CHEMISTRY OF PHOSPHODIESTERS, DNA AND MODELS .6. MICROGONOTROPENS AND THEIR INTERACTIONS WITH DNA .4. SYNTHESIS OF THE TRIPYRROLE PEPTIDES TREN-MICROGONOTROPEN-A AND TREN-MICROGONOTROPEN-B AND CHARACTERIZATION OF THEIR INTERACTIONS WITH DSDNA

The novel concept of attaching a connector to a pyrrole nitrogen of a tripyrrole peptide minor groove binding agent to carry functionalities to the phosphates and major groove of DNA has been extended with the synthesis of tren-microgonotropen-a and -b {6a and 6b; Chart 1}. The tren-microgonotropens are tripeptides of 3-aminopyrrole-2-carboxylic acid where (i) the amino terminus is acetylated; (ii) the terminal carboxyl has an amide linkage to beta-(N,N-dimethylamino)propylamine; (iii) the ring nitrogens of the first and third pyrrole rings are N-methylated; and (iv) the ring nitrogen of the central pyrrole carries the substituents -(CH2)(3)NH(CH2)(2)N{(CH2)(2)NH2}(2) (6a) and -(CH2)(4)NH(CH2)(2)N{(CH2)(2)NH2}(2) (6b). To determine the sequence specificity of binding, complementary strand analysis by DNase I footprinting of 6a,b bound to the 5'- and 3'-[(32)p] labeled 167 bp EcoRI/RsaI restriction fragment of pBR322 was carried out. Results show clear and specific cleavage inhibition patterns at three of the four potential A + T-rich binding sites. Employing Hoechst 33258 (Ht) as a fluorescent titrant, the equilibrium constants for the binding of 6a,b to the hexadecameric duplex d(GGCGCAAATTTGGCGG)/d(CCGCCAAATTTGCGCC) were determined (35 degrees C). The equilibrium constants for the formation of 1:1 (K-L1) and 2:1 (K-L2) complexes with 6a,b and 5a-c (the dien-microgonotropens) exhibit a slight degree of cooperativity {K-L2 > K-L1} The product KL1KL2 was slightly greater for 6a and 6b than for the dien-microgonotropens 5a, 5b, and 5c. In addition, we now show that Ht forms a 1:1 and a 2:1 complex with the hexadecamer not only by fluorescence titration but also by H-1 NMR titrations. The electrophoretic mobilities of phi X-174-RF DNA HaeIII restriction fragments complexed to 6a or 6b revealed a much greater conformational change in the DNA fragments than when distamycin (Dm) was bound to the same fragments and about a 2-fold greater change than generated by the dien-microgonotropens. Complete inhibition of mammalian topoisomerase I with 30 mu M 6b was observed while dien-microgonotropen-b and Dm only partially inhibited topoisomerase I at 150 mu M. Thus, evidence from equilibrium constants for complexation, electrophoretic mobilities, and topoisomerase I assays suggests that 6b alters the conformation of DNA in a manner that is not directly related to the affinity of complexation.