acetyl-3,7-anhydro-2-deoxy-D-glycero-D-ido-octitol with appropriate bifunctional phosphoro-, phosphono- and thiophosphonylation reagents gave, after removal of the base-labile protecting groups, analogues of UDP-Glc containing charged phosphate, neutral alkyl- and arylphosphonate and phosphonothioate linkages. In an analogous manner, GDP-Man analogues, in which guanosine and octitol units are similarly linked via phosphate, alkyl phosphate or phosphotriester bonds, were prepared.

Immobilized and fully protected oligodeoxynucleotides containing a 4-(1,2,4-triazolyl)-thymidine residue at a predetermined position were prepared according to a well-established phosphite triester methodology using 2-cyanoethyl phosphoramidites of a 4-(1,2,4-triazolyl)-substituted thymidine and standard protected nucleosides. Treatment of the immobilized oligomer with methanol, ethanol or n-propanol in the presence of DBU at 50°C gave the corresponding oligonucleotides containing 4-methoxy, 4-ethoxy- or 4-n-propoxythymidine residue.

High-quality oligodeoxynucleotides having an 6-O-alkyl-2′-deoxyguanosine (alkyl = methyl, ethyl, n-propyl or n-hexyl) residue at a predetermined position can be obtained via a solid-phase approach using the respective 2-cyanoethyl N,N-diisopropylphosphoramidites of 5′-O-(4.4′-dimethoxytrityl)-protected 6-O-alkyl-2′-deoxyguanosines having a free exocyclic amino group, and 5′-O-(4-4′-dimethoxytrityl) N-acyl-protected (i.e., benzoyl and isobutyryl for dC/dA and dG, respectively) 2′-deoxynucleosides.

Easily accessible bis(diisopropylamino)alkylphosphines (alkyl is methyl, n-butyl or n-octyl) are convenient reagents for the synthesis of DNA fragments having an alkylphosphonate or alkylphosphonothioate linkage at a predetermined site in solution and on solid support.

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

The naturally occurring RNA-nucleopeptide H-Ala-Tyr[5'-pUUAAAAC-3']-NH2 is prepared via a solid-phase phosphite triester approach using N-SiOMB/O-TBDMS-protected nucleosides. Preliminary 1H-NMR studies show that the peptidyl unit has a remarkable effect on the conformational behaviour of the RNA moiety in the nucleopeptide.

Iodonium ion promoted condensation of properly protected 3′- O -methylthiomethyl or 3′- O -(4-penten-1-oxymethyl)-thymidine with 3′- O -methoxyacetyl-thymidine, was explored. A judicious choice of the iodoinium source and protecting groups led to an efficient preparation of thymidine dimers having internucleosidic-3′5′)-methylene bonds. The latter procedure was utilized towards the synthesis, in solution and on a solid support, of DNA-fragments containing one or more T- CH 2 -T dimers. Further, 5′- O -methylthiomethyl-3′- O -methoxyacetyl-N 3 -benzoyl-thymidine proved to be a suitable donor for the introduction of 5′- O -methylene acetal-linkages between 2,3,4,6-tetra- O -benzyl-D-glucose, benzyl N -benzyloxycarbonyl-L-serine and dibenzyl phosphate.

One-pot coupling of 2′,3′-di-O-benzoyl-uridine and 4,5,6,8-tetra-O-acetyl-3,7-anhydro-2-deoxy-D-glycero-D-ido-octitol with appropriate bifunctional phosphoro-, phosphono- and thiophosphonylation reagents gave, after removal of the base-labile protecting groups, analogues of UDP-Glc containing charged phosphate, neutral alkyl- and arylphosphonate and phosphonothioate linkages. In an analogous manner, GDP-Man analogues, in which guanosine and octitol units are similarly linked via phosphate, alkyl phosphate or phosphotriester bonds, were prepared.

The preparation of the nucleopeptide H-Phe-Tyr-(pATAT)-NH2 could be realized via a solid phase phosphitetriester approach and by using the protected protecting group 2-(tert-butyldiphenylsilyoxymethyl)-benzoyl for the masking of the N6-amino function of deoxyadenosine. The latter protecting group can be removed under mild conditions with fluoride ion.

