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lambda-Tris(3,4,7,8-tetramethyl-1,10-phenanthroline)ruthenium(II) [lambda-Ru(TMP)2+3] was found to be a distinctive molecular tool to examine the local variations in conformation along the strand. The metal complex binds cooperatively to A-form helices of various base sequences under conditions where little or no binding was found to analogous B-form DNAs. Photoactivated DNA cleavage may be coupled to this conformation-specific binding by taking advantage of the photophysical properties of ruthenium(II) complexes. lambda-Ru(TMP)2+3 cleaves preferentially 3H-labeled A-form polynucleotides upon irradiation with visible light. The photoinduced DNA strand scission is likely to be mediated by singlet oxygen, which leads to a preferential cleavage of guanine residues. Comparative mapping of cleavage sites on a linear pBR322 fragment for tris(phenanthroline)ruthenium(II), which binds to B-DNA and cleaves also by sensitization of singlet oxygen, and for Ru(TMP)2+3 shows the selective binding of lambda-Ru(TMP)2+3 to conformationally distinct sites along the fragment. These sites correspond to 5- to 13-base-pair homopyrimidine stretches.

The first enantioselective total synthesis of (-)-asperdiol has been accomplished in 15 steps from epoxygeranyl bromide. It is noteworthy that the trisubstituted epoxide was robust enough to survive the conditions for the nucleophilic displacement reactions which were used to form the C-12, C-13 and the '2-3, C-4 bonds. The epoxide also survived the reductive desulfonylation and the intramolecular Horner-Emmons reaction. Our assumption that the cyclization to the 14-membered ring in the desepoxy series would be more straightfoward than in the epoxy series, which provided the impetus for the synthesis of (+)-desepoxyasperdiol, was shown to be false. A number of the optically pure fragments which were used for the total synthesis of 1, particularly 4 and 7, will be useful for the synthesis of other cembranoids.

The chiral complex tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), lambda-Co(DiP)3(3+), binds to and, with photoactivation, cleaves left-handed DNA helices, thereby providing a unique molecular probe for local DNA conformation. We have mapped the specific left-handed sites where lambda-Co(DiP)3(3+) cleaves in the plasmids pBR322 and pLP32, which is the derivative of pBR322 containing a Z-form d(C-G)16 insert. For pLP32, a primary cleavage is at the insert; for native pBR322, cleavage occurs at four discrete sites: 1.45, 2.3, 3.3, and 4.2 kilobase pairs. These sites correspond to segments of alternating purine-pyrimidines. Moreover, these positions map to the ends of the three distinct coding regions in pBR322: the tetracycline-resistance gene, the origin of replication, and either end of the ampicillin-resistance (beta-lactamase) gene. The locations of these left-handed segments suggest to us that Z-DNA might serve as a conformational punctuation mark to demarcate the ends of genes.

experiments, and the 3537-cm-’ band could be a hydrogen-bound OH stretch. A molecule that matches these characteristics is the radical 5, which would be formed by breaking both the N H and N N bonds; however, this might be expected to be photosensitive and to be destroyed on photolysis. Class 111 absorptions, which are destroyed on photolysis, include bands at 1438 and 1382 cm-’, which became multiplets in mixed oxygen isotope experiments, and bands at 1525, 1517, and 535 cm-’, which displayed no ‘*O shifts. Possible products, involving only N N bond dissociation, are the peroxyamine and nitroxide radicals.

The simple inorganic complex cis-dichlorodiammineplatinum (II) (DDP) inhibits the division of bacterial cells with little effect on their growth. It was subsequently shown that DDP has substantial antitumor activity on a wide variety of animal malignancies. These findings stimulated numerous investigations of diammineplatinum (II) complexes both to establish their mode of action and to design more effective antitumor platinum drugs.

Transition metal chemistry provides a versatile means to develop reagents which with photoactivation cleave DNA either in a sequence-specific or sequence-uniform fashion. In my laboratory we have exploited the photochemistry of tris(phenanthroline) metal complexes and their derivatives in developing such photocleaving molecules.1 The tris(phenanthroline) complexes are illustrated schematically below.

lambda-Tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), a photoactivated DNA-cleaving agent, is a small molecular probe of DNA structure. Because of its chirality, the complex cannot bind to regular right-handed B-form DNA but exhibits site-specific cleavage along the polymer strand at conformationally distinct sites such as those in a left-handed conformation. Both coarse and higher resolution mapping experiments using the chiral cobalt complex indicate intriguing conformational variations along the simian virus 40 genome. Highly specific cleavage is evident in the enhancer and promoter blocks and in the region downstream of 3' termini. A specific cleavage pattern borders an alternating purine/pyrimidine stretch within the enhancer, which was found earlier to bind anti-Z-DNA antibodies. Throughout the simian virus 40 genome, variations in structure delineated with the cobalt complex appear to correlate with regions important for control of gene expression.

