Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bcen-T and iso-tricyclo-T nucleosides

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Mononuclear Tetraamineplatinum(II) Complexes: Synthesis, Anticancer Activity, DNA Binding, and Cellular Uptake

The synthesis of three bis[(tert-butoxy)carbonyl]-protected (tetramine)dichloroplatinum complexes 2a – c of formula cis-[PtCl2(LL)] and of their cationic deprotected analogs 3a – c and their evaluation with respect to in vitro cytotoxicity, intramolecular stability, DNA binding, and cellular uptake is reported. The synthesis comprises the complexation of K2[PtCl4] with di-N-protected tetramines 1a – c to give 2a – c and subsequent acidolysis, yielding 3a – c. The cytotoxicity of the complexes is in direct relation to the length of the polyamine. Complexes 3a – c display a significant higher affinity for CT DNA as well as for cellular DNA in A2780 cells than cisplatin.

Synthesis and Properties of 6′-Fluoro-tricyclo-DNA

The synthesis of the two fluorinated tricyclic nucleosides 6?-F-tc-T and 6?-F-tc-5MeC, as well as the corresponding building blocks for oligonucleotide assembly, was accomplished. An X-ray analysis of N4-benzoylated 6?-F-tc-5MeC reavealed a 2?-exo (north) conformation of the furanose ring, characterizing it as an RNA mimic. In contrast to observations in the bicyclo-DNA series, no short contact between the fluorine atom and the H6 of the base, reminiscent of a nonclassical F···H hydrogen bond, could be observed. Tm measurements of modified oligodeoxynucleotides with complementary RNA showed slightly sequence-dependent duplex stabilization profiles with maximum ?Tm/mod values of +4.5 °C for 6?-F-tc-5MeC and +1 °C for 6?-F-tc-T. In comparison with parent tc-modified oligonucleotides, no relevant changes in Tm were detected, attributing the fluorine substituent a neutral role in RNA affinity. A structural analysis of duplexes with DNA and RNA by CD-spectroscopy revealed a shift from B- to A-type conformation induced by the 6?-F-tc-nucleosides. This is not a specific ?fluorine effect?, as the same is also observed for the parent tc-modifications. The two fluorinated tc-nucleosides were also incorporated into a pure tricyclo-DNA backbone and showed no discrimination in Tm with complementary RNA, demonstrating that 6?-F substitution is also compatible within fully modified tc-oligonucleotides.

Synthesis of cyclopentane amide DNA (cpa-DNA) and its pairing properties

We recently reported on the synthesis and pairing properties of the DNA analogue bicyclo[3.2.1]amide DNA (bca-DNA). In this analogue the nucleobases are attached via a linear, 4-bond amide-linker to a structurally preorganized sugar-phosphate backbone unit. To define the importance of the degree of structural rigidity of the bca-backbone unit on the pairing properties, we designed the structurally simpler cyclopentane amide DNA (cpa-DNA), in which the bicyclo[3.2.1]-scaffold was reduced to a cyclopentane unit while the base-linker was left unchanged. Here we present a synthetic route to the enantiomerically pure cpa-DNA monomers and the corresponding phosphoramidites containing the bases A and T, starting from a known, achiral precursor in 9 and 12 steps, respectively. Fully modified oligodeoxynucleotides were synthesized by standard solid-phase oligonucleotide chemistry, and their base-pairing properties with complementary oligonucleotides of the DNA-, RNA-, bca-DNA-, and cpa-DNA-backbones were assessed by UV melting curves and CD-spectroscopic methods. We found that cpa-oligoadenylates form duplexes with complementary DNA that are less stable by -2.7 degrees C/mod. compared to DNA. The corresponding cpa-oligothymidylates do not participate in complementary base-pairing with any of the investigated backbone systems except with its own (homo-duplex). As its congener bca-DNA, cpa-DNA seems to prefer left-handed helical duplex structures with DNA or with itself as indicated by the CD spectra

Synthesis of a cyclopentane amide DNA analogue and its base pairing properties

cpa-DNA monomers containing the bases adenine and thymine have been synthesized starting from the known compound 1 in 12 steps. Partially and fully modified cpa-thymidine and cpa-adenosine containing oligodeoxynucleotides were synthesized by standard oligonucleotide chemistry. Fully modified homo-cpa-A sequences lead to duplex destabilization by -1.4 degrees C/mod. relative to DNA. As its congener bca-DNA, cpa-DNA prefers left-handed duplex formation where possible

Synthesis and incorporation into a-DNA of a novel conformationally constrained alpha-nucleoside analogue

We describe the synthesis and incorporation into alpha-DNA of a novel conformationally constrained alpha-nucleoside analogue. The carbohydrate part of this analogue was prepared in 4 steps from the known bicyclic precursor 1 via a stereospecific, intramolecular, Et 3B mediated radical addition to a keto-function as the key step. The thus obtained intermediate 4 was transformed stereoselectively into the corresponding alpha-nucleoside analogues 7 and 8 containing the bases adenine and thymine, and were further elaborated into the phosphoramidite building blocks 11 and 12 . Both building blocks were incorporated into alpha-oligodeoxynucleotides and their pairing behavior to parallel complementary DNA studied by UV-melting experiments. Single substitutions of alpha-deoxyribnucleoside units by the new analogues in the center of duplexes were found to be thermally destabilizing by only -0.8 to -3.1›C.

