A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)(2).

The structures of the complexes formed between 9-amino-[N:-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG(5Br)UACG)(2) and d(CGTACG)(2) have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 A. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).

Kinetic and equilibrium studies of the interaction of amsacrine and anilino ring-substituted analogues with DNA.

The kinetic stability of complexes of the clinical antileukemic drug amsacrine and anilino ring-substituted analogues with DNA has been studied by using the surfactant sequestration technique in a stopped-flow spectrophotometer. In addition, viscometric measurements using covalently closed circular DNA and displacement measurements of DNA-bound ethidium have been performed to evaluate helix unwinding angles and association constants, respectively. Amsacrine and its analogues dissociate from DNA by a complex kinetic pathway which involves at least three discernible transiently bound forms of the drug. Dissociation time constants for amsacrine are found to range from less than 1 to 6 ms in buffer of ionic strength 0.1, and the biomolecular association rate constant is greater than 10(6) M-1 s-1. We find that amsacrine forms one of the weakest intercalation complexes among the compounds studied, as judged by the criteria of kinetic stability, affinity, and chromophore-base pair stacking interactions. Unlike other compounds of this broad class (intercalating chromophores bearing sterically-demanding side chains), addition of the bulky side chain has little effect on the kinetic stability of the drug-DNA complexes, suggesting that the acridinesulfonanilides may intercalate into DNA from the less sterically demanding major groove of the helix. Such a binding geometry would facilitate the formation of the previously proposed ternary complex between amsacrine, DNA, and gene-regulatory enzymes (e.g., topoisomerases and repressors) which are known to bind in the major groove.

Sequence specific conformation of a DNA decamer containing an adenine tract studied in solution by H-NMR spectroscopy.

The decanucleotide duplex d(AAAACGTTTT)2 and a variety of phase-sensitive two-dimensional (2D) NMR experiments have been used to investigate the solution conformation of an adenine-tract and its junction with another DNA sequence. 2D nuclear Overhauser effect data confirm that the oligonucleotide has a general B-type DNA morphology but an array of unusual correlations implies that the adenine tract and the 5'-ApC junction have conformations more compatible with the modified X-ray structures recently reported for DNAs of similar sequence (Nelson, H.C.M., Finch, J.T., Luisi, B.F. and Klug, A. (1987) Nature 330, 221-226). The pattern and magnitude of interstrand NOEs from the adenine H2s to the sugar H1's of the complementary base to the 5'-neighbouring residue indicate that the A-T basepairs are highly propeller twisted and that the minor groove is narrowed, showing its greatest compression at the 3'-end of the tract at the 5'-ApC step. Quantifying spin-coupling interactions within the deoxyribose rings by analysing both 1D and high-resolution 2D DQF-COSY data reveals that the conformation of the purines is predominantly C2'-endo, with the pseudorotation phase angle P lying in the range 140-180 degrees. For the pyrimidines, however, there are distortions away from this standard B-type geometry with the data being best described by P values lying in the range 90-130 degrees (i.e., O4'-endo, C1'-exo). The sugar puckers of A1, T9 and T10 are dynamically distorted no doubt as a consequence of their positions at, or close to, the ends of the duplex. Thus the conformation of the adenine and thymine sugars within the oligo(dA) and oligo(dT) strands are different with an abrupt change in sugar puckering occurring at the 5'-ApC (5'-GpT) step. Peculiar chemical shifts values for A4H2, T7CH3 and sugar C5 H1', H2' and H2", together with a number of interresidue NOEs with unusual intensities, imply that there are also substantial modifications to basepair stacking interactions at this step. Taken as a whole, our data are consistent with the view that the conformational dislocation at the 5'-ApC dinucleotide results from a combination of slide and roll manoeuvres and that the junction between the AAAA and CG sequences is a potential nucleation site for DNA bending.

DNA-binding properties and antitumour activity of monofunctional alkylating groups attached to the DNA-intercalating chromophore phenanthridine: n-bromoalkylphenanthridinium bromides.

