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[carbohydrate structure: see text] The design and synthesis of bis 1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI)-pyrrole polyamide conjugates (13, 17) as DNA minor groove binding agents are described.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin have been screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase (RT) on a poly(rA).oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and trans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) and (2) reduced affinity for a "free" TP analog, when compared with the parent drug distamycin. All three drugs lack the ability to inhibit processive incorporation of nucleotide, suggesting drug intervention instead at initiation or termination of processive cycles. The two bis-linked drugs exhibit different kinetic behavior with reverse transcriptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (Ki = 6.5 microM). With nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitive. Gel band mobility shift assays with MPyr indicate that the drug inhibits via entrapment of TP on the enzyme rather than displacement of TP from the enzyme surface. The conformation of nucleic acid is most likely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is considered in relation to enzyme association/dissociation with TP that occurs prior to (-)-DNA strand transfer, and to the structural implications of an enzyme-bound hybrid RNA/DNA nucleic acid.

The interaction between the cobalt(III) complex of a bleomycin functional model (AMPHIS-NET) and the oligonucleotide d(CGCAATTGCG)2 and the structural features of the 1:1 ligand-DNA complex have been determined by high-resolution two-dimensional nuclear magnetic resonance methods and restrained molecular dynamics calculations. The intermolecular nuclear Overhauser effect (NOE) cross-peaks between ligand protons and the DNA minor groove protons suggest that the cobalt(III) complex of AMPHIS-NET binds in the minor groove of DNA at the central AATT site. The NOE connectivities also clearly indicate that the H8 pyridine proton and the H2 imidazole proton in the metal-binding domain interact with the H4' sugar proton of C19 and the H4' sugar proton of A5, respectively, which defines a structure where the metal binding moiety of Co(III).AMPHIS-NET participates in binding to the DNA and extends into the region two base pairs beyond the central AATT site in the minor groove. This binding model is in accord with the consistently observed nondiffusion DNA cleavage in locations two to three residues beyond the end of AT-rich binding sites induced by the corresponding iron(II) complexes of AMPHIS-NET and other AMPHIS-lexitropsin hybrids of the bleomycin functional model compounds.

The imidazole (Im) containing lexitropsin ImPy related to netropsin (Nt) is a sequence reading DNA ligand which, in contrast to Nt, permits binding to a GC base pair. The ImPy induced DNA conformational changes differ significantly from those induced by Nt as monitored by titration viscometry, although interaction modes have also been resolved with boundaries at the same ligand to DNA phosphate ratio, r. Evidently ImPy covers similar binding sites (in the same sequence) as Nt for natural calf thymus DNA at r0.023. This result suggests that the preferred binding sites of ImPy are A tracts (cf. K.E.R. JBSD 9(1993) 973), in agreement with previous data. The respective DNA coil expansion, most probably caused by unbending (l.c.), is similar but smaller compared to the Nt-DNA interaction. These results again suggest that, at low r values, the van der Waals interaction in the narrowed minor groove of AT clusters provides a dominating energy contribution to ImPy binding. At r0.03 the DNA coil expansion increases to extremely high values in that r range where Nt binding (to mixed AT/GC sequences) induces no effect at all owing to steric hindrance with the amino group of guanine. On the basis of many quantitative results for the Nt-DNA systems these effects can be understood in terms of an unbending of intrinsic helix bends (l.c.). They are of considerable interest in connection with the ability of such compounds to influence the direction of the local regulatory relevant DNA curvature.