A convenient method is described for the synthesis of oligodeoxynucleotides 15N labeled at the exocyclic amino group of cytosine. The 4-triazolide derivative of deoxyuridine is used as a building block in the solid phase synthesis of the oligodeoxynucleotide. Deprotection and cleavage from the solid support is achieved by treatment with 15N labeled NH4OH. This treatment also substitutes the 4-triazolide group of the deoxyuridine by an 15N labeled amino group to yield 4-15N cytosine. The method was applied to a DNA fragment corresponding to a glucocorticoid response element. The oligomer was characterised by 1H-15N coherence spectroscopy.

A linear DNA oligomer (M(r) 14,000, 46 nucleotides) was especially designed, chemically synthesized, and studied by means of 1H NMR spectroscopy. The design of the oligomer was guided by the idea that incorporation of three short palindromic sequences, each interspersed by 5'-CTTG-3' motifs at predetermined positions in the oligomer, would give rise to the formation of three stable minihairpin loops [Ippel, J. H., et al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expected to encourage further folding of the strand into a stable four-way junction containing three "hairpin" arms and an open-ended duplex stem as the fourth arm. Linear DNA four-way junctions constructed according to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compared to junctions that are built from two or more separate strands. A stable cruciform conformation was substantiated for the 46-mer in aqueous solution in the presence of Mg2+. Complete sequential 1H NMR assignments of the nonexchangeable protons (except H4', H5', and H5") were obtained with the aid of NOESY and HOHAHA experiments. The NMR data gave evidence for the expected existence of minihairpin-loop structures at the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the interresidual NOEs observed between the protons of the eight central residues revealed the complete stacking pattern of the residues at the branch point.

Two heteronuclear proton-carbon NMR experiments are applied to the DNA-octamer d(TTGGCCAA)2 with carbon in natural abundance. They lead to a complete assignment of the carbon resonances of the sugars and bases. In addition, several heteronuclear coupling constants, proton-carbon as well as proton-phosphorous and phosphorous-carbon, were determined. The information can be obtained in a reasonable measuring time and offers valuable information for a detailed picture of DNA structure.

Condensation of N3-benzoyl-5′-O-dimethoxytrityl-3′-O-(4-penten-1-oxymethyl)-thymidine with N3-benzoyl-3′-O-methoxyacctyl-thymidine, in the presence of N-iodosuccinimide, afforded a fully-protected thymidine dimer having an internucleosidic-(3′-5′)-methylene linkage. The latter was utilized in the preparation, in solution and on a solid support, of DNA-fragments containing one or more T-CH2-T dimer(s).

Easily accessible phenacetyl or benzoyl disulfide proved to be very convenient reagents for a rapid P-sulfurization of phosphite-triesters and H-phosphonate diesters, respectively.

To analyze human tumors for the presence of mutated ras oncogenes, a procedure was developed based on selective hybridization of mutation-specific oligodeoxynucleotide probes to genomic DNA [Bos et al., Nucl. Acids Res. 12 (1984) 9155-9163]. We have improved this procedure both in sensitivity and speed by including an in vitro amplification step of ras-specific sequences. This amplification step has first been described by Saiki et al. [Science 230 (1985) 1350-1353] and results in a more than 10(4)-fold increase in the sequence which might contain the mutation. Furthermore, we have improved the selectivity of our hybridizations. As a result, mutated ras oncogenes can now be detected with a dot-blot screening procedure requiring less than 1 microgram of tumor DNA.

Silylation of a H-phosphonate diester with N, O-bis(trimethylsilyl)acetamide or tert-butyldimethylsilyl chloride affords 3′,5′-internucleotidic phosphite triester intermediates. Arbusow reaction of the latter compounds with appropriate halogenides gives the corresponding phosphonate derivatives.

The coupling reagent pivaloyl chloride reacts slowly with an internucleosidic H-phosphonate function to afford the corresponding acylphosphonate derivative.