We have synthesised an homologous series of n-bromoalkylphenanthridinium bromides and studied their DNA-binding and antitumour properties. Each of these compounds has the capacity both to intercalate and alkylate DNA. Dialysis measurements reveal a relatively high affinity for calf thymus DNA, being about 10(5) M-1 at ionic strength 0.01. Incubating calf thymus DNA-ligand complexes having a ligand-to-basepair ratio of 0.4 at 37 degrees C for 18 h leads to maximum alkylation levels of about one ligand molecule bound irreversibly per 40 basepairs. The reactivity of these compounds towards DNA is chain-length dependent, the n-decyl compound, for example, requiring about 10-times the ligand-to-basepair input ratio of the n-hexyl derivative to reach the same level of alkylation. The limited degree of alkylation is a consequence of conversion of the alkylbromides to the less reactive alkylchlorides in the buffer medium. The results of DNA sequencing experiments indicate that the n-hexyl derivative alkylates at guanines occurring in 5'-GT-3' sequences and in runs of guanines [(Gp)n]. The corresponding n-decyl compound, on the other hand, is highly selective for guanines in 5'-GT-3' sequences only and also reacts weakly with some adenines. None of the phenanthridinium compounds showed significant antitumour activity in the P388 murine leukaemia test system.

A solvent-partition method for measuring the binding of drugs to DNA. Application to the quinoxaline antibiotics echinomycin and triostin A.

The development of a novel solvent-partition method for measuring the interaction between nucleic acids and drugs of limited water solubility is described. Factors relevant to the choice of a suitable water-immiscible solvent are summarised. i-Amyl acetate was selected for studying the binding of echinomycin and triostin A to DNA. Details of the experimental determination of extinction and partition coefficients are given; in the i-amyl acetate/buffer system employed for most experiments, the partition coefficients for echinomycin and triostin A were 111 +/- 4 and 943 +/- 23, respectively. Equilibration of echinomycin between the organic and aqueous phases was 90% complete within a few minutes, and a period of 2 h shaking was found satisfactory to ensure full attainment of equilibrium. Representative results are presented showing specific binding of the quinoxaline antibiotics to DNA, strong preference for double-helical as opposed to heat-denatured or single-stranded DNA, and restricted uptake by closed circular duplex PM2 DNA. The method is potentially applicable, with appropriate modifications, to the study of interactions between other ligands and DNA.

The interaction of substituted and rigidly linked diquinolines with DNA.

Viscometric measurements with circular and sonicated rodlike DNA fragments were used to explore whether ring substituents or conformationally restricted linkers promote bifunctional intercalation amongst a series of binuclear 4-aminoquinolines bridged via their 4-amino group. We find that ligands comprising unsubstituted quinolines and piperazine or pyrazole linkages bisintercalate. Quinoline-substituted alkyl-linked dimers intercalate in either a mixed monofunctional-bifunctional mode or bind with only one of their chromophores intercalated depending on the nature of the substituents. Equilibrium dialysis measurements show that the binding affinity for calf thymus DNA of the compounds studied ranges from (1.2-12) . 10(4) M-1 in buffer of ionic strength 0.1. Both co-operative and antico-operative binding isotherms were obtained and there is evidence for a second binding mode for the piperazine-linked diquinoline at saturating binding levels. For this compound the high-affinity association constant decreases with increasing ionic strength, 3.4 cations being released per bound ligand molecule. Partition dialysis measurements with DNAs of differing base composition indicate that the compounds studied are either AT selective or sequence neutral depending on ligand structure. For example, the pyrazole linker imparts a marked specificity for binding to AT-rich DNA, whereas the piperazine linker does not. Kinetic measurements using the surfactant-sequestration method reveal that DNA-diquinoline complexes dissociate very rapidly by complex mechanisms with rate constants greater than 100 s-1 in buffer of ionic strength 0.1.

Alkyl-linked diquinolines are monofunctional AT-selective DNA-intercalating agents.

The binding of a homologous series of alkyl-linked 4-aminodiquinolines to circular and linear DNAs was studied using viscometric titrations and equilibrium dialysis. The compounds are monofunctional intercalators with the capacity for intercalative binding reaching a peak for the heptane homologue. They show marked AT-base pair selectivity, which suggests that the non-intercalated quinoline ring may lie in the DNA minor groove. Affinities for calf thymus DNA increase as the alkyl chain is lengthened, falling in the range 6 to greater than 250 X 10(4) M-1 in a buffer of I 0.01. The association constant of the heptane diquinoline decreases with increasing ionic strength, 2.1 cations being released per bound dipositive ligand molecule. All the agents are growth inhibitory towards mouse leukemia cells in culture with IC50 values in the range 0.06-18 microM.