There has been considerable attention focused on the optical and electronic properties of ruthenium(II) polypyridyl complexes and on their rich excited-state chemistry because of their possible application in the design of photochemical sensitizers for solar energy conversion. The intense coloration and stability of the ruthenium(II) diimine complexes furthermore provide uniquely sensitive photophysical probes for solids, for surfaces, and, in their own laboratory, for biopolymers. While the detailed characterization of the ground- and excited-state electronic structures of bipyridyl and phenanthroline complexes of ruthenium(II) has proceeded, little attention has been given to other diimine complexes of ruthenium(II). The authors have examined complexes of the phenanthrenequinone diimine ligand to explore new photochemical electron transfer agents and to develop new photophysical probes for biopolymers.

The complex bis(phenanthrenequinone diimine)(bipyridyl)rhodium(III), Rh(phi)_2(bpy)^(3+), cleaves DNA efficiently in a sequence-neutral fashion upon photoactivation so as to provide a novel, high resolution, chemical photofootprinting reagent. Photofootprinting of two crystallographically characterized DNA-binding agents, distamycin, a small natural product which binds to DNA in the minor groove, and the endonuclease EcoRI, which binds in the major groove, gave respectively a 5–7 base pair footprint for the drug at its A_6 binding site and a 10–12 base pair footprint for the enzyme centered at its recognition site (5′-GAATTC-3′). Both footprints agree closely with the crystallographic results. The photocleavage reaction can be performed using either a high intensity lamp or, conveniently, a simple transilluminator box, and the photoreaction is not inhibited by moderate concentrations of reagents which are sometimes required for examining interactions of molecules with DNA. When compared with other popular footprinting agents, the rhodium complex shows a number of distinct advantages: sequence-neutrality, high resolution, ability to footprint major as well as minor groove-binding ligands, applicability in the presence of additives such as Mg^(2+) or glycerol, ease of handling, and a sharply footprinted pattern. Light activated footprinting reactions furthermore offer the possibility of examining DNA-binding interactions with time resolution and within the cell.

Le complexe Co(phen) 3 3+ a faible concentration provoque une scission de l'ADN par irradiation a 254 nm

How is Nature able to recognize and access specifically one bit of information along the DNA strand? It is remarkable to consider the chemical specificity required by DNA-binding proteins and enzymes, which are able to bind a specific small sequence of bases, in the presence of the remaining genomic sequences, and having found the site of interest, carry out a complex series of reactions there. We would like to understand some of the rules which govern these specific processes. Toward that aim, we have been examining the interactions of small transition metal complexes with DNA as models for these site-specific reactions. How do variations in shape and symmetry of the metal complex as well as possible hydrogen-bonding interactions affect specific recognition of DNA sites? Does the local conformation of DNA play a role in its recognition? In order to determine where precisely along the DNA strand a given molecule is bound, it has been useful to couple reactivity to the binding moiety, usually through a metal-mediated redox reaction, which causes cleavage of the DNA strand at the bound site; this site of strand scission may then be determined (for recent reviews of DNA cleaving molecules see Barton 1986; Dervan 1986; sigman 1986). But the reactivity may also have an associated base or sequence selectivity. The resultant reaction, or DNA site targeted, then depends both on the binding characteristics and the cleavage characteristics.

The cationic complex (2-hydroxyethanethiolato)(2,2',2"-terpyridine) platinum(II), [(terpy)Pt( HET)]^+, binds cooperatively to poly(A)•poly(U) by intercalation. The melting temperature of poly(A)•poly(U) in low-salt buffer is increased by 6 °C in the presence of [(terpy)Pt(HET)]^+, indicating stabilization of the duplex structure by the bound platinum reagent. Viscosity measurements provide evidence for comparable lengthening of the polynucleotide in the presence of [(terpy)Pt(HET)]^+ and the intercalating dye, ethidium bromide. Scatchard plots of the binding of [(terpy)Pt(HET)]^+ to poly(A)•poly(U) and poly(I)•poly(C), determined through ultracentrifugation pelleting methods, show large positive curvature, reflecting the strong cooperativity associated with the platinum complex-RNA interaction. The characteristics of the binding isotherms are interpreted in terms of a model where cooperative pair units of [(terpy)Pt(HET)]^+ intercalate into the double-stranded polymer. At saturation, two platinum molecules are bound for every three base pairs. This stoichiometry may be compared with the nearest-neighbor-exclusion binding observed previously in the interaction of [(terpy)Pt(HET)]^+ and the ethidium cation with DNA, in which one intercalator occupies every other interbase-pair site at saturation. The striking differences observed in the interaction of [(terpy)Pt(HET)]^+ with DNA and RNA suggest that drug recognition is sensitive to the constraints imposed by nucleic acid secondary structure.