Synthesis and Thermodynamic and Biophysical Properties of Tricyclo-DNA

The DNA analogue tricyclo-DNA, built from conformationally rigid nucleoside analogues that were linked via tertiary phosphodiester functions, can efficiently be synthesized from the corresponding phosphoramidites by conventional solid-phase cyanoethyl phosphoramidite chemistry. 5'-End phosphorylated tricyclo-DNA sequences are chemically stable in aqueous, pH-neutral media at temperatures from 0 to 90 C. Tricyclo-DNA sequences resist enzymatic hydrolysis by the 3'-exonuclease snake venom phosphodiesterase. Homobasic adenine- and thymine-containing tricyclo-DNA octa- and nonamers are extraordinarily stable A-T base-pairing systems, not only in their own series but also with complementary DNA and RNA. Base mismatch formation is strongly destabilized. As in bicyclo-DNA, the tricyclo-DNA purine sequences preferentially accept a complementary strand on the Hoogsteen face of the base. A thermodynamic analysis reveals entropic benefits in the case of hetero-backbone duplex formation (tricyclo-DNA/DNA duplexes) and both an enthalpic and entropic benefit for duplex formation in the pure tricyclo-DNA series compared to natural DNA. Stability of tricyclo-DNA duplex formation depends more strongly on monovalent salt concentration compared to natural DNA. Homopyrimidine DNA sequences containing tricyclothymidine residues form triplexes with complementary double-stranded DNA. Triple-helix stability depends on the sequence composition and can be higher when compared to that of natural DNA. The use of one tricyclothymidine residue in the center of the self-complementary dodecamer duplex (d(CGCGAAT t CGCG), t = tricyclothymidine) strongly stabilizes its monomolecular hairpin loop structure relative to that of the corresponding pure DNA dodecamer ( T m = +20 C), indicating (tetra)loop-stabilizing properties of this rigid nucleoside analogue.

Distamycin-NA: A DNA Analog with an Aromatic Heterocyclic Polyamide Backbone. Part 1. Synthesis and structural analysis of monomers and dimers containing the nucleobase uracil

The synthesis of the monomeric building block 13 and its constitutional isomer 12 of a new type of DNA analog, distamycin-NA, is presented (Schemes 1 and 2). This building block consists of a uracil base attached to a thiophene core unit via a biaryl-like axis. Next to the biaryl-like axis on the thiophene chromophore, a carboxy and an amino substituent are located allowing for oligomerization via peptide coupling. The proof of constitution and the conformational preferences about the biaryl-like axis were established by means of X-ray analyses of the corresponding nitro derivatives 10 and 11. Thus, the uracil bases are propeller-twisted relative to the thiophene core, and bidentate H-bonds occur between two uracil bases in the crystals. The two amino-acid building blocks 12 and 13 were coupled to give the dimers 15 and 16 using dicyclohexylcarbodiimide (DCC) in THF/LiCl and DMF, respectively. While the dimer 15 showed no atropisomerism on the NMR time scale at room temperature, its isomer 16 occurred as distinct diastereoisomers due to the hindered rotation around its biaryl-like axis. Variable-temperature 1H-NMR experiments allowed to determine a rotational barrier of 19 ± 1 kcal/mol in 16. The experimental data were complemented by AM1 calculations.

Distamycin-NA: A DNA analog with an aromatic heterocyclic polyamide backbone. Part 2. Solid-phase synthesis of distamycin-NAs containing the nucleobase uracil: Unexpected solvent participation in the coupling step

10.1002/hlca.19980810512.abs The synthesis of the Fmoc-protected amino acid 2 is presented. First attempts of amide-bond formation to the homodimer 4 in solution showed only poor coupling yields indicative for the low reactivity of the amino and carboxy groups in the building blocks 1 and 2, respectively (Scheme 1). Best coupling yields were found using dicyclohexylcarbodiimide (DCC) without any additive. The oligomerization of building block 2 adopting the Fmoc ((9H-fluoren-9-ylmethoxy)carbonyl) solid-phase synthesis yielded a mixture of N-terminal-modified distamycin-NA derivatives. By combined HPLC and MALDI-TOF-MS analysis, the N-terminal functional groups could be identified as acetamide and N,N-dimethylformamidine functions, arising from coupling of the N-terminus of the growing chain with residual AcOH or DCC-activated solvent DMF. An improved preparation of building block 2 and coupling protocol led to the prevention of the N-terminal acetylation. However, ‘amidination’ could not be circumvented. A thus isolated tetramer of 2, containing a lysine unit at the C-terminus and a N,N-dimethylformamidine-modified N-terminus, not unexpectedly, showed no complementary base pairing to DNA and RNA, as determined by standard UV-melting-curve analysis.

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.