Kinetic and equilibrium binding studies of amsacrine-4-carboxamides: a class of asymmetrical DNA-intercalating agents which bind by threading through the DNA helix.

Detailed equilibrium and kinetic studies of the DNA interaction of the amsacrine-4-carboxamide class of compounds suggest that they bind by intercalating the acridine chromophore at near-maximal overlap with the base pairs, locating their two dissimilar side chains in specific grooves of the double helix. The first step is a fast bimolecular association to form an outside-bound complex (probably in the major groove). Insertion of the less bulky carboxamide side chain then occurs in a process governed largely by the rate of transient opening of the double helix by natural "breathing" motions and is followed by further monomolecular rearrangements to allow the carboxamide side chain to find its highest affinity binding sites in the minor groove. Dissociation of the complexes are much more ligand structure dependent, but also involve opening of the double helix to allow disengagement. Compounds of this type, which locate their two distinguishable side chains one in each DNA groove, form a unique class of DNA-binding ligand, with considerable potential for regiospecific delivery of reactive functionality to DNA. Although natural products which also have such specific binding modes are known (e.g. nogalamycin), the amsacrine-4-carboxamides discussed here are the first class of readily modified synthetic compounds with this property.

Synthesis and evaluation of DNA-targeted spatially separated bis(aniline mustards) as potential alkylating agents with enhanced DNA cross-linking capability.

DNA-targeted separated bis-mustards were synthesized by attaching aniline mono-mustards at the 4- and 9-positions of the DNA-intercalating ligand 9-aminoacridine-4-carboxamide, with the intention of improving the low cross-link to monoadduct ratio found with most alkylating agents. The geometry of these compounds requires that, when the acridine binds to DNA by intercalation, one alkylating moiety is delivered to each DNA groove. Gel electrophoretic studies show that only one arm of these compounds (probably that attached to the 9-position) alkylates DNA, such alkylation occurring specifically in the major groove at the N7 of guanines. Cell-line studies confirm that the mode of cytotoxicity of these compounds (unlike that of untargeted aniline bis-mustards of comparable reactivity) is due to bulky DNA monoadduct formation. It is concluded that more information is required about the exact orientation of the initial monoadducts before ligands with specific DNA cross-linking ability can be designed.

Activity of platinum(II) intercalating agents against murine leukemia L1210.

Four series of intercalating, square-planar Pt(II) complexes derived from the ligands 2,2'-bipyridine, 2,2':6',2"-terpyridine, 1,10-phenanthroline, and 3,4,7,8-tetramethyl-1,10-phenanthroline were synthesized and aspects of their activity against murine leukemia L1210 cells investigated. The 2,2':6',2"-terpyridine-thiolato complexes are growth inhibitory in culture, with IC50 values in the range 6-32 microM, and cause cell lysis at high concentrations. Of the remaining three series, the 2,2'-bipyridine complexes are the least potent in their effects. There is a general enhancement in activity on moving from the 1,10-phenanthroline complexes to the 3,4,7,8-tetramethyl-1,10-phenanthroline analogues. Flow cytometric analysis on representative complexes shows that they are not cell cycle specific. Alkaline elution experiments indicate no damage to DNA of cells exposed to (thiophenolato)(2,2':6',2"-terpyridine)platinum(II) chloride monohydrate and (ethylenediamine)(1,10-phenanthroline)platinum(II) dichloride dihydrate although (ethylenediamine)(3,4,7,8-tetramethyl-1,10-phenanthroline)platinum(II) dichloride dihydrate causes both single-strand breaks and DNA cross-links. Compounds 2a, 5a, and 6a showed no antitumor activity against L1210 in mice.

Potential antitumor agents. 44. Synthesis and antitumor activity of new classes of diacridines: importance of linker chain rigidity for DNA binding kinetics and biological activity.