Two-dimensional NMR methods were used to model the possible solution structure of an intercalative complex of 9-aminoellipticine (Aell), a polycyclic pyridocarbazolamine, covalently bound to an apurinic ring-opened deoxyribose site of a duplex DNA fragment in the reduced Schiff base form. The required oligonucleotide single strand containing covalently attached aminoellipticine was obtained by reductive amination in the presence of sodium cyanoborohydride. The combined NMR-energy minimization methods were employed to refine the model structures of two distinct forms, intrahelical and extrahelical, of a control 9-mer duplex DNA, d(CGTG.dr.GTGC).d(GCACTCACG), which contains an apurinic site positioned opposite a dT residue on the complementary strand. The model structure of an aminoellipticine conjugate with the same DNA sequence, derivatized via the aforementioned covalent attachment, was also obtained by incorporating intermolecular drug-DNA and intra- and internucleotide NOE-derived proton-proton distance estimates as restraints in energy minimization routines. The indole ring system of aminoellipticine, which is inserted at the apurinic site, intercalates between and is parallel to flanking GC base pairs. The pyridinic ring of aminoellipticine, in protonated form, also stacks between cytidine and thymidine bases on the complementary strand, which is consistent with the observation that the normal sequential NOE connectivity at the 5'-C13-T14 step is broken and indeed diverted through the ellipticine moiety, e.g., C13-Aell-T14 connectivities through the Aell-H4/C5Me protons. Interestingly, the partial stacking of the pyridinic ring is observed only between the 5'-CT step vs an adjacent 5'-TC step, owing to inherently weak stacking interactions associated with the former. In the absence of any potential groups that can participate in electrostatic or hydrogen-bonding interactions with the nucleic acid, pi-pi stacking and hydrophobic contacts at the intercalation site appear to be the important factors in determining stability and conformation of the aminoellipticine-DNA conjugate. Stacking interactions in such a bistranded intercalative complexation of aminoellipticine apparently govern the formation of a single intrahelical form of a right-handed B-type DNA duplex. The overall structural features lead us to propose working models for an enzyme-like DNA cleavage activity of 9-aminoellipticine and the observed inhibition of the AP endonuclease-dependent DNA excision-repair pathway.

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.

A series of hybrid molecules have been synthesized which result from the combination of a DNA sequence-specific ligand (netropsin) coupled to a DNA-interactive structural component of bleomycins (bithiazole). The DNA binding affinities as well as the cytostatic activity and their in vitro activity against a wide variety of viruses have been determined. Most of the new agents retain the DNA binding capacity of netropsin and distamycin, and force field and Pi calculations reveal the important role of the arc of curvature of these compounds in their binding to DNA. Like netropsin, the evaluated molecules did not show significant antiviral activity, but one of them demonstrated enhanced cytostatic activity against both human and murine tumor cell lines.

Ab initio calculations (Hartree-Fock) using the 6-31 G basis set have been performed on two prototype lexitropsins or information-reading molecules. The latter are DNA minor groove binding agents related to the A.T recognizing netropsin in which each of the two N-methylpyrrole moieties is replaced in turn by 1-methylimidazole and which thereby confers the property of recognizing G.C sites.Ab initio treatment was possible by examining composities of separate non-conjugated segments of the molecules. Geometry optimized conformations, energies and distribution of electrostatic charges within the molecules were derived. The ab initio derived parameters of the geometry optimized conformations of these lexitropsins were used to interpret their interaction with different sequences within the minor groove of B-DNA.