The x-ray photoelectron spectrum (XPS) of crystalline cis-diammineplatinum a-pyridone blue, [Pt_2(NH_3)_4(C_5H_4ON)_2]_2(NO_3)_5, has been investigated and the Pt 4f binding energies of this complex found to be similar to those exhibited by Pt(II) complexes such as cis-(H_3N)_2PtCl_2. This result is in accord with the previous assignment of a formal platinum oxidation state of 2.25 in the blue a-pyridone complex. The widths of the Pt 4f peaks (fwhm ~ 1.3) indicate that the binding energies associated with the two geometrically different types of Pt environments in the α-pyridone oligomer are very similar, the implication being that within the Pt4 unit there must be a considerable delocalization of charge. A comparison of the XPS data for the α-pyridone blue with that found for the platinum uracil and acetamide blues shows that these species have similar electronic structures, there being no evidence that they are pure derivatives of Pt(IV). The XPS of some samples of the acetamide blue ("Platinblau") show the presence of small amounts of higher oxidation state contaminants, consistent with the difficulty in preparing this species in a reproducibly pure form. The Pt(III) dimer K_2Pt_2(SO_4)_4·3H_2O possesses Pt 4f binding energies which are much closer to those of coordination complexes of Pt(IV) than Pt(II).

Charge transport (CT) through DNA has been found to occur over long molecular distances in a reaction that is sensitive to intervening structure. The process has been described mechanistically as involving diffusive charge-hopping among low-energy guanine sites. Using a kinetically fast electron hole trap, N 4 -cyclopropylcytosine ( CP C), here we show that hole migration must involve also the higher-energy pyrimidine bases. In DNA assemblies containing either [Rh(phi) 2 (bpy′)] 3+ or an anthraquinone derivative, two highenergy photooxidants, appreciable oxidative damage at a distant CP C is observed. The damage yield is modulated by lower-energy guanine sites on the same or complementary strand. Significantly, the efficiency in trapping at CP C is equivalent to that at N 2 -cyclopropylguanosine ( CP G). Indeed, even when CP G and CP C are incorporated as neighboring bases on the same strand, their efficiency of photodecomposition is comparable. Thus, CT is not simply a function of the relative energies of the isolated bases but instead may require orbital mixing among the bases. We propose that charge migration through DNA involves occupation of all of the DNA bases with radical delocalization within transient structure-dependent domains. These delocalized domains may form and break up transiently, facilitating and limiting CT. This dynamic delocalized model for DNA CT accounts for the sensitivity of the process to sequence-dependent DNA structure and provides a basis to reconcile and exploit DNA CT chemistry and physics.

Here we examine the photooxidation of two kinetically fast electron hole traps, N_4-cyclopropylcytosine (^(CP)C) and N_2-cyclopropylamine-guanosine (CPG), incorporated in DNA duplexes of various sequence using different photooxidants. DNA oxidation studies are carried out either with noncovalently bound [Ru(phen)(dppz)(bpy‘)]^(3+) (dppz = dipyridophenazine) and [Rh(phi)_2(bpy)]^(3+) (phi = phenanthrenequinone diimine) or with anthraquinone tethered to DNA. Because the cyclopropylamine-substituted bases decompose rapidly upon oxidation, their efficiency of decomposition provides a measure of relative hole localization. Consistent with a higher oxidation potential for ^(CP)C versus ^(CP)G in DNA, ^(CP)C decomposes with photooxidation by [Rh(phi)_2(bpy)]^(3+), while CPG undergoes ring-opening both with photoexcited [Rh(phi)_2(bpy)]^(3+) and with [Ru(phen)(dppz)(bpy‘)]^(3+). Anthraquinone-modified DNA assemblies of identical base composition but different base sequence are also probed. Single and double base substitutions within adenine tracts modulate ^(CP)C decomposition. In fact, the entire sequence within the DNA assembly is seen to govern ^(CP)C oxidation, not simply the bases intervening between ^(CP)C and the tethered photooxidant. These data are reconciled in the context of a mechanistic model of conformationally gated charge transport through delocalized DNA domains. Photooxidations of anthraquinone-modified DNA assemblies containing both ^(CP)C and ^(CP)G, but with varied distances separating the modified bases, point to a domain size of at least three bases. Our model for DNA charge transport is distinguished from polaron models. In our model, delocalized domains within the base pair stack form transiently based upon sequence-dependent DNA structure and dynamics. Given these results, DNA charge transport is indeed remarkably sensitive to DNA sequence and structure.