Four classes of diacridines, joined at the 9-position by linker chains of varying length, rigidity, and polarity, were evaluated for DNA-binding properties and antitumor activity. Diacridines linked by flexible chains of varying polarity show relatively fast chromophore exchange kinetics among DNA binding sites but slower dissociation rates, suggesting the potential for considerable "creeping" of the drug along the helix, and are inactive in vivo. The exchange kinetics can be slowed dramatically by inclusion of positive charges in the side chain, but the resulting polycationic drugs are inactive in vivo, possibly due to poor distribution. Diacridines linked by a rigid, polar but neutral dicarbamoylpyrazole chain retain slow exchange kinetics, have a greatly reduced potential "creep rate", and possess good in vitro potency and significant in vivo antileukemic activity.

Relationships between DNA-binding kinetics and biological activity for the 9-aminoacridine-4-carboxamide class of antitumor agents.

The kinetics of dissociation of calf thymus DNA complexes of the new intercalating antitumor drug N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (5) and selected derivatives have been investigated by using the surfactant-sequestration method. The derivatives studied include those where the position (14 and 15) and nature of attachment (20 and 21) of the cationic side chain is modified, those where the distance (16-19) and composition (22-24) of the cationic group are varied, and those in which the chromophore is further substituted (25-31). While all of the compounds dissociate by a mechanism that involves at least three intermediate bound forms, derivatives bearing a 4-CONH(CH2)2NR1R2 side chain (where R1 and R2 are groups that permit the nitrogen to be protonated at neutral pH) have access to an additional binding mode of greater kinetic stability. A positive correlation is found between in vivo antitumor activity, selectivity of binding to GC-rich DNAs, and the presence of this fourth, long-lived transient species. We have interpreted our kinetic findings in terms of a molecular model for acridinecarboxamide-DNA complexes that accounts for the appearance of the fourth component. The acridine chromophore is postulated to intercalate from the narrow groove, its major axis lying at an angle to the major axis of the base pairs so that the CH atoms of positions 5 and 6 protrude into the groove. An important feature of the model is a bifurcated hydrogen bond between the O2 oxygen atom of a cytosine base adjacent to the binding site and the NH atoms of the carboxamide and protonated terminal amino functions of the drug molecule. Since the structural features required to form this bonding interaction are necessary, although not sufficient, conditions for in vivo antitumor activity, it is suggested that the model may describe the essential characteristics of the biologically active form of the bound drug. These findings further attest to the value of investigating the kinetics of DNA-drug interaction in studies of the mode of action of antitumor intercalating agents.

DNA-directed alkylating agents. 2. Synthesis and biological activity of platinum complexes linked to 9-anilinoacridine.

Two different classes of cis-diaminedichloroplatinum(II) complexes linked to the DNA-intercalating chromophore 9-anilinoacridine have been synthesized and evaluated as DNA-targeted antitumor agents. Two different Pt chelating ligands were investigated (based on 1,2-ethanediamine and 1,3-propanediamine), designed to deliver the Pt in an orientation likely to respectively enhance either intrastrand or interstrand cross-linking. Although both sets of ligands were somewhat unstable under neutral or basic conditions with respect to disproportionation, the corresponding Pt complexes, once prepared, appeared to be quite stable. All the Pt complexes were monitored for purity by TLC, HPLC, and FAB mass spectra, and the mode of Pt coordination was established by 195Pt NMR spectroscopy. The complexes appeared to cause simultaneous platination and intercalative unwinding of plasmid DNA. In vitro studies were carried out with both wild-type and cisplatin-resistant P388 cell lines. Whereas cisplatin itself and the ethylenediamine and 1,3-propanediamine complexes used as standards were about 10-fold less active against the resistant line, the ethylenediamine-linked Pt complexes showed no differential toxicity between the two lines and the propanediamine-linked complexes showed significant differentials (up to 8-fold) in favor of the cisplatin-resistant line. However, these were no greater than those shown by the unplatinated ligands themselves. The majority of the acridine complexes were inactive in vivo against the wild-type P388 leukemia. They were very insoluble, and although a suitable formulation was found, this may have been a factor. It is also possible that these compounds bind in such a way as to direct the Pt away from the major groove.

DNA-directed alkylating agents. 5. Acridinecarboxamide derivatives of (1,2-diaminoethane)dichloroplatinum(II).