The novel hybrid duplex alpha-5'-d(TACACA)-3'.beta-5'-r(AUGUGU)-3' was analyzed extensively by 1D and 2D NMR methods. Two forms of the duplex exist in about an 80:20 ratio. Analysis of the exchangeable imino protons of the major component revealed that three AU and one AT base pair are present in addition to two GC base pairs, confirming that the duplex anneals in parallel orientation. The presence of the AT base pair, which can only be accounted for by a parallel duplex, was confirmed by a selective INEPT experiment, which correlated the thymidine imino proton to its C5 carbon. The lesser antiparallel form could be detected by exchangeable and nonexchangeable proton resonances in both strands. An exchange peak was observed in the NOESY spectrum for the thymidine methyl group resonance in both the predominant and lesser conformations, indicating the lifetime of the individual structures was on the millisecond time scale. The nonexchangeable protons of the predominant duplex were assigned by standard methods. The sugar pucker of the ribonucleosides was determined to be of the S type by a pseudorotation analysis according to Altona, with the J-couplings measured from the multiplet components of the phase-sensitive COSY experiment. The NOE pattern observed for the alpha-deoxynucleosides alsomore » suggested an S-type sugar pucker. The adoption of an S-type sugar pucker for both strands indicates that, in contrast to RNA.DNA duplexes formed exclusively from beta-nucleotides, the alpha-DNA.beta-RNA duplex may form a B-type helix. The 31P resonances of the alpha and beta strands have very different chemical shifts in the hybrid duplex and the difference persists above the helix melting temperature, indicating an intrinsic difference in 31P chemical shift for nucleotides differing only in the configuration about the glycosidic bond.« less

The biological activity of many low molecular weight antitumor compounds appear to be related to their mode and specificity of interaction with particular DNA sequences. Such small molecules are of considerable interest in chemistry, biology and medicine. Most of the anticancer drugs employed clinically exert their antitumor effect by inhibiting nucleic acid (DNA or RNA) or protein synthesis. Inhibition can occur for example through cross-linking of bases in DNA or binding to and inactivation of enzymes necessary for the synthetic processes. It is evident that DNA is an important cellular target for many anticancer agents. Much information has been obtained from molecular genetics, i.e. replication of DNA and its transcription to RNA, which provides the template for protein synthesis. DNA is a well-characterized intracellular target but its large size and sequential nature makes it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this context PBDs (pyrrolo[2,1-c][1,4]benzodiazepines), a group of potent naturally occurring antitumor antibiotics produced by various Streptomyces species, are one of the most promising types of lead compounds. They differ in the number, type and position of substituent in both their aromatic A-ring and Py C-rings, and in the degree of saturation of the C-rings which can be either fully saturated or unsaturated at either C2-C3 (endocyclic) or C2 (exocyclic). There is either an imine or carbinolamine methyl ether at the N10-C11 position. This latter is an electrophilic center responsible for alkylating DNA. In the search for compounds with better antitumor selectivity and DNA sequence specificity many PBD analogues have been synthesized in an attempt to increase their potency against tumor cells. We review here recent progress on pyrrolo[2,1-c][1,4]benzodiazepine (PBDs) analogues and their conjugates, also the progress and developments of PBD conjugates with polyamides (information reading molecules in the minor groove of DNA). For example, the cross-linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin and melphalan. A large number of PBD dimers and polyamide conjugates with varying linker lengths and bearing different heterocycles at different positions in the PBD ring synthesized in our group and their pharmacological properties have been reviewed.

The synthesis and properties of an amide isostere of the antibiotic distamycin, thioformyldistamycin 3 is described. Compound 3 exists predominantly in the E conformation of the thioamide group in freshly prepared DMSO solution but is converted into the Z form, predicted by molecular mechanics to be more stable, on standing for 24 h. The coalescence temperature in DMSO is 110 degrees C by 1H-NMR. The thioformyl moiety of 3 is resistant to both peptidase action and acid treatment. Complementary strand MPE footprinting on a EcoRI/Hind III restriction fragment of pBR322 DNA demonstrated that either E or Z forms of 3 give a single set of footprints very similar to that of the parent antibiotic with strongest protection at TAAG and TATTAT with moderately strong protection at ATTT and AAAA. The strength of binding of 3 and distamycin from delta Tm measurements to either poly.d(AT) or calf thymus DNA is comparable. Molecular modeling predicted a preferred conformation for 3 wherein the C = S bond has a torsional angle of 110 degrees with the pyrrole ring. The energy difference between this conformation and the E form is less than 1 kcal/mole. In contrast the E-form has an energy 17.3 kcal/mole greater than the Z and a value of 26.3 kcal/mole was calculated for the energy barrier between the two isomers.