DNA-mediated charge transport (CT) is exquisitely sensitive to the integrity of the bridging pi-stack and is characterized by a shallow distance dependence. These properties are obscured by poor coupling between the donor/acceptor pair and the DNA bridge, or by convolution with other processes. Previously, we found a surprising periodic length dependence for the rate of DNA-mediated CT across adenine tracts monitored by 2-aminopurine fluorescence. Here we report a similar periodicity by monitoring N 2-cyclopropylguanosine decomposition by rhodium and anthraquinone photooxidants. Furthermore, we find that this periodicity is attenuated by consequent back-electron transfer (BET), as observed by direct comparison between sequences that allow and suppress BET. Thus, the periodicity can be controlled by engineering the extent of BET across the bridge. The periodic length dependence is not consistent with a periodicity predicted by molecular wire theory but is consistent with a model where multiples of four to five base pairs form an ideal CT-active length of a bridging adenine domain.

Complexes derived from tris(phenanthro1ine) metal cations possess several features in common which are critical to their application as site-specific DNA binding molecules. Transition metal complexes have been designed which recognize and react at specific conformations along the DNA strand. The complexes, derivatives of tris(phenanthro1ine) metal cations, possess several features in common which are critical to their application as site-specific DNA binding molecules. The complexes all are rigid, chiral, and contain a coordinatively saturated metal ion at their core. The complexes bind to DNA in a non-covalent fashion. Spectroscopic studies have been useful in characterizing both intercalative and groove-bound interactions of the complexes with the DNA helix (ref 1). Because the complexes are rigid, once aspects of the binding interaction with DNA are determined, the orientation of the molecule as a whole with respect to the DNA helix can be considered. The chirality of the complexes is furthermore useful in specifying conformation-selective recognition along the strand (ref 2). The two binding modes to DNA show differing enantiomeric selectivity. In addition, simply, the diastereomeric interaction of the transition metal complexes with the dissymmetric DNA helical structure in itself provides a level of specificity that is usefully exploited. Lastly, the importance of the central metal ion at the core of the structure of the DNA binding molecule is noteworthy. Besides serving as a spectroscopic and photoreactive handle to monitor sensitively the binding of the complex to DNA, the central metal ion provides the core to hold together the ligand scaffolding around it. The metal, though not directly coordinating to the DNA helix, instead defines the shape of the rigid asymmetric structure which may be matched to that of the DNA. Some of the complexes which recognize and react at sites along the DNA are shown below