A series of acridine-2- and -4-carboxamide-linked analogues of PtenCl2 has been prepared and evaluated for biological activity against several tumor cell lines in vitro and in vivo. The platinum complexes were generally more cytotoxic than the corresponding ligands against wild-type P388 leukemia cells in vitro, with acridine-4-carboxamide complexes being the more effective. In contrast to cisplatin and PtenCl2, the complexes were equally active in vitro against both wild-type and cisplatin-resistant P388 lines. The 4-carboxamide complexes showed high levels of in vivo activity (ILS greater than 100%) against wild-type P388 using a single-dose protocol, and one compound was also significantly active in vivo in a cisplatin-resistant line, against which cisplatin and PtenCl2 are inactive.

DNA-directed alkylating agents. 1. Structure-activity relationships for acridine-linked aniline mustards: consequences of varying the reactivity of the mustard.

A series of DNA-targeted aniline mustards have been prepared, and their chemical reactivity and in vitro and in vivo cytotoxicity have been evaluated and compared with that of the corresponding simple aniline mustards. The alkylating groups were anchored to the DNA-intercalating 9-aminoacridine chromophore by an alkyl chain of fixed length attached at the mustard 4-position through a link group X, while the corresponding simple mustards possessed an electronically identical small group at this position. The link group was varied to provide a series of compounds of similar geometry but widely differing mustard reactivity. Variation in biological activity should then largely be a consequence of this varying reactivity. Rates of mustard hydrolysis in the two series related only to the electronic properties of the link group, with attachment of the intercalating chromophore having no effect. The cytotoxicities of the simple mustards correlated well with group electronic properties (with a 200-300-fold range in IC50S). The corresponding DNA-targeted mustards were much more potent (up to 100-fold), but their IC50 values varied much less with linker group electronic properties. Most of the DNA-targeted mustards showed in vivo antitumor activity, being both more active and more dose-potent than either the corresponding untargeted mustards and chlorambucil. These results show that targeting alkylating agents to DNA by attachment to DNA-affinic units may be a useful strategy.

DNA-directed alkylating agents. 3. Structure-activity relationships for acridine-linked aniline mustards: consequences of varying the length of the linker chain.

Four series of acridine-linked aniline mustards have been prepared and evaluated for in vitro cytotoxicity, in vivo antitumor activity, and DNA cross-linking ability. The anilines were attached to the DNA-intercalating acridine chromophores by link groups (-O-, -CH2-, -S-, and -SO2-) of widely varying electronic properties, providing four series of widely differing mustard reactivity where the alkyl chain linking the acridine and mustard moieties was varied from two to five carbons. Relationships were sought between chain length and biological properties. Within each series, increasing the chain length did not alter the reactivity of the alkylating moiety but did appear to position it differently on the DNA, since cross-linking ability (measured by agarose gel assay) altered with chain length, being maximal with the C4 analogue. The in vivo antitumor activities of the compounds depended to some extent on the reactivity of the mustard, with the least reactive SO2 compounds being inactive. However, DNA-targeting did appear to allow the use of less reactive mustards, since the S-linked acridine mustards showed significant activity whereas the parent S-mustard did not. Within each active series, the most active compound was the C4 homologue, suggesting some relationship between activity and extent of DNA alkylation.

cis-bis(pyridine)platinum(II) organoamides with unexpected growth inhibition properties and antitumor activity.

The platinum(II) organoamides [Pt(NRCH2)2L2] (L = pyridine (py), R = p-HC6F4, C6F5,p-IC6F4,p-CIC6F4,p-C6F5C6F4; L = 4-methylpyridine, R = p-HC6F4) and [Pt(NRCH2CH2NR')(py)2] (R = p-HC6F4, R' = C6F5, p-BrC6F4, or p-MeC6F4) inhibit the growth of murine L1210 leukemia cells in culture with ID50 values for continuous exposure in the range 0.6-2.7 microM. Representative complexes are also active against L1210 cells in 2-h pulse exposures, as well as against the cisplatin-resistant variant L1210/DDP and human colonic carcinoma cell lines HT 29 and BE. Three complexes [Pt(NRCH2)2L2] (R = p-HC6F4, C6F5, or p-IC6F4) have good activity (T/C greater than or equal to 180%) against P388 leukemia in mice, and all other compounds tested are active except when R = p-C6F5C6F4, L = py. Although the molecular basis of the biological activity of these complexes is not known, the observation of good activity for amineplatinum(II) compounds with no hydrogen substituents on the nitrogen donor atoms introduces a new factor in the anticancer behavior of platinum(II) complexes.