The DNA binding characteristics of new analogues (2-8) of Hoechst 33258 (1), containing pyridine and benzoxazole units and designed for altered base specificity, were evaluated using UV, fluorescence, and circular dichroism studies. Like Hoechst 33258 the new analogues also bind through the minor groove of B-DNA in a nonintercalative fashion. The interaction of the compounds with poly(dA-dT) is salt independent. The studies with poly(dA-dT), ct DNA, and poly(dG-dC) indicated a decrease in the relative binding strength of the new analogues to DNAs compared with the parent molecule, Hoechst 33258. Compounds 5 and 7 showed acceptance of GC bases adjacent to AT base pairs. None of the compounds studied exhibited affinity for A-DNA, double-stranded RNA, or Z-DNA. Structure-DNA binding relationships of the new analogues compared with their parent molecule, Hoechst 33258, are discussed.

The non-exchangeable 1H-NMR signals of the branch core trinucleotide of the lariat branch site (A2'p5'G3'p5'C, 1) and its derivatives 2 and 3 are completely assigned using one- and two-dimensional NMR techniques including NOE, COSY, NOESY, 1H-1H INADEQUATE and 2D-J-resolved spectroscopy. From the vicinal coupling constants in the individual ribose rings, NOE data and T1 measurements, the following properties of the trimers are deduced. (i) The unique stacking behavior of the trimers is S2'N3'N, and the sugar rings exist predominantly in the N-conformation (3'-endo-2'-exo). (ii) The sugar-base orientations appear to be anti. (iii) The branched trimers exist in solution as single-stranded right-handed conformations resembling A-RNA with stacking between the adenine and guanine residues in aqueous solution at 21 degrees C and pH 7.2. (iv) The calculated values for the torsion angles epsilon t and gamma+ for the trimers are 201-203 degrees and 71-86%, respectively, while the percent beta t values are higher for the guanine (87-92%) than the cytosine residues (73-77%). The computer generated depiction of the triribonucleotide 1 is also shown. These subtle structural features may act as recognition signals for this critical lariat branch site which is essential for the second step in yeast mRNA splicing.

The complementary consensus donor exon intron junction d(ApCpTpTpApCpCpTpG) has been synthesized by a solid phase procedure. The non-exchangeable proton assignments were obtained using one- and two-dimensional NMR techniques including NOE, COSY, NOESY and 1H-1H-INADEQUATE. The non self-complementary nonamer exists as a random coil form in aqueous buffer at 21 degrees C as evidenced by the temperature variable 1H-NMR and NOE measurements. The nonamer was annealed to the primary consensus donor junction d(CpApGpGpTpApApGpT) and confirmation of complete annealing was obtained by detection and assignment of base pair imino protons in D2O/H2O mixtures. Application of one- and two-dimensional NMR techniques permitted the complete assignment of all the non-exchangeable protons in the duplex nonamer. These data, together with determination of vicinal coupling constants in the individual deoxyribose moieties, permits the following conclusions on the structure and conformation of the consensus donor junction: (i) it exists in aqueous solution in a conformation that belongs to the B family (ii) the sugar-base orientations are anti (iii) the deoxyribose units exist predominantly in the S conformation (2'-endo-3'-exo) (iv) the contiguous A.T base pairs d[T(5)-A(6)-A(7)].d[T(12)-T(13)-A(14)], two positions removed downstream from the splice site (5'-CAG decreases GTAAGT-3'), are uniquely propeller twisted. The propeller twisting occurs in the region in which there is partial complementarity with the branch site splice signal TACTAAC. The cross-correlation rates were used to derive the interproton distances between adjacent AH2 protons of 4.00 A in the T(5)-A(6).T(13)-A(14) step and of 3.87 A in the A(6)-A(7).T(12)-T(13) step. This structural and conformational feature if carried over into the primary RNA transcripts may serve as a recognition signal for this critical site in the genome.

