Transition metal promoted reactions. 30. Cyclopropyl anion as an allyl anion synthon. Novel synthesis of butadienes by nickel-catalyzed coupling of cyclopropyl Grignard reagents with dithioacetals

Resolution of the reaction mixture and isolation of the triester product can be accomplished by using HPLC (4.6 X 250 mm Hypersil-ODS with 0.02 M KH2P04, pH 5.5, and a methanol gradient). Modification of the phosphorothioate was observed to be more efficient for the single-stranded d[CGCA(s)GCG] fragment than the self-complementary eicosomer, d[CGTACTAGTT(s)AACTAGTACG] . This difference in reactivity was partially overcome when the reaction mixture was heated at 50 OC. In the absence of the phosphorothioate diester, control reactions using native oligodeoxynucleotides did not result in any significant labeling.'2 T h e unlabeled dodecamer helix, d [CGCA(s) GCG].d[CGCGCG], exhibited a T , of 55 "C, and this was indistinguishable from the T , values obtained for the PROXYL-labeled (a in Figure 1) or drug-labeled (b in Figure 1) helices. The T , value for the self-complementary eicosomer, d[CGTACTAGTT(s)AACTAGTACGI2, with two labels was also largely unchanged (68.5 "C) in comparison to the unlabeled fragment ( T , = 67 "C). The hydrolytic stability of the phosphorothioate triesters is an important practical consideration for the value of such derivatives in many studies. Hydrolysis of the triesters proceeded by desulfurization (monitored by HPLC and confirmed by comparison with authentic standards). We have not yet examined the fate of the reporter group in these reactions. No detectable cleavage of the oligodeoxynucleotide at the point of attachment was observed. This agrees with the results of ethylated or hydroxyethylated derivatives, which result in primarily desulfurization and only very minor amounts of chain cleavage.6 Less than 5% of the Tp(s)T triester carrying the PROXYL spin label was hydrolyzed after 24 h at pH 7. At pH 8 this increased to 28%, and at pH 10 the triester was completely hydrolyzed within 11 h. With longer fragments, the hydrolytic stability of the triester increased [the labeled dodecamer was hydrolyzed <1%, 30%, and 99% at pH values 7, 8, and 10, respectively; the values for the eicosomer were <1%, 2%, and 63% (24 h)]. The triester prepared from a y-brom~cu,P-unsaturated carbonyl (b in Figure 1) exhibited stability similar to that of the PROXYL-labeled derivatives while that resulting from reaction with the aziridinyl sulfonamide (c in Figure 1) was more stable [the Tp(s)T-labeled triester was hydrolyzed < I % (pH 7), 5% (pH 8), and 34% (pH 10) after 24 h at ambient temperature]. It is noteworthy that the triester produced from 1,5-I-AEDANS and Tp(s)T was significantly less stable than the PROXYL-labeled derivative although the triesters formed both resulted from iod0a~etamides.l~ The AEDANS-labeled dimer exhibited 19% (pH 7) and 88% (pH 8) hydrolysis (24 h); it was completely hydrolyzed within 2 h at pH 10. However, the AEDANS-labeled dodecamer (d in Figure 1) exhibited only <1%, 49%, and 99% hydrolysis at the same respective pH values (24 h). The ability to attach reporter groups (covalently) where desired on the DNA backbone should simplify studies involving protein binding, resonance energy transfer, structural analyses and nucleic acid dynamics. By employment of the appropriate functional group, it should additionally be possible to attach a variety of derivatives including, but not limited to, peptides and proteins,