Radiosensitization of mammalian cells in vitro by nitroacridines.

The nitroacridine nitracrine (1-NC) is a DNA intercalator and a hypoxia-selective, electron-affinic radiosensitizer. Sensitization of Chinese hamster fibroblast cultures at 0 degrees C by the nitro positional isomers of 1-NC has now been compared to help establish structure-activity relationships. The des-nitro analog (E(1) at pH 7 = -899 mV) did not sensitize, suggesting that an electron-affinic chromophore is required. All the nitroacridines (E(1) range -376 to -257 mV) sensitized hypoxic cells with a maximum sensitizer enhancement ratio of about 1.7, but with a 200-fold range in potency. When mean intracellular drug concentrations were compared, 2-, 3-, and 4-NC had potencies which were similar, independent of E(1), and no greater than predicted for non-DNA binding nitroheterocycles. Sensitization by these three isomers occurred at intracellular concentrations likely to saturate the potential intercalation sites on DNA. A large fraction of the radical sites sensitized by O2 are apparently inaccessible to these drugs. It is suggested that sensitization results from electron transfer from migrating transient charge carriers of low reduction potential to immobile bound intercalators. An additional sensitizing mechanism may be available to 1-NC, which was 20 times more potent, a potency not accounted for by E(1), cell uptake, or DNA binding affinity. The dissociation kinetics of the DNA-drug complex was faster for 1-NC than for the other isomers. The higher potency of 1-NC may reflect a short mean residence time (less than 1 ms) in its intercalation site, allowing significant mobility on the DNA within the lifetime of relatively stable radiation-induced target radicals.

Differences between central and peripheral rat alpha-adrenoceptors revealed using binuclear ligands.

We have used two homologous series of binuclear ligands, diacridines and diquinolines, and the radioligand receptor assay to compare the topology of alpha 1- and alpha 2-adrenoceptors in rat cerebral cortex and kidney membranes. While the chain length-dependence of affinity of the diacridines, as well as that of the diquinolines, for the alpha 1-adrenoceptors of these central and peripheral tissues are similar, we find marked differences in affinity profiles for interaction with central and peripheral alpha 2-adrenoceptors. In the context of our previously proposed model for the binding of diacridines and diquinolines to alpha-adrenoceptors the results suggest that the surface features of central and peripheral alpha 2-adrenoceptors differ in the area surrounding the noradrenaline binding site. This difference may prove to be of therapeutic relevance.

Interaction of paired homologous series of diacridines and triacridines with deoxyribonucleic acid.

Viscometric measurements using covalently closed circular DNA and sonicated rod-like DNA fragments were performed to investigate unwinding and extension of the DNA helix associated with binding of paired homologous series of diacridines and triacridines. The maximum interchromophore distance for members of the diacridine series spans from 15.1 to 27.5 A, permitting the largest of these ligands to cover up to 4 or 5 base-pairs, allowing for helical twist and local unwinding in a bisintercalated complex lacking severe bending or kinking of the DNA backbone. Helix unwinding angles and increments in DNA contour length are characteristic of bifunctional reaction for all the diacridines studied, the DNA lattice appearing to saturate with one ligand molecule bound per 4 base-pairs. The triacridines, whose maximum end-to-end interchromophore distances are the same as those of their paired diacridines, have maximum central-to-terminal interchromophore distances covering the range 7.5-13.8 A and thus have the potential to form trisintercalated complexes with one or two base-pairs sandwiched between each chromophore. However, helix extension and unwinding parameters for the triacridines are similar to those of the diacridines, and we find no evidence of a transition from bifunctional to trifunctional reaction as the homologous series is ascended. In general, the binding site size appears to be 5 base-pairs for the triacridines. The stereochemical requirements for trisintercalation of triacridines are discussed with reference to the present findings and to the work of others.