The beta-complementary hexamer, beta-d[GTACGC], to the alpha-sequence, alpha-d[CATGCG], was synthesized by the phosphotriester method. The non-exchangeable proton assignments were obtained using 1D- and 2D-NMR techniques, including NOE, COSY and NOESY. The beta-strand exists as a random coil at 21 degrees C; however, at 4 degrees C, it forms an antiparallel self-recognition duplex annealing at positions 1-4. The beta-strand was annealed to the alpha-strand, and confirmation of complete annealing was obtained by detection and assignment of the six base pair imino protons in H2O/D2O solution at 21 degrees C. 1D-NOE experiments of the alpha, beta duplex d[alpha-(CATGCG) X beta-(GTACGC)] reveal that (i) it exists in aqueous solution in a conformation that belongs to the B family, (ii) it is 70 +/- 10% right-handed, (iii) the sugar-base orientations of the beta-strand are anti, and the deoxyribose units exist predominantly in the 2'-endo-3'-exo conformation. NOE measurements of the imino proton signals in the alpha, beta duplex reveal that the duplex exhibits parallel polarity.

The novel deoxyribonucleotide alpha-[d(CpCpTpTpCpC)] and its complement beta-[d(GpGpApApGpG)] were synthesized by the phosphotriester method. 1H-NMR-NOE examination of the alpha-hexamer revealed that the cytosine and thymine bases appear to adopt anti conformations in this strand. In addition the deoxyribose of the thymidine moieties may adopt average conformations approximating to C3'-endo while the cytidine furanose groups are close to C2'-endo conformations. Both hyperchromicity in thermal melting and detection of base paired imino protons in 1H-NMR studies in H2O provide evidence for the annealing of alpha-d[CCTTCC] with its complement beta-d[GGAAGG] in potassium phosphate buffer pH 7.1 containing 10 mM magnesium chloride. Under these conditions thermal melting begins at 38 degrees C and its complete at approximately 45 degrees C. NOE experiments do not permit a decision on the polarity of annealing (predicted to be parallel) for this particular pair of sequences.

Complete 1H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]2. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of 1H and 13C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and 31P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.

The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, three aspects of mitomycin C action on DNA have been studied: (a) cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. Cross-linking of DNA is determined by the return of fluorescence after a heat denaturation step at alkaline pH's. Under these conditions denatured DNA gives no fluorescence. The cross-linking was independently confirmed by St-endonuclease (EC 3.1.4.–) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was shown not to be due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship exists between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation is due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggests that the cross-linking event, to which the primary cytotoxic action has been linked, occurs sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, was shown to induce single strand cleavage as well as monoalkylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhibition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggests that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C is largely due to the free radical [Formula: see text]. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C does not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C is able to cross-link DNA in the absence of reduction at pH 4. This is consistent with the postulated cross-linking mechanisms.

Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.

The consensus acceptor exon:intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20 degrees C as evidenced by temperature variable {sup 1}H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique {sup 1}H-{sup 1}H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of {sup 31}P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon:intron junction adopts a random coil conformation in solution under the experimental conditions employed.

The high field 1H-NMR assignments of a single strand consensus donor exon: intron junction and that of the duplex splice domain has been achieved using 2D-NMR and additional techniques.

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the non-exchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5' regions exist predominantly in the S form (2'endo-3'exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2'exo-3'endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.

