General reaction conditions for the synthesis of aryl(trialkoxy)silanes from aryl Grignard and lithium reagents and tetraalkyl orthosilicates (Si(OR)(4)) have been developed. Ortho-, meta-, and para-substituted bromoarenes underwent efficient metalation and silylation at low temperature to provide aryl siloxanes. Mixed results were obtained with heteroaromatic substrates: 3-bromothiophene, 3-bromo-4-methoxypyridine, 5-bromoindole, and N-methyl-5-bromoindole underwent silylation in good yield, whereas a low yield of siloxane was obtained from 2-bromofuran, and 2-bromopyridine failed to give silylated product. The synthesis of siloxanes via organolithium and magnesium reagents was limited by the formation of di- and triarylated silanes (Ar(2)Si(OR)(2) and Ar(3)SiOR, respectively) and dehalogenated (Ar-H) byproducts. Silylation at low temperature gave predominantly monoaryl siloxanes, without requiring a large excess of the electrophile. Optimal reaction conditions for the synthesis of siloxanes from aryl Grignard reagents entailed addition of arylmagnesium reagents to 3 equiv of tetraethyl- or tetramethyl orthosilicate at -30 degrees C in THF. Aryllithium species were silylated using 1.5 equiv of tetraethyl- or tetramethyl orthosilicate at -78 degrees C in ether.

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Vaccinia DNA topoisomerase forms a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate at a specific target site 5-C ؉5 C ؉4 C ؉3 T ؉2 T ؉1 p2N ؊1 in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N 2 -deoxyguanosine and N 6 -deoxyadenosine positions within the 3-sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the ؉6 and ؉5 base pairs, slowed 4-fold by intercalation between the ؉5 and ؉4 base pairs, and virtually abolished by BcPh intercalation between the ؉4 and ؉3 base pairs and the ؉3 and ؉2 base pairs. Intercalation between the ؉2 and ؉1 base pairs by the ؉2R BcPh dA adduct abolished transesterification, whereas the overlapping ؉1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the ؉1 and ؊1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the ؊1 and ؊2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the ؊2 and ؊3 base pairs had little effect. The anthracycline drug nogalamycin, a noncovalent intercalator with preference for 5-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC 50 of 0.7 M. Nogalamycin was most effective when the drug was preincubated with DNA and when the cleavage target site was 5-CCCTT2G instead of 5-CCCTT2A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.Poxvirus DNA topoisomerase I is important for virus replication (1) and a potential target for drug therapy of smallpox, in light of its unique DNA recognition specificity, compact structure, and distinctive pharmacological sensitivities compared with human topoisomerase I (2-6). Poxvirus topoisomerases are exemplary type IB family members; they cleave and rejoin one strand of the DNA duplex through a transient DNA-(3Ј-phosphotyrosyl)-enzyme intermediate. Vaccinia topoisomerase cleaves duplex DNA at a pentapyrimidine target sequence, 5Ј-(T/C)CCTTp2 (3). (The Tp2 nucleotide is defined as the ϩ1 nucleotide.) Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (6 -10). Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the DNA target (11,12).Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV crosslinking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (13)(14)(15)(16)(17)(18)(19). Re...