Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5'-(CpG) motif: model of a malondialdehyde cross-link.

Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis. MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine. In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F. H. and Goldstein, B. D. Science 1976, 191, 868--869). The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines. An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions. The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link. The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation. This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.

Synthesis and characterization of nucleosides and oligonucleotides bearing adducts of butadiene epoxides on adenine n(6) and guanine n(2).

Butadiene is a major industrial chemical whose genotoxic effects are attributed to the reaction of its oxidized metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), with DNA. Nucleosides and oligonucleotides containing regio- and stereochemically specific adducts of BDO and the BDO2-related compound, butene 3,4-diol 1,2-epoxide (BDE), on guanine [(2R)- and (2S)-N(2)-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N(2)-(2,3,4-trihydroxybut-1-yl), respectively] and on adenine [(2R)- and (2S)-N(6)-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N(6)-(2,3,4-trihydroxybut-1-yl), respectively] have been prepared by nonbiomimetic routes. For guanine adducts, 2-fluoro-O(6)-(trimethylsilylethyl)-2'-deoxyinosine was treated with (2R)- and (2S)-2-amino-3-buten-1-ol to give the BDO adducts and with (2R,3R)- and (2S,3S)-1-amino-2,3,4-butanetriol to produce the BDE adducts; the adducted oligonucleotides were prepared from 11-mer oligonucleotides containing the halopurine. Adenine adducts were prepared in a similar fashion using 6-chloropurine 2'-deoxyriboside as the reactive purine component.

Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine.

Improved methodology has been developed for preparation of oligodeoxynucleotides bearing adducts on the N2 position of guanine in which the adduction reaction is carried out in homogeneous solution rather than while the oligonucleotide is immobilized on a solid matrix. The methodology utilizes a new synthon, 2-fluoro-O6-(trimethylsilylethyl)-2'-deoxyinosine (3). Nucleoside 3 is stable to the conditions of oligonucleotide synthesis, but the O6 protection is eliminated under very mild conditions following displacement of the 2-fluoro group by amine nucleophiles. Oligonucleotides containing 3 could be removed from the solid support by treatment with 0.1 M NaOH (8 h, rt) without disruption of 3. Reaction of the crude, partially deprotected oligonucleotide with (R)-2-amino-2-phenylethanol in homogeneous solution, followed by removal of the remaining protective groups with NH4OH (60 degrees C, 8 h) and then 0.1% acetic acid, gave the adducted oligonucleotide in good purity and yield. Alternatively, fully deprotected oligonucleotide containing 3 could be prepared by use of labile phenoxyacetyl-type protecting groups on the exocyclic amino groups.

Synthesis and serotonin binding site studies of some conformationally restricted indolylethylamine analogues based on 2-amino-3-(3'-indolyl)bicyclo[2.2.2]octane.

The bicycloannulation reaction between cyclohexenone and indolyl enamines yields trans-3-(cyclic amino)-2-(3'-indolyl)bicyclo[2.2.2]octan-5-ones, and these adducts are conformationally restricted analogues of indolylethylamine (tryptamine) which exhibit structure-dependent affinity for the serotonin 5HT2 and 5HT1a receptors. The stereochemistry of the isomeric endo and exo adducts obtained is assigned from the 1H NMR spectra of the specifically deuterated alkenes prepared from the ketones by the Bamford-Stevens reaction. Molecular mechanics calculations indicate that the conformational flexibility of the amino and indolyl groups is restricted through van der Waals interactions with the bridges of the bicyclic unit. These compounds inhibit the binding of [3H]ketanserin to 5HT2 sites in mouse cerebrocortical membranes, and the binding of [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT) to 5HT1a sites in mouse hippocampal membranes. The endo compounds are the most potent, and molecular mechanics calculations indicate that these isomers have a less bulky bicyclo bridge proximate to the amine group and more conformational freedom about the C alpha-C beta-N(+)-H dihedral angle (tau 3). In the 5HT2 assay, endo-trans-3-(N-piperidinyl)-2-(3'-indolyl)bicyclo[2.2.2]octan-5-one (10a) is the most potent, and endo-trans-3-(N-pyrrolidinyl)-2-(3'-indolyl)bicyclo[2.2.2]oct-5-ene (12a) is the most potent in the 5HT1a assay. A phenyl-substituted adduct shows the least affinity in these two assays. These data provide insight into the structural differences between the 5HT1a and 5HT2 receptor sites.