A series of metalloporphyrins linked through basic chains to certain DNA interactive groups has been synthesized. Several of these agents reproduce the characteristic properties of the antitumor glycopeptide bleomycin, including the oxygen-mediated scission of DNA in the presence of thiols, antibiobic activity under aerobic conditions, and activity against human and animal tumor models. Initial screening by scission of PM2-CCC-DNA identified six of the compounds, including those bearing acridine and acodazole intercalating groups, as the most active. The specificity of the oxygen-mediated scission of a 139 base pair HindIII/NciI restriction fragment of pBR322 by these six selected agents was then determined and compared with the action of pancreatic DNase by densitometric scans. All six of these compounds produce uniform base and sequence neutral cleavage of the restriction fragment at each base site. The six active compounds bear either of two types of intercalators, 6-chloro-2-methoxyacridine or acodazole, and with linkages to the ferric binding domain of -NH(CH2)2-, -NH(CH2)3-, -NH(CH2)4-, or -NH(CH2)3NH(CH2)3- and either porphyrin or deuteroporphyrin moieties. Comparison of the Kassoc values for binding to calf thymus DNA suggests that the enhanced binding observed with the linker -NH(CH2)3NH(CH2)3- contributes to the efficiency of sequence neutral DNA scission and may be a factor in the relative anticancer activities of these agents. The iron porphyrins give no evidence of the production of base propenals in DNA degradation, and the autoradiograms clearly indicate that a phosphate group is attached to the 5' end of the oligomer. The scission is partially suppressible by catalase and superoxide dismutase.(ABSTRACT TRUNCATED AT 250 WORDS)

A high-field 1H and 31P-NMR study of the oligomer d[CpGp ApTpCpG]2 was carried out in H22O and water signal suppression was employed in all 1H NMR acquisitions. Particular attention was given to imino proton and 31P assignments. Two dimensional 31P-1H shift correlation contours were particularly useful in 31P assignments and confirming previous 1H assignments. Titrimetric addition of aliquots of the anticancer agent mitoxantrone resulted in selective and progressive chemical shifts with critical changes at stoichiometrics of 1:1 and 2:1 drug to DNA ratios. The results indicate ultimate intercalative binding of the drug at both C.G termini of the oligomer in accord with the previously determined C.G preference and with non-nearest neighbor intercalation.

All 1H-NMR resonances of d-[CATGGCCATG]2 and the 1:1 complex of lexitropsin 1 and the DNA were assigned by the NOE difference, COSY and NOESY methods. Addition of 1 causes the base and imino protons for the sequence 5'-CCAT to undergo the most marked drug-induced chemical shift changes, thereby indicating that 1 is located in this base pair sequence. NOEs confirmed the location and orientation of the drug in the 1:1 complex, with the amino terminus oriented to C(6). The van der Waals interaction between H12a,b of 1 and AH2(8) may be responsible for reading of the 3' A.T base pair in the 5'-CCAT sequence. Exchange NMR effects allow an estimate of approximately equal to 62 s-1 for the intramolecular "slide-swing" exchange of the lexitropsin between two equivalent binding sites with delta G = 58 +/- 5 kJ mol-1 at 301 degrees K.

A group of oligopeptides has been synthesized that are structurally related to the natural antiviral antitumor agents netropsin and distamycin but which bear alkylating functions. Cytostatic activity against both human and murine tumor cell lines as well as their in vitro activity against a range of viruses is reported. The biological activity of these agents is discussed both in terms of their alkylating reactivity and of their structural differences. The incorporation of alkylating moieties into these minor groove DNA binders results in up to 45-fold increase in cytostatic activity compared with netropsin and up to 18 times the activity of distamycin.

The novel hexadeoxyribonucleotides α-d(CpCpTpTpCpC) and α-d(CpApTpGpCpG), in which each glycosidic linkage exhibit the anomeric α-configuration, were synthesized by the phosphotriester method. 1H-NMR and thermal denaturation studies provided evidence for these a-oligonucleotides to exhibit a secondary structure similar to that of the natural nucleic acids.

The mechanism of breakage of apurinic DNA with 3-aminocarbazole was determinated on a short oligonucleotide model. The results founded contrast with those reported in the literature.1

High-field NMR, methods have been developed for assigning proton resonances of duplex oligodeoxyribonucleotides which may be applied to the analysis of their complexes with anticancer agents.