MICROGONOTROPENS AND THEIR INTERACTIONS WITH DNA .2. QUANTITATIVE-EVALUATION OF EQUILIBRIUM-CONSTANTS FOR 1 1 AND 2/1 BINDING OF DIEN-MICROGONOTROPEN-A, DIEN-MICROGONTROPEN-B, AND DIEN-MICROGONTROPEN-C AS WELLAS DISTAMYCIN AND HOECHST-33258 TO D(GGCGCAAATTTGGCGG)/D(CCGCCAATTTGCGCC)/

A quantitative methodology has been introduced to determine equilibriu m constants for minor groove binding by double-stranded DNA oligomers. The method is dependent upon the fact that Hoechst 33258 (Ht) fluores ces when bound in the minor groove of B-DNA while lexitropsins and die n-microgonotropens do not. Equilibrium constants were determined from competitive binding experiments with Ht at 35-degrees-C. Equilibrium c onstants for the 1:1 and 1:2 complexing of the double-stranded DNA hex adecamer d(GGCGCAAATTTGGCGG)/d(CCGCCAAATTTGCGCC) with dien-microgonotr open-a, -b, and -c (5a, 5b, and 5c) have been compared to the same con stants for the complexing of lexitropsins 2 and distamycin (Dm) as wel l as Ht. The following equilibrium constants were determined: K(Ht1) = [DNA:Ht]/[DNA] [Ht];K(Ht2) = [DNA:Ht2]/[DNA:Ht] [Ht];K(L1) = [DNA:L]/ [DNA][L];K(L2) = [DNA:L2]/[DNA: L][L];K(HtL) = [DNA:Ht:L]/[DNA:Ht][L]; and K(LHt) = [DNA:Ht:L]/[DNA:L][Ht]. Anticooperativity for complexing of 2 is marked by K(L2) being an order of magnitude less than K(L1). The first and second bindings of 2 to the hexadecamer are between 1 an d 4 orders of magnitude weaker than the comparable bindings of 5a, 5b, 5c, Dm, or Ht. For the latter, all second association constants (K(Ht 2), K(HtL), K(L2), and K(LHt)) are larger than the first association c onstants by approximately 1-3 orders of magnitude, indicating positive cooperativity. Although for 5a, 5b, 5c, Dm, or Ht the equilibrium con stants for stepwise complexation of one and two L or Ht species varied , the calculated equilibrium constants for formation of DNA:L2 or DNA: Ht2 species (K(L1)K(L2) or K(Ht1)K(Ht2)) were similar [(1-20) X 10(16) M-2] and 10(4) greater than the comparable constant for 2. The order of affinity is 5a is similar to 5b is similar to 5c > Ht > Dm >> 2. Re placement of the triamine substituents of 5a, 5b, and 5c with a methyl group provides 2. Thus it can be seen that the triamine substituents contribute substantially to double-stranded DNA (dsDNA) complexation o f 5a, 5b, and 5c. The temperature dependence of Ht binding to the hexa decamer between 20 and 40-degrees-C shows a critical temperature at ap proximately 32-degrees-C. Cooperativity for Ht binding to the hexadeca mer duplex is 6 orders of magnitude greater below 30-degrees-C (log K( Ht1) = 4.4, log K(Ht2) = 12.3) than above 30-degrees-C (log K(Ht1) = 7 .5, log K(Ht2) = 9.3) even though log K(Ht1)K(Ht2) is essentially unch anged. This is attributed to a marked conformational change in the DNA :Ht1 species. In the special cases of 5a, 5b, and 5c, a 38% quenching of the fluorescence of Ht in the DNA:Ht:L mixed complexes was observed . This has been shown to be due to static quenching by the (CH2)nN{(CH 2)3N(CH3)2}2 substituent (n = 3, 4, 5, for 5a,b,c, respectively).