Although zinc is present in a range of enzymes that bind to DNA [1], the function of the Zn(II) dication either in the DNA binding process or in subsequent enzymic action is uncertain. Recently we have shown that the type II restriction endonuclease EcoRI [2] contains one equivalent tightly bound zinc per monomer and that the zinc ion is essential for activity [3]. In order to more fully characterize the metal center and its interaction with the nucleic acid substrate, we have prepared the spectroscopically active cobalt(II) derivative. Substitution with cobaltous ion yields a particularly active restriction endonuclease which displays a substrate specificity that is comparable to the native zinc enzyme. Cobalt-EcoRI was prepared by dialysis of the native Zn(II)-EcoRI against high concentrations (2.5 mM) cobalt chloride (puratronic grade). Nitrogen was bubbled through the solution continuously to avoid air oxidation to cobalt(III). The possibility that in situ oxidation occurred however cannot be ruled out without spectroscopic examination. The cobalt dialysis buffer was replaced a minimum of six times to achieve complete incorporation. Thereafter dialysis against buffer without cobalt was conducted to eliminate residual free cobaltous ion; again at least six changes were required. Figure 1 shows the metal content of the enzyme over the course of one such dialysis procedure. Both the zinc and cobalt contents of the enzyme were assayed using flameless atomic absorption spectroscopy. Each time the dialysate containing CoCl2 was replaced, an aliquot was removed and dialyzed in parallel against buffer lacking cobaltous ion. As can be seen in Fig. 1, over the course of the experiment a consistent rise in the bound cobalt/enzyme monomer ratio is observed with a concomitant decrease in the intrinsic zinc content. After seven days, stoichiometric cobalt incorporation is found. No subsequent increase in bound cobalt is observed. These data indicate strongly that cobalt ion specifically displaces the enzyme-bound zinc ion in view of i) the parallel incorporation of cobalt with the loss of zinc and ii) the eventual stoichiometric and tightly bound cobalt content. It is noteworthy that no significant decrease in the level of bound zinc is observed with exhaustive dialysis against buffer lacking these high concentratives of cobaltous ion. Furthermore, metal substitution cannot be practically achieved if as little as 0.1 μM zinc ion is available in solution. Quantitative values for the relative binding affinities of zinc(II) and cobalt(II) to the metal site in EcoRI are being determined presently. Cobalt(II)-EcoRI shows activity and a DNA substrate sequence specificity that is comparable to the native enzyme. To assay for activity we examined the fragmentation of the phage λ DNA using agarose gel electrophoresis. Phage λ DNA with a molecular weight of 30 megadaltons, has five restriction sites for native EcoRI of somewhat differing affinities, and upon digestion with EcoRI a characteristic fragmentation pattern is observed [4]. Figure 2 shows the agarose gel electrophoretic pattern of the λ DNA fragments after incubation with the various EcoRI aliquots that differ in levels of cobalt incorporation as was given in Fig.1. It is apparent that Co-EcoRI shows the same substrate specificity as the native enzyme; the identical fragmentation patterns are observed with complete cleavage. Interestingly, we also find identical patterns with partial digestion (not shown) which indicates a comparable hierarchy of site preferences for the cobalt and zinc metalloenzymes. Two additional features in Fig.2 are not noteworthy. Co-EcoRI appears more active or is at least more stable than the native form of the enzyme treated similarly. Dialysis of the native Zn-EcoRi in buffer for 10 days, not surprisingly, decreases activity. This is evident in comparing channel 1, λ DNA after incubation with exhaustively dialyzed enzyme, where only intermediate high molecular weight fragments are visible and channel 8, λ DNA after incubation with untreated Zn-EcoRI, which shows complete digestion. With increasing cobalt substitution, given for channels 1–4, increasing levels of activity are found. Cobalt derivatives of EcoRI maintain high activity in dilute form for at least two months at 4 °C. Whether Co(II)-EcoRI is not only more stable to denaturation but also kinetically more efficient is being tested. Secondly the lack of cleavage in channel 5 illustrates the sensitivity of the endonuclease action to the presence of extraneous metal ions. Metal analyses of the enzyme aliquot used for this incubation revealed only partial removal of the residual cobaltous ion. The 0.5 μM level of free cobalt present in the incubation mixture was sufficient to inhibit EcoRI. We find inhibition of EcoRI with various divalent metal ions at micromolar concentrations [5]. It is likely that these ions compete for the magnesium cofactor site. Characteristics of the metal center of EcoRI are thus beginning to emerge. EcoRI contains at least one tightly bound zinc ion per monomer which is in accessible to solvent and cannot be removed by exhaustive dialysis against anionic chelating agents. The apoenzyme, which is inactive, can however be obtained by extensive dialysis against the neutral ortho-phenanthroline; this perhaps indicates that the metal resides in a hydrophobic pocket. The cobalt derivative is in contrast easily prepared by direct displacement of the bound zinc ion. The small hydrated cobaltous ion gains access to the metal center and like zinc(II) binds tightly and subsequently appears non-dialyzable. Co-EcoRI exhibits high activity and the same substrate specificity as native Zn-EcoRI. Other examples of high activity in cobalt derivatives of zinc enzymes are well known; Co(II)-carboxypeptidase, for instance, displays a three-fold enhancement in peptidase activity over the native enzyme [6]. The similar substrate specificity of Co-EcoRI and Zn-EcoRI likely reflects the similar chemistry of these two metal ions. Co-EcoRI is certainly more hardy than the native enzyme, and derivatization with cobaltous ion may be generally helpful to improve enzyme lifetime for biomedical applications. Other metal derivatives of EcoRI will be important to examine in order to determine the contribution of the metal center to both the specific DNA binding and endonucleolytic steps of enzyme action.

In non-polar solvents, chiral europium complexes provide attractive n. m. r. shift reagents to resolve spectra of optically active cations, and, in particular, for tris(phenanthroline)ruthenium dichloride,^1H n. m. r. shift differences of up to 0.7 p.p.m. between isomers easily permit the determination of absolute enantiomeric purity.